Abstract B013: Exploring the switch II pocket of KRAS(G12D) with mutant-selective monobody inhibitors

Abstract

Abstract The G12D mutation is among the most common KRAS mutations associated with cancer, in particular pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are highly selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over HRAS(G12D). Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and alpha 3 helix. Strikingly, the monobody captured a shape of the S-II pocket distinct from previously reported structures. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of RAS Asp12. This feature closely resembles that of a small-molecule KRAS(G12D) inhibitor, MRTX1133. The monobody also directly interacted with H95, a residue not conserved in HRAS. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a G12D-specific monobody generated hundreds of functional and nonfunctional single-point mutants, from which we identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results demonstrate the feasibility of targeting KRAS(G12D) functions using a mutant selective inhibitor and will guide the development of next-generation KRAS(G12D)-selective inhibitors. Citation Format: Takamitsu Hattori, Padma Akkapeddi, Imran Khan, Akiko Koide, Eliezra Glasser, Gayatri Ketavarapu, Michael Whaby, Mariyam Zuberi, Kai Wen Teng, Julia Lefler, Lorenzo Maso, Injin Bang, Michael C. Ostrowski, John P. O’Bryan, Shohei Koide. Exploring the switch II pocket of KRAS(G12D) with mutant-selective monobody inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B013.