Development of a high-throughput TR-FRET screening assay for a fast-cycling KRAS mutant

Mutations in the small GTPase protein KRAS are one of the leading drivers of cancers including lung, pancreatic, and colorectal, as well as a group of developmental disorders termed "Rasopathies ". Recent breakthroughs in the development of mutant-specific KRAS inhibitors include the FDA approved drug Lumakras (Sotorasib, AMG510) for KRAS G12C-mutated non-small cell lung cancer (NSCLC), and MRTX1133, a promising clinical candidate for the treatment of KRAS G12D-mutated cancers. However, there are currently no FDA approved inhibitors that tar-get KRAS mutations occurring at non-codon 12 positions. Herein, we focused on the KRAS mutant A146T, found in colorectal cancers, that exhibits a "fast-cycling " nucleotide mechanism as a driver for oncogenic activation. We developed a novel high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay that leverages the reduced nucleotide affinity of KRAS A146T. As designed, the assay is capable of detecting small molecules that act to allosterically modulate GDP affinity or directly compete with the bound nucleotide. A pilot screen was completed to demonstrate robust statistics and reproducibility followed by a primary screen using a diversity library totaling over 83,000 compounds. Compounds yielding > 50% inhibition of TR-FRET signal were selected as hits for testing in dose-response format. The most promising hit, UNC10104889, was further investi-gated through a structure activity relationship (SAR)-by-catalog approach in an attempt to improve potency and circumvent solubility liabilities. Overall, we present the TR-FRET platform as a robust assay to screen fast-cycling KRAS mutants enabling future discovery efforts for novel chemical probes and drug candidates.