Structural mechanisms underlying resistance to Switch-II pocket inhibitors in KRAS

Direct targeting of KRAS through the Switch-II pocket is an effective therapeutic strategy, but acquired resistance limits clinical benefit. We examine clinically observed resistance mutations (R68S, H95Q, Y96C, Y96D and Q99L) arising during KRAS G12C inhibitor therapy using biochemical, structural, computational and cellular approaches. Covalent and noncovalent binding measurements reveal mutation- and inhibitor-specific reductions in drug engagement across inhibitor classes. Structural analyses identify two principal resistance mechanisms: Type I, involving loss of key inhibitor contacts, and Type II, involving stabilization of pocket-occluding Switch-II conformations. Y96D exhibits both mechanisms through nucleotide-dependent non-native intramolecular interactions, whereas R68S primarily exhibits Type I resistance with modest Type II effects on pocket architecture and Switch-region dynamics. Y96C, H95Q and Q99L predominantly exhibit Type I resistance. All mutants retain RAF1 binding, whereas PI3Kα binding is selectively reduced by R68S and Q99L. Together, these findings provide structural insights for understanding on-target resistance to Switch-II pocket inhibitors.