Novel Chemoproteomics (KinobeadsTM) and Phenotypic (BioMAP®) Discovery Platforms for the Development of Novel and Safer Kinase Inhibitors for Inflammatory Diseases, presentation by E. Berg at the 17th International Conference of the Inflammation Research Association, Bolton Landing, NY, September 10, 2012

1. Novel Chemoproteomics (KinobeadsTM) and Phenotypic (BioMAP®)
Discovery Platforms for the Development of Novel and Safer Kinase
Inhibitors for Inflammatory Diseases
Bridging the Gap From In Vitro to In Vivo
Ellen L. Berg
Inflammation Research Association Conference
Bolton Landing NY 10 September, 2012
1

2. 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

3. Technologies
 Advanced chemoproteomics tools
 KinobeadsTM platform
 Human phenotypic screening models
 BioMAP® platform
3

4. KinobeadsTM Platform
A 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

5. Kinobeads™ Concept
1 2
Kinobeads™ Kinobeads™
3
(empty) (loaded)
N
Broad spectrum Immobilized on
kinase inhibitors solid support Capture tissue
(ATP mimetics) kinome on matrix
Incubate beads with
cell or tissue lysate
Identify captured proteins
by mass spectrometry
5

6. Kinobeads™ competition binding assays
for mass spec profiling
Cell lysate
Compound Kinobeads™
(empty)
Competition experiment: 100
Compounds compete for PI-Kinobeads™
kinase binding to beads
%
1 2
0 m/z
200 400 600 800 1000 1200
Capture kinases Elution
Kinobeads™
in a compound-dose (loaded)
3 MASS SPECTROMETRY BASED PROFILING
dependent manner
Detection by quantitative mass spectrometry:
Most potent compounds are profiled against 100+
N
kinases
Bantscheff et al, Nat Biotechnol 25, 2007
6

7. Chemoproteomics Platform: Mass Spec Detection
1. Add inhibitor to lysate at 2. Incubate - inhibitor 3. Add Kinobeads™ 4. Elute beads, 5. Mix all 6 samples,
different concentrations binds to targets digest & label run single LC-MS/MS
with stable isotope & quantify in MS/MS
0.0 µM spectrum
(DMSO, TMT 131
Control)
0.04 µM TMT 130
Protein bindsnotdrug: to drug
does to bind
Inhibitor concentration
0.16 µM TMT 129
0.625 µM TMT 128
2.5 µM TMT 127
10 µM TMT 126
TMT =Tandem Mass Tags
7

8. Kinobeads™ and Episphere™-
based competition binding assays
Cell lysate
Immuno-detection on Arrays:
Kinobeads™
Compound
Episphere™
POSITIVE
(empty) CONTROL
Competition experiment:
Compounds compete for Kinobeads™
kinase/epi-target binding Episphere ™
to beads
HITS
1 2
NEGATIVE
Capture kinases/epi-targets Elution CONTROL
Kinobeads™
in a compound-dose
Episphere™ 3
dependent manner (loaded)
Multiplexing:
N Spotting of low volumes of eluate allows preparation of
multiple arrays for detection
 Immunodetection on affinity protein arrays
– high throughput (50,000 cmpds/wk)
– low protein coverage (>10 proteins quantified per sample)
8

9. A chemoproteomic approach to drug discovery:
PI3Kg
Hit ID: library screening
Target
Cell lysate
Kinobeads™
Hit ID
LO: primary assay
PI3Kg PI3Ka
PI3Kb PI3Kg
Lead
[cpd]
PI3Ka
Optimization PI3Kb
(LO) TPI3Kd
mTor DNAPK
Tissue lysate
mTor
DNAPK PI3Kd
PI3Ka
mTor
DNAPK
PI3Kb
PI3Kd
PI3Kg
Late LO/
clinical LO/clinical: broad kinase profiling/cross species comparison
Human blood
Clinical Compounds/Library
Candidate
9

10. From multiplexed screening to selective probes:
PI3Kg inhibitors
Library screening
mTor DNAPK PI3Kd PI3Kg
Compounds
Bergamini et al, NatChemBiol, June 2012
10

11. Identification of a selective PI3Kg inhibitor
Bergamini et al, NatChemBiol, June 2012
11

12. CZC24832 shows efficacy in mouse arthritis model
(CIA)
 Mixed species KinobeadsTM
profiling:
 Potency drop for PI3Kg,
b of 2-5 fold in rodents
 Selectivity unchanged
5,0
Collagen induced arthritis model
4,5
 Dose-dependent efficacy 4,0
Disease
control
mouse CIA model 3,5 CZC24832
Mean±SE Clinical Arthirits Score
3 mg/kg
 Reduction in severity of
3,0
*
* * * * * * *
(Scored 0-5)
2,5 CZC24832 * * CZC24832
* 10 mg/kg
*
arthritis is comparable to 2,0
*
*
* Dex
0.01mg/kg
PI3Kg-/- mice 1,5
1,0
0,5
0,0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Arthritis Day
Bergamini et al, NatChemBiol, June 2012
12

13. Selectivity translates from chemoproteomic profiles to primary
cell phenotypes: a new role of PI3Kg in TH17cell biology
PI3Kg
BioMAP® profile
Bergamini et al, NatChemBiol, June 2012
13

14. BioMAP® Technology Platform
BioMAP Reference Predictive
Assay Systems Profile Database Informatics Tools
Human primary cells Biomarker responses to drugs Specialized informatics tools are
Disease-models are stored in the database used to predict clinical outcomes
30+ systems >3000 drugs
Human Biology Integrated into a Robust, Scalable Platform
14

15. BioMAP® Systems
Human primary cell-based assays
Engineered to model complex human disease biology
 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
15

16. BioMAP® Systems – Cell Types & Co-cultures
BioMAP Systems
Endothelial Cells
Peripheral Blood Mononuclear Cells
B cells
Bronchial Epithelial Cells
Dermal Fibroblasts
Smooth Muscle Cells
 10 primary human cell types Keratinocytes
 12 assay systems Lung Fibroblasts
• Different cell types, co-culture combinations Macrophages
• Different activation conditions – disease models
16

17. BioMAP® Systems – Biology Covered
BioMAP Systems
Vascular Biology
Immune Biology
Lung Biology
Skin Biology
Wound Healing
Th1 Inflammation
Th2 Responses
Th17 Biology
17

18. BioMAP Profile of CZC24832
BioMAP Systems
95% significance envelope
Control (no drug)
Log expression ratio Dose
(Drug/DMSO control) Response
Cytotoxicity Readouts
Readout Parameters (Protein Biomarkers)
18

19. BioMAP Profile of CZC24832
 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
19

20. AS-605240: first published PI3Kg inhibitor with efficacy
in models of inflammation
PI3Kg
Kinobeads AS-605240 DNA-PK
Camps, Nat. Med., 2005
PI3Kg
Kinobeads CZC24832 Bergamini et al, NatChemBiol, June 2012
Williams, Chemistry and Biology (supplement), 2010
20

21. Comparison of CZC24832 and AS605240
 Overlay of CZC24832 (PI3Kg)and AS605240 (less selective PI3Kg
inhibitor)
 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
21

22. 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?
22

23. Case Study: Kinase Inhibitors in RA
Tofacitinib
CP-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

24. BioMAP Profile of Tofacitinib (≤ 370 nM)
IL-2
IL-6 Lin, 2010
Eotaxin-3 TNFa De Paz, 2010
IgG Kutukculer, 1998
 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)
24

25. BioMAP Profile of Tofacitinib (≥ 370 nM)
IL-2
CD69
IL-6 ICAM VCAM MIG
VCAM CD38 HLA-DR IP-10
CD40 TNFa IP-10
Eotaxin-3 IP-10 HLA-DR
MIG IL-17A, F MIG
MIG MIG
HLA-DR IgG
 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
25
and in the clinic

26. Fostamatinib (R788)
R788
Fostamatinib
 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)
26

27. BioMAP Profile of Fostamatinib
TF IL-8 MMP3
uPAR
uPAR uPA uPAR MIP1a
IL-1a IL1a
Eotaxin-3 MCP-1 IL-6
TNFa
MIG
 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)
27

28. Expression of Syk Kinase mRNA in BioMAP Systems
Expression of Syk mRNA in BioMAP Systems (AU units)
3C 4H LPS SAg BT BF4T BE3C CASM3C HDF3CGF KF3CT Myo /Mph
F g
100.4 98.5 133 137.3 550 251.3 165.3 179.1 108.3 87 108.1 733.7
 Fostamatinib is active in systems where syk kinase mRNA is not detected
 Fostamatinib may not be a selective syk inhibitor at clinically relevant doses
28

29. Clinical Standards of Care
SAA
E-sel IL-8 IP-10 MMP-9 IL-8
TNFa IL-8 IL-6
E-sel MCP-1 VCAM MCP-1
IL-2
IL-8 VCAM
IL-17 E-sel
TNFa IgG
 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
29

30. What about side effects?
 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

31. 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)
31

32. 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

33. Fostamatinib Inhibits Prostacyclin Production
BioMAP LPS System
(stable metabolite of PGI2)
Relative [6-keto-PGF1a]
P < 0.01
P < 0.01
10 10 1 10
Tofacitinib Fostamatinib Prednisolone Rofecoxib Control
Jak Syk GR Cox-2
33  Possible relationship to fostamatinib hypertension side effect

34. Rosiglitazone increases Prostacyclin Production
BioMAP LPS System
P < 0.01
(stable metabolite of PGI2)
Relative [6-keto-PGF1a]
10 10 1 10 100
Tofacitinib Fostamatinib Prednisolone Rofecoxib Rosiglitazone Control
Jak Syk GR Cox-2 PPARg
 Possible relationship to blood pressure lowering effect of rosiglitazone
34

35. 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

36. 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

37. Acknowledgements
 BioSeek  Cellzome
 Alison O’Mahony  Oliver Rausch
 Mark A. Polokoff  Giovanna Bergamini
 Dat Nguyen  Gitte Neubauer
 Jennifer Melrose
 Andrew Melton
37

38. Contacts
BioSeek, LLC
Ellen L. Berg, PhD
T: +1-650-416-7600
T: +1-650-416-7621
F: +1-650-416-7625
F: +1-650-416-7625
www.biomapsystems.com
eberg@bioseekinc.com
www.bioseekinc.com
Bridging the Gap From In Vitro to In Vivo
BioSeek, LLC
310 Utah, #100
South San Francisco, CA 94080

39. www.biomapsystems.com
39