High throughput assay to identify inhibitors against UDP‐galactopyranose mutase from eukaryotic pathogens

The flavoenzyme UDP‐galactopyranose mutase (UGM) catalyzes the isomerization of UDP‐galactopyranose to UDP‐galactofuranose, the biosynthetic precursor of galactofuranose (Gal f ). Gal f residues are essential components in the cell wall of pathogens and play vital roles for their virulence. Thus inhibitors of UGM that block the biosynthesis of Gal f could lead to novel therapeutics. Up to date, no eukaryotic UGM inhibitors but only a few prokaryotic UGM inhibitors have been reported. Here we present the synthesis of fluorescently labeled UDP analogs that can be used to develop a high throughput fluorescence polarization (FP) assay to identify specific eukaryotic UGM inhibitors. Our FP binding assay indicates that all these analogs showed tight bindings to prokaryotic Mt UGM, but specific bindings to eukaryotic Af UGM were only obtained from the UDP‐rhodamine analogs 3 and 4 with K d values of 2.2±0.2 and 2.5±0.2 μM, respectively, which are 10 folds lower than that of UDP‐fluorescein analogs 1 and 2 . The Af UGM FP assay using fluorophore 3 was validated against several known prokaryotic UGM ligands, and displayed excellent Z′ factor (0.79±0.01) and good tolerance to DSMO. Our results demonstrate that analogs 3 and 4 can be used as fluorescent probes for a high throughput assay to identify specific inhibitors of Af UGM. This work was supported by a National Institute of Health grant RO1‐AI082542 (R. Tarleton PI).