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The RAS family of proto-oncogenes are the most frequently mutated genes in cancer, in which mutations in KRAS account for approximately 25% of all human cancers. RAS oncogenes impair the ability of RAS to convert from its active GTP-bound form into its inactive GDP-bound state leading to the sustained activation of the MAPK signaling pathway and ultimately driving tumorigenesis. Until recently, KRAS was regarded as undruggable, but newly approved covalent inhibitors of KRASG12C have demonstrated clinical benefit in patients harboring this mutation. However, patients with KRASG12C mutation only represent approximately 10% of the KRAS-mutated or amplified cancer population, and multiple RAS-mediated resistance mechanisms have emerged with these covalent compounds. Here we show that QTX3034 is a potent, highly selective, and orally bioavailable mutant KRAS inhibitor. Using the most common KRAS mutant, KRASG12D, QTX3034 demonstrated picomolar binding affinity (0.6 nM) to the inactive form of KRASG12D and inhibited SOS1/2 mediated nucleotide exchange with picomolar potency (0.2 nM). In a NanoBiT cellular target engagement assay, QTX3034 treatment disrupted KRASG12D:RAF1 protein-protein interaction in a concentration-dependent manner with an EC50 of 17 nM in HEK293T cells. Western blot analyses and CTG proliferation assays illustrated that QTX3034 treatment inhibited downstream ERK phosphorylation and cell proliferation in KRASG12D -driven cancer cell lines. Similar inhibitory effects were observed across KRASG12D and KRASG12V mutant cancer cell lines, albeit less potent against the latter. Notably, QTX3034 produced no effect against NRAS or BRAF mutant cells. In vivo, QTX3034 achieved sustained systemic exposure levels required for efficacy following single oral administration in mice, indicating potential durable inhibition of KRAS signaling in vivo. In a KRASG12D -driven xenograft model, oral administration of QTX3034 twice daily (BID) demonstrated dose-dependent anti-tumor efficacy and achieved tumor regression in 100% of tumors. In a KRASG12V -driven xenograft model, oral administration of QTX3034 showed significant tumor growth inhibition, consistent with its activity against KRASG12V in vitro. QTX3034 was well-tolerated in rodent, repeat dose toxicology studies and exhibited significant exposure in the brain. QTX3034 has good solubility, permeability, and moderate protein binding in human plasma. The systemic clearance in rats and dogs was moderate to high, respectively, with good oral bioavailability. QTX3034 exhibited weak CYP inhibition (direct and time-dependent) with a favorable in vitro off-target selectivity/safety profile. Taken together, this preclinical characterization supports the advancement of QTX3034 into IND-enabling studies for KRAS mutant cancers. Citation Format: Yang W. Zhang, Dave Rominger, Elizabeth Donohue Vo, Jillian M. Silva, Yang J. Zhang, Greg Lee, John Micozzi, Ben Reid, Brooke McDonough, Audrey Hospital, Juan I. Luengo, Cameron Pitt, Hong Lin. Discovery and characterization of QTX3034, a potent, selective, and orally bioavailable allosteric KRAS inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB320.

Oncogenic mutations in the RAS family are the most frequently occurring among human cancers. The recent development of KRAS-targeted covalent inhibitors display efficacy in KRAS-mutant tumors; however, this approach is limited to KRASG12C mutant cancers. Here, we describe a novel drug discovery program to target the full-scope of mutant RAS-driven cancers through allosteric inhibition of the oncogenic KRAS-RAF1 signaling complex. To identify novel allosteric inhibitors, we developed a second harmonic generation (SHG) assay to detect conformational changes in the KRAS-RAF1 membrane-bound complex. Fully processed farnesylated and methylated KRAS4b-G12D protein (KRASG12D-FMe) was complexed with the RAF1 N-terminal RAS-binding domain (RBD) and cysteine rich domain (CRD) and immobilized on a phosphatidylserine-enriched bilayer. The RAF1RBD-CRD protein was rendered SHG-active by chemical conjugation and tethered to the bilayer by KRASG12D-FMe, thus replicating the physiological complex orientation. Ligands that disrupted or altered the orientation of the complex relative to the surface were identified from a curated diversity library of 60,000 chemical compounds based on % SHG signal change relative to baseline. Validated hit compounds were selected for SAR characterization and development wherein newly synthesized molecules were assayed by SHG and inhibition of cellular ERK phosphorylation by HTRF. Quanta (QTX) molecules were confirmed to rapidly inhibit ERK phosphorylation across multiple RAS-mutant cell lines by Western blot. Biochemical cellular target engagement studies reveal inhibition of RAF1:BRAF dimerization downstream of allosteric modulation of the KRAS:RAF1 signaling complex. Mass spectrometry with a photoactive derivative identified modified residues clustered along the KRAS:RAF1 interface, thus providing structural evidence for this unique mechanism of action. In addition to MAPK inhibition, we observe inhibition of cellular proliferation across mutant RAS cell lines. Cellular growth assays in the presence of QTX molecules combined with the SHP-2 inhibitor, RMC4550, displayed synergistic growth inhibition, providing a rationale combination therapy. Moreover, RAS-mutant cell-line derived xenograft tumors treated with QTX inhibitors elicited significant tumor growth inhibition and dose-dependent reductions in the pharmacodynamic MAPK targets, P-ERK and DUSP6, which is consistent with anti-tumor activity. This research illustrates a promising approach towards the development of mutant RAS-targeted inhibitors with a unique mechanism of action and target engagement. Citation Format: Elizabeth Donohue Vo, Juan Luengo, Hong Lin, Jerry Chen, Ben Reid, Brooke McDonough, Norman Fultang, Jillian Silva, Cameron Pitt. Discovery and characterization of oncogenic KRAS:RAF1 conformational modulators with in vitro and in vivo MAPK inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA007.

The Ras protein family consists of small GTPases that are critical signaling transducers regulating cellular processes including proliferation, differentiation, and survival. As monomeric GTPases, Ras proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Oncogenic mutations in one of the Ras isoforms, H-, N-, and K-Ras, impair GTP hydrolysis, thus de-regulating Ras signaling. Ras mutations are found in one-third of human cancers, and tumors bearing these mutations are notoriously difficult to treat. Conventional approaches to inhibit Ras with small molecules have been largely unsuccessful leading many to characterize the Ras family as an “undruggable” target. However, there is renewed enthusiasm towards Ras drug discovery in response to a recent surge of reports revealing structural complexities to Ras proteins that provide opportunity for innovative drug discovery efforts. In light of findings suggesting KRas contains transient and dynamic binding pockets, many of which are undetectable with traditional structural techniques, we used a second harmonic generation (SHG) platform to screen a ~2700 chemically diverse fragment collection for allosteric modulators of KRas. This high throughput SHG platform enables real-time measurement of protein conformational change under physiological conditions. We selected ~50 fragments for validation by SPR, and positive binders will be selected for structural and biochemical characterization. Another advantage of the SHG platform is that we can monitor the conformational dynamics of fully processed, farnesylated KRas bound to a phosphatidylserine-enriched membrane that mimics KRas’ physiological interaction with the plasma membrane. No other biophysical method is suitable to monitor the real-time interactions of small molecules or proteins with membrane-bound KRas. This analysis may reveal surface contacts unavailable in solution-based assays. We have successfully implemented SHG as a drug discovery platform against KRas, and we aim to utilize selected KRas conformational modulators towards the identification of novel binding pockets and towards the development of targeted KRas therapies. Citation Format: Elizabeth Donohue Vo, Gabriel Besserer Mercado, Patrick Alexander, Ben Moree, Que Van, Andrew G. Stephen, Joshua Salafsky, Frank McCormick. Identifying allosteric modulators of KRas using second harmonic generation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4018. doi:10.1158/1538-7445.AM2017-4018

Proteins populate a landscape of conformations that changes upon binding native ligands or drugs, altering protein function. Different conformations reveal distinct potential drug target sites, including allosteric sites distal to the active site of the protein. It has been historically difficult to identify allosteric modulators early in the screening process since the structural methods required to reveal binding sites are relatively low throughput.