Chemical Proteomic Discovery of Isotype‐Selective Covalent Inhibitors of the RNA Methyltransferase NSUN2

Abstract 5‐Methylcytosine (m 5 C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1‐7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C 5 position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell‐active inhibitors of these enzymes are lacking. Here, we use cysteine‐directed activity‐based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross‐reactivity with other human NSUNs and exhibit good proteome‐wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2‐tRNA interactions in cancer cells, leading to a global reduction in tRNA m 5 C content. Our findings thus highlight the potential to create isotype‐selective and cell‐active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine.