04 kurumbail 2012-ny-neurodegeneration-meeting v3
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    04 kurumbail 2012-ny-neurodegeneration-meeting v3 04 kurumbail 2012-ny-neurodegeneration-meeting v3 Presentation Transcript

    • Challenges in Targeting Protein Kinases forNeurodegenerative Diseases – A Case Study onGlycogen Synthase Kinase 3-b for Alzheimer’s Disease Ravi Kurumbail Pfizer 6th Drug Discovery for Neurodegeneration Conference February 14, 2012
    • GSK-3b Project Team 2Structural Biology Chemistry Comp Chem & BiolJeanne Chang Mark Plummer Panos ZagourasHong Wang Mike Chen Ye CheRavi Kurumbail Kim Para Zachary HendschRon Sarver Justine Pine Sridharan SundaramJim Boyd Susan Andrle Veer ShanmugasundaramMark Noe 4 CRO chemists Brajesh RaiSafety, PDM, Pharm. Sci Assays & Biology Project LeaderGreg Cadelina Lori Lopresti-Morrow Ravi KurumbailMichael DePasquale Jose PerezFouad Janat Lorraine LanyonGeorge Chang Pat CosgroveRob Durham Jim Cook Previous GSK-3b projectBrad Enerson Karl Richter team members at PfizerHang Nguyen Charlie NolanChristine Taylor Jeff Asbill Many thanks to PaulHaojing Rong Joel Schachter Galatsis, Travis Wager and XinjunLauren Quattrochi Hou for helpful discussionsPatrick Trapa March 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Overview 3  Background on GSK-3b and its role in Alzheimer’s disease  Development of GSK-3b inhibitors  Chemical target space for CNS compounds and protein kinase inhibitors – what are the opportunities and challenges?  Importance and assessment of kinase selectivity  Cell biology assessment of target safety  Summary and conclusionsMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Protein Kinases as Drug Targets 4  Central role in biology  518 kinases in the human genome - ~1.7% of all genes  Regulation of biological functions through reversible phosphorylation  Involved in nearly every signal transduction cascadeHuman Kinome Map - Science 298:1912  Therapeutic interest  Implicated in disease pathologies  Attractive targets for intervention  Good starting chemical matter or leads for most kinase projects  Precedence in the clinic – approaching 1% of all approved drugs  Potential Issues  Lack of efficacy – redundant pathways  Off-target effects (lack of specificity) March 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Glycogen Synthase Kinase 5 GSK-3b is a serine/threonine protein kinase (46 kDa) ATP Site Substrate- GSK-3a & GSK-3b are ~98% binding Site identical in their catalytic domains Constitutively active in resting cells and gets inactivated upon stimulation (insulin, Wnt etc.) Requires ‘priming’ of substrates for optimal catalytic Axin/FRATTIDE activity docking Site MacAulay, K. & Woodgett, J.R. Expert Opin. Ther. Targets (2008) 12: 1265-1274March 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Role of Glycogen Synthase Kinase-3b in AD – CIR/CIM 6 GSK-3b mediates many aspects of tau neurodegeneration in AD:  One of the kinases involved in hyperphosphorylation of tau which leads to formation of neurofibrillary tangles (NFT) Cytoskeletal support  This leads to destabilization of Axonal transport microtubules which impairs axonal transport and synaptic trafficking Synaptic trafficking  Mediates Ab-induced apoptosis & neuronal cell-death GSK-3b GSK-3b expression/activity is upregulated CDK-5 MARK tau P in the hippocampus of AD patients TTBK1 Over-expression of GSK-3b in transgenic PKA models results in NFT & CAMK2 neurodegeneration Hypothesis: Specific inhibition of GSK-3b could be beneficial for the treatment of AD De-stabilizedMarch 26, 2012 microtubules pTau agg; NFT 6th Drug Discovery for Neurodegeneration Conference
    • Confidence in Safety – The Major Issue for the GSK-3b Project 7 Wnt Signaling Pathway  GSK-3b is a major player in the Wnt Wauwe, J. V. & Haefner, B. signaling pathway – it is an integral Drug News Perspect 16, 557 member of the axin complex (APC, b- (2003) catenin, axin, GSK-3b, & CK1)  Mechanistic link to proliferation – Total or near-total knockdown of GSK-3b could lead to cell proliferation  Mutations in Axin, APC or b-catenin have been linked to colon cancer & other cancers  Key question- Will there be sufficient therapeutic index for a GSK-3b inhibitor?  Set out to evaluate this by developing suitable GSK-3b inhibitors and testing them in cell and animal models – target validation  Important for normal physiology  Control of cellular fate determination & stem cell maintenanceMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Key Challenges/Issues 8  Development of kinase inhibitors that penetrate the blood brain barrier – orthogonal chemical properties for a kinase inhibitor & a CNS drug?  Kinase inhibitors for chronic indications face high safety hurdles – selectivity against other members of the human kinome  Establish the confidence in safety of the target and evaluate the therapeutic index associated with target modulationMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Approved Small Molecule Kinase Drugs 9 N F O HN F F O N H O F N N HN Cl H N N O N O Cl O N O O N N H H N N O N N N N F O H H HN O N NH O N OImatinib (Gleevec; Novartis) Gefitinib (Iressa,; AZ) Erlotinib (Tarceva; OSIP Sorafenib (Nexavar; Onyx/Bayer) Sunitinib (Sutent ; Pfizer)BCR-ABL, c-KIT & PDGF-Rb EGFR; Breast cancer, Genentech); EGFR; RAF1, KDR and PDGF-Rb FLT3, c-KIT, KDR and PDGF-RbCML & GIST (2001) NSCLC (2003) NSCLC, pancreatic cancer RCC, liver cancer (2005) RCC, GIST (2006) (2004) ABS NH N H Cl H HO N N O N N N N N N N NH H N N S O Cl N O HN N N N H N N N HN N Cl F S O N F F N O O S N F O NH2 O N O NH2 Cl F Dasatinib (Sprycel; BMS) Nilotinib (Tasigna; Novartis) Lapatinib (Tykerb ; GSK) Pazopanib (Votrient; GSK) Crizotinib (Xalkori; Pfizer) BCR-ABL, SRC BCR-ABL and 8 others EGFR & Erb2 VEGF, PDGF-R, c-Kit ALK, cMET CML, ALL (2006) CML (2006) Breast cancer (2007) RCC (2009) NSCLC (2011) ABS F Br NH N F H O N HN Cl O H N O N N N S O N N N S O S O F H O N O O N N N N H N N N N N HVandetanib (Caprelsa, AZ) Vemurafenib (Zelboraf; Daiichi Pirfenidone Ruxolitinib (Jakafi; Axitinib (Inlyta; Pfizer) HA-1077 (Fasudil ; AsahiVEGFR, EGFR, RET Sankyo/Roche); BRAF (V600E) (Esbriet; Incyte/Novartis VEGFR, PDGFR, c-Kit Kasei); ROCK-1,2Medullary thyroid cancer Melanoma (2011) Intermune) Jak1, Jak2 RCC (2012) Cerebral vasospasm (1995)(2011) P38g?, TGFb Myelofibrosis (2011) March 26, 2012 IPF (2011) 6th Drug Discovery for Neurodegeneration Conference
    • Property Space for CNS Drugs 10 Property space of high value CNS Drugs:  Molecular weight: 300 (250-350)  logD: 2.5  Polar surface area: 50 (25-75)  # Hydrogen bond acceptors: 4  # Hydrogen bond donors: 1  # Total rings: 4  # Aromatic rings: 2  # Rotatable bonds: 4 (<7)Legend: Black line=mean value, red area=+/-1standard deviation, blue area=min-max CNS MPO Desirability Score (ClogP, logD, Hbond donor, PSA, Mol. Wt, & pKa): 0-6March 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • CNS MPO Desirability as Measure of Drug-likeness Transitional Desirable Undesirable CNS MPO score contribution PF-02545920 1.0 Drug 1.0 Candidate 0.8 PF-03654746 T0_ClogP 0.8 T0_ClogD 4.0 0.6 0.6 5.0 N 0.4 0.4 2.0 3.0 F 0.2 0.2 H F 0 0 N -2 0 2 4 6 -6 -4 -2 0 2 4 6 8 ClogP ClogD O CNS MPO = 4.6 1.0 1.0 CNS MPO = 4.9 0.8 0.8 T0_TPSA T0_MW 0.6 500 0.6 20 120 0.4 360 0.4 40 90 0.2 0.2 PF-03654746 0 0 ClogP 2.4 1.00 PF-02545920 200 300 400 500 600 0 20 40 60 80 100 120 140 ClogP 3.8 0.58 ClogD7.4 0.0 1.00 MW TPSA ClogD7.4 3.5 0.24 TPSA 32.3 0.62 1.0 TPSA 52.8 1.00 1.0 MW 322.4 1.00 0.8 T0_HBD 0.8 T0_pKa MW 392.5 0.77 3.5 HBD 1 0.83 0.6 0.6 HBD 0 1.00 pKa 9.2 0.42 0.4 0.4 8.0 10.0 pKa 4.3 1.00 Desirability Score 4.87 0.2 0.5 0.2 Desirability Score 4.6 0 0 0 1 2 3 4 5 2 4 6 8 10 12 HBD pKa 11Wager TT, Hou X, Verhoest PR, Villalobos A: ACS Chemical Neuroscience (2010) 1(6):435-449.
    • Properties of Protein Kinase Drugs 12 HB- Kinase inhibitor-drug MW ClogP TPSA LogD Donor pKa CNS_MPO cBA Lapatinib_Tykerb 581 5.97 106 3.58 2 6.61 2.17 0.046 Nilotinib_Tasigna 530 5.84 98 5.14 2 5.68 2.25 0.065 Sorafenib_Nexavar 465 5.46 92 5.16 3 1.62 2.34 0.060 Crizotinib_Xalkori 450 4.29 78 2.12 3 10.26 2.82 0.066 Dasatinib_Sprycel 488 2.53 107 3.28 3 6.59 3.06 0.051 Vemurafenib_Zelboraf 490 4.17 92 3.45 2 1.63 3.20 0.085 Vandetanib_Caprelsa 475 5.84 60 2.12 1 9.32 3.29 0.159 Pazopanib_Votrient 438 3.65 119 1.98 3 4.69 3.32 0.072 Imatinib_Gleevec 494 4.53 86 1.76 2 8.03 3.77 0.083 Sunitinib_Sutent 398 3.00 77 0.92 3 9.78 4.00 0.094 Gefitinib_Iressa 447 5.60 69 2.35 1 7.34 4.04 0.194 Axitinib_Inlyta 386 3.33 71 4.15 2 3.35 4.15 0.173 Erotinib_Tarceva 393 4.34 75 0.89 1 5.06 4.92 0.279 HA-1077_Fasudil 291 1.04 62 -1.07 1 9.73 4.97 0.561 Pirfenidone_ Esbriet 185 2.40 22 1.82 0 -0.16 5.10 1.963 Ruxolitinib_Jakafi 306 2.18 83 0.97 1 2.57 5.83 0.506March 26, 2012 Paul Galatsis 6th Drug Discovery for Neurodegeneration Conference
    • Kinase Selectivity Hurdle for CNS Compounds 13  Many kinase inhibitors show a right shift in potency in going from enzyme assay to cellular assays – effect of high cellular concentration of ATP  However, these right shifts are not the same for different kinases (depends on their Km for ATP); this could lead to unanticipated off-target activities  One consequence of limited brain penetration or availability (low brain/plasma ratio) is high relative peripheral concentration of drugs – could lead to on-target or off-target toxicities  Selectivity requirements for CNS kinase inhibitors might be more stringent compared to those for peripheral indicationsMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Optimized GSK-3b Inhibitor 14 Enzyme IC50: 2.3 nM Whole cell IC50: ~450 nM (200X) N Inhibition of pTau inh (CNS): 60-80% N Inhibiton of pGS inh (peripheral): >80% N ClogP: 1.47 LogD @ pH 7.4: 0.0 O N Cl Kinetic solubility >200 mM H No efflux issues (BA/AB – 1.1) N Human hepatic microsomal t1/2: ~80 min O Brain/plasma: ~0.5-1 O PF-367 Safe in cell viability studies (IC50>100 mM) Cardiac safety studies (hERG IC50: >100 mM) LigE: 0.46; LipE: 7.0 Clean in genetox (BiolumeAmes & IVMN) & broad screen panels (CEREP) CNS MPO Desirability score: 5.65 High clearance in rats P450 interaction (DDI effects) Kim Para, Mike Chen, Mark Plummer, Ye CheMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Molecular Interaction of PF-367 with GSK-3b 15 P-Cation Interaction GSK-3b Complex with PF-367 Arg 141 Triazole CH-O H-bond (non-traditional) Buried/interior Glu 137 P- Cation interaction Solvent front Seungil Han, Jeanne Chang Estimated energy: 1-5 Kcal/mol Strong near protein-solvent interfaceMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Kinase Selectivity of PF-367 16 Kinase Selectivity Screen • Profiled in Invitrogen full-kinase panel (252 kinases) • Only nine kinases (except GSK-3a/b) show >40% inhibition @10 mM • ~>1000X specificity for GSK-3a/b over every other kinase tested • Among the elite – strictly ATP competitive yet exquisitely specific forGSK3B (G... CSF1R (FMS) RAF1 (cRAF) Y34... CSNK1E (CK1 epsi... EEF2K KDR (VEGFR2) ALK EGFR (ErbB1) T79... FGFR1 EPHA4 RET GSK-3a/b PF-367 was profiled against 386 unique kinases (442 total) at 10 mM in the Ambit kinome panel (competition binding) - ~75% of the kinome PF-367 ranks among the most selective kinase PF-367 S(35): 0.044 inhibitors that have been reported One of the most selective ATP-competitive kinase inhibitors March 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Central & Peripheral Efficacies of PF-367 nhibitors on phosphoprotein sed as % of control 17 CNS & peripheral efficacies CNS Dose Response nhibitors on phosphoprotein phosphoprotein Effect of GSK-3 inhibitors inhibitors on phosphoprotein Effect of GSK-3 on brain sed as % ofexpressed as % of controlof control control expressed as % immunoreactivity (% of vehicle control) quadriceps pGS (47) 140% 140% pTau (106) (583) 120% 120% N 100% 100% brain brain brain N N (3964) 80% 80% quadriceps quadriceps quadriceps (4956) (1098)89 7 8 6 O 36 36 53 60% 60% N Cl H 3X 02 02 80 48 48 48 40% 40% N -0 -0 -0 O PF PF PF 04-23-08-tau04-23-08-tau O 20% 20% 0% 0% PF-36789 7 8 6 69 37 8 6 569 7 8 6 36e 38e 53 038 36 36 53 l cl 02ic 025 825 hi h 801 80 801 02 02 80 48 e ve v 048 -048 48 48 48 04 -04 -0 -0 -0 -0 PF- PF- PF PF PF PF PF PF 50 mpk s.c Free Brain Exposures shown in parentheses (nM) Cellular IC50 is ~450 nM Richter, Taylor, Chang, Cosgrove March 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • PK/PD Relationship & Time Course of PF-367 18 PK/PD Relationship Time Course Single dose PK (PO & SC) Squares represent efficacy data from Tg4510 mice; all other data points from studies in rats Richter, Taylor, Chang, Hudson, QuattrochiMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Assessment of Target Safety 19 Wauwe, J. V. & Haefner, B.  Activation of Wnt pathway or Drug News Perspect 16, 557 (2003) GSK-3b inhibition leads to b- catenin translocation into nucleus  b-catenin turns on gene transcription events  Proliferative response  Effect of PF-367 on Wnt pathway activation probed by  Nuclear translocation of b- catenin  TopFlash reporter assay that measures gene transcription  Ki67/BrdU markers (cell proliferation) Wnt SignalingMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Cellular Markers of Wnt Pathway Activation – Right-Shifted Relative to Whole Cell IC50 20 b-catenin translocation in HeLa cells Beta-Catenin Nuclear Translocation b-catenin translocation in U2OS cells B -c a te n in T r a n s lo c a tio n As s a y u s in g U2 O S c e lls in HeLa Cells Fold Increase from Control 20 PF-04802367 (6.2 uM) 160000 EC50(mM) PF-04217452 (> 30 uM) 150000 0.11 P F - 0 3 69 4 2 33 Total Counts 15 140000 2.9 P F - 0 4 80 2 3 67 PF-03694233 (0.13 uM) 130000 1.2 P F - 0 4 80 1 5 89 PF-04460611 (~1.99 uM) 120000 10 110000 >10 P F - 0 4 84 0 1 02 PF-04279731 (0.51uM) 100000 PF-04446208 (0.08 uM) 90000 5 80000 PF-367 70000 PF-367 60000 0 50000 0.001 0.01 0.1 1 10 100 -5 -4 -3 -2 -1 0 1 2 uM C onc. (uM)TopFlash Reporter – gene transcription Ki67 expression – proliferation marker Ki 67 Expression in HeLa Cells Fold Increase from Control 4 PF-233 PF-367 PF-04802367 (~9 uM) (EC50: 0.17 mM) (EC50: 20.6 mM) 3 PF-04217452 (> 30 uM) PF-03694233 (0.24 uM) PF-04460611 (~2.3 uM) 2 PF-04279731 (0.8 uM) PF-04446208 (0.14 uM) 1 PF-367 0 0.001 0.01 0.1 1 10 100 uM Lopresti-Morrow & JanatMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Is there a TI for GSK-3b Inhibitor Based on Cell-Based Studies? 21 Whole cell b-catenin b-catenin TopFlash Ki67 marker assay translocation translocation reporter assay (HeLa cells) in HeLa cells in U20S cells (U20S cells) 450 nM 6 mM (13X) 3 mM (6.5X) 21 mM (46X) 9 mM (20X)  Wnt pathway activation can be monitored by multiple downstream events – b-catenin translocation, gene transcription or proliferation markers.  A right shift in GSK-3b inhibitor potency was seen between TAU phosphorylation and WNT signaling.  Assay sensitivity decreased as the output was measured further downstream of GSK- 3b inhibition.  Reporter<Translocation< phospho-Tau<Enzyme  Consistent with previous results from collaborative studies with Epistem  Incorporation of BrdU incorporation into intestinal epithelial cells  Proliferation observed at concentrations ~50-100X cellular IC50s Lopresti-Morrow, Janat, SchachterMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Summary & Conclusions 22  Developed a CNS-active, selective kinase inhibitor scaffold  Identified a highly selective, efficient (LE – 0.46; LipE – 7.0) compound with reasonable oral bioavailability (20%); good aqueous solubility, rapid absorption (Tmax – ~1 hr)  Demonstrated in vivo efficacy as measured by biomarkers (lowering of pTau & pGS biomarker levels ) establishing a PK/PD relationship  Lowered pTau levels in transgenic (Tg4510) mouse model  Demonstrated a right shift of Wnt pathway activation markers from cellular potency for pTau inhibition  Profiled the lead compound in 10-day toxicology studies (up to 250 mpk):  Observed signs of proliferation in liver or kidney at high doses  Does not appear to have the desired therapeutic index (~20X) for the target  CNS-penetrant kinase inhibitors could be developed – requires exceptional kinase selectivityMarch 26, 2012 6th Drug Discovery for Neurodegeneration Conference
    • Thank you!!! 23March 26, 2012 6th Drug Discovery for Neurodegeneration