Identification of eukaryotic UDP-galactopyranose mutase inhibitors using the ThermoFAD assay

• Published in: Biochemical and biophysical research communications Vol. 493; no. 1; pp. 58 - 63
• Main Authors: Martín del Campo, Julia S., Eckshtain-Levi, Meital, Sobrado, Pablo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.11.2017
• Subjects: Aspergillus fumigatus; Aspergillus fumigatus - enzymology; Drug Evaluation, Preclinical - methods; Enzyme Activation; Enzyme Inhibitors - chemistry; Flavins - chemistry; Flavonoids - chemistry; Flavopiridol; Galactofuran; Intramolecular Transferases - analysis; Intramolecular Transferases - antagonists & inhibitors; Piperidines - chemistry; Protein Interaction Mapping; Spectrometry, Fluorescence - methods; Temperature; ThermoFAD; UDP-Galactopyranose mutase; UGM inhibition; UDP-Galactopyranose mutase; Galactofuran; Flavopiridol; Aspergillus fumigatus; ThermoFAD; UGM inhibition
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2017.09.074

Aspergillus fumigatus is a human pathogen responsible for deadly infections in immune-compromised patients. A potential strategy for treating A. fumigatus infections is by targeting the biosynthesis of cell wall components, such as galactofuranase, which is absent in humans. Galactofuranose biosynthesis is initiated by the flavoenzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM requires the reduced form of the flavin for activity, which is obtained by reacting with NADPH. We aimed to identify inhibitors of UGM by screening a kinase inhibitor library using ThermoFAD, a flavin fluorescence thermal shift assay. The screening assay identified flavopiridol as a compound that increased the melting temperature of A. fumigatus UGM. Further characterization showed that flavopiridol is a non-competitive inhibitor of UGM and docking studies suggest that it binds in the active site. This compound does not inhibit the prokaryotic UGM from Mycobacteria tuberculosis. [Display omitted] •ThermoFAD assay was validated for HTS of inhibitors of UGM.•Flavopiridol inhibits eukaryotic but not prokaryotic UGM.•Flavopiridol is non-competitive inhibitor of UGM.•Docking studies suggest that Flavopiridol binds in the in the active site of UGM.