Identification and Characterization of Novel Small-Molecule Enhancers of the CUL3LZTR1 E3 Ligase KRAS Complex

The Ras family of GTPases is among the most frequently mutated proteins in human Cancer, creating a high clinical demand for therapies that counteract their signaling activity. An important layer of regulation that could be therapeutically exploited is the proteostatic regulation of the main Ras GTPases KRAS, NRAS, and HRAS, as well as the closely related members, MRAS and RIT1, by the leucine zipper-like transcriptional regulator 1 cullin 3 RING E3 ubiquitin Ligase complex (CUL3^LZTR1). Genetic inactivation of LZTR1, as observed in different Cancer entities and Noonan syndrome leads to enhanced Ras GTPase abundance and altered MAPK pathway activation state. Novel therapeutic approaches to interfere with hyperactive Ras signaling, thereby complementing existing treatments, are highly sought after. Motivated by the growing arsenal of molecular glue degraders, we report the identification of novel chemical fragments that enhance the protein-protein interaction (PPI) of the KRAS-LZTR1 complex. We established a split-luciferase-based reporter assay that monitors the Ras GTPase-LZTR1 interaction in a scalable format, capable of capturing chemical, as well as mutational perturbations. Using this screening system, in combination with a small fragment library, we identified two fragments, C53 and Z86, that enhance the interaction of the KRAS-LZTR1 complex in a dose-dependent manner. Further orthogonal validation experiments using proximity biotinylation (BioID), thermal shift assays, and NMR spectroscopy demonstrated fragment-dependent enhanced recruitment of endogenous LZTR1 and physical engagement of KRAS. The two fragments, which potentiate the KRAS-LZTR1 interaction, serve as starting points for fragment-based drug discovery. Additionally, the assay we introduced is amenable to high-throughput screening to further explore the pharmacological modulation of the CUL3^LZTR1-RAS GTPase complex.