[WDB178] mM

RAS

pERK

tERK

WDB178 Effects on KRAS Signaling Are G12C-Specific

Western blot analysis of changes in downstream signaling in cell lines treated with WDB178 for 4 hours. Effects on signaling were seen

in KRASG12C mutant cell lines H358 and LU65 and Ras-less MEFs expressing human KRASG12C, but not in non-KRASG12C control cells.

Trametinib (1mM) was used as a positive control for pERK inhibition in the KRAS mutant RAS-less MEF experiment.

tERK

pMEK

pAKT

pS6

RAS

pERK

[WDB178] mM

H358

(G12C)

LU65

(G12C)

A549

(G12S) MEF G12C G12D G12V WT

WDB178 Engages KRASG12C in Cells and Inhibits KRASG12C Signaling

H358 cells were treated with compound for 4 hours prior to

lysate preparation. Recombinant GST-RAF RBD was added

to the lysate and pull down was performed with glutathione

beads

0.5 1 2 4

0.5 1 2 4

0.5 1 2 4

0.5 1 2 4

-2 5

0

2 5

5 0

7 5

1 0 0

1 2 5

-0 .2 5

0 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

T im e (h o u rs )

G1

2C

Cro

ss

-lin

kin

g (

%)

pE

RK

(% C

trl.)

D M S O 0 .3 m M 1 m M 3 m M[W D B 1 7 8 ]

KRASG12C cross-linking and pERK levels were measured in WDB178-

treated H358 cells. G12C peptide cross-linking was measured in cell

lysates by a Mass Spectroscopy target engagement assay2.

Phospho-ERK levels were measured using a 96-well pERK In Cell

Western (ICW) assay and signal was normalized to cell number by

DRAQ5 staining.

KRAS-GTP

Input

GST-RAF

KRAS

RAF

Pull-

Down

WDB178

µM0.1 1 1010

WDB178 Interacts Potently and Selectively with GTP-Bound KRASG12C

via Covalent and Non-Covalent Interactions

kinact (sec-1) KI (µM) kinact/KI (M-1 sec-1)

WDB178 7.2E-03 2.7 2645

ARS-853*2,3 5.0E-02 200 250

ARS-1620*1 > 6.7E-02 > 64 1100

0 1 0 2 0 3 00 .0 0 0

0 .0 0 2

0 .0 0 4

0 .0 0 6

W D B 1 7 8 (µ M )

ko

bs (

se

c-1

)

0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 00

2 0

4 0

6 0

8 0

1 0 0

T im e (s )

% C

ro

ss

-Lin

kin

g

1 m M

2 m M

4 m M

8 m M

1 5 m M

3 0 m M

-20

20

60

100

140

-40 -11 18 47 76 105 134 163 192 221 250

Tim e s

RU

Re

sp

on

se

W0125178-01-001 G12C/New -CypA 3 ug/m l

-10

20

50

80

110

140

-40 -11 18 47 76 105 134 163 192 221 250

Tim e s

RU

Re

sp

on

se

W0125178-01-001 WT/New -CypA 3 ug/ml

-10

20

50

80

110

140

-40 -11 18 47 76 105 134 163 192 221 250

Tim e s

RU

Re

sp

on

se

W0125178-01-001 GDP/New -CypA 3 ug/m l

KRASG12C-GMPPNP

KRASG12C-GDP

KRAS-GMPPNP

Ternary SPR KD

0.26 0.06 mM

> 100 mM

> 100 mM

Mass Spec. Cross-linking

Time (sec.)

Resp

on

se

Resp

on

se

Resp

on

se

*ARS compounds target GDP-KRASG12C

KI values indicate that WDB178 engages in significantly stronger

non-covalent interactions with KRASG12C than ARS compounds

WDB178 Activity in H358(G12C) Cells is CYPA-Dependent

1

1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

5 0

1 0 0

W D B 1 7 8 (n M )

Ce

ll v

iab

ilit

y (

% C

trl.

)

H 3 5 8 (G 1 2 C ) C Y P A K O

H 3 5 8 (G 1 2 C ) C tr l. K O

A 5 4 9 (G 1 2 S )

[WDB178] mM

CYPA KO

RAS

ERK

pERK

Ctrl. KO

WDB178 treatment led to a concentration-dependent decrease in pERK

levels and a concomitant increase in supershifted (‘178 X-linked) RAS in

CYPA expressing, but not in non-CYPA expressing H358 cells. The IC50

for pERK inhibition by WDB178 is 0.65 mM in an ICW assay. H358 cells

were engineered with a cyclophilin A (CYPA KO) or control (Ctrl. KO)

CRISPR vector. Absence of CYPA in CYPA KO cells was verified by

western analysis (inset).

Cells were treated for 5 days in 3D Ultralow attachment

plates (96-well), and cell viability was measured by 3D

CellTiter-Glo.

WDB178 Activity in H358(G12C) Cells is Not Attenuated by

Growth Factor Treatment

+ Growth

factor (e.g.

EGF)

WDB178 ARS-16201 (GDP-targeting)

Figure Adapted from (2) GF Treatment reduces GDP-KRASG12C pool

0 4 0 8 0 1 2 0 1 6 0 2 0 0 2 4 0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

M in u te s

KR

AS

G1

2C

cro

ss

lin

k (

%)

v eh ic le

E G F (1 0 0 n g /m L )

0 4 0 8 0 1 2 0 1 6 0 2 0 0 2 4 0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

M in u te s

KR

AS

G1

2C

cro

ss

lin

k (

%)

v eh ic le

E G F (1 0 0 n g /m L )

KRASG12C

Cross-linking

Mass Spec. target engagement assay2

0 .0 1 0 .1 1 1 0

2 5

5 0

7 5

1 0 0

1 2 5

W D B 1 7 8 (m M )

pE

RK

(%

Ctr

l.)

v eh ic le

E G F (1 0 0 n g /m L )

0 .0 1 0 .1 1 1 0

2 5

5 0

7 5

1 0 0

1 2 5

A R S -1 6 2 0 (m M )

pE

RK

(%

Ctr

l.)

v eh ic le

E G F (1 0 0 n g /m L )

pERK (1h post

treatment)

pERK 96-well ICW

5 day CTG assay in,

3D Ultralow attachment

plates

Viability

0 .0 1 0 .1 1 1 0

0

2 5

5 0

7 5

1 0 0

1 2 5

W D B 1 7 8 (m M )

Ce

ll V

iab

ilit

y (

% C

trl.

) V e h ic le

E G F

H G F

T G F a

0 .0 1 0 .1 1 1 0

0

2 5

5 0

7 5

1 0 0

1 2 5

A R S -1 6 2 0 (m M )

Ce

ll

Via

bil

ity

(%

Ctr

l.)

V e h ic le

E G F

H G F

T G F a

Development of Inhibitors of the Activated Form of KRASG12C

Roy M. Pollock*, Michelle L. Stewart, Nicholas R. Perl, Seung-Joo Lee, Linlong Xue, Minyun Zhou, Jonah Simon, Kathryn M. Luly, Simina Grigoriu, Alex Yuzhakov, Alec Silver, Jason T. Lowe, Alan S.

Mann, Gizem Akcay, Cindy Benod, Gregory L. Verdine, Alan C. Rigby, Mark J. Mulvihill, Earl W. May, Anna Kohlmann, Sharon A. Townson, Meizhong Jin

Warp Drive Bio, a subsidiary of Revolution Medicines, Inc., 400 Technology Square, Cambridge, MA 02139, USA, *email: rpollock@warpdrivebio.com

Summary Results

BackgroundConclusions

• WDB178 is a first in class inhibitor of the activated form of KRASG12C

• WDB178 harnesses cyclophilin A to potently and selectively bind GTP-

KRASG12C via non-covalent and covalent interaction

• WDB178 disrupts RASG12C|RAF interaction and blocks KRASG12C

signaling in cells

• WDB178 effects on KRAS signaling and viability are selective for

G12C-mutant tumor cell lines

• In contrast to GDP-targeting inhibitors, the potency of WDB178 is

maintained in the presence of growth factor treatments

• Targeting the GTP-state of KRASG12C may offer advantages in

developing KRASG12C selective therapeutics

• We are currently evaluating the activity of further optimized inhibitors in

in vivo models

References:

1) Janes, M. R., et al., Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor. Cell 2018, 172, 578-589.

2) Patricelli, M. P., et al., Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State. Cancer Discovery 2016, 6, 316-329.

3) Lito, P., et al., Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science 2016, 351, 604-608.

Disclosures: All authors:

Warp Drive Bio, a subsidiary of Revolution Medicines, Inc. :Employment

RAS oncogenes are mutated in ~ 1/3 of all human cancers. Mutant

RAS proteins exist predominantly in the activated GTP-bound state

leading to aberrant downstream signaling via interaction with

effectors such as RAF. A KRAS mutation in which glycine is mutated

to cysteine (G12C) is particularly common in non-small cell lung

cancer where it is found in ~ 15% of lung adenocarcinomas.

We used a novel drug discovery technology to develop compounds

that selectively bind and inhibit KRASG12C through formation of

ternary complexes. WDB178 is a simplified sanglifehrin derivative

that binds non-covalently with Cyclophilin A (CYPA), an abundant

immunophilin present in all human cells. The CYPA-WDB178 binary

complex is then able to form an inhibitory ternary complex with the

activated (GTP-bound) form of KRASG12C via covalent targeting of the

mutant cysteine. The interaction with KRASG12C is completely

dependent on CYPA and is potent and selective for G12C over WT

KRAS. Formation of the CYPA-WDB178-KRASG12C ternary complex

occludes RAF binding in biochemical and cell based assays.

WDB178 selectively inhibits KRAS signaling and cell viability in

human tumor cell lines bearing a KRASG12C mutation. In contrast to

ARS-1620 – a previously described GDP-targeting KRASG12C

inhibitor1, WDB178 activity is not attenuated by growth factor

treatment.

WDB178 is a covalent warhead containing derivative of Sanglifehrin A

(“SFalog”) that contains a Cyclophilin A (CYPA) binding moiety

WDB178 Inhibits KRASG12C by Forming Ternary Complexes

WDB178

CYPA CYPA GTP-KRASG12C

WDB178

CYPA GTP-KRASG12C

WDB178

1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4

0

2 0

4 0

6 0

8 0

1 0 0

W D B 1 7 8 (M )

% D

isru

pti

on

G 1 2 C + C Y P A

G 1 2 C - C Y P A

W T + C Y P A

RAS-RAF Disruption TR-FRET

WDB178 Disrupts KRASG12C|RAF Interaction

EC50 = 0.021 ± 0.012 μM

APC-α-GST(acceptor)

GST-BRAFHis-KRASG12C

Excitation

Emission

615 nm

Eu-α-His

(donor)

TR-FRET

665 nm

Excitation

Emission

615 nm

CYPA

WDB178

RAF-RBD

WDB

CYPA

KRASG12C

WDB

CYPA

KRASG12C

Cells were treated for 5 days in 3D Ultralow attachment plates (96-well), and

cell viability was measured by 3D CellTiter-Glo.

WDB178 Effects on Cell Viability Are G12C-Specific

0 .1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0G 1 2 C P a n e l

W D B 1 7 8 (n M )

Ce

ll V

iab

ilit

y (

% C

trl.

)

L U 65

H 1 7 92

H 358

H 1 3 73

M IA P A C A 2

H 23

H 2 1 22

L U 99

0 .1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0N o n -G 1 2 C P a n e l

W D B 1 7 8 (n M )

Ce

ll V

iab

ilit

y (

% C

trl.

)

A 5 4 9

A 3 7 5

H 441

H C T 1 16

A S P C 1

H 1 2 99

H 1 7 55

1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0

R a s - le s s M E F s

W D B 1 7 8 (n M )

Ce

ll V

iab

ilit

y (

% C

trl.

) K R A SG 1 2 C

K R A SW T

K R A SG 1 2 V

K R A SG 1 2 D

Cell line KRAS Viability IC50 Cell line KRAS Viability IC50

H358 G12C 0.49 H441 G12V > 10

MiaPaca-2 G12C 0.38 A549 G12S > 10

LU65 G12C 0.31 A375 WT > 10

H23 G12C 0.38 HCT116 G13D > 10

LU99 G12C 1.39 ASPC1 G12D > 10

H2122 G12C 1.49 H1299 WT > 10

H1792 G12C 4.03 H1755 WT > 10

H1373 G12C 2.21