Strain-release alkylation of Asp12 enables mutant selective GTP-state targeting of K-Ras(G12D)

K-Ras is the most commonly mutated oncogene in human cancer, yet direct small-molecule targeting of K-Ras mutants has been mostly unsuccessful until recently. The discovery of an allosteric pocket under Switch-II with covalent cysteine-crosslinking molecules has allowed for the development of targeted therapies that selectively engage the highly reactive acquired cysteine in the K-Ras(G12C) mutation without affecting the wild-type protein. Sotorasib and adagrasib, two advanced Switch-II Pocket inhibitors, have received FDA approval to treat K-Ras(G12C)-driven non-small cell lung cancer. However, the most frequent K-Ras mutation G12D particularly prevalent in pancreatic ductal adenocarcinoma has remained untargetable with covalent drugs due to the poor nucleophilicity of the somatic aspartate residue. Here we present a set of malolactone-based electrophiles which exploit ring strain to crosslink K-Ras(G12D) at the mutant aspartate to form stable covalent complexes. Structural insights from x-ray crystallography and exploitation of the stereoelectronic requirements for attack of the electrophile allowed development of a substituted malolactone which resisted attack by aqueous buffer but rapidly crosslink with the aspartate-12 of K-Ras in both GDP- and GTP-state. The signaling-competent GTP-state targeting allowed effective suppression of downstream signaling and proliferation of cancer cells harboring K-Ras(G12D) mutation, and tumor growth of cell line-derive xenograft in mice. Our results demonstrate the rational design of covalent inhibitors to target a non-catalytic carboxylic acid side chain in K-Ras(G12D) which has resisted traditional drug discovery efforts.