Sjuts Hanno, Vargiu Attilio V, Kwasny Steven M, Nguyen Son T, Kim Hong-Suk, Ding Xiaoyuan, Ornik Alina R, Ruggerone Paolo, Bowlin Terry L, Nikaido Hiroshi, Pos Klaas M, Opperman Timothy J
Institute of Biochemistry, Goethe University Frankfurt, D-60438 Frankfurt, Germany;
Department of Physics, University of Cagliari, I-09042 Monserrato (CA), Italy;
Proc Natl Acad Sci U S A. 2016 Mar 29;113(13):3509-14. doi: 10.1073/pnas.1602472113. Epub 2016 Mar 14.
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.
大肠杆菌AcrAB - TolC外排泵是革兰氏阴性菌中耐药性结节化细胞分裂（RND）转运蛋白的原型。RND型外排泵的过表达是多药耐药（MDR）的主要因素，这使得这些泵成为重要的抗菌药物发现靶点。我们最近开发了新型的基于吡喃吡啶的AcrB抑制剂，其效力比先前已知的抑制剂高出几个数量级。然而，由于缺乏用于合理药物设计的结构信息，此类抑制剂的进一步开发受到了阻碍。虽然只有AcrB的可溶性周质部分结合并转运配体，但AcrB中膜嵌入结构域的存在及其多特异性结合行为使得与药物的共结晶具有挑战性。为了克服这一障碍，我们设计并制备了AcrB的可溶性版本[AcrB周质结构域（AcrBper）]，其结构与全长蛋白的周质部分高度一致，并且能够结合底物和强效抑制剂。在此，我们结合细胞、X射线晶体学和分子动力学（MD）模拟研究，描述了基于吡喃吡啶抑制AcrB的分子基础。吡喃吡啶结合在一个从AcrB的深结合口袋分支出来的富含苯丙氨酸的笼状结构内，在那里它们形成广泛的疏水相互作用。此外，改进抑制剂效力的增加与精细的蛋白质和水介导的氢键网络的形成相关。这些详细的见解为开发使用外排泵抑制剂对抗多药耐药革兰氏阴性病原体的新型联合疗法提供了一个分子平台。
