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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