Transport across two membranes by AcrAB-TolC

描述（由申请人提供）：细菌中抗生素耐药性的发展和传播是对人类和动物的普遍威胁。迫切需要针对多药耐药感染的新疗法。大多数目前可用的抗生素对革兰氏阴性病原体的疗效较低，因为药物通过多药外排转运蛋白从细胞中主动外排。这些转运蛋白是开发小分子外排抑制剂（EPI）的有希望的靶点，所述小分子外排抑制剂可与抗生素组合使用以提高其对革兰氏阴性病原体的功效。我们的长期目标是了解革兰氏阴性菌中药物外排的分子机制，并开发抑制多药外排转运蛋白的方法。在上一个资助期间，使用生物化学，遗传学和生物物理学方法的组合，我们已经重建了一系列的事件，导致主动药物外排复合物的组装，并表征了每个组件在这个过程中的作用。我们的研究结果揭示了多药泵的一个以前未知的弱点，可以在开发新的抑制剂时作为目标。本申请的目的是在分子细节上表征这种脆弱性，并发现革兰氏阴性菌中药物外排的新的有效抑制剂。中心假设是，周质膜融合蛋白控制外排泵从休眠状态到激活状态的转变，并且抑制这种转变是阻断革兰氏阴性菌中多药外排的有效方法。 阴性病原体。实验方法是基于外排复合物的生物化学性质的分子分析，在存在的EPI和合理设计的新的抑制剂。我们将追求三个具体目标：（i）研究多药外排泵的激活;（i）研究药物外排抑制的机制;（iii）鉴定药物外排转运蛋白的新变构抑制剂。拟议研究的预期结果是详细了解多药外排泵如何被激活，以及新的变构EPI如何作用于药物外排的这一关键步骤。这一贡献是重要的，因为预期EPI将恢复现有抗生素的活性，并扩大针对多药耐药的治疗选择。 耐药性感染

DESCRIPTION (provided by applicant): The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals. New therapies against multidrug resistant infections are urgently needed. The majority of currently available antibiotics have low efficacy against Gram-negative pathogens because of active efflux of drugs from cells by multidrug efflux transporters. These transporters are promising targets in development of small molecule efflux inhibitors (EPIs) that could be used in combinations with antibiotics to improve their efficacy against Gram-negative pathogens. Our long-term goal is to understand the molecular mechanism of drug efflux in Gram-negative bacteria and to develop approaches to inhibit multidrug efflux transporters. During the previous funding period, using a combination of biochemical, genetic and biophysical approaches we have reconstructed a sequence of events leading to the assembly of active drug efflux complexes and characterized the roles of each component in this process. Our findings exposed a previously unknown vulnerability of multidrug pumps that could be targeted in development of new inhibitors. The objective of this application is to characterize this vulnerability in molecular details and to discover new effectiv inhibitors of drug efflux in Gram-negative bacteria. The central hypothesis is that periplasmic membrane fusion proteins control the transition in efflux pumps from the dormant to the active state, and that inhibition of this transition is an effective way to block multidrug efflux in Gram negative pathogens. The experimental approach is based on molecular analyses of biochemical properties of efflux complexes in the presence of EPIs and the rational design of new inhibitors. We will pursue three specific aims: (i) to investigate the activation of multidrug efflux pumps; (i) to investigate the mechanisms of drug efflux inhibition; (iii) to identify new allosteric inhibitor of drug efflux transporters. The expected outcome of the proposed studies is detailed understanding of how multidrug efflux pumps are activated and new allosteric EPIs acting on this critical step in drug efflux. This contribution is significant because EPIs are expected to restore activities of already existing antibiotics and expand therapeutic options against multidrug resistant infections.