Chemical alkylation of Asp12 enables mutant selective 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 β-lactone-based electrophiles which exploit ring strain to crosslink K-Ras(G12D) at the mutant aspartate to form stable covalent complexes in cells, effectively blocking Ras-effector interaction and downstream signaling. Structural insights from x-ray crystallography and exploitation of the differential chemoselectivity and stereoelectronic requirements for attack of the β-lactone electrophile allowed development of a substituted β-lactone which resisted attack by aqueous buffer but enabled rapid attack by aspartic acid-12 in K-Ras. 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. ### Competing Interest Statement Z.Z., Q.Z., and K.M.S. are inventors on patents related to covalent K-Ras(G12D) inhibitors reported here. K.M.S. is an inventor on patents owned by University of California San Francisco covering KRAS targeting small molecules licensed to Araxes, Erasca and Novartis. K.M.S. has consulting agreements for the following companies, which involve monetary and/or stock compensation: Revolution Medicines, Black Diamond Therapeutics, BridGene Biosciences, Denali Therapeutics, Dice Molecules, eFFECTOR Therapeutics, Erasca, Novartis, Genentech/Roche, Janssen Pharmaceuticals, Kumquat Biosciences, Kura Oncology, Mitokinin, Type6 Therapeutics, Nested Therapeutics, Vevo, Wellspring Biosciences (Araxes Pharma), Ikena, Initial Therapeutics and BioTheryX.