Small molecule inhibitors of bacterial iron acquisition systems

描述（由申请方提供）：尿路是细菌感染的最常见部位之一，大肠埃希菌是迄今为止感染该部位的最常见菌种。感染通常用抗生素治疗，包括甲氧苄啶-磺胺甲恶唑或喹诺酮类。虽然疫苗开发正在进行中，但目前在美国还没有许可使用的疫苗。面对抗生素耐药性的稳步上升和新抗生素开发的急剧减少，迫切需要分离新型小分子抗菌剂。缺乏这种新的治疗方法将导致尿路感染（UTI）与多重耐药菌株是无法治愈的。使用一系列 通过包括转录组学、蛋白质组学、免疫蛋白质组学和qPCR分析在内的无偏筛选，我们已经将铁获得系统（铁载体介导的和血红素介导的）鉴定为抑制和潜在治疗的易受攻击的靶标。我们的长期研究目标是预防和治疗男性和女性的UTI。目的是鉴定和表征革兰氏阴性细菌病原体中铁摄取的小分子抑制剂。在6298种化合物的中试高通量培养筛选中，我们的初始命中率为2.3%（147种化合物）[总Z'因子=0.81（平均Z'/板=0.9），S/B=6，CV=6%]，截止值设定为低于阴性对照3SD，抑制活性高于30%。通过反筛选、剂量响应曲线和新鲜化学样品的抑制来验证五个命中。对于一种化合物，数据与作为其目标的TonB系统的初始值一致。我们的中心假设是，不依赖于铁螯合的特定小分子抑制剂可以特异性地阻断细菌中的铁获得。这一发现将推动对不同铁获得机制的生物学研究，并补充我们使用铁受体蛋白靶点的疫苗接种工作。我们将通过完成以下具体目标来实现我们的目标：1）开发和实施高通量筛选以鉴定肠外致病性大肠杆菌（ExPEC）铁获取的小分子抑制剂; 2）进行计数筛选，使用新鲜化学样品验证命中，并分析ExPEC铁获取的小分子抑制剂的结构-活性关系（SAR）;和3）确定被每种经验证的小分子抑制剂抑制的铁获取途径所使用的共同靶标。这些目标的预期结果将是确定化合物并开发通过阻止细菌铁同化来抑制细菌生长的衍生物。这些研究的积极影响将是巨大的。我们将从150，000种化合物和27，278种天然产物提取物中鉴定新型细菌铁摄取的小分子抑制剂，用新鲜样品验证这些命中，并通过评估衍生物来改进这些产品。这些知识将为我们提供现有抗生素和潜在疫苗之外的替代治疗药物，以预防复发性UTI妇女和易患首次UTI的妇女的这种公共卫生灾难。

DESCRIPTION (provided by applicant): The urinary tract is among the most common sites of bacterial infection and Escherichia coli is by far the most common species infecting this site. Infections are routinely treated with antibiotics including trimethoprim- sulfamethoxazole or quinolones. While vaccine development is in the pipeline, there are no vaccines currently licensed for use in the U.S. In the face of steadily climbing rates of antibiotic resistance and dramatic reduction in development of new antibiotics, isolation of novel small molecule antimicrobials are desperately needed. Lack of such new treatments will result in urinary tract infections (UTI) with multiply antibiotic-resistant strains that are untreatable. Using a series of unbiased screens including transcriptomic, proteomic, immunoproteomic, and qPCR assays, we have identified iron acquisition systems (siderophore- and heme- mediated) as vulnerable targets for inhibition and potential therapy. Our long-term research goal is to prevent and therapeutically treat UTIs in women and men. The objective is to identify and characterize small molecule inhibitors of iron uptake in gram-negative bacterial pathogens. In a pilot high throughput culture-based screen of 6298 compounds, we had an initial hit rate of 2.3% (147 compounds) [overall Z' factor=0.81 (avg Z' per plate=0.9), S/B=6, CV=6%] with a cutoff set at 3 SD below the negative control and inhibitory activity higher than 30%. Five hits were validated by counterscreens, dose response curves, and inhibition with fresh chemical samples. For one compound, data are consistent with inihibition of the TonB system as its target. Our central hypothesis is that specific small molecule inhibitors that do not depend on iron chelation can specifically block iron acquisition in bacteria. This discovery will drive biological investigationof the diverse iron acquisition mechanisms and complement our vaccination efforts using iron receptor protein targets. We will achieve our objective by completing the following specific aims: 1) Develop and implement a high throughput screen to identify small molecule inhibitors of iron acquisition by extraintestinal pathogenic Escherichia coli (ExPEC); 2) Conduct counter screens, validate hits using fresh chemical samples, and analyze structure-activity relationships (SAR) for small molecule inhibitors of ExPEC iron acquisition; and 3) Determine common targets used by iron acquisition pathways that are inhibited by each validated small molecule inhibitor. The expected outcomes of these aims will be to identify compounds and develop derivatives that arrest bacterial growth by preventing bacterial iron assimilation. The positive impact of these studies will be substantial. We will identify novel small molecule inhibitors of bacterial iron uptake from among 150,000 chemical compounds and 27,278 natural product extracts, validate these hits with fresh samples, and improve upon these products by assessment of derivatives. This knowledge will provide us with alternate therapeutic agents beyond existing antibiotics and potential vaccines to prevent this public health scourge in women with recurrent UTI and those susceptible to their first UTI.