title: Glycosidase inhibition by macrolide antibiotics elucidated by STD-NMR spectroscopy.
abstract: A glycosynthase approach was attempted to glycodiversify macrolide antibiotics, using DesR, a family-3 retaining beta-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-beta-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 microM and Ki = 2 +/- 0.2 microM) with respect to the hydrolysis of pNP-beta-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.
authors.affiliation: College of Pharmacy, Dalhousie University, Halifax, NS, B3H 1E9, Canada.
authors.forename: Ali
authors.initials: A
authors.lastname: Sadeghi-Khomami
authors.forename: Michael D
authors.initials: MD
authors.lastname: Lumsden
authors.forename: David L
authors.initials: DL
authors.lastname: Jakeman
mesh_terms: D000606:Amino Sugars / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004917:Erythromycin / Q000494:pharmacology; D005947:Glucose / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D018942:Macrolides / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008968:Molecular Conformation
chemical_list: D000606:Amino Sugars; D000900:Anti-Bacterial Agents; D018942:Macrolides; C008517:methymycin; C110125:desosamine; D004917:Erythromycin; D006026:Glycoside Hydrolases; D005947:Glucose