Deploying Fluorescent Nucleoside Analogues for High-Throughput Inhibitor Screening.

Area of Science:

• Medicinal Chemistry
• Biochemistry
• Drug Discovery

Background:

• Fluorescence-based assays are crucial for high-throughput screening in drug discovery.
• Existing methods like fluorescent polarization and FRET can yield high false-negative rates and background noise.
• Complex fluorophore labeling can complicate these screening techniques.

Purpose of the Study:

• To synthesize and apply fluorescent nucleoside analogues for competitive binding analyses.
• To develop environment-sensitive (ES) nucleoside probes adaptable to various enzymes.
• To establish a fragment-based lead discovery assay for the TcdB glycosyltransferase (GT).

Main Methods:

• Synthesis of selective fluorescent environment-sensitive (ES) nucleoside analogues.
• Application of these analogues in competitive binding assays.
• Development and benchmarking of an ES-based probe assay for TcdB GT against other screening methods.

Main Results:

• Demonstrated the utility of fluorescent nucleoside analogues for enzyme screening.
• Developed a specific assay for the TcdB GT using a uridine analogue probe.
• The uridine probe exhibited a K_D of 7.2 μm and a >30-fold fluorescence increase upon binding to TcdB GT.

Conclusions:

• Fluorescent nucleoside analogues provide a robust platform for directed competitive binding analyses.
• The developed ES-based probe assay is effective for fragment-based lead discovery.
• This approach offers a more sensitive and specific alternative to traditional fluorescence screening methods.