Disulfide tethering reveals cryptic pockets in oncogenic KRAS

Disulfide tethering is a site-directed method of drug discovery used to identify hits for challenging targets. We applied tethering to target oncogenic KRAS, a small GTPase once considered undruggable due to its high nucleotide affinity and a perceived absence of binding sites. We prepared a library of 2160 disulfide-containing fragments. We screened over 1000 compounds against a panel of 83 engineered cysteine mutants of KRAS G12D in the active conformation and screened the full library for a subset of 30 mutants. For select mutants and hits, we performed 2-mercaptoethanol competition assays (βME-50) to prioritize ligands. Ligandability analysis comparing hit rates across mutant residues enabled the identification of druggable hot spots. Our studies confirmed known binding sites, including the Switch-II / α-helix 3 pocket. In addition, we identified previously undescribed cryptic pockets and validated select hits using computational chemistry and NMR spectroscopy. These pockets represent promising opportunities for future drug discovery campaigns.