Analysis of Orthogonal Efflux and Permeation Properties of Compounds Leads to the Discovery of New Efflux Pump Inhibitors

Optimization of compound permeation into Gram-negative bacteria is one of the most challenging tasks in the development of antibacterial agents. Two permeability barriersthe passive diffusion barrier of the outer membrane (OM) and active drug effluxact synergistically to protect cells from the antibacterial action of compounds. In Escherichia coli (E. coli) and relatives, these two barriers sieve compounds based on different physicochemical properties that are defined by their interactions with OM porins and efflux pumps, respectively. In this study, we critically tested the hypothesis that the best substrates and inhibitors of efflux pumps are compounds that can effectively permeate the OM and are available at relatively high concentrations in the periplasm. For this purpose, we filtered a large subset of the ZINC15 database of commercially available compounds for compounds containing a primary amine, a chemical feature known to facilitate the uptake through E. coli general porins. The assembled library was screened by ensemble docking to AcrA, the periplasmic component of the AcrAB-TolC efflux pump, followed by experimental testing of the top predicted binders for antibacterial activities, efflux recognition, and inhibition. We found that the filtered primary amine library is a rich source of compounds with efflux-inhibiting activities and identified efflux pump inhibitors with novel chemical scaffolds effective against E. coli AcrAB-TolC and efflux pumps of multidrug-resistant clinical isolates of Acinetobacter baumannii. However, primary amines are not required for the recognition of compounds by efflux pumps and their efflux-inhibitory activities.

优化化合物向革兰氏阴性菌（Gram-negative bacteria）的渗透，是抗菌剂（antibacterial agents）研发过程中最具挑战性的任务之一。两类渗透屏障——外膜（Outer Membrane, OM）的被动扩散屏障与主动药物外排（active drug efflux）——协同作用，保护细胞免受化合物的抗菌攻击。在大肠杆菌（Escherichia coli, E. coli）及其近缘菌种中，这两类屏障依据不同的理化性质对化合物进行筛选：前者通过与外膜孔蛋白（OM porins）的相互作用确立筛选标准，后者则通过与外排泵（efflux pumps）的相互作用定义筛选规则。本研究严谨验证了如下假说：外排泵的最优底物与抑制剂，是能够有效穿透外膜、且在周质（periplasm）中浓度相对较高的化合物。为此，我们从商用化合物数据库ZINC15的大型子集中，筛选出含有伯胺（primary amine）的化合物——伯胺是已知可促进大肠杆菌通用孔蛋白介导摄取的化学特征。我们对组装得到的化合物文库开展了针对AcrAB-TolC外排泵的周质组分AcrA的集合对接（ensemble docking）筛选，随后对预测结合活性排名靠前的化合物，进行抗菌活性、外排识别与抑制作用的实验验证。研究结果表明，经筛选得到的伯胺文库是一类富含外排抑制活性化合物的资源库，并鉴定出具有全新化学骨架的外排泵抑制剂：这类抑制剂对大肠杆菌AcrAB-TolC以及多重耐药（multidrug-resistant）临床分离株鲍氏不动杆菌（Acinetobacter baumannii）的外排泵均具有有效活性。不过，外排泵识别化合物及其产生外排抑制活性，并不一定需要伯胺结构。