title: Chromophore-linked substrate (CLS405): probing metallo-β-lactamase activity and inhibition.
abstract: Serine- and metallo-β-lactamases present a threat to the clinical use of nearly all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. Efforts to develop metallo-β-lactamase (MBL) inhibitors require suitable screening platforms to allow the rapid determination of β-lactamase activity and efficient inhibition. Unfortunately, the platforms currently available are not ideal for this purpose. Further progress in MBL inhibitor identification requires inexpensive and widely applicable assays. Herein the identification of an inexpensive and stable chromogenic substrate suitable for use in assays of clinically relevant MBLs is described. (6R,7R)-3-((4-Nitrophenoxy)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5,5-dioxide (CLS405) was synthesised in a three-step protocol. CLS405 was then characterised spectroscopically, and its stability and kinetic properties evaluated. With a Δλmax value of 100 nm between the parent and hydrolysis product, a higher analytical accuracy is possible with CLS405 than with commonly used chromogenic substrates. The use of CLS405 in assays was validated by MBL activity measurements and inhibitor screening that resulted in the identification of N-hydroxythiazoles as new inhibitor scaffolds for MBLs. Further evaluation of the identified N-hydroxythiazoles against a panel of clinically relevant MBLs showed that they possess inhibitory activities in the mid- to low-micromolar range. The findings of this study provide both a useful tool compound for further inhibitor identification, and novel scaffolds for the design of improved MBL inhibitors with potential as antibiotics against resistant strains of bacteria.
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Rd, Oxford, OX1 3TA (UK).
authors.forename: Anne
authors.initials: A
authors.lastname: Makena
authors.forename: Sander S
authors.initials: SS
authors.lastname: van Berkel
authors.forename: Clarisse
authors.initials: C
authors.lastname: Lejeune
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Owens
authors.forename: Anil
authors.initials: A
authors.lastname: Verma
authors.forename: Ramya
authors.initials: R
authors.lastname: Salimraj
authors.forename: James
authors.initials: J
authors.lastname: Spencer
authors.forename: Jürgen
authors.initials: J
authors.lastname: Brem
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002511:Cephalosporins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D013844:Thiazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: C000589334:3-((4-nitrophenoxy)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo(4.2.0)oct-2-ene-2-carboxylic acid 5,5-dioxide; D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013844:Thiazoles; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases
keywords: antibiotics; assays; chromogenic substrates; drug resistance; metallo-β-lactamases; pan inhibitors