!

Power of thought!

32nd Cyprus Noordwijkerhout- Camerino Symposium

Trends in Drug Resarch

Limassol , Cyprus, May 18-22, 2014

!
Power of thought!

Registered under the Cyprus Company Law CAP. 113 as a Private non-for-profit
Company with limited liability by guarantee.
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Reproduction is authorized provided the source is acknowledged

Copyright: Quintessence Enterprises Ltd

The Book of Abstracts of the Trends in Drug Research 32nd Cyprus

NoordwijkerhoutCamerino Symposium was published in May
2014 in 250 copies.
Cover design: Lightblack Solutions Ltd

ISBN 978-9963-9615-3-5

Committees of the 32nd Cyprus-Camerino-Noordwijkerhout

Trends in Drug Research
Symposium
Honorary Chairman
A.Makriyannis
Chairmen of the Symposium
A. Tsotinis (University of Athens)
T. Mavromoustakos (University of Athens)
Organizing Committee
H. Timmerman (VU University, Amsterdam, Netherlands)
R. Leurs (VU University, Amsterdam, Netherlands)
M. Giannella (University of Camerino, Italy)
P. Angeli (University of Camerino, Iataly)
Del Bello Fabio (University of Camerino, Italy)
Gianfabio Giorgioni (University of Camerino, Italy)
C. Petrou (University of Nicosia, Cyprus)
C. Potamitis (National Hellenic Research Foundation, Greece)
T. Tselios (University of Patras, Greece)
A. Afantitis (Nova Mechanics, Greece)
A. Tsantili-Kakoulidou (University of Athens, Greece)
D. Hadjipavlou (University of Thessaloniki, Greece)
P. Koutentis (University of Cyprus, Cyprus)
G. Liapakis (University of Crete, Greece)
Scientific Committee
R. Hubbard (University of York, UK)
D. Sugden (Kings College, U.K.)
P. J. Garratt (University College London, U.K.)
M. Rappolt (University of Leeds, UK)
Alexander Tropsch (University of North Carolina, USA)
G. Kokotos (University of Athens, Greece)
P. Marakos (University of Athens, Greece)
N. Pouli (University of Athens, Greece)
S. Noskov (University of Galgary, Canada)
A. Goutopoulos (EMD Serono Pharmaceutical Company, USA)
S. Durdagi (University of Bahcesehir, Turkey)
S. Golic Grdadolnik (National Institute of Chemistry, Slovenia)
C. Glaubitz (Institute of Biophysical Chemistry Goethe UniversityFrankfurt, Germany)
D. Papahadjis (National Hellenic
Research Foundation, Greece)
M. Koufaki (National Hellenic Research Foundation, Greece)
T. Calogeropoulou (National Hellenic Research Foundation, Greece)
Jean-Paul Renaud, NovAlix, France
Charlton Steven, Novartis, UK
Supporting Organizing Team
D. Ntountaniotis, C. Koukoulitsa, G. Melagraki, T. Kellici, E.
Vrontaki, A. Tsatsaroni

Contents
The address from the Organizers

The Chairman to the meeting

10

Minister of Health address to the meeting

11

Scientific Program

15

Lipidic soft self-assembled drug nanocarriers:

Cubosomes, hexosomes, and in situ formation of cubic and hexagonal liquid
crystalline phases

23

Observed drug-receptor association rates are governed by membrane affinity: The

importance of establishing micro PK/PD Relationships

24

The potential of solid-state NMR in drug research

25

Gating Pathways in Secondary Transporters from NSS family

26

Insights into the molecular basis of action of the AT1 antagonist losartan using a
combined NMR spectroscopy and computational approach.

27

Current Perspectives in Fragment Based Lead Discovery.

34

Honorium to Prof. A. Makriyannis

35

Serotonin 5-ht2 receptor structure-based drug design leads to clinical candidates for
neuropsychiatric disorders.

36

Structural and Dynamical Properties of Indomethacin Molecules embedded within the

Mesopores of Silicates and Metal-Organic Framework Materials: a Solid-State NMR
37
View.
Targeting promising inhibitors for HCV: A multi step chemoinformatics approach
A Scientific Workflow Management System for Virtual Screening in Cancer
Chemoprevention.

44

Molecular Dynamics in Drig Design: Development of Compstatin-based Compounds

for the Regulation of the Key Complement System Protein C3.
46
Interaction of inhibitors with aspartic proteases

47

Structure-Based Design of Non-Peptide Mimetics for the Treatment of Multiple

Sclerosis
Novel approach in Multiple Sclerosis treatment

53

NMR metabolomics in diagnosis and toxicology

NMR structures and cation binding of G-quadruplexes with unique features

60

NMR-assisted discovery of novel inhibitors of protein targets:

owards new therapeutic agents

65

Insights on the conformational plasticity of drug-target proteins

68

Stereoelectronic control of AT2R/AT1R subtype selectivity in angiotensin II

analogues and their potential in cancer therapy

69

Conformational studies of LHRH analogue conjugated with E- cyclodextrin using 2D

NMR and Molecular Modeling
70
NMR structure and function of the mutated forms of the RING-H2 domain of Arkadia 75
Structural NMR Study of four viral Macro Domains

76

Targeting a heteromeric Glutamate/Serotonin GPCR complex involved in psychosis

77

Synthesis of Biological Compounds Against Alzheimer Disease

78

Novel Cannabinergic Ligands

85

Systems Medicine, Applying Systems Biology Approaches for Manipulation of

Altered Biomechanisms towards Multiple Sclerosis Treatment by the Use of Specific
Structured molecules and Antioxidant Vitamins: the PLP10 intervention Paradigm.
86
Natural Products for Cancer Treatment: Tripterygium wilfordii and Amygdalin
Promotes cell death in Cancer Cells but not Normal Cells

103

Poly-aromatic heterocyclic AMPK activators: the new platform for developing of

bifunctional drugs against type two diabetes

104

Effect of metal ions on fetal hemoglobin induction by hydroxyurea in erythroid cell

107
lines
Targeting Transcription factors with DNA interactive small molecules

108

Novel Inhibitors for the Aspartic Protease Endothiapepsin by Combining StructureBased Design, Dynamic Combinatorial Chemistry and STD-NMR Spectroscopy.
109
Dynamic Combinatorial Mass Spectrometry: a powerful tool for quick identification
of selective 2-oxoglutarate oxygenases inhibitors
De Novo Fragment-Based Design of Inhibitors of DXS Guided by Spin-DiffusionBased NMR Spectroscopy
116
Copper Coordination Compounds As Antimicrobial Agents

123

Fraction lipophilicity index (FLI): A Metric for assessing oral drug likeness of
chemical entities

124

New antivascular agents in the combretastatin A-4 series

129

Tocopherol/Tocotrienol Carboxylate Functionalized Conjugated - Compounds

exhibiting Apoptotic/Anticancer Properties.

130

The Design andEvaluation of a Multi- level Cross Flow Filtration Microfluidic

Device for the Separation of Human Blood Cells.

131

Interactions of Silybin A with cyclodextrin derivatives using solid and liquid state
NMR spectroscopy, differential scanning and isothermal titration calorimetry as well
as molecular dynamics simulations.
147
An in silico effort to discover new HCV Replication Inhibitors

148

Stability and Binding Effects of Ag Metal Complexes at LOX-1.

149

New N-alkanoyl-4-methoxybicyclo[4.2.0]octa-1,3,5-trien-7-ethanamines:
melatoninergic action and controlled release from solid pharmaceutical formulations. 150
Zanamivir Conjugates Linked with Anti-inflammatory Agents Exhibit Enhanced
Anti-influenza Activity.

151

Design and synthesis of non-peptide mimetics based on trimolecular complex

HLA/antigen/TCR

152

Molecular Dynamics Simulations and MM-PBSA Binding Free Energy Estimation in

GK241-sPLA2 GIIA complex.
153
Hydrazones of 5-nitro-2-furanecarboxaldehyde with
1-adamantanealkanoyhydrazides with probable trypanocidal activity.

154

Effect of metal ions on fetal haemoglobin induction by hydroxyurea in erythroid cell

lines.
155
The Thalidomide Impact on Patients with Multiple Myeloma. The Revival of an Old
Story through the Histological and Immunohistochemical Study of 100 Cases.
156
Delineation of Molecular Mechanisms of Action of Decitabine for the Treatment of thalassaemia.
157
Novel 1,4-dioxane derivatives as NMDA receptor channel blockers.

158

Developing in silico tools towards the personalized therapy of beta-thalassemia.

159

Rational design and synthesis of potent cyclic Luteinizing Hormone Releasing

Hormone LHRH analogues.

160

In silico High-Throughput screening for Kinase Profiling as a platform for Drug

Discovery.

161

Paediatric vagus nerve stimulation: a retrospective study

163

CURRICULM VITAE
SPONSORS

177

The Organizers Address to the Symposium

It is with great honor and proudness that we, Synnous and Quintessence Enterprises
Ltd, the organizers of this symposium, welcome you all in the island of Cyprus,
coming from all over the world, to share your deep secrets of knowledge and get back
much more from your colleagues.
This medicinal chemistry symposium entitled Trends in Drug Research takes place
for a 32nd time. This year is the turn of Cyprus again to organize this valued CyprusNoordwijkerhout-Camerino Symposium.
The Organizers of this Symposium express their gratitude for your kind eagerness to
cooperate with us and make this scientific event happen.
Gratitude is also attributed, for their contribution to this event, to the sponsors of the
symposium, who, even in times of recession, support research and symposia. In fact,
it is now, more than ever that we need research to be enhanced.
In this 32nd symposium, forty-five announcements are presented orally and sixteen
are posted, thus giving the opportunity to interact, exchange views and philosophies,
preview cutting edge technologies in drug discovery and get glimpse of things to
come.
All announcements are published in the book of abstracts of the symposium. This
book has one hundred eighty pages and is registered with an international ISBN
number for citation purposes. Fifteen of the announcements are published as long
abstracts; an innovation in this book, for the scientists to extrapolate further in their
deep quest.
Synnous, meaning thoughtful, is a think-tank promoting the power of thought. The
members of Synnous are individuals with active role and strong initiative towards
creativity and innovation. Within this context Synnous promotes the sustainable
development of the intellectual entropy, i.e. the development of methodologies to
solve more and more complicated problems.
Synnous, together with Quintessence Enterprises Ltd; a vocational Training Centre,
undertake the organization of this symposium to contribute to the scientific advances
in medicinal chemistry and drug discovery.
The vision of the organizers of this Trends in Drug Research Symposium is to offer
the platform to medicinal chemistry to contribute to the improvement of the quality of
biological and spiritual life. We are strongly committed to continuously support your
scientific work and make this very island the cradle of drug research and medicinal
chemistry innovations.
We hope that this symposium will contribute to the reengineering and make science a
servant of peace and prosperity. We wish this symposium to be a milestone in the
history of medicinal chemistry and the scientific career of each one of you.
The locale of the symposium, Cyprus, and the associated social program are so
chosen and interrelated, in a way to contribute to the whole vision of the organizers.
Tomorrow, Tuesday, we are visiting the town center of Limassol. Under the auspices
of the honorable Mayor of Limassol, the municipality of Limassol will offer a tour to
the old town of Limassol.
After that, we have the gala dinner in Limassol.
On Wednesday we are visiting, Chirokitia an archeological site dated from the
neolithic age, listed as a world heritage site by UNESCO since 1998. The site is
known as one of the most important and best-preserved prehistoric sites of the eastern
Mediterranean. Much of its importance lies in the evidence of an organized functional

society in the form of a collective settlement, with surrounding fortifications for

communal protection.
Then, we go to Nicosia, the capital of Cyprus, to visit the Cyprus Archeological
museum. The museum houses artefacts discovered during numerous excavations on
the island. It is home to the most extensive collection of Cypriot antiquities in the
world.
The visit to Chilokitia and the Museum is offered by the Department of Antiquities of
the Ministry of Communications and Works.
We then have the chance to walk around the narrow streets of the old city of Nicosia,
enjoy the traditional food-meze and drink the local varieties of xynisteri and
maratheftico wines.
It is important to note that the historic center of Nicosia is under construction! We
hope that its division will soon be over. May you are lucky to keep saying, from now
on, that you had the depressive privilege to see the capital of Cyprus still divided, as
soon after, hopefully, is being reunited and thrived; as in the past, when all Cypriots
used to live in peace and prosperity.
May you, the honorable delegates of this symposium, be the precursors and
harbingers of peace and the ambassadors of prosperity in this little country, looking
forward to, Cyprus, being the cradle of this Trends in Drug Research Symposium.
We wish you a fruitful symposium and look forward to enjoying the knowledge and
expertise coming out of this meeting.
Evsevios Hadjicostas
Chairman of the Organizing Committee

TheChairmenaddresstotheSymposium
Thisscientificeventisacontinuation ofaseriesofMedicinalChemistrySymposiainitiatedbyProf.A.
Makriyannisin1983.Theyhavebeenservedasabiennialopportunityforresearchersandmanagement
executivestointeract,exchangeviews,previewcuttingedgetechnologiesinDrugDiscoveryandgeta
glimpseofthingstocome.
Duringtheclosingaddressofthe16thCamerinoNoordwijkerhoutsymposiumin2007,MarioGiannella,
Henk Timmerman and Alexandros Makriyannis announced an agreement of joining the Camerino
Noordwijkerhout Symposia with Conferences, already run in Cyprus, establishing thus the CNC
Symposia. The Symposia became annual, taking place every year, sequentially, in Cyprus, Holland and
Camerino.he29thTrendsinDrugResearchCyprusNoordwijkerhoutCamerinoSymposiumwasheldin
LimassolCyprus, followed by the 30th  NoordwijkerhoutCyprus Camerino Symposium, held in
Amsterdam (May 1317, 2012) and the 31st CamerinoCyprusNoordwijkerhout Symposium held in
Camerino (May 1923, 2013). We have the pleasure this year to address the 32nd Trends in Drug
ResearchCyprusNoordwijkerhoutCamerinoSymposium,whichistakingplaceinLimassolofCyprus.
InthelastSymposiumaquestionnairewasaskedtobecompletedbytheparticipants,whorequested
moreshort15minutestalksbyyoungscientiststobeincludedinourScientificProgramandtoshorten
the duration of the Symposium by one day in order for the guests to have more time to enjoy the
beautiesoftheisland.Bothoftheserequestshavebeenmet.
The venue of the Conference remained the same (Grand Resort Hotel), a seaside fivestar Hotel in
Limassol,Cyprus.Theparticipantshavetheopportunitytoenjoyswimminginthewarmandsandysea
ofCyprusandcombinesciencewithrelaxation.Agaladinnerisofferedtotheparticipantswherethey
canenjoythetraditionalcuisineandbeveragesofCyprus.Anexcursionisplanned,fortheparticipants
to be briefed to the history of the island and enjoy the archaeological treasures of Cyprus. After the
Conferencelectures,theparticipantswillhavetheopportunitytoenjoythebeautifulcityofLimassol.
We believe that the seaside locale of the venue of the Symposium and the islands natural beauty
guaranteeapleasantandproductiveweekforallparticipants.

Thechairmen
AndreasTsotinis
ThomasMavromoustakos



10

Minister of Health address to the meeting

Ladies and gentlemen,
It is with a great sense of honour and elation that I am among you today, given the
opportunity to address such a distinguished audience.
Please allow me to welcome those of you who have travelled long distances to be here among
us and share your valuable knowledge and expertise.
I feel very proud that Cyprus has been actively involved in the organisation of such symposia
since 1983, having already hosted the 26th symposium in 2008, the 29th symposium in 2011
and now the 32nd. The continuity of these events for so many years is a testament to their
success.
The historical ambience and geographical location of Cyprus as well as the dedication of our
local scientists, present a perfect location for the hosting of such events. I trust that the
knowledge and expertise shared here today will pave new and exciting paths in humanitys
quest for treating illness as well as advancing and promoting science, a common goal shared
by the scientific community throughout the world.
It is this very understanding of the interplay between biological activity and chemical
structure that intrigues the participants of this symposium that has allowed us to make great
advances by introducing novel medicines for the treatment of illnesses that in turn have
played a key role in the improvement of both the quality of life and life expectancy.
Currently, the field of healthcare in Cyprus is in a state of flux and facing numerous
challenges. The current economic crisis has revealed many inherent weaknesses that need to
be firmly and permanently addressed. We view this crisis not just as a setback but also an
opportunity for deep introspection and to bring forth the necessary changes and adjustments
in order to move forward. Within this context, we envisage the implementation of the
National Healthcare Insurance Scheme as a measure that will enable the seamless provision
of high quality health-care to all citizens of Cyprus keeping in mind that the emerging public
health needs of a perpetually evolving and transforming society need to be met in an effective
and efficient manner where broad changes will require swift implementation.
It is in the scientific advances, new technologies and perspectives such as the ones to be
presented and discussed in this symposium, which we look upon to draw not just knowledge
but also inspiration and vision. I anticipate that this discourse will help create, sustain and
propagate this vision for the benefit of public health.
I would like to conclude my address by congratulating everyone involved in the successful
organisation of this symposium. I, further, extend my gratitude to all for this commendable
effort.
I wish you a successful symposium and look forward to welcoming you again on the
subsequent meetings to be held in Cyprus.
Philippos Patsalis
Minister of Health

11

SCIENTIFIC PROGRAM

10

Scientific Program
Sunday 18.5.2014
16:00-18:30

Registration and Poster Set-Up

18:30-18:55

Welcome

18:55-19:00

A.Keramidas,AcommunicationplatformfortheChemistsin
Cyprus

19:00-19:30

C. Barlos, The history of a global peptide manufacturing and

technology company (CBL) and drug synthesis, Greece.

19:30-20:00

N. Peristianis, Education in Cyprus

20:00-22:00

Welcome Reception

Monday 19.5.2014
8:00-8:30

Registration

8:30-8:45

Welcome by the Chairmen

BIOPHYSICAL METHODOLOGIES AND DRUG DISCOVERY, Part I

Chair

C. Glaubitz and A.Yagmhur

8:45-9:30

A.Yaghmur, Lipidic Soft self-assembled drug nanocarriers:

cubosomes, hexosomes, and in situ formation of cubic and hexagonal
liquid crystalline phases, Denmark.

9:30-10:15

S. Charlton, Observed drug-receptor association rates are governed by

membrane affinity: The importance of establishing micro PK/PD
Relationships, Novartis, UK.

10:15-10:45

Coffee Break

10:45-11:30

C. Glaubitz, The potential of solid-state NMR in drug research:

Examples from GPCRs and Multidrug Effliux Pumps,
IBMRZ, Germany.

11:30-12:15

S. Noskov, Gating Pathways in Secondary Transporters from NSS

family, Canada

12:15-12:30

M. Zervou, Insights into the molecular basis of action of the AT1

antagonist losartan using a combined NMR spectroscopy and
computational approach, Greece.

12:30 15:00 Poster Session - Lunch Time

15

BIOPHYSICAL METHODOLOGIES AND DRUG DISCOVERY-Part II

Chair

R. Hubbard and S. Charton

15:00-15:45

R. Hubbard, Current perspectives in fragment-based drug

discovery, UK.

15:45-16:00

T. Mavromoustakos, Honorium to Prof. A. Makriyannis

16:00-16:30 Coffee Break

16:30-17:15

Raymond G. Booth, Serotonin 5-HT2 Receptor Structure-Based Drug

Design Leads to Clinical Candidates for Neuropsychiatric Disorders,
USA.

17:15-18:00

Gregor Mali, Structural and dynamical properties of indomethacin

molecules embedded within the mesopores of silicates and metalorganic framework materials: a solid-state NMR view, Slovenia.

Tuesday 20.05.2014
IN SILICO STUDIES FOR RATIONAL DRUG DESIGN
Chair

S. Noskov, G. Archontis

9:00-9:30

G. Melagraki, A. Afantitis, Targeting promising inhibitors for HCV: A

multi step chemoinformatics approach, Cyprus.

9:30-10:00

V. Promponasa, C. Pattichis, A. Constantinou, Scientific Workflow

Management System for Virtual Screening in Cancer
Chemoprevention, Cyprus.

10:00-10:30 Coffee Break

10:30-11:00

G.Archontis, Molecular Dynamics in Drug Design: Development of

Compstatin-based Compounds for the Regulation of the Key
Complement System Protein C3,Cyprus.

11:00-11:30

M.G. Papadopoulos, The mechanism of binding of aspartic

proteases, Greece.

11:30-11:45

M. P. Yannakakis, T.Tselios, Structure-based design of non-peptide

mimetics for the treatment of Multiple Sclerosis, Greece.

11:45-12:00

A. Tapeinou , T. Tselios, Novel approach in Multiple Sclerosis

treatment, Greece.

12:00-14:00

Poster Session- Lunch Time

16

NMR AND MEDICAL APPLICATIONS

Chair

E. Mikros and J. Plavec

14:00-14:05

E. Mikros, A brief overview of the NMR status in the medical

applications University of Athens, Greece.

14:05-14:35

E. Mikros, NMR metabolomics in diagnosis and toxicology, Greece

14:35-15:20

J. Plavec, NMR structures and cation binding of G-quadruplexes with

unique features National Chemistry Institute, Slovenia.

15:20-15:50 Coffee Break

15:50-16:20

S. S. Golic Grdadolnik, NMR-assisted discovery of novel inhibitors of

protein targets: towards new therapeutic agents, Slovenia.

16:20-16:50

G. Spyroulias, Insights on the conformational plasticity of drug-targets

proteins University of Patras,Greece.

16:50-17:20

A. Tzakos, Sculpting stereoelectronic tuning to analogues selectively

targeting the AT2 receptor and their potential as very potent agents in
breast cancer, Greece.

17:20-17:30

G. Kordopati , Conformational studies of LHRH analogue conjugated

with cyclodextrin using 2D NMR and Molecular Modeling, Greece.

17:30-17:45

M. Birkou, C. Chasapis, Bentrop Detlef, Herrmann Torsten, V.

Episkopou, G.Spyroulias, NMR structure and function of the mutated
forms of the RING-H2 domain of Arkadia, Greece.

17:45-18:00

A. Tsika, D. Ntonti, S. Melekis, C. Chasapis, I. Margiolaki, N.

Papageorgiou, B. Coutard, D. Bentrop, G. Spyroulias, Structural nmr
study of FOUR VIRAL macro domains, Greece.

20:30 GALA DINNER

Wednesday 21.05.2014
COMPARING MAJOR AND NEGLECTED DISEASES
Chair

D. Logothetis and P. Koutentis

8:30-9:15

D. Logothetis, Targeting a heteromeric Glutamate/Serotonin GPCR

complex involved in psychosis,USA.

17

9:15-9:45

P. Koutentis, Synthesis of biological compounds against Alzheimer

disease, Cyprus.

9:45-10:15

S. Nikas, Novel Cannabinergic Ligands, USA.

10:15-10:45

Coffee Break

10:45-11:15

I. Patrikios, Systems Medicine, Applying Systems Biology

Approaches for Manipulation of Altered Biomechanisms towards
Multiple Sclerosis Treatment by the Use of Specific Structured
olecules and Antioxidant Vitamins: the PLP10 intervention
Paradigm, Cyprus.

11:15-11:45

A. Stephanou, Natural Products for Cancer Treatment: Tripterygium

wilfordii and Amygdalin Promotes cell death in Cancer Cells but not
Normal Cells, Cyprus.

12:30

EXCURSION

Thursday 22.05.2014
SELECTED TOPICS
Chair

A. Gruzman and M. Phylactides

8:00-8:45

A. Gruzman, Poly-aromatic heterocyclic AMPK activators: the new

platform for developing of bifunctional drugs against type two
diabetes, Israel.

8:45-9:15

M. Phylactides, Effect of metal ions on fetal haemoglobin induction

by hydroxyurea in erythroid cell cultures, Cyprus.

9:15-10:00

Khondaker Miraz Rahman, Targeting Transcription Factors with DNA

Interactive Small Molecules, UK.

10:00 -10:30 Coffee Break

Chair

M.R. Khondaker and M. Zervou

10:30-10:45

C. Potamitis, Novel Inhibitors for the Aspartic Protease

Endothiapepsin by Combining Structure-Based Design, Dynamic
Combinatorial Chemistry and STD-NMR Spectroscopy.,Greece.

10:45-11:15

M.Demetriades, Dynamic Combinatorial Mass Spectrometry:

a powerful tool for quick identification of selective 2-oxoglutarate
oxygenases inhibitors, U.K.

11:15-11:30

T. Masini, De novo fragment-based design of inhibitors of DXS

guided by Spin-diffusion-based NMR spectroscopy, Netherlands.

18

11:45-12:00

B.Miroslaw, Copper Coordination Compounds As Antimicrobial

Agents, Poland.

12:00-12:15

A. Tsantili-Kakoulidou, Fraction Lipophilicity Index (FLI):

A metric for assessing oral drug likeness of ioniazable chemical
entities, Greece.

12:15-12:30

O. Provo, New antivascular agents in the combretastatin A-4

series, France.

12:30-13:00 A. Keramidas, C. Drouza, E. Maltezou, A. Odysseos,

Tocopherol/Tocotrienol Carboxylate Functionalized Conjugated
Compounds with Apoptotic/Anticancer Properties, Cyprus
13:00- 13:30 . Zach Odeh, 
 

13:30-13:35

First prize poster award.

13:35-13:40

Second prize poster award.

13:40-13:45

Third prize poster award.

19

ORAL PRESENTATIONS

17

LIPIDIC SOFT SELF-ASSEMBLED DRUG NANOCARRIERS:

CUBOSOMES, HEXOSOMES, AND IN SITU FORMATION OF CUBIC AND
HEXAGONAL LIQUID CRYSTALLINE PHASES
Anan Yaghmur
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen, Denmark
Email: anan.yaghmur@sund.ku.dk

In this contribution, the main attention is to present our recent investigations on the formation, the
characterization, and the potential pharmaceutical applications of lipidic lyotropic non-lamellar
liquid crystalline phases and their corresponding nanostructured aqueous dispersions. The nonlamellar liquid crystalline phases in both the non-dispersed and the dispersed states represent a
unique class of nanomaterials that holds a promise in meeting the needs for efficient nanocarriers
that can be designed to control drug release. The fundamental research approach focuses an
approach that includes the structural characterization under non-equilibrium conditions upon the
exposure of these self-assembled nanocarriers to the biological environment, and the combination
of relevant in vitro and in vivo investigations. The main goal is to address the prominent factors
affecting the structural characteristics and the drug release properties of in situ formed highly
viscous non-lamellar liquid crystalline depots. These stimuli-triggered in situ forming drug delivery
systems are attractive due to their sustained release properties and they may offer also various
advantages including ease of administration, the use of less invasive small needles, and the
possibility of improving patient compliance due to a reduced frequency of administration. Besides
covering recent studies on the in situ formation of promising lipidic drug delivery systems, we aim
at presenting our recent studies on the use of nanoparticlulate formulations based on lyotropic liquid
crystalline particles (cubosomes and hexosomes) as drug nanocarriers. Our interest is also to focus
on designing cubosomes and hexosomes that can be labeled for use as imaging agents for
SPECT/CT and further investigating the in vivo performance of theranostic soft nanocarriers based
on cubosomes and hexosomes.

23

Observed drug-receptor association rates are governed by membrane affinity:

The importance of establishing micro PK/PD Relationships
Steven J Charlton
Novartis Institutes for Biomedical Research, Horsham, UK

Current pharmacological models for determining affinity and kinetics of drugs for membrane
receptors assume the interacting molecules are homogeneously distributed in the bulk aqueous
phase. The phospholipid membrane can, however, provide a second compartment into which
drugs can partition, especially lipophilic/basic compounds. This is particularly important for
drugs that have been specifically designed to have tissue affinity in order to enhance duration of
action after topical delivery, for example the inhaled 2-adrenoceptor agonists used to treat asthma
and COPD. To examine the effect that membrane affinity may have on observed pharmacology
we have measured and compared phospholipid interactions and receptor binding kinetics of
several clinically relevant 2-adrenoceptor agonists and antagonists. We find that the degree of
phospholipid interaction is directly related to the observed kinetic association rate (kon) and
affinity (KD), but not the dissociation rate (koff) from the target, presumably by concentrating
drug in the local environment around the receptor. When the local drug concentration is
accounted for, the kon is comparable across the cohort and the corrected KD is directly related to
the koff. We have also applied this approach to other receptors and shown that this relationship is
not unique to the 2-adrenoceptor. In conclusion, we propose a new approach to determining the
pharmacology of drugs for membrane targets that accounts for differences in local drug
concentration brought about by direct affinity for phospholipids, establishing micro PK/PD
relationships for drugs.


24

The potential of solid-state NMR in drug research

Clemens Glaubitz
Institute for Biophysical Chemistry
Centre for Biomolecular Magnetic Resonance (BMRZ)
Goethe University Frankfurt
glaubitz@em.uni-frankfurt.de
www.bmrz.de
Solid-state NMR and in particular magic angle sample spinning (MAS) is a potentially
powerful method to support the process of drug discovery and development starting from
compound design and characterisation, via binding site mapping and bioavailability analysis
to quality control of active pharmaceutical ingredients e.g. through polymorphism studies 1. In
particular, solid-state NMR is applied to problems involving membrane embedded target
proteins, which are more difficult to access by other methods. In the first part, it is
demonstrated how the membrane interaction of lipophilic drugs can be screened by MASNMR. Examples include antibiotics, which are substrates of P-glycoprotein 2, COX-2
inhibitors 3 and lipid-ceramide interactions. The second part will focus on the interaction of
small molecules with membrane protein targets as demonstrated for the case of the human
bradykinin B-2 receptor, a major drug target 4. The structure of its native agonist bradykinin
has been determined, which could serve as a lead structure for synthetic ligands. The
examples presented here will be discussed in the context of latest technical innovations such
as dynamic nuclear polarisation, which offers a sensitivity enhancement for solid-state NMR
by orders of magnitude.
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25

Gating Pathways in Secondary Transporters from NSS family

Sergei Noskov
Centre for Molecular Simulations and Department of Biological Sciences
University of Calgary, Calgary, Alberta, Canada

Members of the NSS family are being increasingly implicated in a variety of psychiatric disorders1.
They are important targets for antidepressant medications but have also been identified as targets for
drugs of abuse. The recent publication of several crystal structures for bacterial homologue LeuT and
dopamine transport (DAT) in complex with tricyclic antidepressants and selective-serotonin
inhibitors provided fresh impetus for understanding how small molecules affect the dynamics of NSS
transporters. It has been shown previously that Na+ binding can stabilize the outward-open
conformation of prokaryotic NSSs to facilitate extracellular substrate access to the substrate site(s).
In this work, we used combination of the free energy and molecular dynamics (MD) simulations to
document a transition to an outward-open state similar to that of the inhibitor-stabilized structure
(PDB: 3F3A2) by starting from the outward-occluded state of LeuT (PDB: 2A653) and investigating
the evolution of the system in the presence of bound Na+ but in the absence of any substrate. We
showed that the degree of such openness is modulated by Na+ binding at the Na1 and Na2 sites in
the central binding pocket, as well as the protonation state of the negatively charged residues in the
proximity of binding pocket. We were able to show that this mechanism also exists in hSERT
transporters (ELife, 2014, in review). This is most likely achieved through reconfiguration of a
network of dynamically-coupled structural motifs and microdomains that can propagate a local
structural perturbation, such as that resulting from ion binding, to impact the entire protein4. Such
local configurational changes labeled here as Allosteric Interaction Networks (AIN) then cause
changes in the global conformational propagation and dynamics involved in traversing the various
transporter states during the transport cycle. Complimentary FRET analysis of LeuT transporter
gating dynamics suggests that ion-specific effects are propagated from the binding site through a
cluster of aromatic residues closely associated with the substrate-binding (S1) site, resulting in
different configurations in the middle of TM10. Thus, short-range local changes produced in the
region of the ion binding sites, and the resulting long-range critical allosteric changes link the
substrate binding sites to the intracellular gate region as described by us previously. By using newly
developed analysis protocols (in collaboration with the H. Weinstein and L. Shi labs at Weill Cornell
Med) specifically designed to handle micro-seconds long MD simulations, we will be able to
quantify significant alterations in global conformation and associated local pairwise residue
interactions, and deduce the identity and dynamics of the AIN in each case.

REFERENCES
1.
Lee, S.H., et al. Genetic relationship between five psychiatric disorders estimated from genome-wide
SNPs. Nature Genetics 45, 984-+ (2013).
2.
Noskov, S.Y. Molecular mechanism of substrate specificity in the bacterial neutral amino acid
transporter LeuT. Proteins-Structure Function and Bioinformatics 73, 851-863 (2008).
3.
Yamashita, A., Singh, S.K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial
homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 437, 215-223 (2005).
4.
Zhao, C.F., et al. Ion-Controlled Conformational Dynamics in the Outward-Open Transition from an
Occluded State of LeuT. Biophysical Journal 103, 878-888 (2012).


26

Insigghts into the

t moleccular basis of action
n of the AT1
A antaggonist losaartan
ussing a com
mbined N
NMR spectroscopy and comp
putationaal approacch
Maria Z
Zervoua, Zooe Courniab, Constantin
nos Potamittisa, Georgee Patargiasb, Serdar Durrdagia,1,
a
S
Simona
Gollic Grdadolnnik c,d, Thom
mas Mavrom
moustakosa,e
a

National Hellenic Research Foundaation, Institutte of Biology

y, Medicinal Chemistry &
b
Biotechhnology, Vaas. Constantinnou 48, 116335 Athens, Greece;
G
Biom
medical Reseearch Foundaation of
the Accademy of Athens,
A
4, Sorranou Efesioou, 11527 Athhens, Greecee; c Laboratorry of Biomollecular
Structture, Nationaal Institute off Chemistry, Hajdrihova 19, POB 30,, SI-1115 Lju
ubljana, Slovvenia; d
e
EN-FIS
ST Centre off Excellence, Dunajska 1556, SI-1000 Ljubljana,
L
Sllovenia; Ch
hemistry Deppartment
of Natioonal Capodisstrian Univerrsity, Zographhou, Athens 15784, Greeece; 1Currentt address: Baahcesehir
University, Faculty of Medicine,
M
Deepartment of Biophysics, 34353 Istan
nbul, Turkey.
Emaill: mzervou@
@eie.gr

The drrug:membraane interacttions for the

t
antihyppertensive AT1
A
antago
onist losarttan, the
prototyppe of the saartans class, were studieed using an integrated approach
a
[1
1].
Throughh studyingg a. the drug
d
pharm
macophore arrangemennt in soluution enviroonments
mimickking biologiical conditions and b. drug: mem
mbrane inteeractions in modelled micellar
aggregaates and DP
PPC bilayeers with thee AT1 receeptor embeedded, we provide siggnificant
insightss into the bioactive conformatio
c
on of losarrtan, an atoomic-level descriptionn of the
losartann-membranee interactionns and partiitioning intoo the membbrane as welll as investiigate the
potentiaal pathwayss of entrancee towards th
he AT1 actiive site.
The connformationaal features and
a dynamics of the drrug were invvestigated in
i DMSO, D2O and
SDS m
micelles. Usiing intermoolecular ROE interactioons and NM
MR relaxation probes we
w have
further elucidated the
t drug toppography inn the micellar aggregatte and the preferred oriientation
flexible pharrmacophorees.
of the fl
MD sim
mulations inn both micelllar and lipid bilayers with
w the AT
T1 receptor embedded
e
p
provided
strong eevidence i for the spoontaneous innsertion of losartan in the lipidic core. Moreover, a
long exxploratory unbiased
u
M run (58
MD
80 ns) in the
t membraane:receptorr modelledd system
enabledd the moniitoring of the diffusio
on pathwayys of the drug and the interm
molecular
interacttions.
The vallidity of ouur integratedd approach is evidenceed by the reemarkable convergenc
c
ce of the
experim
mental and the theoreticcal data.
The aqqueous-mem
mbrane "waalk" of losaartan at atoomic resolu
ution was implemented as a
continuuation to ou
ur previous studies on the interacttions of thee amphiphilles AT1 anttagonists
with meembranes and
a the posssible impliccation of the latter in the
t effective interactioon of the

drug wiith its transm

membrane target
t
[2-4]..
REFER
RENCES
[1]. Zerrvou, M., et al,
a Biochim. Biophys.
B
Actta-Biomembaanes 1838, 10311046, 2014.
2
[2]. Fottakis, C., et al,
a J. Phys. Chem.
C
B 1155 (19), 6180-6192, 2011.
2012.
[3]. Fottakis, C., et al,
a Biochim. Biophys. Acta-Biomembanes 1818, 31073120,
3
[4]. Pottamitis, C., ett al, J. Chem
m. Inf. Modeel. 49, 726739, 2009.

27

Insights into the molecular basis of action of the AT1 antagonist losartan
using a combined NMR spectroscopy and computational approach
Maria Zervoua, Zoe Courniab, Constantinos Potamitisa, George Patargiasb, Serdar Durdagia,1,
Simona Golic Grdadolnik c,d, Thomas Mavromoustakosa,e
a

National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry &

Biotechnology, Vas. Constantinou 48, 11635 Athens, Greece; b Biomedical Research Foundation of
the Academy of Athens, 4, Soranou Efesiou, 11527 Athens, Greece; c Laboratory of Biomolecular
Structure, National Institute of Chemistry, Hajdrihova 19, POB 30, SI-1115 Ljubljana, Slovenia; d
EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia; e Chemistry Department
of National Capodistrian University, Zographou, Athens 15784, Greece; 1Current address: Bahcesehir
University, Faculty of Medicine, Department of Biophysics, 34353 Istanbul, Turkey.
Email: mzervou@eie.gr

Introduction
Sartans are pharmaceuticals, which modulate the renin-angiotensin-aldosterone
system. They antagonize with high selectivity the binding of the vasoconstrictor hormone
Angiotensin II (ANG II) at the AT1 receptor. Losartan potassium is a paradigm of successful
rational drug design since its molecular structure is based on the mimicry of the C-terminal
part of ANG II [1].
The flexible pharmacophore segments of losartan (Scheme 1) allow it to adopt many
low energy conformers. In particular, the tetrazole and imidazole moieties may adopt an anti
or syn orientation with respect to the A phenyl ring plane. Superimposition studies with the
C-terminal segment of sarmesin, a competitive peptide antagonist of ANG II, showed an
excellent fit with the anti configuration of the tetrazole and imidazole rings [2]. Both
orientations have been determined in the crystals of losartan's anionic or neutral form [3].
The lack of crystallographic data for the human AT1 (hAT1) receptor which belongs
to the class A G-protein-coupled receptors (GPCRs) and the notable flexibility of losartan has
led to a discrepancy regarding the specific binding site and bioactive modes of the drug. The
ligand binding site has been determined based on homology modelling of the hAT1 or the rat
AT1 receptors using structural models of rhodopsin photointermediates and site-directed
mutagenesis studies [4].
Our previous studies using DSC, 13C MAS and 31P CP SS NMR spectroscopy in
DPPC bilayers loaded with losartan have confirmed the interaction of losartan with the
interface region of the bilayer while molecular modeling studies revealed favorable
electrostatic interactions between the hydroxyl and tetrazole groups of the drug with the polar
headgroups and diffusing water molecules [5]. ESR studies also confirmed the interaction of
the drug with phospholipid membranes leading to the speculation that losartan may exert
some of its effects through interaction with the lipids of the membrane bilayer besides its
direct antagonistic action on the receptor [6]. This data is consistent with our proposed
diffusion model according to which the favorable insertion of the drug into the interface is
required for its diffusion towards the transmembrane active site of the AT1 receptor [5a].
We have applied an integrated approach using NMR spectroscopy and computational
studies aiming to investigate the impact of membranes in losartan's pathway towards the AT1
receptor. Through studying a. the drug pharmacophore arrangement in solution environments

28

mimicking biological conditions and b. drug: membrane interactions in modelled micellar

aggregates and DPPC bilayers with the AT1 receptor embedded, we provide significant
insights into the bioactive conformation of losartan, an atomic-level description of the
losartan-membrane interactions and partitioning into the membrane as well as investigate the
potential pathways of entrance towards the AT1 active site [7].
Experimental part
NMR spectroscopy: The conformational features of the drug were investigated by ROESY
NMR spectroscopy in water, in the lower dielectric constant amphiphilic DMSO solvent
mimicking the membrane head-group/water interface, and ultimately in SDS micellar
solution, which probes the hydrophobic interior of the bilayers.
2D DOSY NMR spectroscopy was used to follow the association of losartan with micelles.
ROESY spectroscopy determined drug:micelle intermolecular interactions.
Paramagnetic spin relaxation agents 5-doxylstearate (5-DSA) and 16- doxylstearate (16DSA) were introduced to the micelle solution to study the topographical sites of the drug.
Molecular Modeling: We have applied
x Stochastic dynamics simulations of losartan with NMR-derived distance restraints in
implicit solvent accounting for DMSO, water, and micellar environments
(Macromodel 9.9)
x All-atom MD simulation of losartan in solvated SDS micelles (Desmond 3.1)
x All-atom MD simulation of losartan and AT1 in a solvated DPPC lipid bilayer
(GROMACS 4.5.4)
Results and Discussion
Conformational flexibility of the drug in the different environments. The conformational
flexibility of the drug in DMSO and micellar environments emerges to be very similar
displaying a plethora of ROEs between the butyl chain protons and the A ring as well as
between the chain protons and the spacer methylene, suggesting a predominant arrangement
of the chain in a close proximity with the A ring. On the other hand, an enhanced
conformational mobility of the chain was observed in the aqueous environment in agreement
also with crystallographic studies of losartan potassium.
The experimentally derived interatomic distances could not determine unambiguously
whether the anti or syn configuration of the drug is the predominant one in solution. MD
simulations in implicit solvation models driven by ROE interatomic distances support the
interconversion between the anti and syn conformation , which are allowed by the
energetically favourable rotation around the critical torsion angle 3, which bridges the
biphenyl tetrazole with imidazole.

29

Scheme 1

Figure 1. Heat map illustrating the energy landscape (kcal mol -1) resulting from the grid scan search
by varying the dihedrals 2 and 3. Low energy conformers bearing the anti (A and B) or the syn (C
and D) conformation are depicted. The energy minima are located symmetrically at 2=600, 1200
indicating the unobstructed fluctuation of the phenyl-tetrazoyl moiety around the vertical position
with respect to the neighboring A phenyl ring.

Diffusion experiments Diffusion experiments confirmed the association of the drug

molecules with the micellar aggregate. Inspection of a single set of 1H resonance lines as well
as the observation of a single series of DOSY traces corroborate with a fast chemical
exchange between the free and the micelle-bound states of the drug. Moreover, DOSY
spectroscopy provided evidence for a concentration dependent self-assembly behavior in
aqueous solution which is possibly associated
with the charged tetrazole moiety of the drug and the presence of K+ counterions.
Losartan localization in the micellar environment. 5-DSA spin label has a nitroxide near
the head of the stearate and induces the paramagnetic broadening of the NMR signals of the
drug moieties in the proximity of the micelle surface while 16-DSA spin label has a nitroxide
near the end of the alkyl chain of the stearate and affects the resonances of losartan protons
located deep in the hydrophobic region of the micelles.
The obtained results support a location of losartan near the micellar surface since the
interaction with 5-DSA was notably more prominent than the one with 16-DSA. Furthermore,
evidence of the preferred orientation of losartans pharmacophores was also provided.
Specifically, the hydroxymethyl group and B ring proton H22 adjacent to the tetrazole face
the micelle-water interface, whereas the butyl chain and the B ring protons H19 and H20 are
interacting with the hydrophobic center of the micelle

30

Probing drug: micelles intermolecular interactions. Strong intermolecular ROEs are

observed between the drug protons and SDS protons S3-11, which are attached to the third
carbon after the sulfate group till the penultimate one, The obtained results indicate clearly
that losartan is distributed in the
vicinity of the hydrocarbon chains.
Furthermore, the protons of the A
ring as well as the spacer mrthylene
appear to be in contact with SDS
proton S2 attached to the second
carbon after the sulfate group,
providing evidence that losartan is
also interacting with the head groups
of the micelle. The lack of NOEs
between the B aromatic ring and
hydroxymethyl protons with S12
further supports their orientation
towards the polar headgroup of the
micelles.
Figure 2: ROE interactions between the drug and the micelle. Overlaid are expanded regions
from the spectra of losartan in SDS-d25 (red traces) indicating the drug intra- NOE
connectivities and of losartan in SDS-H25 (grey traces) corresponding to both intra- and
inter- interactions.
MD simulations of losartan
loaded
SDS.
Losartan
anionic form initially was
placed randomly ~5 away
from the surface of SDS
aggregates in the aqueous
Figure 3
environment.
The
drug
approaches within the first 2 ns the micellar surface from the aqueous phase and then it is
immersed within the micelle and diffuses between the sulfate group and the upper part of the
dodecyl chains. This localization favors the formation of hydrogen bond interactions between
the hydroxyl group of losartan and the sulfate group of SDS. The simulation points to the anti
conformation as being most favorable in the micellar environment (Figure 4).

31

Figure 4. The heat maps depict the distribution of losartan torsion angles 2 and 3
throughout the simulation time in SDS micelles for the anti (A) and the syn (B) initial
conformers. The anti conformation is characterized by values of 2 and 3 bearing the same
sign while the syn conformation by values of 2 and 3 of opposite signs.

Figure 5

MD simulations of losartan in a DPPC lipid

bilayer with the AT1 receptor embedded.
Losartan molecules were initially placed randomly
in the water and lipid bilayer phases at a minimum
distance of 20 from the AT1 receptor. Within
the first few ns all losartan molecules partition
spontaneously in the DPPC bilayer from the water
phase with the exception of one molecule, which
interacts with the extracellular part of the AT1
receptor. Likewise, all losartan molecules initially
placed within the lipid bilayer remain there for the
rest of the simulation time. Losartan molecules
diffuse freely along the membrane plane remaining
close to lipid head-groups and the water:bilayer.
Hydrogen bond analysis and calculation of the
radial pair distribution function g(r) confirmed that
tetrazole moieties interact mostly with the DPPC
glycerol backbone while hydroxyl methyls interact
predominantly with the phosphate group of DPPC.
Drug molecules do form hydrogen bonds with
water molecules that are close to or have
penetrated the lipid bilayer. The imidazole moiety
is buried under the hydroxymethyl and positioned
around the area of the glycerol backbone while it
exhibits occasionally polar interactions with water.

Convergence of the experimental and the simulation results is provided by: a) The
interatomic distances for losartan calculated from the MD simulations in micelles and in the
DPPC bilayer are in excellent agreement with the NMR measured values. b) The diffusion
coefficient of losartan calculated from the MD simulations in DPPC bilayer is D = 3.02 x 10 11
m2 s-1, is in excellent agreement with the diffusion constant derived from DOSY NMR
experiments in the micellar environment (D= 4.3 x10-11 m2 s-1). c) The spontaneous insertion
of losartan in the lipid core is confirmed by MD simulations in both micelles and lipid bilayer
with the AT1 embedded. The drug diffuses between the polar head-groups and the upper part
of the alkyl chain of the lipids, in agreement with NMR findings.

32

Conclusions
Overall, in silico and NMR approaches support the assumption that the membrane
environment serves as a pool of drug molecules locally trapped and potentially diffusing
towards the embedded AT1 receptor active site. This localisation restricts the drug flexibility
and probably
contributes to the effective interaction with the receptor while it could also probe alternative
pathways of entrance towards the AT1 active site beyond the random encounter at the
extracellular part.
Furthermore, it has been reported that the free drug molecules with high membrane affinity
could be trapped inside the membrane bilayer and consecutively bind to the same target
and/or targets nearby, even when their concentration in the bulk aqueous phase has already
dropped to insignificant levels [8]. In this respect, monitoring drug interaction with
membranes can provide valuable information for designing new long acting drugs.
The aqueous-membrane walk of losartan at atomic resolution was implemented as a
continuation to our previous studies [5a, 9] on the interactions of the amphiphiles AT1
antagonists with membranes and the possible implication of the latter in the effective
interaction of the drug with its target.
REFERENCES
[1] R.R. Wexler, W.J. Greenlee, J.D. Irvin, M.R. Goldberg, K. Prendergast, R.D. Smith, P.B.M.W.M.
Timmermans, J Med Chem, 1996, 39, 625.
[2] T. Mavromoustakos, A. Kolocouris, M. Zervou, P. Roumelioti, J. Matsoukas, R. Weisemann, J
Med Chem, 1992, 42, 1714.
[3] a) X.R. Hu, Y.W. Wang, J.M. Gu, Acta Crystal. E, 2005, 61, M1686, b) D. Fernandez, D. Vega,
J.A. Ellena, G. Echeverria, Acta Crystal.C, Crystal structure communications, 2002, 58, m418, c) L.
Tessler, I. Goldberg, Acta Crystal. E, 2004, 60, o1830.
[4] a) M.A. Bhuiyan, M. Ishiguro, M. Hossain, T. Nakamura, M. Ozaki, S. Miura, T. Nagatomo, Life
Sci, 2009, 85, 136, b) T. Tuccinardi, V. Calderone, S. Rapposelli, A. Martinelli, J Med Chem, 2006,
49, 4305, c) C. Baleanu-Gogonea, S. Karnik, J. Mol. Model., 2006, 12, 325.
[5] a) P. Zoumpoulakis, I. Daliani, M. Zervou, I. Kyrikou, E. Siapi, G. Lamprinidis, E. Mikros, T.
Mavromoustakos, Chem. Phys. Lip, 2003, 125, 13, b) C. Fotakis, D. Christodouleas, P.
Chatzigeorgiou, M. Zervou, N.P. Benetis, K. Viras, T. Mavromoustakos, Biophys. J, 2009, 96, 2227
[6] E. Theodoropoulou, D. Marsh, Biochim. Biophys. Acta, 1999, 1461,135.
[7] M. Zervou, Z. Cournia, C. Potamitis, G. Patargias, S. Durdagi, S. Golic Grdadolnik , T.
Mavromoustakos, Biochim. Biophys. Acta 2014, 1838, 1031.
[8] G. Vauquelin, S.J. Charlton, Br. J. Pharmacol. 2010, 161, 488.
[9] a) C. Fotakis, D. Christodouleas, P. Zoumpoulakis, E. Kritsi, N.P. Benetis, T. Mavromoustakos,
H. Reis, A. Gili, M.G. Papadopoulos, M. Zervou, J. Phys. Chem. B, 2011, 115, 6180, b) C. Fotakis,
G. Megariotis, D. Christodouleas, E. Kritsi, P. Zoumpoulakis, D. Ntountaniotis, M. Zervou, C.
Potamitis, A. Hodzic, G. Pabst,M. Rappolt, G. Mali, J. Baldus, C. Glaubitz, M.G. Papadopoulos, A.
Afantitis, G. Melagraki, T. Mavromoustakos, Biochim. Biophys. Acta , 2012, 1818, 3107, c) C.
Potamitis, M. Zervou, V. Katsiaras, P. Zoumpoulakis, S. Durdagi, M.G. Papadopoulos, J.M. Hayes,
S.G. Grdadolnik, I. Kyrikou, D. Argyropoulos, G. Vatougia, T. Mavromoustakos, J Chem Inf Model,
2009, 49, 726.

33

Current Perspectives in Fragment Based Lead Discovery

Roderick Hubbard
Fragment-Based Lead Discovery (FBLD) is now maturing as an approach to hit
identification, with many compounds now in clinical trials and the first compound on
the market. The central feature is that the drug discovery process begins with
identification (usually by biophysical methods) of small (<250 MW), weakly binding
(affinity of 100s of M) compounds which are then optimised to drug candidates by
structure-guided design. The advantages are that a small library can sample a
potentially large chemical diversity to generate novel lead compounds and that hits
can be identified for new classes of target for which existing compound collections
cannot provide a hit.
I will discuss recent developments in the methods and their application with a focus
on fragment to hit evolution. This includes such issues as deciding which fragments
to progress; combining fragments with HTS and literature data; and how to work with
challenging targets, such as protein-protein interactions.

34

Honorium to Prof. A. Makriyannis

National Kapodistrian University of Athens, Laboratory of Organic Chemistry,
Panepistimiopolis, Zografou 11571
T. Mavromoustakos
A short talk will be given in which it will be outlined my collaboration with Prof. A.
Makriyannis who initiated this scientific event since 1983 and served as a biennial opportunity
for researchers and management executives to interact, exchange views and philosophies,
preview cutting edge technologies in Drug Discovery and get a glimpse of things to come.
During the closing address of the 16th Camerino-Noordwijkerhout symposium in 2007 Mario
Giannella, Henk Timmerman and Alexandros Makriyannis announced an agreement of joining
the Camerino-Noordwijkerhout symposium and Cyprus Conference. The symposium became
annual and included the three countries namely Cyprus, Italy and Camerino. The three leaders of
the Conferences agreed to shift every year the responsibility of organizing it in the three
countries.
Emphasis will be given in the examples related to drug:membrane interactions and their
significant in the rational drug design.

Localisation of TCV-116 (left) and olmesartan (right) between two adjacent DMPC phospholipids.

35

SEROTONIN 5-HT2 RECEPTOR STRUCTURE-BASED DRUG DESIGN

LEADS TO CLINICAL CANDIDATES FOR NEUROPSYCHIATRIC DISORDERS
RAYMOND G BOOTH
Center for Drug Discovery, Northeastern University, Boston, Massachusetts USA Ra.booth@neu.edu
Psychotic disorders affect ~3% of the population and most existing antipsychotic medications interact
with dopamine D2 receptors. Approximately 2/3 of psychotic patients, however, are noncompliant or
cease taking their medication due to serious side effects (weight gain, diabetes, movement disorders,
sedation, emotional dampening) and limited efficacyin this regard, second-generation antipsychotics
do not have superior efficacy compared to their first-generation predecessors.
Targeting the serotonin (5-hydroxytryptamine, 5-HT) system, and precisely the 5-HT2C G proteincoupled receptor (GPCR), represents an alternative approach to pharmacotherapy of psychoses. The
predominant expression of 5-HT2C receptors is in the central nervous system, thus, compounds that
specifically target 5-HT2C receptors should have limited impact in peripheral tissues. In the brain, the 5HT2C receptor is expressed in several neural systems (frontal cortex, limbic system, striatum) affected
in psychotic disorders. Moreover, 5-HT2C receptors modulate dopamine release, 5-HT2C receptor
knockout mice possess enhanced baseline dopamine levels and behavioral hypersensitivity to
dopamine-releasing agents. Also, induced-overexpression of dopamine D2 receptors increases
expression of 5-HT2C receptors, further corroborating a physiological link between 5-HT2C receptors
and central dopamine function. In clinical trials, a 5-HT2C agonist (vabicaserin) showed proof-ofconcept for treating schizophrenia.
5-HT2C agonists also decrease appetite and the 5-HT2C receptor-preferring agonist lorcaserin
significantly reduce weight relative to placebo in clinical trials and approved in the US to treat obesity.
One common problem with most existing, selective 5-HT2C agonists, including vabicaserin and
loracaserin, however, is that they also activate 5-HT2A and/or 5-HT2B receptors, which can lead to
hallucinations and cardiac valvulopathy, respectively.
Our laboratory built molecular models of each of the serotonin 5-HT2 GPCR subtypes and performed
ligand docking with molecular dynamics experiments to design high-affinity ligands. Computational
studies together with mutagenesis experiments were undertaken in an iterative fashion to delineate the
molecular determinants for activation vs. inactivation of 5-HT2 receptor subtype signaling. A novel and
potent 5-HT2C-specific agonist, ()-trans-(2S,4R)-4-(3' [meta]-bromophenyl)-N,N-dimethyl-1,2,3,4tetrahydronaphthalen-2-amine (4-m-Br-phenyl-2-dimethylaminotetralin, m-Br-PAT; MBP) was
developed as a promising lead. ()-MBP activates only the 5-HT2C receptor subtype and behaves as
an inverse agonist and competitive antagonist of 5-HT at 5-HT2A and 5-HT2B receptors.
In preclinical translational studies, ()-MBP demonstrated antipsychotic activity in 3 rodent models,
without altering locomotion. ()-MBP also negatively modulated methamphetamine-induced
psycholocomotor behaviors and decreased self-administration of ethanol in rats. Moreover, ()-MBP
negatively modulates food consumption in a rodent model of binge-eating/compulsive feeding. ()-MBP
is active after oral administration and has a pharmacological half-life of at least 6-hours. Several PAT
analogs underwent profiling for GI absorption and brain bioavailability, plasma stability and protein
binding, metabolic characterization in 5 species, CYP enzyme inhibition, off-target binding and function
(including hERG and kinases), and cytotoxicity evaluation using human hepatocytes. Data indicate
PATs are safe and effective after oral administration. Accordingly, a 5-HT2C receptor-specific agonist
such as ()-MBP is an attractive candidate for clinical development to treat psychostimulant-induced
and endogenous psychoses, methamphetamine and ethanol abuse/addiction, without liability for
obesity, hallucinations, heart disease, sedation or motoric disorders. See Canal et al., J Pharmacol Exp
Ther 349:19, May 2014. Supported by the US National Institute of Mental Health Grant R01MH081193 and National Institute on Drug Abuse Grants R01-DA023928 and R01-DA030989.

36

Structural and Dynamical Properties of Indomethacin Molecules embedded

within the Mesopores of Silicates and Metal-Organic Framework
Materials: a Solid-State NMR View
Gregor Mali, Toma endak
National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia.
e-mail: gregor.mali@ki.si

Mesoporous silicates and metal-organic materials comprise two extremely interesting families of
materials that exhibit promising properties for several fields of application including catalysis, gas
separation and storage, and heat storage. In the field of pharmaceutical science and technology these
materials are especially interesting as matrices for the drug-delivery systems, which can improve
release of poorly soluble drugs. While many studies exist that test the feasibility of preparation of
delivery systems from various mesoporous matrices and various model drugs, the atomic-scale studies
of drugs embedded within the mesopores are rather rare. In this presentation we will try to
demonstrate that solid-state NMR spectroscopy is a unique tool for studying the structural properties
of the mesoscopically confined drug and for studying the drug-drug and drug-matrix interactions. We
will also try to explain that knowledge about drug-matrix interactions is very important, because the
interactions crucially determine the rate of the release of drug from the delivery system.
The presentation will focus on our investigation of model drug-delivery systems prepared from
SBA-15 mesoporous silicate matrix, and MIL-101 and MIL-53 metal-organic frameworks loaded with
different amounts of indomethacin. In the SBA-15-based drug-delivery systems NMR spectroscopy
indicates that only when concentration of indomethacin within the mesopores exceeds a certain value,
hydrogen bonds between the drug molecules become abundant. Nitrogen sorption analysis and
comparison of 1H spin-lattice relaxation times in progressively loaded SBA-15 matrices suggest that
above such loading concentration rigid nanoparticles that extend throughout the entire mesopore crosssection start to form. 1H-13C CPMAS NMR spectrum of indomethacin embedded within the mesopores
of SBA-15 closely resembles the spectrum of the bulk amorphous indomethacin and does not allow
drawing firm conclusions about the molecular conformation and the packing of the drug molecules
within the pores. On the contrary, variable-temperature 1H spin-lattice relaxation measurements show
that the mesoscopically confined indomethacin is significantly different from the bulk amorphous
indomethacin. It does not become rubbery and is, regarding the mobility of the drug molecules, much
more similar to the crystalline form of indomethacin. In MIL-101- and MIL-53-based drug-delivery
systems the NMR measurements also show that the solvent molecules, used during the drug-loading
procedure, attach to the frameworks by hydrogen bonds and cannot be removed from the pores by
gentle drying. This makes the mesoporous metal-organic matrices less convenient for the drug
delivery than mesoporous silicates.

Figure 1. NMR spectroscopy uses atomic nuclei as probes that provide information about their local
environment. In this way it enables the inspection of the drug-drug and drug-matrix interactions in
drug-delivery systems based on mesoporous silicates and metal-organic materials.

37

Structural and Dynamical Properties of Indomethacin Molecules embedded

within the Mesopores of Silicates and Metal-Organic Framework
Materials: a Solid-State NMR View
Gregor Mali, Toma endak
National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia.
e-mail: gregor.mali@ki.si

Abstract
Ordered mesoporous matrices, such as mesoporous silicates and metal-organic framework
materials, are promising drug carriers that can improve delivery of drugs that are poorly
soluble in physiological media. Knowing the arrangement of drug molecules within the
mesopores of these drug carriers and identifying drug-matrix interactions is very important for
the understanding of the performance of new drug-delivery systems. In this contribution we
try to show, through several examples, that such knowledge can be gathered by relatively
simple solid-state nuclear magnetic resonance measurements.
Introduction
Mesoporous silicate and metal-organic materials are two extremely interesting families of
materials that exhibit promising properties for several fields of application including catalysis,
gas separation and storage, and heat storage. In the field of pharmaceutical science and
technology these materials are especially interesting as matrices for the drug-delivery
systems, which can improve release of poorly soluble drugs [1]. While many studies exist that
test the feasibility of preparation of delivery systems from various mesoporous matrices and
various model drugs, the atomic-scale studies of drugs embedded within the mesopores are
rather rare [2]. In this presentation we will try to demonstrate that solid-state NMR
spectroscopy is a unique tool for studying the structural properties of the mesoscopically
confined drug and for studying the drug-drug and drug-matrix interactions. The knowledge
about drug-matrix interactions is very important, because the interactions crucially determine
the rate of the release of drug from the delivery system [3]. The presentation will focus on our
investigation of model drug-delivery systems prepared from SBA-15 mesoporous silicate
matrix [4] and MIL-101 metal-organic matrix [5] loaded with different amounts of
indomethacin.
Materials and Methods
The discovery of SBA-15 was reported in 1998 by the researchers from University of
California, Santa Barbara [6]. SBA-15 can be synthesized in acidic media using different
amphiphilic poly(alkylene oxide) triblock copolymers as structure directing agents. Obtained
mesoporous silicates have well-defined systems of parallel channels with hexagonal p6mm
symmetry (Figure 1). Depending on the method of synthesis, diameters of pores range from
4.6 up to 30 nm, pore volume fraction can be as high as 0.85, and BET surface areas vary
between 600 and 1000 m2/g. Thickness of the SBA-15 walls is between 3.1 and 6.4 nm and is
crucial for high hydrothermal stability of SBA-15. Out of many possible mesoporous silicas
SBA-15 was chosen as a drug-delivery matrix due to its favourable pore structure, high
thermal stability and also due to relatively simple preparation and obtainability in large
enough quantities.
MIL-101, originally synthesized as Cr3F(H2O)2O[(O2C)-C6H4-(CO2)]3nH2O (n~25) by
Ferey et al. [7], is a metal-organic framework material with extremely large unit-cell volume

38

(~700 nm3). Aluminium and iron analogues of the original chromium MIL-101 can be
prepared as well [8]. MIL-101(Al)-NH2 is built of inorganic trimers that consist of three AlO6
octahedra and are arranged in the corners of the supertetrahedra. The six edges of the
supertetrahedron are six 2-amino terephthalic acid anions (BDC-NH2). The microporous
supertetrahedra are connected one to another through common vertices and form porous
framework with two types of cages, one delimited by 20 and another by 28 supertetrahedra
(Figure2). The cages have internal free diameters of ~2.9 nm and ~3.4 nm, and accessible
pore volumes of ~12.7 nm3 and ~20.6 nm3. Extremely high surface area (~6000 m2/g) makes
MIL-101 a suitable candidate for various applications, including drug delivery.

Figure 1. Mesoporous materials used as drug carriers and their basic building units:
mesoporous silicate SBA-15 (basic building units are SiO4 tetrahedra) (left) and mesoporous
metal-organic material MIL-101(Al)-NH2 (basic building units are trimers of AlO6 octahedra
and BDC-NH2 linkers) (right).
Drug loading was realized by immersing empty and dry mesoporous matrices into the
solution of indomethacin (IMC) in tetrahydrofuran (THF). Subsequently the impregnated
particles were filtered out of the solution and dried in a ventilation dryer and vacuum dryer at
323 K.
The obtained model drug-delivery systems, denoted as SBA-15/IMC and MIL-101(Al)NH2/IMC, were inspected by solid-state NMR spectroscopy. This technique is very
convenient for the analysis of solids and motifs within solids that do not exhibit long-range
order thus it is also very convenient for the inspection of indomethacin molecules embedded
within the mesopores of the drug carriers. An example showing the advantage of solid-state
NMR spectroscopy over the X-ray diffraction technique is presented in Figure 2. In case of
bulk crystalline indomethacin both, diffraction and NMR spectroscopy, give rise to the
pattern/spectrum with well resolved sharp diffraction maxima/spectral lines. Once
indomethacin is incorporated into the mesopores, it no longer exhibits long-range order, and
diffraction cannot provide any information about these molecules. It only sees the periodic
arrangement of the mesopores. Solid-state NMR spectroscopy, however, still yields a
spectrum with strong resonances, which are, however, broadened compared to the ones
observed for the crystalline material.

39

Figure 2. Comparison of X-ray diffraction patterns and 13C NMR spectra of bulk crystalline
indomethacin and of indomethacin embedded within the mesoporous SBA-15 matrix. X-ray
diffraction, depending on long-range order, provides no information on indomethacin within
the pores, whereas NMR spectroscopy, being a technique that inspects local structure, still
yields a resolved spectrum. The crystalline indomethacin used in this example is IMC-beta, a
solvate of IMC in THF.
Results and Discussion
H and 13C magic-angle spinning (MAS) NMR spectra of crystalline IMC-beta, SBA-15/IMC,
MIL-101(Al)-NH2/IMC, and empty MIL-101(Al)-NH2 are shown in Figure 3. In case of the
crystalline IMC-beta, NMR resonances are narrow and well resolved. One can easily detect
contributions of THF molecules, which are part of this IMC-beta solvate, and determine the
IMC/THF ratio of 2 for the composition of the asymmetric unit of IMC-beta. 1H NMR
spectrum clearly shows a single resonance that can be assigned to protons in a hydrogen bond.
It seems that in IMC-beta there is a single type of hydrogen bond and that this bond is a
bidentate bond between two carboxylic groups of two IMC molecules that form a dimer.
Because SBA-15 is a pure silicate matrix, the only contribution to the 13C MAS NMR
spectrum of SBA-15/IMC is from the embedded indomethacin molecules. The signals are
broader than the ones in the 13C MAS NMR spectrum of IMC-beta, but appear at the same
positions. It is interesting to note that there are no signals of THF in the spectrum of SBA15/IMC, but there are such signals in the 13C MAS NMR spectrum of MIL-101(Al)NH2/IMC. Integration of the latter signals shows that in this delivery system there are more
THF molecules per one molecule of IMC than in the IMC-beta solvate. Obviously, equivalent
drying procedure was able to entirely remove the THF solvent from SBA-15/IMC but not
from MIL-101(Al)-NH2/IMC. The comparison of the 13C and 1H MAS NMR spectra of empty
and loaded MIL-101(Al)-NH2 provides also some information about the mesoporous matrix
itself.
1

40

Figure 3. 13C and 1H MAS NMR spectra of the crystalline IMC-beta, of indomethacin
embedded within the mesopores of SBA-15, and of the empty and loaded MIL-101(Al)-NH2.
Simple NMR spectroscopy can provide some information on the composition of the drug
delivery system, i.e. on the amount of the drug and the solvent molecules, as well as about the
order of these molecules. But NMR can provide also information about the dynamics of the
embedded molecules. As an example one can compare temperature dependence of spin-lattice
relaxation times in several bulk crystalline polymorphs of indomethacin, in the bulk
amorphous indomethacin, and in the indomethacin embedded within the mesopores of SBA15. Although 13C MAS NMR spectrum of indomethacin embedded within the mesopores of
SBA-15 closely resembles the spectrum of the bulk amorphous indomethacin (not shown) and
does not allow drawing firm conclusions about the molecular conformation and the packing of
the drug molecules within the pores, variable-temperature 1H spin-lattice relaxation
measurements show that the mesoscopically confined indomethacin is significantly different
from the bulk amorphous indomethacin. It does not become rubbery and is, regarding the
mobility of the drug molecules, much more similar to the crystalline forms of indomethacin.

Figure 4. Temperature dependence of 1H spin-lattice relaxation times of two polymorphs of

indomethacin, IMC-alpha and IMC-gamma, of the crystalline solvate IMC-beta, of the bulk
amorphous IMC, and of IMC embedded within the mesopores of SBA-15 (left). NMR
relaxation and heteronuclear correlation measurements along with measurements of porosity

41

indicate that when increasing the concentration of IMC within the mesopores, at certain
critical concentration particles of IMC start to form, which extend over the entire pore cross
section. These particles resemble the crystalline particles much more than the amorphous
particles (scheme on the right).
NMR, via homonuclear and heteronuclear correlation spectroscopy, enables studies of
proximities among nuclei even in non-crystalline solids. As an example of such spectroscopy
the 1H-1H homonuclear correlation spin-diffusion spectrum of MIL-101(Al)-NH2/IMC is
shown in Figure 5. This spectrum, together with the 1H-13C heteronuclear correlation
spectrum (not shown), indicates that protons in hydrogen bonds (resonance at about 12.5
ppm) see protons from the THF molecules (resonance at about 3 ppm). The heteronuclear
correlation spectrum indicates that the hydrogen-bonding protons must be the framework
protons, most probably the protons that belong to the Al-OH-Al bridging hydroxyl groups of
the inorganic framework trimers. This further means that in the MIL-101(Al)-NH2/IMC drug
delivery system THF molecules (hydrogen-bond acceptors) attach to the framework inorganic
units through hydrogen bonds, which is why these molecules cannot be removed from the
pores by gentle drying.

Figure 5. 1H-1H homonuclear correlation spin-diffusion spectrum of MIL-101(Al)-NH2/IMC

and schematic presentation of the arrangement of THF solvent molecules (small red ellipses)
and IMC drug molecules (larger green ellipses) within this drug-delivery system.
Conclusions
The presented examples show that NMR spectroscopy is a valuable tool for studying the
arrangement and the interactions of molecules embedded within the mesopores of silicate and
metal-organic drug-carriers.
REFERENCES
[1] Wang, S. Ordered Mesoporous Materials for Drug Delivery. Microporous Mesoporous Mater.
2009, 117, 19.
[2] Skorupska, E.; Jeziorna, A.; Kazmierski, S.; Potrzebowski, M.J. Recent progress in solid-state
NMR studies of drugs confined within drug delivery systems. Solid State Nucl. Magn. Reson.
2014, 57-58, 216.
[3] Ukmar, T.; Maver, U.; Planinek, O.; Kaui, V.; Gaberek, M.; Godec, A. Understanding
Controlled Drug Release from Mesoporous Silicates: Theory and Experiment. J. Controlled
Release 2011, 155, 409417.

42

[4] Ukmar, T.; endak, T.; Mazaj, M.; Kaui, V.; Mali, G. Structural and Dynamical Properties of
Indomethacin Molecules Embedded within the Mesopores of SBA-15: A Solid-State NMR View.
J. Phys. Chem. C 2012, 116, 26622671.
[5] endak, T.; unkovi, E.; Ukmar, T.; Mazaj, M.; Zabukovec Logar, N.; Mali, G.Indomethacin
Embedded into MIL-101 Frameworks: A Solid-state NMR Study. J. Phys. Chem. C 2014, 118,
61406150.
[6] Zhao, D.; Feng, J.; Huo, Q.; Melosh, N.; Fredrickson, G.H.; Chmelka, B.F.; Stucky, G.D.
Triblock copolymer syntheses of mesoporous silica with periodic 50 to 300 angstrom pores.
Science 1998, 279, 548552.
[7] Frey, G.; Mellot-Draznieks, C.; Serre, C.; Millange, F.; Dutour, J.; Surbl, S.; Margiolaki, I. A
Chromium Terephthalate-Based Solid with Unusually Large Pore Volumes and Surface Area.
Science 2005, 309, 20402042.
[8] Serra-Crespo, P.; Ramos-Fernandez, E. V.; Gascon, J.; Kapteijn, F. Synthesis and
Characterization of an Amino Functionalized MIL-101(Al): Separation and Catalytic Properties.
Chem. Mater. 2011, 23, 25652572.

43

Targeting promising inhibitors for HCV: A multi step

chemoinformatics approach
Georgia Melagraki,a Eleni Vrontaki,a, b Thomas Mavromoustakos,b Antreas Afantitisa
a

Department of Chemoinformatics, NovaMechanics Ltd, Nicosia, Cyprus

Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Athens

15771, Greece.
Email:info@novamechanics.com Website: www.novamechanics.com

Chronic hepatitis C virus (HCV) is a major disease and constitutes a significant

worldwide health concern. Globally, up to 3% of the worlds population is chronically
infected with HCV and 3 to 4 million persons are newly infected every year. Due to
its serious detrimental effects in the health and its extent of infections, HCV has
become the paramount target of antiviral protease inhibitor research, especially the
genotype 1. Pegylated alpha Interferon (-PEG-IFN), alone or in combination with
Ribavirin (RBV), is a current effective treatment but only in limited cases since it has
serious disadvantages. Thus, the development of new therapeutic strategies to treat
HCV-associated hepatitis is a major challenge.
Towards this goal we have for long been involved in the in silico exploration of
various molecular patterns for the identification of novel HCV inhibitors. For this
purpose we have combined data mining, molecular docking and 3D-QSAR CoMSIA
with similarity search and virtual screening techniques to understand the structural
characteristics that affect the binding of different patterns to HCV NS5B polymerase
[1,2]. Several databases such as CheMBL and PubChem were used as the initial pool
of compounds that were in silico explored. Our workflows succeeded to identify the
most promising compounds from a pool of new analogues and to prioritize a list of
compounds for screening. The approach revealed several promising chemistry driven
compounds with potential high activity. The encouraging results indicate that the
proposed methodology can be generalized as a rational drug discovery tool for the
identification of new leads for a broad spectrum of diseases.
ACKNOWLEDGEMENT
This work is supported by funding under the Seven Research Framework Programme of the
European Union. Project SYSPATHO (ex. PATHOSYS HEALTH-F5-2010-260429)

REFERENCES
1. Vrontaki E, Melagraki G*, Mavromoustakos T, Afantitis A*Exploiting ChEMBL database
to identify indole analogs as HCV replication inhibitors. Methods. 2014 Mar 27. pii: S10462023(14)00123-6. doi: 10.1016/j.ymeth.2014.03.021
2. G. Melagraki* & A. Afantitis* Ligand and structure based virtual screening strategies for
hit-finding and optimization of hepatitis C (HCV) inhibitors Curr. Med Chem. 2011 18(17)
2612-2619

44

A Scientific Workflow Management System for Virtual Screening in Cancer

Chemoprevention
Vasilis J. Promponasa, Constantinos S. Pattichisb, Andreas I. Constantinoua
a

Deaprtment of Biological Sciences and b Department of Computer Science

University of Cyprus, P.O. Box 20537, 1678, Nicosia, Cyprus
email: vprobon@ucy.ac.cy

Computer-aided methods are an essential component of modern pipelines for

discovering and optimizing chemical or natural compounds with desired biological
activities. Virtual screening tools, aim to reduce the large number of candidate
molecules available in chemical databases into a tractable number of promising lead
compounds amenable to more detailed in vitro and in vivo characterization.
Along these lines, we have developed the Life Science Informatics system (LiSIs) [1],
a web-based platform offering tools for developing and executing virtual screening
experiments within a scientific workflow framework based on Galaxy [2]. LiSIs also
enables sharing workflows along with relevant data with selected users, thus
facilitating interdisciplinary research towards the development of new
pharmaceuticals. Other freely available/open access tools provide additional
functionality to LiSIs, including RDKit [3] for supporting chemoinformatics related
features, Pybel [4] a Python wrapper to the OpenBabel chemoinformatics toolkit [4],
R [5] a statistical environment providing data mining and machine learning support
and AutoDock Vina [6] for supporting the execution of docking experiments.
In this presentation, we will give an overview of the LiSIs system, the underlying
architecture and capabilities provided to end-users. More specifically, we will
highlight currently available tools for (i) loading and preprocessing input chemical
data, (ii) processing chemical data (e.g. compound similarity, substructure matching,
docking, QSAR models) and (iii) post-processing and generating output. We will
conclude with a case study highlighting its application in the domain of discovering
promising compounds for cancer chemoprevention.
LiSIs
This work has been partially supported through the EU-FP7 GRANATUM project,
A Social Collaborative Working Space Semantically Interlinking Biomedical
Researchers, Knowledge and data for the design and execution of In Silico Models
and Experiments in Cancer Chemoprevention, contract number ICT-2009.5.3.
REFERENCES
[1].

[2].
[3].
[4].
[5].
[6].

Kannas C.C., Achilleos K.G., Antoniou Z., Nicolaou C.A., Pattichis C.S., Kalvari I.,
Kirmitzoglou I. and Promponas V.J., Proceedings of the 13th IEEE International
Conference on BioInformatics and BioEngineering, 439-446, IEEE Computer Society,
Los Alamitos, CA, USA, 2012.
Goecks J., Nekrutenko A., Taylor J., Galaxy Team, Genome Biology, 11, R86, 2010.
Landrum, G.: RDKit: Open-source cheminformatics. http://www.rdkit.org/
OBoyle N.M., Morley C., Hutchison G.R. Chemistry Central Journal 2, 5, 2008.
R Development Core Team: R: A Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Vienna, Austria (2008). ISBN 3900051-07-0.
Trott O., Olson A.J. Journal of Computational Chemistry 31, 455-461, 2010.

45

Molecular Dynamics in Drig Design: Development of Compstatinbased Compounds for the Regulation of the Key Complement System
Protein C3.
Georgios Archontis1, Ph. Tamamis1, Aliana Lopez de Victoria2, Roland Gorham Jr. 2, Meghan
L-Bellows Peterson3, Panagiota Pierou1, Christodoulos Floudas3, Dimitrios Morikis2
1
Department of Physics, University of Cyprus, PO20537, CY1678, Cyprus
2
Department of Bioengineering, University of California Riverside, CA92521, USA
3
Department of Chemical and Biological Engineering, Princeton University, Princeton NJ
08544, USA
The complement system provides the first line of defense against foreign pathogens.
Nevertheless, its inappropriate or excessive activation is associated with a range of
pathological diseases, including age-related macular degeneration, asthma, rejection of
xenotransplantation, asthma and heart attack. The peptide compstatin and its derivatives
inhibit the complement-component protein C3 in primate mammals and are potential
therapeutic agents against unregulated activation of complement in humans. Over the past
several years we have employed a combination of computational, rational-design and
experimental methods to design compstatin-based derivatives and study their effect on C3
regulation. Compstatin is active against primate C3 but inactive against non-primate C3. We
elucidated this species-specificity by Molecular Dynamics (MD) simulations of complexes
between compstatin and human or rat C3 [1]. In the rat simulations, the protein underwent
reproducible conformational changes, which eliminated or weakened specific interactions and
reduced the complex stability. Using additional simulations, we proposed that the
introduction of a small number of human C3 substitutions at key positions of the mouse
sequence may create a transgenic protein able to bind compstatin [2]. Furthermore, using a
combination of computational and rational-design methods, we studied new generations of
potential compstatin-based inhibitors of C3 [3]. The criteria for the new design were: (i)
optimization for C3 affinity and solubility, and (ii) development of dual specificity, humanrat/mouse C3 inhibitors. Three of the new analogs possessed strong and novel binding
characteristics and are promising candidates for further optimization. More recently [4], we
used a novel human retinal pigmented epithelial (RPE) cell assay to evaluate the efficacy of
newly designed compstatin-based inhibitors of the complement system, in a model that that
mimics drusen biogenesis and the pathobiology of macular degeneration. Our study
demonstrated that the RPE cell assay has higher discriminatory capability for measuring the
potency of inhibitory peptides, compared to biochemical assays, in addition to being operative
in a macular disease environment.
REFERENCES
1.

P. Tamamis, D. Morikis, C.A. Floudas and G. Archontis (2010). Species Specificity of the
Complement Inhibitor Compstatin Investigated by All-atom Molecular Dynamics simulations.
Proteins; Structure, Function and Bioinformatics 78:2655-2667.

2.

P. Tamamis, P. Pierou, C. Mytidou, CA. Floudas, D. Morikis and G. Archontis (2011). Design of a
Modified Mouse Protein with Ligand Binding Properties of its Human Analog using Molecular
Dynamics Simulations: The Case of C3 Inhibition by Compstatin. Proteins: Structure, Function and
Bioinformatics 79:3166-3179.

3.

P. Tamamis, A. L. de Victoria, R. D. Gorham, M. L. Bellows-Peterson, P. Pierou, C. A. Floudas, D.

Morikis, G. Archontis (2012). Molecular Dynamics in Drug Design: New Generations of Compstatin
Analogs. Chemical Biology & Drug Design 79:703-718.

4.

R. D. Gorham, D. L. Forest, P. Tamamis, A. Lopez de Victoria, M. Kraszni, C. A. Kieslich, C. D.

Banna, M. L. Bellows-Peterson, C. K. Larive, C. A. Floudas, G. Archontis, L. V. Johnson, D. Morikis
(2013). Novel Compstatin Family Peptides Inhibit Complement Activation by Drusen-like Deposits in
Human Retinal Pigmented Epithelial Cell Cultures. Experimental Eye Research 116:96-100.

46

Interaction of inhibitors with aspartic proteases

Manthos G. Papadopoulos
Institute of Biology, Pharmaceutical Chemistry and Biotechnology
National Hellenic Research Foundation
48 Vas. Constantinou Ave.
Athens 116 35, Greece

We have selected two proteases, the human immunodeficiency virus type 1 protease
(HIV-1 PR) and renin as well as a series of inhibitors to study their interaction. HIV1 PR is one of the main targets toward AIDS therapy, while renin participates in the
renin-angiotensin-aldosterone system which is considered as an important regulatory
pathway for several metabolic processes. We will consider the following applications:
(a) We have selected the potent drug darunavir and a weak inhibitor (a fullerene
analog) as HIV-1 PR substrates to compare proteases conformational features upon
binding [1].
(b) Molecular mechanics (MD) PoissonBoltzmann surface area (MMPBSA) freeenergy calculations and inhibition assays for canagliflozin, an antidiabetic agent
verified its effective binding to both proteins [2]. Moreover, the drugs aliskiren (a
renin inhibitor) and darunavir (an HIV-1 PR inhibitor) showed high affinity for HIV-1
PR, respectively. This study suggests that canagliflozin, aliskiren, and darunavir may
induce profound effects toward dual HIV-1 PR and renin inhibition.
(c) Saquinavir, the first inhibitor against HIV-1 protease, offers the most extensive
clinical data regarding resistance mutations [3]. We examine L10I, G48V, L63P,
A71V, G73S, V82A, and I84V single mutant HIV-1 PR strains in complexes with
saquinavir to elucidate drugprotease interactions and dynamics.
(d) We employed MD calculations and free energy (MMPBSA and thermodynamic
integration) analyses on wild-type (WT) and mutated HIV-1 PR complexes with
darunavir, amprenavir, indinavir, and saquinavir to clarify the mechanism of
resistance due to the I50V flap mutation [4].
(e) We investigate the binding mechanism in renin complexes, involving three drugs
(remikiren, zankiren and enalkiren) and four lead compounds, which were selected
after screening the ZINC database employing appropriate criteria [5]. For this
purpose, we used: (i) Several ab initio methods: the effective fragment potential
(EFP) approach, the variational perturbation theory, the Su and Li method to
calculate the interaction energy of selected fragments and the atoms-in-molecules
approach in order to find the energy of several hydrogen bonds, (ii) docking, (iii) MD
and (iv) the MMPBSA technique. Biological assays for two lead compounds have
been performed to verify the accuracy of the theoretical findings.
1.
2.
3.
4.
5.

G. Leonis, et al., J. Chem. Inf. Model. 52, 1542 (2012).

H. Tzoupis, et al., J. Med. Chem., 55, 5784 (2012).
H. Tzoupis, et al., J. Chem. Theory Comput. 9, 1754 (2013).
G. Leonis, et al., | J. Chem. Inf. Model. 53, 2141 (2013).
H. Tzoupis, et al., submitted for publication.

47

Interacction of in
nhibitors with
w
aspa
artic protteases
Manthoos G. Papaddopoulos
Insstitute of Bioology, Pharm
maceutical Chhemistry and
d Biotechnoloogy
N
National
Helllenic Researcch Foundatio
on
48 Vas. Constantinoou Ave.
Greece
Athenns 116 35, G

Exttended Absstract
We have selected two prooteases, the human imm
munodeficiency virus type 1 prottease
V-1 PR) annd renin as well
w as a seeries of inhiibitors to stuudy their innteraction. HIVH
(HIV
1 PR
R is one off the main targets
t
towaard AIDS thherapy, whille renin paarticipates inn the
reniin-angiotenssin-aldosterrone system
m which is considered
c
as an imporrtant regulaatory
pathhway for sevveral metabbolic processes. We willl consider the
t followinng applications:

Figu
ure 1. The structure of the considdered system
ms.
(a) We have compared
c
thhe conformaational propperties and binding
b
beh
havior of HIIV-1
darunavir) or
o an
PR upon entraapping into the catalyttic cavity eiither a poteent drug (d
V-1 PR stru
ucture appeeared
ineert fullerenne derivativve [1]. Thee darunavir--bound HIV
veryy stable thhroughout thhe simulatiion with loow rms deeviations frrom the cryystal
struucture (Figu
ure 1). In contrast, thhe protease appeared significantly
s
y flexible uupon
fulleerene bindinng, mainly due to an inncrease in mobility
m
of the
t flap in chain
c
A (Ilee50).
Thee flaps in daarunavirHIIV-1 PR weere stabilizeed by hydro
ogen-bonds (HBs) betw
ween
Ile550 and Gly
y51 as welll as betweeen Ile50 andd Ile50, which
w
were mediated via
v a
water moleculle. The latter interacttion was not
n observeed in the fullerene-bo
f
ound
mplex or in the free protease,
p
thu
us denotingg the imporrtance of a H2O-bridgge in
com

48

effeective ligandd-binding. Darunavir was

w also sttabilized intto HIV-1 PR
P via a strrong
HB network. Although significannt van derr Waals innteractions and nonppolar
o solvationn acted favvorably tow
ward fullerreneHIV-11 PR com
mplex
conttribution to
form
mation, MM
MPBSA calculations
c
showed thhat the fulllerene is a poor prottease
inhiibitor mainlly due to its destabilizinng electrosttatic contrib
bution.

Figu
ure 2. Stru
uctural alignnment of HIIV-1 PR (yeellow) and renin
r
(green
n).
(b)
Two asppartic proteeases, HIV-1 PR andd renin, posssessing sevveral structtural
2 have beeen comparedd regardingg their modde of
charracteristics in commonn (Figure 2)
action and conformatio
c
onal properrties uponn binding [2]. Moleecular docking
nhibitor mayy be
calcculations suuggested thaat canaglifllozin, an anntidiabetic, SGLT2 in
alsoo an efficiennt inhibitorr for both proteases.
p
A
Additionally
y, darunavirr and aliskiren,
twoo potent dru
ugs associateed with HIV
V-1 PR andd renin, resp
pectively, were
w
exchannged
betw
ween proteaases to inveestigate whhether the structural sim
milarities of
o the proteeases
resuult in simiilar conform
mational changes
c
uppon bindingg to the same
s
inhibbitor.
Cannagliflozin induced
i
strructural chaanges that accompany
a
binding too both proteeins.
Thee darunavir
renin com
mplex resultted in a staable structu
ure and pressented a strrong
hyddrogen bondding
netw
work involvving the druug and maiinly loop reesidue Thr7
77 and activ
ve site residdues
Aspp215 and Gly217.
G
Sim
milarly, the aliskirenH
HIV-1 PR complex was
w involveed in
freqquent HB in
nteractions between
b
alisskiren and flap
f residuess (Gly48), as
a well
as w
with active site residuees (Asp29). Furthermorre, a water molecule
m
brridged the flaps
f
of thhe protease via hydroggen bonds. These
T
findinngs implicatted canaglifflozin, aliskiren,
and darunavir as effectivve, dual inhhibitors of both proteein systemss. Additionnally,
MPBSA freee energy calculations
c
s described the bindingg modes off HIV-1 PR and
MM
reniin in a quan
ntitative waay. The totaal free enerrgy of binding for all complexes was
preddicted to bee adequate, with canagliflozin servving as a veery effectiv
ve dual inhibbitor
Thiss finding iss of paramoount importtance since it is well-kknown thatt AIDS patiients
whoo follow traaditional theerapy eventuually develoop diabetes.. Thus, canaagliflozin-based

49


2

drugg design may

m lead too the elimination of this probleem. The most favorrable
conttributions to the binnding energgy were mainly
m
due to van der
d Waals and
elecctrostatic innteractions involving active site and flaap residuess. Importanntly,
experimental confirmation
c
n of our caalculations has
h been offfered by innhibition asssays
P and aliskkirenHIV--1 PR comp
plexes. Thiss study strongly
for canaglifloziinHIV-1 PR
c
u
used
for the
t
treatm
ment of AIIDS,
sugggested thaat particulaar drugs currently
hyppertension, and
a diabetees could seerve as duaal inhibitorss of HIV-1 PR and reenin.
Exaamples incluude the aliiskiren-relatted hyperteension treattment that may also help
agaiinst AIDS and the use
u of canaagliflozin too induce pronounced effects tow
ward
protteases inhibbition.

Figu
ure 3. The structuress of HIV-1 PR/saquiinavir com
mplex (left) and saquinnavir
(righht).
(c) The conforrmational prroperties annd binding modes
m
of eiight saquinavirHIV-11 PR
mplexes in the wild-tyype and sinngle mutantt forms, L10I, G48V, L63P, A771V,
com
G733S, V82A, and I84V
V (Figure 3),
3 have been investiigated by means of MD
simuulations andd
bindding free-ennergy (MM
MPBSA an
nd TI) calcuulations [3]]. Binding energy
e
anallysis
show
wed that mutations
m
G
G48V,
L63P
P, and I84V
V significanntly deterioorate saquinnavir
bindding compaared to the wild-type
w
a other mutated
and
m
com
mplexes. In contrast, A71V
A
had a negligiblle effect on the binding
g affinity off saquinavirr. An extenssive comparrison
a dynamics for all systems indiicated the m
main
of sstructural prroperties, ennergetics, and
reassons that paarticular muutations con
nfer resistaance to saquuinavir: (i) Mutations that
induuce flexibillity to the flaps
f
of thee protease (e.g.
(
G48V
V) lead to th
he reduction of
bindding.(ii) Co
onformationnal changess of saquinavir may not
n affect binding
b
(V882A,
A711V), provideed that its structure eveentually stabbilizes, as observed
o
in the wt.
(iii) Saquinavirr-related HB
B interactio
ons with thee active sitee (Asp25/255/29/29/30//30)
o HIV-1 PR
R should bee present to hold the drrug into thee binding caavity
and the flaps of
c
on. Mutationns that resuult in majorr resistance (e.g. L63P
P) do
in a compact conformatio
not preserve faavorable intteractions with
w the acttive site an
nd the flapss. (iv) A waaternteraction between
b
saqquinavir andd the region
n of the actiive site (Aspp29)
meddiated HB in
is of major impportance (e..g. wildtypee, A71V). Mutants
M
thatt lack this innteraction (e.g.,
(

50


3

G488V, I84V) showed siignificantly lower (exxperimental and theorretical) binnding

affinnities. (v) Enhanced
E
vaan der Waalls interactions and electtrostatics arre the main
conttributions too the bindinng energy.

Figu
ure 4. The structure of HIV-1 PR
R bound to thhe drug ampprenavir.
(d) A systemattic approachh on the meechanism off the I50V (Figure
(
4) drug
d
resistaanceutation in HIV-1
H
PR has been attempted by meanss of moleccular
assoociated mu
dynnamics (M
MD), moleecular meechanics PoissonBoltzmann surface area
(MM
MPBSA), and thermoodynamic in
ntegration (TI) computtational metthodologiess [4].
Thee conformattional featuures and binnding modees of four marketed, potent
p
prottease
inhiibitors, daruunavir, am
mprenavir, inndinavir, and saquinaavir, have been
b
comppared
betw
ween wild-ttype (WT) andI50V mutated
m
forrms of the protease to
t elucidatee the
mecchanism of resistance due to thee flap mutattion. Confoormational analysis
a
on WT
and mutated complexes
c
w
with
PIs reevealed thatt the WT structures
s
a more sttable
are
mpared to thhe mutated ones,
o
with the
t only excception of amprenavir
a
complexes that
com
appear equally flexible in both formss of the prootease. Desspite the rellative proxim
mity
t flaps inn all compleexes, it waas shown thhat there is a great diffference in flap
of the
flexxibility betw
ween WT and mutateed forms: the flaps in most I550V complexes
appearincreasinngly mobilee compared to the WT.. Hydrogen
n-bonding analysis
a
shoowed
a I50V) with
w signifiicant
thatt the drugs were stabiilized insidee all complexes (WT and
inteeractions, prrimarily invvolving resiidues at the active site, such as thhe side chaiin of
Aspp25/25 andd the backkbone atom
ms of Aspp29/29/30/3
30.MMPB
BSA and more
m
rigoorous TI callculations verified
v
the experimenttal results annd implicatted that the loss
in bbinding eneergy is moostly enthallpically driven. The change
c
in van
v der W
Waals
inteeractions haas been ideentified as the majorr componen
nt of the binding
b
ennergy
diffference betw
ween all WT
T and mutatted complexxes.

51


4

Figu
ure 5. Watter mediatedd hydrogen--bond betweeen remikiren and Tyr114.
(e) We eluciddate the mechanism
m
o inhibitor binding too renin (Figure 5). For this
of
taskk, we have selected three commeercially avaiilable drugss (remikirenn, zankiren and
enallkiren), andd 4 potentiaal lead com
mpounds, aft
fter screenin
ng the ZINC database [5].
Thiss set of derivatives
d
allows forr a comparative inveestigation on
o the binnding
mecchanism in renin.
r
MD simulations
s
s revealed siimilar patteerns in all reenin compleexes.
Thee conformational analyses showedd that no maj
ajor changess are induceed in renin upon
u
drugg binding, with
w the struucture of the protease remaining
r
relatively
r
staable throughout
the simulationss. In agreem
ment with prrevious obsservations, it
i was show
wn that the three
t
a extensivee HB netw
work with reenin. The proposed
p
leead compouunds
druggs create an
creaate a less broad HB neetwork, how
wever, interractions wiith the activve site andd the
flapp are conseerved. MMPBSA calculations off the bindinng free enerrgy showed that
in aall renin com
mplexes, thee most impo
ortant contrribution arisses from thee van der W
Waals
inteeractions. Thhe interactioon energy of
o the reninremikiren system has been compputed
by the
t effective fragment potential approach.
a
It has been found
f
that thhe environm
ment
H 2O
O/Na+ has a rather littlee effect on thhe reninrem
mikiren inteeraction.
1.
2.
3.
4.
5.

G. Leoniss, et al., J. Chhem. Inf. Mo

odel. 52, 15442 (2012).
H. Tzoupiis, et al., J. Med.
M Chem. 55, 5784 (20012).
H. Tzoupiis, et al., J. Chem.
C
Theorry Comput. 9,
9 1754 (2013).
G. Leoniss, et al., | J. Chem.
C
Inf. Moodel. 53, 21441 (2013).
H. Tzoupiis, et al., submitted for puublication.

52


5

Structure-Based Design of Non-Peptide Mimetics for the Treatment

of Multiple Sclerosis
Mary Patricia P. Yannakakis, Theodore V. Tselios
University of Patras, Department of Chemistry, Rion Patras, 26504, Greece
e-mail: yannakakism@gmail.com

Multiple Sclerosis (MS) is an immunologically controlled, inflammatory,

demyelinating disease, characterized by destruction of the white matter (myelin) of
the Central Nervous System (CNS), leading to serious medical conditions and
paralysis [1]. It is believed that MS is an autoimmune disease in which T-cell
response is directed to the immunodominant epitopes of the myelin proteins. T-cell
response is triggered by the formation of the trimolecular complex between the Major
Histocompatibility Complex (MHC), known as Human Leukocyte Antigen (HLA) in
humans, the immunodominant myelin protein epitopes and the T-cell receptor (TCR)
[2].
In this project, a detailed mapping of the interactions that occur during the creation of
the trimolecular complex HLA-peptide antigen-TCR was carried out (pdb file:
1YMM). The mentioned procedure provides the conformational characteristics that
are essential for the triggering of the disease and the rational design of non-peptide
inhibitors. The MBP83-97 epitope was specifically chosen for the rational design
because it is identified as the main immunodominant epitope in MS patients [3, 4].
The aim of this study was the rational design of non-peptide mimetic molecules that
bind to the TCR receptor, avoiding the interaction with the HLA-antigen. The new
molecules were designed to prevent the formation of the trimolecular complex and the
further multiplication and activation of T-cells that are involved in MS [5, 6].
Several pharmacophore models were developed and the search for new candidates
was performed using the ZINC database. The structure-based design was achieved
with the MOE and LigandScout softwares in a LINUX operating system.

REFERENCES
[1]. Mantzourani E.D. et al, Curr. Med. Chem., 12, 1521-1535, 2005
[2]. Mantzourani E.D. et al, J. Med. Chem., 49, 6683-6691, 2006
[3]. Hahn M. et al, Nature Immunology, 6, 490-496, 2005
[4]. Wucherpfennig K.W. et al, Molecular Immunology, 40, 1009-1017, 2004
[5]. Wucherpfennig K.W. et al, Journal of Immunology, 185, 63897126, 2010
[6]. Li Y. et al, The EMBO journal, 24, 2968-2979, 2005

53

Structure-Based Design of Non-Peptide Mimetics for the Treatment

of Multiple Sclerosis
Mary Patricia P. Yannakakis, Theodore V. Tselios
University of Patras, Department of Chemistry, Rion Patras, 26504, Greece
e-mail: yannakakism@gmail.com

Abstract:
Multiple Sclerosis (MS) is an immunologically controlled, inflammatory,
demyelinating disease, characterized by destruction of the white matter (myelin) of
the Central Nervous System (CNS), leading to serious medical conditions and
paralysis. It is believed that MS is an autoimmune disease in which T-cell response is
directed to the immunodominant epitopes of the myelin proteins. T-cell response is
triggered by the formation of the trimolecular complex between the Major
Histocompatibility Complex (MHC), known as Human Leukocyte Antigen (HLA) in
humans, the immunodominant myelin protein epitopes and the T-cell receptor (TCR).
In this project, a detailed mapping of the interactions that occur during the creation of
the trimolecular complex HLA-peptide antigen-TCR was carried out (pdb file:
1YMM). The mentioned procedure provides the conformational characteristics that
are essential for the triggering of the disease and the rational design of non-peptide
inhibitors. The MBP83-96 epitope was specifically chosen for the rational design
because it is identified as the main immunodominant epitope in MS patients. The aim
of this study was the rational design of non-peptide mimetic molecules that bind to the
TCR receptor, avoiding the interaction with the HLA-antigen. The new molecules
were designed to prevent the formation of the trimolecular complex and the further
multiplication and activation of T-cells that are involved in MS. Several
pharmacophore models were developed and the search for new candidates was
performed using the ZINC database. The structure-based design was achieved with
the MOE and LigandScout softwares in a LINUX operating system.
Key Words: Molecular Modeling, Non-Peptide Mimetics, Trimolecular Complex,
Multiple Sclerosis, Myelin
Introduction:
Multiple Sclerosis (MS) is an autoimmune, demyelinating disease of the Central
Nervous System (CNS), in which the encephalitogenic T-cells play a significant role
in the induction of the disease1. It is known that T-cell response is directed to the
immunodominant epitopes of the myelin proteins. T-cell response is triggered by the
formation of the trimolecular complex between the Major Histocompatibility
Complex (MHC), known as Human Leukocyte Antigen (HLA) in humans, the
immunodominant myelin protein epitopes and the T-cell receptor (TCR)2.
The first crystal structure of a T cell receptor from a human autoimmune disease
bound to its self-peptide/MHC target was determined by Hahn et al (the
Wucherpfennig lab)3. This TCR (termed Ob.1A12) originated from a patient with
relapsing-remitting multiple sclerosis (MS), and transgenic mice that express this
TCR and the human HLA class II molecule (HLA-DR2) develop spontaneous CNS
autoimmunity. The structure showed a highly unusual topology of peptide/HLA

54

binding4 that had not been observed for human TCRs directed against infectious
agents. All other human TCRs that had been crystallized with their peptide-HLA
ligand had shown a very similar topology in which the TCR is positioned in a
diagonal orientation over the center of the peptide/HLA surface. In contrast, the
Ob.1A12 TCR contacts only the N-terminal segment of the human myelin basic
protein5 peptide and makes highly asymmetrical interactions with the HLA helices
(Fig. 1). This highly unusual binding mode6 is dominated by the hypervariable CDR3
loops of the TCR which make the majority of contacts to the HLA helices as well as
the peptide. The unconventional topology for this autoimmune TCR is probably the
result of distinct selection pressures exerted on autoimmune T cells. During
repertoire development in the thymus, T cells with an optimal fit for a selfpeptide/HLA complex are the most likely to be deleted. In contrast, T cells whose
TCR has an optimal fit for a microbial peptide bound to HLA are not systematically
deleted in the thymus and have a competitive advantage over other T cells in an antimicrobial immune response. This structure thus provides an explanation for the
finding that autoreactive T cells are present in every subject, despite elaborate
mechanisms for the elimination of such cells. In this study, a detailed mapping of the
interactions that occur during the creation of the trimolecular complex HLA-peptide
antigen-TCR was carried out (pdb file: 1YMM). The mentioned procedure provides
the conformational characteristics that are essential for the triggering of the disease
and the rational design of non-peptide inhibitors7. The MBP83-96 (E83-N-P-V-V-H-F-FK-N-I-V-T-P96) epitope was specifically chosen for the rational design because it is
identified as the main immunodominant epitope in MS patients8.

Figure 1: The trimolecular Complex HLA-MBP83-96-TCR (1ymm). The receptors are

presented with ribbons (HLA, blue and TCR, green). The MBP83-96 epitope is
presented in magenta sticks.
Results and Discussion:
The whole design of the non-peptide inhibitors was based on the conformational study
of the complex HLA-MBP83-96-TCR, the development of pharmacophore models and
the optimization of the proposed molecules for better binding with the receptor and
easier synthesis. The goal of this study was the rational design of non-peptide mimetic
molecules that bind to the TCR receptor, avoiding the interaction with the HLA
molecule. The new molecules were designed to prevent the formation of the

55

trimolecular complex and the further multiplication and activation of T-cells that are
involved in MS. The above essay has led us in finding molecules that not only bind
strongly with the TCR, but that are also placed deeply in the pockets P-1 and P2, a
placement very important for the biological potency. This strong binding is due to the
inhibitors small size compared to the large size of the natural antigen. The nonpeptide inhibitors seem to apply strongly with the TCR due to the formation of pistacking interactions, a fact that defines the binding as well as the biological effect of
a pharmaceutical molecule. The molecules are well protected in the active center and
their exposed surface is limited, making their interaction with the HLA less possible.
The proposed inhibitors agree with Lipinskis Rule of Five, so they could be absorbed
by the human organism and possibly be used as pharmaceutical products for the
treatment of Multiple Sclerosis. The retro-synthetic analysis showed that the synthesis
of the proposed non-peptide inhibitors is quite simple, followed by basic reactions of
organic synthesis. Previous studies3 demonstrated that P2 His, P3 Phe, and P5 Lys are
the key residues for TCR recognition of the HLAMBP peptide complex (Fig. 2).
Residue P2 His of the MBP peptide packs against Hisb81, whereas side chain P3 Phe
nestles in a hydrophobic shelf of the a2 helix. The side chains of P2 His and P3 Phe
interact and bind with the TCR (Fig. 3).

Figure 2: The MBP83-96 residues that are placed into the TCR pockets (red and
yellow) and the HLA pockets (blue).

Figure 3: Selected region of the MBP83-96 in the TCR. The main residues and the
pockets they insert respectively are presented.
P1 Val and P4 Phe are primary HLA contact residues for human MBP 8396-specific T
cell clones since substitution of either P 1 Val or P4 Phe reduces the HLA binding.
Valine at the P1 position of the MBP peptide occupies the hydrophobic P 1 pocket of
HLA. A large, primarily hydrophobic P4 pocket was found to be a prominent feature
of the HLA peptide binding site. This pocket is occupied by a Phe of the MBP peptide
that makes an important contribution to the binding of the MBP peptide to HLA. The

56

HLAMBP crystal structure shows that these peptide side chains are solvent exposed
and available for HLA binding (Fig. 4).

Figure 4: The main residues of MBP83-96 (green) that interact i) with the TCR (pink)
and ii) with the HLA (orange, blue).
At first, a mapping of the active center of the TCR took place. We reported the main
binding pockets of the peptide-antigen and the TCR amino acids that interact with it.
The natural peptide MBP8396 does not insert deeply into the TCR, since it is placed on
the TCR surface, due to its size and the fact that it is bound to the HLA. The TCR
interaction residues are limited for the natural antigen.
Experimental Session:
Several pharmacophore models were developed (Ligand Scout software). The model
that was chosen included the key residues (F, pharmacophore spheres) that interact
with the TCR and the important residues (V, volume exclusion spheres) that interact
with the HLA. The pharmacophore features were defined and the search for new
candidates was performed using the ZINC database which includes over 4million
drug-alike molecules. The small organic molecules bind to the TCR but do not
interact with the HLA, preventing the formation of the trimolecular complex. The
structure-based design was achieved with the MOE and Ligand Scout softwares in a
LINUX operating system. When the pharmacophore search was completed, all the
possible hits were checked, based on their interactions with the TCR, the way they
bind, the depth their insert into the TCR pockets as well as their physical and
chemical properties. The molecule that was selected as lead-drug was docked to the
TCR (Molecular Docking, MOE) and then we performed Molecular Dynamics for
10ns in water (explicit solvent, MOE). The lead drug was further optimized to
increase the interactions and the binding energy with the TCR. The molecules that
where designed were according to the Lipinskis Rule of Five for possible
pharmaceutical candidates. Retro-synthetic analysis was performed so that the
molecules to be synthesized and evaluated in vitro and in vivo for their efficacy (Fig.
5).

57

Figure 5: The main experimental steps followed for the rational drug design.
ACKNOWLEDGEMENTS
This work is financially supported by the Cooperation program 09SYN- 609-21, (O. P.
Competitiveness & Entrepreneurship (EPAN ), ROP Macedonia - Thrace, ROP Crete and
Aegean Islands, ROP Thessaly Mainland Greece Epirus, ROP Attica).
REFERENCES
1. (a) Steinman, L., A molecular trio in relapse and remission in multiple sclerosis. Nat Rev
Immunol 2009, 9 (6), 440-447; (b) Mantzourani, E. D.; Mavromoustakos, T. M.; Platts, J. A.;
Matsoukas, J. M.; Tselios, T. V., Structural requirements for binding of Myelin Basic Protein
(MBP) peptides to MHC II: Effects on immune regulation. Current medicinal chemistry
2005, 12 (13), 1521-1535.
2. Wucherpfennig, K. W.; Gagnon, E.; Call, M. J.; Huseby, E. S.; Call, M. E., Structural
Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and
Initiation of Signaling. Csh Perspect Biol 2010, 2 (4), 1-14.
3. Hahn, M.; Nicholson, M. J.; Pyrdol, J.; Wucherpfennig, K. W., Unconventional topology of
self peptide-major histocompatibility complex binding by a human autoimmune T cell
receptor. Nat Immunol 2005, 6 (5), 490-496.
4. Sethi, D. K.; Schubert, D. A.; Anders, A. K.; Heroux, A.; Bonsor, D. A.; Thomas, C. P.;
Sundberg, E. J.; Pyrdol, J.; Wucherpfennig, K. W., A highly tilted binding mode by a selfreactive T cell receptor results in altered engagement of peptide and MHC. J Exp Med 2011,
208 (1), 91-102.
5. Madsen, L. S.; Andersson, E. C.; Jansson, L.; krogsgaard, M.; Andersen, C. B.; Engberg,
J.; Strominger, J. L.; Svejgaard, A.; Hjorth, J. P.; Holmdahl, R.; Wucherpfennig, K. W.;
Fugger, L., A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell
receptor. Nature Genetics 1999, 23 (3), 343-347.
6. Hemmer, B.; Archelos, J. J.; Hartung, H. P., New concepts in the immunopathogenesis of
multiple sclerosis. Nat Rev Neurosci 2002, 3 (4), 291-301.
7. (a) Mantzourani, E. D.; Platts, J. A.; Brancale, A.; Mavromoustakos, T. M.; Tselios, T. V.,
Molecular dynamics at the receptor level of immunodominant myelin basic protein epitope
87-99 implicated in multiple sclerosis and its antagonists altered peptide ligands: Triggering
of immune response. J Mol Graph Model 2007, 26 (2), 471-481; (b) Spyranti, Z.; Tselios, T.;

58

Deraos, G.; Matsoukas, J.; Spyroulias, G. A., NMR structural elucidation of myelin basic
protein epitope 83-99 implicated in multiple sclerosis. Amino Acids 2010, 38 (3), 929-936.
8. Wucherpfennig, K. W.; Catz, I.; Hausmann, S.; Strominger, J. L.; Steinman, L.; Warren, K.
G., Recognition of the immunodominant myelin basic protein peptide by autoantibodies and
HLA-DR2-restricted T cell clones from multiple sclerosis patients - Identity of key contact
residues in the B-cell and T-cell epitopes. J Clin Invest 1997, 100 (5), 1114-1122.

59

Novel approach in Multiple Sclerosis treatment

Anthi Tapeinou, Theodore V. Tselios
Department of Chemistry, University o Patras, 26504 Rion Patras, Greece
e-mail: atapeinou@upatras.gr

Multiple Sclerosis (MS) is a chronic, progressive disease of the Central Nervous System
(CNS), characterized by focal inflammation. MS is generally considered to be an autoimmune
disease involving CD4+, CD8+ T and B cells [1,2]. This is strongly supported by the
association of the Major Histocompatibility Complex class II (MHC II) with the disease and
the ability of Th1 and Th17 (T helpers) CD4+ T lymphocytes to lead the disease to an animal
model for MS called Experimental Autoimmune Encephalomyelitis (EAE) [3]. Candidate
autoantigens for MS include constituents of the myelin sheath, such as Myelin Basic Protein
(MBP), Proteolipid Protein (PLP) and Myelin Oligodendrocyte Glycoprotein (MOG) [3].
Modern therapeutical approaches against MS involve the design and synthesis of peptide
analogues of disease-associated myelin epitopes in order to induce tolerance in CNS specific
T cells. Mutated linear or conformational restricted cyclic peptide analogues, derived from
proteins of the myelin sheath, have been designed and synthesized in order to inhibit the
Experimental Autoimmune Encephalomyelitis (EAE, the best animal model of MS). The
peptide analogues have been rationally designed using a combination of Nuclear Magnetic
Resonance (NMR) and Molecular Dynamic (MD) calculations. Moreover, a number of
peptides were conjugated to mannan (polymannose) in its reduced or oxidized form via Schiff
bases [4,5]. Mannan has successfully been used as a carrier to target T cell antigens to the
mannose receptor of macrophages and dendritic cells leading to the induction of Th1 or Th2
immune responses in cancer immunotherapy protocols. The ability of mannan to induce
different T cell cytokine profiles depending on the mode of conjugation is of great importance
to our research. The synthesized analogues were evaluated in vivo and in vitro, ex vivo using
the EAE animal model and blood derived from patients with MS respectively.

REFERENCES
[1].
[2].
[3].
[4].
[5].

Steinman, L., Cell, 85, 299-302, 1996

Martin, R. et al, Ann. Rev. Immun., 10, 153-187, 1992
Mantzourani E.D. et al, Curr. Med. Chem., 12, 1521-1535, 2005
Katsara M. et al, Mol. Immunol., 45(13), 3661-3670, 2008
Katsara M. et al, Immunology, 128, 521-533, 2009

60

Novel approach in Multiple Sclerosis treatment

Anthi Tapeinou, Theodore V. Tselios
Department of Chemistry, University o Patras, 26504 Rion Patras, Greece
e-mail: atapeinou@upatras.gr

Abstract:
Multiple Sclerosis (MS) is a chronic, progressive disease of the Central Nervous System
(CNS), characterized by focal inflammation. MS is generally considered to be an autoimmune
disease involving CD4+, CD8+ T and B cells. There is a relation between the Major
Histocompatibility Complex class II (MHC II) and the disease. The MHC molecules play a
significant role in the stimulation of T cells through the formation of trimolecular complex
(TCR- antigen- MHC). Candidate autoantigens for MS include constituents of the myelin
sheath, such as Myelin Basic Protein (MBP), Proteolipid Protein (PLP) and Myelin
Oligodendrocyte Glycoprotein (MOG). Modern therapeutical approaches against MS involve
the design and synthesis of peptide analogues of disease-associated myelin epitopes in order
to induce tolerance in specific T cells. Mutated linear or conformational restricted cyclic
peptide analogues, derived from proteins of the myelin sheath, have been designed and
synthesized in order to inhibit the Experimental Autoimmune Encephalomyelitis, EAE, the
best animal model of MS. The peptide analogues have been rationally designed using a
combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamic (MD)
calculations. Moreover, peptides were conjugated to mannan (polymannose) in its reduced or
oxidized form via Schiff bases. Mannan has successfully been used as a carrier to target T cell
antigens to the mannose receptor of macrophages and dendritic cells leading to the induction
of Th1 or Th2 immune responses in cancer immunotherapy protocols. The ability of mannan
to induce different T cell cytokine profiles depending on the mode of conjugation is of great
importance to this research. The synthesized analogues were evaluated in vivo and in vitro
using the EAE animal model and blood derived from patients with MS respectively.
Introduction
Multiple Sclerosis (MS) is a slowly progressive, immunologically mediated disease of the
Central Nervous System (CNS), characterized by inflammation of white matter in the brain
and spinal cord [1-2]. MS is usually considered to be a T-helper 1 (Th1) mediated disease,
based mainly on pathological resemblance to a delayed-type hypersensivity response in the
CNS as well as on observations made when investigating the EAE. The disease is
characterized by local CD4+ and CD8+ T cell and macrophage infiltrates perivascular
inflammation, foci of demyelination and loss of neurologic function. This is a widely spread
disease in Europe and USA (100 patients in 10000 people). The disease typically manifests
between the ages of 20 and 40 and affects women twice as often as men. An association
between Major Histocompatibility Complex (MHC) / human leukocyte antigen (HLA) class II
alleles and the disease has been observed in MS patients, in particular HLA-DR1, HLA-DR2
and HLA-DR4 [3]. Although the pathology of MS remains unclear, there is evidence that T
cells which recognize immunodominant epitopes of myelin proteins, such as Myelin Basic
Protein (MBP), Proteolipid Protein (PLP) or Myelin Oligodendrocyte Glycoprotein (MOG)
play a pathogenic role in the induction of MS [4]. Modern approaches towards the therapeutic
management of MS involve the design and use of peptide analogues of disease-associated
myelin epitopes to induce tolerance in CNS specific T cells and, more recently, to actively
induce antigen-specific regulatory T cells which have the ability to suppress inflammation.
EAE represents an invaluable in vivo system for the evaluation of such therapeutic approaches

61

towards the treatment of MS. EAE is a Th1 CD4+ T cell driven disease that can be induced by
immunisation with MBP, PLP or MOG proteins or their appropriate peptide epitopes.
The main experimental protocols for the induction of the appropriate immune response for
disease (EAE or MS) prevention are: i) the use of conformationally restricted cyclic Altered
Peptide Ligands (APLs) based on the immunodominant myelin epitopes [5-8] and ii) the use
of oxidized or reduced form of mannan (polymannose) conjugated with linear peptide
analogues via a (Lys-Gly)5 bridge. Mannan has successfully been used as a peptide carrier to
the mannose receptor of macrophages and dendritic cells (DCs). Certainly, experiments have
shown that proteins conjugated with the oxidized or reduced form of mannan lead to Th1 or
Th2 immune response respectively [9-11]. The ability of mannan to induce different cytokine
profiles depending on the mode of conjugation is of great importance to the current proposal.
Results and Discussion
The backbone cyclization of peptides has been demonstrated to increase biological activity, in
vivo stability and to reduce conformation freedom. More specifically, cyclic peptides have: (i)
greater bioavailability and higher resistance to proteolytic degradation making them better
potential candidates for therapeutic drugs, (ii) more precise conformation and increased
receptor selectivity and specificity. The lack of conformational flexibility, which is an
important characteristic of linear counterparts, confirms or eliminates the bioactive
conformation. NMR and Molecular Modelling studies (Restrained Molecular Dynamics based
on NMR data, Homology Modelling, Pharmacophore Analysis) on linear counterparts will be
used in order to design potent cyclic APLs with crucial TCR or MHC/HLA amino acids
substitutions. The cyclic mutated peptides cyclo(87-99)[Arg91, Ala96]MBP87-99 or cyclo(9199)[Ala96]MBP87-99 were designed using the Arg and Ala at positions 91 and 96 respectively.
These amino acids are presented as primary TCR contacts and play a significant role in the
stimulation of encephalitogenic T cells via the formation of trimolecular complex TCRantigen-MHC (HLA). Acute EAE was prevented in Lewis rats by the co-administration of
cyclo(87-99)[Arg91, Ala96]MBP87-99. In contrast, co-administration of cyclo(9199)[Ala96]MBP87-99 resulted in a delay in the onset of the clinical signs [5-8].
The mannan-peptide (MOG35-55) conjugation (Figure 1) has the ability to induce T cell
tolerance and resistance to chronic EAE. The peptide conjugated to mannan in its oxidized
form, but not unconjugated peptides or mannan, strongly protected mice against EAE when
administered to mice. The mannan- peptide analogue protects the animals by the induction of
EAE through a mechanism that maybe modifies the antigen presenting properties of dendritic
cells (DCs, the main antigen presenting cells in the body) and stops the activation of
encephalitogenic T cells which are responsible for the MS.

Figure 1: Schematic representation of linear MOG35-55 peptide conjugated with mannan via
(LysGly)5 bridge.

62

Experimental Session
Synthesis of linear and cyclic peptide analogue
The peptide was synthesized by Fmoc/tBu methodology using the acid sensitive 2-chlorotrityl
chloride (CLTR-Cl) resin (0.6-1.0 mmolCl-/g) and Na-Fmoc (9-fluorenylmethyloxycarboxyl)protected amino acids. The cyclization was achieved using O-benzotriazol-1-yl-N,N,N,Ntetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxy-7-azabenzotriazole, 2,4,6collidine allowing fast reaction and high-yield cyclization product. The final product was
further purified using Semi Preparative Reverse Phase-HPLC (Figure 2). The purity of
peptide was >95% as determined by Analytical Reverse Phase-HPLC. The peptide was
synthesized using (Lys-Gly)5 at the N-terminus that acts as a linker between the peptide and
mannan. The crude peptide product was further purified by Semi-Preparative Reverse Phase
(RP-HPLC) and it was indentified using ElectronSpray Ionization Mass Spectrometry (ESIMS).
Cl
Cl

1.Fmoc-Pro-OH / DIPEA
2. 20% piperidine/DMF

Pro

Fmoc Deprotection
20% piperidine/DMF

Coupling
HOBt/DIC
Fmoc-AA-OH

Val-His(Trt)-Phe-Phe-X1-Asn-Ile-Val-Thr(tBu)-Y1-Arg(Pbf)-Thr(tBu)-Pro-O
Cleavage
DCM/TFE
7:3

Val-His(Trt)-Phe-Phe-X1-Asn-Ile-Val-Thr(tBu)-Y1-Arg(Pbf)-Thr(tBu)-Pro-OH
Cyclization
TBTU, HOAt,
Collidine in dry DMF

Val-His(Trt)-Phe-Phe-X1-Asn-Ile-Val-Thr(tBu)-Y1-Arg(Pbf)-Thr(tBu)-Pro-OH
Deprotection
90% TFA in DCM,
scavengers

Val-His-Phe-Phe-X-Asn-Ile-Val-Thr-Y-Arg-Thr -Pro
X1: Lys(Boc), Arg(Pbf)
X: Lys, Arg
Y, Y1: Pro, Ala

Figure 2: Synthetic procedure of myelin cyclic peptides cyclo(87-99)[Arg91, Ala96]MBP87-99

and cyclo(91-99)[Ala96]MBP87-99 using the Fmoc/tBu methodology.
Conjugation of lineal peptide with Mannan
14mg/ml mannan (0.1M phosphate buffer pH 6.0) was oxidized to a poly-aldehyde by treating
with 100l 0.1M sodium periodate, for 1 hour at 4oC. 10l ethylene glycol is added for 30
minutes at 4oC to stop the oxidation. The mixture was passed through a PD-10 column and the

63

mannan fraction is collected. The PD-10 column pre-calibrated with 0.1M carbonate buffer
pH 9.0. 1mg of peptide (figure 2) was reacted with oxidized mannan overnight at RT. The
conjugation was used with no further purification.
ACKNOWLEDGMENTS
This work is financially supported by the Cooperation program 09SYN- 609-21, (O. P.
Competitiveness & Entrepreneurship (EPAN ), ROP Macedonia - Thrace, ROP Crete and Aegean
Islands, ROP Thessaly Mainland Greece Epirus, ROP Attica).
REFERENCES
[1]. Steinman, L., Multiple sclerosis: a coordinated immunological attack against myelin in the central
nervous system. Cell 1996, 85 (3), 299-302
[2]. Martin, R. McFarland HF, McFarlin DE, Immunological aspects of demyelinating diseases. Ann.
Rev. Immun. 1992, 10, 153-187
[3]. Lincoln M.R., Montpetit A., Cader M.Z., Saarela J., Dyment D.A., Tiislar M., Ferretti V., Tienari
P.J., Sadovnick A.D., Peltonen L., Ebers G.C., Hudson T.J., A predominant role for the HLA class II
region in the association of the MHC region with multiple sclerosis. Nature Genetics 2005, 37 (10),
1108-1112
[4]. Mantzourani ED, Mavromoustakos TM, Platts JA, Matsoukas JM, Tselios TV, Structural
requirements for binding of myelin basic protein (MBP) peptides to MHC II: effects on immune
regulation. Curr. Med. Chem. 2005, 12 (13), 1521-1535
[5]. Tselios T., Apostolopoulos V., Daliani I., Deraos S., Grdadolnik S., Mavromoustakos
T., Melachrinou M., Thymianou S., Probert L., Mouzaki A., Matsoukas J., Antagonistic effects of
human cyclic MBP(87-99) altered peptide ligands in experimental allergic encephalomyelitis and
human T-cell proliferation. J. Med. Chem. 2002, 45 (2), 275-83
[6]. Tselios T., Daliani I., Deraos S., Thymianou S., Matsoukas E., Troganis A., Gerothanassis
I., Mouzaki A., Mavromoustakos T., Probert L., Matsoukas J., Treatment of experimental allergic
encephalomyelitis (EAE) by a rationally designed cyclic analogue of myelin basic protein (MBP)
epitope 72-85. Bioorg. Med. Chem. Lett 2000, 10 (24), 2713-7
[7]. Tselios T., Daliani I., Probert L., Deraos S., Matsoukas E., Roy S., Pires J., Moore G., Matsoukas
J., Treatment of experimental allergic encephalomyelitis (EAE) induced by guinea pig myelin basic
protein epitope 72-85 with a human MBP(87-99) analogue and effects of cyclic peptides. Bioorg.
Med. Chem. Lett 2000, 8 (8), 1903-9
[8]. Tselios T., Probert L., Daliani I., Matsoukas E., Troganis A., Gerothanassis I.P., Mavromoustakos
T., Moore G.J., Matsoukas J.M., Design and synthesis of a potent cyclic analogue of the myelin basic
protein epitope MBP72-85: importance of the Ala81 carboxyl group and of a cyclic conformation for
induction of experimental allergic encephalomyelitis. J. Med. Chem. 1999, 42, 1170-7
[9]. Katsara M., Yuriev E., Ramsland P.A., Deraos G., Tselios T., Matsoukas J., Apostolopoulos V.,
Mannosylation of mutated MBP83-99 peptides diverts immune responses from Th1 to Th2. Mol.
Immunol. 2008, 45 (13), 3661-3670
[10]. Katsara M., Yuriev E., Ramsland P.A., Tselios T., Deraos G., Lourbopoulos A., Grigoriadis
N., Matsoukas J., Apostolopoulos V., Altered peptide ligands of myelin basic protein ( MBP87-99 )
conjugated to reduced mannan modulate immune responses in mice. Immunology 2009, 128 (4), 521533
[11]. Katsara M., Deraos G., Tselios T., Matsoukas M.T., Friligou I., Matsoukas J., Apostolopoulos
V., Design and synthesis of a cyclic double mutant peptide (cyclo(87-99)[A91,A96]MBP87-99)
induces altered responses in mice after conjugation to mannan: implications in the immunotherapy of
multiple sclerosis. J. Med. Chem. 2009, 52 (1), 214-218

64

NMR metabolomics in diagnosis and toxicology

Emmanuel Mikros, Dimitra Benaki, Kostantinos Tsiagkas, Anastasia Papachristodoulou, Alkistis
Milioni
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, National and Kapodistrian University of
Athens, Panepistimiopolis, 15771 Zografou Greece

NMR spectroscopy is a powerful non-targeted technique that has been used successfully in the
analysis of body fluids especially in the field of metabolomics. It allows rapid analysis of the
complex metabolic profiles of biological samples taking advantage of the degree of inherent
biochemical similarities. In combination with multivariate chemometric analysis has been used
successfully in the fields of drug toxicology and disease diagnosis. Applications of metabolomics
provide real biological endpoints as fingerprints that portray the perturbations of endogenous
metabolites caused by diseases, toxins, drugs, etc. Capabilities of NMR based metabolomics for the
discovery of new biomarkers will be presented through different examples.
The first example provides the analysis of 120 newborn urine samples collected and analyzed
previously by screening based mainly on MS for the diagnosis of inborn errors of metabolism.
Metabolites usually not detected with other techniques (formate, dimethylamine, N-oxide
trimethylamine) can be identified and monitored with NMR. Complex cases with more than one
deficiencies were detected by NMR where conventional screening techniques failed. Typical
examples are a) detecting increased urocanic acid in a patient with diagnosed methylmalonic aciduria
b) in samples of a patient with ornithine transcarbamoylase deficiency in addition to the expected
increased levels of uracil, and orotic acid increased uridine has been also determined. More
importantly metabolites never been described previously in newborn errors of metabolism have been
detected and identified using 2D techniques namely gluconolactone, and 4-hydroxyphenyllactate.
The second example demonstrates the NMR study of biological responses induced after Adriamycin
(doxorubicinDXR) administration notably cardiotoxicity and resistance development. DXR is a
commonly used antineoplastic agent, but its use is limited by the occurrence of dose dependent
cardiotoxicity. In acute toxicity experiments in animals 1H NMR spectra of aqueous myocardium
extracts showed alteration in heart energy metabolism and biomarkers like acetate and succinate
relate to toxicity. In chronic toxicity experiments the evaluated metabolic profile exhibit an overall
disturbance of protein metabolism.
Related to DXR the NMR based study of the metabolome alterations during the step-wise acquisition
of cancer cells resistance to doxorubicin (DXR) will be presented. Metabolic profiling of
osteosarcoma cell lines that were increasingly resistant DXR concentrations suggested that protein
biosynthesis and glutathione metabolism, are reduced in resistant cells while other biochemical
pathways like pyrimidine and purine metabolisms are also perturbed. The study depicts how cellular
metabolomics may provide valuable information shedding light to complex biological mechanisms.
In a final case the metabolic determinants of the response of patients to clopidogrel and notably the
possibility to predict development of resistance to the drug through a pharmacometabonomic
analysis will be exemplified. Pharmacometabonomics utilizes metabolic profiling to predict
individual response to drugs and has important implications for personalized medicine applications.
ACKNOWLEDGEMENT

The present work was funded by SYNERGASIA 2009 PROGRAMME. This Programme is cofunded by the European Regional Development Fund and National Resources. (Project code:
09SYN-12-827)

65

NMR structures and cation binding of G-quadruplexes with unique

features
Janez Plavec

Slovenian NMR Center, National Institute of Chemistry, Ljubljana, Slovenia

EN-FIST Center of Excellence, Ljubljana, Slovenia
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
janez.plavec@ki.si

G-rich nucleic acid sequences exhibit potential to fold into four stranded assemblies called
G-quadruplexes. These structures are comprised of G-quartets, the basic structural motifs held
together by eight Hoogsteen hydrogen bonds. G-rich tracts within a G-quadruplex are linked
by residues that adopt loops of various lengths and topologies. Stability of G-quadruplexes
depends on a number of stacked G-quartets as well as sequence details of loops that connect
guanines involved in G-quartets. In addition to being constitutive elements of G-quartets,
guanine residues can be involved in loops thus contributing to the variety of their lengths and
orientations.
NMR has been successful in determining 3D structures of several unique architectures of
G-quadruplexes. NMR studies have not only helped to determine high-resolution structures of
several G-quadruplex structures of various origins including telomeric and promoter regions,
but have also contributed insights into structural polymorphism and its thermodynamic and
kinetic implications. Our recent NMR study on d[TAG3CG3AG3AG3A2] originating from the
first intron of the N-myc gene demonstrated formation of G-quadruplex in K+ ion containing
aqueous solution. The dimeric G-quadruplex is characterized by consecutive stacking of six
G-quartets. Quadruplex adopted by LNA-modified VEGF aptamer RNV66 adopts a parallel
topology with three G-quartet planes and unique loop orientations. Inclusion of five
consecutive guanine residues in the G-quadruplex core is accommodated by a no-residue
propeller-type loop, whereas a two-residue D-shaped intrastrand propeller-type loop connects
residues of the outer G-quartets within the same G-rich strand and is followed by edgewise
and propeller loops.
G-quadruplexes are stabilized by coordinated metal ions in the central cavity of their
structures. In general, cations can be found between two G-quartets or in the plane of a
G-quartet along the central cavity of a G-quadruplex. However, different stacking properties
of G-quartets and other structural features make cation-binding sites unequal. As a
consequence, cation-binding sites can be occupied by cations or, alternatively, they can be
temporarily vacant or occupied by water molecules. Solution-state NMR studies have
demonstrated that cations undergo dynamic exchange between coordination sites in the
interior of a G-quadruplex and bulk solution. Dynamics of 15NH4+ ion movement occurs on a
range of timescales and has been shown to correlate with structural features of
G-quadruplexes. Structures formed by d(TG3T) and its analogs containing a 5-5 or 3-3
inversion of polarity sites revealed that the inter-quartet cavities at the inversion of polarity
sites bind ammonium ions less tightly.
Recent references from the lab: Angew. Chem. Int. Ed. 2014, doi: 10.1002/anie.201400531. Nucleic
Acids Res. 2013, 41, 9524. J. Am. Chem. Soc. 2012, 134, 4132. Nucleic Acids Res. 2012, 40, 6946. J.
Phys. Chem. C 2012, 116, 23821. Nucleic Acids Res. 2012, 40, 11047.

66

NMR-assisted discovery of novel inhibitors of protein targets:

towards new therapeutic agents
Simona Goli Grdadolnik
National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia,
e-mail: simona.grdadolnik@ki.si

Despite the large efforts in private companies and academia in the past decades the output in
discovering novel therapeutic agents is rather low. Several failures in the development of
effective therapeutic agents can be attributed to the lack of knowledge about the relationship
between inherent molecular flexibility and drug-like behavior and to the unexplored influence
of dynamic processes on the interaction of biologically active molecules with protein targets.
High resolution NMR spectroscopy is a method of choice for investigation of dynamic
processes, because it can monitor and discriminate dynamic events on a broad range of time
scales from fast (< ns) to slow ( s-ms to days) internal motions without perturbing chemical
and structural equilibria. This technique provides atomic resolution insight into both
molecular structure and dynamics and offers complementary information to the rigid crystal
structure studies. Probably in the structure-based methods, which are commonly used in lead
design strategies, too much expectation has been placed on the rigid molecular structures
determined by the X-ray diffraction.
The result of our recent investigation of novel inhibitors of muramyl ligaze D (MurD) and
sterol 14- demethylases (Cyp51) by application of NMR and molecular modeling methods
will be used to present the capabilities of NMR methods for detection and characterization of
the ligand-receptor interactions and their ability to provide a combined structure-dynamic
insight into ligand binding modes. The Mur enzymes are attractive targets for development of
antibacterial drugs, while the human and fungal Cyp51 orthologs enable development of
selective antifungal drugs. New therapeutic agents of both classes of drugs are urgently
required, because the bacterial and fungal resistances are becoming critical all over the world.
We discovered fast domain motions of MurD, which affect the conformation and flexibility of
bound ligands, stability of binding interactions and the adaptability of the MurD binding site.
Conformational flexibility of novel inhibitors at the receptor binding site was related to the
differences in their inhibitory activities. The influence of complex dynamic processes on the
ligand binding modes was found to be one of the important reasons for insufficient biological
activity of inhibitors, which were designed as transition state analogues. Considering a
combined structure-dynamic aspect of binding inhibitors with novel structure scaffolds were
discovered, which without any further optimization possess a comparable in-vitro inhibitory
activities as the multistep optimized transition state analogues.

67

Insights on the conformational plasticity of drug-target proteins

Georgios A. Spyroulias
Department of Pharmacy, University of Patras, GR-26504, Patras, Greece.

Modern Biomolecular NMR is a powerful technique for the investigation of the

conformational dynamics and structure-activity correlation of drug targets such as peptides,
proteins and their complexes. To achieve this goal it is required the concerted action of
molecular biology, spectroscopy and computational biology for the high-yield production of
proteins in suitable forms for NMR studies (uniform labeling in 2H, 13C, 15N nuclei, and
specific labeling of residues or chemical groups in 13C, 15N), the acquisition a series of NMR
data and the determination of the 3D structures in solution along with the ellucidation of the
relaxation properties of proteins with molecular weight ranging from 7 to >25 kDa.
Among our current research interests is the NMR-driven conformational dynamics study of
disease-related enzymes and proteins, such as: (i) various E3 ubiquitin ligases involved in
ubiquitination pathway, which are enzymes that catalyzes the ubiquitination of proteins
targeted for destruction through the proteasome, like ARKADIA tumor suppressor [1-2], (ii)
extracellular domains of ligand-gated ion channels of the cys-loop family (nAChR is the
prototype) that involved in various neurodegenerative disorders [3-4], (iii) Zn(II)-proteases
that proteolyse signaling polypeptides regulating thus vital processes during the cell-cycle [56], (iv) RNA binding proteins [7], etc.
REFERENCES
1. Kandias N.G., Chasapis C.T., Bentrop D., Episkopou V., Spyroulias G.A. BBRC 378, 498-502
(2009).
2. Chasapis C.T., Kandias N.G., Episkopou V., Bentrop D., Spyroulias G.A. Proteins 80, 1484-9
(2012).
3. Chasapis C.T., Argyriou A.I., Corringer P.-J., Bentrop D., Spyroulias G.A. Biochemistry 50,
9681-3 (2011).
4. Dimitropoulos N., Papakyriakou A., Dalkas G.A., Chasapis C.T., Poulas K., Spyroulias G.A.
Proteins 79, 142-52 (2011).
5. Gkazonis PV, Dalkas GA, Chasapis CT, Vlamis-Gardikas A, Bentrop D, Spyroulias GA.
BBRC 396, 643-7 (2010)
6. Dalkas G.A., Chasapis C.T., Gkazonis P.V., Bentrop D., Spyroulias G.A. Biochemistry, 49,
10767-9 (2010).
7. Apostolidi M., Vourtsis D., Chasapis C.T., Stathopoulos C., Bentrop D., Spyroulias G.A.
Biomol NMR Assign (2014)
Acknowledgments: FP7-REGPOT-2011 SEE-DRUG (nr. 285950) & EU FP7-INFRA BIO-NMR
(nr. 261863)










68

Stereoelectronic control of AT2R/AT1R subtype selectivity in angiotensin II

analogues and their potential in cancer therapy
Francesca Magnani, Charalampos G. Pappas, Tim Crook, Vassiliki Magafa,A Paul Cordopatis,A
Susumu Ishiguro, Naomi Ohta, Jana Selent, Sanja Bosnyak, # Emma S Jones, # Ioannis P.
Gerothanassis, Masaaki Tamura, Robert E Widdop, # Andreas G. Tzakos*

Medical Research council, Laboratory of Molecular Biology, Cambridge, UK Department of

chemistry, University of Ioannina, GR 45110, Greece, Division of Cancer research, University of
Dundee, Dundee, DD1 9SY, UK, ADepartment of Pharmacy, University of Patras, Greece,

Department of Anatomy & Physiology, Kansas State University College of Veterinary Medicine,

Manhattan, Kansas 66506, USA, Computer-Assisted Drug Design Lab, Research Programme on
Biomedical Informatics (GRIB), Universitat Pompeu Fabra, Barcelona, Spain, # Department of
Pharmacology, Monash University, Clayton Victoria 3800, Australia
G Protein Coupled Receptor (GPCR) subtypes posses distinct functional and pharmacological
profiles, thus development of subtype selective ligands has immense therapeutic potential. We
describe a strategy to fine-tune ligand selectivity for the GPCR AT2R/AT1R subtypes through
electronic control of ligands aromatic-prolyl interactions. Through this strategy an AT2R high
affinity agonist analogue, Ki=3 nM, with 18.000-fold higher selectivity versus the AT1R was
obtained. This is the first time that a strategy is described to control ligand-receptor subtype
selectivity via delicate tuning of ligand aromatic electronics. Finally, the discovery that AII receptor
subtype selectivity could be precisely sculpted by tuning the electronic character of a simple
substitution in AII, was only made possible due to a combined experimental, chemical synthesis and
computational approaches. The effectiveness of this analogue to attenuate specific types of cancer
progression will be presented.

69

Conformational studies of LHRH analogue conjugated with cyclodextrin

using 2D NMR and Molecular Modeling
Golfo Kordopati,a Theodore Tselios,a Tahsin Kellici,b Franci Merzel,c Thomas
Mavromoustakos,b Simona Golic Grdadolnik,c,d Gerasimos Tsivgoulisa
a

Department of Chemistry, University of Patras,26504, Patras, Greece

Department of Chemistry, University of Athens, 15771, Athens, Greece
c
Laboratory of Biomolecular Structure, National Institute of Chemistry, 1001, Ljubljana, Slovenia
d
EN-FIST Centre of ExcellenceDunajska 156, 1000 Ljubljana, Slovenia
e-mail:gkordopati@upatras.gr
b

Cyclodextrins (CDs) are a family of cyclic oligosaccharides containing -D-glucose units

connected through -1,4 glucosidic bonds. Cyclodextrins have the ability to encapsulate into
their hydrophobic cavity via host/guest complexation, a variety of compounds[1] and based on
this property cyclodextrins have been proposed as candidates that will improve bioavailability
of certain drugs.[2] Luteinizing hormone-releasing hormone (LHRH) is a linear decapeptide
produced in the hypothalamus under the control of neurotransmitter type compounds and it is
the central regulator of fertility.[3] Leuprolide is a peptide agonist analogue, widely used for
the treatment of fertility and hormone dependent cancer.[4,5] However, its amino acid skeleton
is sensitive to proteolysis and LHRH analogues have low intestinal absorption and
bioavailability.[6] In this study, we aimed the reduction of the proteolytic degradation of
Leuprolide using a covalently attached beta-cyclodextrin unit. Since spatial interactions
between the peptide and the cyclodextrin unit are crucial for our goal, a detailed
conformational study of the conjugate was performed, using high field (800 MHz) NMR
techniques such as TOCSY, DQF COSY, NOESY and HSQC spectroscopy in combination
with Molecular Dynamics simulation. All the hydrophobic molecular segments included
intramolecularly in the cyclodextrin cavity were identified and will be presented.

REFERENCES
[1]. G.Wenz, Angewandte Chemie International Edition in English, 33 (8), 803, 1994.
[2]. R.L. Carrier, L.A. Miller, I. Ahmed, Journal of Controlled Release, 123, 78, 2007
[3]. D.W. Lincoln, Endocrynology Philadelphia, 142, 1997.
[4]. A.V. Schally, A. Arimura, Y. Baba, R.M.G. Nair, H. Matsuo, T.W. Redding, L. Debeljuk, W.F.
White, Biochemicaland Biophysical Research Communication, 43, 393, 1971.
[5]. D.K. Laimou, M. Katsara, M.-T.I. Matsoukas, V. Apostolopoulos, A.N. Troganis, T.V. Tselios,
Amino Acids, 39 (5), 1147, 2010.
[6].D. Laimou, T. Katsila, J. Matsoukas, A. Schally, K. Gkountelias, G. Liapakis, C. Tamvakopoulos,
T. Tselios, European Journal of Medicinal Chemistry, 58, 237, 2012
Angrit,. Clitw. lnt. Ed. Enxi. 1994. 33. 803-822

70

Conformational studies of LHRH analogue conjugated with

cyclodextrin using 2D NMR and Molecular Modeling
Golfo Kordopati,a Theodore Tselios,a Tahsin Kellici,b Franci Merzel,c Thomas
Mavromoustakos,b Simona Golic Grdadolnik,c,d Gerasimos Tsivgoulisa
a

Department of Chemistry, University of Patras, 26504, Patras, Greece

Department of Chemistry, University of Athens, 15771, Athens, Greece
c
Laboratory of Biomolecular Structure, National Institute of Chemistry, 1001, Ljubljana,
Slovenia
d
EN-FIST Centre of ExcellenceDunajska 156, 1000 Ljubljana, Slovenia
b

Introduction
Cyclodextrins (CDs) are cyclic oligosaccharides consisted of (D-1,4)-linked D-Dglucopyranose units. CDs contain a hydrophobic cavity that is capable to include a
variety of hydrophobic compounds via host-guest complexation.[1] This property has
been extensively exploited in the past to change the physicopharmaceutical properties
of lipophilic drugs such as water-solubility, bioavailability, improved stability, and
effectiveness.[2] D-cyclodextrin may typically complex low molecular weight
molecules or compounds with aliphatic side chains, -cyclodextrin can complex
aromatic and heterocyclic molecules, and J-cyclodextrin can form inclusion
complexes with molecules as large as macrocycles, steroids and C60.[3,4]
The Gonadotropin-releasing hormone (GnRH) known as Luteinizing-hormonereleasing hormone (LHRH) stimulates the secretion of gonadotropins via binding to
receptors on the surface of gonadotrophin cells of the anterior pituitary. It is a
decapeptide, amide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) produced in
the hypothalamus and adjusts the steroidogenesis and gametogenesis. The
gonadotropins (LH, luteinizing hormone and FSH, follicle-stimulating hormone)
regulate the production of gametes and steroid sex hormones from the gonads (ovaries
and testes).[5,6]
Over the last years, for the treatment of hormone dependent cancer and fertility
have been synthesized a various LHRH peptide analogues (agonists or antagonists).
Leuprolide, (pGlu-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NHEt), is a synthetic altered
peptide ligand (APL) of LHRH.[7,8] Leuprolide has an aminoacid skeleton and thus is
sensitive to proteolysis.It is well known that peptides suffer from chemical and
enzymatic instability, poor absorption through biological membranes, rapid plasma
clearance, peculiar dose response curves and immunogenicity. 4t is reported in the
literature that CDs, based on the their complexation properties, can protect peptides
and proteins against enzymatic as well as chemical degradation.[4,9]
Aiming to reduce the proteolysis sensitivity of Leuprolide, we report here, for the
first time, the synthesis and conformational study of a CD LHRH analogue conjugate.
Synthesis
The LHRH analogue has DLeu at position 6, pyroglutamic acid at the amino end and
glycine at the carboxy end. The carboxyl group of glycine is an attractive candidate
for the attachment of the CD unit. However, to reduce any aggregation and steric
hindrance effects induced by the direct grafting of the peptide onto the CD, a nonnatural and flexible amino acid, H-aminohexanoic acid, (HAhx) was added to glucine.

71

Therefore the final molecule was synthesized via coupling of HAhx with a modified
cyclodextrin in which one hydroxyl group has been replaced by an amino group (3monoamino--CD) (Sheme 1).

Scheme 1. Structure of the synthesized LHRH analogue coupled with E-CD.

Initially the synthesis of the analogue was performed by the Fmoc/tBu methodology,
utilizing the 2-chlorotrityl chloride (CLTR-Cl) resin. The side chains of the amino
acids were protected as follows: trityl group (Trt) for His, 2,2,4,6,7pentamethyldihydrobenzofurane group (Pbf) for Arg, tert-butyl group (tBu) for Ser
and Tyr. The protected peptide was cleaved from the resin using the cleavage mixture
CH2Cl2/Trifluoroethanol (7/3).
In the next step, the protected peptide was coupled with 3-monoamino-E-CD in
DMF as solvent. In this coupling step, a reaction took place between the amino group
of 3-monoamino-E-CD and the activated carboxylic acid. The reagents for activation
of the carboxylic group were HOBt/DCC.
The final products (peptide and the conjugated peptide) prepared by removal of all the
protected groups present by treatment with a mixture of TFA/CH2Cl2 in the present of
scavengers. Purification of the final products realized with semi preperative RPHPLC.

72

Scheme 2. Total synthesis of the LHRH analogue (2) and its conjugate with E-CD
(3).
NMR conformational analysis
The NMR experiments obtained at 298 K using Direct Drive Varian 800 MHz. For the
experiments were used 2 mM synthetic peptide 2 or conjugated peptide 3 in 0.7 mL of
a mixture 9:1 H2O/DMSO-d6.
In NOESY spectrum of 2 mM conjugated peptide, clear NOEs between the internal
H-3 and H-5 protons of E-CD (CD-H3, CD-H5) and the aromatic protons of Tyr and
Trp are eminent. These clear NOEs depict spatial vicinity of the interior lipophilic
core of E-CD with the aromatic protons. In order to distinguish if this interactions
between the peptide moieties and E-CD were intramolecular or intermolecular, extra
experiments were performed with mixture of peptide 2 and E-CD at different
concentrations.
Molecular dynamics
MD simulation was performed taking into account the results of the NMR
experiments. The obtained results were confirmed using Desmond MD calculations.
An extended conformation of the peptide backbone was manually constructed and
constraints between Tyr, Trp and E-CD were applied.

73

ACKNOWLEDGEMENTS
The NMR experiments were accomplished at Laboratory of Biomolecular Structure, National
Institute of Chemistry, Ljubljana, Slovenia. Financial support was provided by the Bio-NMR
(EU FP7) program.
REFERENCES
[1] Wenz, G., Angew. Chem., 1994, 106, 851-870 .
[2]
Albers, E.; Mller, B. W., Critical Reviews in Therapeutic Drug Carrier
Systems, 1995, 12, 311-337.
[3]
J. Krlov, Z. Kejk, T. Bza, P. Poukov, A. Krl, P. Martsek, V. Krl, J.
Med. Chem., 2010, 53, 128138.
[4]
Irie T., Uekama K., Adv. Drug Deliv. Rev., 1999, 36, 101-123.
[5]
Schally A. V.,. Arimura A, Baba Y., Nair R. M., Matsuo H., Redding T.W.,
Debeljuk L., White W. F., Biochem. Biophys. Res. Commun., 1971, 43, 393-399
[6]
Millar R. P., Anim. Reprod. Sci,. 2005, 88, 5-28.
[7]
Tan M.M., Corley C.A. & Stevenson C.L, Pharm. Res, 1998,19, 1442- 1448
[8]
Meyer J.D., Manning M.C., Vander Velde D.G., J. Peptide Res., 2002, 60,
159-168
[9]
J. Krlov, Z. Kejk, T. Bza, P. Poukov, A. Krl, P. Martsek, V. Krl, J.
Med. Chem., 2010, 53, 128138.

74

NMR structure and function of the mutated forms of the RING-H2

domain of Arkadia
Birkou Maria1, Chasapis T. Christos1, Bentrop Detlef2, Herrmann Torsten 3, Episkopou
Vasso4, Spyroulias A. Georgios1
Department of Pharmacy, University of Patras, GR-26504, Greece.
2
Institute of Physiology II, U of Freiburg, D-79104 Freiburg, Germany.
3
CRMN, Universit de Lyon, UMR 5280 CNRS, Lyon, France.
4
Imperial College, School of Medicine, London W12 0NN, UK.
G.A.Spyroulias@upatras.gr
1

The ubiquitin- proteasome pathway is a major pathway for the targeted degradation of
proteins. In general, protein ubiquitination is catalyzed by a cascade of enzymes,
including an ubiquitin- activating enzyme E1, an ubiquitin conjugating enzyme E2
and an ubiquitin ligase E3. E3 ubiquitin ligases are crucial in the selective recognition
of target proteins and in the subsequent protein degradation by the 26S proteasomes
[Inoue and Imamura 2008]. Arkadia is an E3 ubiquitin ligase with a characteristic
RING domain in its C-terminus. Arkadia is the first example of an E3 ligase that
positively regulates TGF family [Episkopou et al. 2007].
The Arkadia RINGs, were cloned and expressed in their Zn-loaded form and studied
through NMR Spectroscopy [Kandias et al. 2009]. The 3D NMR solution structure of
Arkadia RING was determined and deposited in PDB (2KIZ). Additionally, NMRdriven titration studies were also performed to probe the interaction interface of
Arkadia RING and the partner E2 (UbcH5B) enzyme and the RING-E2 complex was
constructed through an NMR-driven docking [Chasapis et al. 2012].
Additionally, this study resulted to the identification of Arkadias RING functionally
important residues, such as the conserved Trp972. Trp972 is considered as one of the
key residues for E2 recognition and binding [Huang et al. 2009]. According to recent
experimental evidence, the mutation of the Trp972 to Arg abolishes the ability of
Arkadia to amplify TGF-b-Smad2/3 signaling responses in tissue culture transcription
assays [Episkopou, et al. 2011]. Various Arkadia Trp mutants are now being studied
through NMR in order to obtain an atomic-level insight about the structural base of
Arkadia capability to selectively interact with the appropriate E2.
ACKNOWLEDGEMENT

EU FP7-INFRA BIO-NMR (nr. 261863) & FP7-REGPOT-2011 SEE-DRUG (nr.

285950)
REFERENCES
[1]. Inoue Y., Imamura T., 2008. Cancer Science, 99 (11), 2107-2112
[2] Mavrakis K.J., Andrew R.L., Lee K.L., Petropoulou C., Dixon J.E., Navaratnam N.,
Norris D. P., Episkopou V., 2007. Plos Biology, 5(3): 586-603
[3]. Kandias N.G., Chasapis C.T., Detlef B., Episkopou V., Spyroulias G.A., 2008.
Biochemical and Biophysical Research Communications 378(3):498-502
[4]. Chasapis C.T., Kandias N.G., Episkopou V., Bentrop D., Spyroulias G.A., 2012.
Proteins, 80, (5):1484-9
[5]. Huang A., N. de Jong R., Wienk H., Winkler S. G., Timmers M. H. Th., Boelens R.,
2009. Journal of Molecular Biology 385: 507-519

[6]. Vikas S., Antonacopoulou G Anna, Tanaka S., Panoutsopoulos A.A., Bravou V.,
Kalofonos H., Episkopou V., 2011. Cancer Science, 71(20): 6438- 49

75

STRUCTURAL NMR STUDY OF FOUR VIRAL MACRO DOMAINS

Tsika A.1, Ntonti D.1, Melekis S.1, Chasapis C.T.1, Margiolaki I.2, Papageorgiou N.3, Coutard
B.3, Bentrop D.4, Spyroulias G.A.1
1

Department of Pharmacy, University of Patras, GR-26504, Patras, Greece.

Department of Biology, University of Patras, GR-26504, Patras, Greece.
3
AFMB, UMR7257 CNRS/Aix Marseille Universit, Marseille CEDEX 9, France.
4
Institute of Physiology II, University of Freiburg, D-79104 Freiburg, Germany.
2

Macro domains are evolutionarily conserved in eukaryotic organisms, bacteria and archaea,
indicating important basic biological function. Furthermore, they are found in nonstructural
proteins (nsPs) of several positive-strand RNA viruses, including hepatitis E virus, rubella
virus and coronaviruses, as well as alphaviruses. There are limited information on the viral
macro domains but it has been suggested that they act as an ADP-ribose-binding module,
while they are also involved in ADP-ribose metabolism and post-translation modification.
In this study we apply NMR spectroscopy to investigate the conformational properties and
dynamics of four macro domains: (a) two from New World alphavirus (Mayaro &
Venezuelan equine encephalitis virus), (b) one Old world alphavirus (Chikungunya virus) and
(c) one from the Hepevirus genus (HEV-1).
The four macro domains are cloned and expressed with Poly(His)tag, in high yields in E.
Coli.. All the protein constructs are soluble and, using E.coli culture supplements prior to
induction in typical (M9) minimal media, the bacteria growth rates and protein yield were
generally increased. Initial 1D 1H & 2D 1H-15N HSQC NMR experiments suggest that all
four macro domains are folded in solution. Acquisition and analysis of 2D/3D
homo/heteronuclear NMR data are underway.
ACKNOWLEDGEMENT
EU FP7-REGPOT-2011 SEE-DRUG (nr. 285950)

REFERENCES
[1]. Nikolas Papageorgiou,, Yves Watier, Lucy Saunders, Bruno Coutard, Violaine Lantez, Ernest A.
Gould, Andrew N. Fitch, Jonathan P.Wright, Bruno Canard and Irene Margiolaki, Preliminary
insights into the non structural protein 3 macro domain of the Mayaro virus by powder diffraction, Z.
Kristallogr. 225 (2010) 576-580
[2]. Georgios I. Karras, Georg Kustatscher, Heeran R Buchecha, Mark D Allen, Celine Pugieux ,
Flons Sait, Mark Bycroft and Andreas G. Ladurner, The macro domain is an ADP-ribose binding
module, The EMBO Journal (2005) 1911-1920
[3]. Weidong Han, Xiaolei Li, Xiaobig Fu, The macro domain family: Structures, functions, and their
potential thetapeutic implications

76

Targeting a heteromeric Glutamate/Serotonin GPCR complex involved in

psychosis
D. E. Logothetis1, L. Baki1, M. Fribourg3, J. Younkin1, A. Ellaithy1, T. Jin1, J. M. Eltit1, M. Cui1,
F. Guarnieri1, J. Gonzalez-Maeso3, Jose L. Moreno3, S. Sealfon3, D. Iliopoulos4, S. Gaitonde2,
M. Dukat2, and R. A. Glennon2
1

Department of Physiology and Biophysics, Virginia Commonwealth University, School of Medicine,

Richmond, VA, USA
2
Department of Medicinal Chemistry, Virginia Commonwealth University, School of Pharmacy,
Richmond, VA, USA
3
Departments of Psychiatry, Neurology and Neuroscience, Icahn School of Medicine at Mount Sinai,
New York, NY, USA
4
Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles,
CA, USA

Schizophrenia is a complex disorder afflicting ~1% of the population worldwide with symptoms
such as psychosis (hallucinations and delusions), motivational impairment (avolition or
amotivation), affective dysregulation (depression, mania) and alterations in information
processing (cognitive impairment). Although the introduction of antipsychotics since the mid1950s has dramatically reduced the hospitalization requirements for schizophrenic patients,
pharmacological treatments of this devastating disease have not substantially improved outcomes
for most patients. The worldwide market for schizophrenia drugs has grown tenfold since the
introduction of atypical antipsychotics such as risperdal (risperidone) in the early 90s, reaching
in the course of 15 years prescriptions of 50 million and drug sales of 10 billion dollars annually.
Unlike typical antipsychotics that target the Dopamine 2 Receptor (D2R), atypical antipsychotics
act as inverse agonists with highest affinity for the serotonin, 5-HT2A G protein-coupled receptor
(GPCR), a key player in the management of schizophrenia. Although these drugs have been
effective in managing this mental disorder in 70% of those affected, a significant fraction of
patients face serious side effects. The 30% of patients for whom there is no treatment coupled
with the underlying lack of knowledge on the biological basis of schizophrenia underscore the
need for new approaches to treat the disease.
The 5-HT2A receptor (or simply 2AR) forms functional heteromeric complexes with the
metabotropic glutamate type 2 receptor (mGlu2R) (Gonzalez-Maeso et al., 2008 Nature 452:937). We previously characterized signaling through the mGlu2/5-HT2A receptor complex
(mGlu2R/2AR) in a model cell expression system and showed that it results from the relative
conformations of the two receptors which are inversely coupled, such that drugs that stabilize a
given conformation in one receptor (active or inactive) cause the opposite conformation in the
partner receptor (Fribourg et al., 2011 Cell 147:1011-23). This inverse coupling, shown to hold
in native tissues and in behavioral models of psychosis, led us to develop a metric we call the
Balance Index (BI) that assesses a given drugs signaling output through this receptor complex
and predicts its psychoactive behavioral effects. Our recent efforts are focused on a) using the
BI to develop a High-Throughput assay to identify novel antipsychotics; b) developing bivalent
ligands that specifically recognize heteromeric over homomeric receptors. In this seminar, I will
summarize our published and unpublished work.

77

Synthesis of Biological Compounds Against Alzheimer Disease

Panayiotis. A. Koutentis
Department of Chemistry, University of Cyprus, PO Box 20537, 1678 Nicosia, Cyprus.
koutenti@ucy.ac.cy

The development of synthetic methods for heteroatom rich heterocycles leads often to novel and
unnatural structures. This area of study compliments the search for increased structural chemical
diversity from natural sources. Many unusual or unnatural heterocycles, have interesting biological
and physical properties and have uses in the pharmaceutical and material sciences.
Compounds that display interesting biological behavior towards A-amyloid aggregation inhibition
and/or inhibition of AChE/BChE were discovered during our ongoing study on the synthesis and
chemistry of 1,2,3-dithiazoles and areno fused 1,2,4-triazines. These compounds are, therefore, useful
candidates for studies against Alzheimers Disease.
In particular, benzotriazinone I and triazafluoranthenone II, selected owing to their suitable aqueous
solubility and A aggregation inhibition, were submitted to a time course kinetic assay followed with
thioflavin T (ThT) spectrofluorimetry, circular dichroism (CD) and transmission electron microscopy
(TEM). Experimental data indicated that I acted at a low concentration ratio (10 M I vs. 50 M II),
stabilizing the unstructured A peptide and inhibiting fibrillogenesis, and that II also acted as
fibrillization inhibitor, but likely enhancing and stabilizing the -sheet arrangement of A to yield
protofibrillar species as detected by TEM (Figure 1).[1]
A

Figure 1. Effects of inhibitors I and II on A1-40 aggregation. A1-40 (50 M) was incubated under
fibrillization conditions alone or in the presence of 10 M or 50 M . Aliquots of each reaction
were assayed by TEM (39,000-fold magnification): for control reaction at time point zero (A) and
after 7 d (B); for co-incubated A/ (C) and A/ (D) after 7 d.[1]

REFERENCES
[1]. Catto, M., Berezin, A.A., Lo Re, D., Loizou, G., Demetriades, M., De Stradis, A.,
Campagna, F., Koutentis, P.A., Carotti, A. Eur. J. Med. Chem., 58, 84-97, 2012.

78

Synthesis of Biological Compounds Against Alzheimers Disease

Panayiotis. A. Koutentis
Department of Chemistry, University of Cyprus, PO Box 20537, 1678 Nicosia, Cyprus.
koutenti@ucy.ac.cy

The development of synthetic methods for heteroatom rich heterocycles leads often to novel
and unnatural structures. This area of study compliments the search for increased structural
chemical diversity from natural sources. Many unusual or unnatural heterocycles, have
interesting biological and physical properties and find uses in the pharmaceutical and material
sciences.
Compounds that display interesting biological behavior towards A-amyloid aggregation
inhibition and/or inhibition of AChE/BChE were discovered during our ongoing study on the
synthesis and chemistry of 1,2,3-dithiazoles and areno fused 1,2,4-triazines. These
compounds are, therefore, useful candidates for studies against Alzheimers Disease.
In particular, benzotriazinone I and triazafluoranthenone II, selected owing to their suitable
aqueous solubility and A-amyloid aggregation inhibition, were submitted to a time course
kinetic assay followed with thioflavin T (ThT) spectrofluorimetry, circular dichroism (CD)
and transmission electron microscopy (TEM). Experimental data indicated that I acted at a
low concentration ratio (10 M I vs. 50 M II), stabilizing the unstructured A peptide and
inhibiting fibrillogenesis, and that II also acted as fibrillization inhibitor, but likely enhancing
and stabilizing the -sheet arrangement of A to yield protofibrillar species as detected by
TEM (Figure 1).[1]
A

Figure 1. Effects of inhibitors I and II on A1-40 aggregation. A1-40 (50 M) was incubated
under fibrillization conditions alone or in the presence of 10 M or 50 M . Aliquots of
each reaction were assayed by TEM (39,000-fold magnification): for control reaction at time
point zero (A) and after 7 d (B); for co-incubated A/ (C) and A/ (D) after 7 d.[1]
The study initially began with the screening of a small library of heterocyclic quinonimines
(Figure 2) for A1-40 aggregation by colleagues Dr. Marco Catto and Prof. Angelo Carotti at
the University of Bari. While the study indicated that the phenazinone 1, the phenathiazinone
1

79

2 and the fused carbazolone 3 were more active, we chose to develop the chemistry of the
bezotriazinone 4 for two reasons: First, the triazinones were much more readily functionalized
and secondly, the synthetic chemistry of the benzotriazinone was less well explored.

Figure 2. Mean % A inhibition values @ 100 mM of core heterocyclic quinonimines 1-4.

1,3-Diphenylbenzo[e][1,2,4]triazin-7(1H)-one (4) is a potentially useful heterocyclic scaffold
that is highly coloured (max 544 nm), and supports a quinonimine moiety. It was originally
prepared in low yield (15%) by Neugebauer et al.,[2] preventing an in depth study of its
potentially rich chemistry. However, by treating amidrazone 5 with KMnO4,[3] or
benzotriazinyl radical 6 with MnO2,[4] the benzotriazinone 4 can be prepared in high yields of
82 and 84%, respectively (Scheme 1).

Scheme 1. Synthesis of 1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-one (4).

Some chemistry of the benzotriazinone 4 has recently been reported:[5],[6] Benzotriazinone 4
undergoes nucleophilic addition regioselectively at C-6, and electrophilic substitution
regioselectively at the C-8 position,[5] however, direct condensations on the carbonyl at C-7
using primary amines or active methylenes failed. Oxidative and nonoxidative silver-mediated
palladium catalyzed cyclizations at C-8 afford the triazafluoranthenone 7 that is structurally
similar to the canthinone alkaloids.[7],[8]

A range of non-metal catalyzed cyclization reactions also afforded linear and angular fused
benzotriazinones, some of which were unusual zwitterionic compounds (e.g., compound
2

80

8).[9] Interestingly, new radical entities could also be prepared from the benzotriazinone 4
such as the -extended fused imidazolo-, oxazolo- and thiazolobenzotriazinyls 9.[10],[11]

We also recently targeted an improved synthesis of benzotriazinones sporting a wide variety

of substituents at both N-1 and C-3. The classical route to benzotriazinyl radicals 6 involved
accessing the amidrazone 5 which was difficult to prepare in high yield or obtain very pure
owing to its oxidative instability that also made it difficult to store. As such, we developed a
two-step route to 1,3-disubstituted benzo and pyrido-fused 1,2,4-triazinyl radicals that
involved the N-(2-nitroarylation) of easily prepared N-(het)arylhydrazides via nucleophilic
aromatic substitution of 1-halo-2-nitroarenes, which in most cases gaves N-(het)aryl-N-[2nitro(het)aryl]hydrazides 10 in good yields. Mild reduction of the nitro group followed by an
acid-mediated cyclodehydration gave the fused triazines, which upon alkali treatment
afforded the desired radicals, subsequent mild oxidation of which gave the desired
quinonimines (Scheme 2).[12]

Scheme 2. Improved synthesis of benzotriazinones 4 via N-(het)aryl-N-[2nitro(het)aryl]hydrazides that avoids the preparation of amidrazone 5.
With the diverse chemistry of the scaffold developed, we were able to provide a wider array
of triazinones for analysis of their biological properties. The 8-halo 1,3-diphenyl substituted
benzotriazinones 11-13 strongly inhibited A1-40 aggregation and led to IC50 values ranging
between 6.1 and 9 M (Figure 3).

81

Figure 3. Mean % A inhibition values @ 100 M of 8-halobenzotriazinones 11-13 and IC50

values.
The inhibition of A1-40 aggregation could also be manipulated by modifying the N-1 and C-3
substituents to give inhibition as high as 89% for the N-1 Ph, C-3 CF3 analogue 17 for which
an IC50 value of 11 M was determined. Interestingly, ring fusion to give the carbazolone 7
also afforded a highly active analogue with an IC50 value of 6.1 M (Figure 4).

Figure 4. Mean % A inhibition values @ 100 M of benzotriazinones 4, 7, 14-17 and IC50

values for compounds 7 and 17.
However, the most interesting behavior was found when the C-6 position of either scaffold 4
or 7 was substituted by dialkylamines. After screening a wide range of amines, compounds 18
and 19 were identified that showed sub M inhibition values for A inhibition while
compound 20 was found to not only to inhibit A inhibition but also acted as a selective sub
M inhibitor of BChE (Figure 5).

82

Figure 5. Mean % A inhibition values @ 100 M of benzotriazinones 18-20 and their IC50
values. Also, the IC50 value for BChE inhibition is presented for compound 20.
Conclusion: The facile chemistry of 1,2,4-benzotriazinones has enabled the preparation of a
diverse library of analogues some of which display impressive biological activities for A
aggregation inhibition and selective inhibition against BChE which are important behaviors
for studies related to Alzheimers disease. At this stage the most active compounds have yet
to be discovered and additional work is required to elucidate the mode of action. These
studies are currently in progress.
REFERENCES
[1].

Catto, M.; Berezin, A. A.; Lo Re, D.; Loizou, G.; Demetriades, M.; De Stradis, A.; Campagna,
F.; Koutentis, P. A.; Carotti, A. Eur. J. Med. Chem. 2012, 58, 84.
[2]. Neugebauer, F. A.; Umminger, I. Chem. Ber. 1980, 113, 1205.
[3]. Koutentis, P. A.; Lo Re, D. Synthesis 2010, 2075.
[4]. Constantinides, C. P.; Koutentis, P. A.; Krassos, H.; Rawson, J. M.; Tasiopoulos, A. J. J. Org.
Chem. 2011, 76, 2798.
[5]. Koutentis, P. A.; Krassos, H.; Lo Re, D. Org. Biomol. Chem. 2011, 9, 5228.
[6]. Koutentis, P. A.; Loizou, G.; Lo Re, D. J. Org. Chem. 2011, 76, 5793.
[7]. Ioannidou, H. A.; Martin, A.; Gollner, A.; Koutentis, P. A. J. Org. Chem. 2011, 76, 5113.
[8]. Gollner, A.; Koutentis, P. A. Org. Lett. 2010, 12, 1352.
[9]. Ioannou, T. A.; Koutentis, P. A.; Krassos, H.; Loizou, G.; Lo Re, D. Org. Biomol. Chem. 2012,
10, 1339.
[10]. Berezin, A. A.; Constantinides, C. P.; Drouza, C.; Manoli, M.; Koutentis, P. A. Org. Lett. 2012,
14, 5586.
[11]. Berezin, A. A.; Constantinides, C. P.; Mirallai, S. I.; Manoli, M.; Cao, L. L.; Rawson, J. M.;
Koutentis, P. A. Org. Biomol. Chem. 2013, 11, 6780.
[12]. Berezin, A. A.; Zissimou, G.; Constantinides, C. P.; Beldjoudi, Y.; Rawson, J. M.; Koutentis, P.
A. J. Org. Chem. 2014, 79, 314.

83

aggregation inhibition and selective inhibition against BChE which are important behaviors
for studies related to Alzheimers disease. At this stage the most active compounds have yet
to be discovered and additional work is required to elucidate the mode of action. These
studies are currently in progress.
REFERENCES
[1].

Catto, M.; Berezin, A. A.; Lo Re, D.; Loizou, G.; Demetriades, M.; De Stradis, A.; Campagna,
F.; Koutentis, P. A.; Carotti, A. Eur. J. Med. Chem. 2012, 58, 84.
[2]. Neugebauer, F. A.; Umminger, I. Chem. Ber. 1980, 113, 1205.
[3]. Koutentis, P. A.; Lo Re, D. Synthesis 2010, 2075.
[4]. Constantinides, C. P.; Koutentis, P. A.; Krassos, H.; Rawson, J. M.; Tasiopoulos, A. J. J. Org.
Chem. 2011, 76, 2798.
[5]. Koutentis, P. A.; Krassos, H.; Lo Re, D. Org. Biomol. Chem. 2011, 9, 5228.
[6]. Koutentis, P. A.; Loizou, G.; Lo Re, D. J. Org. Chem. 2011, 76, 5793.
[7]. Ioannidou, H. A.; Martin, A.; Gollner, A.; Koutentis, P. A. J. Org. Chem. 2011, 76, 5113.
[8]. Gollner, A.; Koutentis, P. A. Org. Lett. 2010, 12, 1352.
[9]. Ioannou, T. A.; Koutentis, P. A.; Krassos, H.; Loizou, G.; Lo Re, D. Org. Biomol. Chem. 2012,
10, 1339.
[10]. Berezin, A. A.; Constantinides, C. P.; Drouza, C.; Manoli, M.; Koutentis, P. A. Org. Lett. 2012,
14, 5586.
[11]. Berezin, A. A.; Constantinides, C. P.; Mirallai, S. I.; Manoli, M.; Cao, L. L.; Rawson, J. M.;
Koutentis, P. A. Org. Biomol. Chem. 2013, 11, 6780.
[12]. Berezin, A. A.; Zissimou, G.; Constantinides, C. P.; Beldjoudi, Y.; Rawson, J. M.; Koutentis, P.
A. J. Org. Chem. 2014, 79, 314.

6


84

Novel Cannabinergic Ligands

Spyros P. Nikas,a,c Rishi Sharma,a Marsha D Souza,a Demetris P. Papahatjis,b Marion
Schimpgen,b Shashank Kulkarni,a Alexandros Makriyannis.a
a

Center for Drug Discovery, Northeastern University, 116 Mugar Life Sciences Building, 360
Huntington Avenue, Boston, MA, 02115, USA. bInstitute of Organic and Pharmaceutical Chemistry,
National Hellenic Research Foundation, 48 Vass. Constantinou, Athens 11635, Greece. cE-mail:
s.nikas@neu.edu

The endogenous lipids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) as well as the
plant derived (-)-9-THC produce their biological effects by acting primarily on CB1 and
CB2 cannabinoid receptors, two G-protein coupled receptors (GPCRs) that are currently
being targeted for a number of conditions including pain, inflammation, CNS disorders and
cancer. The structures of AEA, 2-AG and THC are suitable templates for the development of
biological and pharmacological tools to explore the role of the endocannabinoid system and to
generate cannabinergic leads for drug discovery. In ongoing work we have focused on AEA
and 2-AG analogs with methyl groups at the bis-allylic carbons [1, 2]. The novel compounds
possess high CB receptor binding affinities and efficacies and exhibit enhanced metabolic
stabilities to the action of oxidative and hydrolytic enzymes. On the other hand, the
exogenous CB1 agonist 9-THC (as Marinol) has been approved to treat nausea and vomiting
in patients undergoing cancer chemotherapy and to stimulate appetite in HIV/AIDS patients.
However, this drug suffers from undesirable pharmacokinetic/pharmacodynamic (PK/PD)
properties including, high lipophilicity, generation of biologically active metabolites,
unpredictable time course of action, and low oral bioavailability. To address the undesirable
PK/PD profile of THC we have designed carboxy-cannabinoids with controlled
deactivation/detoxification through a soft drug/controlled release approach [3, 4].

ACKNOWLEDGEMENT
NIDA, DA 009158, DA007215, DA09046.
REFERENCES
[1]. Papahatjis D.P., Nahmias V. R., Nikas S. P., Schimpgen M., Makriyannis A. Chem. Eur. J., 16,
4091-4099, 2010.
[2]. Nikas, S. P., DSouza, M., Makriyannis, A. Tetrahedron, 68, 6329-6337, 2012.
[3]. Sharma, R., Nikas, S. P., Paronis, C. A., Wood, J-A. T., Halikhedkar, A., Guo, J. J., Thakur, G. A.,
Kulkarni, S., Benchama, O., Raghav, J. G., Gifford, R. S., Jrbe, T. U. C., Bergman, J., Makriyannis,
A. J. Med. Chem., 56, 10142-10157, 2013.
[4]. Sharma, R., Nikas, S. P., Guo, J. J., Mallipeddi, S., Wood, J-A. T., Makriyannis, A. ACS, Med.
Chem. Lett, ASAP, 2014.

85

Systems Medicine, Applying Systems Biology Approaches for Manipulation of

Altered Biomechanisms towards Multiple Sclerosis Treatment by the Use of
Specific Structured molecules and Antioxidant Vitamins: the PLP10
intervention Paradigm
Ioannis S. Patrikios*a,b, George .N. Loukaidesb, Evangelia E. Ntzanic, Marios C. Pantzarisb
a

School of Medicine, European University Cyprus, Nicosia, Cyprus bThe Cyprus Institute of Neurology
and Genetics, Nicosia, Cyprus, cUniversity of Ioannina School of Medicine, Ioannina, Greece.
Presenting author: I.Patrikios@euc.ac.cy

Introduction Multiple sclerosis (MS) treatments are products of reductionism, partially effective with no
(re)myelinating/neuroprotective abilities associated with significant side-effects. We aimed to assess
whether our novel interventions, formulated based on systems medicine (SM), comprising specific
polyunsaturated fatty acids (PUFA) and vitamins reduce disease activity in patients with relapsing
remitting (RR) MS who were either treated with disease modifying treatment (DMT) or untreated.
Methods We contacted a 30-month randomized, double-blind, placebo-controlled, proof-of-concept
clinical study at the CING. Of a total of 80 patients, 20 were randomly assigned to receive intervention A
(docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) (3:1 w/w) omega-3, linoleic acid
(LA)/gamma()-linolenic acid (GLA) (2:1 w/w) omega-6 fatty acids, omega-3/omega-6 (1:1 w/w), other
specific PUFA, monounsaturated fatty acids (MUFA), minor quantity of specific saturated fatty acids
(SFA), vitamin A and vitamin E), 20 to receive -tocopherol, intervention C, 20 to receive the
combination of A and C, intervention B (PLP10) and 20 to receive placebo, as an oral solution, once
daily. The primary end point was the annualized relapse rate (ARR) and the key secondary end point was
the time to disability progression. ISRCTN87818535.
Results PLP10 reduced the ARR by 70% (p=0.003), in relation to the baseline ARR and the placebo
increased by 46% (p=0.354). For the primary end point, PLP10 reduced the ARR by 58% (95% CI 0.10
to 0.79, p=0.016) and for the secondary end point, significantly reduced the risk of sustained progression
of disability by 86% over the 2-year period (Hr, 0.11; 95% CI 0.01-0.97, p=0.047) vs. placebo. More
patients in the PLP10 group (72%) vs. placebo group (20%) were free from new or enlarging T2weighted lesions on brain MRI scans over the 2-year study. No adverse events were reported.
Interventions A and C showed no significant efficacy.
X axis: Annual Relapse Rate

Discussion PLP10 treatment significantly reduced the ARR, and the risk of sustained disability
progression without any adverse or significant side effects. This is the first clinical study of SM approach
medical nutrient formula that holds strong promise as an effective treatment for RRMS.

86

Systems Medicine, Applying Systems Biology Approaches for Manipulation of

Altered Biomechanisms towards Multiple Sclerosis Treatment by the Use of
Specific Structured olecules and Antioxidant Vitamins: the PLP10
intervention Paradigm
Ioannis S. Patrikios1,3,4*, George N. Loukaides1,3, Evangelia E. Ntzani2 & Marios C. Pantzaris1,3*
Current available multiple sclerosis (MS) treatments are products of reductionism, partially
effective with no (re)myelinating or neuroprotective abilities and mostly associated with adverse
and/or significant side effects. We aimed to assess whether our novel oral interventions (PLP10,
A and C) formulated based on systems medicine concept, comprising specific fatty acids and
vitamins reduce disease activity in patients with relapsing remitting (RR) MS who were either
treated with disease modifying treatment (DMT) or untreated versus placebo. We contacted a 30month randomized, double-blind, placebo-controlled, parallel design, phase II proof-of-concept
clinical study. Here we show that PLP10 treatment significantly reduced the annual relapse rate
(ARR), and the risk of sustained disability progression without any adverse or significant side
effects. PLP10 has characteristics like no any other intervention or medication tried before with
unique efficacy abilities through different mechanisms of action, for innovative more effective
approach on MS treatment and with (re)myelination and neuroprotection potential.
This study is registered as an International Standard Randomized Controlled Trial, number
ISRCTN87818535.
Multiple sclerosis (MS) is a multifactorial complex disease that results from interplay between as
yet unidentified environmental factors and susceptibility genes. Together, these factors trigger a
cascade of events, including the engagement of immune system, inflammatory injury of myelin,
axons and glia, functional recovery and structural repair, gliosis, and neurodegeneration.
Multiple variables dynamically interact and many different complex interrelated processes are
simultaneously orchestrated for MS pathogenesis. Pathogenic mechanisms involved are immunemediated inflammation, oxidative stress and excitotoxicity. 1 The onset with relapsing remitting
(RR) MS is seen in 85% of cases. About 60-70% of these individuals will eventually enter a
progressive phase of the disease. The increasing prevalence of MS, the limited efficacy and the
side effects of the existing treatments urge the clinical need for the development of new,
innovative, more effective, safe, and preventive treatment strategies.
Specific PUFA and antioxidant deficiencies along with decreased cellular antioxidant defense
mechanisms have been reported in MS patients.2 PUFA, such as the omega-3 EPA and DHA are
the most abundant structural components of neural tissue. The PUFA composition of membrane
1

Neurology Clinic C, The Cyprus Institute of Neurology and Genetics (CING), Nicosia, Cyprus.
Clinical and Molecular Epidemiology Unit, Department of Hygiene and Epidemiology,
University of Ioannina School of Medicine (UISM), Ioannina, Greece. 3 PALUPA Medical Ltd.,
CING, Nicosia, Cyprus. 4 School of Medicine, European University Cyprus, Nicosia, Cyprus.
*
Correspondence should be addresses to e-mail: I.Patrikios@euc.ac.cy or pantzari@cing.ac.cy
2

87

phospholipids plays a direct role in immune and non-immune related inflammation,

neuroprotection and remyelination. The fatty acid composition of phospholipids determines the
biophysical and functional characteristics of membranes such as membrane fluidity, transport,
polarity, and plays an important role in overall membrane organization, cellular integrity, and
intra and intercellular signaling.3 Vitamin E (meaning the alpha-tocopherol isoform) and tocopherol (another form of vitamin E) are both efficiently implicated in trapping reactive
oxygen (ROS) and nitrogen species (RNS) and both exert non-antioxidant properties, including
modulation of cell signaling and immune function, regulation of specific gene transcription, and
induction of apoptosis.4,5
In vitro, in vivo, and ex vivo studies have demonstrated that diet EPA, DHA, LA, and GLA can
be implicated and modulate almost all known complex pathways repertoire in MS
pathophysiology. Anti-inflammatory properties of omega-3 and omega-6 PUFA include
competitive inhibition of arachidonic acid and its metabolites that can be involved in promoting
inflammation;6 production of anti-inflammatory prostaglandins, thromboxanes and inhibition of
pro-inflammatory cytokines.7 They can produce lipoxins and cause alteration of cytoskeletal
components, so inhibiting the ability of leukocytes for migration (an essential part of the MS
pathogenesis).8,9 Omega-3 PUFAs regulate the expression of the nuclear transcription factor -B,
which is very important in the production of pro-inflammatory cytokines, chemokines and
adhesion molecules and controls the expression of antiapoptotic genes that might protect
inflammatory leukocytes from programmed cell death.10-13 Resolvins and protectins derived from
omega-3 PUFA promote control of inflammation in neural tissues by inhibition of neutrophil
migration, reduction of tumor necrosis factor expression, interferon-alpha production and T-cell
apoptosis. 14,15,16 T-cell proliferation in acute and chronic inflammation can be reduced by
supplementation with either omega-6 or omega-3 PUFA.17 PUFAs are also shown to modulate
major histocompatibility complex (MHC) conformation, vertical orientation, lateral organization,
and trafficking.18
Omega-3 fatty acids electrophilic derivatives formed by cyclooxygenase-2, in activated
macrophages, stimulate the nuclear respiratory factor (Nrf2) inducing the transcription of
neuroprotective and antioxidant related genes, and activate the peroxisome proliferator-activated
receptor (PPAR) for anti-inflammatory response.19 Omega-3 PUFA and their eicosanoid
derivatives are thought to have strong PPAR-alpha-agonist properties of T-cells in humans; a
neuroprotective event in experimental animal models.20,21
Retinol X receptor (RXR) is a positive regulator of endogenous oligodendrocyte precursor cell
differentiation and remyelination.22 EPA and DHA are endogenous ligands of RXRs and PPAR
and increase neurogenesis.23,24 DHA has neurotrophic and neuroprotective properties against
excitotoxicity, inflammation and oxidative stress.24 DHA supplementation results to
neuroprotection with reduced neuronal and oligodendrocyte cell apoptosis and loss.24,25 It blocks

88

depolarization-induced increased glutamate efflux and induces glutamate receptors-activation

excitotoxicity, preventing neuronal damage and death.26 In vitro, omega-6 PUFA can alter
oligodendrocytes membrane composition and membrane depolarization affecting
phosphorylation of myelin basic protein, an important event in (re)myelination.27,28 EPA and
DHA interfere with the production of certain matrix metalloproteinases (MMPs). EPA and DHA
significantly decrease MMP-9 activity (an activity that can cause disruption of the blood brain
barrier (BBB)) and so inhibit T cell migration into the central nervous system (CNS).29 In animal
studies, both EPA and DHA stimulate the production of molecules involved in
(re)myelinogenesis.30,31 Based on the above, specific PUFA and dietary antioxidants fulfill the
criterion of biologic plausibility and have the potential to diminish MS symptoms severity and
activity, even promoting recovery (remyelination). 1
We are studying for first time a novel oral cocktail intervention PLP10, designed and formulated
based on the disease multifactorial nature and through systems medicine (SM) model approach
for MS treatment; where understanding and treating disease translates into identifying and
simultaneously manipulating global affected and interconnected pathogenic networks/pathways
rather than focusing only on solitary failing component(s). PLP10 comprises mainly different
specific structured fatty acids within specific ratio, quantity, quality, and structural form
including specific antioxidant vitamins and gamma-tocopherol; with each one of the agents able
to efficiently interfere with different pathophysiological process involved in MS and all together
potentially able to affect the system holistically to a desirable outcome.
In our phase II, single-center, double-blind, randomized, placebo-controlled clinical trial we
intended to evaluate the therapeutic ability of PLP10 and of two other interventions consisting of
PLP10 constituent partial fractions versus placebo, when used in patients with RRMS.
RESULTS
Study population
From July 2007 through December 2010 (including the 12-month extended period), a total of 80
MS patients were randomly assigned to a study group at The Cyprus Institute of Neurology and
Genetics (CING), a tertiary neurological center.
Among the 80 patients, 20 patients were randomly assigned to each of the three groups to receive
the interventions and 20 to receive placebo. Baseline characteristics of both the ITT and the perprotocol populations were similar across groups (Table 1). Total drop-out patients completed
follow-up until study completion and considered for the ITT analyses. Five patients were totally
lost to follow before their first scheduled visit and two patients dropped-out before their first
scheduled visit progressed to secondary progressive MS. Fifteen patients dropped-out without
successfully completing the normalization period including five pregnancies. Another 17
patients dropped-out early after entry baseline. Seven patients that dropped out were given

89

monoclonal antibody treatment (natalizumab). Overall, a total of 41 (51%) patients completed

the 30 month study (July 2007 through December 31st 2009) where one patient from group A and
two from the placebo group transferred on natalizumab, and 39 (49%) patients either withdrew
(drop-out) or lost to follow.
Efficacy
Relapses
Regarding the per-protocol analysis, during the first year of treatment, the relapse rate was 0.80,
0.40, 0.78 and 0.83 for the four intervention groups respectively. During the second year, the
relapse rate was 0.90, 0.40, 0.67 and 1.25 for the four intervention groups respectively. Overall,
for the two year primary end point, 8 relapses were recorded for the 10 patients in PLP10 group
(0.40 ARR) vs. 25 relapses for the 12 patients in placebo (1.04 ARR), a 64% adjusted relative
rate reduction (RRR) for the PLP10 group (RRR 0.36, 95% confidence interval (CI) 0.15-0.87,
p=0.024) (Tables 2 and Fig. 1A, 1C). Excluding patients on monoclonal antibody (natalizumab)
treatment, the observed adjusted RRR became stronger (72%) over the two years (RRR 0.28,
95% CI 0.10-0.79, p=0.016, Table 2). Pair-wise comparisons for the other two groups against
placebo did not yield statistically significant results. The proportion of patients with 1 relapse
for the two years on-study was higher in the PLP10 group than in the placebo group (90% vs.
42%, p=0.030, Table 2). Seeking to investigate further the observed difference, we compared the
relapse rate during the 24 months before entry to the study to the 24 months on-treatment for
each intervention group. We observed a statistically significant relative reduction in the ARR
(70%) only in the PLP10 group (RRR 0.30; 95% CI 0.14-0.65, p=0.003); within-group
comparisons for the three other groups ARR reduction was not significant and remained not
significant when natalizumab treated patients were further excluded from the analysis. The effect
of PLP10 through time at different time-windows versus placebo for all-time on-study patients is
shown in figures 1a to 1d. PLP10 reached maximum effect within a year on-treatment and
remained stable at an ARR of 0.4, displaying a steadily reduced ARR with long free-relapse
time-windows. Finally, during the 12 month post-study extended period (January 1st 2010 to
December 31st 2010) all-time on-study patients that received PLP10, showed persistent benefit in
relapse rates compared to placebo (six relapses for PLP10, 0.6 ARR vs. 19 for the placebo group,
1.58 ARR) indicating a statistically significant 62% adjusted relative rate reduction in the ARR
for the PLP10 group (RRR 0.38, 95% CI 0.12-0.99, p=0.046).
Regarding the ITT analysis, within PLP10 group, none of the nine drop-out patients changed to
natalizumab and two out of nine discontinued because of pregnancy, whereas two out of seven
drop-out patients from the placebo group changed to natalizumab (a total of four patients within
total placebo arm population were on natalizumab, including the two patients that transferred
while all-time on-study vs. none within PLP10 group). As reported, natalizumab reduces ARR by
68%, decreases the possibility of disability progression by 43% with 57% of patients free of new
or enlarging T2 lesions on MRI scans compared to 15% on placebo.32 No statistically significant

90

differences in the ARR were observed for the comparison of any group vs. placebo for the 24
months on-treatment, with a 0.75 ARR within PLP10 (30 relapses) and 1.03 ARR within placebo
(41 relapses) group, a 27% ARR reduction. Interestingly, despite the high non-adherence rate,
there was a statistically significant difference for the comparison of the ARR in the 24 months
before entry baseline to the 24 months on-treatment for the PLP10 group (RRR 0.45, 95% CI
0.26 to 0.78, p0.005).
Disability progression
Regarding the per-protocol analysis, at two years, the time to disability progression, with
confirmation after six months (secondary end-point) was significantly longer only with PLP10.
The cumulative probability of disability progression was 10% in the PLP10 group and 58% in
the placebo group (p=0.019). After excluding patients on natalizumab, there was a statistically
significant difference between the PLP10 and the placebo group for the same analysis (p=0.006)
(Fig. 2). At two years, the cumulative probability of progression was 10% in the PLP10 group
and 70% in the placebo group, which represents a decrease of 60 percentage points or a relative
86% decrease in the risk of sustained progression of disability within PLP10 group (adjusted
hazard ratio, 0.11; 95% CI 0.01-0.97, p=0.047). One vs. seven out of ten patients progressed to
confirmed disability in the PLP10 and the placebo groups respectively when patients on
natalizumab were excluded. No statistically significant difference was observed for any
comparison of the other two groups compared to placebo (Fig. 2).
Regarding the ITT analysis, at two years, the cumulative probability of progression was 10% in
the PLP10 group and 35% in the placebo group (p=0.052), which represents a decrease of 25
percentage points or a relative 71% decrease of the PLP10 for the risk of sustained progression
of disability (adjusted hazard ratio 0.22, 95% CI 0.04 -1.07, p=0.06). Two vs. seven out of the
total randomized patients progressed to confirmed disability in the PLP10 and the placebo
groups respectively. No significant differences were observed for groups A or C against placebo.
MRI
Over two years, the MRI results support the overall conclusion from the study that PLP10 has a
positive effect on disease activity since only 29% from the PLP10 group as opposed to 67% from
the placebo group developed new or enlarging T2 lesions (57% relative risk reduction).
Excluding patients on natalizumab there is an increased relative risk reduction (64%) between
PLP10 as opposed to placebo with 29% of patients on PLP10 and 80% on placebo with
development of new or enlarging T2 lesions (Table 2).

Safety
Over the course of the 30 month study no significant adverse events were reported from any
group. According to a questioner procedure the only aetiology for drop-outs was the palatability

91

and smell of the formula preparations. Nausea was reported by two patients. No abnormal values
observed on any of the biochemical and haematological blood tests. No allergic reactions
reported.

PLP10

1,4

Placebo

1,2

1,17

0,8

0,8
0,67

0,6
0,4
0,2

0,27

0
Entry
Baseline
PLP10 Group

0-6mo

0-12mo

Group A

PLP10 group

0-18mo

Group C

0,1

0-24mo

0
0-6mo

Placebo Group

Placebo group

6-12mo

1,04

0,4

0,4

Number of Relapses

0,83

6-24mo

PLP10 group
Placebo group
Linear (PLP10 group)
Linear (Placebo group)

3,5

1,35

0.83

6-18mo

2,5
2
1,5
1
0,5
0

Entry Baseline

1st year

Total 2-year

7 9 11 13 15 17 19 21 23
Month on Treatment

Figure 1ARR of all-time on-study population at different time-window periods. (a) ARR
of all-time on-study patients during the 24mo pre-treatment (baseline ARR) and at different onstudy intervals (6, 12, 18, 24 mo) per treatment arm.** (b) ARR of all-time on-study population
per 6 months intervals per treatment-arm, between 0-6, 6-12, 6-18, and 6-24 mo period intervals,
of PLP10 vs. placebo group.** (c) ARR of the all-time on-study population of PLP10 vs. placebo
group at baseline, during 1st year, and during the 2-year on-treatment.** (d) Dispersion of

92

relapses throughout the 2-year period of all-time on-study (excluding patients on natalizumab) of
PLP10 (n=10) vs. placebo (n=10). Placebo shows an irregular dispersion of relapses in relation to
PLP10 group with linear increasing trend wile PLP10 shows a stabilized linear trend. By using
the per-protocol model where patients on natalizumab are excluded, we could compare the
number of relapses on a same number of patients.
**Including the patients on natalizumab.

DISCUSSION
In this proof-of-concept clinical trial assessing the safety and efficacy of three cocktail
intervention formulas in RRMS, we observed a significant benefit for the novel PLP10
intervention compared to placebo for both the relapse rate and the progression to disability. Our
results include analyses pertaining to a total of 42 months study collected data, including the 12month, free of intervention treatment, extension period. We focused on the per-protocol data
analysis as a much more reliable method to answer the proof of concept trial-addressed
question.33 We thus present our main per-protocol analysis, as well as a subgroup analysis
excluding patients on natalizumab. We have found a statistically significant reduction in the
ARR and the disability progression comparing not only patients on PLP10 versus placebo but
also comparing the ARR of the PLP10 patients in the 24-month period prior to the study to the
ARR of the 24 months on- study; the observed differences became larger when patients that
received natalizumab (the most potent disease modifier) were excluded. The ARR decreased
within a year on PLP10 and significantly remained stable until study completion. Statistically
significant difference of ARR between patients on PLP10 versus placebo continued for the
additional 12 month extended period (persistent effect) without significant difference on
DMT.34 These clinical findings are supported by the results regarding the MRI analysis where
the proportion of patients free from new or enlarging brain T2 lesions was also higher in PLP10
group versus placebo. This study also provides important 30-month, placebo-controlled
information about the safety of PLP10 and the interventions A and C, where no any severe or
significant side-effects have been reported or associated with these regimen preparations.

93

100
Group
A
B (PLP10)
C
D (Placebo)

80

p=0.006 for PLP10 vs Placebo

60

40

20

0
0
Number at risk
Group: A
9
Group: B (PLP10)
10
Group: C
9
Group: D (Placebo)
10

12

15

18

21

24

27

30

Time to First Progression (Month)

9

10

10

10

10

10

10

Figure 2KaplanMeier plots of the time to sustained progression of disability among alltime on-study patients, excluding patients on natalizumab, receiving intervention A, PLP10
and C as compared with placebo. PLP10 reduced the risk of sustained progression of disability
by 86% over two years (p=0.006). Intervention formula A reduced the risk of sustained
progression of disability by 53% (p=0.266) and intervention formula C by 67% (p=0.061).

Following the concept of SM, PLP10 formulation rational innovatively combines all up-to-date
knowledge of the network relationships in MS pathophysiology of how environment, diet,
inflammation, (auto)immunity, antioxidant deficiency, oxidative stress, demyelination,
remyelination, genetics, and other factors possibly interrelate and influence each other's action;
able to modify, correct, and probably halt the pathogenic processes. PLP10 ingredients are
associated with treatment effective abilities to all known specific cascade of events and/or
pathophysiological mechanisms involved in MS and even disease recovery. It is the only
treatment preparation with remyelinating and neuroprotective potential.
Different factors and molecular entities appear to be part of the possible aetiology for MS but
specific PUFA and antioxidants are key substances found to be related to all known pathogenic
and recovery mechanisms. MS patients are characterized with significant deficiencies in PUFA

94

levels in blood, cellular membranes of mononuclear and immune cells, and various other tissues,
with the cause not entirely clear that may involve metabolic alterations and nutritional habit
variations.
The intervention daily dose was aiming and believed to be high enough to restore/amplify
organisms antioxidant ability and ensure cellular membranes lipid profile (specific essential
PUFA content) normalization and simultaneously potentiate involvement of the ingredients in
the anti-inflammatory and recovery mechanisms; as therapeutic and medical treatment agents.
Diet fatty acid molecules need about six months period to exert their beneficial effect and this
essential parameter was for the first time under consideration in our study design. If this
chronotherapy parameter is not included in the clinical protocol design then the possibility of
misleading result evaluation greatly increases.
The maintenance of myelin requires continued turnover of its components throughout life.35,36 In
addition to the EPA, DHA, LA, and GLA, PLP10 was containing limited quantities of other
structural PUFA, specific MUFA (mostly oleic acid) and SFA (palmitic and stearic acid),
specifically to provide a direct source for neuronal cell phospholipids, for (re)myelination and
neuroprotection as they are all major components and building blocks of any new physiological
myelin and cellular membranes in general. Assembly of the correct molecules into myelin
membrane may be especially critical during active synthesis. Possibly, if critical constituents
arent available or are metabolically blocked, amyelination, dysmyelination or demyelination
may ensue.37
Free radicals and RNS produced during inflammation play an important role in MS pathogenesis
and have been found in high concentrations within inflammatory MS lesions. Gamma-tocopherol
is a strong nitrogen oxide quencher among all natural known bioavailable lipophylic vitamins
and as a direct non-enzymatic antioxidant can actively interfere with the oxidative stress and trap
the elevated quantities of reactive species. Gamma-tocopherol is also a strong metalloprotein
quencher and protects the BBB disruption, a function process that is supported by the specific
PUFA contained in PLP10.38 Vitamin E (alpha-tocopherol) due to its dynamic free radical
scavenging, metal chelating and radical chain reaction-breaking properties, is important in
neutralizing ROS and can protect neuronal cells against glutamate induced excitotoxicity as well
as peroxidation of accumulated tissue and plasma lipid/PUFA levels, as a result of the PLP10
consumption.
Pathological stimuli in general (e.g. energy deprivation) and abnormal increased content of SFA
in membranes can cause disruption of the normal endoplasmic reticulum (ER) function, a state
known as ER stress resulting in the unfolded protein response (UPR) with accumulation of
unfolded protein in the ER, that can lead to the oligodendrocyte cell apoptosis.39 As a novel
hypothesis we believe that by normalizing membranes PUFA to SFA ratio content we probably

95

balance/reduce the ER stress and UPR. This will result in survival signals through activation of
the Nrf2 pathway that prevents cell death following the ER stress.
The established safety of the ingredients used and the protocol guidelines allowed us to proceed
with the clinical study even though with limitation on the pre-estimation of required trial sample
size as discussed above.
Our observations are consistent with the idea that simultaneous availability of specific omega3/omega-6 fatty acids and other major membrane and myelin lipid building blocks in
combination with specific antioxidants, within optimum quantity, quality, ratio and structural
form can possibly result to a more appropriate holistic therapy reducing MS disease activity.
This is probably succeeded through synergistic and/or simultaneous effect on the interactions and
dynamics of the most probable environmental and biological disease causing factors that induce
complex biological network of events for disease pathogenesis and evolution; as well as on the
protective and reparative mechanisms. The nature of the formula cannot be prohibitive for its use
as preventive regimen and does not preclude probable positive efficacy on the other types of MS,
but has to be further investigated. A larger size multicenter clinical trial will better establish
PLP10 place in the armamentarium of treatments for MS.
Environment acts long before MS becomes clinically evident and this suggests the existence of a
prodromal asymptomatic phase of the disease; a window of opportunity to potentially interfere,
regulate and perhaps halt the disease processes before it is clinically evident. 40 PLP10 might be
the only regimen able to serve such a preventive purpose and for delaying the RRMS evolution
to progressive MS that is of a major importance in the treatment of the disease.
The present study, for the first time provides strong link evidence between dietary, metabolic,
immunological, and neurobiological aspects of MS after three quarters of a century of
unsuccessful scientific efforts. This might probably be the beginning of opening new horizons
and new avenues in the approach of MS prevention and treatment, and possibly of other
multifactorial chronic diseases, including neurodegenerative and autoimmune as well.

10

96

Table 1 Demographics and baseline disease characteristics.

total Group A
(n=20)

Characteristics
of
randomized population

Group B Group C
(n=20)
(n=20)

Placebo
(n=20)

Pvalue

15 (75)

15 (75)

1.000

Sex
Female - no. (%)

15 (75)

15 (75)

Age (yr)
Mean Standard Deviation 38.011.9
38.110.9
0.982
36.98.4
37.78.7
(SD)
Median (Range)
38.0 (22 37.0 (25 36.5 (24 36.0 (21
65)
61)
54)
58)
Treatment history
Patients on DMT- no. (%)
Pre-treatment
duration (yr)
Mean SD

11 (55)

9 (45)

12 (60)

10 (50)

0.875

9.07.6

8.64.8

8.65.3

7.75.7

0.909

disease

Median (Range)

7.5 (2 37) 8.0 (2 20) 8.0 (3 24)

6.5 (2 25)

Mean SD

2.331.68

2.411.73

2.311.66

2.101.32

Median (Range)

2.0 (1 6)

2.0 (1 7)

2.0 (1 6)

2.0 (1 4)

ARR

1.17

1.21

1.16

1.05

Patients -% with 1 relapse

40

45

40

35

Mean SD

2.521.23

2.151.05

2.421.21

2.39 0.93

Median (Range)

2.5
5.5)

Pre-treatment relapses
0.946
0.946

Baseline EDSS score

(1.0 2.0
4.0)

Characteristics of all-time on- Group A

study population
(n=10)

(1.0 2.5
5.0)

(0.0 2.5
4.0)

0.775

(1.0

Group B
(n=10)

Group C
(n=9)

Placebo
(n=12)

Pvalue

5 (50)

7 (70)

6 (66.6)

10 (83.3)

0.419

Mean SD

36.613.5

34.805.4

40.98.1

39.813.2

0.572

Median (Range)

34.5

Sex
Female - no. (%)
Age (yr)

(22 34.5

(26 40.0

(29 37.5

(21

11

97

65)

43)

54)

58)

Patients on DMT- no. (%)

6 (60)

4 (40)

6 (67)

6 (50)

0.949

Pre-treatment
duration (yr)
Mean SD

9.710.0

8.35.3

11.36.1

8.7 7.1

0.807

Treatment history
disease

Median (Range)

7.5 (2 37) 8.0 (2 20) 8.0 (4 24)

5.5 (2 25)

Mean SD

2.201.47

2.701.25

1.780.66

1.671.37

Median (Range)

2.0 (1 6)

2.5 (1 4)

2.0 (1 3)

1.5 (1 4)

ARR

1.10

1.35

0.89

0.83

Patients -% with 1 relapse

30

20

33

50

Mean SD

2.651.37

2.401.12

2.111.02

2.160.96

Median (Range)

2.8
5.5)

Pre-treatment relapses
0.241

Baseline EDSS score

(1.0 2.5
4.0)

(1.0 2.0
4.0)

(1.0 2.2
3.5)

0.698

(1.0

Table 1 reports the demographics and baseline disease characteristics for total randomized and
all-time on-study population by treatment arm. There were no significant between study-group
differences at baseline for any characteristic.
PLP10 group
Available data at Entry Baseline (n=18 for group A, n=17 for group B, n=19 for group C, n=19
for group D)

12

98

Table 2 Clinical end points, according to study group for all-time on-study population.
Characteristics*

Group A
(n=10)

Group B
PLP10
(n=10)

Group
C
(n=9)

Placebo
(n=12)

Annual relapse rate over 1year**

0.80

0.40

0.78

0.83

Total number of relapses**

10

P-value
Group
B
vs
Placebo

Primary end points

Annual relapse rate over 2 years 0.85
(95% CI)**

0.40
0.87)

(0.15- 0.72

Total number of relapses**

17

13

25

Excluding patients on natalizumab

(n=9)

(n=10)

(n=9)

(n=10)

Annual relapse rate over 2 years 0.83

(95% CI)

0.40
0.79)

Total number of relapses

13

19

10 (1/10)

24

58 (7/12)

15

(0.10- 0.72

1.04

0.95

0.024

0.016

Secondary end points

Cumulative probability of sustained
progression increase by1 point on
EDSS confirmed after 6 mo, over 2 43
years -% **

0.019

Excluding patients on natalizumab

cumulative probability of sustained

13

99

progression increase by1 point on

EDSS confirmed after 6 mo, over 2 33
years -%

10 (1/10)

24

70 (7/10)

0.006

Patients proportion with 1 relapse

over 2 years -% **
50 (5/10)

90 (9/10)

56 (5/9)

42 (5/12)

0.030

MRI
Patients proportion with new or
enlarging T2 lesions-% **
----

29 (2/7)

----

67 (4/6)

29 (2/7)

----

80 (4/5)

60 (6/10)

67 (6/9)

75 (9/12)

Exploratory Results

Excluding patients on natalizumab

Patients proportion with no new or ---enlarging T2 lesions-%
DMT (interferons, glatiramer
acetate) and natalizumab
Patients proportion on DMT and
natalizumab at the end of 2 years-% 80
**
(8/10)

0.747

* CI denotes confidence interval.

** Including patients on natalizumab

1out of 10 on natalizumab

2 out of 12 on natalizumab

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102

Natural Products for Cancer Treatment: Tripterygium wilfordii and

Amygdalin Promotes cell death in Cancer Cells but not Normal Cells
Ioannis S Patrikios, Constantine G Hadjileontis, Anastasis S Stephanou
School of Medicine, European University Cyprus, Nicosia, Cyprus
Presenting author: A.Stephanou@euc.ac.cy
Plant extracts have a long history as a natural source of anticancer activity by inducing cell death.
Tripterygium wilfordii Hook F, an ivy-like vine also known as Thunder of God Vine, belongs to the
Celastraceae family and has been used as a natural medicine in China for hundreds of years [1].
Amygdalin (vitamin B17 also called Laetrile), is found in seeds of Prunus persica (L.) and also been
implicated in having anti-cancer activity [2]. The precise target of action for these plant-base anti-cancer
agents may be distinct, but all lead to identical cell death pathways. Importantly, studies performed with
Tripterygium wilfordii and amygdalin have not precisely indicated whether these anti-cancer plant-base
agents have any effects on normal cells. Therefore, in order to determine the direct effects of
Tripterygium wilfordii leaf or its root extract as well as amygdalin, we investigated their death-promoting
ability in cancer cell lines compared to normal cells. We treated neuroblastoma cell line SK-N and normal
primary fibroblasts cells with 1mg/ml of methanol extracted Tripterygium wilfordii (leaf and root extract)
or methanol extracted amygdalin and assessed morphological changes under inverted light microscope.
Following 48hrs treatment with Tripterygium wilfordii (leaf and root extract) or amygdalin, SK-N cells
detached from the tissue culture wells and showed morphological changes of apoptotic cell death. In
contrast, treatment with Tripterygium wilfordii (leaf and root extract) or amygdalin in normal fibroblast
cell did not have any affect after 48 hrs and this continued after 7 days treatment. Thus these results
strongly indicate that Tripterygium wilfordii (leaf and root extract) or amygdalin have death promoting
activity in cancer cells but not normal cells. All these data indicated the therapeutic potential of
Tripterygium wilfordii and amygdalin in cancer therapy.

REFERENCES
[1]. Kitzen JJ, et al, Eur J Cancer, 45: 176472, 2009.
[2]. Hyun-Kyung C, et al, Biol. Pharm. Bull, 29 (8) 15971602, 2006.

103

Poly-aromatic heterocyclic AMPK activators: the new platform for

developing of bifunctional drugs against type two diabetes
Ella Meltzer-Mats, Naomi Rozentul, Gali Babai, Lily Pasternak, Neta Uritsky, Tamar
Getter, Olga Viskind,, Jrgen Eckel, Erol Cerasi, Hanoch Senderowitz, Shlomo
Sasson and Arie Gruzman.
Division of Medicinal Chemistry, Department of Chemistry, Faculty of Exact Sciences,
Bar Ilan University, Ramat Gan, 52900, Israel Department of Pharmacology, Institute for Drug
Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, 91120,
Jerusalem, Israel German Diabetes Center, Integrative Physiology, Paul-Langerhans-Group,
40225, Dsseldorf, Germany The Endocrinology and Metabolism Service, Department of
Medicine, Hadassah- Hebrew University Medical Center, Jerusalem, 91120, Israel
A major pathophysiological factor in diabetics type two (T2DM) is decreased glucose utilization
in peripheral tissues, such as skeletal muscles and fat depots and insulin resistance. As a result of
decreased utilization of glucose, the former accumulates in blood and lead to hyperglycemia (high
level of glucose in the blood). The discovery of drugs that increase the rate of glucose entry to
skeletal muscles, even in the face of hyperglycemia, in non insulin dependent mechanism, is
therefore considered most promising for the development of novel antidiabetic therapeutic agents.
The enzyme AMPK, which acts as a sensor of cellular energy status, plays a central role in the
regulation of glucose transport in skeletal muscles. Activated AMPK promotes the translocation
of GLUT4-containing vesicles (in insulin sensitive tissues) to the plasma membrane, thus
increases glucose influx in a non-insulin dependent manner. Several classes of direct and indirect
activators of AMPK have been reported to date. However, side effects, such as lactic acidosis,
fluid retention, weight gain and development of tolerance to long-term use, emphasize the need
for more potent, less toxic and better tissue specific AMPK activators. In our work we aimed at
designing and synthesizing novel potent activators of AMPK in skeletal muscles. Based on
medicinal chemistry insight, a pharmacophore model and structure-activity relationship (SAR)
study of previously synthesized by our group compounds [1,2,3], we have prepared a series of
poly-aromatic heterocyclic derivatives. These were screened for their glucose uptake augmenting
effects and activation of AMPK in L6 myotubes. The lead compound dose and time dependently
augmented glucose uptake in L6 myotubes, increased phosphorylation of AMPK (Thr172) and its
downstream substrates such as, ACC (Ser79) and AS-160 (Thr642) and amplified the amount of
GLUT4 transporter on plasma membrane. I addition, the lead compound increased the insulin
secretion in rat islets. Finally, the antihyperglycemic potential of the lead compound was
evaluated in diabetic STZ-treated and KKAy mice. The blood glucose level and peripheral insulin
resistance in such mice was significantly reduced already after two days of treatment. Now our
ongoing work is focusing on improvement of a bioavailability of the lead compound.
REFERENCES
[1] Gruzman, A.; Shamni, O.; Ben Yakir, M.; Sandovski, D.; Elgart, A.; Alpert, E.; Cohen,

G.; Hoffman, A.; Cerasi, E.; Katzhendler, J.; Sasson, S. Novel D-xylose derivatives
stimulate muscle glucose uptake by activating AMP-activated protein kinase. J. Med.
Chem., 2008, 51, 80968108.
[2] Gruzman A.; Elgart A.; Viskind O.; Billauer H.; Dotan S.; Cohen G.; Mishani E.;
Hoffman A.; Cerasi E. and Sasson S. Antihyperglycaemic activity of 2,4:3,5dibenzylidene-D-xylose-dithioacetal in mouse models of type 1 and type 2 diabetes. J. Cell.
Mol. Med., 2012, 16(3), 593-603
[3] Meltzer-Mats E.; Babai G.; Pasternak L.; Uritsky U.; Getter T.; Viskind O.; Eckel J.;Cerasi E.; Senderowitz H.; Sasson S.;
Gruzman A. Synthesis and mechanism of antihyperglycemic activity of benzothiazole derivatives. J. Med. Chem., 2013, 56
(13):53355350.

104

Poly-aromatic heterocyclic AMPK activators: the new platform for

developing of bifunctional drugs against type two diabetes
A major pathophysiological factor in diabetics type two (T2DM) is decreased glucose
utilization in peripheral tissues, such as skeletal muscles and fat depots and insulin
resistance. As a result of decreased utilization of glucose, the former accumulates in
blood lead to hyperglycemia (high level of glucose in the blood) and as a result of that to
severe diabetic complications. The introduction of drugs that are able to increase the rate
of glucose entry to skeletal muscles, even in the face of hyperglycemia, in non insulin
dependent mechanism and simultaneously to augment the insulin secretion is therefore
considered most promising for the development of novel antidiabetic therapeutic agents.
Such compounds are not yet included in the broad spectrum of existing antidiabetic
therapy possibilities.
AMP-activated kinase (AMPK) is a member of a large family of highly conserved
heterotrimeric serinethreonine kinases. The main role of AMPK is to fine tune the cells
energy needs by monitoring the availability of ATP and quickly responding to different
environmental signals. Activated AMPK lowers the cells energy expenditure allowing
the cell to undergo mild adaptations to accomplish permanent changes in bodys
homeostasis. AMPK plays a central role in the regulation of glucose transport in skeletal
muscles. Activated AMPK promotes the translocation of GLUT4-containing vesicles (in
insulin sensitive tissues) to the plasma membrane, thus increases glucose influx in a noninsulin dependent manner.
A lot of research was done on the structure-, ligand- and fragment-based designs of
potential AMPK activators and an impressive structural basis for developing AMPK
activators and AMPK targeted drug discovery has been achieved. Several classes of
direct and indirect activators of AMPK have been reported to date. However, side effects,
such as lactic acidosis, fluid retention, weight gain and development of tolerance to longterm use, emphasize the need for more potent, less toxic and more tissue specific AMPK
activators.
In our work we aimed at designing and synthesizing novel potent activators of
AMPK in skeletal muscles and -cells. Although the beneficial role of AMPK in -cells
function is not clear. Based on medicinal chemistry insight, a pharmacophore
model and structure-activity relationship (SAR) study of previously synthesized by our
group compounds [1,2,3], we have prepared a series of poly-aromatic heterocyclic
derivatives. These compounds were screened for their glucose uptake augmenting effects
and stimulation of insulin secretion in -cells. Additionally, the activation of AMPK in
both types of the cells were investigated. The lead compound (phenylchromane
derivative) dose- and time-dependently augmented glucose uptake in L6 myotubes,
increased phosphorylation of AMPK (Thr172) and its downstream substrates such as,
ACC (Ser79) and AS-160 (Thr642) and amplified the amount of GLUT4 transporter on
plasma membrane. I addition, the lead compound increased the insulin secretion in INS1E -cells line and rat islets. Additionally, several ADME parameters: P450 inhibition,
mouse liver microsome stability and the possible inhibition of "Human ether-a-go-gorelated" gene (hERG)
were also investigated (in vitro models). Finally, the
antihyperglycemic potential of the lead compound was evaluated in diabetic STZ-treated
and KKAy mice. The blood glucose level and peripheral insulin resistance in such mice

105

was significantly reduced already after two days of treatment. Gross pathological
examination did not detect abnormal external findings or macroscopic alterations in
internal and external organs. Now our ongoing work is focusing on improvement of a
bioavailability and other pharmacokinetic parameters of the lead compound.
[1] Gruzman, A.; Shamni, O.; Ben Yakir, M.; Sandovski, D.; Elgart, A.; Alpert, E.; Cohen,
G.; Hoffman, A.; Cerasi, E.; Katzhendler, J.; Sasson, S. Novel D-xylose derivatives
stimulate muscle glucose uptake by activating AMP-activated protein kinase. J. Med.
Chem., 2008, 51, 80968108.
[2] Gruzman A.; Elgart A.; Viskind O.; Billauer H.; Dotan S.; Cohen G.; Mishani E.;
Hoffman A.; Cerasi E. and Sasson S. Antihyperglycaemic activity of 2,4:3,5dibenzylidene-D-xylose-dithioacetal in mouse models of type 1 and type 2 diabetes. J. Cell.
Mol. Med., 2012, 16(3), 593-603
[3] Meltzer-Mats E.; Babai G.; Pasternak L.; Uritsky U.; Getter T.; Viskind O.; Eckel J.;
Cerasi E.; Senderowitz H.; Sasson S.; Gruzman A. Synthesis and mechanism of
antihyperglycemic activity of benzothiazole derivatives. J. Med. Chem., 2013, 56 (13):5335
5350.

106

EFFECT OF METAL IONS ON FETAL HAEMOGLOBIN

INDUCTION BY HYDROXYUREA IN ERYTHROID CELL
LINES
S. Voskoua, M. Phylactidesa, GJ. Kontoghiorghesb, and M. Kleanthousa.
a

Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and

Genetics, Nicosia, Cyprus;b Postgraduate Research Institute of Scince Technology,
Environment and Medicine, Limassol, Cyprus
mphylact@cing.ac.cy

-thalassaemia, arising from reduced or absent synthesis of -globin, is an inherited

haemoglobinopathy resulting in severe anaemia. Treatment usually involves blood
transfusions and chelation therapy. -globin chain imbalance is at the root of the
pathology of the disease. Oxidative stress, resulting from globin chain degradation
and release of iron, causes ineffective erythropoiesis and red blood cell haemolysis
which are responsible for the anaemia seen in patients.
Hydroxyurea (HU) is the only FDA approved drug for the treatment of sickle cell
disease (SCD), another haemoglobinopathy resulting from a -globin gene mutation.
HU is only used sporadically for the treatment of -thalassaemia due to variability in
the responsiveness of the patients. HU has been shown to increase the level of fetal
haemoglobin (HbF), which can substitute for the reduced or mutant adult
haemoglobin in both diseases. A number of mechanisms have been proposed for the
mode of action of HU.
HU also exhibits chelating activity. It is anticipated that in a complexed form, many
of its biological, pharmacological and therapeutic properties are altered. It is,
therefore, possible that the presence of high levels of iron seen in heavily transfused,
inadequately chelated -thalassaemia major patients could affect HbF induction by
HU.
To study the effect of iron, as well as other metal ions on the HbF inducing ability of
HU, the erythroleukaemic cell line K562 and human primary erythroid cultures were
treated with HU in combination with increasing concentrations of Fe(II), Fe(III),
Cu(II), Zn(II) and Al(III). Cytotoxicity, haemoglobin and HbF levels were
determined.
Our results indicate that in both cell culture systems iron decreased HUs HbF
inducing activity without reducing HbF production on its own. Additionally, in the
concentration range tested, iron is not cytotoxic, nor does it affect HUs cytotoxic
characteristics. This lends support to the hypothesis that excess amounts of free iron
in -thalassaemia patients may interact with HU altering its therapeutic properties and
could in part explain the variability in response to HU treatment observed among thalassaemia patients. If proven, this would suggest the need for a reduction in iron
levels in patients in order to enhance the efficacy of HU treatment.

107

TARGETING TRANSCRIPTION FACTORS WITH DNA

INTERACTIVE SMALL MOLECULES
Khondaker Miraz Rahmana,b and David E Thurstona,b
a

Institute of Pharmaceutical Science, Kings College London, Britannia House, London SE1
1DB;
b

Transcriptgen Limited, 7 Trinity Street, London SE1 1DB; E-mail:

k.miraz.rahman@kcl.ac.uk
.

Transcription factors are regulatory macromolecules that induce profound and sustained
effects in cells by interacting with, and modulating the expression of, genes responsible
for critical cellular processes1. Interaction of a small molecule with the consensus DNA
sequence can prevent a transcription factor from recognizing its cognate sequence, thereby
preventing expression of genes associated with the transcription factor. Transcription
factor inhibition is an exciting new area of drug discovery, and is considered by some
experts to represent the next wave of cancer therapeutics following the kinase inhibitors
(which have now reached maturity) and the antibody-based approaches which are now in
the ascendancy. There are few approved drugs at present that work by selectively
inhibiting transcription factors, and so there is significant clinical and commercial
potential. From a scientific/clinical perspective, this approach has the advantage that a
selective and potent inhibitor would act at the ultimate signalling point of gene expression
(i.e., the promoter region of a gene) thus directly modulating the expression of genes
carrying the cognate DNA recognition site of the targeted transcription factor.
Furthermore, there are sub-families of transcription factors (e.g., STAT1 and STAT3), and
it may prove possible to target these independently, thus achieving fine-control over
transcription2. This is an important distinction from the kinase inhibitors that also
modulate gene expression, but have multi-pathway downstream targets and are rarely
highly selective, as their kinase target will usually control a range of transcription factors.
The talk will explore innovative DNA targetting therapeutic approaches to develop low
molecular weight druggable molecules that can be targeted to unique transcription
factor recognition sites in the human genome. Various mechanisms can be used including
the inhibition of protein-protein interactions (PPIs) and protein-DNA interaction (PDIs). A
number of duplex-DNA and promoter G- Quadruplex-binding agents are being developed
to target the PDI interaction3,4, and some examples will be presented.
REFERENCES
[1] Darnell, J. E. Nature Reviews Cancer 2002, 2, 740.
[2] Turkson, J. Expert opinion on therapeutic targets 2004, 8, 409.
[3] Rahman, K. M.; Jackson, P. J. M.; James, C. H.; Basu, B. P.; Hartley, J. A.; de la
Fuente, M.; Schatzlein, A.; Robson, M.; Pedley, R. B.; Pepper, C.; Fox, K. R.; Howard, P. W.;
Thurston, D. E. Journal of Medicinal Chemistry 2013, 56, 2911.
[4] Rahman, K. M.; Reszka, A. P.; Gunaratnam, M.; Haider, S. M.; Howard, P. W.; Fox, K. R.;
Neidle, S.; Thurston, D. E. Chemical Communications 2009, 4097.

108

Novel Inhibitors for the Aspartic Protease Endothiapepsin by Combining

Structure-Based Design, Dynamic Combinatorial Chemistry and STDNMR Spectroscopy.
Milon Mondala, Nedyalka Radevab, Helene Ksterb, Ahyoung Parkb, Constantinos Potamitisc,
Maria Zervouc, Gerhard Klebeb, and Anna K. H. Hirscha
a

Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The
Netherlands, bInstitute of Pharmaceutical Chemistry, Marbach Weg 6, 35032 Marburg, Germany,
c
Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation,
48 Vas. Constantinou Ave., 11635 Athens, Greece
E-mail: potamitis@eie.gr
Dynamic Combinatorial Chemistry (DCC) has proven to be an effective and innovative strategy
towards the faster identification of novel ligands for a selected biological target. In a Dynamic
Combinatorial Library (DCL) the building blocks are in dynamic equilibrium with their directly
synthesized products. The composition of a DCL responds to the addition of a biological target that
binds one or more library members, thus selecting and amplifying member(s) from the DCL [1].
Based on the crystal complexes of a series of chemotypes with endothiapepsin [2], a model system for
the pepsin-like aspartic proteases that are involved in numerous diseases such as hypertension,
Alzheimers disease and malaria, we designed a library of potential inhibitors (acylhydrazones).
This structure-based design in combination with DCC resulted in the selection of the best binders from
a DCL generated from five aldehydes and five hydrazides by the target. Saturation-Transfer
Difference (STD) NMR spectroscopy, an efficient NMR technique for the direct characterization of
targetligand interactions in solution, has been applied to identify DCL members bound to the target
[3].
The identified ligands were synthesized and their inhibitory potency was evaluated by an enzymebased fluorescence assay, exhibiting IC50 values in the low micromolar range. Subsequent co-crystal
structure determinations validated the predicted binding modes. Our results constitute a proof-ofconcept that the combination of de novo structure-based design, DCC and STD-NMR could be an
efficient methodology for hit identification and optimization [4].

REFERENCES
[1]. Lehn, J.-M., Ramstrm, O., Nat. Rev. Drug Discov., 1, 2636, 2002.
[2]. Kster, H., et al., J. Med. Chem. 54(22), 7784-7796, 2011.
[3]. Meyer, B., Peters, T., Angew. Chem. Int. Ed., 42, 864 890, 2003.
[4]. Mondal, M., Radeva, N., Kster, H., Park, A., Potamitis, C., Zervou, M., Klebe, G., Hirsch,
A.K.H., Angew. Chem. Int. Ed., in press.

109

Novel Inhibitors for the Aspartic Protease Endothiapepsin by Combining

Structure-Based Design, Dynamic Combinatorial Chemistry and
STD-NMR Spectroscopy
Milon Mondala, Nedyalka Radevab, Helene Ksterb, Ahyoung Parkb, Constantinos Potamitisc,
Maria Zervouc, Gerhard Klebeb, and Anna K. H. Hirscha
a

Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The
Netherlands, bInstitute of Pharmaceutical Chemistry, Marbach Weg 6, 35032 Marburg, Germany,
c
Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation,
48 Vas. Constantinou Ave., 11635 Athens, Greece
E-mail: potamitis@eie.gr

Introduction
In the recent years, dynamic combinatorial chemistry (DCC) [1] has been proven an effective
and innovative strategy towards the faster identification of novel ligands for a selected
biological target.[2] DCC allows the formation of a dynamic combinatorial library (DCL), in
which the building blocks are in dynamic equilibrium with their directly synthesized products.
The composition of a DCL responds to the addition of a biological target that binds one or
more library members, thus selecting and amplifying member(s) from the DCL[3]. SaturationTransfer Difference (STD) NMR spectroscopy, an efficient NMR technique for the direct
characterization of targetligand interactions in solution, has been applied to identify DCL
members bound to the target.[4] In the present study, we have used de novo structure-based
design (SBD) in combination with acylhydrazone-based DCC and 1H-STD-NMR
spectroscopy to identify a new family of potent hits for endothiapepsin, member of the
challenging class of aspartic proteases.[5] Finally, we have validated the proposed binding
mode of the inhibitors by X-ray crystallography.
Endothiapepsin belongs to the family of pepsin-like aspartic proteases, which are
involved in a wide range of diseases such as hypertension and malaria. [5] Endothiapepsin has
been used as a model enzyme for mechanistic studies [6] as well as for the development of
renin[7] and b-secretase[8] inhibitors. Eukaryotic aspartic proteases comprise two structurally
similar subunits, each contributing an aspartic acid residue to the catalytic dyad (Asp35 and
Asp219 in endothiapepsin) that cleaves the peptide bond of the substrate using a bound water
molecule.
The formation of acylhydrazones in aqueous environment has the right kinetic and
thermodynamic balance [9] and despite its pH dependence, this type of reversible linkage has
started to attract attention. This linkage requires the target protein to be stable at room
temperature for one week (pH 7.2),[10] the use of aniline as a nucleophilic catalyst (pH 6.2),[11]
or an acidic buffer system (pH<6). Acylhydrazones are attractive for biological DCC given
that the building blocks are readily available, afford an amide-type linkage that offers both
hydrogen-bond donor and -acceptor sites for molecular recognition by the target and are
sufficiently stable both at acidic and physiological pH values to enable direct analysis of the
DCL. The optimum pH value of endothiapepsin is 4.5, and we have shown that the enzyme is
stable under these conditions at room temperature for more than 20 days, thus making it an
ideal target enzyme for an SBD project that exploits acylhydrazone-based DCC.

110

Results and Discussion

We have used two crystal structures of endothiapepsin (PDB: 3PBD and 3PI0) that
represent two alternative binding modes: with and without a crystallographically localized
water molecule. We designed a series of acylhydrazones to address the catalytic dyad through
hydrogen-bonding interactions, by using the molecular-modeling software MOLOC[12] and
the FlexX docking module in the LeadIT suite.[13] The acylhydrazone moiety appeared to be a
suitable central scaffold, as modeling suggested it was anchored to the active site through a
strong hydrogen-bond network with the catalytic dyad. We introduced an a-amino group that
can form charge-assisted hydrogen bonds to the catalytic dyad as well as to Asp81, Asp33,
Gly221, or Thr222.
We modeled two alternative binding poses, in which the acylhydrazone addresses the
catalytic dyad either directly or via the catalytic water molecule (Figure 1). During the
modeling study, the E isomers emerged as the most suitable scaffolds, displaying two vectors
pointing towards the S2, S1, S1', and S2' pockets. Inspection of the cocrystal structures of
endothiapepsin with 11 fragments,[14] as well as hot-spot[15] analyses of the active site of
endothiapepsin showed that both aromatic and aliphatic substituents can be accommodated in
the S2, S1, S1', and S2' pockets surrounding the catalytic dyad.

Figure 1. Superposition of MOLOC-generated molecular models of potential inhibitors

featuring a) direct hydrogen bonding with the catalytic dyad in the active site of
endothiapepsin (PDB code: 3PBD) and b) hydrogen bonding with the catalytic dyad in the
active site of endothiapepsin through the catalytic water molecule (PDB code: 3PI0). Color
code: protein backbone: gray; inhibitor heavy atoms: C: green, violet, purple, blue; N: dark
blue; F: cyan; O: red; water molecule: red sphere.
According to our modeling study, both mono- and bicyclic aromatic moieties can be
accommodated by the S1'/S2' pocket, whereas the hydrophobic S2 pocket is best occupied by
a mesityl substituent. The S1/S1' pocket can host an indolyl, isobutyl, or phenyl moiety. We
selected a series of acylhydrazone-based inhibitors, retrosynthesis of which led to five
hydrazides H1H5 and five aldehydes A1A5 (Scheme 1).
We used 1H-STD-NMR experiments to identify the best component associations bound to
endothiapepsin, as this approach enables bound ligands to be monitored and requires only
small amounts of unlabeled protein.[16] To facilitate the analysis, we divided the whole library

111

into five sublibraries, each consisting of five hydrazides and one aldehyde, thus resulting in
the formation of five potential acylhydrazones (ten isomers, including E/Z isomers) in
equilibrium with the initial building blocks. The potential formation of imine products with
the free amino group of the hydrazides H1H4 or a surface exposed Lys side chain can be
ruled out because of the use of acidic conditions.[17] After addition of the target enzyme to the
pre-equilibrated libraries, we identified bound acylhydrazones by analyzing mainly the iminetype proton signals of acylhydrazones in the 1H-STD-NMR spectra, as in the case of the two
acylhydrazones H3-A4 and H4-A4 (Figure 2). In total, we identified a total of eight binders
from the five sublibraries (Scheme 1).

Scheme 1. Dynamic formation of an acylhydrazone library and enzymatic selection of the

best inhibitors by 1H-STD-NMR analysis.
g
f
e
d
c
b
a

112

Figure 2. Two acylhydrazones (H3-A4 and H4-A4) were identified by 1H-STD-NMR

spectroscopy from the DCL generated from H15 + A4 by assignment of the imine-type
proton signals of the bound
acylhydrazones indicated by vertical dashed red lines. The aromatic region of a) 1H-STDNMR spectrum of H15 + A4, b) 1H-NMR spectrum of H15 + A4, c) 1H-NMR spectrum of
H3+A4, d) H4+A4 (2 singlets correspond to the E/Z isomers), e) H1+A4, f) H2+A4 and g)
H5+A4.
To differentiate specific from nonspecific binding, we added the potent inhibitor saquinavir
(Ki=48 n) to the H15 + A4 sublibrary, thereby leading to the appearance of its signals at
the expense of the acylhydrazones signals in the 1H-STD-NMR spectrum, thus confirming
specific binding of the acylhydrazones H3-A4 and H4-A4 (data not shown).
To investigate the biological activity of the hits identified by 1H-STD-NMR, we performed
inhibition studies using the acylhydrazones (S)-H1-A1, (S)-H1-A3, (S)-H2-A1, (R)-H3-A1,
(R)-H3-A4, (R)-H3-A5, (S)-H4-A4, and H5-A1 synthesized from their corresponding
aldehyde and hydrazide precursors. We determined their inhibitory potency using the mixture
of E/Z isomers by applying a fluorescence-based assay adapted from the HIV-protease
assay.[18] The enzyme-inhibition assay confirmed the result of the 1H-STD-NMR experiments.
All eight acylhydrazones indeed inhibit endothiapepsin with IC 50 values in the range of 13 to
365 M. The most potent inhibitors, acylhydrazones (S)-H4-A4 and (R)-H3-A5, feature IC50
values of 12.8 M and 14.5 M, respectively To validate the predicted binding mode from
SBD, we soaked crystals of endothiapepsin with the two most potent inhibitors and were able
to determine crystal structures of (R)-H3-A5 (PDB code: 3T7P) and (S)-H4-A4 (PDB code:
4KUP) in complex with endothiapepsin at 1.25 and 1.31 resolution, respectively. During
the soaking experiments, the enzyme selects the E isomer from the mixture of E/Z isomers.
The cocrystal structure of (R,E)-H3-A5 shows two ligands in the active site: one binds to the
catalytic dyad, whereas the second is oriented towards the solvent. As a result of solvent
exposure and lack of protein contacts, the portion of this ligand molecule not visible in the
electron density map is most likely highly mobile and scattered over various conformational
states. The (S,E)-H4-A4 complex also exhibits two ligand molecules in the binding pocket.
Of these, only the first binds to the catalytic dyad, whereas the second ligand molecule is
solvent-exposed and, by analogy to the previous case, only fractionally visible in the electron
density and partially occupied (63%; Figure 3).

Figure 3. X-ray crystal structures of endothiapepsin cocrystallized with ligands: a) Overview

of the two molecules of (R,E)-H3-A5 (C: green, water molecules: green spheres) bound in the

113

active site (PDB code: 3T7P). The central ligand binds through the catalytic water molecule to
Asp35 and Asp219. b) Overview of both molecules of (S,E)-H4-A4 (C: yellow, water
molecule: yellow sphere; PDB code: 4KUP). The central ligand addresses Asp35 and Asp219
directly through its a-amino group. c) Superposition of (R,E)-H3-A5 and (S,E)-H4-A4. Only
the surface of the (R,E)-H3-A5 complex is shown.
The detailed binding modes of both ligands are shown in Figures 3 and 4. The amide NH
group of (R,E)-H3-A5 forms a hydrogen bond to the dyad, mediated by the catalytic water
molecule. The imine-type N atom of the acylhydrazone linker forms a hydrogen bond with the
backbone amide NH group of Gly80. Another hydrogen bond is observed between the
carbonyl group of the acylhydrazone and the backbone amide NH group of Asp81. The
hydroxy group of the naphtholyl portion of the second ligand donates a hydrogen bond via its
hydrogen atom to the outer oxygen atom of the side chain of Asp219. Furthermore, the aamino group of the ligand forms a weak hydrogen bond to an adjacent water molecule, with
the remainder of the ligand being bound in the S1' and S2 pockets and oriented towards the
solvent.
(S,E)-H4-A4 interacts differently with the catalytic dyad as it uses its a-amino group
to form direct hydrogen bonds to Asp35 and Asp219; the a-amino group occupies virtually
the same position as the catalytic water molecule in the complex with (R,E)-H3-A5. While
the amide-type NH group of the acylhydrazone linker forms a hydrogen bond to the hydroxy
group of the Thr222 side chain, the indolic NH group donates its proton to form a hydrogen
bond to the carboxylate group of Asp81. (R,E)-H3-A5 and (S,E)-H4-A4 occupy different
regions of the binding pocket of endothiapepsin. Clearly, the different binding poses are
provoked by the inverted stereochemistry at Ca. This allows (S,E)-H4-A4 to address the S1
pocket with its indolyl moiety benefiting from CHp interactions with Phe116 and Leu125
and to form a salt bridge with the catalytic dyad. Furthermore, (R,E)-H3-A5 places its
hydrophobic phenyl group in the S1 pocket and is engaged in CHp and pp interactions with
Leu125 and Phe116, but its stereochemistry does not allow an interaction with the dyad.
Instead the water-mediated contact through its acylhydrazone linker is achieved. This ligand
benefits from CHp interactions with Ile77 and Leu133 through its naphtholyl moiety in the
S2' pocket, whereas (S,E)-H4-A4 experiences some hydrophobic contacts with Ile300 and
Ile304 in the S2 pocket.

Figure 4. Full binding mode of a) H3-A5 and b) H4-A4 in the catalytic active site.

114

Conclusions
We have demonstrated for the first time that the combination of de novo SBD and DCC is a powerful
technique for the rapid identification of novel hits that inhibit the aspartic protease endothiapepsin. We
have exploited 1H-STD-NMR spectroscopy to identify the binders directly from the DCL. Among the
hits identified, the best ones exhibit IC50 values in the low micromolar range. Subsequent cocrystal
structure determination confirmed our in silico prediction that either direct or water-mediated
interactions with the catalytic dyad can be achieved. We have reported the first example of
acylhydrazone-based inhibitors of endothiapepsin and aspartic proteases in general. Our synergistic
combination of methods holds great promise for the acceleration of the drug-discovery process, not
only for the notoriously challenging class of aspartic proteases but for a wide range of drug targets. By
enabling the identification and concomitant optimization of novel inhibitors, its potential would appear
to be greatest when applied during the early stages of the drug discovery process.
This work has been published: Mondal, M., Radeva, N., Kster, H., Park, A., Potamitis, C., Zervou,
M., Klebe, G. and Hirsch, A. K. H. (2014), Structure-Based Design of Inhibitors of the Aspartic
Protease Endothiapepsin by Exploiting Dynamic Combinatorial Chemistry. Angew. Chem. Int. Ed.,
53: 32593263.
REFERENCES
[1] a) J.-M. Lehn, Chem. Soc. Rev. 2007, 36, 151160, b) P. T. Corbett, J. Leclaire, L. Vial, K. R.
West, J. Wietor, J. K. M. Sanders, S. Otto, Chem. Rev. 2006, 106, 36523711, c) J. Li, P. Nowak, S.
Otto, J. Am. Chem. Soc. 2013, 135, 92229239.
[2] a) O. Ofori, J. Hoskins, M. Nakamori, C. A. Thornton, B. L. Miller, Nucleic Acids Res. 2012, 40,
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K. Yeoh, Y. Tian, T. D. W. Claridge, P. J. Ratcliffe, E. C. Y. Woon, C. J. Schofield, Angew. Chem.
2012, 124, 67766779; Angew. Chem. Int. Ed. 2012, 51, 66726675.
[3] Lehn, J.-M., Ramstrm, O., Nat. Rev. Drug Discov. 2002, 1, 2636.

[4] Meyer, B., Peters, T., Angew. Chem. Int. Ed. 2003, 42, 864 890.
[5] J. B. Cooper, Curr. Drug Targets 2002, 3, 155173.
[6] L. Coates, P. T. Erskine, S. Mall, R. Gill, S. P. Wood, D. A. A. Myles, J. B. Cooper, Eur. Biophys.
J. 2006, 35, 559566.
[7] J. Cooper, W. Quail, C. Frazao, S. I. Foundling, T. L. Blundell, C. Humblet, E. A. Lunney, W. T.
Lowther, B. M. Dunn, Biochemistry 1992, 31, 81428150.
[8] S. Geschwindner, L. Olsson, J. S. Albert, J. Deinum, P. D. Edwards, T. de Beer, R. H. A. Folmer,
J. Med. Chem. 2007, 50, 59035911.
[9] M. Sindelar, T. A. Lutz, M. Petrera, K. T. Wanner, J. Med. Chem. 2013, 56, 13231340.
[10] V. T. Bhat, A. M. Caniard, T. Luksch, R. Brenk, D. J. Campopiano, M. F. Greaney, Nat. Chem.
2010, 2, 490497.
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[12] P. R. Gerber, K. Mller, J. Comput.-Aided Mol. Des 1995, 9, 251268.
[13] BioSolveIT GmbH, Sankt Augustin. http://www.biosolveit.de, LeadIT, version 2.1.3.
[14] H. Kster, T. Craan, S. Brass, C. Herhaus, M. Zentgraf, L. Neumann, A. Heine, G. Klebe, J. Med.
Chem. 2011, 54, 77847796.
[15] H. Gohlke, M. Hendlich, G. Klebe, Perspect. Drug Discov. Des. 2000, 20, 115144.
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115

Dynamic Combinatorial Mass Spectrometry: a powerful tool for quick

identification of selective 2-oxoglutarate oxygenases inhibitors
Marina Demetriades
Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, OX1 3TA, Oxford, UK,
marina.demetriades@chem.ox.ac.uk

Dynamic combinatorial chemistry (DCC) involves the interaction of a number of compounds that
react reversibly to form large dynamic combinatorial libraries (DCLs) (Figure 1). Introduction of
template molecules, such as proteins, changes the composition of the products in the library with the
products stabilized by the template being amplified. Analyses and characterisation of the amplified
products (hits) leads in a quick identification of good binders of the template. The first analytical
methods applied in DCC were high pressure liquid chromatography (HPLC) and nuclear magnetic
resonance (NMR); the introduction of mass spectrometry (MS) later has insured high sensitivity and
high speed DCC assays. [1, 2] In the last decade, dynamic combinatorial mass spectrometry (DCMS)
with protein targets has emerged as a promising method for the identification of enzyme-inhibitors.
The number of reversible reactions that have found use in protein-targeted DCMS, over the years, is
limited. [3, 4] We developed a protein-directed DCMS employing for the first time the reversible
reaction between boronic acids and alcohols to form boronate esters. [5] Moreover we applied this
method as well as other DCMS reactions with enzymes of the Fe(II) and 2-oxoglutarate dependent
dioxygenases (2OG oxygenases) family for the identification of potent inhibitors. [4, 6]
Enzymes of the 2OG oxygenases family are involved in important biological processes and are related
to many diseases such as anaemia, stroke and obesity. The discovery of potent and selective inhibitors
of human 2OG oxygenases is essential for pharmaceutical intervention. [7, 8]

Figure 1. The principle of dynamic combinatorial chemistry.

Overall, the work described validates the use of DCMS methods for the development of potent and
selective inhibitors for 2OG oxygenases, and by implication of other enzyme families.
REFERENCES
[1].
[2].
[3].
[4].
[5].
[6].
[7].
[8].

Huc, I. and J.M. Lehn, P Natl Acad Sci USA, 94, 2106-10, 1997.
Scott, D.E., G.J. Dawes et al., Chembiochem, 10, 2772-9, 2009.
Poulsen, S.A., J Am Soc Mass Spectr, 17, 1074-80, 2006.
Lienard, B.M.R., Hueting R. et al., J Med Chem, 51, 684-8, 2008.
Demetriades, M., Leung, I.K.H. et al., Angew Chem Int Edit, 51, 6672-5, 2012.
Woon, E.C.Y., Demetriades, M., Bagg, E.A.L., et al., J Med Chem, 55, 2173-84, 2012.
Prescott, A.G. and Lloyd M.D., Nat Prod Rep, 17, 367-83, 2000.
Ryle, M.J. and R.P. Hausinger, Curr Opin Chem Biol, 6, 193-201, 2002.

116

Dynamic Combinatorial Mass Spectrometry: a powerful tool for quick

identification of selective 2-oxoglutarate oxygenases inhibitors
Marina Demetriades
Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, OX1 3TA, Oxford, UK,
marina.demetriades@chem.ox.ac.uk

Iron and 2-oxoglutarate dependent dioxygenases (2OG oxygenases) are non-haem enzymes
which depend on 2-oxoglutarate (2OG) and FeII for their catalytic activity.1-3 Enzymes of the
2OG oxygenase family are present in many living organisms where they catalyse a wide
variety of biologically important reactions. In humans, 2OG oxygenases are involved in
important biological processes such as DNA repair and the hypoxic response. Therefore, a
number of 2OG oxygenases are linked to disease onset and progression, and are consequently
considered therapeutic targets.
All 2OG oxygenases bind the co-factors FeII and 2OG and share a common mechanism of
action, albeit on different substrates (Figure 1).2,3 These shared properties are due in part to
structural similarities between their active sites, which makes the selective inhibition of a
single 2OG oxygenase by small molecules very challenging. To develop 2OG oxygenase
selective inhibitors, analysis of the differences within their active sites was required. In order
to achieve this, suitable molecules that can occupy each enzymes active site selectively were
identified using dynamic combinatorial chemistry with mass spectrometric detection
(DCMS). Selective inhibitors for two 2OG oxygenases, AlkB and PHD2 were successfully
designed.
Dynamic combinatorial chemistry (DCC) involves the interaction of a number of compounds
that react reversibly to form large dynamic combinatorial libraries (DCLs). Introduction of
template molecules, such as proteins, changes the composition of the products in the library
with the products stabilized by the template being amplified. Stabilised species may then be
identified by an analytical technique. We used native mass spectrometry (MS) as the
analytical method due to its relatively high sensitivity, high speed and low sample
consumption. MS analysis of the amplified products (hits) allows for quick identification of
binders of the template.4, 5

117

Figure 1 Structural overlay of the active site residues of four 2OG oxygenases with a co-factor analogue
(1).

AlkB is a bacterial (Escherichia coli) 2OG oxygenase that is involved in the repair of
alkylated DNA and RNA bases.6, 7 AlkB is structurally and catalytically similar to its human
homologs, AlkBH2/AlkBH3, which are involved in DNA repair in vivo.7,

AlkB was

therefore targeted for inhibition as an AlkBH2/AlkBH3 model.

After screening for suitable ligands, N-oxalyl-L-cysteine was chosen as a suitable support
ligand for DCMS on AlkB. A DCL of N-oxalyl-L-cysteine with a library of 37 thiols was
formed in the presence of AlkB, from which three AlkB binders were identified (Figure 2).
Stable analogues of the three binders, which lack the labile disulfide group, were
synthesised as AlkB inhibitors. Inhibition properties and crystallographic studies of these
inhibitors with AlkB were then undertaken, which enabled the design of a fourth inhibitor that
displayed improved activity against AlkB (sub-micromolar IC50) compared to the first
generation of inhibitors. Inhibition studies of the final inhibitor with three other biologically
relevant 2OG oxygenases (PHD2, FTO and PHF8) revealed selectivity of this inhibitor for
AlkB.9

118

Figure 2 Representation of AlkB based DCC with (a) support ligand (4b) and (b) support ligand
disulfides.

Prolyl hydroxylase domain 2 (PHD2), is considered the key oxygen sensor in humans; in
hypoxia, PHD2 is inhibited leading to survival of HIF1 and activation of the hypoxia
response (HR) pathway. Therefore inhibition of PHD2 under normal oxygen levels has
potential for the treatment of various diseases; promising results have been reported for the
treatment of anaemia10, 11 and ischaemic diseases12 including heart disease13, 14 and stroke.15, 16
The role of PHD2 in disease has led to a number of investigations to identify PHD2 selective
inhibitors. For studies with PHD2, a reversible reaction was applied for the development of a
DCL; the formation of boronate esters from boronic acids (Figure 3). It is known that boronic
acids react with 1,2- and 1,3-cis-diols,17 -hydroxy carboxylic acids18,19 and dicarboxylic
acids19 and sugars.18 Even though these reversible reactions are well characterised they have
not been used before in DCC, with only one attempt by NMR.20

Figure 3 Boronic acid/ boronate ester DCMS.

119

Two boronic acid substituted pyridyl compounds with iron chelating properties were used as
support ligands for the development of the DCMS against PHD2. A DCMS assay using
these boronic acids with a library of 40 diols was developed, yielding in the identification of
seven hit structures for PHD2. Synthesis of stable analogues provided four inhibitors.
Inhibition studies and binding by NMR against PHD2 validated the boronate ester DCMS
approach. These pyridyl inhibitors were further modified to introduce a hydrogen bond with
PHD2 active site residues and hence improve their binding strength. The new inhibitors were
highly potent against PHD2; their activity over PHD2 was improved 10000 times compared to
the lead compounds. Moreover, one of the improved inhibitors derived from the DCMS hit
structures was found to be roughly 1000 times more selective for PHD2 over other 2OG
oxygenases compared to a PHD2 inhibitor currently in clinical trials against anaemia.21
Overall, this work describes the identification of selective inhibitors of two 2OG oxygenases,
which was achieved with the use of DCMS and crystallographic analyses. The DCMS method
was enriched after the development of a novel DCL using the reversible reaction between
boronic acids and diols. This boronic acid/boronate ester reaction may be applied for future
DCC studies with multiple templates.

REFERENCES
1. Costas, M., Mehn, M.P., Jensen, M.P., and Que, L., Dioxygen activation at mononuclear
nonheme iron active sites: Enzymes, models, and intermediates. Chem Rev, 2004, 104, 93986.
2. Hausinger, R.P., Fe(II)/alpha-ketoglutarate-dependent hydroxylases and related enzymes.
Crit Rev Biochem Mol, 2004, 39, 21-68.
3. Zhou, J., Kelly, W.L., Bachmann, B.O., Gunsior, M., Townsend, C.A., and Solomon,
E.I., Spectroscopic studies of substrate interactions with clavaminate synthase 2, a
multifunctional alpha-KG-dependent non-heme iron enzyme: Correlation with mechanisms
and reactivities. J Am Chem Soc, 2001, 123, 7388-98.
4. Huc, I. and Lehn, J.M., Virtual combinatorial libraries: Dynamic generation of molecular
and supramolecular diversity by self-assembly, P Natl Acad Sci USA, 1997, 94, 2106-10.
5. Poulsen, S.A., Davis, R.A., Keys, T.G., Screening a natural product-based combinatorial
library using FTICR mass spectrometry. Bioorgan Med Chem, 2006, 14, 510-5.
6. Falnes, P.O., Bjoras, M., Aas, P.A., Sundheim, O., and Seeberg, E., Substrate specificities
of bacterial and human AlkB proteins. Nucleic Acids Res, 2004, 32, 3456-61.
7. Trewick, S.C., Henshaw, T.F., Hausinger, R.P., Lindahl, T., and Sedgwick, B.,
Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage.
Nature, 2002, 419, 174-8.

120

8. Ringvoll, J., Nordstrand, L.M., Vagbo, C.B., Talstad, V., Reite, K., Aas, P.A., Lauritzen,
K.H., Liabakk, N.B., Bjork, A., Doughty, R.W., Falnes, P.O., Krokan, H.E., and Klungland,
A., Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing
1meA and 3meC lesions in DNA. Embo J, 2006, 25, 2189-98.
9. Woon, E.C.Y., Demetriades, M., Bagg, E.A.L., Aik, W., Krylova, S.M., Ma, J.H.Y.,
Chan, M.C., Walport, L.J., Wegman, D.W., Dack, K.N., McDonough, M.A., Krylov, S.N.,
and Schofield, C.J., Dynamic Combinatorial Mass Spectrometry Leads to Inhibitors of a 2Oxoglutarate-Dependent Nucleic Acid Demethylase. J Med Chem, 2012, 55, 2173-84.
10. Yan, L., Colandrea, V.J., and Hale, J.J., Prolyl hydroxylase domain-containing protein
inhibitors as stabilizers of hypoxia-inducible factor: small molecule-based therapeutics for
anemia. Expert Opin Therap Pat, 2010, 20, 1219-45.
11. Muchnik, E. and Kaplan, J., HIF prolyl hydroxylase inhibitors for anemia. Expert Opin
Inv Drug, 2011, 20, 645-56.
12. Fraisl, P., Aragones, J., and Carmeliet, P., Inhibition of oxygen sensors as a therapeutic
strategy for ischaemic and inflammatory disease. Nat Rev Drug Discov, 2009, 8, 139-52.
13. Eckle, T., Kohler, D., Lehmann, R., El Kasmi, K.C., and Eltzschig, H.K., Hypoxiainducible factor-1 is central to cardioprotection - A new paradigm for ischemic
preconditioning. Circulation, 2008, 118, 166-75.
14. Myllyharju, J., HIF Prolyl 4-Hydroxylases and their Potential as Drug Targets. Curr
Pharm Design, 2009, 15, 3878-85.
15. Bergeron, M., Gidday, J.M., Yu, A.Y., Semenza, G.L., Ferriero, D.M., and Sharp, F.R.,
Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat
brain. Ann Neurol, 2000, 48, 285-96.
16. Prass, K., Ruscher, K., Karsch, M., Isaev, N., Megow, D., Priller, J., Scharff, A., Dirnagl,
U., and Meisel, A., Desferrioxamine induces delayed tolerance against cerebral ischemia in
vivo and in vitro. J Cerebr Blood F Met, 2002, 22, 520-5.
17. Lorand, J.P. and Edwards, J.O., Polyol Complexes and Structure of the Benzeneboronate
Ion. J Org Chem, 1959, 24, 769-74.
18. Friedman, S., Pace, B., and Pizer, R., Complexation of Phenylboronic Acid with
Lactic-Acid - Stability Constant and Reaction-Kinetics. J Am Chem Soc, 1974, 96, 5381-4.
19. Leung, I.K.H., Brown, T., Schofield, C.J., and Claridge, T.D.W., An approach to enzyme
inhibition employing reversible boronate ester formation. Medchemcomm, 2011, 2, 390-5.
20. Friedman, S. and Pizer, R., Mechanism of Complexation of Phenylboronic Acid with
Oxalic-Acid - Reaction Which Requires Ligand Donor Atom Protonation. J. Am. Chem. Soc.,
1975, 97, 6059-62.
21. Demetriades, M., Leung, I.K.H., Chowdhury, R., Chan, M.C., McDonough, M.A.,
Yeoh, K.K., Tian, Y.M., Claridge, T.D.W., Ratcliffe, P.J., Woon, E.C.Y., and Schofield, C.J.,
Dynamic Combinatorial Chemistry Employing Boronic Acids/Boronate Esters Leads to
Potent Oxygenase Inhibitors. Angew Chem Int Edit, 2012, 51, 6672-5.

121

De Novo Fragment-Based Design of Inhibitors of DXS Guided by

Spin-Diffusion-Based NMR Spectroscopy
T. Masinia, J. Pilgerb, B.S. Kroezena, B. Illarionovc, M. Fischerc, C. Griesingerb,
A.K.H. Hirscha
a

Stratingh Institute for Chemistry, University of Groningen, 9747 AG, Groningen, NL.
Max-Planck-Institute for biophysical Chemistry, Am Fassberg 11, Gttingen, Germany.
c
Institut fr Lebensmittelchemie, Grindelallee 117, 20146, Hamburg, Germany.

t.masini@rug.nl
Pathogens such as Mycobacterium tuberculosis, use the non-mevalonate pathway for the biosynthesis
of the precursors of the essential isoprenoids, whereas humans exclusively utilise the alternative
mevalonate pathway.[1] Due to the emergence of resistant strains of these pathogens, there is an urgent
need for the development of new anti-infective agents.[2]
We chose 1-deoxy-D-xylulose-5-phosphate synthase (DXS) as the target of a structure-based design
project, exploiting de novo fragment-based design to target the cofactor-binding pocket and an
innovative combination of NMR techniques to validate the binding mode, circumventing the need for
protein crystallography.
DXS catalyses the first and rate-limiting step of the non-mevalonate pathway, employing thiamine
pyrophosphate (TPP) as a cofactor. Because of the low dissociation constant for TPP
(Kd = 114 13 nM), the development of cofactor-competitive inhibitors is challenging. We designed
fragment 1 de novo to occupy the TPP-binding pocket and determined the inhibition in vitro against
D. radiodurans DXS (IC50 = 1.81 0.48 mM, ligand efficiency = 0.33). Using a combination of NMR
experiments, we validated the predicted binding mode of 1. Saturation transfer difference (STD)
NMR[3] revealed the protein-bound parts of the ligand, whose bound conformation was established
using transfer-NOE NMR. Using the INPHARMA method,[4] we derived the binding mode of 1
relative to a ligand with known binding mode (2, IC50 = 434 68 M). For the INPHARMA method to
work, the two molecules have to target the same binding pocket and should overlap, so that
magnetisation transfer from one to the other can be observed.

Fragment
growing

(a)
(b)
(c)
Figure 1. (a) Fragment 1 and reference molecule 2 for INPHARMA. (b) Validated binding mode of 1
in the same binding pocket as 2. (c) General structures of derivatives, which have been synthesised to
improve the inhibitory potency of 1.
Our approach enables future design cycles in the absence of co-crystal structures, which are often
precluded due to weak affinity or solubility issues. Based on the validated binding mode of 1, we
designed a series of derivatives with significantly improved inhibitory potency.
REFERENCES
[1]. van der Meer, J. Y., Hirsch, A. K. H., Nat. Prod. Rep. 29, 721-728, 2012.
[2]. Koul, A., Arnoult, E., Lounis, N., Guillemont, J., Andries, K., Nature, 469, 483490, 2011.
[3] . Mayer, M., Mayer, B. Angew. Chem. Int. Ed., 38, 17841788, 1999.
[4]. Orts, J., Tuma, J., Reese, M., Grimm, S. K., Monecke, P., Bartoschek, S., Schiffer, A., Wendt, K. U.,
Griesinger, C., Carlomagno, T. Angew. Chem. Int. Ed., 47, 77367740, 2008.

122

Copper Coordination Compounds As Antimicrobial Agents

Barbara Miroslawa, Anna E. Koziola, Joanna Stefanskab, Daniel Szulczykc, Marta Strugac
a

Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland; bDepartment of

Pharmaceutical Microbiology, Medical University, 02-007 Warszawa, Poland; cDepartment of
Pharmacogenomics, Faculty of Pharmacy, Medical University, 02-097 Warszawa, Poland
e-mail: barbara.miroslaw@poczta.umcs.lublin.pl

The emergence of bacterial and fungal resistance to commercial drugs is an issue of global
importance. There are many generations of antimicrobial agents starting from low molecular-weight
molecules like ciprofloxacin or fluconazole, through cephalosporins, ending with voluminous
glycopeptide antibiotics, such as vancomycin etc. However, the development of new classes of
compounds to control the virulence of the pathogens is still required.
Coordination compounds based on small molecules are the attractive approach to overcome the
problem of drug-resistant microorganisms. The toxicity of heavy metals on microorganisms was
widely studied previously [1]. The metallodrugs are already successfully applied in medicine as
antimalarial or anticancer agents [2,3].
The present work refers to comparison of antimicrobial activity of newly synthesized unsymmetrically
substituted thioureas and their copper(II) complexes. The ligand molecules show antibacterial activity
against Gram-positive cocci but, as most of antibiotics, they are not effective against fungi [4]. The
performed studies indicate that introduction of copper ions to the structure of the maternal organic
ligands broaden their activity to antifungal too. This may be a good strategy to develop antimicrobial
agents toxic simultaneously to bacteria and fungi.

REFERENCES
[1].
[2].
[3].
[4].

Nies, D.H., Appl. Microbiol. Biotechnol., 51, 730750, 1999.

Patra, M.; Gasser, G.; Metzler-Nolte, N., Dalton Trans., 41, 63506358, 2012.
Hartinger, C. G.; Dyson, P. J., Chem. Soc. Rev., 38, 391401, 2009.
Stefanska, J; Szulczyk, D.; Koziol, A.E.; Miroslaw, B.; Kedzierska, E.; Fidecka, S.;
Busonera, B., Sanna, G.; Giliberti, G.; La Colla, P.; Struga, M., Eur. J. Med. Chem., 55,
205213, 2012.

123

FRACTION LIPOPHILICITY INDEX (FLI): A METRIC FOR ASSESSING

ORAL DRUG LIKENESS OF IONIZABLE CHEMICAL ENTITIES
Maria Chatzopoulou,a Theodosia Vallianatou,b Vassilis J. Demopoulos,a and
Anna Tsantili-Kakoulidou*b
a

Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of

Thessaloniki, 54124 Thessaloniki, Greece
b
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens,
Panepistimiopolis, Zografou, 157 71 Athens, Greece

In the present work we developed Fraction Lipophilicity Index (FLI), a metric for
assessing oral drug likeness of ionizable chemical entities, considering that a
drawback of the ubiquitous Rule of Five (Ro5) is that it does not explicitly take into
account the ionization potential of a molecule.
The basic concept of FLI is to allocate lipophilicity to a pH dependent neutral
fraction of the molecule and to establish borders for either too high or too low
lipophilicities. FLI is expressed by the following equation, where the neutral fraction
is referred at physiological pH=7.4.
FLI = log[P/fN] = logP-log fN = logP-log[D7.4/P] = 2xlogP-logD7.4
In establishing the width and the borders of FLI we considered the oral drugs
introduced worldwide in the years 2003-2012, based on the To Market, To Market
sections of the respective Annual Reports in Medicinal Chemistry, in combination
with the ChEMBL data base. The number of oral molecular entities thus identified,
were 138. In the next step, the partition coefficients (logP) and the distribution
coefficients (logD7.4) were calculated using the freeware MedChem Designer (i.e.
S+logP and S+logD7.4 respectively). The compounds were further divided into highly
ionized (>50% ionization at pH=7.4), and into low ionized, zwitterions, and neutrals.
In this way, 59 highly ionized oral drugs were identified, from which 39 (66%) were
bases and 20 (34%) were acids. From the diagram it is apparent that the majority
(93%) of FLI values falls in the range of 1
to 8. Among the drugs outside this span,
two with FLIs>8 are reported to have poor
absorption, although administered orally.
The performance of FLI metric was further
compared to the Ro5. We found out that 3
highly ionizable drugs with good
absorption violated simultaneously two
criteria of the Ro5 (MW and Lp), however
they have acceptable FLIs. Furthermore, in
the same set of compounds, 5 violate one
criterion (Lp), but the majority of them (4 out of 5) have acceptable FLIs. Finally, we
recalculated the FLI values by replacing S+logP with ClogP. We observed a similar
distribution of FLI values with a positive shift of approximately 0.1.
In conclusion, we could state that FLI stands as a promising addition to the metrics
array, targeting the oral drug likeness of highly ionic chemical entities. Work is under
way to expand the database by including more years of drug introductions.

124

FRACTION LIPOPHILICITY INDEX (FLI): A METRIC FOR

ASSESSING ORAL DRUG LIKENESS OF IONIZABLE
CHEMICAL ENTITIES
Maria Chatzopoulou,a Theodosia Vallianatou,b Vassilis J. Demopoulos,a and Anna
Tsantili-Kakoulidou b *
a

Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of

Thessaloniki, 54124 Thessaloniki, Greece
b
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens,
Panepistimiopolis, Zografou, 157 71 Athens, Greece

Introduction
Drug development is currently struggling against high attrition rates and rising costs;
clinical trials are becoming larger and more complex, and developing a drug from
concept to market costs staggering amounts, even up to as much as $11 billion.
Therefore, it is in a medicinal chemists best interests to assess the potential of a drug
as early as possible. In this respect, Lipinskis, now ubiquitous, Rule of Five (Ro5)
defines four simple rules that apply for the majority of compounds with good oral
absorption1. According to Ro5, cut off values are suggested for molecular weight
(MW<500), lipophilicity (ClogP<5), counts of hydrogen bond donor (HD<5) and
acceptor sites (HA<10). Twofold violation of Ro5 may indicate bioavailability
problems. A drawback of Ro5 is that it specifically does not take into consideration
the ionization degree of a molecule, and it often fails in cases of ionizable compounds
2,3
. Using Caco-2 permeability data, Waring proposed molecular weight dependent
lower limits of logD 4. In the present work we developed a metric for assessing oral
drug likeness of ionizable chemical entities, combining both logP of the neutral
species and logD at physiological pH.
The basic concept was to allocate lipophilicity to a pH dependent neutral
fraction of the molecule; in this way the known problematic pharmacokinetic profile
of ionic compounds having either too high or too low lipophilicities will be
established and the borders quantified. Partition coefficient in octanol/water was used
as the numeric expression of lipophilicity, while the neutral fraction was referred to
physiological pH=7.4. We called this metric fraction lipophilicity index (FLI) and it is
expressed in logarithmic scale as follows:
FLI = log[P/fN] = logP-log fN = logP-log[D7.4/P] = 2xlogP-logD7.4 (1)
Methodology
The oral drugs introduced worldwide in the years 2003-2012, based on the To
Market, To Market sections of the respective Annual Reports in Medicinal
Chemistry5 in combination with the ChEMBL data base6, we considered in
establishing the width and the borders of FLI. The number of oral molecular entities
thus identified were 138.
Partition coefficients (logP) and distribution coefficients at pH 7.4 (logD7.4) were
calculated using the freeware MedChem Designer7 (i.e. S+logP and S+logD7.4
respectively). FLI(S) values were calculated according to equation (1). FLI(C) values

125

2
were calculated by replacing S+logP with ClogP8 while keeping the method for
calculating logfN (i.e. S+logD7.4 - S+logP).
Results and Discussion
The compounds were divided into highly ionized (>50% ionization at pH=7.4), and
into low ionized, zwitterions, and neutrals. Calculation of FLI values was performed
for the highly ionized drugs. Two drugs, hydrolyzed in the gastrointestinal tract to the
active principle as well as one drug reported to be substrate /inhibitor/inducer of pglycoprotein, were not included in the FLI study. In this way, 59 highly ionized oral
drugs were identified, from which 39 (66%) were bases and 20 (34%) were acids. In
Table 1 descriptive statistics are presented. They include the minimum, maximum
and mean values of S+logP, S+logD7.4 and their difference =(S+logP)-(S+logD7.4) as
well as the corresponding data of ClogP. The latter shows a small shift to higher
values, which is reflected also in the generated FLI(C) values. FLI(S) values range
from -0.28 to 9.24 with mean = 4.99, while FLI(C) values derived using ClogP range
from 1.11 to 9.78 with mean = 5.05 (Table1). We further observed that if the
molecule combines low lipophilicity (i.e. logP close or lower than 1) and negative
logD7.4 values, the difference did not exceed 1.5. This observation is based on 6 out
of the 59 compounds.
Table 1. Descriptive statistics
Min.

Max.

Mean

S+logP

-0.72

7.86

3.42

S+logD7.4

-1.72

7.01

1.85

.37

3.90

1.57

ClogP

0.32

8.57

3.49

FLI(S)

-0.28

9.24

4.99

FLI(C)

1.11

9.78

5.05

The distribution of FLI(S) is shown in Figure 1.

Figure 1. Distribution of FLI(S)

126

3
The generated FLI(S) values follow almost a normal distribution and the majority
(93%) of the values falls in the range of 1 to 8. It is worth noting that among the three
drugs outside this span (FLI(S)>8, Table 2, entries 1-3), two (one base and one acid)
are reported to have poor absorption, although they are administered orally.
From another perspective, we compared the performance of the FLI metric
with the Ro5. Detailed data are given in Table 2 for the ten compounds which needed
special comments. As shown in Table 2, we found out that, from the studied highly
ionic oral drugs, three bases violate simultaneously two criteria of the Ro5 (molecular
weight and lipophilicity). However, they present FLI(S) values <8 (Table 2, entries 46). Entry 1 (Table 2) with poor absorption and FLI >8, as already commented, shows
also a twofold violation of the Ro5. Furthermore, five drugs (one acid and four bases)
(Table 2, entries 3, 7-10) violate one criterion (lipophilicity); the four bases, however,
have FLI(S) within the defined range.
Table 2. Detailed data for ten compounds
Drug / year of type Ro5 violation
approval
1
Lomitapide
B
V2: MW/lip
mesylate / 2012*
2
Eltrombopag
A
V1: Lip
/2009**
3
Tamibarotene/
A
V1: Lip
2005
4
Bedaquiline/2012
B
V2: MW/lip
5
Ponatinib/ 2012
B
V2: MW/lip
6
Dronedarine/2009
B
V2: MW/lip
7
Fingolimod
B
V1: Lip
hydrochloride
/2010
8
Dapoxetine/2009
B
V1: Lip
9
Blonanserin/2008
B
V1: Lip
10
Cinacalcet/2004
B
V1: Lip
*33% absorption **50% absorption

FLI(S)
8.70

FLI(C) FLI(C)
scaled
8.26
8.84

8.45

8.45

8.59

9.24

9.78

9.39

7.61
5.53
7.46
5.22

8.82
6.28
9.56
6.47

7.73
5.61
7.58
5.29

5.97
6.23
7.44

6.16
6.68
7.64

6.06
6.33
7.56

Since lipophilicity calculation in Ro5 is based on ClogP, FLI(C) was also

considered. The distribution of FLI(C) shows a shift of high frequencies to lower
values. This is evident also from the plot of FLI(C) versus FLI(S) which shows a
tendency for curvature in low values (Figure 2A). In Figure 2B the distribution of
FLI(C) is presented. In the case of FLI(C) more drugs exceed the border value of 8.
However a 1:1 correlation is obtained between FLI(C) and FLI(S), expressed by
equation 2.
FLI(C) = 1.02(0.05) FLI(S) -0.04(0.27)
n=59, r=0.936, s=0.785

(2)

If equation 2 is used to scale FLI(C), analogous results as for FLI(S) are obtained
(Table 2).

127

4
10

F LI(C)

0
-2
N t

0
Thi i

2
b

10

FLI(S)

Figure 2. A: Plot of FLI(S) versus FLI(C). B: Distribution of FLI(C)

In conclusion, we could state that FLI stands as a promising addition to the
metrics array, targeting the oral drug likeness of highly ionic chemical entities. Work
is under way to expand the database by including more years of drug introductions.
REFERENCES
1) Lipinski C.A., Lombardo F., Dominy B.W., Feeney P.J. Experimental and
Computational Approaches to Estimate Solubility and Permeability in Drug
Discovery and Development Settings. Adv. Drug. Deliv. Rev. 23, 3-25, 1997
2) Martin Y. A bioavailability score. J. Med. Chem. 48, 31643170, 2005
3) Vistoli G., Predretti A., Testa B. Assessing Drug-Likeness. What are we missing?
Drug Discov. Today, 13, 285-294, 2008
4) Waring M.J.Defining optimum lipophilicity and molecular weight ranges for drug
candidates.- Molcular weight dependent lower logD limits based on permeability.
Biorg.Med.Chem.Let. 19, 2844-2851, 2009
5) Annual Reports in Medicinal Chemistry, Volumes 39-48 (2004-2013)
6) https://www.ebi.ac.uk/chembldb/
7) MedChem DesignerTM. Version 2.5.0.9, Copyright 2011-2013 Simulations Plus,
Inc., http://www.simulations-plus.com/
8) ClogP for Windows Version 4.0, Copyright 1995-1999 BioByte Corp.,
http://www.biobyte.com/

128

New antivascular agents in the combretastatin A-4 series

O. Provot*, M. Alami.
University Paris-Sud, CNRS, BioCIS-UMR 8076, Laboratoire de Chimie Thrapeutique,
Facult de Pharmacie, 5 rue J.-B. Clment, Chtenay-Malabry, F-92296, France

Combretastatin A-4 (CA-4) is arguably the most biologically significant member of a group of cis-stilbenes
isolated from the South African tree Combretum caffrum extracts used in traditional medecines. CA-4 displays
strong antiproliferative and antimitotic properties that have led to its examination as antivascular disrupting
agent. Indeed, a phosphate prodrug form CA-4P, and a serinamido-derivative AVE-8062 are now undergoing
(phase III) clinical evaluation in USA and Europe as drugs for the treatment of cancers (thyrod, colon,).
Nevertheless, besides its poor hydrosolubility, the major drawback of CA-4 is its ability to isomerize during
storage and administration to the more stable and inactive trans-isomer.

In an ongoing project aimed at developing novel anticancer molecules, we have studied the structure-activity
relationships around CA-4 by replacing the isomerizable 1,2-diarylethylene double bond by several stable
linkers. To this end, we have prepared after docking studies and measurement of the dihedral angle between
the aromatic rings, various derivatives 1-8 according to eco-friendly procedures.

The preparation of these CA-4 analogues, and their biological properties (cytotoxicity, inhibition of tubulin
assembly, apoptosis and antivascular properties) will be discussed. IsoCA-4 has been identified as a very
promising candidate for preclinical results. Its metabolism8 and in vivo preliminary results of this compound
will be also presented.
1

Bioorg. Med. Chem. Lett. 2008, 18, 3266.

Tetrahedron 2010, 66, 3775.

J. Med. Chem. 2009, 52, 4538 and ChemMedChem 2009, 4, 1912.

Tetrahedron 2006, 62, 11994.

ChemMedChem 2011, 6, 488.

Eur. J. Med. Chem. 2013, 28.

Eur. J. Med. Chem. 2012, 52, 22.

ChemMedChem 2011, 6, 1781.

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143

POSTER PRESENTATIONS

67

Interactions of Silybin A with cyclodextrin derivatives using solid

and liquid state NMR spectroscopy, differential scanning and
isothermal titration calorimetry as well as molecular dynamics
simulations
T. Kellicia,b, D. Ntountaniotisa, G. Leonisc, M. Chatziathanasiadoub, A. Tzakosb, J.
Baldusd, C. Glaubitzd, G. Valsamie, E. Archontakia, K. Virasa, M. G. Papadopoulosc,
T. Mavromoustakosa
a

Chemistry Department, National and Kapodistrian University of Athens, Panepistimiopolis

Zografou 15771, Greece
b
University of Ioannina, Chemistry Department, 45110 Ioannina, Greece
c
Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research
Foundation
d
Institute of Biophysical Chemistry Goethe University Frankfurt, Max-von-Laue-Str. 9,
60438 Frankfurt, Germany
e
Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis
Zografou 15771, Greece
Milk thistle has been used since ancient times to treat a range of liver and gallbladder
disorders, such as hepatitis, cirrhosis as well as other gastrointestinal problems. The bioactive
phytochemical of this plant is silymarin while the main ingredient of the silymarin complex is
silybin, which is present as a mixture of two diastereomers A and B. Silymarin/silybin is
considered to be a very safe compound and there are only few reports on its adverse effects. It
has lately received attention due to its anticancer and chemopreventive actions. Additionally,
references in bibliography have mentioned neuroprotective and cardioprotective activities. As
the interest on this compound keeps growing, the limited bioavailability as well as its low
water solubility remain the main problems for wider use. The utilization of cyclodextrins
(CDs) and their synthetic derivatives have been extensively exploited to improve certain
properties of the drugs, such as solubility and bioavailability. The enhanced drug activity and
selective transfer or the suppression of side effects can be achieved by inclusion complex
formation. Since Silybin possesses five hydroxyl groups and three aromatic rings, CDs are
particularly suitable for the increase in solubility as well as for the protection of the
compound. In the present study we have used both solid state and high resolution liquid NMR
spectroscopy as well as Molecular Dynamics calculations to detect the molecular interactions
of silybin A with -CD and 2-hydroxypropyl--CD. A description of the (silybin-CD) system
is offered in the right side of the following scheme.

The toroidal shape of -CD (left) and the proposed complex of silybin A (in green) with cyclodextrin (gray) (right).

147

An in silico effort to discover new HCV Replication Inhibitors

E. Vrontaki,a,b G. Melagraki,a T. Mavromoustakos,b A. Afantitisa
a

Department of Chemoinformatics, NovaMechanics Ltd, Nicosia, Cyprus

Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Athens
15771, Greece
info@novamechanics.com

A combination of computational methods: (i) Molecular Docking, (ii) 3D-QSAR

CoMSIA, (iii) Similarity Search and (iv) Virtual Screening workflow using ChEMBL
was applied for 41 compounds to identify new N-substituted indole analogues acting
as inhibitors of HCV replication. The inhibitors were docked into the Palm II
allosteric site of the crystal structure of the enzyme HCV RNA-dependent RNA
polymerase (NS5B GT1b) [1-3]. Then, the CoMSIA fields were generated through a
receptor-based alignment of docking poses to build a validated and stable 3D-QSAR
CoMSIA model. This can be utilized (i) to get insight into the molecular features that
promote their bioactivity and (ii) estimate activity of novel potential bioactive
compounds prior to their synthesis and biological tests, within a virtual screening
procedure.

ACKNOWLEDGEMENTS
This work is supported by funding under the Seven Research Framework Programme of the
European Union. Project SYSPATHO (ex. PATHOSYS HEALTH-F5-2010-260429)
REFERENCES
[1].
[2].
[3].

Karelson M. et al., Current Computer-Aided Drug Design, 8, 5561, 2012.

Barreca M.L. et al., Future Medicinal Chemistry, 3, 10271055, 2011.
Chen G. et al., Bioorganic Medicinal Chemistry Letters, 23, 39423946, 2013.

148

Stability and Bind

ding Effeccts of Ag Metal
M
Complexes at
a LOX-11
E Vrontakia, G. Leonisb, A. Avram
E.
mopoulosb, M.G.
M Papaddopoulosb, M.
M Simic, S.
c, d
e
e
Goli Grdadolnik
G
, A. Afanttitis , G. Meelagraki , S.K. Hadjikaakouf, T.
Maavromoustakkosa
a

Laaboratory of Organic Cheemistry, Dep

partment of Chemistry,
C
U
University
of Athens, Athhens
1
15771,
Greecce
b
Innstitute of Biiology, Mediicinal Chemistry and Biotechnology, National Heellenic Reseaarch
Foundation, Vas. Consstantinou 48,, Athens 116
635, Greece
c
EN-FIST Centre off Excellence, Dunajska 156,
1 SI-1000 Ljubljana, Slovenia
S
d
Laboratory off Biomolecullar Structure,, National Innstitute of Ch
hemistry, Hajjdrihova 19, SI1001 Ljubljana,
L
Sllovenia
e
Depaartment of Chhemoinformaatics, NovaM
Mechanics Lttd, Nicosia, Cyprus
C
f
S
Section of Innorganic and Analytical Chemistry,
C
D
Department
of Chemistry,, University oof
Ioannina, 45110
4
Ioanniina, Greece
evron
ntaki@chem.uoa.gr

An anti-inflammatory com
mplex of Agg(I), namelyy [Ag(tpp)3(asp)](dmf)
(
[tpp=tripheenylN,N-dimethyylformamidde], was syn
nthesized inn an
phoosphine, asppH=aspirin and dmf=N
attem
mpt to develop noveel metallottherapeutic molecules.. Moleculaar docking and
dynnamics weree used to exxamine if this
t
compleex binds to LOX-1 [1,,2]. Our stuudies
werre complem
mented by Saaturation Trransfer Diff
fference (ST
TD) 1H NM
MR experimeents.
Expperiments without
w
or with sonicaation were performedd in order to increasee the
soluubility of Ag(I)
A
complex in the enzyme ennvironment. The 1H NMR
N
spectrra in
bufffer TRIS/D2O betray the
t existencce of two coomplexes; the
t complexx of aspirinn and
the complex of salicylicc acid thaat is produuced after deacetylatiion of asppirin.
t STD speectra show that only thhe complexx of salicyliic acid is boound
Nevvertheless, the
to thhe enzyme. Molecular docking an
nd dynamicss were usedd to provide information on
the molecular interactions
i
s of the two complexess at the activve site. Thee Ag complexes
d inside a large LOX-1 cavity byy establishinng a netwoork of hydroogen
werre stabilized
bonnds and sterric interactions. The co
omplex witth salicylic acid was more
m
favoraable.
Thee in silico reesults explaain the experimental ressults while the compleex with saliccylic
acidd predominaates in the binding
b
caviity.

REF
FERENCES
S
[1].
[2].

Banti C.N
N. et al., Metaallomics, 4, 545560,
5
2012.
Poyraz M. et al., Inorgganica Chim
mica Acta, 375, 114121, 2011.

149

New N-alkanoyl-4-methoxybicyclo[4.2.0]octa-1,3,5-trien-7-ethanamines:
melatoninergic action and controlled release from solid pharmaceutical
formulations
M. Vlachou*a, V. Ioannidoua, M. Vertzonia, A. Tsotinisb, P. Afroudakisb, S. Konstantinidoub, S.
Pantazopoulosc and D. Sugdend
a

Faculty of Pharmacy, Department of Pharmaceutical Technology, Panepistimioupoli-Zografou, 15771 Athens, Greece; bFaculty
of Pharmacy, Department of Pharmaceutical Chemistry, Panepistimioupoli-Zografou, 15771 Athens, Greece; Current address:
School of Biomedical Sciences, Department of Pharmacy, Franklin-Wilkins Building, King's College London, London SE1 1UL,
U.K.; cKonstantopouleio General Hospital N. Ionias, S. Olgas 3-5, . onia, 14233 Athens, Greece;
d
School of Medicine, Division of Women's Health, Room 2.12N Hodgkin Building, King's College London, London SE1 1UL,
U.K.

Melatonin (N-acetyl 5-methoxytryptamine, MT), a hormone synthesised by the pineal gland and released at night,
has a regulatory role in the onset of sleep in mammals, including humans. It has been shown to have a hypnotic
action in animals and humans, and it has been used as an agent for restoring circadian rhythms disturbed by jet-lag,
shift-work or ageing. The physiological actions of melatonin in regulating seasonal and circadian rhythms are
mediated through a family of specific, high affinity G protein-coupled membrane receptors. There have been
numerous studies directed towards the understanding of how melatonin binds to and activates these receptors using
both indole and non-indole derivatives.1 The present work refers to the second category of melatoninergics and in
particular to the N-alkanoyl-4-methoxybicyclo[4.2.0]octa-1,3,5-trien-7-ethanamines (I) and (II). Both of these
compounds are potent melatonin receptor agonists in the Xenopus laevis malanophore assay2 and thus information
about their oral absorption profile will be very useful in future in vivo studies. To this end, dissolution experiments
were conducted in gastric and intestinal-like fluids, as an initial attempt to probe their release characteristics from
solid pharmaceutical formulations.

% release

150

MT pH 1.2
MT pH 7.4
I pH 1.2
I pH 7.4
II pH 1.2
II pH 7.4

100

50

200

400

600

time (min)

I: R = Me; pEC50 = 7.81 0.07

II: R = Et; pEC50 = 8.53 0.04

Melatonin (MT); pEC50 = 8.58 0.03

Fig: Release profiles of MT, I and II at pH 1.2 and 7.4

The synthesis of compounds (I) and (II) involved the sequence of [2+2]-cycloaddition between 4methoxybromobenzene and 1,1-dimethoxyethylene, chromatographic separation of the isomeric diacetals formed,
hydrolysis of the requisite diacetal to the ketone, conversion to the a,-unsaturated nitrile, under Wittig reaction
conditions, and concomitant reduction and acylation (H2, Raney-Ni (RCO)2O, 55 psi, ) with the appropriate acid
anhydride. Experimental details will be reported elsewhere.
The dissolution experiments involved flat tablets (10 mm diameter, 200 mg weight, and 8-10 kp hardness)
comprised of 2 mg of the active substance, HPMC K15 (16 mg), Sodium Alginate (144 mg), Lactose Monohydrate
(16 mg), Avicel PH 102 (20 mg) and MgStr (2 mg). The tablets were stirred at 100 rpm in a USP XXIII dissolution
apparatus II (Pharmatest, Hainerp, Germany) containing 450 ml of either gastric or intestinal fluid at 370.5 oC.
Samples (5 ml) were withdrawn at predetermined time intervals, filtered and analyzed at max=280 nm using a
PerkinElmer UV spectrophotometer (Norwalk, CT). All experiments were performed in triplicate.
The results obtained suggest that the new compounds, albeit being more lipophilic (R=Me: logP=1.48; R=Et:
logP=2.14) than melatonin (logP=0.46), follow Case II order-like release profiles in both pH 1.2 and 7.4. At pH 1.2,
approximately 50% of the total amount of the active substances (I: R=Me and II: R=Et) was released within 300
minutes. At pH 7.4, within the same time period, the entire amount of the active substances (I: R=Me and II: R=Et)
was released.
REFERENCES
[1] Garratt, P.J.; Tsotinis, A., Mini-Rev. Med. Chem., 2007, 7, 1075-1088.
[2] Faust, R.; Garratt, P. J.; Jones, R.; Yeh, L.-K.; Tsotinis, A.; Panoussopoulou, M.; Calogeropoulou, T.; Teh, M.T.; Sugden, D., J. Med. Chem. 2000, 43, 1050-1061.

150

Zanamivir Conjugates Linked with Anti-inflammatory Agents Exhibit

Enhanced Anti-influenza Activity
Jim-Min Fanga, b
a

Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan.

b
The Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan.
E-mail: jmfang@ntu.edu.tw

Influenza is a respiratory infection that causes severe health problems. In the influenza virus
infected patients, the uncontrolled virus-induced cytokines can cause high mortality. Our antiinfluenza drug discovery program has focused on the inhibition of the activity of the
neuraminidase on the surface of influenza virus. Monotherapy with a single antiviral drug for
influenza may be limited in efficacy due to the rapidly developed drug-resistance.1 In a study,
we explored the bifunctional therapeutic drug comprising a neuraminidase inhibitor
conjugated to an anti-inflammatory agent for simultaneous inhibition of influenza virus
neuraminidase and suppression of pro-inflammatory cytokines.2 We shall present the
synthetic methods for preparation of these enhanced anti-influenza conjugate drugs. Their
activities against various influenza viruses were evaluated by enzymatic and cell-based
assays. The survival rates in the mice challenged with H1N1 or H5N1 viruses were greatly
improved by treatment with the zanamivir conjugates, in comparison with the combination
treatments3 with zanamivir and anti-inflammatory drugs.


REFERENCES
[1].
[2].
[3].

Baz, M.; Abed, Y.; Papenburg, J.; Bouhy, X.; Hamelin, M.-.; Boivin, G. N. Engl. J. Med. 361,
22962297, 2009.
Liu, K.-C.; Fang, J.-M.; Jan, J.-T.; Cheng, T.-J. R.; Wang, S.-Y.; Yang, S.-T.; Cheng, Y.-S. E.;
Wong, C.-H. J. Med. Chem. 55, 84938501, 2012.
Zheng, B. J.; Chan, K. W.; Lin, Y. P.; Zhao, G. Y.; Chan, C.; Zhang, H. J.; Chen, H. L.; Wong,
S. S.; Lau, S. K.; Woo, P. C.; Chan, K. H.; Jin, D. Y.; Yuen, K. Y. Proc. Natl. Acad. Sci. USA
105, 80918096, 2008.

151

New N-alkanoyl-4-methoxybicyclo[4.2.0]octa-1,3,5-trien-7-ethanamines:
melatoninergic action and controlled release from solid pharmaceutical
formulations
M. Vlachou*a, V. Ioannidoua, M. Vertzonia, A. Tsotinisb, P. Afroudakisb, S. Konstantinidoub, S.
Pantazopoulosc and D. Sugdend
a

Faculty of Pharmacy, Department of Pharmaceutical Technology, Panepistimioupoli-Zografou, 15771 Athens, Greece; bFaculty
of Pharmacy, Department of Pharmaceutical Chemistry, Panepistimioupoli-Zografou, 15771 Athens, Greece; Current address:
School of Biomedical Sciences, Department of Pharmacy, Franklin-Wilkins Building, King's College London, London SE1 1UL,
U.K.; cKonstantopouleio General Hospital N. Ionias, S. Olgas 3-5, . onia, 14233 Athens, Greece;
d
School of Medicine, Division of Women's Health, Room 2.12N Hodgkin Building, King's College London, London SE1 1UL,
U.K.

Melatonin (N-acetyl 5-methoxytryptamine, MT), a hormone synthesised by the pineal gland and released at night,
has a regulatory role in the onset of sleep in mammals, including humans. It has been shown to have a hypnotic
action in animals and humans, and it has been used as an agent for restoring circadian rhythms disturbed by jet-lag,
shift-work or ageing. The physiological actions of melatonin in regulating seasonal and circadian rhythms are
mediated through a family of specific, high affinity G protein-coupled membrane receptors. There have been
numerous studies directed towards the understanding of how melatonin binds to and activates these receptors using
both indole and non-indole derivatives.1 The present work refers to the second category of melatoninergics and in
particular to the N-alkanoyl-4-methoxybicyclo[4.2.0]octa-1,3,5-trien-7-ethanamines (I) and (II). Both of these
compounds are potent melatonin receptor agonists in the Xenopus laevis malanophore assay2 and thus information
about their oral absorption profile will be very useful in future in vivo studies. To this end, dissolution experiments
were conducted in gastric and intestinal-like fluids, as an initial attempt to probe their release characteristics from
solid pharmaceutical formulations.

% release

150

MT pH 1.2
MT pH 7.4
I pH 1.2
I pH 7.4
II pH 1.2
II pH 7.4

100

50

200

400

600

time (min)

I: R = Me; pEC50 = 7.81 0.07

II: R = Et; pEC50 = 8.53 0.04

Melatonin (MT); pEC50 = 8.58 0.03

Fig: Release profiles of MT, I and II at pH 1.2 and 7.4

The synthesis of compounds (I) and (II) involved the sequence of [2+2]-cycloaddition between 4methoxybromobenzene and 1,1-dimethoxyethylene, chromatographic separation of the isomeric diacetals formed,
hydrolysis of the requisite diacetal to the ketone, conversion to the a,-unsaturated nitrile, under Wittig reaction
conditions, and concomitant reduction and acylation (H 2, Raney-Ni (RCO)2O, 55 psi, ) with the appropriate acid
anhydride. Experimental details will be reported elsewhere.
The dissolution experiments involved flat tablets (10 mm diameter, 200 mg weight, and 8-10 kp hardness)
comprised of 2 mg of the active substance, HPMC K15 (16 mg), Sodium Alginate (144 mg), Lactose Monohydrate
(16 mg), Avicel PH 102 (20 mg) and MgStr (2 mg). The tablets were stirred at 100 rpm in a USP XXIII dissolution
apparatus II (Pharmatest, Hainerp, Germany) containing 450 ml of either gastric or intestinal fluid at 370.5 oC.
Samples (5 ml) were withdrawn at predetermined time intervals, filtered and analyzed at max=280 nm using a
PerkinElmer UV spectrophotometer (Norwalk, CT). All experiments were performed in triplicate.
The results obtained suggest that the new compounds, albeit being more lipophilic (R=Me: logP=1.48; R=Et:
logP=2.14) than melatonin (logP=0.46), follow Case II order-like release profiles in both pH 1.2 and 7.4. At pH 1.2,
approximately 50% of the total amount of the active substances (I: R=Me and II: R=Et) was released within 300
minutes. At pH 7.4, within the same time period, the entire amount of the active substances (I: R=Me and II: R=Et)
was released.
REFERENCES
[1] Garratt, P.J.; Tsotinis, A., Mini-Rev. Med. Chem., 2007, 7, 1075-1088.
[2] Faust, R.; Garratt, P. J.; Jones, R.; Yeh, L.-K.; Tsotinis, A.; Panoussopoulou, M.; Calogeropoulou, T.; Teh, M.T.; Sugden, D., J. Med. Chem. 2000, 43, 1050-1061.

152

Molecular Dynamics Simulations and MM-PBSA Binding Free Energy

Estimation in GK241-sPLA2 GIIA complex
S. Vasilakaki1, G. Leonis2, M. G. Papadopoulos2, T. Mavromoustakos1, G. Kokotos1
1

Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece

Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research
Foundation, 48 Vas. Constantinou Ave., Athens 11635, Greece
svasilak@chem.uoa.gr

Inflammation is the complex biological response of the immune system of an organism to

conditions such as cell damage and infection by pathogens. Potent mediators of inflammation
are eicosanoids arising from arachidonic acid. Phospholipase A2 is a superfamily of enzymes
that plays a key role in the hydrolysis of sn-2 bond of glycerophospholipids producing mainly
arachidonic acid and lysophospholipids [1]. Selective inhibition of enzymes belonging to this
family has been an aim for pharmaceutical purposes. Recently, Kokotos and coworkers
developed a novel -ketoamide derivative of valine (GK241) with potent inhibitory activity
toward sPLA2 GIIA. In order to understand and improve the binding properties of GK241
towards the synthesis of novel analogs, we performed molecular dynamics simulations with
the AMBER program [2]. The trajectory obtained through MD simulation time was used to
study conformational properties, hydrogen bonds between the key aminoacids of the active
site and the inhibitor as well as possible interactions with Ca2+ in the active site, during the
simulation time. The estimation of the binding free energy of the complex performed with
MM-PBSA [3].


GK241



ACKNOWLEDGMENDS
This research has been co-financed by the European Union (European Social Fund ESF) and Greek
national funds through the Operational Program "Education and Lifelong Learning" of the National
Strategic Reference Framework (NSRF) - Research Funding Program: Heracleitus II. Investing in
knowledge society through the European Social Fund.
REFERENCES
[1].
[2].
[3].

Dennis, E.A.; Cao, J.; Hsu, Y. H.; Magrioti, V.; Kokotos, G. Chem. Rev. 111, 6130-6185, 2011.
Salomon-Ferrer, R.; Case, D.A.; Walker, R.C.; WIREs Comput. Mol. Sci. 3, 198-210 2013.
Honig, B.; Nicholls, A. Science 268, 11441149, 1995.

153

Hydrazones of 5-nitro-2-furanecarboxaldehyde with

1-adamantanealkanoyhydrazides with probable trypanocidal activity
Angeliki-Sofia Foscolosa, Ioannis Papanastasioua, Georgios Foscolosa*, Andrew Tsotinisa,
Tahsin Kellicib, Thomas Mavromoustakosb
a

Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Athens,

Panepistimioupoli-Zografou, 157 71 Athens, Greece.
b
Faculty of Chemistry, Department of Organic Chemistry, University of Athens, PanepistimioupoliZografou, 157 71 Athens, Greece.
papanastasiou@pharm.uoa.gr

Trypanosomiasis is spread nowadays with population movements from endemic to nonendemic countries such as North America, the western Pacific region and, more recently,
Europe [1]. Nitroheterocyclic drugs, such as nifurtimox and benznidazole, have been used for
the treatment of Human American Trypanosomiasis for more than 40 years [2,3]. The
synthesis of the title hydrazones is described and their Trypanososoma brucei brucei and their
Trypanosoma cruzi trypanocidal activity is under evaluation. Moreover, conformational
analysis studies on the new molecules are currently underway.

REFERENCES
[1].
[2].
[3].

Coura, JR, Vias, PA, Nature, 465, S6-7, 2010.

Wilkinson, SR, Kelly, JM, Expert Rev. Mol. Med., 11, e31, 1, 2009.
Lpez-Muoz, R, Fandez, M, Klein,S, Escanilla, S, Torres, G, Lee-Liu, D, Ferreira, J,
Kemmerling, U, Orellana, M, Morello, A, Ferreira, A, Maya, DA, Exp. Parasitol., 124, 16771, 2009.

154

EFFECT OF METAL IONS ON FETAL HAEMOGLOBIN INDUCTION

BY HYDROXYUREA IN ERYTHROID CELL LINES
S. Voskoua, M. Phylactidesa, GJ. Kontoghiorghesb, and M. Kleanthousa.
a

Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics,
Nicosia, Cyprus
b
Postgraduate Research Institute of Scince Technology, Environment and Medicine, Limassol,
Cyprus
stellav@cing.ac.cy

Pharmacological reactivation of the -globin gene for the production of fetal haemoglobin
(HbF) presents an attractive strategy for the treatment of -thalassaemia and sickle cell
disease (SCD). Although, several compounds have been found to possess an HbF-inducing
activity, hydroxyurea (HU) is the only FDA (Food and Drug Administration) approved drug
for the treatment of SCD. However, a major drawback in HU therapy is the variability in
response among patients and the limited use of hydroxyurea in thalassaemia major.
Recent findings suggest that HU can act as a chelator [1, 2] and form complexes with copper
and iron ions [3]. The metal complexes of HU are expected to have altered chemical and
biological properties and thus, the therapeutic activities of HU may be affected when high
concentrations of such metal ions are present [3]. Therefore, our main hypothesis is that the
iron overload presented in SCD and thalassaemia patients, as well as the excess amount of
other metal ions, may in part explain the high variability in response to HU treatment and the
limited success of HU therapy in the highly transfused and iron overloaded thalassaemia
major patients.
In the present study, K562 cells and human erythroid cultures were treated with a specific
concentration of HU (100M) in combination with increasing concentrations (50, 100, 200,
and/or 400M) of different metal ions [Fe(II), Fe(III), Cu(II), Zn(II), and Al(III)].
Cytotoxicity, in both culture systems, was defined using trypan blue solution. In K562 cells
Hb production was determined with benzidine staining, whereas in human erythroid cultures
the amount of HbF was measured with high performance liquid chromatography (HPLC).
Based on our results so far, only iron can support the above hypothesis. Iron was found to
decrease HUs HbF-inducing activity without reducing HbF production on its own, in both
culture systems. Additionally, in the concentration range tested, iron does not appear to be
cytotoxic nor does it have an effect on HUs cytotoxic properties.
Therefore, excess amounts of free iron in SCD and thalassaemia patients may interact with
HU altering its therapeutic properties. This could possibly explain the variability in response
to HU treatment observed among patients and necessitate reduction of iron levels prior to HU
treatment for increased response.
REFERENCES
[1]. Italia K, Colah R, Ghosh K, Plos One, 8, e82928, 2013.
[2]. Kontoghiorghes GJ, Efstathiou A, Ioannou-Loucaides S, Kolnagou A, Hemoglobin, 32, 217-227,
2008.
[3]. Konstantinou E, Pashalidis I, Kolnagou A, Kontoghiorghes GJ, Hemoglobin, 35, 237-246, 2011.


155

The Thalidomide Impact on Patients with Multiple Myeloma. The Revival of

an Old Story through the Histological and Immunohistochemical Study of 100
Cases.
Constantine Hadjileontisa, Vassiliki Kaloutsib, Kostas Zervasc, Anastasis Stephanoua and Ioannis
Patrikiosa.
a

Biomedical lab, School of Medicine, European University Cyprus, Nicosia, Cyprus; b Pathology dept,
Medical School, Aristotle University, Thessaloniki, Greece. c Hematology Clinic, Theagenion Cancer
Hospital, Thessaloniki, Greece.
Presenting author: C.Hadjileontis@euc.ac.cy

Introduction: Despite the fact that thalidomide is a drug of known anti-angiogenic activity [1], its actual
impact on patients with multiple myeloma (MM), has not been studied yet; especially when combined with
dexamethasone, or vincristine-adriamycin-dexamethasone (VAD).
Patients and methods: 100 patients with MM were randomized into four groups: Group A (20 patients)
receiving thalidomide / dexamethasone; Group B (20 patients) receiving thalidomide / VAD; Group C (30
patients) receiving melphalan/ prednisolone; and Group D (30 patients) receiving VAD. 50 subjects with
normal bone marrow biopsies were used as the control group. We investigated the histological and
immunohistochemical bone marrow biopsies from all enrolled population at the time of diagnosis, as well
as after treatment. The primary end point was to evaluate the angiogenic alteration before and after
treatment by quantitative estimation of the microvascular density (MVD) in the cellular bone marrow,
through counting the number of microvessels expressing the CD34 antibody per mm2 at X400
magnification.
Results: The MVD alterations of the control group subjects showed a narrow frame of variation, ranging
between 2.3-3.8 microvessels (mvs) per mm2 of marrow surface, expressing a mean value of 3.06
mvs/mm2. In contrast, for the patients with MM, the MVD was significantly elevated at the time of
diagnosis, with a variation between 9.56-30.91 mvs/mm2 and a mean value of 19.03 mvs/mm2 (p<0.0001).
This mean value decreased to 15.63 mvs/mm2, ranging between 7.89-29.76 mvs/mm2 (p<0.001) after
treatment, regardless of the pharmaceutical scheme, with an exception of 20 cases where the mean value
increased. For the 19 out of the 20 exception cases the percentage of bone marrow infiltration increased
after treatment (p< 0.01), but for the one out of the 20 exception cases the percentage of infiltration
remained the same before and after treatment. By comparing the alterations of MVD for each
pharmaceutical scheme individually, it was found that the combinations containing thalidomide were the
ones associated with the highest anti-angiogenic efficacy (p< 0.01). Nevertheless, MVD did not show any
statistically significant correlation with the survival rate of the patients (p< 0.56).
Conclusions: The MVD levels in the bone marrow of patients with MM depend on the activity of the
disease and vary accordingly to its progression, thus expressing a sensitive biological marker for follow-up
evaluation. Furthermore, our findings are in accordance with the reported anti-antiogenic effectiveness of
thalidomide for the treatment of patients with MM, especially the resistant ones; but without prognostic
significance for the patients overall survival.
REFERENCES
[1]. Dredge K, et al, Br J Cancer, 4:87 (10):1166-72. 2002

156

Delineation of Molecular Mechanisms of Action of Decitabine for the

Treatment of -thalassaemia
A. Theodoroua,b, Marios Phylactidesa, Swee Lay Theinb and Marina Kleanthousa
a

Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics,
Cyprus; bSchool of Medicine, Kings College London, UK

eta-thalassaemia, a global health burden, is characterised by reduced beta globin chain production.
Due to the lack of curative therapeutic approaches for -thalassaemia, current management involves
regular blood transfusions in combination with regular iron chelation therapy. Epidemiological data
have shown that pharmacological reactivation of -globin gene for production of foetal haemoglobin
ameliorates the clinical symptoms of the disease. Currently, agents being investigated are limited by
low efficacy and specificity while others are potentially toxic or carcinogenic [1]. Furthermore,
pharmacological attempts to find a suitable HbF inducer are limited since none of the mechanisms of
action of these agents in activating -globin expression is fully understood. Therefore, the aim of this
study is the delineation of the molecular mechanism of action of 5-aza-2-deoxycytidine (Decitabine)
a known and effective HbF inducer [2,3], that will provide a new mechanism-based approach to the
reactivation of HbF in -thalassaemia [4]. Decitabine was initially tested on Primary erythroid
cultures from 14 healthy individuals and 12 thalassaemic patients and the effect was assessed using
HPLC and real-time PCR after 3 and 6 days of induction with the agent. The molecular mechanism of
action of Decitabine was investigated further by quantitative real-time PCR where the gene expression
levels of transcription factors such as GATA1, BCL11A and KLF1, known factors that are associated
with the molecular mechanism of globin gene expression [5], are quantified before and after induction
with the agent. Although there is a large variability in the response observed between individuals,
Decitabine increased the HbF levels by an average of 14% and 66% after 3 and 6 days in healthy
individuals and on average by 34% and 43% in thalassaemic patients respectively. Furthermore,
Decitabine was shown to increase the -globin gene expression by an average of 2.7 and 3.7 fold in
healthy and thalassaemic cultures respectively. Interestingly, there was a concurrent increase in the globin gene expression but not in the -globin gene both in healthy and thalassaemic cultures with
greater effect observed in healthy individuals. Preliminary results show that treatment of primary
erythroid cultures from healthy donors with Decitabine results in an increase in BCL11A and a
decrease in KLF1 gene expression levels after 6 days while showing an opposite effect in primary
erythroid cultures from thalassaemic patients. Moreover, there is an increase in the expression of Sp1
transcription factor in all cultures. Since the results are somewhat contradictory to the literature based
on the effect observed on BCL11A and KLF1, confirmatory experiments are currently underway
where expression of the above factors will be investigated at the RNA level in a larger number of
cultures from healthy and thalassaemic individuals as well as potentially at the protein level with
western blot analysis. Furthermore, the effect observed might be due to the fact that Decitabine might
act through alternative epigenetic mechanisms that bypass the BCL11A and KLF1 dependent -globin
gene expression.
REFERENCES
1. Gambari R. and Fibach E., Curr Med Chem 14:199-212, 2007
2. De Simone J et al, Blood, 99(11):3905-3908, 2002
3. Saunthararajah Y. et al, Blood, 102(2):3865-3870, 2003
4. Higgs D. R. et al, Lancet 379(9813):373-83, 2011
5. Goodwin A. J. et al, J Biol Chem 276:26883-26892, 2001

157

Novel 1,4-dioxane derivatives as NMDA receptor channel blockers

F. Del Bello1, A. Bonifazi1, W. Quaglia1, A. Piergentili1, M Giannella1, M. Pigini1, V.
Mammoli1, G. Giorgioni1, C. Cimarelli2, M. Pellei2, B. Wnsch3, D: Schepmann3
1

School of Pharmacy, Medicinal Chemistry Unit, University of Camerino,

2
School of Science and Technology, University of Camerino,
3
Institut fr Pharmazeutische und Medizinische Chemie, Westflische Wilhelms Universitt Mnster
fabio.delbello@unicam.it

NMDA receptors are glutamate-gated cation channels with high calcium permeability that play
important roles in many aspects of the biology of higher organisms. They are critical for the
development of the central nervous system (CNS), generation of rhythms for breathing and
locomotion, and the processes underlying learning, memory, and neuroplasticity. Therefore, NMDA
receptors are important therapeutic targets for many CNS disorders [1]. To date, clinically drugs
targeting the PCP binding site of NMDA receptors have had only limited success due to poor efficacy
and unacceptable side effects, including hallucinations, catatonia, ataxia, nightmares, and memory
deficits [2].
Recently, it has been demonstrated that the piperidine ring of the potent PCP antagonists dexoxadrol
and etoxadrol are not necessary for high NMDA receptor affinity since its replacement with
aminomethyl or methylaminomethyl chains led to potent NMDA receptor antagonists [3]. Ring and
side chain homologues of aminomethyl analogues of dexoxadrol and etoxadrol resulted in compounds
showing high NMDA receptor affinity. The derivative 1 behaved as the most potent NMDA
antagonist with an affinity value similar to those of dexoxadrol and etoxadrol, [3].

In the present work a series of regioisomers of 1, bearing the 1,4-dioxane nucleus, were prepared. The
biological profiles of the novel compounds were assessed using binding assays at PCP binding site of
the NMDA receptor. The functional activity of the most affine compounds 2-4 was investigated on
L(tk-)-cells stably expressing the NMDA receptor subunits GluN1a and GluN2A by inhibition of the
citotoxicity induced by the activation of NMDA receptors with (S)-glutamate and glycine. Compound
3, showing binding affinity and functional activity not significantly different from those of (S)-(+)ketamine, displayed a promising therapeutic potential. Moreover, it also showed a cytotoxic activity
significantly higher than that of (S)-(+)-ketamine on MCF7 human breast cancer cell lines, expressing
NMDA receptors.
REFERENCES
[1].
Lemoine, D.; Jiang, R.; Taly, A. et al. Chem. Rev. 112, 6285-6318, 2012.
[2].
Blank, M. L; Van Dongen, A. M. Activation mechanisms of the NMDA receptors, Chapter 3,
CRC press, 2009.
[3].
Utech, T.; Khler, J.; Wnsch, B. Eur. J. Med. Chem. 46, 2157-2169, 2011 and references
therein.

158

Developing in silico tools towards the personalized therapy of betathalassemia

Eleni Vrontaki, Georgia Melagraki and Antreas Afantitis
Department of Chemoinformatics, NovaMechanics Ltd, Nicosia, Cyprus
Email: info@novamechanics.com Website: www.novamechanics.com
Thalassaemia is a severe hereditary anaemia with monogenic inheritance and a
complex systemic phenotype. In -thalassaemias, mutations of the -globin gene or its
regulatory regions cause absence or reduced synthesis of -globin chains, and this
impairment leads to an excess of the complementary - globin chains. Ultimately, the
precipitation of the excess -globin chains produced causes apoptosis of erythroid
precursors in the bone marrow and at extramedullary sites and shortens survival of
RBCs in the peripheral blood. The disease is associated with morbidity and mortality
due to severe chronic anaemia, iron overload and treatment- related complications.
In the course of our efforts towards thalassemia treatment, we have been involved in
several international, European and local consortia with the aim to identify and
develop alternative treatments. We are currently participating in an EU FP7 funded
grant, entitled THALAssaemia Modular Stratification System for personalized
therapy of beta-thalassemia (THALAMOSS) that will, for the first time, collate and
analyze jointly data from four independent international medical centers on patient
characteristics and pertaining molecular data, potential therapeutic responses and
critical aspects of medical regimens. THALAMOSS aims in the development of a
universal set of markers and techniques for the stratification of -thalassaemia
patients into treatment subgroups. Within this framework we develop clustering-based
and machine-learning-based stratification techniques applied to selected clinical data
and marker subsets. The produced models will subsequently be tested for accurate
class predictions with an independent set of samples, and agreement of results will be
assessed. For this goal we build KNIME workflows (analyzing data and automatically
reporting) to support customized needs of the project. We use existing and develop
new custom-made KNIME nodes, drawing on the user friendly and comprehensive
open-source data integration of KNIME to develop a customised THALAMOSS
processing, analysis, and exploration platform, in line with their extant KNIME nodes
(called Enalos KNIME nodes http://www.novamechanics.com/knime.php) for the
validation of in silico models.

ACKNOWLEDGEMENT
This work is supported by funding under the Seven Research Framework Programme of the
European Union. Project THALAMOSS (HEALTH.2012.1.2-1 Grant agreement no: 306201).

159

Rational design and synthesis of potent cyclic Luteinizing Hormone

Releasing Hormone LHRH analogues
Despina Laimou1, Theodora Katsila2, John Matsoukas1, Kostas Gkountelias3, George
Liapakis3, Constantin Tamvakopoulos2 and Theodore Tselios1
1
Department of Chemistry, University o Patras, 26504 Rion Patras, Greece
Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation,
Academy of Athens, Athens 11527, Greece
3
Department of Pharmacology, Faculty of Medicine, University of Crete, Heraklion, Crete,
Greece.
e-mail: ttselios@upatras.gr
2

Luteinizing Hormone - Releasing Hormone, LHRH (A.V. Schally, R.Guillemin Nobel

Prize 1977) was first isolated from mammalian hypothalami as the decapeptide (pGluHis-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH2) [1]. LHRH is synthesized by
gonadotropic cells of the hypothalamus and is the central regulator of the reproductive
system. LHRH interacts with high affinity the G-protein-coupled receptor localized
on anterior pituitary gland and cancer cells stimulating the biosynthesis and the
releasing of luteinizing (LH) and follicle-stimulating (FSH) hormones. Many
diseases, such as prostate and breast cancer, endometriosis and uterine leiomyomas,
are stimulated by steroid hormones. LHRH agonistic analogues, Leuprolide [2],
Triptorelin, Buserelin, Goserelin, Nafarelin and Histrelin, have achieved widespread
clinical use for the control of reproduction [3]. Following the elucidation of the amino
acid sequence of LHRH native hormone, modifications to the sequence were
developed in the expectation of greater potency and improved receptor-binding. This
study reports [4] the design and synthesis of novel Leuprolide analogues, cyclized at
the C- and N- terminal, retaining the basic backbone sequence of leuprolide. The
analogues synthesized were: cyclo(1-10)[Pro1, DLeu6, BABA10]LHRH (1), cyclo(110)[Pro1, Tyr(OMe)5, DLeu6, Aze9, BABA10]LHRH (2), cyclo(1-11)[Pro1, DLeu6,
BABA10, Acp11]LHRH (3), cyclo(1-11)[Pro1, Tyr(OMe)5, DLeu6, BABA10,
Acp11]LHRH (4), cyclo(1-10)[Pro1, Tyr(OMe)5, DTrp6, Aze9]LHRH (5). The Nterminal, pGlu was replaced by Pro which bears the available amino group for
cyclization with the C- terminal carboxyl group of 3-aminobutyric acid (BABA) or
aminocaproic acid (Acp) as the C- terminal amino acids in the position of ethylamide.
BABA or Acp were used as anchors for amide linked cyclization with the Pro Nterminal residue and for their methylene lipophilic groups as in the ethylamide of
leuprolide. In analogues 3 and 4 the backbone size was increased compared to the
cyclic analogues 1 and 2 by extending the backbone length using Acp. The rational of
our approach is that novel LHRH peptide analogues with a profound pharmacokinetic
advantage that is particularly valuable with respect to local/direct effects can serve as
therapeutic alternatives for the treatment of endocrine disorders and hormone
dependent cancers.
REFERENCES
[1]. Schally A.V. et al, Biochem. Biophys. Res. Commun. 43, 393399, 1971
[2]. Laimou D. et al, Amino acids 39(5), 1147-1160, 2010
[3]. Scambia, G. et al, Anticancer Res. 8, 187-190, 1988
[4]. Laimou D. et al, Eur. J. Med. Chem., 58, 237-247, 2012

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In silico High-Throughput screening for Kinase Profiling as a platform for

Drug Discovery
Gaia Corso and Rosella Ombrato.
R&D Computational Chemistry Lab., Angelini Research Center
ACRAF S.p.A., P.le della Stazione, snc, I-00040 Santa Palomba, Pomezia (RM) Italy
G.Corso@angelini.it

In the Drug Discovery process, the hit optimization is usually harder to achieve than the hit
identification, and the knowledge of the compound profile is of primary importance for the drug
development to have success [1]. The selectivity issue is straightforward when considering the human
kinome and, at this regard, at the Angelini Research Center the computational endorsement has been
the building of a platform for in silico kinase profiling.
The platform includes a built-in kinase collection composed of human ATP-binding proteins retrieved
from the UniProt Knowledge repository [2]. The database is constantly updated by means of a KNIME
[3] workflow that implements several Python and shell scripts, and uses several Schrdinger tools [4].
The platform can be used to evaluate the protein sequence homology of a given target toward the
kinase collection. When human ATP-binding proteins holo x-ray structures are available in the RCSBPDB [5], the platform is devised to perform the docking of the hit compound and the molecular
interaction fingerprints.
The outcome of the in silico profile can be used to drive the in vitro screening (e.g., selectivity, offtarget).
It should be noted that the present kinase profiling platform can be extended for other purposes such as
hit identification, drug repositioning, and potential orphan drugs mode of action elucidation.

REFERENCES
[1]. Huggins, D. J.; Sherman, W.; Tidor, B. J. Med. Chem., 55(4), 1424-44, 2012.
[2]. The UniProt Consortium, Nucleic Acids Res., 40, D71-D75, 2012.
[3]. Berthold, M.; Cebron, N.; Dill, F.; Gabriel, T.; Ktter, T.; Meinl, T.; Ohl, P.; Sieb, C.;
Thiel, K.; Wiswedel, B., KNIME: The Konstanz Information Miner. In Data Analysis,
Machine Learning and Applications, pp 319-326, Springer Berlin Heidelberg, 2008.
[4]. Schrdinger Release 2013-1: Epik, version 2.4, Schrdinger, LLC, New York, NY,
2013.
[5]. Bernstein, F.C.; Koetzle, T.F.; Williams, G.J.; Meyer Jr., E.E.; Brice, M.D.; Rodgers,
J.R.: Kennard, O.; Shimanouchi, T.; Tasumi, M. J. Mol. Biol., 112, 535-542, 1977.

161

CURRICULUM VITAE

97

Afantitis Antreas, PhD, MBA: He is the director of NovaMechanics Ltd. Dr.

Antreas Afantitis is an experienced consultant in chemoinformatics and applied drug
discovery projects. Dr Afantitis has a strong scientific background in the field of
chemoinformatics, bioinformatics and medicinal chemistry. He has been included in
the most productive authors in the field of Chemoinformatics (bibliometric analysis
performed by Prof. Peter Willet) and according to Scopus his h-index is 18. He is a
reviewer in leading scientific journals (more than 40), member of Editorial Boards
(more than 4) and an evaluator of international competitive grants. Recently he has
joined Combinatorial Chemistry & High Throughput Screening as the Section Editor
for Combinatorial/ Medicinal Chemistry. He has developed and applied several
virtual screening methodologies to drug discovery problems and based on the results 3
patents were filled and several articles have been published in top scientific journals.
Dr. Afantitis is actively involved (PI or co-PI) in 3 FP7 EU projects, 2 Cost Actions, 1
Eureka and 4 National Funded R&D projects.
Archontis Georgios: Associate Professor, Department of Physics, University of
Cyprus. Email:archonti@ucy.ac.cy; webpage: www.biophys.ucy.ac.cy; Tel: +357 22
892822; Fax: +357 22 89 28 21. Brief Description of Research: My research aims to
understand the properties of biomolecular systems by means of computational
methodologies (molecular dynamics simulations with atomic-detail or implicitsolvent models) and theoretical concepts, (based on statistical mechanics, continuum
electrostatics). I am among the developers of the widely used.
Birkou Maria, BSc & MSc in Biology. She is a PhD student at University of Patras,
Pharmacy Department. Her research area is Molecular and Structural Biology. Her
interests include the study of structure and function of metalloproteins, proteinprotein interactions and specificity of protein-protein/substrate assembly. Specifically,
in her PhD thesis she is studying the Structure- Function correlation of E3 ubiquitinprotein ligase Arkadia.
Booth G. Raymond, PhD, Professor and Director of Laboratories for Medicinal
Chemistry and Molecular and Behavioral Pharmacology Center for Drug Discovery,
Northeastern University, Boston, Massachusetts, USA. Raymond Booth has been
involved in academic-based drug discovery and development research since 1990. His
area of expertise concerns drugs targeting G protein-coupled receptors involved in the
pathophysiology and pharmacotherapy of neuropsychiatric and neurodegenerative
disorders. Recently, his lab has developed small orally-active molecules that target
serotonin 5-HT2-type receptors in the brain with unique multifunctional
pharmacology, i.e., 5-HT2C agonism together with 5-HT2A/2B inverse agonism.
These drug candidates are progressing through preclinical studies and are being
positioned for clinical development to treat psychoses without liability for weightgain.
Charlton J Steven; Novartis Institutes for Biomedical Research, Horsham, United
Kingdom. For the past 15 years Dr Charlton has worked in the pharmaceutical
industry, both at SmithKline Beecham and Novartis, and has experience ranging from
target validation through to leading full lead optimisation programmes to successful
clinical proof of concept. He is currently Director of Molecular Pharmacology at
Novartis, Horsham (UK), leading an assay development and compound profiling team
of 30 scientists providing expert opinion and support for GPCR, ion channel and
enzyme projects. He is interested in all aspects of the quantitative assessment of

165

ligand-receptor interactions, with a particular interest in the kinetics of ligand binding

and signaling. Dr Charlton collaborates widely with academia, has published over 50
papers and serves as an editor of the British Journal of Pharmacology. He is actively
engaged in training new pharmacologists and works closely with the British
Pharmacological Society to organise scientific symposia and teaching workshops. Dr
Charlton was awarded Novartis Leading Scientist in 2007 and Fellowship of the
British Pharmacological Society in 2013.
Demetriades Marina. After finishing my BSc studies in the Department of
Chemistry at the University of Cyprus in 2009, She started her PhD degree at the
University of Oxford, in the division of Organic Chemistry. The main focus of her
PhD was inhibitor design for enzymes of the 2OG oxygenase family mainly with the
assistance of dynamic combinatorial chemistry. Currently she works towards the
investigation of the mechanism of action and inhibition of DNA and RNA
demethylases as a Post-Doctorial research asssistant at the Chemical Biology
department of the same university.
Gaia Corso joined Angelini as a Computational Chemistry Scientist in 2008. Since
then she has been involved in several Drug Design projects using both Structurebased and Ligand-based approaches. Her main research activities include the
identification of new hit compounds and hit to lead optimization in the core
therapeutic areas, the identification of new possible therapeutic indications for
marketed drugs, and the automation of modelling processes by means of the
development of scripts and workflows. She received the Degree in Chemistry and the
Ph.D. in Chemical Science at the University of Rome La Sapienza.
Tel: +39 06 91045289 Fax: +39 06 91984597 Email: G.Corso@angelini.it |
www.angelinipharma.com
Glaubitz Clemens Dr. Prof. Born 18.4.1969, Leipzig, Germany. Study of physics
(1989-1994, Leipzig University). Rhodes Scholar at University of Oxford (19941998). Doctor of Philosophy (1998), Biochemistry, University of Oxford, with Prof.
A. Watts. Postdoc (1998-2000), University of Oxford, National Facility for Biological
Solid State NMR. Postdoc (2000-2001) with Prof. M. Levitt, Stockholm University,
Emmy Noether Fellowship. Emmy Noether Group Leader, Research Institute for
Molecular Pharmacology, Berlin. Since 2002: University Professor for Biophysical
Chemistry, Goethe University Frankfurt. Principal Investigator at Cluster of
Excellence Frankfurt: Macromolecular Complexes. Managing Director Centre of
Biomolecular Magnetic Resonance.
Contact Details: Institute for Biophysical Chemistry, Centre for Biomolecular
Magnetic Resonance (BMRZ); Goethe University Frankfurt. Max-von-Laue-Str. 9.
60438 Frankfurt am Main Germany. Tel: +49 (0) 69- 798-29927 (direct); -28
(secretary) Fax: +49 (0) 69- 798-29929 Email: glaubitz@em.uni-frankfurt.de.
www.bmrz.de
Golic Grdadolnik Simona Dr, Prof., is a senior researcher at National Institute of
Chemistry (NIC) and associate professor at University of Nova Gorica (UNG). She is
an expert on biomolecular NMR spectroscopy, working in the field of structural
chemistry. Here expertise includes development of methodology for determination of
structurally dependent NMR parameters in solution and investigation of structure,
dynamics and interactions of molecules in relation to their biological activity. Her

166

current research projects are focused (i) on the development of novel inhibitors of
enzymes involved in the membrane biogenesis of bacterial cells, which lead to design
of novel antibacterial agents, (ii) on the development of novel selective inhibitors of
fungal sterol 14-demethylase, which lead to design of selective antifungal drugs in
medicine and fungicides in agriculture and, (iii) on the investigation of non-covalent
interactions in unfolded proteins and model linear peptides, which are important for
understanding the mechanisms of protein folding, amyloid-fibril formation etc. She
has co-authored more than 90 peer-reviewed publications in international scientific
journals and books and she has delivered numerous contributions in international
conferences including several invited lectures. She has leading and performed more
than twenty national and international basic research projects as well as development
and industrial projects for pharmaceutical industry, among them she was responsible
researcher for the partnership of NIC within ARCADE (Advancement of Research
Capability for the Development of New Functional Compounds) EU project (FP7REGPOT-2009-1 Support action) and she was leading Drug design project within
EN-FIST Centre of excellence. She is supervising diploma, master and PhD students.
She is a president of the scientific council of NIC and a member of the Program
Committee of Slovenian National NMR Centre.
Gruzman Arie, B.Sc., Chemistry Bar-Ilan University, Ramat Gan, Israel (1993
1995), Ph.D (Summa cum Laude), Department of Medicinal Chemistry, School of
Pharmacy, Hebrew University of Jerusalem, Israel (1997 2003), Post-Doctoral
Research Fellow, Department of Physiology, Medical School, University of
California, and Research Institute of Pacific Medical Center at San Francisco, U.S.A.
(2004 2007). Assistant Professor at Bar-Ilan University, Department of Chemistry,
Bar-Ilan University, Ramat Gan, Israel (From 2009). In 2011 Arie was elected as a
Vice-president of the Israel Medicinal Chemistry Society. Main research topics:
development of new drugs against diabetes type two and Amyotrophic Lateral
Sclerosis (ALS).
Hubbard E. Roderick, Professor, University of York and Senior Fellow, Vernalis
(R&D) Ltd. Rod Hubbard has been working with methods for analysis and
exploitation of protein structure for over 30 years. In the 1980s, he developed
molecular modelling methods; in the 1990s he helped build the Structural Biology
Lab at the University of York. Since 2001, he has spent varying amounts of his time
with Vernalis, establishing and applying structure and fragment-based methods for
drug discovery.
Keramidas Anastasios was borned in Nikea of Pireaus Greece in 1965. He graduated
from the University of Ioannina in 1988. He did his Ph.D. on the Bioinorganic
Chemistry of Vanadium at the same University. From 1994 up to 1997 he worked as
Post Doc. on Antidiabetic Vanadium Based Drugs at Colorado State University. In
1997 he appoited the position of Lecturer at the University of Cyprus and from 2005
is Associate Proffesor in the Chemistry Department of the same University.
Kordopati Golfo is a PhD student at the Department of Chemistry, University of
Patras under the supervision of Assis. Prof. Gerasimos Tsivgoulis since January 2014.
Her thesis is related to synthesis and study of bioactive molecules and their conjugates
with cyclodextrins. She received her B.Sc.degree in Chemistry in 2011 and M.Sc.

167

degree in Medicinal Chemistry: Drug Discover and Design in 2013 at University of

Patras.
Koutentis A. Panayiotis, CChem, FRSC; Associate Professor in Organic Chemistry
Department of Chemistry, University of Cyprus, Nicosia, Cyprus. Research interests
Four areas of interest are currently under investigation. 1) The synthesis and reactivity
of novel and unusual S,N-heterocyclic compounds; 2) The design and synthesis of
organic magnets based on radical and diradical polyazaheterocyclic systems; 3) The
design and synthesis of novel conjugated and non conjugated polymers; 4) The design
and synthesis of biologically active heteroarenes. Publication record ca. 90
Publications and 4 Book Chapters on sulfur nitrogen heterocycles (Thiazoles, 1,2,5Thiadiazoles and 1,3,4-Thiadiazoles).
Logothetis E. Logothetis, Northeastern University, Boston, MA, B. A, (1980), Physics;
Northeastern University, Boston, MA (M.A.) (1981), Psychology; Harvard University,
Cambridge, MA, Ph.D (1987), Physiology & Biophysics. Present Position (2008 to
date) Professor and John D. Bower Chair of Physiology and Biophysics, Virginia
Commonwealth University (VCU) School of Medicine (SOM), Richmond, VA.
My laboratory has studied for well over two decades regulation of ion channel activity
by signaling mechanisms that involve protein-protein and protein-lipid interactions. We
have aimed to understand the physiology of the regulation of channel activity by
phospholipids and G protein subunits, especially by exploiting structural information
available only in the past decade. Bringing together theoretical studies that have yielded
dynamic structural models with experimental tests to validate or reject the models has
allowed us to gain greater insights into mechanisms of regulation of ion channel
activity. We were recently able to decipher the functional details of signaling through
the heteromeric G Protein-Coupled Receptor (GPCR) complex, the mGlu2R and 2AR.
Our ability to quantify the signaling changes triggered by binding of different ligands of
this complex, allowed us to create a metric, the Balance Index (BI), that predicted the
psychoactive behavioral effects elicited by these drugs. The BI provided a great
opportunity to develop a cell-based assay for High-Throughput screening studies of
small molecule libraries that would be predicted to have antipsychotic effects. In my
seminar I will review our published (Fribourg et al., 2011 Cell 147:1011-23) and
unpublished work aiming to take our mechanistic studies in a direction where
identification of novel antipsychotic drugs has the potential of helping the many
schizophrenic patients for whom current drug therapy is problematic.
Mali Gregor is a senior researcher at the National Institute of Chemistry in Ljubljana
and an associated professor of physics at the University of Nova Gorica. He is
working on the development and application of solid-state NMR methods for the
investigation of structure and function of porous materials. He uses NMR
spectroscopy also for the investigation of promising new materials for lithium
batteries and for structural analyses of solid pharmaceuticals from the local
pharmaceutical companies.
Mavromoustakos Thomas. Born in Cyprus, graduate Chemist, University of Athens,
Msc-Medicinal Chemistry, University of Connecticut, Ph.D, in 1990. B.Sc-Theology
School of Athens University-2007. Research director of Laboratory of Molecular
Analysis at the Institute of Organic and Pharmaceutical Chemistry (IOPC) of National
Hellenic Research Foundation (1991-2007) where he is continuing exerting research

168

activity as scientific collaborator. Associate Professor at the University of Athens

since 2007. Since 2012 he is a Full Professor.
Melagraki Georgia PhD, MBA: Dr. Melagraki is an experienced consultant in
chemoinformatics and applied drug discovery projects. Her work is focused on the
development and implementation of in silico methods to guide decisions in the design
and selection of promising drug candidates for a wide range of therapeutic targets. Dr
Melagraki Georgia has a strong scientific background and publication record in the
field of chemoinformatics, bioinformatics and medicinal chemistry (h-index 18). She
is a member of the Editorial Board and reviewer in leading scientific journals. She has
participated in large scaled scientific projects targeting nanomaterials toxicity,
systems biology and personalized medicine.
Mikros Emmanuel, Chemist, Ph.D. is Professor in Pharmaceutical Chemistry
University of Athens. He obtained his Ph.D. in Chemistry from Universit Paris-Sud
(Paris XI) in 1988 and then he joined University of Athens. Research Fellow at the
Institut fr Chemie Medizinische Universitt zu Lbeck, (Germany), with Prof. T.
Peters at 1996 and at the Laboratoire Ingenierie Moleculaire, INRA, Centre de
Recherches de Nantes, (France) with Dr. S. Perez (1993 and 1994). He was awarded
the DAAD (Germany, 1996) and Marie Curie (European 1994) Fellowships. He is coauthor of 80 peer reviewed scientific publications (1600 citations, h-factor 22). He has
participated in over 20 research and educational projects funded by National and EU
organisms, the most recent are: FP7- Research Potential-2007, FP7-PEOPLEIndustry-Academia Partnerships and Pathways-2008 Marie Curie Actions, FP7Knowledge Based Bio-Economy-2009-3-1-04. He is member of several national and
international scientific societies (EFMC, ACS) and vice president of Hellenic Society
of Medicinal Chemistry. Invited speaker in more than 20 international meetings, he
has also participated in the organising committee of 4 international and national
scientific congresses. Prof. Mikros leads a research group focused on NMR
spectroscopy, NMR based Metabonomics Structure elucidation of biomolecules,
natural products and drugs, as well as Drug Discovery, Structure Based Drug Design,
Molecular and in silico screening.
Miroslaw Barbara is an assistant professor at the Department of Crystallography
(Faculty of Chemistry, Maria Curie-Sklodowska University in Lublin, Poland) since
2009. Her research interests focus on synthesis and characterization of transition
metal complexes with low molecular weight organic ligands to obtain new
compounds having the desired biological activity. The other field of her research are
structural studies on heterometallic coordination compounds of 3d-4f metals with
Schiff base ligands and search for new magnetic and/or luminescent materials. She
specializes in synthesis of coordination compounds, X-ray single crystal structure
analysis, spectroscopy (IR, RAMAN, XAS) and quantum chemical calculations.
Nikas P. Spyros, Research Associate Professor, Center for Drug Discovery,
Northeastern University. Spyros Nikas has been working with the design and
synthesis of cannabinergic analogs for over 13 years. His major goal is to explore the
role of these compounds as biological/pharmacological tools and as potential drug
candidates for pain, inflammation and central nervous system disorders.
Noskov Sergei is an Associate Professor of Biophysical Chemistry with the
Department of Biological Sciences, University of Calgary. The principal research goal

169

of his research is to understand how small molecules regulate membrane proteins

function and how cells at the level of single membrane protein can be controlled. As
PI or co-Investigator on a number of federal and provincial Canadian and USAfunded grants, he laid the groundwork for the studies of ion transport by developing
effective computations for single-channel recordings, ion channels and transporters
function as well as mechanisms of drug action on membrane proteins.
Papadopoulos G Manthos has developed and applied methods for: (a) The
calculation of the linear and nonlinear optical (NLO) properties of microscopic
(molecules) and macroscopic (crystalline materials, thin films, molecular liquids)
forms of matter. At the molecular level benchmark computations have been reported
taking into account all important contributions to the NLO properties, that is due to
the correlation of electrons, vibrational motion and relativistic corrections (if heavy
atoms are involved in the molecule).(b) The design of novel inhibitors for HIV-1PR
and (c) The study of the interaction of proteins with medium size molecules and
particles.
Patrikios S. Ioannis, Professor, was born in Cyprus in 1960. Prof. Patrikios received
his BSc degree in Pre-Medical studies/Biochemistry from the City College of the City
University of NY. He completed his PhD studies in 1994, specialized on Immunology
and Lipids/Lipidomics and post-doctoral studies specialization in Medical
Biochemistry at the City College of NY, next to the world-known Professor CS
Russell. Professor Patrikios went through several different fellowships including
research specializations /collaborations with different high reputation institutions
including, Mount Sinai NY and was awarded advanced Immunology specialization
courses at Scuola Superiore d'Immunologia Ruggero Ceppellini, Italy. He served and
continues to serve as a Research Scientific Consultant for Industry and higher
education. His broad research interests include studies of new therapeutic approaches
of chronic diseases by the use of Systems Medicine, through Systems Biology and
Nutritional Systems Biology; Lipid Hemagglutinins, Lectins, Immunology and the
use of novel interventions against Metastatic Tumor Cells. As from 2004, Professor
Patrikios got involved in the research of innovative, pioneer, holistic therapeutic
approaches for chronic multifactorial Neurodegenerative diseases, specifically of
Multiple Sclerosis. He is affiliated as a Graduate Studies Mentor with the Institute of
Brain Chemistry of London. During the last decade he has obtained many competitive
research grants, participated in several different collaborative European scientific
research-projects and he organized several large European Consortium Platforms for
research and Clinical Trials. His 2002 research findings on the effect of frying oils as
human hemagglutinins got an international interest; and have been discussed by
several different National Food and Drug Administrations, including UK, and through
articles in high impact Magazines such as the New Scientist. He has been appointed
as a reviewer of several international scientific journals in the field of Medicine, MedBiochemistry, Pharmaceuticals-Therapeutics and Neurology and as a reviewer for
research grants. He is the solid author of University Books in Biochemistry, Medical
Biochemistry and Med-Chemistry and he has authored numerous peer reviewed,
limited co-authored original publications, in high impact factor scientific journals.
Professor Patrikios is a member of several International associations and bodies
including Sigma Xi. He is the chief scientific investigator of the team which lately
invented and patented the nutraceutical formula PLP10 as a new therapeutic
intervention for multiple sclerosis. He is the co-founder and Director of PALUPA

170

Medical Ltd (a research and innovation company), that performed and successfully
completed the phase II clinical trial for PLP10 and the Chief Executive Officer of the
P. Essential Oils Enterprises. Now he is the scientific coordinator of the MINERAL
phase III multicenter clinical trial for PLP10. At present he is a Professor, Faculty of
Medicine, Chair and Acting Deputy Dean at the School of Medicine of the European
University of Cyprus and affiliated as a research collaborator at the Cyprus Institute
of Neurology and Genetics.
Phylactides Marios, Dr, completed his undergraduate studies in the field of
Biochemistry at Imperial College and obtained his PhD at University College
London, in the area of molecular immunology. For his first post-doctoral position he
was involved in research in the regulation of the Cystic Fibrosis Conductance
Regulator gene. Currently, he is working at the Cyprus Institute of Neurology and
Genetics, carrying out research in the field of drug therapy for beta-thalassaemia, the
regulation of globin gene expression and genotype/phenotype correlation studies for
thalassaemic patients.
Plavec Janez is head of Slovenian NMR centre at National Institute of Chemistry and
Professor of Structural Biology at University of Ljubljana, Faculty of Chemistry and
Chemical Technology. He received his B.Sc. and M.Sc. degrees in chemistry at
University of Ljubljana. His Ph. D. degree was conferred by Uppsala University,
Sweden. He was recipient of Fulbright fellowship at Georgia Institute of Technology
in Atlanta, USA. His research interests include the structure and dynamics of
(modified) nucleic acid constituents with the use of NMR spectroscopy.
Potamitis Constantinos, is a postdoctoral researcher at the Institute of Biology,
Medicinal Chemistry and Biotechnology of the National Hellenic Research
Foundation (NHRF) since 2010. His research field and scientific experience are
focused on NMR spectroscopy and in silico methodologies towards structure-based
and ligand-based drug discovery. He received his bachelors degree in Chemistry
from the University of Athens in 2003. In collaboration with the NHRF, he obtained
his PhD in Physical Chemistry from the same University in 2009. His research work
has resulted in 17 publications at international journals.
Rahman Khondaker Miraz Dr, graduated as a pharmacist from the Faculty of
Pharmacy of University of Dhaka, Bangladesh in 1996. He worked for 3 years as a
research and development pharmacist at SK&F Pharmaceutical before moving to
academia in April 2001 and joined the Pharmacy department of University of Asia
Pacific as a Lecturer. In October 2003, he was appointed as a Lecturer in
pharmaceutical chemistry at Dhaka University and was promoted to Assistant
Professor in June 2005. He completed his PhD research in Medicinal Chemistry at the
London School of Pharmacy (UCL) in 2009 and joined the CRUK Protein-Protein
Interaction Research Group as a CRUK Research Fellow in July 2009. He was
appointed as a Lecturer in Medicinal Chemistry at Kings College London in May
2012. Dr Rahmans research activities focus on the application of synthetic medicinal
chemistry and chemical biology techniques to the design, synthesis and evaluation of
novel anticancer and antibacterial agents, along with studies to understand their
molecular and cellular mechanisms of action.
Spyroulias A. Georgios PhD., Department of Pharmacy, Associate Professor, Contact
details: Tel: +302610 962350-1-2, Email: G.A.Spyroulias@upatras.gr, Web page:

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http://bionmr.upatras.gr & http://www.seedrug.upatras.gr. G.A. Spyroulias (Chemist,

Aristotle University of Thessaloniki, 1989) performed his PhD in Chemistry
(University at Crete, 1995) and has been as a Marie-Curie Researcher at Center of
Magnetic Resonance at University of Florence since early 2000. He is currently the
Principal Investigator of the UPATs Biomolecular NMR Group at the Department of
Pharmacy (since 2003). The team have a long-term experience in advanced techniques
of NMR Spectroscopy applied in the conformational & dynamical study of
biomolecules, expression/purification of uniformly or selectively/specifically labeled
in 2H, 13C & 15N proteins and other physicochemical characterization. The group has
recently installed a high-field NMR instrument in Greece and exploit the capabilities
of a 700MHz NMR, equipped with cryoprobe suitable for biomolecular NMR studies
towards the experimental & computational study of the conformational dynamics of
biomolecules and their complexes, such as protein-RNA/protein & protein-drug
complexes. He is the author or the co-author of 77 articles (citations >1150, h-index =
20) in peer-reviewed journals.
Stephanou Anastasis, Professor was born in 1962 in London UK. He gained his BSc
on Applied Biology in 1985 at the University of East London and a MSc (Molecular
Biology) in 1988 at Birkbeck College London. He then completed his PhD at the
Westminster and Charing Cross Medical School, University of London in 1992. He
then did his Post-doctoral training (1992-1995) in the Department of Endocrinology,
Cincinnati Children's Hospital, USA, working on transcriptional gene regulation. In
1995, he returned to London as a postdoctoral fellow to the laboratory led by
Professor David Latchman at the Windeyer Institute of Medical Sciences, University
College London where he studied the regulation of heat shock proteins and their
cytoprotective properties. During his postdoctoral work, he developed his interest in
the Signal Transducers and Activators of Transcription (STATs) factors as key
regulators of apoptosis. In 2002, Dr Stephanou became a Lecturer at UCL and in 2005
was promoted to an Associate Professor at University College London, in the Medical
Molecular Biology Unit (Faculty of Medicine) and leads a group in the Medical
Molecular Biology Unit, Faculty of Medicine. His main research interests are
opposing roles of STAT1 and STAT3 in regulating processes such as apoptosis, cell
cycle regulation and autophagy in disease models such as cardiac ischaemia
reperfusion injury and also in cancer. He has recently edited a book entitled "JAKSTAT Pathway in Diseases" and is an author for over 100 peer-reviewed articles. He
is Editor-in-Chief for the Journal JAK-STAT and also receiving editor for Cell Death
& Diseases and a frequent reviewer for journals such as PNAS, JBC, Oncogene and
Cell Death & Differentiation. Dr Stephanou has been successful in grant awards of
about 2Million English Pounds, over the last 10 years and supervised over 10 PhD
students. Another major interest is in collaborating with a colleague in the Mechanical
Engineering Department at UCL, who has developed a novel technique called bioelectrospraying (BES) for deposition and controlled jetting of primary neonatal
cardiac myocytes, primary cardiac and endothelial cells, as well as creating a beating
cardiac tissue graft and are hoping to use such protocols for transplantation and
treatment of severe heart failure models. Moreover, his research activities include the
following areas (with UK and International collaborators): examination of the
relationship between constitutive ERK activity and STAT1 levels in leukaemia; the
role of IL-17 in the ischaemic myocardium; interactions of p73 with STAT1; STAT1
and STAT3 inhibitory peptides as therapeutic agents in the isolated heart; and the role

174

of STAT1 and STAT3 in models of stroke. He is currently a visiting Professor at the

School of Medicine of the European University of Cyprus.
Tapeinou Anthoula is a PhD student at the Department of Chemistry in the
University of Patras under the supervision of Assis. Prof. T. Tselios since October
2012. Her research interests focus on the protein expression and the design, synthesis
and analysis of peptide - polysaccharide conjugations that are involved in the
immunotherapy of Multiple Sclerosis. She received the BSc degree in Chemistry in
2010 and MSc degree in Medicinal Chemistry: Drug Discover and Design in 2012
at the University of Patras.
Tzakos Andreas, studied Chemistry at the University of Ioannina (1995-1999).
During his MSc (2000-2002) and PhD (2002-2004) studies he performed several long
term scientific visits in research labs in Utrecht (Bijvoet center of Biomolecular
Research) and Florence (PARABIO) working on the application of multidimensional
NMR and other biophysical techniques in biomolecular systems. After almost four
years postdoctoral research in structural and molecular biology at the MRC
Laboratory of Molecular Biology, Cambridge, UK, he became (since 2009) a Lecturer
of Organic Chemistry at the University of Ioannina and from 2013 he was promoted
to an Assistant Professor in the same Department. He has 50 publications in
International Journals. He is a member of the editorial board of three international
scientific Journals. He has been the lead scientist and inventor of novel and selective
AT2R analogues (patent WO 2013091883 A2) with MRCT, UK and has submitted
four more patents in collaboration with pharmaceutical companies. He has developed
a software for rapid identification of natural products in plant extracts. He is a
committee member in two Laboratory Units/Centers within the Network of Horizontal
Laboratory Units and Centers in the University of Ioannina: a) Human Cancer
Biobank Center and b) NMR Center. His lab integrates biophysical tools for
biomolecular screening (isothermal titration calorimetry, robotics NMR, fluorimetry),
synthesis (peptides and non peptide molecules, natural products - biotransformations)
as also biochemical tools (cell based screening assays) in a platform for smallmolecule screening with relevance to disease areas including cancer, diabetes,
hypertension and atherothrombosis. He is in the process of establishing a MS-based
pharmacokinetic facility in his lab after receiving a relevant award. He received
numerous fellowships and awards including the Greek State Scholarship Foundation
(1997-1999); Marie Curie (Chemistry Depart., Univ. Florence); FEBS Summer
Fellowship (Univ. Utrecht, Netherlands); Leonidas Zervas Award (2004); EMBO
long term fellowship (MRC, LMB, Cambridge, UK); Medicinal Research Council
fellowship, LMB, Cambridge, Gardiki-Kouidou Award (2007), Aristeia II (2013).
Tsika Aikaterini is a MSc student at the Department of Pharmacy, School of Health
Sciences, University of Patras since 2013. Her B.Sc thesis as an undergraduate
student (2011-2013) focused on Expression and Structural studies of Alphaviruses
macro domains (MAYARO, CHIKV,VEEV). Her research interests and experience
concern Molecular Biology (PCR, gene cloning, DNA recombinant techniques,
Protein expression), Protein Chemistry Protein purification (FPLC, affinity
chromatography) and Isotopic protein labeling (15N ,13C, 2H) in E.Coli for NMR
structural studies.
Tsantili-Kakoulidou Anna was borm in Athens, Greece. She studied Pharmacy in
the Univsristy of Athens. She obtained her PhD from the same University in 1976.

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She performed postdoc research in the Free University of Amsterdam, the

Netherlands, during 1982-83, where she specialized on QSAR. In April-June 1985,
June 1987. May-June 1989, July 1994 she jointed as Visiting Scientist the
Department of Pharmacy, University of Lausanne, Switzerland. Her current position
is Professor of Pharmaceutical Chemistry- Pharmaceuticall Analyis in the Department
of Pharmaceutical Chemistry, School of Pharmacy, University of Athens. She has
published 112 research papers and review articles in international peer-reviewed
scientific journals which have attracted more than 1500 citations. She is President of
the Hellenic Society of Medicinal Chemistry, member of the Council of the European
Federation of Medicinal Chemistry and Vice-President of the Hellenic Pharmaceutical
Society. She is editor of the Greek bilingual scientific journal Pharmakeftiki.
Voskou Stella obtained a BA in Biology from the Kapodistrian University of Athens.
Her dissertation titled Research of the mechanisms induced by ongogenic signals
was completed at the N.H.R.F., Athens, and part of this work was published at
Molecular Cancer Research (2008) 6:1071-1083. She subsequently obtained an
MRes in Applied Fish Biology from the University of Plymouth, UK, where she
studied the Immunological effects of carbon and titanium nanoparticles. She is
currently performing her PhD at the CING, Department of Molecular Genetics
Thalassaemia. The main focus of her work is the pharmacologic induction of foetal
haemoglobin.
Vrontaki Eleni was born on Crete, Greece, in 1987 and received her Bachelors
Degree (B.Sc.) in Chemistry from the National and Kapodistrian University of Athens
(UoA), Greece in 2010. Furthermore, she attained a Certificate of Enology from the
inter-university program of the University of Athens and the Agricultural University
of Athens. In 2012, she received a Masters Degree (M.Sc.) in Organic Synthesis and
Applications in Chemical Industry (Organic Chemistry) from the UoA under the
direction of Prof. Thomas Mavromoustakos. Since June 2012, she is a PhD student in
Organic Chemistry at the same university, and by November of that year is a
researcher in NovaMechanics Ltd, Chemoinformatics & Bioinformatics Solutions,
Nicosia, Cyprus.
Vlachou Marilena is an assistant Professor of Pharmaceutical Technology in the
sector of Physical Pharmacy. PhD in Pharmaceutical Technology Department of
Pharmacy, University of Athens (1992). BSc in Pharmacy Department of Pharmacy,
University of Athens (1988). Professional License in Pharmacy (Chartered Pharmacist
- 1988). BSc in Pedagogy (Didactics) (1985) Teachers College, Athens.
Phone: +30 210 7274674 (office), Fax: +30 210 7274674 (office).
Email:vlachou@pharm.uoa.gr Web page: http://www.pharm.uoa.gr/an8ropinodynamiko/melh-dep-kai-biografika/dep-farmakeytikis-texnologias/blaxoymarilena.html
Yaghmur Anan is associate professor at the Department of Pharmacy (Faculty of
Health & Medical Sciences, University of Copenhagen, Denmark) since 2009. His
research interests focus on the formulation and characterization of soft self-assembled
drug nanocarriers. He worked as a young scientist between 2006-2009 in Peter
Laggners research laboratory at IBN (the Austrian Academy of Sciences, Graz,
Austria). He had also a postdoctoral position between 2002-2005 in Otto Glatters
research laboratory (University of Graz, Austria). Anan earned his PhD in applied

174

chemistry under the supervision of Prof. Nissim Garti at The Hebrew University of
Jerusalem in 2003.
Yannakakis Mary Patricia was born in 1989 in Chios, Greece. She obtained her
diploma (Chemistry) in 2011 and MSc in Medicinal Chemistry, entitled: "Design of
peptide mimetics based on the conformational demands and interactions between the
T-cell receptor that is involved in Multiple Sclerosis and the immunodominant
epitope of the Myelin Basic Protein", in 2013 from the Department of Chemistry,
University of Patras. She has joined the School of Chemistry, Cardiff University, UK
(2010) with the ERASMUS program, as a part of her studies and the Department of
Chemistry, University of Florence, Italy (2012) as a visiting scientist. At present, she
is a PhD student at the Department of Chemistry, University of Patras in the field of
Molecular Modeling and Rational Drug Design.
Zervou Maria, is an Assoc Researcher in the Institute of Biology, Medicinal
Chemistry and Biotechnology, National Hellenic Research Foundation. She has
received her Bachelor /M.Sc. in Physics ( University of Athens, 1989/1992), and her
PhD in Chemistry from the University of Patras (2002). Her research interests
comprise (a) Drug design by applying NMR spectroscopy and in silico studies; (b)
bio-NMR applications and (c) NMR- and LC-MS based metabolomics She is a coauthor of 49 publications in peer-review journals, 6 chapters in international edition
books, and several written/oral conference participations.

175

PAEDIATRIC VAGUS NERVE STIMULATION: A RETROSPECTIVE

STUDY
Sargsyan N a b, Niotakis G b, Valentin A b, Hughes E b, McCormick D b,
a
b

St. Georges University Medical School, Nicosia Campus

Kings College Hospital, Department of Neurophysiology
Email: sargsyan.n@live.sgul.ac.cy

Introduction: VNS is a treatment option for paediatric epilepsy patients, with epilepsy refractory to
medication and not amenable to intracranial surgery.
The VNS system consists of a pulse generator and a bipolar lead that transmits this electrical
stimulation to the left mid-cervical vagus nerve, which conveys the current via ascending fibres to the
brain. After implantation, the generator is activated and adjusted individually for output current
frequency, pulse width, and signal on/off time. A portable magnet that triggers additional stimuli from
the generator, is also provided in an attempt to minimize acute seizures.
Aims / Objectives: At Kings College Hospital, from 1995 until 2008, 131 VNS implantations took
place in children. This is one of the three largest groups of such patients in Europe. We conducted a
retrospective study to review the efficacy and safety of VNS in this group. We aim to define the
population and compare outcomes to the literature.
Results: 104 cases of the total 131 implantations were eligible for inclusion. The mean age at
implantation was 12.23 years (5.93 - 17.54). The mean follow up period was 4.75 years. Table1 shows
the distribution in regards to the epilepsy type/syndrome and the underlying pathology shows.
According to the literature, VNS has very promising results for seizure frequency reduction, with other
parameters like the duration of the seizures and the post-ictal recovery are also improved.
Improvements in Quality of Life (QoL) appear to be another benefit of VNS implantation. Alertness,
mood, behaviour, memory, seizure clustering and verbal communication were independently
improved. VNS proved to be a safe procedure: infection risk has been approximately 3%, implant
removal required in 1%, lead breakage recorded up to 2.7%. Transient side effects including coughing,
voice changes, hoarseness, headache, nausea, dyspnoea, and neck spasms, occur only at the time of
stimulus delivery and are completely reversible.

Conclusions: The group studied here is of large size with a significant follow-up period. 131 children
have been implanted with VNS for refractory epilepsy at KCH, with approximately 100 available for
study over a mean follow-up period of nearly five years. Focal onset epilepsies predominate in this
group and the majority have learning and behavioural difficulties. The results showed great
improvement in QoL irrespective to remission of seizures. Clinical effectiveness seems higher in
children with short epilepsy history, suggesting a precocious useful role for VNS.
REFERENCES
Helmers S.L., et al. European Journal of Paediatric Neurology, 16 (5) , pp. 449-458. 2012

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