An Optimized Microscale Thermophoresis Method for High- Throughput Screening of DNA Methyltransferase 2 Ligands

Developing methyltransferase inhibitors is challeng-ing, since most of the currently used assays are time-consuming and cost-intensive. Therefore, efficient, fast, and reliable methods for screenings and affinity determinations are of utmost importance. Starting from a literature-known fluorescent S- adenosylhomocysteine derivative, 5-FAM-triazolyl-adenosyl-Dab, developed for a fluorescence polarization assay to investigate the histone methyltransferase mixed-lineage leukemia 1, we herein describe the applicability of this compound as a fluorescent tracer for the investigation of DNA-methyltransferase 2 (DNMT2), a human RNA methyltransferase. Based on these findings, we established a microscale thermophoresis (MST) assay for DNMT2. This displacement assay can circumvent various problems inherent to this method. Furthermore, we optimized a screening method via MST which even indicates if the detected binding is competitive and gives the opportunity to estimate the potency of a ligand, both of which are not possible with a direct binding assay.

Automated summary

This study explores the applicability of a novel fluorescent tracer for human RNA methyltransferase DNMT2 and establishes a new screening method using microscale thermophoresis. The results overcome some limitations of traditional methods and provide additional information.