Elucidação Estrutural e

Metabolómica na Indústria
Farmacêutica
Brian J Goodfellow

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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The Pharmaceutical Industry

Dream
Health is our dream. Because we believe that
dreams should be pursued, we are researching and
developing new drugs to offer a longer and better
life. In Portugal and worldwide. Caring for your
Health. And there are dreams that come true...

We have a challenging and inspiring mission: to

GlaxoSmithKline improve the quality of human life by enabling
people to do more, feel better and live longer.

As a global biopharmaceutical
company, our activities touch many
people’s lives

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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The Pharmaceutical Industry

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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How do we get new drugs ?
What is a drug molecule?

๏ A drug molecule has one or more functional groups positioned in 3D space

on a structural framework that enables the molecule to bind to a targeted
biological macromolecule, the receptor

+ biological response

“drug-like” molecule “druggable” target

Properties: low Mwt, not too Properties: usually protein, leads to

lipophilic, not too hydrophilic, needs biological response, does not cause
functional groups toxicity

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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New Drugs - recent trends

Reasons:
๏ fundamentally difficult
๏ high failure rate (4% of compounds become a drug and of these 89 % fail
clinical trials)
๏ expensive > 800 million dollars and can take 15 years
๏ cost minimization in industry leads to fewer drugs
๏ Mainly based on high throughput screening (HTS) or structure based drug
design (SBDD)
Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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New Drugs - SBDD and HTS
๏ Requires 3D structure of protein target to design drugs to bind to active
site, therefore need rapid methods for structure determination of targets

http://www.kubinyi.de/lectures.html

๏ Corporate databases (100,000s of compounds) are screened automatically

by robots - identify high-affinity binders but produce false leads - need to
identify lead compounds that interact with the target in a biologically
relevant mechanism
Chemical Microarrays (Graffinity, Heidelberg)

! ca. 100,000 fragments linked onto chip

! parallel detection of weak, specific interactions
! confirmation of hits by functional testing and X-ray/NMR

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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New Drugs - How can NMR help ?
The diversity of NMR enables the technique
to be applied at multiple stages along the
drug discovery pathway

NMR is being used for:

๏ protein structure determination

๏ lead discovery and optimization

๏ evaluating in vivo selectivity and

efficacy

๏ analyzing drug toxicity profiles and

identifying new drug discovery
targets

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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New Drugs - How can NMR help ?
NMR can:

๏ rapidly determine protein and protein-ligand structures

๏ efficiently screen fragment-based libraries to identify biological relevant

ligand interactions and identify new therapeutic targets

๏ monitor changes in the metabolome from biofluids and cell lysates to

explore in vivo drug activity

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Protein structure determination - NMR

NOE

H 5Å H

C C
1 Å = 1x10-10 m

Input: intercity distance table Output: map of USA

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Protein structure determination - NMR

NOE

H 5Å H

C C
1 Å = 1x10-10 m

100 VAL H 100 VAL HB 3.32 #peak 288 #SUP 0.96 #QF 0.96
21 LEU H 21 LEU HG 3.97 #peak 289 #SUP 0.98 #QF 0.93
189 LYS H 189 LYS HB2 3.71 #peak 290 #SUP 0.86 #QF 0.86
100 VAL H 100 VAL QG2 3.97 #peak 292 #SUP 1.00
188 VAL QG1 189 LYS H 4.32 #peak 293 #SUP 0.84 #QF 0.84
45 GLU H 46 VAL H 4.60 #peak 297 #SUP 0.96 #QF 0.96
131 TYR QD 189 LYS H 4.55 #peak 300 #SUP 0.79 #QF 0.79
45 GLU H 45 GLU QG 3.96 #peak 321 #SUP 0.98 #QF 0.98
45 GLU H 45 GLU HB2 3.30 #peak 323 #SUP 0.91 #QF 0.91
45 GLU H 45 GLU HB3 3.30 #peak 324 #SUP 0.95 #QF 0.95
71 ASP H 114 ALA H 3.83 #peak 330 #SUP 0.99 #QF 0.99
72 LYS H 114 ALA H 4.37 #peak 337 #SUP 0.82 #QF 0.82
138 TYR QD 139 ALA H 4.10 #peak 339 #SUP 0.96 #QF 0.96
32 GLU HB2 33 GLU H 4.52 #peak 467 #SUP 0.94 #QF 0.94
23 THR QG2 33 GLU H 3.83 #peak 470 #SUP 0.31 #QF 0.31
99 LYS HB3 101 TRP HE1 5.13 #peak 482 #SUP 0.44 #QF 0.44
84 PHE HB2 85 ALA H 4.01 #peak 493 #SUP 0.98 #QF 0.98
85 ALA H 101 TRP HD1 4.05 #peak 497 #SUP 0.99 #QF 0.99
85 ALA H 168 TYR QD 4.95 #peak 499 #SUP 0.94 #QF 0.94
………..

Input: 1H-1H distance table Output: structure of protein

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Protein structure determination - NMR
๏ But to obtain a structure we need to
identify every peak
๏ get a ca. peak from each 1H in the
protein
๏ but 150 residue protein has ~1000 1H
signals methyl

๏ incomplete dispersion of frequencies

๏ need to separate resonances in
another frequency dimension
aliphatic

many peaks overlapped

aromatic
amide

Lehninger, Nelson & Cox, 3rd ed.

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Protein structure determination - 2D NMR

1D
Fourier
Transform

2D
2D Fourier
Transform
t1 !M

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Protein structure by NMR - going to nD

๏ faster structure determination needs higher dimensions (and isotopic

enrichment of the protein)

๏ But a 5D experiment for a 30 sec FID would take (128x32x16x8x4).......

around 2 years !

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Protein structure by NMR - example using up to 3D
๏ a mathematical
p22HBP a 22 kDa proteincalculation that converts a table of
distances into a map or structure
๏ Used 1851 distances and took ca. 1 year from
start to finish

Backbone atoms of the final

family of 20 conformers 173-180

Dias et al. J Biol Chem (2006) vol. 281 (42) pp. 31553-31561
Dias et al. J Biomol NMR (2005) vol. 32 (4) pp. 338-338

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Protein structure by NMR - speed up data collection
๏ Robotic can be used for automated production of protein samples for NMR
and x-ray

๏ New NMR pulse sequences shorten the time required to collect a set of
NMR spectra: automated projection spectroscopy (APSY); G-matrix Fourier
transform NMR (GFT-NMR) are two examples

๏ Dimensionality of traditional multi-dimensional triple resonance experiments

is reduced = shorter acquisition time. Works by joint sampling of multiple
chemical shifts in a single indirect dimension

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Protein structure by NMR - speed up data collection
๏ Robotic can be used for automated production of protein samples for NMR
and x-ray

๏ New NMR pulse sequences shorten the time required to collect a set of
NMR spectra: automated projection spectroscopy (APSY); G-matrix Fourier
transform NMR (GFT-NMR) are two examples

๏ Dimensionality of traditional multi-dimensional triple resonance experiments

is reduced = shorter acquisition time. Works by joint sampling of multiple
chemical shifts in a single indirect dimension

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Protein structure by NMR - Structural genomics

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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New Drugs - How can NMR help ?
NMR can:

๏ rapidly determine protein and protein-ligand structures

๏ efficiently screen fragment-based libraries to identify biological relevant

ligand interactions and identify new therapeutic targets

๏ monitor changes in the metabolome from biofluids and cell lysates to

explore in vivo drug activity

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Ligand binding by NMR

๏ NMR techniques for ligand binding

๏ where/how does the ligand bind ? use the fact that the ligand, when
bound, has properties similar to the
large target or use magnetization
๏ which ligand of a series binds ? transfer between target and ligand

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Ligand binding by NMR - which ligand(s) binds ?

๏ Saturation Transfer Difference is

based on a transfer of
magnetization from the protein to
saturate baseline
the bound molecule

saturate protein

difference
[ligand] = 1 mM
[protein] = 35μM
expt time 1-4 hours
STD experiment for p38 kinase domain

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Ligand binding by NMR - which ligand(s) binds ?
INPHARMA [ligand] = 0.1-0.5 mM
[protein] = 25 μM
expt time 20 hours

๏ LA binds so that HA to HT transfer

occurs
๏ LA dissociates and LB enters
๏ HT to HB transfer now occurs
๏ Result is a NOE peak between HA and
HB mediated by HT

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Ligand binding by NMR - which ligand(s) binds ?
๏ Diffusion Ordered SpectroscopY (DOSY)

Cromatografia em camada fina TLC

DOSY
Chromatograpy, TLC

slow
lento
Polarity

Diffusion
Polaridade Difusão

rápido
fast " 1H

Desvio químico
chemical shift

Adapted from EJ Cabrita, Presentation, UA 2008

Edição espectral
Spectra porby
editing coeficiente de difusão
diffusion constant

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Ligand binding by NMR - which ligand(s) binds ?

slow
lento

Host (H)
Diffusion
Difusão

Guest (G)
rápido
fast

slow
lento

H+G HG
Diffusion
Difusão

rápido
fast

chemical shift
Desvio químico Adapted from EJ Cabrita, Presentation, UA 2008

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Ligand binding by NMR - where/how does the ligand bind ?
๏ NMR chemical shift mapping
identifies ligand binding sites

๏ binding site obtained rapidly and

the process can be automated
(robotic sample-changers and flow-
probes)

๏ HSQC = Heteronuclear Single

Quantum Correlation
๏ one peak for each NH gives a
“fingerprint” of the protein
๏ need to label the protein with 15N
and or 13C
๏ fast experiment 5-20 mins

Ileal bile acid binding protein, 14 kDa

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Ligand binding by NMR - where/how does the ligand bind ?

[ligand] = 0.5-1 mM
[protein] = 0.5-1 mM
expt time 1-4 hours

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Ligand binding by NMR - where/how does the ligand bind ?

hydrophobic interaction PPIX-induced shift perturbations

between p22HBP and heme small negative shifts
large negative shifts
small positive shifts (0.05-0.1ppm)
large positive shifts (>0.1ppm)

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Summary of some NMR methods for binding studies

Approach Observe Use Description

1º screening
Perturbs chem shifts of
Shift mapping target Hit validation
target near binding site
Binding site
Identifies compounds that
1º screening bind weakly.
STD ligand
Hit validation Bulid up curves identify
FGs
Identifies compounds that
waterLOGSY ligand 1º screening bind via water mediated
NOEs

Mesures differences in
1º screening
DOSY ligand diffusion rates for ligands
Hit validation
in bound versus free state

Detects protein mediated

Fragment based DD
SAR by ILOE ligand-to-ligand ligand-ligand interactions
Lead optimization
(occupying adjacent sites)
Detects protein mediated
ligand-ligand interactions
INPHARMA ligand-to-ligand Lead characterization
(competition for same
site)

Eluciadçao Estrutural e Metabolomica na Indústria Farmacêutica

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New Drugs - How can NMR help ?
NMR can:

๏ rapidly determine protein and protein-ligand structures

๏ efficiently screen fragment-based libraries to identify biological relevant

ligand interactions and identify new therapeutic targets

๏ monitor changes in the metabolome from biofluids and cell lysates to

explore in vivo drug activity

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Metabolomics by NMR
๏ Basically, a 1D 1H NMR spectrum of a lysed cell or biofluid captures its
metabolic state (NMR only observes the most abundant metabolites)

Genomics (DNA)
25,000 genes
Transcriptomics (RNA)
100,000 mRNAs

Proteomics (proteins)
Proteins
1,000,000 proteins

Metabolomics (metabolites)
2,500 metabolites (small
molecules)

The metabolome reflects the

general cellular state

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Metabolomics by NMR
Metabolomics definition

“the quantitative measurement of the dynamic multiparametric metabolic

response of living systems to pathophysiological stimuli or genetic
modification” (Imperial College, London, 1999)

Non-perturbed Perturbed
system system
- Disease
- Xenobiotics
- Genetic modification
- Diet
- Environmental effects Metabolic adjustments
to mantain homeostasis
๏ Changes in metabolic profiles

Fingerprint of NMR of:

Biofluids (plasma, urine, etc.)
biochemical perturbation Tissues
Cells

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Metabolomics by NMR - multivariate analysis
๏ Comparing 1 or 2 spectra by eye is easy - 40 is harder…..
๏ Use multivariate analysis (PCA) to identify differences between spectra
๏ Always need many spectra to filter out external/experimental variables

some samples have a lot more

trehaolse than others

other differences harder to see!

expt time = 5 mins x Nº samples

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Metabolomics by NMR - multivariate analysis
๏ Scores plot identifies grouping of samples - similar spectra group together
๏ Loadings plot identifies differences between grouped spectra
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!"#"(% !"#"*% !"#")% !"#"$% "% "#"$% "#")% "#"*% "#"(% cells frozen in N2(l) then extracted
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freeze dried and buffer added

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expt time = 5 mins x Nº samples ,"#$ +"#$ *"#$ )"#$ #"#$ ("#$ '"#$ &"#$ %"#$ !"#$

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Metabolomics by NMR - in vivo models

cells frozen in N2(l) then

ground and buffer added

๏ The effect of the drug 8-

azaxanthine on the target urate
oxidase can be seen

๏ The drug specifically inhibits

urate oxidase

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Metabolomics by NMR - in vivo, biofluids
phosphate buffer added to urine
Rat urine

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Metabolomics by NMR - in vivo, toxicity

The COMET project: 5 pharmaceutical companies + Imperial College (UK)

Approach
๏ Animals (rodents) subjected to treatments generating typical toxicological
lesions and their biofluids analysed by NMR.

Achievements
๏ Generation of a comprehensive database of NMR spectra of biofluids
treated with various toxicants (147 studies; >30000 samples)
๏ Predictive screening methodologies that both detect abnormal metabolic
states and classify target organ toxicity (mainly liver and kidney)

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Metabolomics by NMR - in vivo, know your subject

๏ Differences in metabolite
profiles for Aito Town,
Chicago, and Guangxi
population revealed
differences related to
genetic, dietary, and gut
microbial factors

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Metabolomics by NMR - disease

๏ Can distinguish malformations from controls

in diffusion edited spectra of AF

๏ 22 metabolites were found to change

significantly in the AF of malformed fetuses,
compared to controls, together with four
unassigned resonances

๏ Results confirmed that malformed fetuses

suffer altered energy metabolism and kidney
underdevelopment

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Metabolomics by NMR - the future
๏ Personalized medicine Disease Co-/pre-
administration of
Gut microbiota other drugs

Nutritional
status

Genotype Metabolic
Phenotype
Ph
s ar
ic m
om ac
gen o-
m
- et
a co ab
m on
har om
P ics

Prediction of how different individuals will

respond to a particular drug/dose combination

Elucidação Estrutural e Metabolómica na Indústria Farmacêutica

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Conclusion
The diversity of NMR enables the technique to be applied at multiple stages
along the drug discovery pathway

๏ lead discovery and optimization helped:

! by rapidly determining protein and protein-ligand structures
! efficiently screening fragment-based libraries to identify biological
relevant ligand interactions and new therapeutic targets
๏ Evaluation of in vivo selectivity and efficacy helped by:
! monitoring changes in the metabolome from biofluids and cell lysates to
explore in vivo drug activity and by analyzing drug toxicity profiles and
identifying new drug discovery targets

๏ Protein structure (UAveiro, ITQB, FCT-UNL, UCoimbra)

๏ Ligand binding (UAveiro, ITQB, FCT-UNL, UCoimbra)

๏ Metabonomics (UAveiro - diseases)

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