Shaheen A, Afridi W A, Mahboob S, Sana M, Zeeshan N, Ismat F, Mirza O, Iqbal M, Rahman M
Drug Discovery and Structural Biology group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, 44000 Pakistan.
Department of Biochemistry and Biotechnology, University of Gujrat, Hafiz Hay at Campus, Gujrat, 50700 Pakistan.
Mol Biol (Mosk). 2019 Jul-Aug;53(4):674-684. doi: 10.1134/S0026898419040128.
Acriflavine resistance protein B (AcrB) serves as prototype for multidrug resistance (MDR) efflux transporters of resistance nodulation division (RND) superfamily. AcrB has been proven as potential drug target with many synthetic and natural inhibitors have been identified such as those belonging to pyranopyridine, naphthamide and pimozide classes. The plant derived alkaloid inhibitors represented by reserpine has been found to inhibit both ATP binding cassette and major facilitator efflux transporters. In this study we report the reserpine induced inhibition of RND transporter AcrB. The preliminary docking analysis hints that reserpine shares its binding site with ciprofloxacin, a known substrate of AcrB and could possibly act as competitive inhibitor. For in vitro validation, AcrB from Salmonella typhi was cloned under the control of tac promoter and resulting vector was introduced into E. coli C41(DE3). Under autoinduced conditions, cells overexpressing AcrB transporter were subjected to combined dose of ciprofloxacin and reserpine. The combined exposure resulted in enhanced ciprofloxacin-induced growth inhibition of cells expressing AcrB transporter as compared to control cells transformed with vector of backbone sequence. Time kill analysis further confirmed these findings. To the best of our knowledge, this is first study to show that exposure to reserpine induces inhibition of AcrB. The assay developed in this study allows simple and reproducible detection of substrate/inhibitor effects upon AcrB and related efflux transporters.
吖啶黄素抗性蛋白B（AcrB）是抗性结瘤分裂（RND）超家族多药抗性（MDR）外排转运蛋白的原型。AcrB已被证明是潜在的药物靶点，已鉴定出许多合成和天然抑制剂，如属于吡喃吡啶、萘酰胺和匹莫齐特类的抑制剂。以利血平为代表的植物源性生物碱抑制剂已被发现可抑制ATP结合盒和主要易化子外排转运蛋白。在本研究中，我们报道了利血平对RND转运蛋白AcrB的诱导抑制作用。初步对接分析表明，利血平与AcrB的已知底物环丙沙星共享其结合位点，可能作为竞争性抑制剂。为了进行体外验证，在tac启动子的控制下克隆了伤寒沙门氏菌的AcrB，并将所得载体导入大肠杆菌C41(DE3)。在自诱导条件下，将过表达AcrB转运蛋白的细胞用环丙沙星和利血平联合给药。与用骨架序列载体转化的对照细胞相比，联合暴露导致环丙沙星诱导的表达AcrB转运蛋白的细胞生长抑制增强。时间杀菌分析进一步证实了这些发现。据我们所知，这是第一项表明暴露于利血平会诱导AcrB抑制的研究。本研究中开发的检测方法允许简单且可重复地检测底物/抑制剂对AcrB和相关外排转运蛋白的影响。
