BridgeBio Pharma : BBIO – KRAS Cancers – 2023 AACR-NCI-EORTC – GTP in Active-state KRASG12C Inhibitors

Fig. 5: Phospho-ERKinhibition in an engineered HeLa KRASG12C/A59G

Use of the Natural Nucleotide, GTP, is Essential for the Identification of Potent, Active-State KRASG12C Inhibitors That Bind in the Switch II Pocket

Bin Wang1, Alok K. Sharma2, James P. Stice1, Brian Smith2, Marcin Dyba2, Devansh Singh1, John Paul Denson2, Dana Rabara2, Erik Larsen2, Yue Yang3, Felice C. Lightstone3, Andrew Stephen2, Dwight V. Nissley2, Frank McCormick1,2, Eli Wallace1, Anna E. Maciag2, Pedro J. Beltran1

1BridgeBio Pharma Inc., Palo Alto, CA; 2Frederick National Laboratory for Cancer Research, Frederick, MD; 3Lawrence Livermore National Laboratory, Livermore, CA

1

BACKGROUND

Figure 1: Potency Profiles of KRASG12C

Inhibitors

Figure 3: Covalent Labeling Findings Correlate With a PPI Assay: Only Compounds With GppNHp Activity

Show Potency and GppNHp Overestimates Potency vs GTP

Figure 5: In a KRAS GTP "Locked" G12C/A59G Mutant,

Compounds With Higher GTP Labeling Display Greater Potency

Mutations in codon 12 of KRAS are observed in many human cancers. KRASG12C mutations are found in ~15% of non-small cell lung cancers and in a low percentage of colorectal and pancreatic adenocarcinomas1. These activating mutations in KRAS push cellular balance towards its active, GTP- bound state that signals downstream and drives cellular transformation2. Recently approved inhibitors of KRASG12C that bind and sequester the oncogenic protein in its inactive, GDP-bound state, have demonstrated clinical efficacy in patients with KRASG12C cancers, including NSCLC, CRC and pancreatic adenocarcinoma3-6. However, duration of response has been shorter than expected from the potent inhibition of a driver oncogene7,8 . This quick emergence of acquired resistance has been attributed to reactivation of MAPK

A.	0.05
30min		0.04
μM)	0.03
pERK,	,
50
(IC	0.02
H358		0.01
		0.00
		AMG-510
B.
	120

MRTX-849

-
GDC6036-	Cmpd2	Cmpd3 Cmpd1
AMG-510

A.

μ(M)	100
50
IC	10
-PPIRAF1 GTP-
1

Cmpd 1

B.		150								150
	%Activity	100							%Activity	100
														KRAS G12C GppNHp
AMG-510		50								50						KRAS G12C GTP
MRTX-849		0								0
GDC-6036
	0.0001	0.001	0.01	0.1	1	10	100		0.0001	0.001	0.01	0.1	1	10	100
				AMG-510 (log μM)				150		Cmpd 1 (log μM)
		150
	%Activity	100							%Activity	100						KRAS G12C GTP
																KRAS G12C GppNHp
		50								50
		0								0
		0.0001	0.001	0.01	0.1	1	10	100		0.0001	0.001	0.01	0.1	1	10	100
				MRTX-849 (log μM)						Cmpd 2 (log μM)
		150								150

		Cmpd 2 (μM)			AMG-510 (μM)			MRTX-849 (μM)
DMSO	10	3	1	0.3	10	3	1	0.3	10	3	1	0.3

KRAS
pERK
Total ERK
Vinculin
					GDC-6036 (μM)			Cmpd 1 (μM)				Cmpd 3 (μM)
	DMSO	10	3	1	0.3	10	3	1	0.3
DMSO 10	3	1	0.3

KRAS pERK

signaling through multiple mechanisms, including RTK signaling and KRASG12C gene amplification, resulting in increased active, GTP-bound KRASG12C 9-12. To address this unmet need, we have developed a series of dual state inhibitors and here summarize their activity against the active and inactive states of the KRASG12C protein.

Adapting to a KRASG12C-			KRASG12C prefers the
Increased RTK drive		GTP state. GTP
GDP inhibitor can be
achieved by making small			levels are 10x of
		GDP in cells
modifications	GEFs	Gene
KRASG12C-	Amplification

2hr	100
%pERK,	80
60
40
H358
20
	0
	0.01	0.1
C.
Day7	120
CTG),(
100
80
Viability
60
40
3D%	20
0
H358
0.01	0.1

1	10	100	1000
	nM

1	10	100
nM

MRTX-849		H358 (KRASG12C)
GDC-6036
Cmpd 1		pERK	3D Viability
Cmpd 2
	(IC50,nM)	(IC50,nM)
Cmpd 3
	AMG-510	40.0	3.7
	MRTX-849	37.8	2.1
	GDC-6036	1.2	0.2
AMG-510	Cmpd 1	0.9	0.2
MRTX-849
GDC-6036	Cmpd 2	0.7	0.2
Cmpd1
Cmpd 2	Cmpd 3	0.5	0.2
Cmpd 3

G12C	0.1	Cmpd 2					C. %Activity	100							%Activity	50
											100
KRAS																				KRAS G12C GppNHp
								50														KRAS G12C GTP
		Cmpd 3
	0.01							0								0
							0.0001	0.001	0.01	0.1	1	10	100
								0.0001	0.001	0.01	0.1	1	10	100
										GDC-6036 (log μM)						Cmpd 3 (log μM)
	0.01	0.1	1	10	100
													IC50, μM
									Compound			KRAS G12C GppNHp -	KRAS G12C GTP - RAF1
			G12C										RAF1 RBD					RBD
		KRAS	PPI-RAF1 IC50 (μM)
					AMG-510					>30					>30
				-GppNHp					MRTX-849					>30					>30
								GDC-6036					>30					>30
									Cmpd 1						0.122					3.186
									Cmpd 2						0.027					0.102
									Cmpd 3						0.011					0.043

Total ERK

Vinculin

cell line. HeLa cells that inducibly express a KRASG12C/A59G double mutant were tested at 2 hours for their activity on KRAS, phospho-ERK(Thr202/204), total ERK and vinculin as a loading control, using 0.3 to 10μM of selected compounds. Compound 3 demonstrated the greatest inhibition on pERK followed by compound 2 and 1, whereas compounds without GTP activity (AMG-510, MRTX-849, GDC-6036)had no inhibition of phosphorylated ERK.

RESULTS

GDP	Does		Tumor
(inactive)
	Progression
not	KRASG12C-
	via MAPK,
	signal	GTP
	PI3K/Akt
		(active)
AMG-510		Pathways
MRTX-849

Fig. 1: Phospho-ERK and 3D Viability IC50s across a series of KRASG12C inhibitors. IC50s of a series of compounds profiled at 30min (A) or at 2 hours (B) for phospho-ERKby HTRF or at day 7 for 3D viability (C). Compounds 1, 2, 3 as well as GDC-6036show equivalent potency and are approximately 40-foldmore potent on pERK and 10 to 15-foldmore potent on 3D viability than AMG-510or MRTX-849.

Figure 2: Covalent KRASG12C Labeling

Fig. 3: RAF1-KRASG12C protein-protein interactions show loss of activity in presence of GTP vs. GppNHp. Compounds were profiled for potency in the disruption of KRASG12C with RAF1 by FRET. IC50s of each compound were compared under GTP vs GppNHp conditions are plotted on the left (A) and representative curves (B) of a select number of compounds and a table (C) of their IC50s are shown on the right. The majority of compounds demonstrated loss of potency on GTP vs. GppNHp. Similar to results seen in MALDI, Cmpd 3>Cmpd 2>Cmpd 1 in terms of correlation, whereas AMG-510, MRTX-849,and GDC-6036had no activity in the PPI assay.

In order to overcome active KRASG12C-driven resistance, we have developed direct KRASG12C small molecule inhibitors that inhibit both the active, GTP-bound and inactive, GDP-bound forms of KRASG12C through interactions

GDC-6036	Intrinsic
hydrolysis
D-1553
New
ARS-1620	KRASG12C
JDQ443	synthesis
LY3537982
BI-1823911
JAB-21822	Mechanisms to adapt to KRASG12C-GDP inhibitors

KRASG12C-GDP Inhibitors Target a "Dead" Protein With No Signaling or Transforming Potential. KRASG12C GDP inhibitors rely on the conversion to active RAS to show efficacy. For inactive state (GDP) inhibitors, a number of mechanisms could result in a loss of potency, including increased RTK drive, KRASG12C amplification, as well as newly synthesized KRASG12C leading to reactivation of the MAPK and PI3K pathways resulting in tumor progression. Active state inhibitors may overcome these resistance mechanisms and this rationale provided the impetus for the development of our potent series

OBJECTIVES

▪	Characterize the potency of a series of KRASG12C inhibitors using a variety
	of biochemical and cell-based assays that measure GDP and GTP
	activity.
▪	Demonstrate that the GTP analog, GppNHp, versus the natural substrate

Reveals That the Potency of "Active State" Inhibitors can be Overestimated When Using GppNHp

A.	100		Cmpd 3
	AMG-510	Cmpd 1
(15min)
	Cmpd 2
	GDC-6036
	80
	MRTX-849
GDP	60	R2= 0.8405
40
%MALDI
20
	0

0	20	40	60	80	100
1

Figure 4:

A.

50	100
ICto
80
RASRAF- TimeAMG510,-
60
	40
	20
of
%	0
	0

Inhibition of RAS-RAF Binding Shows a Stronger Correlation Between Modification of GTP- Bound vs GppNHp-Bound KRAS

					C.	120
AMG-510											AMG-510
MRTX-849
						100						MRTX-849
GDC-6036
				RAF-RAS							GDC-6036
					80
				Cmpd 1								Cmpd 1
						60
											Cmpd 2
					%	40						Cmpd 3
R2= 0.7343				Cmpd 2		20
				0
				Cmpd 3
20	40	60	80	100		0	10	20	30	40	50	60

with the switch II pocket, and independently of any other partner proteins. Mass spectrometry analysis of KRASG12C covalent engagement shows complete modification of both KRASG12C active, GTP-bound and inactive, GDP-bound proteins, while sotorasib (AMG-510), adagrasib (MRTX-849), and divarasib (GDC-6036) only modify the inactive, GDP-bound protein. As expected, our active state inhibitors also show potent inhibitory activity in an effector (RAF1) disruption assay where inactive, GDP-bound inhibitors demonstrate no measurable potency. Interestingly, during our work assessing the potency of these direct KRASG12C inhibitors of the active state, we discovered that employing the broadly used non-hydrolyzable GTP nucleotide analog GppNHp as a surrogate for the natural nucleotide GTP results in overestimation of potency. These differences in potency between GppNHp and GTP were biologically meaningful as only compounds with strong activity against GTP- bound KRASG12C were able to demonstrate cellular activity consistent with inhibition of the active, GTP-bound state which leads us to identify a series of potent dual KRASG12C inhibitors.

GTP substantially overestimates the potency of KRASG12C inhibitors in

assays that measure the GTP-bound KRASG12C activity.

MATERIALS AND METHODS

• Homogeneous Time Resolved Fluorescence (HTRF) pERK Assay: H358 cells were seeded at 30,000 cells/well in a 96- well plate and allowed to adhere overnight. The next day, cells were treated with compound for 30min-2hours and samples were processed with a CisBio Phospho-ERK1/2 (Thr202/204) cellular kit, HTRF (Cat. No. 64ASPEG).
• CellTiter-Glo3D Viability Assay: H358 cells were seeded 1,000 cells/well in an ultra-lowattachment 3D plate.and allowed for spheroids to form for 2 days. Cells were then treated with compound for 7 days and samples were processed with a Promega CellTiter-Glo®3D Cell Viability Assay (Cat. No. G9683).
• Mass Spectrometry-Based Covalent Engagement Assay: 1 µM solutions of GTP, GppNHp and GDP-loadedKRAS4b (amino acids 1-169)-G12C/C118Sprotein were prepared and dispensed onto plates and then 30 nL of tested compounds from 1 mM DMSO stocks were added to the appropriate wells. At 15 minutes, 2 µL of each reaction mixture was pipetted into 15 µL MALDI matrix solution deposited onto plates. The resulting solution was mixed by aspiration, centrifuged at 2000 g for 1 minute, and then 1.5 µL aliquots were dispensed on pre-treatedMALDI target. MALDI-TOFmeasurements were performed on Bruker Daltonics rapifleX Tissuetyper TOF-TOFmass spectrometer using linear mode and mass range from 18.6 to 21.6 kDa. Percent modification was calculated as a ratio of peak height for protein modified by compound to sum of peak height of remaining protein plus peak height for protein modified by compound. The mean percent modifications from 17 experiments are shown.
• PPI: Avi-KRASG12C (amino acids 2-169)GTP/GppNHp and RAF1 RBD-3xFLAG(amino acids 51-131)and HTRF reagents were mixed and dispensed onto plates and then incubated for 1 hour at room temperature with shaking. Plates were analyzed on an Envision plate reader. Data was reported as percentage of activity with DMSO as 100% and plotted and
  analyzed using Graph Prism 8. Representative results are shown and the mean IC50 values from 6 experiments was calculated.
• RAS-RAFELISA: MiaPaca-2cells were seeded 280,000 cells/well and allowed to adhere overnight. Cells were treated with 1μM of compound for 5,15,30, and 60 minutes and lysate were processed using a commercially available ELISA kit (Abcam# ab134640). Briefly, lysates collected and incubated with a RAF-RASbinding domain conjugated to bottom of ELISA plate, followed by a KRAS detection ab and luciferase conjugated secondary antibody. Luciferase signal was measured on a Clariostar plate reader.
• HeLa A59G Western: HeLa Tet-ON KRASG12C/A59G Western blot: HeLa cells were engineered to express the KRASG12C/A59G under control of doxycycline-inducedpromoter. Cells were transduced with lentivirus and selected with 1ug/mL puromycin for several passages. Cells were plated at 1.25e6 cells in 10 cm dish into media containing 200 ng/ml doxycycline, allowed to attach for 24h, then treated for 2 hours with various doses of compound. Following treatment, cells lysates were collected and processed for Western blot using phospho-ERK(Thr202/204), total ERK, KRAS, and vinculin. All antibodies were procured from Cell Signaling Technologies.

B.		%MALDI GppNHp (15min)
	100					Cmpd 3
(15min)						Cmpd 2
	R2= 0.5355
GTP
50
%MALDI						Cmpd 1
AMG-510
MRTX-849
	0	GDC-6036
	0	20	40	60	80	100
C.		%MALDI GppNHp (15min)
		Compound	GTP	GppNHp		GDP
			5'/15'/30'/60'	5'/15'/30'/60'	5'/15'/30'/60'
		AMG-510	1/3/4/10	0/0/0/0	87/88/88/89
		MRTX-849	0/1/4/10	0/0/0/0	81/82/85/87
		GDC-6036	1/2/4/10	0/4/7/15	85/86/88/90
		Cmpd 1	19/38/53/71	88/97/100/100	89/93/96/100
		Cmpd 2	77/89/96/100	100/100/100/100 94/100/100/100
		Cmpd 3	96/100/100/100	100/100/100/100 100/100/100/100

Fig. 2: Correlation between covalent labeling of GppNHp vs GDP or GTP in a series of KRASG12C GDP and GTP inhibitors. A series of compounds was run on MALDI for 15 minutes and analyzed for KRASG12C modification in the presence of GTP, GppNHp, or GDP and analyzed for their linear correlation. AMG-510,MRTX- 849 and GDC-6036showed no labeling in the presence of GTP or GppNHp and were removed for linear regression analysis. GppNHp displayed a stronger correlation to GDP (A) than GTP (B) on the series of compounds tested. A select number of compounds (C) are highlighted to demonstrate agreement between GppNHp and GTP (Cmpd 3>Cmpd 2) and those with large discrepancy (Cmpd 1).

B.		%MALDI GppNHp (15min)		Minutes
		D.
50	100	AMG-510
ICto	MRTX-849
RAS-RAF510,-AMGTime	80				MiaPaca-2
20		Cmpd 3	100	1.0
	0.5
	GDC-6036	Compound	MALDI-TOF%	RAS-RAF	% of AMG-510,
	60	Cmpd 1		GTP, 15min	Minutes to 50%	Time to IC50
					Inhibition
			AMG-510	0	24.6	100.0
	40		MRTX-849	0	26.3	106.9
		GDC-6036	0	17.8	72.3
			Cmpd 1	38	16.1	65.4
		Cmpd 2	Cmpd 2	89	4.1	16.8
% of		R2= 0.8888
0		Cmpd 3
	0	50	100

% MALDI GTP (15min)

Fig. 4: Cell-BasedRAS-RAF ELISA disruption shows stronger correlation to MALDI GTP vs GppNHp. The potency of various compounds on the disruption of the RAS-RAFinteraction was measured in MiaPaca-2cells using a commercially available ELISA kit. Results comparing the IC50 from the ELISA to the MALDI labeling on GppNHp (A) vs GTP (B) are shown on the left. To the right are the representative curves (C) for a select number of compounds in the RAS-RAFELISA along with a table (D) comparing their labeling by MALDI in the presence of GTP along with their time to inhibition in the RAS-RAFELISA. In summary, RAS-RAFresults had a stronger correlation to MALDI GTP vs GppNHp MALDI labeling and Cmpd 3 showed the fastest inhibition followed by compound 2, 1, then GDP inhibitors GDC-6036, MRTX-849,and AMG-510.

CONCLUSIONS

1. • Inhibiting the active, GTP-bound state of KRASG12C is possible with switch II pocket binders.
   • Using the natural, physiological nucleotide, GTP, in biochemical assays is indispensable to identify compounds with corresponding cellular activity that is differentiated from molecules that target the inactive, GDP- bound state of KRASG12C.
2. Nassar AH, Adib E, and Kwiatkowski DJ. Distribution of KRASG12C Somatic Mutations Across Race, Sex, and Cancer Type. N Engl J Med 2021; 384(2):185-187.
3. Moore AR, Rosenberg SC, McCormick F et al. RAS-targeted therapies: is the undruggable drugged? Nat Rev Drug Discov 2020; 19(8):553-552.
4. Ostrem JM, Peters U, Sos ML et al. KRAS (G12C) Inhibitors Allosterically Control GTP Affinity and Effector Interactions. Nature 2013; 503(7477):548-51.
5. Lito P, Solomon M, Li LS, Hansen R, Rosen N. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science 2016;351(6273):604-608.
6. Canon J, Rex K, Saiki AY, et al. The clinical KRAS(G12C) inhibitor AMG 510 drives antitumour immunity. Nature 2019;575(7781):217-223.
7. Hallin J, Engstrom LD, Hargis L, et al. The KRAS(G12C) Inhibitor MRTX849 Provides Insight Toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer Discov 2020;10(1):54-71.
8. Skoulidis F, Li BT, Dy GK, et al. Sotorasib for Lung Cancers with KRAS p.G12C Mutation. N Engl J Med 2021;384(25):2371-2381.
9. Fakih MG, Kopetz S, Kuboki Y, et al. Sotorasib for previously treated colorectal cancers with KRASG12C mutation (CodeBreaK100): a prespecified analysis of a single-arm, phase 2 trial. Lancet Oncol 2022;23(1):115-124.
10. Awad MM, Liu S, Rybkin, II, et al. Acquired Resistance to KRAS(G12C) Inhibition in Cancer. N Engl J Med 2021;384(25):2382-2393.
11. Ryan MB, Fece de la Cruz F, Phat S, et al. Vertical Pathway Inhibition Overcomes Adaptive Feedback Resistance to KRAS(G12C) Inhibition. Clin Cancer Res 2020;26(7):1633-1643.
12. Tanaka N, Lin JJ, Li C, et al. Clinical acquired resistance to KRASG12C inhibition through a novel KRAS switch-II pocket mutation and polyclonal alterations converging on RAS-MAPK reactivation. Cancer Discov 2021;11(8):1913-1922.
13. Xue JY, Zhao Y, Aronowitz J, et al. Rapid non-uniform adaptation to conformation-specific KRAS(G12C) inhibition. Nature 2020;577(7790):421-425.