Oncogenic mutations of Ras are among the most common genetic alterations in human cancer, with an estimated disease burden of >3 million new patients per year worldwide. Despite widespread appreciation of the importance of Ras in cancer, direct binding ligands, which block downstream signaling, were not reported until 2013 due to the lack of obvious drug binding pockets in the protein. The clinically approved K-Ras inhibitors are mutant-selective as they rely on covalent recognition of the highly nucleophilic somatic cysteine residue of K-Ras(G12C). Recent preclinical reports of noncovalent K-Ras binding inhibitors have emerged, which lack mutant specificity and exhibit varying degrees of biochemical preference for mutant K-Ras over the wild-type. An adjacent glycine-13 mutation, p. G13C, particularly abundant in lung, colorectal, and pancreatic cancer, has not been targeted with an approved therapeutic molecule. Here, we report a series of targeted electrophiles designed to covalently modify Cys13 in K-Ras(G13C), overcoming the structural challenge posed by its shifted position relative to Cys12 in K-Ras(G12C). These inhibitors effectively alkylate K-Ras(G13C) in both GDP- and GTP-bound states, block effector interactions, and suppress the growth of K-Ras(G13C)-mutation cancer cell lines. Our findings expand the landscape of covalent K-Ras inhibitors beyond G12 mutations, providing a new therapeutic strategy for K-Ras(G13C)-driven cancers.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common cancer. More than 90% of PDAC tumors are caused by KRAS mutations, with the majority expressing the K-Ras(G12D) oncoprotein. Despite extensive drug discovery efforts across academia and industry, there are no approved drugs directly targeting K-Ras(G12D) in a mutant-selective manner. We report a series of α-diazoacetamide compounds that form covalent bonds via denitrogenative alkylation of acquired aspartic acid at the mutation site. The lead molecule allosterically inhibits the mitogen-activated protein kinase pathway downstream of K-Ras and therefore inhibits the growth of KRASG12D-driven cancer cell lines but not non-G12D mutation cancer cell lines. Our results show that the diazo-carboxy ligation spares not only the unreactive Gly12 residue in the K-Ras wild-type protein but also strong nucleophiles such as the Cys12 residue in K-Ras(G12C). The preference for a weak nucleophile carboxylic acid over canonically stronger nucleophiles provides the basis to expand the covalently targetable proteome to aspartic and glutamic acids.

The clinical success of covalent KRASG12C inhibition prompts further expansion of the concept to target non-cysteine oncogenic mutation sites as in KRASG12D. This endeavor was hampered by the lack of suitable electrophiles for the selective, covalent engagement of aspartate. Thanks to the recent discovery of β-lactone-bearing covalent inhibitors, new opportunities are emerging. Based on X-ray crystallographic insights and quantum chemical calculations, we herein describe the elucidation of structure–activity and -stability correlations to advance such electrophiles for drug discovery. Guided by predictions of transition state barrier heights for the attack of aspartate 12 at the β-lactone electrophile and structure-based design, we generated substituted β-lactones aiming to balance specific reactivity and chemical and metabolic stability. Our optimization strategy is driven by MS-based and cellular covalent target occupancy assays and PD marker analysis, proteome-wide profiling, and synthetic chemistry. With this work, we aim to expand the use of β-lactones as chemoselective electrophiles in medicinal chemistry.

Development of a malolactone electrophile that contains sufficient ring strain to counteract the weak nucleophilicity of aspartate enables covalent targeting of K-Ras-G12D, which is commonly found in ...