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. Disulfide tethering screening is applied to address the challenge of targeting oncogenic KRAS, historically deemed undruggable due to its high nucleotide affinity and perceived lack of binding sites. Here, the authors developed a disulfide library and a set of 83 cysteine KRAS mutants to perform tethering screens towards the oncogenic KRAS, while describing the discovery of fragment-binding pockets in KRAS G12D, identified by this screening approach.