Challenges in Fragment Based Drug Discovery For Protein Kinases 2013

Challenges
in Fragment Based
Drug Discovery for
Protein Kinases
Stephen K. Burley, M.D., D.Phil.
Center for Integra@ve Proteomics Research
Department of Chemistry and Chemical Biology
Rutgers, The State University of New Jersey
Cancer Ins@tute of New Jersey

February 7th 2013
Outline
Presenta@on Goals and Background Informa@on
Useful Concepts/Tools for Drug Discovery Teams
Introduc@on to Fragment-Based Drug Discovery
FBDD/SBDD Case Study: MET Inhibitor
Non-Confidential Presentation
Copyright 2012 Stephen K. Burley M.D.
Presenta@on Goals
Understand some of the challenges in small-
molecule drug discovery
Understand some of the advantages oered by
fragment-based drug discovery
Appreciate the possibility of unforeseeable
piValls
Distinct Target
Hypotheses
Molecular

Drug Discovery Strategies

Biochemical
Fragment
Drug Discovery
Target Directed
Screening
Molecules built
for purpose
Repurpose/modify
existing molecules
HT Crystallography
SPR
HDX
Fragment libraries
High conc. assays
Gene family platforms

Diversity/iterative screening (HTS)
Compound libraries
Computational models/informatics
Structural Biology
Cellular and biochemical assays
Cellular
Biological Systems
Hypotheses
In Vivo
Phenotypic
Drug Discovery
Uncover molecule signatures
High Content Imaging

Advanced informatics
Alternative diversity
Advanced cellular assays
Stem cells
SCORE
Zebra fish
In vivo imaging
Causes of AWri@on in Pharma
How can MedChemists help address ~1/3 of Pharma Attrition?

Kola and Landis(2004)
Statement of the Problem

# CNOF-containing Cpds with MW<500 ~ 1060
Subset with Pharmacologic Activity ~ <1026
Subset with Good ADMET Properties ~ 10??

Lipinski et al. (1997) Poor Absorp@on or Permeability more likely if
# H-bond don>5, # H-bond acc>10, MW>500, cLogP>5
Broad Adop@on of the Rule of 5
Medicinal Chemists need to bias designGreen Subset
Hydrophobic Eect
G = H - T S
+
Ligand displacement of par@ally ordered waters from a

hydrophobic surface feature is favorable!
Lipophilicity does contribute to target binding
Too much lipophilicityUnwanted o-target binding
Why Lipophilicity MaWers (cLogP<3)!

Median cLogP versus Promiscuity 2,133 Cpds in 200 Cerep
assays
Promiscuity = # Cpds with >30%
inhibi@on at [10 M]

Sigmoidal rela@onship (r = 0.84)

cLogP<3 more favorable!
Leeson and Springthorp (2007)

LLE (Lipophilic Ligand Eciency)

LLE: Potency without too much Grease!
LLE= Log(IC50) cLogP
Exemplar Values: LLE>5; IC50<10nM and cLogP<3
Adding grease is an easy way to accrue bogus Potency
Must consider the eciency of each lipophilic addi@on
LLE allows iden@ca@on/tracking of good Potency
(i.e., not driven by greasehigh protein binding)
Leeson and Springthorp (2007)

Why Size MaWers (MW<400)!

Lower MW Cpds have superior Pr(oral agent approval)!
450
440
430
420
410
400
390
380
370
Phase 1
Phase 2
Phase 3
Launch
LEAN: Drive for Potency at the Right Price!

LEAN= log(IC50)/n [n=# of non H atoms]
Exemplar Values: LEAN>0.27; IC50<10nM and MW<400
Wenlock et al. (2003); Hopkins et al. (2004)
10
LEAN versus LLE Trends

Op@miza@on of both LEAN and LLE in drive from LEADCS
Example: Kinase Project
Lean vs. LLE
Lead
Zone
Target Values:
Lead:
Candidate
Zone
MW400/IC50 100nM L
LEAN ~0.23
cLogP3/IC50 100nM E
LLE~4-5
A
Candidate:
MW400/IC50 10nM
LEAN ~0.27
cLogP3/IC50 10nM
LLE ~5-8
Dot Color Scheme: RLM - Green <33%; Orange >33%, <66%; Red >66%
LLE
11
Guidelines for Oral Drug Candidates
MW < 400
cLogP < 3
Lean > 0.27
LLE > 5
These are the gold zone targets
There is a gray zone but be mindful that
operating there may come with more risk
Make Drugs looks like Natural Products
12
Fragment-Based Drug Discovery

Fragment library screening Hits
Exploit literature precedents
H1
Structure-guided triage process

Pick promising opportunities
H3
Drive Potency/SelectivityCandidate
H2
mM-M Fragment Hit
Perceived/Real Challenges
Lower affinity starting points
Modest fragment libraries (1000s)
Hydrophobic Effect: Friend or Foe?
Lipophilicity Matters!
Size does Matter!
Structure is essential
nM Drug Candidate
Are fragments selective per se?
13
mM-M Anity Star@ng Points

Gtotal = Gintrinsic + Grigid (Grigid ~3.5-5 kcal/mol)
Grigid penalty due to loss of entropy
# Heavy
Atoms/MW
A
Kd

Gtotal
(kcal/mol)
Gintrinsic
(kcal/mol)
Scaold A
11/140Da 100M
-5.4
-9 to -10
CandidateAC
30/400Da 3nM
-11.4
-15 to -16
MW ~3-foldAffinity ~33000-fold
Scaffold A contributes significantly to Gintrinsic!
Only well anchored scaffolds show up in fragment screens
Murray and Verdonk (2002)
14
Crystallographic Fragment Screening
Crystal soaking and

data collection
Br
Hit rate~1-5%
Fragment library (1500-2000 cpds) is divided into pools of 10 shape diverse

compoundssoaked into preformed crystals
Bound fragment is iden@ed from shape of electron density features
En@re library can be screened within a few days at a synchrotron
15
Fragment Library Design/Diversity/U@lity

~2000 fragments (scaolds)
Scaolds have ~3 Handles
amenable to modica@on
1000s-10000s of commercial
R groups/Handle
Poten@al Diversity Enormous
R
O
N
N H
Br
Linear Library 1 (LL1)

Handle
1 (H1)
H3C
O
N
Br
(H2)
N H
(H3)
H
2x103*100032x1012
2x103*1000032x1015
CHFNOcpds(<400Da)~1030
LL3
LL2
H3C
O
N
H3C
O
N
N H
R
N
Br
Fragment Libraries support rapid

hit SAR explora@on with
automated synthesis
16
FBDD Generates Mul@ple Opportuni@es

Multiple Targets
Multiple Binding Sites
Multiple Chemotypes
Active
Site
Allosteric
Site
MEK1
Wealth of Opportunities!
17
MET Receptor Tyrosine Kinase Inhibitor

MET (HGFR) is the receptor for
hepatocyte growth factor/scaWer
factor
Ac@va@ng muta@ons occur in
various human cancers
V13L
I316M
N375S
S
Hereditary Papillary Renal Cell

Carcinoma (HPRCC)
Sporadic, e.g., PRCC, non-small cell
lung cancer (NSCLC)
Ac@va@ng MET muta@ons have been
observed in metastases
MET gene amplica@on seen in

other tumors
Gastric cancer
EGFR inhibitor-resistant lung cancer
Extracellular
Domain
E168D
I638L
V941L
Kinase
Domain
R988C
P1009S
S1058P
V1110I
H1112Y
H1124D
M1149T
V1206L
L1213V
V1238I
D1246N
Y1248C
K1262R
M1268T
Gastric Cancer
Lung Cancer
Kidney Cancer
18
Fragment Hit Evalua@on/Lead Op@miza@on

N N
N
N
N
S
HO
N N
N
N
MET IC50 = 712 nM

GTL16 IC50 = 1.37 M
MW = 302; LEAN = 0.28
cLogP 3.0; LLE = 3.1
Good ligand efficiency AND

Novel binding mode
MET IC50 (nM)
156
114
GTL16 IC50 (nM)
558
179
MW
343
337
cLogP
3.5
3.6
LEAN
0.27
0.27
LLE
3.3
3.3
19
Achieving LEAN>0.27/LLE>5
N
N
N
H
N
N
N
N
N
MET IC50 (nM)
25
61
GTL16 IC50 (nM)
14
151
MW
412
341
Exploring the Cleft
cLogP
2.5
1.5
Goal: Reduce cLogP
LEAN
0.25
0.28
5.1
5.7
LLE
Design: Phenyl variants
and Heterocycles
20
Reducing Clearance (<10mL/min/kg)

Benzylic center known
site of oxidation
Compound
SGX523
N
N
N
N
GTL16
In vitro Cl
IC50 (nM)
T1/2
(mL/min/kg)
(hr)
CH2
151
High
No Data
CF2
20
5.1
2.2
CH(CH3)
38
11
1.7
23
7.2
2.7
21
SGX523: In-vitro Proling

Enzyme Assay: IC50
SGX523
MET Cytoplasmic portion
4 nM
Cell Assay: IC50

GTL16 cell proliferation (XTT)
BaF3/TPR-MET (pMET ELISA)
Cell assay specificity control
24 nM
17 nM
> 10,000 nM
Liver Microsome Metabolism: T1/2

Rat
Human
41 min
279 min
CYP Inhibition (IC50)

1A2
2C9
2C19
2D6
3A4
hPXR 3A4 induction
> 10 M
10 M
> 10 M
> 10 M
> 10 M
> 10 M
Safety Pharmacology
hERG
AMES/Micronucleus
> 10 M
Neg./Neg.
22
SGX523: Unique Binding Mode
RON is METs nearest neighbor

Active site: 1 residue difference Y1248L
RON Inhibition [1M] = 9%
23
SGX523: Human Kinome Proling I

%Inhibition @ 1M for 213 kinases
<50% Inhibition
>50% Inhibition
MET and MET M1250T
Kinase Tested
MET
BRAF V599E
RAF1 (cRAF)
ABL
MAPK14 (p38)
ABL1 Y253F
% Inhibition
(1 M SGX523)
92
36
27
26
25
20
IC50 (nM)
4
>66,667
>66,667
>7,407
>200,000
>7,407
B a r C h a rt
K ina s e T e s te d
24
SGX523: Human Kinome Proling II

Active site
residues in
common
Non-Confidential
25 Presentation
25
SGX523: MET versus MER

MET (blue) inhibited by SGX523 (yellow)
Homology model of MER (magenta)
MET-AlaMER-Ser blocks binding
26
SGX523: MET versus AXL

MET (blue) inhibited by SGX523 (yellow)
Homology model of AXL (green)
MET-AlaMER-Ser and MET-LeuAXL-Met block binding
27
Active in two mouse models

Human gastric carcinoma cell
xenograft (GTL16 cell implant)
Activated MET xenograft (TPR-MET
BaF3 cell implant)
Average Tumor Vol (mm3)
SGX523: In vivo Ecacy

1000
900
Inhibition of pharmacodynamic marker

(pMET)
GTL16
800
700
600
500
400
300
200
100
0
0
6
8 10 12 14
Time (days post tumor implant)
16
10
18
Average Tumor Volume (mm3)
Time (days post implant)
500
450
400
350
300
250
200
150
100
50
0
TPR-MET
Vehicle ctrl
10 mg/kg bid
20 mg/kg bid
X
X
30 mg/kg bid
60 mg/kg qd
100 mg/kg bid
SGX523 PO 6 Hours (GTL16)
10
10
Time (days post tumor implant)
Time (days post implant)
Vehicle
100 mg/kg
mg/kg
6 Hours 10 mg/kg 20 mg/kg 30
30 mg/kg
mg/kg 60 mg/kg 100
12
12
pMET
28
20
SGX523: Phase I Clinical Outcome

Lowest dose group tolerated 40 mg per day
>80 mg dosing
Rapid onset of renal failure
Dosing suspended
Kidney func@on in aected pa@ents restored

Obstruc@ve crystal nephropathy observed in monkeys
Representative photomicrograph of renal histology: Crystals observed

29
SGX MET Inhibitor Summary

TZP LeadsLEAN>0.27, LLE>5
SGX523
MET IC50 = 4 nM
> 1000-fold selec@vity for MET
versus 211 human protein kinases
Human/primate metabolite
crystals in distal tubule
obstruc@ve nephropathy
Other SGX TZPs Lilly

TZP variant Drug Candidate

30
Useful References!
Cavalli et al. (2002) J. Med. Chem. 45, 3844

Fink et al. (2005) Angew. Chem. Int. Ed. 44. 1504
Gleeson (2008) J. Med. Chem. 51, 817
Henkel et al. (1999) Angew. Chem. Int. Ed. 38, 643
Hopkins et al. (2004) Drug Disc.Today 9, 430
Hughes et al. (2008) BMCL 18, 4872
Johnson et al. (2009) BMCL 19, 5560
Kelder et al. (1999) Pharm. Res. 16, 1514
Kola and Landis (2004) Nature Rev./Drug Disc. 3, 711
Leeson and Springthorp (2007) Nature Rev./Drug Disc. 6, 881
Leeson and St-Gallay (2011) Nature Rev./Drug Disc. 10, 749
Lipinski et al. (1997) Adv. Drug Del. Rev. 23, 3-25
Lovering et al. (2009) J. Med. Chem. 52, 6752
Paolini et al. (2006) Nature Biotech. 24, 805
Ploemen et al. (2004) Exp. Toxic Pathol. 55, 347
Price et al. (2009) Expert Opin. Drug Metabol. Toxicol. 5, 921
Varma et al. (2010) J. Med. Chem. 53, 1098
Waring (2009) BMCL 19, 2844
Waring (2010) Expert Opin. Drug Discov. 5, 235
Waring et al. (2006) BMCL 17, 1759
Wenlock et al. (2003) J. Med. Chem. 46, 1250
31

Challenges in Fragment Based Drug Discovery For Protein Kinases 2013

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Challenges

Gene family platforms

High Content Imaging

Causes of AWri@on in Pharma

How can MedChemists help address ~1/3 of Pharma Attrition?

Statement of the Problem

Subset with Pharmacologic Activity ~ <1026

Subset with Good ADMET Properties ~ 10??

Ligand displacement of par@ally ordered waters from a

Why Lipophilicity MaWers (cLogP<3)!

Leeson and Springthorp (2007)

LLE (Lipophilic Ligand Eciency)

Leeson and Springthorp (2007)

Why Size MaWers (MW<400)!

LEAN: Drive for Potency at the Right Price!

LEAN versus LLE Trends

Guidelines for Oral Drug Candidates

Fragment-Based Drug Discovery

Structure-guided triage process

mM-M Fragment Hit

Are fragments selective per se?

mM-M Anity Star@ng Points

Crystallographic Fragment Screening

Crystal soaking and

Fragment library (1500-2000 cpds) is divided into pools of 10 shape diverse

Fragment Library Design/Diversity/U@lity

Linear Library 1 (LL1)

Fragment Libraries support rapid

FBDD Generates Mul@ple Opportuni@es

Multiple Binding Sites

MET Receptor Tyrosine Kinase Inhibitor

Hereditary Papillary Renal Cell

MET gene amplica@on seen in

Fragment Hit Evalua@on/Lead Op@miza@on

MET IC50 = 712 nM

Good ligand efficiency AND

MET IC50 (nM)

GTL16 IC50 (nM)

MET IC50 (nM)

GTL16 IC50 (nM)

Exploring the Cleft

Goal: Reduce cLogP

Reducing Clearance (<10mL/min/kg)

SGX523: In-vitro Proling

MET Cytoplasmic portion

Cell Assay: IC50

Liver Microsome Metabolism: T1/2

CYP Inhibition (IC50)

SGX523: Unique Binding Mode

RON is METs nearest neighbor

SGX523: Human Kinome Proling I

SGX523: Human Kinome Proling II

SGX523: MET versus MER

SGX523: MET versus AXL

Active in two mouse models

Average Tumor Vol (mm3)

SGX523: In vivo Ecacy

Inhibition of pharmacodynamic marker

Average Tumor Volume (mm3)

Time (days post implant)

SGX523 PO 6 Hours (GTL16)

Time (days post tumor implant)

Time (days post implant)