Deploying Fluorescent Nucleoside Analogues for High-Throughput Inhibitor Screening.

High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used for this high-throughput screen has a KD value of 7.2 μm with the TcdB GT and shows a >30-fold increase in fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two other screening approaches.