Discovery and development of RND pump inhibitors from marine microbial sources

 描述（由申请人提供）：由于药物发现管道中缺乏新的抗生素，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性革兰氏阴性病原体。多药转运蛋白是这些努力的关键目标。具体地，转运蛋白的抗性-结瘤-细胞分裂（RND）家族主要负责降低的细胞内渗透性和抗生素的保留。因此，我们建议开发新的小分子抑制剂，选择性地靶向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，以恢复抗生素效力，逆转耐药性，并显著降低革兰氏阴性临床分离株的耐药性发展率。我们对逆转耐药性分子的创新研究极大地受益于对生物体之间化学相互作用的自然历史的理解，并利用了我们内部的天然产物化学库，其中包括来自海洋细菌，浮游植物和真菌的4，400多种粗提取物。我们的方法的可行性已经确定，已经确定了36个海洋分离株能够从~1300个粗提取物的初始筛选中将抗生素的MIC降低4倍或更高。此外，我们已经分离出3，4-二溴吡咯-2，5-二酮，一种新的假定EPI从异养海洋细菌，降低了7种抗生素的MIC 2和16倍之间的菌株过表达三个原型RND转运蛋白（AcrAB-TolC，MexAB-OprM，和MexXY-OprM），并证明EPI功能的积累/流出试验。在目标1中，针对MDR试验菌株的持续筛选工作将鉴定出具有抗生素增效能力的其他海洋分离株，这些分离株将在含和不含抗生素的稀释系列中进一步进行测试，以评估提取物协同作用。在目标2中，优先的线索将针对 放大生产并进行生物测定引导的分级分离以获得足够量的纯化合物用于结构解析/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蓄积/外排试验评价先导化合物的EPI功能。将进行反筛选以排除质子载体和膜透化剂，并将进行棋盘测定以获得部分抑制浓度（FIC），以鉴定与抗生素的化合物特异性协同相互作用。R33阶段只有在我们定义的里程碑实现后才会启动。在目标4中，符合R21阶段选择标准的化合物将被靶向用于微尺度化学修饰，以基于结构-活性关系测试来改进化学支架，并优化以实现最小毒性、增强效力和溶解度。在最终目标中，我们将评估最有希望的候选EPI在携带导致RND泵合成增加的突变的革兰氏阴性临床分离株中的体外有效性，并确定这些先导物是否能够在相关感染模型中杀死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.