Nanomole Scale Screening of Fluorescent RNA‐Methyltransferase Probes Enables the Discovery of METTL1 Inhibitors

Abstract RNA methylation is a metabolic process validated for its association with various diseases, and thus, RNA methyltransferases (MTases) have become increasingly important in drug discovery. Yet, most frequently utilized RNA MTase assays are limited in their throughput and hamper this rapidly evolving field of medicinal chemistry. In this study, we describe a modular nanomole scale building block system that allowed the identification of tailored fluorescent MTase probes to unlock a broad selection of MTase drug targets for fluorescence‐based binding assays. Probe candidates were initially prepared on a 4 nanomole scale and could be tested directly from crude reaction mixtures to allow rapid probe identification and optimization. Using an alkyne‐azide click late‐stage functionalization strategy and in silico protein databank mining, we established a selection of fluorescent probes suitable for relevant drug targets from the METTL and NSUN families, as well as bacterial and viral MTases. Using this concept, a high‐throughput screening on the unexplored drug target METTL1 discovered three hit compounds with micromolar potency providing a (1 H ‐pyrazol‐4‐yl)pyridine‐based starting point for METTL1 drug discovery.