A boronic-acid-based probe for fluorescence polarization assays with penicillin binding proteins and β-lactamases

Penicillin binding proteins (PBPs) and beta-lactamases are involved in interactions with beta-lactam antibiotics connected with both antibacterial activity and mediation of bacterial p-lactam resistance. Current methods for identifying inhibitors of PBPs and beta-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 beta-lactamases are needed. We report the development of a fluorescence polarization (FP) assay for PBPs and serine beta-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 mu M) and more tightly to the TEM1 serine beta-lactamase (K(d) = 109 nM). beta-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 beta-lactamases and potentially with other related enzymes. (C) 2011 Elsevier Inc. All rights reserved.