South African Medical Research Council Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Bellville, South Africa.
School of Pharmacy, University of the Western Cape, Bellville, South Africa.
PLoS One. 2018 Nov 26;13(11):e0207605. doi: 10.1371/journal.pone.0207605. eCollection 2018.
Mycobacterial efflux pumps play a major role in the emergence of antimycobacterial drug resistance. Of particular interest is the proteinaceous multi-drug efflux pump protein Rv1258c that encodes a tetracycline/aminoglycoside resistance (TAP-2)-like efflux pump which is active in susceptible and drug resistant Mycobacterium tuberculosis. Rv1258c is implicated in drug resistance to numerous antimycobacterials including first line drugs rifampicin and isoniazid as well as fluoroquinolone and aminoglycoside antibiotic classes. To date, compounds like verapamil and piperine have been shown to inhibit Rv1258c but no direct evidence for binding or mode of action exist. Therefore in the present study we generated an accurate 3D model of Rv1258c using MODELLER and validated its structure using molecular dynamic simulation studies with GROMACS software. The 3D-structures of Rv1258c and the homologous template 1pw4 were simulated within a POPE/POPG lipid bilayer and found to behave similar. Another important finding was the identification of one local energy minima state of the apo protein, which speaks to the flexibility of the protein and will be investigated further. Extraction of one of the open channel conformations of Rv1258c and blind docking of various structurally diverse putative inhibitors and substrates, allowed for the identification of a probable binding site. Spectinamide was found to bind to a different location on the outside surface of the protein suggesting its ability to avoid the efflux channel. We further identified 246 putative compounds that showed higher binding affinity values to Rv1258c compared to piperine and verapamil. Interaction analysis of the top 20 purchasable compounds identified crucial hydrogen bond interactions with Ser26, Ser45 and Glu243 as well as a pi-pi stacking interaction with Trp32 that accounted for the strong affinity of these compounds for Rv1258c. Future studies will entail purchasing a number of compounds for in vitro activity testing against Mycobacterium tuberculosis.
分枝杆菌外排泵在抗分枝杆菌药物耐药性的出现中起着重要作用。特别有趣的是蛋白质多药物外排泵蛋白 Rv1258c，它编码四环素/氨基糖苷类耐药（TAP-2）样外排泵，在敏感和耐药结核分枝杆菌中均有活性。Rv1258c 与许多抗分枝杆菌药物的耐药性有关，包括一线药物利福平异烟肼以及氟喹诺酮类和氨基糖苷类抗生素。迄今为止，已经证明维拉帕米和胡椒碱等化合物可以抑制 Rv1258c，但不存在结合或作用模式的直接证据。因此，在本研究中，我们使用 MODELLER 生成了 Rv1258c 的准确 3D 模型，并使用 GROMACS 软件的分子动力学模拟研究对其结构进行了验证。在 POPE/POPG 脂质双层内模拟了 Rv1258c 的 3D 结构和同源模板 1pw4，发现它们的行为相似。另一个重要发现是鉴定出apo 蛋白的一个局部能量最小状态，这说明了该蛋白的灵活性，并将进一步进行研究。提取 Rv1258c 的一个开放通道构象，并盲目对接各种结构不同的潜在抑制剂和底物，确定了一个可能的结合位点。 spectinamide 被发现结合在蛋白质外表面的不同位置，这表明它有能力避免外排通道。我们进一步鉴定了 246 种可能的化合物，它们与 Rv1258c 的结合亲和力值高于胡椒碱和维拉帕米。对前 20 种可购买化合物的相互作用分析确定了与 Ser26、Ser45 和 Glu243 的关键氢键相互作用以及与 Trp32 的 pi-pi 堆积相互作用，这解释了这些化合物与 Rv1258c 的强亲和力。未来的研究将包括购买一些化合物进行体外抗结核分枝杆菌活性测试。
