Noncovalent interaction of mutant KRAS inhibitors

Oncogenic mutations in KRAS (mKRAS) are the main drivers for many human lung, pancreatic, and colorectal cancers. For many years, these oncogenic mKRAS have been considered undruggable, creating a significant unmet need. Recently, advancements in chemistry have led to the development of novel covalent inhibitors of the KRAS G12C proteins that target KRAS G12C-driven tumors. The encouraging outcomes of these G12C inhibitors have led to the accelerated FDA approval of sotorasib and adagrasib for adult patients with NSCLC. Many other mKRAS inhibitors targeting G12D and other mKRAS are also under development and evaluation in various clinical trials. Many chemo-proteomic approaches have established the exquisite specificity of various G12C inhibitors for Cys12 of the mKRAS proteins. However, the extent to which these inhibitors target other non-KRAS proteins through non-covalent interactions is unclear. To address this gap, we performed MS-based proteome-wide folding stability measurements that showed that divarasib does have non-covalent interactions with other proteins, such as RBM39, that affect drug response and environmental resistance. This work aims to identify and validate non-covalent interacting proteins of mKRAS inhibitors. In Aim 1, we will utilize two different mass spectrometry-based proteomics techniques for making large-scale measurements of protein folding stability to identify non-covalently interacting protein targets of four KRAS inhibitors, Sotorasib, Divarasib, MRTX1133, and RMC-6236 using cancer cells bearing the targeted mKRAS. The shared and compound-specific non-covalent candidate interacting proteins of these inhibitors will be further prioritized and validated to confirm the direct interaction of these inhibitors with their newly identified protein targets using conventional protein-ligand binding assays. In Aim 2, we will determine the effects of these inhibitors on the levels and activities of these non-KRAS interactors. Furthermore, we will elucidate the functional effects of these interacting proteins by their influence on sensitivity and KRAS pathway activities with or without environmental resistance. The completion of the proposed study will elucidate the role of non-covalent and non-mKRAS interacting proteins of mKRAS inhibitors that may explain the response heterogeneity, mitigate resistance, and enhance response to mKRAS inhibitors. Project Number: 1R21CA297690-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Jen-Tsan Chi | Institution: DUKE UNIVERSITY, DURHAM, NC | Award Amount: $411,152 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZCA1 RTRB-Z (M1)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11221158