Selective and noncovalent targeting of RAS mutants for inhibition and degradation

Kai Wen Teng,Yingkai Zhang,Steven T Tsai,Chao Yang,Benjamin G Neel,Carmine Fedele,Shohei Koide,Takamitsu Hattori,Akiko Koide,John P O’Bryan,Xuben Hou

doi:10.1038/s41467-021-22969-5

Abstract

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.

Highlights

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors
Proteolysis targeting chimeras (PROTACs) are an emerging drug modality, bi-functional molecules that direct a protein of interest (POI) to the E3 ligase for ubiquitination and subsequent degradation by the proteasome[21,22]
To mutants with certain small side chains over the wild type (Affinity: G12C > G12V >> G12A and G12S >> G12). These mutations collectively account for a large fraction of the KRAS mutations found in cancers with poor 5-year survival rates, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC)[1]

Summary

Introduction

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. To develop drug-like molecules for proof-ofconcept purposes, many binding proteins targeting the GTPbound state of RAS mutants have been developed with a hope that larger binding surfaces of proteins coupled with very large sequence diversity afforded by molecular display technologies could achieve high selectivity[12,13,14,15,16] These RASbinding proteins reported to date are either not selective for mutants (over wild-type (WT) KRAS) or not effective in inhibiting RAS-mediated signaling in cells. In a previous application to RAS, we have developed a monobody called NS1 that inhibits RAS-mediated signaling through targeting the α4–α5 surface, which has established an approach to control RAS functions[20] Another important unanswered question in RAS drug discovery is whether or not one can selectively degrade an endogenous RAS mutant in a noncovalent manner. There remain many challenges in the development of degraders against KRAS mutants

Methods

Results

Conclusion