1. Kinase Inhibitors:
Bench Top to Clinic
Christopher J. Larson
Associate Director, Biology
Kemia, Inc.

2. Kinases and Drug Discovery
• 518 Kinases in human genome
• 214 Kinases implicated in disease
• >30% of drug discovery programs
target kinases
• 240 compounds targeting protein
kinases were in development in
05/2004
– 145 in preclinical development
– 27 in PI
– 45 in PII
– 24 in PIII
• Compounds in clinical trials target
about 20 different kinases
– Oncology focused
Manning et al., Science, 6 December 2002
2

3. Kinases Are Validated Therapeutic Targets
Product Company Kinase Target(s) Approved Indications
Gleevec§ BCR/ABL, PDGFR, CML, gastrotintestinal stromal
Novartis
(imatinib) KIT tumors
Nexavar§ Raf, VEGFR-2,
Bayer/Onyx VEGFR-3, KIT, FLT- renal cell carcinoma
(sorafenib) 3, PDGFR-ß
Sutent VEGFR, PDGFR, renal cell carcinoma,
Pfizer
(sunitinib) KIT, FLT-3 gastrointestinal stromal tumors
Tarceva OSI/Genentech/
EGFR NSCLC, pancreatic cancer
(erlotinib) Roche
Iressa
AstraZeneca EGFR NSCLC
(gefitinib)
Sprycel
BMS BCR/ABL, SRC CML, ALL
(dasatinib)
Eril cerebral vasospasm resulting from
Asahi Kisei ROCK
(fasudil) subarachnoid hemorrhage (Japan)
§Binds to the inactive, “DFG-out” conformation of the target kinase(s)
3

4. p38 MAP Kinase as a Drug Target
• MAP kinases integrate, process large number of
extracellular signals
• 3 distinct MAPK pathways
– ERK
• Activated by mitogenic, proliferative stimuli
– JNK
– p38
• Both activated by environmental stress
– Includes inflammatory cytokines
– 60-70% identical
• Differ mainly in sequence, size of activation loop
4

5. Regulation of Cellular Responses by p38
• p38 regulates gene transcription by direct
phosphorylation of transcription factors
• p38 regulates mRNA stability by activating downstream
kinases
– Phosphorylation of AU-rich binding proteins stabilizes IL-1, COX-
2, other inflammatory transcripts
• p38 regulates mRNA translation by activating
downstream kinases
– Translational control proteins
– Major mechanism of p38 effects on TNF
• p38 regulates histone 113 phosphorylation
– NF-kB binding sites upstream of IL-8, MCP-1, other genes
accessible
5

6. p38 Inhibition as a Strategy to Attack Chronic
Inflammatory States
LPS
IL-1β
β
TNFαα
MKK3
MKK6
P38 Kinase
p38 Inhibitors
Inflammation
MAPKAP K2
TRANSLATIONAL
REPRESSION RELEASE
IL-1β
β
Pre-IL-1β
β TNFαα
Pre-TNFαα
6

7. Rationale for p38 Inhibitors in Treatment of RA
and Other Diseases
• p38 regulates cytokine production at transcriptional and
translational levels
• p38 regulates chemotaxis at level of chemokine
expression and cellular chemotactic response
• Variety of chemotypes active in various preclinical
models
– AA and CIA in rodents
– Streptococcal cell wall-induced arthritis
– LPS challenge
– Ischemia/reperfusion in heart, liver, lung
– Cardiac hypertrophy
• Anti-TNF and anti-IL-1 biologics’ efficacy in RA, psoriasis,
Crohn’s disease
7

8. p38 Inhibitors Discontinued From Clinical
Development
• VX745
– 12 weeks 250 mg BID
– ACR20 benefits
– Liver enzyme elevations, other signs
– CNS effects in dog reported
• BIRB796
– Elevated liver enzymes reported in Phase 1 studies
– ~2 uM EC50 in ex vivo LPS challenge
– Reported no efficacy in Crohn’s trial
• RO-3201195
– 75% inhibition of ex vivo LPS-induced IL-1β production by
750 mg BID in 28 day study
8

9. p38 Inhibitors in Clinical Development
• Previous molecules have been dose-limited
by adverse events
– LFT abnormalities
– Rash
– GI irritation
– CNS toxicity
– QTc prolongation
• Lack of unifying toxicity implies chemotype
rather than target
• Strategies that increase selectivity to target
may increases chances of clinical success
9

10. p38 Inhibitors in Clinical Development
• Hypothesis: “safe enough” p38 inhibitor will be medically useful in
RA and other autoimmune/inflammatory conditions driven by IL-1β,
TNFα
• Publicly available data from Vertex in 12 week RA studies
ACR20
10mg VX702 5mg VX702 placebo
40% 38% 30%
250 mg bid VX745 placebo
43% 17%
10

11. Kemia’s Approach To The Challenges in
Kinase Drug Discovery
• Challenges
– Crowded chemical intellectual property space focusing on ATP-
competitive scaffolds
– Poor selectivity of inhibitors
– Clinical toxicities
• Kemia’s Approach
– Target novel chemical space distant from the typical “purine-like”
chemistries
– Target inactive kinase conformations that are incompatible with
ATP-binding
– Utilize slow off-rates to optimize PK/PD relationships that
increase therapeutic indices.
11

12. Many Kinases are Potential Targets for DFG-Out
Inhibitors
• Crystal structures with the inactive DFG-out
conformation have been solved for several kinases
– Tyr kinases - INSR, VEGFR-2, Tie-2, MUSK, IGF1R, ABL,
SRC, FLT3
– Ser/Thr kinases - PKB, Akt-2, p38, RAF
• Additional kinases have the potential to adopt the DFG-
out conformation
• Multiple examples of inhibitors targeting the DFG-out
domain (Gleevec, Nexavar, etc.) provide a motivation
for designing inhibitors targeting kinases of therapeutic
interest
12

13. DFG-In Versus DFG-Out Kinase Inhibitors
• Type I Inhibitors
• Bind in the region normally occupied by the adenine ring of ATP and make similar
contacts to the “hinge” region
• Bind ubiquitous sites that make the design of highly selective inhibitors
problematic
• Bind to the “active” conformation of the kinase similar to that seen with ATP
bound
• Represent the majority of programs that have targeted protein kinases (crowded
IP space)
• Type II Inhibitors
• Bind to regions adjacent to the ATP binding site although can make contacts to
the “hinge” region
• Bind sites that contain significant structural variation that allow for the design of
highly selective inhibitors
• Bind to and stabilize an “inactive” conformation of the kinase with a distinct
(“DFG-out” or “Phe-out”) conformation of the activation loop
• In some cases have very slow off rates (long duration of action)
• Represent minority kinase drug discovery programs to date (greater freedom to
operate)
13

14. Kémia’s Chemistries
• >3000 compounds have been designed and synthesized as
Type II binders for kinases
– Represent several chemical scaffolds
• Chemical scaffolds have been optimized to limit DMPK or
toxicity liabilities
– Solubility
– PAMPA, CACO2
– HLM stability
– Plasma stability
– Plasma protein binding
– CYP inhibition
– hERG inhibition
• Strong intellectual property position
14

15. Targeting p38 for Inhibition
• Publicly available co-crystal structures
– DFG-in
– DFG-out
• Molecular Modeling
• Conventional moieties tied together by a
variety of cores to target
– DFG-out pocket
– Specificity pocket
– Hinge region
15

16. KC706 Summary of In Vitro Results
• Potent, selective p38α inhibitor targeting the Phe-out
pocket
– IC50 = 60 nM (kinase assay)
– IC50 = 50 nM (LPS-stimulated TNFα secretion from THP-1 cells)
– ~10-fold selective vs p38ß, very weak inhibitor of p38γ and
p38δ
– Excellent selectivity profile versus a panel of off-target kinases
• Prevents p38α phosphorylation/activation by upstream
kinases (MKK3/6)
• Slow off-rate/long duration of action (biochemical and
cell-based assays)
• Inhibits LPS-stimulated TNFα and IL-1ß production in
human/rat whole blood
16

17. Time-Dependent Inhibition of p38α Enzymatic
Activity by KC706
100
Preincubation IC50
% Inhibition
75 Time (nM)
0 302
50 t = 0 min 30 38
t = 30 min 60 14
25 t = 60 min 120 8
t = 120 min
0
10 -9 10 -8 10 -7 10 -6 10 -5
[KC706] (M)
Inhibition of enzymatic activity of recombinant human p38α.
Preincubations at 37ºC.
17

18. KC706 Exhibits Time-Dependent IC50 Shift
Characteristic of Some Type II Inhibitors
50 KC706
BIRB796
40 SB-203580
IC50 Ratio*
VX745
30
IC 50 @ 0 min
* IC 50 Ratio =
IC 50 @ time = t
20
10
0
0 50 100
Time
Inhibition of recombinant active p38α
18

19. Type II Inhibitors of p38α Exhibit Long Duration
of Binding
Biacore Analysis
Binding kon koff KD t1/2 Relative
Compound
Mode (M-1sec-1) (sec-1) (nM) (min) Offrate*
SB-203580 Type I 6.1 x 106 0.171 28 <0.1 1
Kémia Series
Type II 0.94 x 104 3.9 x 10-4 41 ~30 438-fold
A Example
Kémia Series
Type II 1.13 x 104 6.2 x 10-4 55 ~19 276-fold
B Example
Studies utilized recombinant, activated p38α at 25°C
*Relative off-rate = t1/2 for indicated compound / t1/2 for SB-203580
19

20. KC706 Wash-out Studies Indicate Half-life of
TNFα Inhibition at Least 10 Hours
BIRB796 SB-203580 KC706
100 60
100
50
75 75
% Inhibition
40
50 30 50
20
25 25
10
0 0 0
10 -8 10 -7 10 -6 10 -5 10 -4 10 -8 10 -7 10 -6 10 -5 10 -4 10 -8 10 -7 10 -6 10 -5 10 -4
[BIRB796] (M) [SB-203580] (M) [KC706] (M)
no washout t=3 hr
t=0 hr t=6 hr
t=1 hr t=8 hr
Leave compound on (“No Wash”), add LPS, incubate, measure TNFα
THP-1 cells; 1hr + compound
Wash out compound Wait 0-8hrs, add LPS, incubate, measure TNFα
20

21. KC706 Inhibition Exhibits Long Duration of Binding,
Stabilization of DFG-out Conformation
• Slow inhibitor binding kinetics
• Indirect evidence for Type II-like mode of
action
– Modeling fits best to DFG-out conformation
– Inhibition of phosphorylation of p38 under
“short” assay conditions
21

22. KC706 Prevents p38α Phosphorylation & Activation
By Upstream Kinases (MKK3/6)
• p38 activated by dual phosphorylation on
Thr180 and Tyr182
• Upstream kinases MKK6 and MKK3
phosphorylate these residues in response to
signals upstream of them
• Phospho-specific antibody detection of
phosphorylation of TGY motif standard method
of detecting p38 activation
• Distinct from MAPKK-independent mechanisms
such as TAB1 and the Lck-ZAP70 mechanism
described by Prof. Miceli
22

23. DFG-Out Inhibitors Function Differently
From ATP-Competitive Inhibitors
Activation PO4 PO4
ATP
Phe Phe Phe
MKK3/MKK6
Inactive protein
“Active” p38
alternates between Phe- ATP-competitive inhibitors
in and Phe-out bind in this conformation
conformations
“Phe-out”
Phe
Inhibitor
PO 4
Phe Phe
“Inactive” p38 Conformation
Phe-out
Inhibitor
p38 locked in Phe-out Phospho-p38 locked in Phe-out
PO4 by MKK3/6 inhibited does not bind ATP
“Inactive” p38 “Inactive” p38
23

24. Targeting Active Versus Inactive Conformations
(DFG-Out) of Kinases
Phe Phe
Activation loop
Activation loop
• Traditional kinase inhibitors (left) compete for binding of ATP to the active conformation
• Allosteric inhibitors (right) stabilize an inactive conformation that cannot bind ATP
24

25. KC706 Inhibits LPS-Induced Phosphorylation
of p38α in Human Whole Blood
100
p38 Phosphorylation
(% Inhibition) 80
60
40
20
0
10 -8 10 -7 10 -6 10 -5
[KC706] (M)
1. Human whole blood pretreated 30 min with KC706
2. Add LPS, incubate 20 min
3. Fix and permeabilize cells
4. Stain with anti-pp38 antibody and control antibodies
5. Flow cytometry
25

26. Selectivity of KC706 Across 45 Kinases (Cerep)
p38α
Potency: Red > Black > Green
BIRB796 KC706 SB203580
BIRB796 KC706 SB203580
26

27. KC706 Inhibits LPS-Induced TNFα Production
in Human Whole Blood
120
KC-706
TNFα Secretion 100 BIRB796
(% Inhibition)
80
60
40
20
0
10 -8 10 -7 10 -6 10 -5
[Compound] (M)
1. Human whole blood diluted 1:1 with RPMI-1640
2. Treat 4 hrs with LPS
3. Quantitate TNFα in supernatant
27

28. KC706 Inhibits LPS-Induced TNFα and IL-1β
Production in Human Whole Blood
TNFα IL-1β
% Inhibition IL-1beta Response
% Inhibition TNF Response
100 100
50 50 IC50 ~70 nM
IC50 ~1300 nM
0 0
-9 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4
Concentration KC706 (logM) Concentration KC706 (logM)
1. Human whole blood diluted 1:1 with RPMI-1640
2. Treat 4 hrs with LPS
3. Quantitate TNFα and IL-1β in supernatant
28

29. KC706 Non-Clinical Pharmacology and
Pharmacokinetics
• Active in acute and sub-chronic models of inflammation
– Carrageenan paw edema (CPE; paw edema and IL-1ß mRNA
induction)
– LPS-stimulated TNFα production
– Collagen induced arthritis (CIA; mice and rats)
• Good pharmacokinetic profile in rats
– Oral bioavailability (%F) ~75 %
– Clearance (Cl) ~19 mL/min/kg
– Terminal half-life (t1/2) ~3-4 hrs
– Volume of distribution (Vss) ~5 L/kg
29

30. Orally Administered KC706 Reduces LPS-
Induced TNFα Levels In Vivo
N=10
6000
5000
pg/ml TNFα
4000
3000
N=6
2000
1000
N=10
0
S
PS
e
in
LP
/L
al
/
6*
le
/s
c
e
70
hi
cl
C
hi
ve
K
ve
In vivo LPS challenge in rat
*30 mg/kg PO
30

31. KC706 Reduces Carrageenan-Induced IL-1ß
mRNA Induction In Vivo
400 No Carrageenan
(Arbitrary Units) Carrageenan
IL-1β mRNA
300
200
100
0
e
)
kg
cl
hi
g/
Ve
0m
(3
6
Rats given vehicle or KC706 PO at t = -2 hrs
70
C
Carrageenan injection at t = 0
K
Sacrifice and isolate total RNA from paw at t = 6 hrs
Quantitative RT-PCR
31

32. Acute Anti-inflammatory Efficacy in Rat
Carrageenan-Induced Paw Edema by KC706
EFFECT OF ORALLY ADMINISTERED KR-002524 ON CARRAGEENAN-INDUCED PAW
EDEMA IN RATS
2.00
1.80
CHANGE IN PAW VOL. (ml)
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20 Vehicle 3m g/kg 10m g/kg 30m g/kg Indom ethacin
0.00
0 2 4 6
TIME (hr)
Rats given vehicle or KC706 PO at t = -2 hrs
Carrageenan injection at t = 0
N = 6 animals/group
32

33. Dose-Dependent Reversal of Signs of
Collagen-Induced Arthritis by KC706
Ankle Diame ter Ove r T ime - KC706
* p ≤0.05 t-test to Arthritis+Vehicle
0.3 45 Normal + V ehicle
Arthritis + V ehicle
KR -00 252 4 30 mg/kg
0.3 35 KR -00 252 4 8 mg/kg
KC706
KR -00 252 4 2 mg/kg
0.3 25 KR -00 252 4 0.4 mg/kg
D ex 0.0 75 mg/kg
0.3 15 E nbrel 10 mg/kg
0.3 05 Bolder BioPATH, Inc.
N=4 rats: Normal Controls
0.2 95 * * *
N=8 rats/treatment group
* * *
* *
0.2 85
* * *
0.2 75 *
* * *
* * * *
0.2 65 *
0.2 55 * 2 * * * * *
Day 0 *
Day 1 *
Day *
Day 3 *
Day 4 *
Day 5 *
Day 6 Day 7
*
Study Day
33

34. KC706 in Clinical Trials
• Initial Phase I trials have been completed
– Highest dose in excess of expected therapeutic
dose
– Excellent bioavailability and dose proportionality
– No drug-related adverse events
– No liver toxicities observed
– Minimal food effect (top single dose)
– Unconjugated bilirubin elevations from partial
UGT1A1 inhibition guided Phase II dosing to
300mg and below
34

35. KC706 Phase 1 Ex Vivo LPS Challenge
Confirms Anti-inflammatory Effect in Man
• Ex vivo LPS challenge assays anti-inflammatory
effect on peripheral blood cells
– Blood sample before and after drug administration
– Blood samples exposed to LPS (bacterial toxin)
– Immune response measured by IL-1ß, TNFα, or
other marker
– Effect assayed by comparing LPS-stimulated
inflammatory cytokines from pre- and post-drug
blood samples
35

36. KC706 Phase 1 Ex Vivo LPS Challenge
Dose-Dependent Inhibition of IL-1β Response
Day 12 Sample (1 hr timepoint)
300
% Baseline IL-1 Response
200
100 *
* *
0
Placebo 80 160 320 640
Dose (mg)
* Statistically significant effect (p <0.01)
36

37. KC706 Inhibition of IL-1β Response to Ex Vivo
LPS Challenge: Long Duration of Action
320 mg Day 12
250
% Baseline IL-1b Response
200 *
150
*
100 *
50
* % Baseline normalized
0
placebo 1hr 6hr 12 hr 24hr
Time
* Statistically significant effect (p <0.05)
37

38. KC706 Current Status
• Challenges in kinase drug discovery
– Crowded chemical intellectual property space focusing on ATP-
competitive scaffolds
– Poor selectivity of inhibitors
– Clinical toxicities
• Kémia’s Approach
– Target novel chemical space distant from the typical “purine-like”
chemistries
– Target kinase conformations that minimize ATP binding
– Utilize slow off-rates to optimize PK/PD relationships that
increase therapeutic indices
• Phase 2a trials with KC706 in RA, Dyslipidemia and
Pemphigus Vulgaris
38

39. p38 Team
• Chemistry
– Antonio Garrido Montalban, Eddine Saiah, Erik Boman, Susana
Conde Ceide, Russell Dahl, David Dalesandro, Nancy G.J.
Delaet, Eric Erb, Justin Ernst, Jeff Kahl, Hiroshi Nakanishi, Ed
Roberts, Robert Sullivan, Zhijun Wang, Nathan Kroll
• Biology
– Stephen G. Miller, Christopher J. Larson, Linda Kessler, Andrew
Gibbs, Jeff Kucharski
• Pharmacology
– Jan Lundstrom, Alison Bendele, Phil Bendele, Sean O’Neill,
Valerie Lowe
• ADMET
– Chau-Dung Chang, Marianne Quintos, Barbara Winningham,
Arnie Garcia, Pauline Chai
• Clinical Development
– Bernard D. King, Constance Crowley, David Shapiro, Bonnie
Hepburn
39

40. Kinase Inhibitors: Benchtop
to Clinic
40