Discovery and development of RND pump inhibitors from marine microbial sources

 描述(申请人提供)：在药物发现过程中缺乏新的抗生素，再加上抗生素耐药性的加速演变，需要实施一种新的方法来对抗耐药的革兰氏阴性病原体。多药转运体是这些努力的关键目标。特别是，抗性结瘤细胞分裂(RND)转运蛋白家族是导致细胞内渗透性降低和抗生素滞留的主要原因。因此，我们建议开发新的小分子抑制剂，选择性地将RND泵作为抗生素的佐剂。事实上，我们的长期目标是将外排泵抑制剂(EPI)与现有抗生素联合使用，恢复抗生素效力，逆转耐药性，并大幅降低革兰氏阴性临床分离株的耐药性发展速度。我们对逆转耐药性的分子的创新搜索极大地得益于了解有机体之间化学相互作用的自然历史，并利用了我们内部的天然产品化学库，该库由海洋细菌、浮游植物和真菌的4,400多种粗提物组成。我们方法的可行性已经得到证实，从最初筛选的约1300种粗提物中鉴定出36株海洋分离株，能够将抗生素的MIC降低4倍或更多。此外，我们从异养海洋细菌中分离出了3，4-二溴吡咯-2，5-二酮，这是一种新的EPI，它能将七种抗生素对三种原型RND转运体(AcrAB-TolC、MexAB-OprM和Mexxy-OprM)的MIC降低2到16倍，并在蓄积/外排试验中证明了EPI的功能。在目标1中，针对MDR测试菌株的持续筛选工作将确定更多具有抗生素增强能力的海洋菌株，这些菌株将在使用和不使用抗生素的稀释系列中进一步测试，以评估提取物的协同作用。在目标2中，按优先级排列的销售线索将针对以下目标 扩大生产并进行生物测定指导分级，以获得足够数量的纯化合物，用于结构鉴定/去复制和下游应用。在目标3中，将通过荧光/放射性标记化合物的累积/外排分析来评估先导化合物的EPI功能。将进行反筛选以排除原生团和膜渗透剂，并将进行棋盘试验以获得分数抑制浓度(FICS)，以确定化合物与抗生素的特定协同作用。只有在达到我们定义的里程碑时，才会启动R33阶段。在目标4中，在R21阶段符合选择标准的化合物将被定位于微尺度化学修饰，以基于结构-活性关系测试来提炼化学支架，并针对最小的毒性、增强的效力和溶解性进行优化。在最终目标中，我们将评估最有希望的候选EPI在革兰氏阴性临床分离株中的体外有效性，这些突变导致RND泵的合成增加，并建立这些领导是否能够在相关的感染模式中杀死MDR病原体。

 DESCRIPTION (provided by applicant): With the lack of new antibiotics in the drug discovery pipeline, coupled with accelerated evolution of antibiotic resistance, a new approach to combating drug resistant Gram-negative pathogens needs to be implemented. Multidrug transporters are a key target in these efforts. In particular, the Resistance-Nodulation-Cell-Division (RND) family of transporters are primarily responsible for the decreased intracellular permeability and retention of antibiotics. We therefore propose to develop new small molecule inhibitors that selectively target RND pumps as adjuvants to antibiotics. Indeed, our long-term goal is to use efflux pump inhibitors (EPIs) in combination with existing antibiotics, to restore antibiotic potency, reverse resistance, and dramatically reduce the rates of resistance development in Gram-negative clinical isolates. Our innovative search for molecules to reverse drug resistance has benefited enormously from understanding the natural history of chemical interactions among organisms, and makes use of our in-house natural product chemical library consisting of over 4,400 crude extracts from marine bacteria, phytoplankton, and fungi. The feasibility of our approach has been established having identified 36 marine isolates able to decrease an antibiotic's MIC by 4-fold or greater from an initial screening of ~1300 crude extracts. Furthermore, we have isolated 3,4-dibromopyrrole-2,5-dione, a new putative EPI from a heterotrophic marine bacteria, which decreased the MICs of seven antibiotics between 2 and 16-fold against strains overexpressing three archetype RND transporters (AcrAB-TolC, MexAB-OprM, and MexXY-OprM), and demonstrated EPI functionality in accumulation/efflux assays. In Aim 1, continued screening efforts against MDR test strains will identify additional marine isolates with antibiotic potentiation capabilities, that will be further tested in dilution series ith and without antibiotics to assess extract synergy. In Aim 2, prioritized leads will be targeted for scaled up production and undergo bio- assay guided fractionation to obtain pure compounds of sufficient quantity for structural elucidation/de- replication and downstream applications. In Aim 3, EPI functionality of lead compounds will be evaluated by accumulation/efflux assays with fluorogenic/radiolabeled compounds. Counterscreens will be performed to rule out protonophores and membrane permeabilizers, and checkerboard assays will be conducted to obtain fraction inhibitory concentrations (FICs) to identify compound-specific synergistic interactions with antibiotics. The R33 phase will be initiated only if our defined milestones are achieved. In Aim 4, compounds meeting selection criteria in the R21 phase will be targeted for micro-scale chemical modification to refine chemical scaffolds based on structure-activity relationship testing, and optimized for minimal toxicity, enhanced potency and solubility. In the final Aim, we will assess in vitro effectiveness of the most promising candidate EPIs in Gram-negative clinical isolates harboring mutations leading to increased synthesis of RND pumps, and establish whether these leads enable killing of MDR pathogens in relevant models of infection.