Development and Validation of a Universal High-Throughput UDP-Glycosyltransferase Assay with a Time-Resolved FRET...

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• Glycosyltransferases are vital enzymes involved in diverse metabolic and regulatory processes.
• These enzymes are significant therapeutic targets for diseases like cancer and metabolic disorders.
• Assay development for glycosyltransferases is challenging due to substrate diversity and detection limitations.

Purpose of the Study:

• To develop a broadly applicable high-throughput screening (HTS) assay for glycosyltransferase inhibitors.
• To utilize the detection of free uridine 5"-diphosphate (UDP) as a generic method for enzyme activity measurement.
• To establish a robust assay for screening enzymes utilizing nucleotide diphosphate (NDP) sugars.

Main Methods:

• Developed a Transcreener assay for immunodetection of UDP using time-resolved Förster resonance energy transfer (TR-FRET).
• Optimized the assay for NDP sugar concentrations ranging from 10 μM to 1 mM.
• Validated the assay through orthogonal pooled screening of 8,000 compounds against polypeptide N-acetylgalactosaminyltransferase T3.

Main Results:

• Achieved Z' values of 0.6 or higher, indicating assay robustness.
• Demonstrated stable reagent and signal performance for over 8 hours at room temperature.
• Successfully identified and confirmed inhibitor hits using orthogonal readouts.

Conclusions:

• The TR-FRET-based UDP detection assay offers a broadly applicable method for screening glycosyltransferases.
• This assay facilitates the discovery of inhibitors targeting UDP-sugar-utilizing enzymes.
• The assay's stability and performance are suitable for automated HTS environments.