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PMCIdRS
Présentation de l’aspect
moléculaire de la découverte du
médicament
Jean A. Boutin
Gilles Ferry, Olivier Nosjean
PMCIdRS
700 to 1000 m€
Up to 15 000 patients 2~50 m€
~50 people
PMCIdRS
Un élément peu connu: l’attrition
PMCIdRS
Target Identification
• Receptor
• Chanel
• Enzyme
• Protein-Protein
interaction
A
C
T
I
O
N
P
L
A
N
• Molecular modelling
• Screening
Drug discovery process
IND
Package
First
administration
to Man
C
L
I
N
I
C
A
L
C
A
N
D
I
D
A
T
E
L
E
A
D
Lead optimisation
• Medicinal Chemistry
• Pharmacology
• Modelisation
• ADMET
• Patent
•Molecular and cellular
Pharmacology
Taget validation Optimisation Stade A Stade B
Target/program
choice
High throughput
Screening:
chemicals
Drug
choice
Clinical stages
PMCIdRS
Diversity
chemistry
Compound
library
Gene
Cell line
Protein
Assay
development
Assay
automation
HTS
Hit triage
Orthogonal
assays
Biophysics
Structural
biology
Molecular
modeling
12 – 30 months for a discovery program
Les différents secteurs de notre organisation
PMCIdRS
Chemical neighbour
selection and tests
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
-150 -80 -55 -30 -5 20 45 70 95 120 165
Score 2
Score 1
Score 0
Score -1
Score -2
Seuil manuel
Produits
Assay development
HEK-GLP1 clone 2
lyse : 1h35 (2431)
-14 -13 -12 -11 -10 -9-20
0
20
40
60
80
100
GLP1 (2000cells)
GLP1 (4000cells)
GLP1 (6000cells)
GLP1 (10000cells)
Log [ GLP1 7-36 ] (M)
Eff
et
ag
on
iste
(% f
ull
ag
o)
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000
9.000
0 8 16 24 32 40 48
Sig
nal
Signal total :
HBSS + BSA
0.1%
Signal non
spécifique :
GLP1(7-36)a
10nM
1 2 3 4 5 6
High throughput screening in its context
Workplan:
Definition, needs
1K
2K
3K4K
5K
6K
7K0
luc+
Neom
ycin
Ampicil l inCMV prom
otor
SV40 promotor
SV
40
pA
pcDNA3.1(+)/LUC+
Cells and/or protein
production
Compound plates
Screnning campaign
NH
OH
OH
NH
O
CH3
CH3
CH3
ClH
OH
NH NH
OH
O
O
diastereoisomere 1 racemique
N
N
N
CH3
N
O
CH3
CH3
O
N
CH3 OH
OH
OH
OH
OOH
O
Hits list
Orthogonal test:
characterization of the main hits.
Biophysical qualification
- 1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
R U
Ré
po
ns
e
T e m p s e n s
- 1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
R U
Ré
po
ns
e
T e m p s e n s
P r o té in e s e u le1 .5 6 µ M3 .1 2 µ M6 .2 5 µ M1 2 .5 µ M2 5 µ M5 0 µ M1 0 0 µ M
- 6 .0 -5 . 5 - 5 .0 - 4 .5 -4 . 0 - 3 .5- 1 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0S 7 1 6 1 7 - 1
B O T T O M
T O P
L O G E C 5 0
H IL L S L O P E
E C 5 0
0 .0
1 0 0 . 0
- 4 . 8 9 3
1 .1 8 9
1 .2 8 1 e - 0 0 5
lo g [ S 7 1 6 1 7 -1 ] M
% in
hib
itio
n
Biophysics NMR, X-rays
Not-a-real-compound
Biochemical/cellular assay
Chemical optimisation
(Med Chem)
Step 1Production of the
target
PMCIdRS
L’Homme
Willy Ronis
Access to biological material from Human origin
Tissues Cells Genome
primo-cultures/cancer cells/
Stem cells
biopsies ADN / mARN
Production
Of human proteins
Functionnal tests
validation & screen
PMCIdRS
Chemical neighbour
selection and tests
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
-150 -80 -55 -30 -5 20 45 70 95 120 165
Score 2
Score 1
Score 0
Score -1
Score -2
Seuil manuel
Produits
Assay development
HEK-GLP1 clone 2
lyse : 1h35 (2431)
-14 -13 -12 -11 -10 -9-20
0
20
40
60
80
100
GLP1 (2000cells)
GLP1 (4000cells)
GLP1 (6000cells)
GLP1 (10000cells)
Log [ GLP1 7-36 ] (M)
Eff
et
ag
on
iste
(% f
ull
ag
o)
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000
9.000
0 8 16 24 32 40 48
Sig
nal
Signal total :
HBSS + BSA
0.1%
Signal non
spécifique :
GLP1(7-36)a
10nM
1 2 3 4 5 6
High throughput screening in its context
Workplan:
Definition, needs
1K
2K
3K4K
5K
6K
7K0
luc+
Neom
ycin
Ampicil l inCMV prom
otor
SV40 promotor
SV
40
pA
pcDNA3.1(+)/LUC+
Cells and/or protein
production
Compound plates
Screnning campaign
NH
OH
OH
NH
O
CH3
CH3
CH3
ClH
OH
NH NH
OH
O
O
diastereoisomere 1 racemique
N
N
N
CH3
N
O
CH3
CH3
O
N
CH3 OH
OH
OH
OH
OOH
O
Hits list
Orthogonal test:
characterization of the main hits.
Biophysical qualification
- 1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
R U
Ré
po
ns
e
T e m p s e n s
- 1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
R U
Ré
po
ns
e
T e m p s e n s
P r o té in e s e u le1 .5 6 µ M3 .1 2 µ M6 .2 5 µ M1 2 .5 µ M2 5 µ M5 0 µ M1 0 0 µ M
- 6 .0 -5 . 5 - 5 .0 - 4 .5 -4 . 0 - 3 .5- 1 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0S 7 1 6 1 7 - 1
B O T T O M
T O P
L O G E C 5 0
H IL L S L O P E
E C 5 0
0 .0
1 0 0 . 0
- 4 . 8 9 3
1 .1 8 9
1 .2 8 1 e - 0 0 5
lo g [ S 7 1 6 1 7 -1 ] M
% in
hib
itio
n
Biophysics NMR, X-rays
Not-a-real-compound
Biochemical/cellular assay
Chemical optimisation
(Med Chem)
Step 2
Test assesment
PMCIdRS
Flowchart of HTSBasic
Project Launch
Planning
Assay design
Assay development
Automation
HTSSecondary/Counter screens
Data analysis
IC50 of actives ICR data export
Complementary studies
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS HTS planning
Resource scheduling
Planning
Assay design
Assaydevelopment
Automation
IC50
Complement.studies
Partnership /Subcontract.
HTS
Data analysis
Excel spreadsheet for planning of team members agenda
Next slot: week 16
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
Update: week 27
PMCIdRS Target classes
Membrane Proteins
TyrK-R
Ionchannels
Soluble Enzymes
Intracellular Proteins
Transporters
GPCR
SecretedProteins
Nuclear Receptors
Protein families
Planning
Assay design
Assaydevelopment
Automation
IC50
Complement.studies
Partnership /Subcontract.
HTS
Data analysis
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS
Flowchart of HTSInteractions with IdRS actors
Planning
Assay design
Assay development
Automation
HTSSecondary/Counter screens
Data analysis
Methods/materialsTransfert
(Pharmacology division)
Project Launch
IC50 of actives ICR data export
Complementary studies
PA2 protocols(GIS/IT)
DANA template(GIS/IT)
Compound plates(BdP)
Compound plates(BdP)
Biological materials(BM/BC; Protein chemistry)
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS Assay development validation steps
"Check list"
Consumables (culture supports, assay plates) …………………………………………………………………………………….
Data acquisition parameters (wavelength, sampling...) ………………………………………............……………………
Optimal reagent concentrations (cells, proteins, probes, substrates, …) ………………………………………………..
Kinetics (pre-incubation, incubation, reading, …) ………………………………………………………………………………..
Solvent tolerance........………………………………………………………………………………………………………………………
Pharmacology of validation……………………………………………………………………………………………………………….
Automation………………………………………………………………………………………………………………………………………
Strength (Z’) …………………………………………………………………………………………………………………………………..
4
5
6
7
8
4 5 6 7 8
pIC50 (FP)
pIC
50
(fu
nc
tio
na
l)
Z' = 0.63 à 6H d' incubation
0
20
40
60
80
100
120
0 10 20 30 40 50
Alexa Fluor 488 - incubation 6H
0 1 2 3 4 5 6 7 8 9 10 11
0102030405060708090
100110120
BTX 10nM
+Epibatidine 100µM
DMSO %
mP
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
T ime ( h)
MIN
MAX
HEK hGPR119 clone 13
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4-40
-20
0
20
40
60
80
100
EC50
w /o BLR 7.320e-009
EC50
BLR 1 nM 1.483e-008
EC50
BLR 3 nM 1.084e-008
EC50
BLR 10 nM 1.605e-008
EC50
BLR 30 nM 2.740e-008
EC50
BLR 100 nM 2.638e-008
EC50
BLR 300 nM 8.258e-009
EC50
BLR 1000 nM 3.602e-008
Log([BLR100])
% E
ffect
Assay design
Assaydevelopment
Automation
IC50
Complement.studies
Partnership /Subcontract.
HTS
Data analysis
Planning
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS
Integration contribution to assay robustness
Cell seeding
Incubation 24hrs @ 37°C, CO2
Switch from culture medium to
DMEM buffer (40 µL)
DW 384 – Probe
50 µM (10X)
Compound plate
(10X)
5 µL
5 µL
Incubation 20 min @ 37°C, CO2
Incubation 60 min @ 37°C, CO2
Double wavelength data acquisition
Washes (x3)
Robotics_v1 Robotics_v2
Reagent
dispenser
Plate
washer
CybiWell
CybiWell
Incubator
Plate
washer
Incubator
Plate reader
Reagent
dispenser
Plate reader
FDSS
FDSS
Incubator
Plate
washer
Incubator
Plate
washer
Inte
gra
ted
TH
ER
MO
-HA
MA
MA
TS
U w
ork
sta
tion
Automation (1) Eg. ClC7 (PVS)
Assay design
Assaydevelopment
Automation
IC50
Complement.studies
Partnership /Subcontract.
HTS
Data analysis
Planning
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS
0
500
1000
1500
2000
2500
3000
3500
-150 -80 -55 -30 -5 20 45 70 95 120 165
Score 2
Score 1
Score 0
Score -1
Score -2
Seuil manuel
Produits
Inhib (%)Hit rate = 4%
Robotics_v2
Automation (2)
lysosome acidification
0
500
1000
1500
2000
2500
3000
3500
4000
-150 -80 -55 -30 -5 20 45 70 95 120 165
Score 2
Score 1
Score 0
Score -1
Score -2
Seuil manuel
Produits
Inhib (%)
« Hit rate » = 14%
Robotics_v1
Assay design
Assaydevelopment
Automation
IC50
Complement.studies
Partnership /Subcontract.
HTS
Data analysis
When handling cells on a lengthy protocol, assay automation increases data quality
Planning
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS Data processing
Integrated process
Channel
DANA
Activity Base
Pipeline Pilot
GIS / IT Templates
Compoundlibrary database
Study declaration
Experimental procedure declaration
Data analysis
Data correlation
Campaign analysisPipeline Pilot
IdRS database
Present process
Channel
SimplicityVersatility
Decision desk
+
or
or
GIS / IT Templates
Assay design
Assaydevelopment
Automation
IC50
Complement.studies
Partnership /Subcontract.
HTS
Data analysis Excel/uHTE/XE
ICR
Planning
ICR
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS
Campaign analysis
Eg. Cancer target
Inhibition of Protein homodimerisation
Assay design
Assaydevelopment
Automation
IC50
Complement.studies
Partnership /Subcontract.
HTS
Data analysis
30000
25000
20000
15000
10000
5000
00 25 50-25-
50 Inhibition (%)
Num
ber
of com
pounds
99547 cpds @ 10 µM or 10 µg/mL = 632 assay plates (n=2)
Selection of 666 actives
Assay with good statistics:
narrow Gaussian dispatch of activitiesGood n1 / n2 reproducibility
548 available @ BdP
Evaluation of reproducibility and Gaussian distribution of activities => assay quality
Definition of a threshold for active compounds
Planning
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS
Applies to ACTIVES from HTS and small size (<700) focused libraries:
Determination of IC50s
0
25
50
75
100
0 1 2 3 4 5 6 7 8 9 10
pIC50
Nu
mb
er
of
co
mp
ou
nd
s
270
Most compounds are active in the micromolar range, depending on the target class
ACTIVES HITS
IC50 of actives
Assay design
Assaydevelopment
Automation
IC50
Complement.studies
Partnership /Subcontract.
HTS
Data analysis
Planning
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS
Complementary studiesDevelopment of complementary assays
Eg. GPR ZZZ
[3H]-BLR100 bindingKi determination of validated compounds
Calcium mobilization AssayInsulin secretion potency of validated compounds
GsAC+
ATP
cAMP+ Pi
[3H]-BLR100
GPR ZZZ
+
Ca2+
+
KATP VDC
MIN6c4 cells
cAMP dosage HTS at CEREP
Assay design
Assaydevelopment
Automation
IC50
Complement.studies
Partnership /Subcontract.
HTS
Data analysis
CRE-Luc
Reporter gene assay HTS at SIMM
Planning
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS
Flowchart of HTSScreening externalization
Outsourcingof
HTS
Planning
Assay design
Assay development
Automation
HTS
PA2 protocols(GIS/IT)
DANA template(GIS/IT)
Compound plates(BdP)
Compound plates(BdP)
Biological materials(BM/BC; Protein chemistry)
Transferttechnics/materials
(Pharmacology division)
Project Launch
IC50 of actives
Complementary studies
Eg. GPRZZZ
CEREP: Integration of the externalization in
our process
SIMM: Fully independent
externalization
Assay development and HTS team – SdP PMC – 09/12/2009PMCIdRS
PMCIdRS
Chemical neighbour
selection and tests
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
-150 -80 -55 -30 -5 20 45 70 95 120 165
Score 2
Score 1
Score 0
Score -1
Score -2
Seuil manuel
Produits
Assay development
HEK-GLP1 clone 2
lyse : 1h35 (2431)
-14 -13 -12 -11 -10 -9-20
0
20
40
60
80
100
GLP1 (2000cells)
GLP1 (4000cells)
GLP1 (6000cells)
GLP1 (10000cells)
Log [ GLP1 7-36 ] (M)
Eff
et
ag
on
iste
(% f
ull
ag
o)
0.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000
9.000
0 8 16 24 32 40 48
Sig
nal
Signal total :
HBSS + BSA
0.1%
Signal non
spécifique :
GLP1(7-36)a
10nM
1 2 3 4 5 6
High throughput screening in its context
Workplan:
Definition, needs
1K
2K
3K4K
5K
6K
7K0
luc+
Neom
ycin
Ampicil l inCMV prom
otor
SV40 promotor
SV
40
pA
pcDNA3.1(+)/LUC+
Cells and/or protein
production
Compound plates
Screnning campaign
NH
OH
OH
NH
O
CH3
CH3
CH3
ClH
OH
NH NH
OH
O
O
diastereoisomere 1 racemique
N
N
N
CH3
N
O
CH3
CH3
O
N
CH3 OH
OH
OH
OH
OOH
O
Hits list
Orthogonal test:
characterization of the main hits.
Biophysical qualification
- 1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
R U
Ré
po
ns
e
T e m p s e n s
- 1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
1 0 0 0
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
R U
Ré
po
ns
e
T e m p s e n s
P r o té in e s e u le1 .5 6 µ M3 .1 2 µ M6 .2 5 µ M1 2 .5 µ M2 5 µ M5 0 µ M1 0 0 µ M
- 6 .0 -5 . 5 - 5 .0 - 4 .5 -4 . 0 - 3 .5- 1 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0S 7 1 6 1 7 - 1
B O T T O M
T O P
L O G E C 5 0
H IL L S L O P E
E C 5 0
0 .0
1 0 0 . 0
- 4 . 8 9 3
1 .1 8 9
1 .2 8 1 e - 0 0 5
lo g [ S 7 1 6 1 7 -1 ] M
% in
hib
itio
n
Biophysics NMR, X-rays
Not-a-real-compound
Biochemical/cellular assay
Chemical optimisation
(Med Chem)
« Last » StepHit qualification
PMCIdRS From target to chemical lead
Screening
CompoundsS. BERGER
Hit selection
From hit to lead
Proteins
Antibodies Analytocal
techniques
Structural
Biology
SubcontractingN. MOULHARAT
Purification
TargetM. ANTOINE
Qualification
AActivity
PMCIdRS Using Biophysics to build up a drug
Kinase inhibitor, anticancer agent
DG = -RTln(1/KD) = DH -TDS
Signature thermodynamique de la mérioline
-20000
-15000
-10000
-5000
0
5000
ca
lori
me
/mo
le
DG
DH
-TDS
Enthalpy-driven interaction
0.0 0.5 1.0 1.5 2.0
-16.00
-14.00
-12.00
-10.00
-8.00
-6.00
-4.00
-2.00
0.00
2.00-0.80
-0.70
-0.60
-0.50
-0.40
-0.30
-0.20
-0.10
0.00
0.100 30 60 90 120 150
Time (min)
µcal/sec
Data: Data1_NDH
Model: OneSites
Chi^2/DoF = 8.517E4
N 0.899 ±0.00330 Sites
K 7.95E7 ±1.36E7 M-1
DH -1.447E4 ±102.6 cal/mol
DS -12.4 cal/mol/deg
Molar Ratio
KC
al/M
ole
of
Inje
cta
nt
ITC* Isothermal Titration Calorimetry
Attraction and repulsion forceslead
to thermodynamical signature of
the compound
Repulsion due
to solvants
Hydrophobicity
-30
-25
-20
-15
-10
-5
0
5
10
15
20
-30 -25 -20 -15 -10 -5 0 5 10 15 20
VVI IV
I
IIIII
Entropy
En
tha
lpy
III
S 51175-1
S 52319-1S 52312-1
AKY33
“Thermodynamic Optimal Plot”
VVI IV
I
II
S 51175-1
S 52319-1S 52312-1
AKY33
TDS
DH
Attraction due
to hydrogen
bonds
Target
Ligand
PMCIdRS
Cristallogenesis
of the target
Search for a new kinase inhibitor
Expression in E.
Coli
12 constructions
PurificationProduction of crystals
350 conditions
Diffraction at
Synchrotron Data
analyses
Structure finale Resolution
Atomic coordonates
PMCIdRS
Compound A, inhibiteur de la Glucokinase
Spécificité d’une protéase.
Sun et al, Prot Sci 2010 19:2240
-> accès aux informations clés de
la relation entre un inhibiteur ( ) ou un
substrat ( ) et sa cible.
Possibilité de modulation de ces interactions
PMCIdRS
Concluons????
1/ L’enzymologie représente une petite part de la chimie des protéines qui
représente une petite part de la recherche de découverte de médicaments
qui représente une petite part de la mise au point d’une nouvelle
substance médicamenteuse
2/ Les enzymes forment la deuxième ou la troisième famille de cible des médicaments
(derrière les récepteurs à 7TM et les canaux)
3/ L’enzymologie permet de comprendre l’activité enzymatique. Elle n’est ‘rien’ sans le
clonage, l’expression, la purification, la biologie structurale, la qualification de la protéine
purifiée, la possibilité de faire des mutants, etc..
4/ Elle nécessité aussi de chercher de nouvelles approches à la mesure de l’activité catalytique:
RMN, stopflow, spectrométrie de masse, etc..
5/ La chimie, en apportant des variations ponctuelles des substrats, co-substrats et inhibiteurs
des enzymes est aussi capitale à la compréhension (et donc au contrôle) de l’activité
catalytique. (ergo: ne pas négliger la compréhension simplifiée de la chimie)
6/ ça coûte…. cher.
PMCIdRS
We accept all missions
even those seemingly impossible
