Abstract A084: Use of the natural nucleotide, GTP, is essential for the identification of potent, active-state KRASG12C inhibitors that bind in the switch II pocket
Authors: Wang Bin , Sharma Alok , Stice James , Smith Brian , Dyba Marcin , Singh Devansh , Denson John-Paul , Rabara Dana , Larsen Erik , Yang Yue , Lightstone Felice C , Stephen Andrew , Nissley Dwight , McCormick Frank , Wallace Eli , Maciag Anna E , Beltran Pedro J 
Published: December 2023

Background: Mutations on codon 12 of KRAS are observed in many human cancers. KRASG12C mutations are found in ~15% of non-small cell lung cancers and in a low percentage of colorectal and pancreatic adenocarcinomas. These activating mutations in KRAS push cellular balance towards its active, GTP-bound state that signals downstream and drives cellular transformation. Recently approved inhibitors of KRASG12C that bind and sequester the oncogenic protein in its inactive, GDP-bound state, have demonstrated clinical efficacy in patients with KRASG12C cancers, including NSCLC, CRC and pancreatic adenocarcinoma. However, duration of response has been shorter than expected from the potent inhibition of a driver oncogene. This quick emergence of acquired resistance has been attributed to reactivation of MAPK signaling through multiple mechanisms, including RTK signaling and KRASG12C gene amplification, resulting in increased active, GTP-bound KRASG12C.

Materials and Methods: Mass spectrometry was used to measure covalent modification of KRASG12C. Inhibition of the active state of KRASG12C was measured biochemically using a protein:protein interaction or RAS:RAF1 ELISA assays. Downstream consequences of inhibiting active-state KRASG12C was performed by western blotting.

Results:  In order to overcome active KRASG12C-driven resistance, we have developed direct, small molecule inhibitors of KRASG12C that inhibit both the active, GTP-bound and inactive, GDP-bound forms of KRASG12C through interactions with the switch II pocket, and independently of any other partner proteins. Mass spectrometry analysis of KRASG12C covalent engagement shows complete modification of both KRASG12C states, while sotorasib, adagrasib, and divarasib only modify the inactive, GDP-bound state. As expected, our active state inhibitors also show potent inhibitory activity in an effector (Raf1) binding assay where inactive, GDP-bound inhibitors demonstrate no measurable potency. Interestingly, during our work assessing the potency of these direct KRASG12C inhibitors of the active state, we discovered that employing the broadly used non-hydrolysable GTP nucleotide analogue GppNHP as a surrogate for the natural nucleotide results in overestimation of potency. We found that potency against the GppNHP-bound form of KRASG12C was consistently higher (3-10x) than when using GTP. These differences in potency between GppNHP and GTP were biologically meaningful as only compounds with strong activity against GTP-bound KRASG12C were able to demonstrate cellular activity consistent with inhibition of the active, GTP-bound state.

Conclusion: We demonstrate here that inhibiting the active, GTP-bound state of KRASG12C is possible with switch II pocket binders and that using the natural, physiological nucleotide, GTP, in biochemical assays is indispensable to identify compounds with promising cellular activity that is differentiated from the inactive, GDP-bound molecules.

Citation Format: Bin Wang, Alok Sharma, James Stice, Brian Smith, Marcin Dyba, Devansh Singh, John-Paul Denson, Dana Rabara, Erik Larsen, Yue Yang, Felice C Lightstone, Andrew Stephen, Dwight Nissley, Frank McCormick, Eli Wallace, Anna E Maciag, Pedro J Beltran. Use of the natural nucleotide, GTP, is essential for the identification of potent, active-state KRASG12C inhibitors that bind in the switch II pocket [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A084.