0

1

This powerpoint slide deck is also available as oral presentation held by Dr. Ekaterina Kuznetsova

Follow link belowhttps://youtu.be/TuObnovpqoE

2

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

2

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.

7

• 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

8

• 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

9

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

10

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

12

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

13

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

14

• 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

15

• 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

16

• 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

17

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

18

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

19

• 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

20

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

21

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

22

• 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

23

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

24

• 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

25

• 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

26

Thank you!