Download Chemoproteomic applications in kinase drug discovery

Chemoproteomic
applications in kinase drug
discovery
di
Kinase 2014: past, present
and beyond
y
Douglas Thomson
Overview
– Introduction and Overview of the Cellzome Proteomic Platform
– Application of the Platform to the Discovery of a PI3K in vivo Probe
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Cellzome’s technologies use cell or tissue lysate
drug
g
resin
Proteome expression
effects:
Measure proteome
dynamics upon drug
treatment
Target deconvolution:
Identify targets of active compounds
from disease relevant screens
Target class profiles:
Test compounds against
entire target classes
(e.g. KinobeadsTM)
Pathway effects:
Follow changes in
signaling upon drug
treatment
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Target Class Profiling
– Binding
g affinityy measurement of compounds
p
to native kinases directly in cellular or tissue
lysate
– No recombinant protein used
Lysates can be prepared
that allow acess to
activated kinases
– Access to difficult targets e.g. lipid kinases
Itk
ZAP
70
Lck
Directly from tissue –
e.g. mouse brain gives acess to other proteins
T-cell
NFAT
GATA-3
STAT6
– One experiment can simutaneously screen
against whole captured proteome
– Kinases -KinobeadsTM
– Epigenetic targets -EpisphereTM
– Rapid assay development and screening
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Compound target profiling
Inhibito
or concentration
1. Add inhibitor to cells or
cell lysate over a range
of concentrations
2. Incubate - inhibitor binds
to targets in cells or
lysate
3. Add affinity matrix
to lysates
4. Elute beads, digest, label
with TMT6 (Tandem Mass Tag)
0 0 µM
0.0
(DMSO,
Control)
TMT 131
0.039 µM
TMT 130
5. Mix, run LC-MS/MS and
quantify in MS/MS spectrum
Protein does
bindsnot
to drug
bind to drug
0.156 µM
TMT 129
0.625 µM
TMT 128
2 5 µM
2.5
M
TMT 127
10 µM
TMT 126
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Cellzome Chemoproteomic Platform (screening)
Matrix
Capture
compounds
on beads
compound
library
Total protein extracts
are prepared from cell
or tissue samples
Pulldown step: Reduction
of target binding to beads
in the presence of excess
"free" compound
IC50 values
for target
Eluted material is spotted
on microarray membrane
detection of target proteins on
dot-microarrays by antibody
Beads are separated and Beadbound proteins are eluted by SDS
elution buffer
Screen > 100 000 compounds
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Kinobeads™
Combination of broad
spectrum ATP mimetics
Approx. 50 kinase inhibitor probes
Bantscheff et al. (2007) Nat Biotechnol 25:1035-44
•
•
•
•
> 350 human protein kinases
>15 lipid kinases (PI3K, PI4K, PIP5K)
Many other purine-binding proteins
Exact number depends on lysate
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Selectivity profiles of clinical kinase inhibitors
Glivec
p(IC50)
p
Dasatinib
Bosutinib
Bantscheff et al. (2007) Nat Biotechnol 25:1035-44
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Kinase protein complexes are preserved
Glivec, Dasatinib, Bosutinib
– Complex partners are quantified in proteomic experiment
– These indirect binders to matrix have similar apparent pIC50 to direct
binders
Kruse et al., Leukemia 2011, 25: 89-100
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Application of Platform to the Discovery of
a PI3K in vivo Probe
PI3K inhibition has anti-inflammatory effects
COPD
Thomas, M. et al, Eur. J.
Immunol., 2005
Allergic Rhinitis
Pinho et al, J. Leukoc. Biol. 2005
Asthma
Pinho et al, J. Leukoc. Biol. 2005
Rheumatoid Arthritis
Camps et al
al, Nat
Nat. Med
Med, 2005
Sepsis
Martin et al, Am J Respir Crit
C
Care
M
Med.
d 2010
Systemic Lupus
Erythrematosus (SLE)
Barber et al., Nat. Med. 2005
ANCA-induced
Glomerulonephritis
Inflammatory Bowel
Disease (IBD):
Schreiber et al
al,
kidney intern. 2009
Gonzalez Garcia,
Gonzalez-Garcia
Gastroenterology, 2010
PI3K -/- mice published 2000 (Hirsch et al Science)
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Selectivity is key: off-target effects mediated by PI3K
family members
PI3K
PI3K
Heart: contractile dysfunction
Lu, Circulation, 2009
PI3K
Insulin resistance
Knight, Cell, 2006
PI3K
PI3K
Eosinophilic infiltration in
different organs, particularly
gastritis
Ji, Blood, 2007
Reproductive organs:
Testes reduced in size,
degeneration of germ cells
Ciraolo, Mol Cell Biol, 2010
PI3K
Increased IgE production
Zhang, J Allergy Clin Immunol
2008
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Establising a Chemoproteomic Assay
Cell lysate
3 Sources of the
proteins were
established
Human blood
immobilize
probe
PI3K
mTOR
Tissue lysate
design
probe
PI3K
identify
cell source
PI3K
DNAPK
format
binding
assay
Multiplex assay setup
allowing simultaneous
analysis of 6 closely
related kinases
PI3K
screen
lead
opt
Antibody validated for
each
h target
t
t enabling
bli
dot-microarray readout
Know PI3K inhibitors
PIK-93 and LY294002
were linked to beads
Hit ID: library
screening 16,146
compounds
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From multiplexed screening to selective probes: PI3K
inhibitors
Library screening
DNAPK PI3K
PI3K
Compoun
nds
mTor
Bergamini et al, NatChemBiol, June 2012
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Identification of a selective PI3K inhibitor
CZC24832
MW
364
LogD
1.36
Solubility
65 M
LE
0.4
PPB
(h/m)
34%/24%
Cl in Liver microsomes
(h/m/r)
0/1/0.4 l/min/mg
hERG
No inhibition at 100 M
CYP1A, 2C19, 2C9, 2D6
and 3A4
No inhibition at 25 mM
PK in rat: 0.2 mg/kg iv and 10 mg/kg po
 Cl: 14 ml/min/kg
 F: 37%
Bergamini et al, NatChemBiol, June 2012
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KinobeadsTM Profiling of CZC24832
PI3K
F
HN
N
O
N N
S
O
NH2
N
Bergamini et al, NatChemBiol, June 2012
Protein
pKdapp
PI3K
7.6
PI3K
6.1
PI3K
51
5.1
PI3K
<5
PIP4K2C
5.8
CK1
5.5
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Affinity Purification
 Imoblised analogue was used as matrix
 CZC24832 was used as competitor
p
at 280 nM
 Either HL-60 or PBMC cells were used as lysate source
No additional proteins were captured and competed
Bergamini et al, NatChemBiol, June 2012
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KinobeadsTM Profoling of CZC24832 in Different Species
 Potency drop for PI3K
PI3K,  of 2-5
2 5 fold in rodents
 Selectivity maintained
Bergamini et al, NatChemBiol, June 2012
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CZC24832 shows efficacy in mouse RA model
Collagen induced arthritis model
50
5,0
4,5
Disease
control
Mean±SE Clinical Arthritis Sco
ore
(Scored 0-5)
4,0
35
3,5
CZC24832
3 mg/kg
*
3,0
*
2,5
CZC24832
*
20
2,0
*
*
*
*
*
*
*
*
*
*
*
CZC24832
10 mg/kg
Dex
0.01mg/kg
*
1,5
1,0
05
0,5
0,0
0
1
2
3
4
5
6
7
8
Arthritis Day
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10
11
12
13
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– Dose-dependent
Dose dependent efficacy mouse CIA model
– Reduction in severity of arthritis is comparable to PI3K-/- mice
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Summary
– An in vivo probe (CZC24832) for PI3K was discovered using our Chemoproteomic
Platform
– CZC24832 is potent for PI3K in both proteomic competition binding assays and cell
based assays
– CZC24832 has excellent selectivity for PIK3 within the PIK3 family and the rest of kinome
– CZC24832 can be used for the further pharmacological investigation and validation of
PI3K
– However CZC24832 has limitations that stop
p it´s further p
progression
g
as a drug
g candidate
– Low solubility
– Non-linear PK at higher doses
– Moderate efficacy
y in CIA models
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Thank you!
All Cellzomers past and
presentt
In particular
Gitte Neubauer
Giovanna Bergamini
Andrew Cansfield
Kathryn Bell
Thilo Werner
Marcus Bantscheff
Gerard Drewes
Our collaborators in particular:
Emilio Hirsch
Alison O‘Mahoni
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