A novel fluorescent probe for retaining galactosyltransferases.

Area of Science:

• Biochemistry
• Enzymology
• Chemical Biology

Background:

• Glycosyltransferases (GTs) are crucial carbohydrate-active enzymes in both prokaryotes and eukaryotes, regulating vital processes like cell adhesion and carcinogenesis.
• GTs represent significant molecular targets for drug discovery and chemical biology applications.
• Development of efficient and broadly applicable GT bioassays, particularly for inhibitor screening, is essential to harness their therapeutic potential.

Purpose of the Study:

• To develop novel, fluorescent UDP-galactose (UDP-Gal) derivatives as donor analogues for galactosyltransferases (GalTs).
• To establish a new, modular, and operationally simple bioassay for high-throughput screening of GT inhibitors.
• To demonstrate the utility of these fluorescent probes in ligand-displacement assays for GalT inhibitor identification.

Main Methods:

• Synthesis of novel fluorescent derivatives of UDP-galactose.
• Characterization of these derivatives as donor analogues for retaining galactosyltransferases (GalTs).
• Utilizing fluorescence quenching upon enzyme binding as a readout in ligand-displacement assays.

Main Results:

• Novel fluorescent UDP-Gal analogues were successfully synthesized and recognized by several retaining GalTs.
• A specific fluorescent derivative exhibited fluorescence quenching upon binding to individual GalTs.
• This quenching effect was effectively used as a readout in ligand-displacement experiments for inhibitor screening.
• The developed assay principle is modular, operationally simple, and facilitates inhibitor screening.

Conclusions:

• Novel fluorescent UDP-Gal probes are effective tools for developing new GalT bioassays.
• The assay's modularity and simplicity significantly aid in inhibitor screening processes.
• The minimal structural differences between natural UDP-Gal and the probes suggest broad applicability to various GalTs and nucleotide-binding proteins.