Abstract
KRAS, the most frequently mutated oncogene in human cancers, was considered "undruggable" until the identification of small molecules that bind irreversibly to the mutant reactive cysteine at residue 12. Despite the encouraging anticancer activity of KRASG12C inhibitors in clinical trials, identification of more potent drugs is expected to achieve the maximal clinical benefit, which is hindered by the low sensitivity or throughput of current biochemical approaches. To overcome these limitations, a biotin-streptavidin-enhanced enzyme-linked immunosorbent assay (BA-ELISA) based on the competitive interaction of biotin-labeled probe and the test compound with KRASG12C was developed. Compared with reported assays, less protein was used in BA-ELISA, which significantly improves the resolution of inhibitor potency, thus contributing to the identification of highly potent inhibitors. Furthermore, BA-ELISA can also be expanded to determine the cellular potency of the inhibitors using KRASG12C mutant living cells. Using three previously disclosed compounds, ARS-1620, AMG 510, and MRTX849, we demonstrated that BA-ELISA is a highly sensitive, specific, and robust method for high-throughput screening of KRASG12C inhibitors.