Pharmacophore Model Development

f the
for th Identification
Id tifi ti off Novel
N l
Acetylcholinesterase
y Inhibitors

Ed i Kamau
Edwin K

Dept. Chem.
Dept Chem & Biochem.
Biochem Kennesa
Kennesaw State Uni
University,
ersit Kennesa
Kennesaw, GA
GA, 30144
30144.
Bioengineering & Bioinformatics Summer Institute, Dept. Computational Biology, University of
Pittsburgh, PGH, PA 15260.
 Background
Acetylcholinesterase
‰ Acetylcholinesterase(AChE) is a multi-
functional enzyme found in cholinergic
system
‰ Function:
9 Terminates nerve impulses
p of
acetylcholine origin
9 Promotes production of β-Amyloid
p
protein
‰ Associated with pathogenesis of Alzheimer's
Disease (AD)
9 Impairs cholinergic system—Reduced
amount of acetylcholine
9 Promote formation of Plaques—
Insoluble β-Amyloid protein http://www cnsforum com
http://www.cnsforum.com
 Alzheimer's Disease
‰ Age related chronic neurodegenerative dementia
‰ Affects 20 million people worldwide
‰ Develops in stages—Early to advanced
‰ Available drugs only able to treat certain stage only

‰ Drugs have associated side effects

‰ N d for
Need f therapeutic
th ti measures withith ability
bilit to
t treat
t t allll stages
t
of AD with less or no side effects
 Structure
‰ Model structure of
Torpedo californica
AChE (TcAChE)
‰ Peripheral Anionic
Site (PAS)
( )
¾ Trp 279
‰ Catalytic
y Anionic Site
(CAS)
¾ Trp 84
‰ Lining off aromatic
residues
‰ 54 X
X-ray
ray structures
with bound ligands Xu. et al. Induced fit or preexisting equilibrium dynamics?
2008,17:601-5
 Inhibition
ˆ Binding activity at CAS well understood while that
of PAS still elusive
ˆ PAS site associated with formation of plaques
ˆ Treatment at all stages of AD ineffective due to
drug inhibition only at the CAS site
ˆ Inhibition at both sites is key to effective treatment
ˆ Hence using available generated dual-inhibitors of
TcAChE will g generate more effective inhibitors
 Aims of this Study
9 Develop a molecule-
molecule-derived pharmacophore
model (MOE) using known PAS and CAS
inhibitors

9 Use ppharmacophore
p model to identifyy
structurally diverse active compounds by virtual
screening
 Strategy
(LBDD) (SBDD)
Start TcAChE

Training set  
Top 5 Hits

Pharmacophore 
Model Development
Experimental  
testing (TBD)

Pharmacophore 
Model Validation

3D Database 
screening
Lipinski`s  Rule‐of‐
Five Criteria 

Top 25 Hits
ACTIVE DUAL INHIBITORS
Training set
Pharmacophore Query Generation

‰ Training set
set: 6 compounds
compo nds
‰ Features defined:
™ Hydrogen-bond
H drogen bond acceptor (ACC)
™ Hydrogen-bond donor (DON)
™ Hydrophobic
H d h bi (HYD)
™ Aromatic (ARO)
‰ Conformer
C f generation
ti 4 kcal/mol
k l/ l
 Queries
z 3 Queries generated:

Tolerance
Query Molecule(s) Features a
Radius

ReF_Vol Donepezil 1.74 HYD| ARO| 3Vols

Ref_No _Vol Donepezil 1.4 HYD| ARO

All training
Consensus
HYD| ACC|DON|
set 1.4
ARO
molecules
.
aACC,hydrogen-bond acceptor; DON, hydrogen-bond donor; HYD, hydrophobic, ARO, ring aromatic, Vols,
excluded volumes
 Reference Queries

Reference query without excluded volumes generated using Donepezil the meshed spheres
represent pharmacophoric features .

TRP 279

TRP84

PHE330

Reference query with excluded volumes generated using Donepezil. The meshed spheres are
the pharmacophore features (ARO, HYD). Solid spheres (red, blue and cyan colored) represents excluded
volumes.
 Consensus Query
Bis5 tacrine
Bis10 Hupyridone
Aminoquinoline28
Bw284c51
Decamethonium
Donepezil

Fig 4: Flexible aligned training set showing defined features scheme used to generate the query

Consensus query generated from flexible aligned training set. The spheres are the pharmacophore features
(Aro, Acc, Hyd, Don) that novel pharmacophores were aligned. Molecules matching those features satisfied
the query and were therefore hits
 Validation
TEST SET: 8 active compounds

All 8 molecules satisfied the three queries

Database Preparation & Search
(MOE)
ZINC
‰ ~ 2.8 M total number of compounds
‰ ~ 900,000 lead-like compounds
9 27,491 compounds screened
‰ Created conformation database: 703,313
compoundsd
 Database Search Results and Sorting
Q
Query R fV l
Ref:Vol R f N Vol
Ref:No V l C
Consensus

Number of hits 211 378 329

Sort top 100 100 100 100

Common hits
68

S l ti b
Selection basedd on llowestt rmsd
d 25
value (0.06-0.3 Å)
 Sorted Hits (cont.)

Consensus compounds generated by all queries and their respective RMSD values.
Sorted Hits (2
(2D Structures)
 Virtual Screening
g
ƒ Flexible Docking with Molegro Virtal
Docker
ƒ Full side-chain flexibility

ƒ Cavity detection – dynamically – used during the

docking process

ƒ Improved scoring function – hydrogen bond directionality

taken into account

Table 1: Accuracy of selected docking programs. A binding mode is regarded as

correctly identified if the RMSD (to the native co-crystallized ligand) is less than
p
2.0Å. The dataset consists of the 77 complexes

*Thomsen R, Christensen M H, J Med Chem 2006; 49:3315-3321

Docking Results

Top 5 ZINC compounds docked in

the binding sites of TcAChE
 Docking Results (cont.)

E2020 (Donepezil) and ZINC 3785268 from different view points. Visual of how well the zinc compounds aligned in the binding site
similar to Donepezil
 Conclusion
‰ Using a combination of ligand based and
structure based drug design approaches we
have identified structurally diverse dual inhibitor
candidates for TcAChE

Future
F t Work
W k
‰ Virtually screen the remaining lead-like
lead like
compounds of the ZINC database
‰ Prioritize hits for experimental testing (i.e.
binding free energy; types of interactions in the
active site)
 Acknowledgements
‰ Dr Gabriela Mustata
‰ Dr Jeffry Madura
‰ BBSI Program
‰ NIH & NSF
 References
1. Lin A. Overview of p
pharmacophore
p Application
pp
in MOE.. Chemica Computing group
MOE group..
http:://www
http //www..chemcomp.
chemcomp.com/journal /ph
/ph44.htm
2. Irwin and Stoichet
Stoichet.. J. Chem.
Chem. Info.
Info. Model.
Model.
(2005
2005)); 45
45((1): 177-
177-182
3. Molegro Virtual Docker.
Docker. www.
www.molegro.
molegro.com
4. Xu. et al.
Xu. al. induced fit or preexisting equilibrium
dynamics? Protein Science (2008
2008),), 17
17:: 601-
601-5