Efficient assays for measuring inhibition of penicillin binding protein 3 (PBP3) transpeptidases.

Abstract

β-Lactam antibiotics, which are amongst the most highly used anti-infective medicines, function via inactivating covalent reaction with the nucleophilic serine residue of transpeptidases (penicillin binding proteins, PBPs). Few methods for monitoring the rate of PBP acylation by β-lactams are reported, limiting the development of new inhibitors. We describe a fluorescence-based method employing release of umbelliferone from a cephem (FC5) for the efficient determination of the second order rate constant (k inact /K I) for PBP inhibition. The method is shown to be suitable for efficient generation of inhibition rate constants for multiple PBP3s. The FC5 probe used is also suited for β-lactamase assays, allowing multiple enzyme (sub)classes to be screened using a single reporter compound. The results of mass spectrometry-based assays measuring the efficiency of covalent reaction with PBP3s compared to meropenem support the results obtained from the fluorescence-based assays. X-ray crystallographic analyses of P. aeruginosa PBP3 following reaction with ertapenem, doripenem and biapenem inform on interactions leading to difference in the rates of their reactions with PBP3. The PBP3 methods will be useful in efforts to develop both novel β-lactam and non-β-lactam transpeptidase inhibitors with improved activity and β-lactamase resistance properties.

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