Glycosidase Inhibition by Macrolide Antibiotics Elucidated by STD-NMR Spectroscopy Original Research Article

A glycosynthase approach was attempted to glycodiversify macrolide antibiotics, using DesR, a family-3 retaining β-glucosidase involved in the self-resistance mechanism of methymycin production. STD-NMR was used to probe enzyme-substrate interactions. Analysis of competitive STD-NMR experiments between erythromycin A and a chromogenic substrate (pNP-β-d-glucose) with the hydrolytically inactive nucleophile mutants led us to discover a family of unprecedented glycosidase inhibitors. Analysis of kinetic data with wild-type DesR determined that erythromycin is a competitive inhibitor of the glucosidase (IC50 = 2.8 ± 0.3 μM and Ki = 2 ± 0.2 μM) with respect to the hydrolysis of pNP-β-d-glucose. Comparable inhibitory data was obtained for clarithromycin; however, the inhibitory effect of azithromycin was weak and no significant inhibition was observed with methymycin or d-desosamine. This report documents significant inhibition of glycosidases by macrolide antibiotics and provides insight into the design of novel glycosidase inhibitors based on the macrolactone ring of macrolide antibiotics.