Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives
Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives
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DOI：
10.1073/pnas.1602472113
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发表时间：
2016-03-29
影响因子：
11.1
通讯作者：
Opperman, Timothy J.
中科院分区：
文献类型：
--
作者：
Sjuts, Hanno;Vargiu, Attilio V.;Opperman, Timothy J.
大肠杆菌acrabb - tolc外排泵是革兰氏阴性菌耐药结瘤细胞分裂（RND）出口者的原型。rnd型外排泵的过度表达是多药耐药（MDR）的重要因素，使其成为重要的抗菌药物发现靶点。我们最近开发了新的吡喃吡啶基AcrB抑制剂，其比以前已知的抑制剂强几个数量级。然而，由于缺乏合理药物设计的结构信息，这些抑制剂的进一步发展受到阻碍。尽管AcrB仅可溶的质周部分结合并输出配体，但AcrB中膜嵌入结构域的存在及其多特异性结合行为使其与药物共结晶具有挑战性。为了克服这一障碍，我们设计并生产了一种可溶性的AcrB [AcrB周质结构域（AcrBper）]，它在结构上与全长蛋白的周质部分高度一致，能够结合底物和有效的抑制剂。在这里，我们通过结合细胞、x射线晶体学和分子动力学（MD）模拟研究，描述了吡喃吡啶基抑制AcrB的分子基础。吡喃吡啶结合在富含苯丙氨酸的笼子内，这个笼子从AcrB的深层结合口袋中分叉，在那里它们形成广泛的疏水相互作用。此外，改进抑制剂的效力增加与微妙的蛋白质和水介导的氢键网络的形成有关。这些详细的见解为开发利用外排泵抑制剂对抗多重耐药革兰氏阴性病原体的新型联合疗法提供了分子平台。
The Escherichia coli AcrAB-TolC efflux pump is the archetype of the resistance nodulation cell division (RND) exporters from Gram-negative bacteria. Overexpression of RND-type efflux pumps is a major factor in multidrug resistance (MDR), which makes these pumps important antibacterial drug discovery targets. We have recently developed novel pyranopyridine-based inhibitors of AcrB, which are orders of magnitude more powerful than the previously known inhibitors. However, further development of such inhibitors has been hindered by the lack of structural information for rational drug design. Although only the soluble, periplasmic part of AcrB binds and exports the ligands, the presence of the membrane-embedded domain in AcrB and its polyspecific binding behavior have made cocrystallization with drugs challenging. To overcome this obstacle, we have engineered and produced a soluble version of AcrB [AcrB periplasmic domain (AcrBper)], which is highly congruent in structure with the periplasmic part of the full-length protein, and is capable of binding substrates and potent inhibitors. Here, we describe the molecular basis for pyranopyridine-based inhibition of AcrB using a combination of cellular, X-ray crystallographic, and molecular dynamics (MD) simulations studies. The pyranopyridines bind within a phenylalanine-rich cage that branches from the deep binding pocket of AcrB, where they form extensive hydrophobic interactions. Moreover, the increasing potency of improved inhibitors correlates with the formation of a delicate protein-and water-mediated hydrogen bond network. These detailed insights provide a molecular platform for the development of novel combinational therapies using efflux pump inhibitors for combating multidrug resistant Gram-negative pathogens.