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This powerpoint slide deck is also available as oral presentation held by Dr. Ekaterina Kuznetsova
Follow link belowhttps://youtu.be/TuObnovpqoE
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A contract research organization
providing
services and integrated partnership
for preclinical drug discovery
3
202020192018201420122010200620032001
Founded in Freiburg,
Germany
FLT3 kinase inhibitor
discovery with 4SC
Integrated discovery
of an AML drug
CDK8 inhibitor development with
MerchaChem (started 2008)
Joined the European Consortium for Tumour Angiogenesis Research
Founded in Malvern,
PA, USA
Launched HotSpot
Kinase Profiling
Service
Acquisition of
ProQinase
Aquisition Ion Channel
Screening Platform
Part of the chemical consortium for epigenetic
inhibitor discovery
2015
ChinaEquityInvestment
Launched Epigenetic
Profiling Service
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Kinases
Epigenetics
Ion channels
GPCRs
Proteases, phosphatases
and many more
Cell proliferation assays
Soft Agar assay
3D Tumor Spheroid
Metastasis assays
Cell-line generation
Cell-based Angiogenesis
assay
Surface plasmon
resonance
ITC
Microscale
thermophoresis
Thermal shift assay
Hollow Fiber Model
Xenograft tumor
models
Syngeneic tumor
models
Kinases
Epigenetic proteins
Substrates
Inhibitors
Custom-tailored
protein production
Cardiac Safety
Panel
CYP inhibition
PK/PD studies
Maximum-tolerated
dose
Cell-line tumor models
Tissue-transfer tumor models
Subcutaneous, subQperior,
orthotopic and metastasis models
3
5
RAS Family:
• small GTPases: KRas, HRas and NRas
• known oncogenes that are frequently mutated in a large
percentage of cancers
• Alterations contribute to 20-30% of all human
cancers
• Mutated in ~90% percent in pancreatic cancer
• associated with poor disease prognosis
• Aberrant activation leads to increased RAS signaling that
promote cancer development
RAS
Upstream activators
RAS
RAF PI3K TIAM1
Oncogenic transcription; cell survival,
growth and metabolism; cell migration
and motility
“ON”“OFF”
6
• Ras cycles between inactive GDP-bound and active GTP-
bound states
• GEFs reduce nucleotide affinity, enable exchange
• GAPs stimulate GTPase activity
• Conformational change in GTP-bound state enables
effector interactions
• Mutated KRas is locked in the activated GTP bound state
that facilitates enhanced Ras signaling in cancer cells.
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• KRas has been a desirable drug target
o No good druggable pocket
o pM GTP affinity
• Several new binding sites have been identified
• Shallow pocket between switch I and II regions
• Covalent binding of molecules to G12C mutant in
switch II pocket
• Alternative approaches
• Inhibition of exchange factor proteins
• Inhibition of Interaction with effectors
• Inhibition of downstream signaling kinases
• KRas expression inhibition (siRNA)
• Inhibition of KRas localization
Switch II Switch I
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• KRas has been a desirable drug target
o No good druggable pocket
o pM GTP affinity
• Several new binding sites have been identified
• Shallow pocket between switch I and II regions
• Covalent binding of molecules to G12C mutant in
switch II pocket
• Alternative approaches
• Inhibition of exchange factor proteins
• Inhibition of Interaction with effectors
• Inhibition of downstream signaling kinases
• KRas expression inhibition (siRNA)
• Inhibition of KRas localization
Switch II Switch I
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1 189/189
P loop Switch I Switch II Hypervariable region
G12
2-1696xHis
2-169GST
2-1698xHis BSP Biotin
220
160
120
100
90
80
70
60
50
40
30
25
20
15
10
kDa
6x HIS GST 8x HIS/BSPWT KRas
Currently available: wt, G12C, G12D, G12V
In production: G13C, G13D
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RhoGEF PH RasGEFN RasGEF
1 1333
SOS1
aa 564-1049
RhoGEF PH RasGEFN RasGEF
1 1332
SOS2
aa 562-1047
SH2 PH C2 RasGAP
1 1047
RASA1
aa 714-1047
SH3 SH2
C1
RBC protein:
RBC protein:
RBC protein:
RBD Protein kinase
1 648
cRAF
Reaction Biology protein: aa 2-303
SOS1 SOS2 RASA1 cRAF
220
160
120
100
90
80
70
60
50
40
30
25
20
15
10
kDa
11BI-2852
BAY-293
ARS-1620
AMG-510
RASGDP
“Off”
RASGTP
G12C
RASGDP
“Off” GTP
GDP
SOS1
RASGTP
RASGDP
“Off” GTP
GDP
SOS1
RASGTP
cRAF
SOS1
SOS1
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RASGDP*
SOS1
RASGTP
GTP GDP*
Fluorescence based detection
Kras G12C SOS1 mediated
nucleotide exchange
0 250 500 750 1000 1250 1500 1750 20000.00.10.20.30.40.50.60.70.80.91.01.11.2
no SOS1
1
0.25
0.125
0
uM ARS-1620
Time, sec
No
rmali
zed
sig
nal
• monitors SOS1/2 mediated exchange of fluorescently labeled GDP (GDP*) to GTP.
• SOS1/2 independent exchange can be measured in the presence EDTA
• main application of the assay is to identify compounds that lock KRas in inactive “OFF” state by
preventing GTP exchange.
• several types of fluorescently labelled GDP molecules can be used in NEA (ex Mant-GDP, Bodipy-
GDP)
• decrease in bodipy-GDP fluorescence is observed upon its dissociation from KRas
SOS1
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KRas G12C specific compounds
-10 -9 -8 -7 -6 -5 -4-10
0102030405060708090
100110120
G12C: ARS-1620
G12C: AMG-510
HILLSLOPE
EC50
G12C: ARS-1620
-1.527
9.855e-008
G12C: AMG-510
-1.595
2.521e-008
WT: ARS-1620
WT: AMG-510
log[compound], M
% e
xch
an
ge
5V9U
• Compound screening
• Selectivity profiling
KRpep-2D
-10 -9 -8 -7 -6 -5 -40
102030405060708090
100110120
WT
G12C
G12D
G12V
HILLSLOPE
EC50
WT
-0.7778
2.446e-006
G12C
-0.7100
2.071e-006
G12D
-0.6190
7.063e-008
G12V
-0.6796
1.073e-006
log[compound], M
% e
xch
an
ge
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• Assessment of time-dependence
• SOS1 is used at concentrations where almost complete exchange occurs within ~2 min
Effect of pre-incubation time on AMG-510 inhibition
-10 -9 -8 -7 -6 -5-10
0102030405060708090
100110120
1 min
5 min
10 min
30 min
HILLSLOPE
EC50
1 min
-1.379
2.202e-007
5 min
-1.450
1.008e-007
10 min
-1.235
5.469e-008
30 min
-1.402
3.134e-008
log[AMG-510], M
% e
xch
an
ge
0 250 500 750 1000 1250 1500 1750 20000
25000
50000
75000
100000
125000
150000
175000rapid
regular
time, sec
RF
U
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• SOS1 specific compound screening
• SOS1 is used at concentrations where linear rate of exchange is observed
• Difference in BAY-293 IC50 among different mutants is due to variable required concentration of SOS1 in assay
0 250 500 750 1000 1250 1500 1750 200025000
50000
75000
100000
125000
150000
175000regular
slow
time, sec
RF
U
Effect of BAY-293 on SOS1 mediatednucleotide exchange
-10 -9 -8 -7 -6 -5-10
0102030405060708090
100110
WT
G12C
G12D
G12V
HILLSLOPE
EC50
WT
-1.699
2.399e-008
G12C
-1.626
2.044e-008
G12D
-1.890
8.227e-008
G12V
-1.658
4.258e-008
log[compound], M
% e
xch
an
ge
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• SOS1/SOS2 selectivity
profiling
• Effect of compounds is
independent of mutation
status of KRas
Kras wt / SOS1
-10 -9 -8 -7 -6 -5 -4-10
0102030405060708090
100110120
BAY-293
BI-2852
HILLSLOPE
EC50
BAY-293
-1.910
1.723e-008
BI-2852
-1.500
6.310e-006
log[compound], M
% e
xch
an
ge
Kras wt / SOS2
-10 -9 -8 -7 -6 -5 -40
102030405060708090
100110120
BAY-293
BI-2852
HILLSLOPE
EC50
-1.829
5.723e-006
BI-2852
log[compound], M
% e
xch
an
ge
Kras G12C / SOS1
-10 -9 -8 -7 -6 -5 -4-10
0102030405060708090
100110120
BAY-293
BI-2852
HILLSLOPE
EC50
BAY-293
-1.467
2.265e-008
BI-2852
-0.9431
1.129e-005
log[compound], M
% e
xch
an
ge
Kras G12C / SOS2
-10 -9 -8 -7 -6 -5 -40
102030405060708090
100110120
BAY-293
BI-2852
HILLSLOPE
EC50
-1.244
7.325e-006
BI-2852
log[compound], M
% e
xch
an
ge
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SOS1
GTP*
GDP
RASGDP
GST
RASGTP*
GST
HTRF detection
• Monitors the increase in HTRF signal observed upon DY-647P1 labelled GTP binding to KRas
• Can be used if compounds have fluorescence interference with regular NEA
• Must be performed at lower GTP concentrations
• Can evaluate various modes of nucleotide exchange inhibition
Kras G12C
-10 -9 -8 -7 -6 -5 -4-10
0102030405060708090
100110
ARS1620
AMG510
BI 2852
BAY-293
HILLSLOPE
EC50
ARS1620
-1.226
7.598e-008
AMG510
-1.376
5.389e-009
BI 2852
-1.335
5.333e-007
BAY-293
-1.480
2.030e-008
log10 [Comp, M]
% e
xch
an
ge
Kras G12D
-10 -9 -8 -7 -6 -5 -4-10
0102030405060708090
100110
HILLSLOPE
EC50
BI 2852
-1.610
7.168e-007
BAY-293
-2.098
2.930e-008
ARS1620
AMG510
BI 2852
BAY-293
log10 [Comp, M]
% e
xch
an
ge
SOS1
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RASGDP
SOS1 SOS1RAS
GDP
KRas (G12C)::SOS1
-10 -9 -8 -7 -6 -5 -40
10
20
30
40
50
60
70
80
90
100
110
HILLSLOPE
EC50
-0.7397
2.780e-008
BAY-293
log[compound], M%
bin
din
g
B-KRas WT::SOS1
-10 -9 -8 -7 -6 -5 -40
10
20
30
40
50
60
70
80
90
100
110BAY-293
HILLSLOPE
EC50
BAY-293
-1.064
1.958e-008
log[compound], M
% b
ind
ing
Orthogonal method to NEA for identification of SOS1 specific compounds
HTRF detection
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• Monitor the disruption of interactions with cRAF effector protein
RAS
GTP
cRAF
B-KRas G12C::GTP titration
0 50 100 150 200 2500
10000
20000
30000GTP loaded
GDP loaded
Kras, nM
HT
RF
sig
nal
Effect of BI-2852 on KRasGTP::cRAFinteraction
-10 -9 -8 -7 -6 -5 -40
102030405060708090
100110120
G12D
G12C
HILLSLOPE
EC50
G12D
-0.7753
1.871e-006
G12C
-0.8589
1.244e-006
LOG10 [BI-2852, M]
% b
ind
ing
KRas G12C:GTP
-11 -10 -9 -8 -7 -6 -5 -4-10
0102030405060708090
100110120
ARS-1620
AMG-510
BI-2852
LOG10 [Comp,M]
% B
IND
ING
cRAF
RAS
GTP
HTRF detection
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RASGDP
GTP
GDP
+/-SOS1
RASGTP
cRAF
GDP loaded KRAS G12C: effect onSOS1 dependent GTP binding
-10 -9 -8 -7 -6 -5 -4-10
0102030405060708090
100110120
ARS-1620
HILLSLOPE
EC50
ARS-1620
-3.120
1.665e-007
AMG-510
-3.021
1.478e-008
BI-2852
-1.058
6.270e-006
AMG-510
BI-2852
LOG10 [Comp,M]
% B
IND
ING
GDP loaded KRAS G12C: effect onSOS1 independent GTP binding
-10 -9 -8 -7 -6 -5 -4-10
0102030405060708090
100110120
ARS-1620
AMG-510
BI-2852
HILLSLOPE
EC50
ARS-1620
-1.359
3.295e-008
AMG-510
-1.431
3.521e-009
BI-2852
-0.9388
3.316e-007
LOG10 [Comp,M]
% B
IND
ING
• “All in one” end point assay
• SOS1 independent reaction is less efficient; also affected by compounds
+/-SOS1
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DTm Kras wtKras G12C
Tm1Kras G12C
Tm2Kras
G12DKras
G12V SOS1 SOS2
25 uM ARS-1620 0 4.75 11.75 0.5 0.25 0 -0.25
25 uM AMG-510 0.5 3 16.25 0.5 0.25 0 0
25 uM BI-2852 0 0.25 x 1 0.75 0 0
25 uM BAY-293 -0.5 0 x 0.5 0 3 0
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• Assessment of direct binding of molecules to KRas
General Kinetics model
Immobilized ligand
Analyte1:1 binding ka
(1/Ms)kd (1/s) KD (M)
1:1 binding wt Kras KRpep-2d 5.49e+5 1.03e-2 1.87e-8
1:1 binding G12D KRpep-2d 7.36e+5 2.07e-3 2.81e-9
1:1 binding G12C KRpep-2d 3.86e+5 3.62e-3 9.37e-9
1:1 binding G12V KRpep-2d 8.17e+5 8.22e-3 1.01e-8
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General Kinetics model
Immobilized ligand Analyte1:1 binding ka
(1/Ms)kd (1/s) KD (M)
1:1 binding SOS1 BAY-293 4.77e+6 3.47e-2 7.28e-9
• Two Biacore 8K instruments
available
• Library screening
• Binding kinetics
• Assessment of small molecules,
peptides and proteins
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• Reaction Biology has the largest collection of kinase assays
• over 700 proteins for selectivity profiling and screening using radioisotope
based miniaturized assay
• Many of the KRas upstream and downstream kinases are available for
testing
Reaction Biology’s HotSpotSM
• Miniaturized filter binding assay
• 33P-ATP substrate
• Enables direct measurement of substrate phosphorylation
• Does not require use of modified substrates or other
reagents
RAF
MEK
ERK
RAS*GTP
PI3K
AKT
mTOR
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• Bioluminescence Energy Transfer (BRET) assay
• Evaluation of apparent binding affinity and cell
permeability of test compound
• HTS compatible
• Kinase is fused to a luminescent reporter
(NanoLuc)
• Fluorescent kinase probe is included in the media
• Compound and probe compete for kinase
binding sites leading to a reduction in BRET signal
• 170 targets are already available, additional
kinases are being added continuously
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Thank you!