Bridging the Gap From In Vitro to In Vivo

Novel Chemoproteomics (KinobeadsTM) and Phenotypic (BioMAP®)

Discovery Platforms for the Development of Novel and Safer Kinase

Inhibitors for Inflammatory Diseases

1

Ellen L. BergInflammation Research Association Conference

Bolton Landing NY 10 September, 2012

Challenges of Kinase Drug Discovery

Difficulty of obtaining selective inhibitors

Kinase gene family members (> 500 members) are highly related

Conserved active sites

Biochemical ≠ cellular efficacy

Challenge of defining optimal target selectivity

Limited knowledge about target biology

Individual kinases can play a role in multiple pathways - complex signaling

Unclear significance of secondary activities

There will be secondary targets at a high enough concentration

Are secondary targets affecting safety and/or efficacy?

2

Technologies

Advanced chemoproteomics tools

KinobeadsTM platform

Human phenotypic screening models

BioMAP® platform

3

KinobeadsTM PlatformA chemoproteomics approach to drug discovery

Works exclusively with native proteins directly in cell

or tissue lysate under close to physiological settings

Protein targets are in their “natural“ environment, with

right

Modifications

Protein complexes

Provides comprehensive profile of targets and off-

targets of drugs in tissue of relevance

One assay plaform can be used throughout entire drug

discovery process and across different species

4

Kinobeads™ Concept

Kinobeads™

(empty)

Kinobeads™

(loaded)

1 2

3

NBroad spectrum kinase inhibitors(ATP mimetics)

Immobilized on solid support

Incubate beads with cell or tissue lysate

Capture tissue kinome on matrix

Identify captured proteinsby mass spectrometry

5

Kinobeads™ competition binding assays

for mass spec profiling

Kinobeads™

(loaded)

1 2

3

N

Kinobeads™

(empty)

Cell lysate

Competition experiment:Compounds compete for kinase binding to beads

Capture kinases in a compound-dose dependent manner

PI-Kinobeads™

Elution

MASS SPECTROMETRY BASED PROFILING

Detection by quantitative mass spectrometry:Most potent compounds are profiled against 100+ kinases

Compound

200 400 600 800 1000 1200m/z0

100

%

6Bantscheff et al, Nat Biotechnol 25, 2007

TMT =Tandem Mass Tags

3. Add Kinobeads™1. Add inhibitor to lysate at different concentrations

4. Elute beads, digest & labelwith stable isotope

Inh

ibit

or

con

cen

trat

ion

10 µM

0.625 µM

0.16 µM

0.0 µM(DMSO,Control)

0.04 µM

2.5 µM

TMT 126

TMT 128

TMT 129

TMT 131

TMT 130

TMT 127

5. Mix all 6 samples,run single LC-MS/MS& quantify in MS/MSspectrum

Protein does not bind to drug

2. Incubate - inhibitor binds to targets

Protein binds to drug:

Chemoproteomics Platform: Mass Spec Detection

7

Multiplexing:Spotting of low volumes of eluate allows preparation of multiple arrays for detection

Kinobeads™ and Episphere™-

based competition binding assays

8

Immuno-detection on Arrays:Cell lysate

Compound

Kinobeads™

Episphere™

(loaded)

1 2

3

N

Kinobeads™

Episphere™

(empty)

Competition experiment:Compounds compete for kinase/epi-target bindingto beads

Capture kinases/epi-targets in a compound-dose dependent manner

Kinobeads™

Episphere ™

Elution

NEGATIVECONTROL

POSITIVECONTROL

HITS

Immunodetection on affinity protein arrays

– high throughput (50,000 cmpds/wk)

– low protein coverage (>10 proteins quantified per sample)

A chemoproteomic approach to drug discovery:

PI3Kg

9

Late LO/clinical

Lead Optimization(LO)

Hit ID

Target

Clinical Candidate

Kinobeads™

LO/clinical: broad kinase profiling/cross species comparison

Hit ID: library screening

Compounds/Library

Human blood

Tissue lysate

Cell lysate

LO: primary assayPI3Kg PI3Ka

PI3Kb

mTor

DNAPK PI3Kd

[cp

d]

PI3Kg

PI3Ka

PI3Kb

TPI3Kd

DNAPK

mTor

PI3

Kg

PI3

Ka

PI3

Kb

PI3

Kd

DN

AP

K

mTo

r

From multiplexed screening to selective probes:

PI3Kg inhibitors

10

mTor DNAPK PI3Kd PI3Kg

Co

mp

ou

nd

s

Library screening

Bergamini et al, NatChemBiol, June 2012

Identification of a selective PI3Kg inhibitor

11Bergamini et al, NatChemBiol, June 2012

CZC24832 shows efficacy in mouse arthritis model

(CIA)

12

Mixed species KinobeadsTM

profiling:

Potency drop for PI3Kg,b of 2-5 fold in rodents

Selectivity unchanged5,0

*

**

**

**

* * * * * * * *

Disease control

CZC24832 10 mg/kg

Dex0.01mg/kg

CZC24832 3 mg/kg

Collagen induced arthritis model

CZC24832

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Arthritis Day

Mea

n±S

E C

linic

al A

rth

irit

s Sc

ore

(Sco

red

0-5

)

Bergamini et al, NatChemBiol, June 2012

Dose-dependent efficacy mouse CIA model

Reduction in severity of arthritis is comparable to PI3Kg-/- mice

Selectivity translates from chemoproteomic profiles to primary

cell phenotypes: a new role of PI3Kg in TH17cell biology

BioMAP® profile

13

PI3Kg

Bergamini et al, NatChemBiol, June 2012

14

BioMAP® Technology Platform

BioMAP

Assay Systems

Reference

Profile Database

Predictive

Informatics Tools

Human primary cells

Disease-models

30+ systems

Biomarker responses to drugs

are stored in the database

>3000 drugs

Specialized informatics tools are

used to predict clinical outcomes

Human Biology Integrated into a Robust, Scalable Platform

Human primary cell-based assays Engineered to model complex human disease biology

BioMAP® Systems

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Physiologically relevant assay conditions

Mixtures of stimulation factors, co-cultures of cells

Signaling networks that reflect in vivo tissue states

Clinically validated disease biomarkers

Quantitative and reproducible

Robust readouts (proteins, mediators); standardized assays, QMP, SOPs

Donor variation is managed

Validated with known drugs

BioMAP® Systems – Cell Types & Co-cultures

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BioMAP Systems

B cells

Endothelial Cells

Bronchial Epithelial Cells

Keratinocytes

Smooth Muscle Cells

Dermal Fibroblasts

Lung Fibroblasts

Peripheral Blood Mononuclear Cells

Macrophages

10 primary human cell types

12 assay systems

• Different cell types, co-culture combinations

• Different activation conditions – disease models

BioMAP® Systems – Biology Covered

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BioMAP Systems

Vascular Biology

Wound Healing

Th1 Inflammation

Skin Biology

Th2 Responses

Immune Biology

Th17 Biology

Lung Biology

BioMAP Profile of CZC24832

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95% significance envelope

Control (no drug)

Readout Parameters (Protein Biomarkers)

Cytotoxicity Readouts

BioMAP Systems

Log expression ratio(Drug/DMSO control)

DoseResponse

BioMAP Profile of CZC24832

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Key activities of CZC24832

• Selective inhibition of T-cell-dependent B cell activation (BT system)

• Reduction in IL-17A, IL-6, TNFa; modest inhibition of B cell proliferation (grey

arrow)

• Consistent with reduced progression and and severity of arthritis in a

mouse model of CIA

AS-605240: first published PI3Kg inhibitor with efficacy

in models of inflammation

Williams, Chemistry and Biology (supplement), 2010

Camps, Nat. Med., 2005Kinobeads AS-605240 DNA-PK

PI3Kg

20

PI3Kg

Kinobeads CZC24832 Bergamini et al, NatChemBiol, June 2012

Comparison of CZC24832 and AS605240

21

Overlay of CZC24832 (PI3Kg)and AS605240 (less selective PI3Kginhibitor)

CZC24832 is selectively active in the BT system

BioMAP profile of AS605240 is dominated by activities in other systems

Compounds show cell-type selectivity in anti-proliferative effects (thick grey arrows)

Minor shared activities are indicated by thin black arrows

Kinase Inhibitors in Development

Tofacitinib (CP-690,550)

Jak 3 kinase inhibitor – FDA review for rheumatoid arthritis

Fostamatinib (R788)

Syk kinase inhibitor – Phase III for rheumatoid arthritis

What can BioMAP profiling tell us?

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Case Study: Kinase Inhibitors in RA

TofacitinibCP-690,550

Jak 3 kinase selective inhibitor Kinase activity: EC50s: Jak3 (1 nM); Jak2 (20 nM); Jak1 (112 nM); Rock-II (3.4 mM) and Lck

(3.8 mM) (Changelian, Science 2003, 302:875). Binding: JAK3 (2.2 nM) and Jak2 (5 nM)

(Karaman, Nat Biotech 2008, 26:127).

Safe and effective in rheumatoid arthritis Kremer, Arthr & Rheum 2012, 60:1895; Fleischmann, Arth & Rheum 2012, 64:619

In review at the FDA for rheumatoid arthritis (Pfizer)23

BioMAP Profile of Tofacitinib (≤ 370 nM)

Key activities of Tofacitinib (CP-690,550 ) ≤ 370 nM

• Inhibition of IL-4 dependent signaling in endothelial cells (4H system)

• Selective inhibition of T-cell-dependent B cell activation (BT system)

• Several activities consistent with clinical efficacy biomarkers (in red)

Eotaxin-3

IgG

TNFa

IL-2

IL-6

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IgG

TNFaIL-6

De Paz, 2010Lin, 2010

Kutukculer, 1998

BioMAP Profile of Tofacitinib (≥ 370 nM)

Many additional activities at higher doses (≥ 370 nM):• Most are clinical efficacy biomarkers for RA (Kuan, 2010; Klimiuk, 2002; Kutukcular,

1998; Dolhain, 1998; Metawi, 2011) (in red)

Activities are consistent with clinical effects and dosing• Van Gurp, Transpl. 2009, 87:79

• Cmax in one clinical study was ~1 microM (Cohen, BJCP 2010, 69:143)

Higher dose (less selective) profile is more “efficacious” in BioMAP

and in the clinic

Eotaxin-3

IgG

TNFa

IL-2

MIGHLA-DR

VCAM CD38

MIG

CD40IL-17A, F

MIG

HLA-DRMIG

HLA-DRICAM

IP-10

MIGIP-10IP-10

VCAMIL-6

CD69

25

IgG

TNFa

MIG

VCAM CD38

MIG IL-17A, FMIG

MIGIP-10IP-10

VCAMIL-6 ICAM

IP-10MIG

Fostamatinib (R788)

Syk kinase inhibitor

Kinase activity: IC50= 41 nM (active form). Braselmann, JPET 2006, 319:998

Well tolerated in Phase II studies for rheumatoid arthritis

Genovese, Arth & Rheum 2011, 63:337

Currently in Phase III clinical trials for RA (Rigel/AstraZeneca)

R788Fostamatinib

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BioMAP Profile of Fostamatinib

Key activities of Fostamatinib (R788) • Broadly active in endothelial cells, leukocytes, epithelial cells and SMC

• Inhibition of monocyte activation, T cell activation, and T-dependent B cell activation

• Many activities (in red) consistent with clinical efficacy biomarkers (Szekanecz, 1997;

Klimiuk, 2002, 2005; Kutukculer, 1998; Kaun, 2010)

Consistent with Clinical effects• Peak serum concentrations of > 800 ng/ml (1.4 mM) reported in clinical studies

(Podolanczuk, Blood 2009, 113:154)

Eotaxin-3 TNFa

TFuPAR

MIG

uPAR

IL-6

IL1auPAR

MCP-1

MIP1auPAIL-8IL-1a

27

MMP3

TNFa

uPAR

MIG

uPAR

IL-6

uPAR

MCP-1

IL-8 MMP3

Expression of Syk Kinase mRNA in BioMAP Systems

Expression of Syk mRNA in BioMAP Systems (AU units)

Fostamatinib is active in systems where syk kinase mRNA is not detected

Fostamatinib may not be a selective syk inhibitor at clinically relevant doses

3C 4H LPS SAg BT BF4T BE3C CASM3C HDF3CGF KF3CT MyoF

/Mphg

100.4 98.5 133 137.3 550 251.3 165.3 179.1 108.3 87 108.1 733.7

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Clinical Standards of Care

E-sel

IgG

TNFaIL-8

E-sel

IL-17

IL-2

TNFa

IP-10IL-8

MMP-9IL-6

MCP-1 VCAM

IL-8

MCP-1VCAM

E-sel

IL-8

SAA

Rheumatoid Arthritis - Clinical Standards of Care• Methotrexate – inhibitor of DHFR

• Selective inhibitor of T cell dependent B cell activation

• Prednisolone – corticosteroid

• Inhibition of macrophage > monocyte activation; T cell activation

• Remicade – TNF antagonist

• Inhibition of monocyte > macrophage activation; myofibroblast activation

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How do we apply BioMAP systems for

investigating side effect mechanisms?

Are there BioMAP profile features that are associated with

particular side effects?

Hypertension

A side effect of NSAIDS and fostamatinib but not tofacitinib

What about side effects?

BioMAP Profile of Fostamatinib

Vascular Effects

• Strong activities in endothelial cell- and smooth muscle cell-containing

systems (brackets) – non syk dependent??

• Inhibition of endothelial and smooth muscle cell proliferation (grey arrows)

• Effect on prostaglandins (black arrow)

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Prostacyclin (PGI2)

Vasodilator with anti-thrombotic and anti-proliferative

effects

Produced by vascular cells, platelets

Regulated by inflammatory settings

In several BioMAP systems, PGI2 levels (6-keto-PGF1a) is

increased by inflammatory stimuli

Effects of selective Cox-2 inhibitors on PGI2 has been

proposed as one potential mechanism underlying the

cardiovascular side effects of these drugs

Yu, Sci Transl Med., 2012, 4:132 (PMID: 22553252)

32

Fostamatinib Inhibits Prostacyclin Production BioMAP LPS System

33

Tofacitinib

Jak

Fostamatinib

Syk

Prednisolone

GR

Rofecoxib

Cox-2

Control

Rel

ativ

e [6

-ket

o-P

GF

1a]

(sta

ble

met

abol

ite o

f PG

I 2)

Possible relationship to fostamatinib hypertension side effect

P < 0.01

P < 0.01

10 10 1 10

Rosiglitazone increases Prostacyclin Production BioMAP LPS System

34

Rel

ativ

e [6

-ket

o-P

GF

1a]

(sta

ble

met

abol

ite o

f PG

I 2)

Tofacitinib

Jak

Fostamatinib

Syk

Prednisolone

GR

Rofecoxib

Cox-2

Rosiglitazone

PPARg

Control

10 10 1 10 100

Possible relationship to blood pressure lowering effect of rosiglitazone

P < 0.01

Summary

New target-based discovery platform, KinobeadsTM

enabled the discovery of a highly selective PI3Kg

inhibitor, CZC24832

CZC24832 is selectively active in inhibiting T cell dependent B cell

activation responses, including decreasing IL-17A

BioMAP systems of primary human cell based assays

can be useful for characterizing novel kinase inhibitors

Discovery new biology or confirm known biology

Comparison to standards of care

Testing combination therapies

Investigation of side effect mechanisms

35

Summary

Current clinical stage kinase inhibitors for rheumatoid

arthritis are not selective

Tofacitinib and fostamatinib are broadly acting agents

Opportunity for next generation compounds

Further study of the control of blood pressure by

inflammatory mechanisms may help identify new drugs

with reduced hypertension side effects

36

Acknowledgements

BioSeek

Alison O’Mahony

Mark A. Polokoff

Dat Nguyen

Jennifer Melrose

Andrew Melton

Cellzome

Oliver Rausch

Giovanna Bergamini

Gitte Neubauer

37

Bridging the Gap From In Vitro to In Vivo

Contacts

BioSeek, LLC310 Utah, #100South San Francisco, CA 94080

BioSeek, LLC

T: +1-650-416-7600

F: +1-650-416-7625

www.biomapsystems.com

www.bioseekinc.com

Ellen L. Berg, PhD

T: +1-650-416-7621

F: +1-650-416-7625

eberg@bioseekinc.com

www.biomapsystems.com

39