1. 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. GSK-3b Project Team
2
Structural Biology Chemistry Comp Chem & Biol
Jeanne Chang Mark Plummer Panos Zagouras
Hong Wang Mike Chen Ye Che
Ravi Kurumbail Kim Para Zachary Hendsch
Ron Sarver Justine Pine Sridharan Sundaram
Jim Boyd Susan Andrle Veer Shanmugasundaram
Mark Noe 4 CRO chemists Brajesh Rai
Safety, PDM, Pharm. Sci Assays & Biology Project Leader
Greg Cadelina Lori Lopresti-Morrow Ravi Kurumbail
Michael DePasquale Jose Perez
Fouad Janat Lorraine Lanyon
George Chang Pat Cosgrove
Rob Durham Jim Cook Previous GSK-3b project
Brad Enerson Karl Richter team members at Pfizer
Hang Nguyen Charlie Nolan
Christine Taylor Jeff Asbill Many thanks to Paul
Haojing Rong Joel Schachter Galatsis, Travis Wager and Xinjun
Lauren Quattrochi Hou for helpful discussions
Patrick Trapa
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

3. 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 conclusions
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

4. 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 cascade
Human 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

5. 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: 1265-1274
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

6. 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-stabilized
March 26, 2012 microtubules pTau agg; NFT
6th Drug Discovery for Neurodegeneration Conference

7. 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 maintenance
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

8. 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 modulation
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

9. 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 O
Imatinib (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-Rb
CML & 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
H
Vandetanib (Caprelsa, AZ) Vemurafenib (Zelboraf; Daiichi Pirfenidone Ruxolitinib (Jakafi; Axitinib (Inlyta; Pfizer) HA-1077 (Fasudil ; Asahi
VEGFR, EGFR, RET Sankyo/Roche); BRAF (V600E) (Esbriet; Incyte/Novartis VEGFR, PDGFR, c-Kit Kasei); ROCK-1,2
Medullary 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

10. 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=+/-1
standard deviation, blue area=min-max
CNS MPO Desirability Score (ClogP, logD, Hbond donor, PSA, Mol. Wt, & pKa): 0-6
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

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

12. 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 0.506
March 26, 2012
Paul Galatsis
6th Drug Discovery for Neurodegeneration Conference

13. 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 indications
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

14. 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 Che
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

15. 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 interface
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

16. 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 for
GSK3B (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

17. 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-367
89
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

18. 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, Quattrochi
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

19. 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 Signaling
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

20. 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 & Janat
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

21. 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, Schachter
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

22. 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 selectivity
March 26, 2012 6th Drug Discovery for Neurodegeneration Conference

23. Thank you!!!
23
March 26, 2012 6th Drug Discovery for Neurodegeneration