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2008 Zing Small Molecule Drug Discovery Talk
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2008 Zing Small Molecule Drug Discovery Talk

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    2008 Zing Small Molecule Drug Discovery Talk 2008 Zing Small Molecule Drug Discovery Talk Presentation Transcript

    • Kinase Inhibitors: Bench Top to Clinic Christopher J. Larson Associate Director, Biology Kemia, Inc.
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • Selectivity of KC706 Across 45 Kinases (Cerep) p38α Potency: Red > Black > Green BIRB796 KC706 SB203580 BIRB796 KC706 SB203580 26
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • Kinase Inhibitors: Benchtop to Clinic 40