Abstract LB321: Discovery and characterization of QTX3046, a potent, selective, and orally bioavailable non-covalent KRASG12D inhibitor
Authors: Vo Elizabeth Donohue , Zhang Yang W. , Rominger Dave , Silva Jillian M. , Zhang Yang J. , Lee Greg , Micozzi John , Reid Ben , McDonough Brooke , Hospital Audrey , Luengo Juan I. , Lin Hong , Pitt Cameron 
Published: April 2023

Abstract
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. While recently approved covalent KRAS inhibitors have demonstrated clinical benefit in lung, colorectal, pancreatic, and other solid tumors, the requirement for the cysteine mutant limits potential applications to the approximately 10% subset of KRAS patients bearing a KRASG12C mutation. As the most prevalent KRAS mutation, KRASG12D –driven malignancies represent a high unmet need, attracting immense attention in drug discovery. However, direct KRASG12D inhibitors described in the literature to date lack oral bioavailability.
Here we show that QTX3046 is a potent, highly selective, and orally bioavailable non-covalent KRASG12D inhibitor. QTX3046 demonstrated picomolar binding affinity (0.01 nM) to the inactive form of KRASG12D by SPR, &gt; 400-fold affinity over the inactive KRASWT protein, and inhibited SOS1/2-mediated nucleotide exchange with picomolar potency (0.1 nM). QTX3046 also displayed nanomolar binding affinity to the GppNHp-bound “ON”-state of KRASG12D by SPR. Allosteric activity of QTX3046 selectively disrupted KRASG12D:RAF1 protein-protein interaction in a concentration-dependent manner using both biochemical and cell-based (NanoBiT) target engagement assays, while showing weak or no activity against KRASWT and other KRAS mutants. Western blot analyses and CTG proliferation assays illustrated that QTX3046 treatment inhibited downstream ERK phosphorylation and cell proliferation selectively in KRASG12D -driven cancer cell lines. KRASG12D selectivity was further confirmed in panels of isogenic SW48 human colorectal cancer and mouse embryonic fibroblast (MEF) cell lines harboring various single RAS mutations.
QTX3046 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 QTX3046 twice daily (BID) achieved tumor regression in 100% of tumors and was well-tolerated. The physiochemical profile for QTX3046 is generally favorable with good solubility. The systemic clearance in rats and dogs was high to moderate, respectively and despite low apparent permeability, demonstrated good oral bioavailability in each species. The current PK profile and preclinical proof-of-concept data support advancement of QTX3046 into IND enabling studies to support potential clinical investigation in KRASG12D -driven solid tumor indications.
Citation Format: Elizabeth Donohue Vo, Yang W. Zhang, Dave Rominger, Jillian M. Silva, Yang J. Zhang, Greg Lee, John Micozzi, Ben Reid, Brooke McDonough, Audrey Hospital, Juan I. Luengo, Hong Lin, Cameron Pitt. Discovery and characterization of QTX3046, a potent, selective, and orally bioavailable non-covalent KRASG12D 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 LB321.