School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University of South Australia, SA 5000, Australia.
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China.
Biochim Biophys Acta Biomembr. 2018 Apr;1860(4):878-886. doi: 10.1016/j.bbamem.2017.08.024. Epub 2017 Sep 8.
Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl β-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.
多药外排蛋白复合物，如大肠杆菌中的 AcrAB-TolC，在革兰氏阴性菌的多药耐药中起着至关重要的作用，并且还与其他过程如毒力和生物膜形成有关。因此，作为逆转临床相关病原体中抗菌耐药性的一种手段，外排泵抑制剂（EPIs）的抑制作用在过去二十年中得到了越来越多的关注。AcrB 的结构和功能分析的显著进展为外排泵抑制剂的选择提供了信息。然而，缺乏一种准确的方法来确定外排泵抑制的动力学。在这项研究中，我们对表面等离子体共振（SPR）进行了标准化和优化，以探测底物和抑制剂与 AcrB 结合的动力学。SPR 方法还与荧光药物结合方法相结合，通过该方法，使用与 SPR 相同的条件和对照来确定两种荧光 AcrB 底物的亲和力。将荧光测定的结果与 SPR 测定的结果进行比较，结果显示出极好的相关性，并为 SPR 所用的方法和条件提供了验证。随后确定了 AcrB 与底物（阿霉素、新生霉素和米诺环素）结合的动力学参数。最后，还探测了两种已建立的抑制剂（苯丙氨酸精氨酸β-萘基酰胺和 1-1-萘基甲基-哌嗪）和三种新型 EPI（4-异丁氧基-2-萘甲酰胺（A2）、4-异戊氧基-2-萘甲酰胺（A3）和 4-苄氧基-2-萘甲酰胺（A9）对 AcrB 抑制的动力学。获得的动力学数据可以与抑制剂的功效和作用机制相关联。这项研究是定量分析临床重要的 RND 类多药外排泵抑制动力学的第一步，将允许设计出改进的和更有效的药物外排泵抑制剂。本文是由 Ute Hellmich、Rupak Doshi 和 Benjamin McIlwain 编辑的特刊“超越膜蛋白的结构-功能范围”的一部分。
