O-GlcNAcase fragment discovery with fluorescence polarimetry

Vladimir S BORODKIN, Karim Rafie, Nithya Selvan, Tonia Aristotelous, Iva Navratilova, Andrew T. Ferenbach, Daan M F van Aalten

Index: 10.1021/acschembio.8b00183

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Abstract

The attachment of the sugar D-N-acetylglucosamine (GlcNAc) with to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer’s and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.