04 kurumbail 2012-ny-neurodegeneration-meeting v3
TRANSCRIPT
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
6th Drug Discovery for Neurodegeneration Conference
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
6th Drug Discovery for Neurodegeneration Conference
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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
6th Drug Discovery for Neurodegeneration Conference
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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)
6th Drug Discovery for Neurodegeneration Conference
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
8
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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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
6th Drug Discovery for Neurodegeneration Conference
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
6th Drug Discovery for Neurodegeneration Conference
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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
6th Drug Discovery for Neurodegeneration Conference
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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
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
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
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
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
6th Drug Discovery for Neurodegeneration Conference
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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)
6th Drug Discovery for Neurodegeneration Conference
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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
6th Drug Discovery for Neurodegeneration Conference
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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
6th Drug Discovery for Neurodegeneration Conference
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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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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
6th Drug Discovery for Neurodegeneration Conference
Thank you!!!
6th Drug Discovery for Neurodegeneration
Conference
23March 26, 2012