title: A boronic-acid-based probe for fluorescence polarization assays with penicillin binding proteins and β-lactamases.
abstract: Penicillin binding proteins (PBPs) and β-lactamases are involved in interactions with β-lactam antibiotics connected with both antibacterial activity and mediation of bacterial β-lactam resistance. Current methods for identifying inhibitors of PBPs and β-lactamases can be inefficient and are often not suitable for studying weakly and/or reversibly binding compounds. Therefore, improved ligand binding assays for PBPs and β-lactamases are needed. We report the development of a fluorescence polarization (FP) assay for PBPs and "serine" β-lactamases using a boronic-acid-based, reversibly binding "tracer." The tracer was designed based on a crystal structure of a covalent complex between a boronic acid and PBP1b from Streptococcus pneumoniae. The tracer bound to three different PBPs with modest affinity (K(d)=4-12 μM) and more tightly to the TEM1 serine β-lactamase (K(d)=109 nM). β-Lactams and other boronic acids were able to displace the tracer in competition assays. These results indicate that fluorescent boronic acids are suited to serve as reversibly binding tracers in FP-based assays with PBPs and β-lactamases and potentially with other related enzymes.
authors.affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Inglis
authors.forename: Matthias
authors.initials: M
authors.lastname: Strieker
authors.forename: Anna M
authors.initials: AM
authors.lastname: Rydzik
authors.forename: Andréa
authors.initials: A
authors.lastname: Dessen
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D001897:Boronic Acids / Q000737:chemistry*; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D046915:Penicillin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000737:chemistry / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*; D047090:beta-Lactams / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001897:Boronic Acids; D005456:Fluorescent Dyes; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; C480727:PBP1a protein, Streptococcus pneumoniae; D010458:Peptidyl Transferases; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1