Abstract B006: Screening a disulfide library against engineered cysteine RAS mutants reveal cryptic binding pockets

Disulfide tethering is a site-directed method for identifying hits of challenging drug targets. We describe the creation of a screening library of over 2000 disulfide fragments. We have screened 1000+ fragments from this library against 83 engineered KRAS cysteine mutants to probe the surface of KRAS for binding sites. In addition, for a selected subset of 30 mutants, the full 2000+ library was screened. The screens were performed with βME to ensure reversible binding conditions and that non-covalent binding drives labeling. Mass spectrometry was used to determine percent labeling. We grouped the residues by proximity to pockets and calculated a ligandability score for each mutant. Furthermore, through a pocket-by-pocket analysis, we identified the most “ligandable” binding sites: the two pockets with the highest ligandability score were the two pockets that have known ligands, the switch II – helix 3 pocket and switch I – switch II pocket. For select pockets, the binding of disulfide-fragment hits were further characterized with dose response curves, NMR spectra, and computational methods (molecular docking and molecular dynamics). From this analysis we identified a ligand that binds an under studied binding site, whose binding mode we validated using computational and NMR results. Citation Format: Trent E. Balius, Marcin Dyba, Vandana Kumari, John-Paul Denson, Ming Yi, Alok K. Sharma, Priyanka Prakash, Emily Alberico, Simon Messing, Vanessa Wall, Albert H. Chan, Dhirendra K. Simanshu, Dwight V. Nissley, Frank McCormick, Dominic Esposito, Anna E. Maciag, David M. Turner. Screening a disulfide library against engineered cysteine RAS mutants reveal cryptic binding pockets [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr B006.