Systematic cysteine scanning identifies a druggable pocket in oncogenic KRAS.

The discovery of druggable pockets within proteins that lack traditional active sites remains a significant challenge in the development of therapeutics. To address this, we developed Cysteine Mapping of Accessible Pockets (CysMAP), a method for identifying druggable pockets in proteins. CysMAP employs systematic pooled cysteine (Cys)-variant libraries screened against diverse covalent compound libraries by intact LC-MS. We applied CysMAP to 189 KRAS(G12D) variants, purifying KRAS Cys-variants and screening them against 47 covalent compounds, quantifying accessibility, and reactivity across KRAS(G12D). We discovered previously unidentified ligand-bound states of Cys-variants surrounding the KRAS switch-II pocket. Structural studies of the D92C variant in complex with the compound BI-1830 uncovered a distinct novel binding pocket, highlighting the inherent plasticity of the region between switch-II and α3, that can accommodate diverse chemical entities in various conformations. This method holds significant potential for advancing drug discovery efforts against elusive targets such as oncogenic RAS mutants.