O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• Protein O-GlcNAcylation, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), influences critical cellular processes.
• Dysregulation of O-GlcNAc is associated with diseases including diabetes, cancer, and neurodegenerative disorders.
• OGA is a potential drug target for Alzheimer's and cardiovascular diseases, as elevated O-GlcNAc levels may offer protection.

Purpose of the Study:

• To synthesize a novel fluorescent probe for a fluorescence polarization assay.
• To facilitate the discovery of specific, potent, and brain-bioavailable OGA inhibitors.
• To establish an orthogonal high-throughput assay platform for OGA inhibitor screening.

Main Methods:

• Synthesis of a novel fluorescent probe.
• Development of a fluorescence polarization based assay.
• Structural characterization of Clostridium perfringens OGA (CpOGA) in complex with a ligand.

Main Results:

• The novel probe is effective for assaying human OGA, bacterial CpOGA, and lysosomal hexosaminidases HexA/B.
• Structural analysis of CpOGA-ligand complex was achieved using the developed assay.
• The synthesis method is adaptable for creating probes for other glycoside hydrolases.

Conclusions:

• A versatile fluorescent probe and assay platform for OGA inhibitor discovery has been established.
• The developed methodology supports the identification of potential therapeutic agents for OGA-related diseases.
• This approach can be extended to the development of assays for other glycoside hydrolase enzymes.