How KRAS Mutations Impair Intrinsic GTP Hydrolysis: Experimental and Computational Investigations

Oncogenic KRAS mutations impair GTP hydrolysis and increase the active GTP-bound KRAS population, which leads to growth-factor-independent cell proliferation and survival of cancer cells. Despite notable successes of small-molecule inhibitors in the treatment of KRASG12C cancer, many of these small-molecule inhibitors preferentially bind to inactive (GDP-bound) mutant KRAS, whose availability is limited by the slow rate of intrinsic GTP hydrolysis. A better understanding of how KRAS mutations impair intrinsic hydrolysis is important for designing more effective small-molecule therapeutics. In this work, experimental and computational approaches were utilized to investigate how the most important oncogenic mutations affect the intrinsic hydrolysis of GTP. We found that Q61H, G12V, and G12R mutations impair intrinsic hydrolysis by around 7-fold, 9-fold, and more than 20-fold, respectively, whereas G12A, G12C, G12D, and G13D have less effect. Based on mechanistic investigations, we propose that KRAS mutations impair intrinsic hydrolysis by disrupting the interactions needed to align the nucleophilic water molecule with GTP for nucleophilic attack. These results can assist small-molecule inhibitor design and also benefit the development of other therapeutic strategies, such as rescuing hydrolysis.