Alok K. Sharma,Jun Pei,Yue Yang,Marcin Dyba,Brian Smith,Dana Rabara,Erik Larsen,Felice C. Lightstone,Dominic Esposito,Andrew G. Stephen,Bin Wang,Pedro J. Beltran,Eli Wallace,Dwight V. Nissley,Frank McCormick,Anna E. Maciag
Journal of Biological Chemistry, 2024
Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient), and state 2 (active, effector binding enabled). Here we use 31P NMR to delineate the differences in state 1 and state 2 populations present in wild-type (WT) and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analogue GppNHp. Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in the state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RBD (RAS Binding Domain) binding and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small molecule inhibitor, BBO-8956, which is effective against both GDP and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs the state 1 - state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1 RBD is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action (MOA) for this novel, active-conformation inhibitor.
