O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry

Vladimir S. Borodkin (Lead / Corresponding author), Karim Rafie, Nithya Selvan, Tonia Aristotelous, Iva Navratilova, Andrew T. Ferenbach, Daan M.F. Van Aalten (Lead / Corresponding author)

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Abstract

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) 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, and 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.

Original languageEnglish
Pages (from-to)1353-1360
Number of pages8
JournalACS Chemical Biology
Volume13
Issue number5
Early online date11 Apr 2018
DOIs
Publication statusPublished - 18 May 2018

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Polarimeters
Fluorescence
Assays
Hexosaminidase B
Cardiovascular Diseases
Hexosaminidases
Clostridium
Clostridium perfringens
Proteins
Fluorescence Polarization
Acetylglucosamine
Glycoside Hydrolases
Threonine
Transcription
Medical problems
Fluorescent Dyes
Sugars
Neurodegenerative Diseases
Pharmaceutical Preparations
Serine

Cite this

Borodkin, Vladimir S. ; Rafie, Karim ; Selvan, Nithya ; Aristotelous, Tonia ; Navratilova, Iva ; Ferenbach, Andrew T. ; Van Aalten, Daan M.F. / O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry. In: ACS Chemical Biology. 2018 ; Vol. 13, No. 5. pp. 1353-1360.
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O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry. / Borodkin, Vladimir S. (Lead / Corresponding author); Rafie, Karim; Selvan, Nithya; Aristotelous, Tonia; Navratilova, Iva; Ferenbach, Andrew T.; Van Aalten, Daan M.F. (Lead / Corresponding author).

In: ACS Chemical Biology, Vol. 13, No. 5, 18.05.2018, p. 1353-1360.

Research output: Contribution to journalArticle

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T1 - O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry

AU - Borodkin, Vladimir S.

AU - Rafie, Karim

AU - Selvan, Nithya

AU - Aristotelous, Tonia

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AU - Ferenbach, Andrew T.

AU - Van Aalten, Daan M.F.

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AB - The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) 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, and 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.

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