Kinase Inhibitors:

Bench Top to Clinic

Christopher J. Larson

Associate Director, Biology

Kemia, Inc.

2

• 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

Kinases and Drug Discovery

Manning et al., Science, 6 December 2002

3

Kinases Are Validated Therapeutic Targets

CML, ALLBCR/ABL, SRCBMSSprycel

(dasatinib)

cerebral vasospasm resulting from

subarachnoid hemorrhage (Japan)ROCKAsahi Kisei

Eril

(fasudil)

NSCLCEGFRAstraZenecaIressa

(gefitinib)

NSCLC, pancreatic cancerEGFROSI/Genentech/

Roche

Tarceva

(erlotinib)

renal cell carcinoma,

gastrointestinal stromal tumors

VEGFR, PDGFR,

KIT, FLT-3Pfizer

Sutent

(sunitinib)

renal cell carcinoma

Raf, VEGFR-2,

VEGFR-3, KIT, FLT-

3, PDGFR-ß

Bayer/OnyxNexavar§

(sorafenib)

CML, gastrotintestinal stromal

tumors

BCR/ABL, PDGFR,

KITNovartis

Gleevec§

(imatinib)

Approved IndicationsKinase Target(s)CompanyProduct

§Binds to the inactive, “DFG-out” conformation of the target kinase(s)

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

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

6

p38 Inhibition as a Strategy to Attack Chronic

Inflammatory States

IL-1ββββ

TNFαααα

LPS

IL-1ββββ

TNFααααMKK3

MKK6P38 Kinase

Pre-IL-1ββββ

Pre-TNFαααα

p38 Inhibitors

MAPKAP K2TRANSLATIONAL

REPRESSION RELEASE

Inflammation

IL-1ββββ

TNFαααα

LPS

IL-1ββββ

TNFααααMKK3

MKK6P38 Kinase

Pre-IL-1ββββ

Pre-TNFαααα

p38 Inhibitors

MAPKAP K2TRANSLATIONAL

REPRESSION RELEASE

Inflammation

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 chemokineexpression 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

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

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 chemotyperather than target

• Strategies that increase selectivity to target may increases chances of clinical success

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

10mg VX702 5mg VX702 placebo

40% 38% 30%

250 mg bid VX745 placebo

43% 17%

ACR20

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.

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

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)

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

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

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

17

Time-Dependent Inhibition of p38α Enzymatic

Activity by KC706

8120

1460

3830

3020

IC50

(nM)

PreincubationTime

Inhibition of enzymatic activity of recombinant human p38α.Preincubations at 37ºC.

10 -9 10-8 10-7 10 -6 10-5

0

25

50

75

100

t = 0 min

t = 30 min

t = 60 min

t = 120 min

[KC706] (M)

% Inhibition

18

KC706 Exhibits Time-Dependent IC50 Shift

Characteristic of Some Type II Inhibitors

0 50 100

0

10

20

30

40

50KC706

BIRB796

SB-203580

VX745

Time

IC50 Ratio*

ttimeIC

ICRatioIC

==

@50

min0@5050*

Inhibition of recombinant active p38α

19

Type II Inhibitors of p38α Exhibit Long Duration

of Binding

~19

~30

<0.1

t1/2(min)

276-fold

438-fold

1

Relative

Offrate*

55

41

28

KD(nM)

6.2 x 10-4

3.9 x 10-4

0.171

koff(sec-1)

Type II

Type II

Type I

Binding

Mode

1.13 x 104Kémia Series

B Example

0.94 x 104Kémia Series

A Example

6.1 x 106SB-203580

kon(M-1sec-1)

Compound

Studies utilized recombinant, activated p38α at 25°C

*Relative off-rate = t1/2 for indicated compound / t1/2 for SB-203580

Biacore Analysis

20

10 -8 10-7 10 -6 10-5 10 -40

25

50

75

100

[BIRB796] (M)

% Inhibition

BIRB796 SB-203580 KC706

10 -8 10 -7 10 -6 10-5 10 -40

10

20

30

40

50

60

[SB-203580] (M)

no washout

t=0 hr

t=1 hr

t=3 hr

t=6 hr

t=8 hr

THP-1 cells; 1hr + compound

Leave compound on (“No Wash”), add LPS, incubate, measure TNFα

Wash out compound Wait 0-8hrs, add LPS, incubate, measure TNFα

10-8 10-7 10 -6 10-5 10-40

25

50

75

100

[KC706] (M)

KC706 Wash-out Studies Indicate Half-life of

TNFα Inhibition at Least 10 Hours

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

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

23

PO 4

Phe

Phe

Inactive protein

alternates between Phe-

in and Phe-out

conformations

“Active” p38

Phe

Phe-out

Inhibitorp38 locked in Phe-out

PO4 by MKK3/6 inhibited

“Inactive” p38

Activation PO4

Phe

PO4ATP

Phe

DFG-Out Inhibitors Function Differently

From ATP-Competitive Inhibitors

MKK3/MKK6

Phe

Phospho-p38 locked in Phe-out

does not bind ATP

“Inactive” p38

“Phe-out”

Inhibitor

ATP-competitive inhibitors

bind in this conformation

“Inactive” p38 Conformation

24

Targeting Active Versus Inactive Conformations

(DFG-Out) of Kinases

• Traditional kinase inhibitors (left) compete for binding of ATP to the active conformation

• Allosteric inhibitors (right) stabilize an inactive conformation that cannot bind ATP

PhePhe

Activation loop

Activation loop

25

KC706 Inhibits LPS-Induced Phosphorylation

of p38α in Human Whole Blood

10-8 10-7 10-6 10-5

0

20

40

60

80

100

[KC706] (M)

p38 Phosphorylation

(% Inhibition)

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

26

p38α

BIRB796 KC706 SB203580

BIRB796 KC706 SB203580

Potency: Red > Black > Green

Selectivity of KC706 Across 45 Kinases (Cerep)

27

KC706 Inhibits LPS-Induced TNFα Production

in Human Whole Blood

10 -8 10-7 10-6 10-5

0

20

40

60

80

100

120KC-706

BIRB796

[Compound] (M)

TNFαα αα Secretion

(% Inhibition)

1. Human whole blood diluted 1:1 with RPMI-1640

2. Treat 4 hrs with LPS

3. Quantitate TNFα in supernatant

28

-9 -8 -7 -6 -5 -40

50

100

Concentration KC706 (logM)

% Inhibition TNF Response

-9 -8 -7 -6 -5 -40

50

100

Concentration KC706 (logM)

% Inhibition IL-1beta Response

1. Human whole blood diluted 1:1 with RPMI-1640

2. Treat 4 hrs with LPS

3. Quantitate TNFα and IL-1β in supernatant

KC706 Inhibits LPS-Induced TNFα and IL-1β

Production in Human Whole Blood

TNFα IL-1β

IC50 ~1300 nMIC50 ~70 nM

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

30

Orally Administered KC706 Reduces LPS-

Induced TNFα Levels In Vivo

vehicle/saline

vehicle/LP

S

KC706*/LPS

0

1000

2000

3000

4000

5000

6000

pg/ml TNFαα αα

N=10

N=10

N=6

In vivo LPS challenge in rat

*30 mg/kg PO

31

KC706 Reduces Carrageenan-Induced IL-1ß

mRNA Induction In Vivo

Vehicle

KC706 (30m

g/kg)

0

100

200

300

400 No Carrageenan

Carrageenan

IL-1ββ ββ mRNA

(Arbitrary Units)

Rats given vehicle or KC706 PO at t = -2 hrs

Carrageenan injection at t = 0

Sacrifice and isolate total RNA from paw at t = 6 hrs

Quantitative RT-PCR

32

Acute Anti-inflammatory Efficacy in Rat

Carrageenan-Induced Paw Edema by KC706

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0 2 4 6

TIME (hr)

CHANGE IN P

AW V

OL. (m

l)

Vehicle 3mg/kg 10mg/kg 30mg/kg Indomethacin

EFFECT OF ORALLY ADMINISTERED KR-002524 ON CARRAGEENAN-INDUCED PAW

EDEMA IN RATS

Rats given vehicle or KC706 PO at t = -2 hrs

Carrageenan injection at t = 0

N = 6 animals/group

33

Dose-Dependent Reversal of Signs of

Collagen-Induced Arthritis by KC706

Ankle Diameter Over Time - KC706

0.255

0.265

0.275

0.285

0.295

0.305

0.315

0.325

0.335

0.345

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Study Day

Normal + VehicleArthritis + VehicleKR-002524 30 mg/kgKR-002524 8 mg/kgKR-002524 2 mg/kgKR-002524 0.4 mg/kgDex 0.075 mg/kgEnbrel 10 mg/kg

Bolder BioPATH, Inc.

N=4 rats: Normal Controls

N=8 rats/treatment group

* p≤0.05 t-test to Arthritis+Vehicle

** *

**

**

**

**

*** ******

* * * * * ********

KC706

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

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

36

KC706 Phase 1 Ex Vivo LPS Challenge

Dose-Dependent Inhibition of IL-1β Response

Day 12 Sample (1 hr timepoint)

Placebo 80 160 320 6400

100

200

300

Dose (mg)

**

*

% Baseline IL-1 Response

* Statistically significant effect (p <0.01)

37

320 mg Day 12

placebo 1hr 6hr 12 hr 24hr0

50

100

150

200

250

Time

% Baseline IL-1b R

esponse

KC706 Inhibition of IL-1β Response to Ex Vivo

LPS Challenge: Long Duration of Action

% Baseline normalized

* Statistically significant effect (p <0.05)

*

*

*

*

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

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

40

Kinase Inhibitors: Benchtop

to Clinic