04 kurumbail 2012-ny-neurodegeneration-meeting v3

Challenges in Targeting Protein Kinases for

Neurodegenerative Diseases – A Case Study on

Glycogen Synthase Kinase 3-b for Alzheimer’s

Disease

Ravi Kurumbail

Pfizer

6th Drug Discovery for

Neurodegeneration Conference

February 14, 2012

2

March 26, 2012

GSK-3b Project Team

Structural BiologyJeanne ChangHong WangRavi KurumbailRon SarverJim BoydMark Noe

ChemistryMark PlummerMike ChenKim ParaJustine PineSusan Andrle4 CRO chemists

Comp Chem & BiolPanos ZagourasYe CheZachary HendschSridharan SundaramVeer ShanmugasundaramBrajesh Rai

Assays & BiologyLori Lopresti-MorrowJose PerezLorraine LanyonPat CosgroveJim CookKarl RichterCharlie NolanJeff AsbillJoel Schachter

Safety, PDM, Pharm. SciGreg CadelinaMichael DePasqualeFouad JanatGeorge ChangRob DurhamBrad EnersonHang NguyenChristine TaylorHaojing RongLauren QuattrochiPatrick Trapa

Previous GSK-3b project team members at Pfizer

Many thanks to Paul

Galatsis, Travis Wager and Xinjun

Hou for helpful discussions

Project Leader

Ravi Kurumbail

6th Drug Discovery for Neurodegeneration Conference

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March 26, 2012

Overview

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 conclusions


Protein Kinases as Drug Targets

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 cascade

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)

Human Kinome Map - Science 298:1912

March 26, 2012

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March 26, 2012

Glycogen Synthase Kinase

GSK-3b is a serine/threonine

protein kinase (46 kDa)

GSK-3a & GSK-3b are ~98%

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

activity

ATP Site Substrate-

binding Site

Axin/FRATTIDE

docking Site

MacAulay, K. & Woodgett, J.R.

Expert Opin. Ther. Targets

(2008) 12: 1265-1274


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March 26, 2012

Role of Glycogen Synthase Kinase-3b in AD – CIR/CIM

GSK-3b mediates many aspects of

neurodegeneration in AD:

One of the kinases involved in

hyperphosphorylation of tau which leads to

formation of neurofibrillary tangles (NFT)

This leads to destabilization of

microtubules which impairs axonal

transport and synaptic trafficking

Mediates Ab-induced apoptosis & neuronal

cell-death

GSK-3b expression/activity is upregulated

in the hippocampus of AD patients

Over-expression of GSK-3b in transgenic

models results in NFT &

neurodegeneration

Hypothesis: Specific inhibition of GSK-3b

could be beneficial for the treatment of AD

tau

Cytoskeletal support

Axonal transport

Synaptic trafficking

tau P

GSK-3b

CDK-5

MARK

TTBK1

PKA

CAMK2

De-stabilized microtubules pTau agg; NFT


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March 26, 2012

Confidence in Safety – The Major Issue for the GSK-3b Project

GSK-3b is a major player in the Wnt

signaling pathway – it is an integral

member of the axin complex (APC, b-

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

Wnt Signaling Pathway

Important for normal physiology

Control of cellular fate determination &

stem cell maintenance

Wauwe, J. V. & Haefner, B.

Drug News Perspect 16, 557

(2003)


Key Challenges/Issues

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 modulation

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March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

Approved Small Molecule Kinase Drugs

Gefitinib (Iressa,; AZ)EGFR; Breast cancer, NSCLC (2003)

Sunitinib (Sutent ; Pfizer)FLT3, c-KIT, KDR and PDGF-Rb

RCC, GIST (2006)

Erlotinib (Tarceva; OSIP Genentech); EGFR; NSCLC, pancreatic cancer (2004)

Imatinib (Gleevec; Novartis)BCR-ABL, c-KIT & PDGF-Rb

CML & GIST (2001)

Sorafenib (Nexavar; Onyx/Bayer)RAF1, KDR and PDGF-Rb

RCC, liver cancer (2005)

Dasatinib (Sprycel; BMS)BCR-ABL, SRCCML, ALL (2006)

N

N

NH

N

NH

O

N

N

NH

N

N

O

O

Cl

F

N

ON

NH

NO

O

O

O

O

N

NH

O

NH

NH

OCl

FF

F

NH

NH

O

N

O

NH

F

N N

N

NOH

NH

N

S

NH

O Cl

Nilotinib (Tasigna; Novartis)BCR-ABL and 8 othersCML (2006)

Pazopanib (Votrient; GSK)VEGF, PDGF-R, c-KitRCC (2009)

Lapatinib (Tykerb ; GSK)EGFR & Erb2Breast cancer (2007)

N

N

N

NHNH

NO

NFF

F

N

NNN

N

NH

SO

O

NH2

NH

N

N

O

Cl

O

NH

S

O O

F

Crizotinib (Xalkori; Pfizer)ALK, cMETNSCLC (2011)

ABS

N

NH2

O

NN

Cl

Cl

F

NH

N

N

NH

F Br

O

O

N

Vandetanib (Caprelsa, AZ)VEGFR, EGFR, RETMedullary thyroid cancer(2011)

N NH

O

F

FNH

S

O

O

Cl

Vemurafenib (Zelboraf; Daiichi Sankyo/Roche); BRAF (V600E)Melanoma (2011)

NO

ABS

N

N NH

NN

N

NH

NS

N

NH

O

S

N

O O

NH

N

Ruxolitinib (Jakafi; Incyte/NovartisJak1, Jak2Myelofibrosis (2011)

Pirfenidone(Esbriet; Intermune)P38g?, TGFb

IPF (2011)

Axitinib (Inlyta; Pfizer)VEGFR, PDGFR, c-KitRCC (2012)

HA-1077 (Fasudil ; Asahi Kasei); ROCK-1,2Cerebral vasospasm (1995)

March 26, 2012

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March 26, 2012

Property Space for CNS Drugs

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=+/-1 standard deviation, blue area=min-max

CNS MPO Desirability Score (ClogP, logD, Hbond donor, PSA, Mol. Wt, & pKa): 0-6


T0

_C

log

P

Transitional

Undesirable

ClogP

0

0.2

0.4

0.6

0.8

1.0

-2 0 2 4 6

3.0

5.0

Desirable

Drug

Candidate

0

0.2

0.4

0.6

0.8

1.0

-6 -4 -2 0 2 4 6 8

T0

_C

log

D

ClogD

4.0

2.0

0

0.2

0.4

0.6

0.8

1.0

0 20 40 60 80 100 120 140

40

120

TPSAT

0_

TP

SA

20

90

0

0.2

0.4

0.6

0.8

1.0

2 4 6 8 10 12

T0

_p

Ka

10.08.0

pKa

T0

_H

BD

HBD

0

0.2

0.4

0.6

0.8

1.0

200 300 400 500 600

MW

T0

_M

W 500

360

0

0.2

0.4

0.6

0.8

1.0

0 1 2 3 4 5

3.5

0.5

CNS MPO Desirability as Measure of

Drug-likeness

ClogP 2.4 1.00

ClogD7.4 0.0 1.00

TPSA 32.3 0.62

MW 322.4 1.00

HBD 1 0.83

pKa 9.2 0.42

4.87

PF-03654746

Desirability Score

N

HN

O

F

F

PF-03654746

CNS MPO = 4.9CNS MPO = 4.6

PF-02545920

ClogP 3.8 0.58

ClogD7.4 3.5 0.24

TPSA 52.8 1.00

MW 392.5 0.77

HBD 0 1.00

pKa 4.3 1.00

4.6

PF-02545920

Desirability Score

CN

S M

PO

sco

re c

on

trib

uti

on

Wager TT, Hou X, Verhoest PR, Villalobos A: ACS Chemical Neuroscience (2010) 1(6):435-449. 11

Properties of Protein Kinase Drugs12

March 26, 2012

Kinase inhibitor-drug MW ClogP TPSA LogD

HB-

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 0.506

Paul Galatsis6th Drug Discovery for Neurodegeneration Conference

Kinase Selectivity Hurdle for CNS Compounds

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 indications

13

6th Drug Discovery for Neurodegeneration ConferenceMarch 26, 2012

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March 26, 2012

Optimized GSK-3b Inhibitor

O

N

Cl

O

NH

O

NN

NEnzyme IC50: 2.3 nM

Whole cell IC50: ~450 nM (200X)

Inhibition of pTau inh (CNS): 60-80%

Inhibiton of pGS inh (peripheral): >80%

ClogP: 1.47

LogD @ pH 7.4: 0.0

Kinetic solubility >200 mM

No efflux issues (BA/AB – 1.1)

Human hepatic microsomal t1/2: ~80 min

Brain/plasma: ~0.5-1

Safe in cell viability studies (IC50>100 mM)

Cardiac safety studies (hERG IC50: >100 mM)

Clean in genetox (BiolumeAmes & IVMN) &

broad screen panels (CEREP)

High clearance in rats

P450 interaction (DDI effects)

PF-367

LigE: 0.46; LipE: 7.0

CNS MPO Desirability score: 5.65

Kim Para, Mike Chen, Mark Plummer, Ye Che


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March 26, 2012

Molecular Interaction of PF-367 with GSK-3b

Seungil Han, Jeanne Chang

Buried/interior

Solvent front

P- Cation interaction

CH-O H-bond (non-traditional)

GSK-3b Complex with PF-367P-Cation Interaction

TriazoleArg 141

Glu 137

Estimated energy: 1-5 Kcal/mol

Strong near protein-solvent interface6th Drug Discovery for Neurodegeneration Conference

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March 26, 2012

Kinase Selectivity of PF-367

Kinase Selectivity Screen

GSK3B (G... CSF1R (FMS) RAF1 (cRAF) Y34... CSNK1E (CK1 epsi... EEF2K KDR (VEGFR2) ALK EGFR (ErbB1) T79... FGFR1 EPHA4 RET

• 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 for

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

inhibitors that have been reported

One of the most selective ATP-competitive kinase

inhibitors

PF-367

S(35): 0.044


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March 26, 2012

Central & Peripheral Efficacies of PF-367

Richter, Taylor, Chang, Cosgrove

O

N

Cl

O

NH

O

NN

N

(1098)

3X

CNS & peripheral efficacies

Effect of GSK-3 inhibitors on phosphoprotein

expressed as % of control

0%

20%

40%

60%

80%

100%

120%

140%

vehicle

PF-

0480

1589

PF-

0480

2367

PF-

0480

2368

PF-

0488

0536

imm

un

ore

acti

vit

y (

% o

f veh

icle

co

ntr

ol)

brain

quadriceps

04-23-08-tau



0%

20%

40%

60%

80%

100%

120%

140%

vehicle

PF-

0480

1589

PF-

0480

2367

PF-

0480

2368

PF-

0488

0536

imm

un

ore

acti

vit

y (

% o

f veh

icle

co

ntr

ol)

brain

quadriceps

04-23-08-tau



0%

20%

40%

60%

80%

100%

120%

140%

vehicle

PF-

0480

1589

PF-

0480

2367

PF-

0480

2368

PF-

0488

0536

imm

un

ore

acti

vit

y (

% o

f veh

icle

co

ntr

ol)

brain

quadriceps

04-23-08-tau



0%

20%

40%

60%

80%

100%

120%

140%

vehicle

PF-

0480

1589

PF-

0480

2367

PF-

0480

2368

PF-

0488

0536

imm

un

ore

acti

vit

y (

% o

f veh

icle

co

ntr

ol)

brain

quadriceps

04-23-08-tau

50 mpk s.c

pGSpTau

Free Brain Exposures shown in parentheses (nM)

Cellular IC50 is ~450 nM

(47)(106)

(583)

(3964)(4956)

CNS Dose Response

PF-367


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March 26, 2012

PK/PD Relationship & Time Course of PF-367

Squares represent efficacy data

from Tg4510 mice; all other

data points from studies in rats

Richter, Taylor, Chang, Hudson, Quattrochi

PK/PD Relationship Time Course

Single dose PK (PO & SC)


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March 26, 2012

Assessment of Target Safety

Activation of Wnt pathway or

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)

Wauwe, J. V. & Haefner, B.

Drug News Perspect 16, 557

(2003)

Wnt Signaling


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March 26, 2012

Cellular Markers of Wnt Pathway Activation – Right-Shifted

Relative to Whole Cell IC50

b-catenin translocation in HeLa cells b-catenin translocation in U2OS cellsBeta-Catenin Nuclear Translocation in HeLa Cells

0.001 0.01 0.1 1 10 1000

5

10

15

20PF-04802367 (6.2 uM)

PF-04217452 (> 30 uM)

PF-03694233 (0.13 uM)

PF-04460611 (~1.99 uM)

PF-04279731 (0.51uM)

PF-04446208 (0.08 uM)

uM

Fo

ld In

crease f

ro

m C

on

tro

l

PF-367

-5 -4 -3 -2 -1 0 1 250000

60000

70000

80000

90000

100000

110000

120000

130000

140000

150000

160000P F-03694233

P F-04802367

P F-04801589

P F-04840102

B -ca ten in T rans loca tio n Assay u s ing U2O S ce lls

C onc. (uM)

To

tal

Co

un

ts

PF-367

TopFlash Reporter – gene transcription

PF-367

(EC50: 20.6 mM)

PF-233

(EC50: 0.17 mM)

Ki67 expression – proliferation marker Ki 67 Expressionin HeLa Cells

0.001 0.01 0.1 1 10 1000

1

2

3

4PF-04802367 (~9 uM)

PF-04217452 (> 30 uM)

PF-03694233 (0.24 uM)

PF-04460611 (~2.3 uM)

PF-04279731 (0.8 uM)

PF-04446208 (0.14 uM)

uM

Fo

ld In

cre

ase f

rom

Co

ntr

ol

PF-367

Lopresti-Morrow & Janat

EC50(mM)

0.11

2.9

1.2

>10


21

March 26, 2012Lopresti-Morrow, Janat, Schachter

Is there a TI for GSK-3b Inhibitor Based on Cell-Based Studies?

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

Whole cell

assay

b-catenin

translocation

in HeLa cells

b-catenin

translocation

in U20S cells

TopFlash

reporter assay

(U20S cells)

Ki67 marker

(HeLa cells)

450 nM 6 mM (13X) 3 mM (6.5X) 21 mM (46X) 9 mM (20X)


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March 26, 2012

Summary & Conclusions

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 selectivity


Thank you!!!

6th Drug Discovery for Neurodegeneration

Conference

23March 26, 2012

Technology

04 kurumbail 2012-ny-neurodegeneration-meeting v3