Format

Send to

Choose Destination
Anal Biochem. 2012 Jan 1;420(1):41-7. doi: 10.1016/j.ab.2011.08.036. Epub 2011 Aug 26.

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

Author information

1
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK.

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.

PMID:
21925482
DOI:
10.1016/j.ab.2011.08.036
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center