Development of split luciferase complementation probes sensing KRAS/effector interaction

Abstract

KRAS is the most frequently mutated oncogene in human cancers. Clinically effective therapy targeting KRAS, however, has not been developed to date despite the decades of intensive efforts. Among various therapeutic approaches taken, blocking the interaction between KRAS and the effector proteins is one of the effective strategies to inhibit KRAS signaling in diseases. Here we have developed the highly-sensitive split ELuc (Emerald Luc, an enhanced luciferase from click beetle) probes which detect the interaction between KRAS (wild-type, G12C and G12D active mutants) and the effector proteins such as BRAF, CRAF, and three RalGEFs (RalGDS, RGL2, and RGL3). The probe pairs transiently co-transfected into 293T cells yielded significantly strong luminescence signals in all combination of KRAS and effector probes but not with negative control probes, indicating the specificity and the sensitivity of the probes. As expected, KRAS G12C and G12D active mutant probes yielded higher signals than KRAS wild-type probe in any combination with effector probes. The novel probe systems described here will be useful tools for the discovery of KRAS genotype-or effector-specific inhibitors.