Abstract 6990: Profiling Ras inhibitors for effects on GTP hydrolysis using the Transcreener GDP assay

Abstract Ras family GTPases are the most frequently mutated proteins in human cancers, most notably missense mutations at G12 and G13 that prolong the activated, GTP-bound state of the protein. Inhibitors that shift the equilibrium toward GDP-bound Ras, such as sotorasib, are proving effective clinically, and efforts to expand upon this strategy are widespread. Current strategies for biochemical screening and analysis of Ras inhibitors rely on the use of fluorescent GTP and GDP analogs to detect effects on GDP release and GTP binding. Though robust and widely used, these methods do not provide information on GTP hydrolysis, a critical part of the GTPase signal transduction process. We have developed methods for detecting GTP hydrolysis by small GTPases using the Transcreener GDP2 assay, a homogenous, competitive immunoassay for GDP with far-red fluorescence readout. Because GDP release is generally rate limiting, stimulation by GEFs can also be measured by monitoring GDP formation. We have optimized these assays for initial velocity detection of GTPase activity for wild type (WT) and mutant Ras proteins with robustness suitable for high throughput screening and tested a number of compounds reported to inhibit GTP and/or GDP binding. Consistent with expectations, the Ras G12C-specific covalent inhibitor, Sotorasib, potently inhibited KRas G12C (IC50 = 9.6 nM) and did not inhibit the wild-type enzyme. MRTX1133, which binds noncovalently to GDP-bound KRas G12D, inhibited the G12D mutant with an IC50 of 8.4 nM, and was less potent with the G12V and WT enzymes; IC50 = 48 nM and 69 nM, respectively. BI-2865, a pan KRas inhibitor, showed much weaker inhibition of all KRas variants tested, with IC50 values of 427 nM, 228 nM and 236 nM for WT, G12D and G12V KRas. We note that these GTPase assays require nanomolar concentrations of protein, and therefore cannot be used to accurately determine the potency of covalent inhibitors, such as sotorasib, that bind with picomolar affinity. However, for non-covalent inhibitors, such as MRTX1113 and BI-2865, measuring GTPase activity is a robust approach for selectivity profiling and may yield mechanistic insights different from those gleaned from guanine nucleotide binding/release assays or biophysical methods, e.g. SPR. Citation Format: Camdon Yemm, Robert Lowery. Profiling Ras inhibitors for effects on GTP hydrolysis using the Transcreener GDP assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6990.