Targeting RNA methyltransferases with small molecules as inhibitors or tool compounds is an emerging field of interest in epitranscriptomics and medicinal chemistry. For two challenging RNA methyltransferases that introduce the 5-methylcytosine (m5C) modification in different tRNAs, namely DNMT2 and NSUN6, an ultra-large commercially available chemical space was virtually screened by physicochemical property filtering, molecular docking, and clustering to identify new ligands for those enzymes. Novel chemotypes binding to DNMT2 and NSUN6 with affinities down to KD,app = 37 µM and KD,app = 12 µM, respectively, were identified using a microscale thermophoresis (MST) binding assay. These compounds represent the first molecules with a distinct structure from the cofactor SAM and have the potential to be developed into activity-based probes for these enzymes. Additionally, the challenges and strategies of chemical space docking screens with special emphasis on library focusing and diversification are discussed.
Keywords: DNMT2; NSUN6; RNA methyltransferases; chemical spaces; molecular docking; ultra-large molecular libraries; virtual screening.