Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK.
Anal Biochem. 2012 Jan 1;420(1):41-7. doi: 10.1016/j.ab.2011.08.036. Epub 2011 Aug 26.
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.
青霉素结合蛋白（PBPs）和β-内酰胺酶与β-内酰胺抗生素相互作用，与抗菌活性和介导细菌β-内酰胺耐药性有关。目前鉴定 PBPs 和β-内酰胺酶抑制剂的方法可能效率低下，并且通常不适合研究弱结合和/或可逆结合的化合物。因此，需要改进用于 PBPs 和β-内酰胺酶的配体结合测定法。我们报告了使用基于硼酸的可还原结合“示踪剂”开发用于 PBPs 和“丝氨酸”β-内酰胺酶的荧光偏振（FP）测定法。该示踪剂是根据硼酸与肺炎链球菌 PBP1b 的共价复合物的晶体结构设计的。该示踪剂与三种不同的 PBPs 具有中等亲和力（Kd=4-12 μM），与 TEM1 丝氨酸β-内酰胺酶的亲和力更强（Kd=109 nM）。β-内酰胺类药物和其他硼酸能够在竞争测定中置换示踪剂。这些结果表明，荧光硼酸适合作为 PBPs 和β-内酰胺酶以及其他相关酶的 FP 测定中的可还原结合示踪剂。
