NSUN6 inhibitor discovery guided by its mRNA substrate bound crystal structure.

NSUN6 preferentially catalyzes the methylation of cytosine nucleotides in mRNA substrates, which enhances transcription. Dysregulation of NSUN6 catalysis drives the oncogenesis of certain cancers. In this study, we determined the crystal structure of human NSUN6 in complex with its S-adenosyl-L-methionine analog and a bound NECT-2 3'-UTR RNA substrate at 2.9 Å resolution. The complex structure reveals how NSUN6 recognizes the specific CUC[CU]A consensus motif of the substrate and facilitates the methyl transfer from S-adenosyl-L-methionine (SAM) to mRNA. By combining the structural data with nuclear magnetic resonance (NMR)-based fragment screening, a virtual screening, and a further comprehensive biochemical verification, we identified thiamine disulfide as a non-SAM analog lead compound that competes with the CUC[CU]A substrate for binding to NSUN6. Our findings pave the way for the discovery of potent inhibitors for the treatment of NSUN6-driven cancers in the future.