KRAS(G12D) can be targeted by potent inhibitors via formation of salt bridge

Zhongwei Mao,Hongying Xiao,Xin Li,Yonghui Zhang,Jing Xue,Yanan Du,Yuying Zhang,Yunyun Yang,Chun-Chi Chen,Yanguo Shang,Rey-Ting Guo,Lilan Zhang,Yu Yang,Panpan Shen

doi:10.1038/s41421-021-00368-w

Abstract

KRAS mutation occurs in nearly 30% of human cancers, yet the most prevalent and oncogenic KRAS(G12D) variant still lacks inhibitors. Herein, we designed a series of potent inhibitors that can form a salt bridge with KRAS’s Asp12 residue. Our ITC results show that these inhibitors have similar binding affinity with both GDP-bound and GTP-bound KRAS(G12D), and our crystallographic studies reveal the structural basis of inhibitor binding-induced switch-II pocket in KRAS(G12D), experimentally confirming the formation of a salt bridge between the piperazine moiety of the inhibitors and the Asp12 residue of the mutant protein. Among KRAS family proteins and mutants, both ITC and enzymatic assays demonstrate the selectivity of the inhibitors for KRAS(G12D); and the inhibitors disrupt the KRAS–CRAF interaction. We also observed the inhibition of cancer cell proliferation as well as MAPK signaling by a representative inhibitor (TH-Z835). However, since the inhibition was not fully dependent on KRAS mutation status, it is possible that our inhibitors may have off-target effects via targeting non-KRAS small GTPases. Experiments with mouse xenograft models of pancreatic cancer showed that TH-Z835 significantly reduced tumor volume and synergized with an anti-PD-1 antibody. Collectively, our study demonstrates proof-of-concept for a strategy based on salt-bridge and induced-fit pocket formation for KRAS(G12D) targeting, which warrants future medicinal chemistry efforts for optimal efficacy and minimized off-target effects.

Highlights

The oncogenic impacts of the KRAS gene were first reported in 1980s, making KRAS one of the first identified oncogenes[1]
One major breakthrough for KRAS inhibition was the discovery of an allosteric switch-II pocket (S-IIP) that is induced by covalent inhibitors of KRAS bearing the G12C driver mutation[8]
We successfully developed a series of small molecule inhibitors of KRAS (G12D), which function by inducing an allosteric S-IIP and forming a salt bridge bonding with Asp[12] residue, as confirmed by crystallographic studies

Summary

Introduction

The oncogenic impacts of the KRAS gene were first reported in 1980s, making KRAS one of the first identified oncogenes[1]. There is consensus that the difficulty in developing direct KRAS inhibitors relates on the one hand to the picomolar affinity of GTP and GDP to KRAS (the intracellular concentrations of these metabolites are much higher), and on the other hand to an absence of suitable deep pockets for allosteric regulation. Studies have shown that induction of S-IIP results from covalent bond formation between the electrophilic acryloyl moieties of these inhibitors and the nucleophilic thiol moiety of the Cys residue at position 129–16.

Methods

Results

Conclusion