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。迫切需要针对耐多药感染的新疗法。目前大多数可用的抗生素对革兰氏阴性病原体的疗效较低，因为药物通过多药物外排转运体从细胞中主动外排。这些转运体是开发小分子外排抑制剂（EPIs）的有希望的靶点，可以与抗生素联合使用，以提高其对革兰氏阴性病原体的疗效。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，并开发抑制多药物外排转运体的方法。在之前的资助期内，我们结合了生物化学、遗传和生物物理方法，重建了导致活性药物外排复合物组装的一系列事件，并描述了每个组件在该过程中的作用。我们的发现揭示了多药泵以前未知的脆弱性，这可能是开发新抑制剂的目标。本应用程序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有效抑制剂。核心假设是质周膜融合蛋白控制外排泵从休眠状态到活跃状态的转变，抑制这种转变是阻断革兰氏多药外排的有效途径

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.