Abstract B37: Development of inhibitors of the activated form of KRAS G12C
Authors: Stewart Michelle L. , Perl Nicholas R. , Lee Seung-Joo , Xue Linlong , Zhou Minyun , Simon Jonah , Luly Kathryn M. , Grigoriu Siminia , Yuzhakov Alex , Silver Alec , Lowe Jason T. , Benod Cindy C. , Mann Alan S. , Verdine Gregory L. , Rigby Alan C. , Mulvihill Mark J. , May Earl W. , Kohlmann Anna , Townson Sharon A. , Pollock Roy M. , Jin Meizhong 
Published: June 2021

Activating mutations in RAS proteins occur in ~1/3 of human cancers. These mutations impair the ability of the protein to hydrolyze GTP to GDP. As a result, mutant RAS proteins exist predominantly in the GTP-bound state, which directly activates aberrant downstream signaling via interaction with effectors such as RAF. Most RAS mutations occur at glycine 12 of the KRAS isoform. One such mutation, KRAS G12C, is particularly common in non-small cell lung cancer where it is found in ~15% of lung adenocarcinomas. Recent efforts have targeted KRAS G12C in the GDP-bound state; however, direct pharmacologic inhibition of active, GTP-bound KRAS G12C has proved challenging. Here, we deployed a novel SMARTTM (Small Molecule Assisted Receptor Targeting) platform to advance covalent compounds that selectively inhibit GTP-bound KRAS G12C. Using a mechanism reminiscent of the natural products rapamycin and cyclosporine, these compounds promote formation of a novel inhibitory ternary complex consisting of cyclophilin A (CypA, an abundant immunophilin present in all human cells), the SMART inhibitor, and GTP-KRAS G12C. Structure-based design of the SMART inhibitor yielded potent covalent inhibitors of GTP-KRAS G12C that exhibit &gt;100-fold selectivity for mutant KRAS G12C over WT KRAS. Structural analysis of the ternary complex revealed that the covalent linkage between the SMART inhibitor and the mutant cysteine of KRAS occurred in the context of extensive interactions between CypA, the SMART inhibitor, and GTP-KRAS G12C that provide significant binding affinity (KI = 2.5 μM). The GTP-KRAS G12C|Inhibitor|CypA complex directly occluded effector binding, and as such, the compounds disrupted the KRAS-RAF interaction in biochemical assays. This activity was dependent on CypA, underlining the importance of the KRAS G12C| CypA protein-protein interaction in driving target engagement. In cell-based studies, SMART inhibitors crosslinked KRAS G12C and potently inhibited ERK phosphorylation and cell growth in G12C mutant tumor cell lines but had no effect on non-G12C bearing tumor cells. CRISPR knockout of cellular CypA confirmed that these activities were dependent on the presence of endogenous CypA. Importantly, SMART inhibitors bind directly to active, GTP-KRAS G12C and thus, their activity does not rely on trapping KRAS G12C in the inactive GDP-bound state. As a result, the cellular potency of SMART inhibitors with respect to crosslinking, pERK inhibition, and growth inhibition was maintained in the presence of growth factor treatments that reduce the cellular GDP-KRAS G12C pool. In contrast, we found that the activity of a previously described GDP-KRAS G12C targeting inhibitor was attenuated by growth factor treatment. To our knowledge, these are the first examples of mutant-selective KRAS inhibitors that target the active, GTP-bound state of KRAS G12C. We are currently optimizing the drug-like properties of these SMART inhibitors and evaluating their activity in in vivo models.

Citation Format: Michelle L. Stewart, Nicholas R. Perl, Seung-Joo Lee, Linlong Xue, Minyun Zhou, Jonah Simon, Kathryn M. Luly, Siminia Grigoriu, Alex Yuzhakov, Alec Silver, Jason T. Lowe, Cindy C. Benod, Alan S. Mann, Gregory L. Verdine, Alan C. Rigby, Mark J. Mulvihill, Earl W. May, Anna Kohlmann, Sharon A. Townson, Roy M. Pollock, Meizhong Jin. Development of inhibitors of the activated form of KRAS G12C [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B37.