Small molecule inhibitors of bacterial iron acquisition systems

描述(申请人提供)：尿路是最常见的细菌感染部位之一，而大肠杆菌是目前为止感染该部位最常见的物种。感染通常使用抗生素治疗，包括甲氧氨嘧啶-磺胺甲恶唑或喹诺酮类药物。虽然疫苗的开发正在进行中，但目前还没有疫苗获准在美国使用。面对抗生素耐药率的稳步攀升和新抗生素开发的急剧减少，迫切需要分离出新的小分子抗菌剂。缺乏这种新的治疗方法将导致尿路感染(UTI)，导致无法治疗的多重耐药菌株。使用一系列 通过包括转录组、蛋白质组、免疫蛋白质组和qPCR分析在内的无偏筛选，我们发现铁获取系统(铁载体和血红素介导的)是易受抑制和潜在治疗的目标。我们的长期研究目标是预防和治疗女性和男性的尿路感染。目的是鉴定和表征革兰氏阴性细菌中铁摄取的小分子抑制剂。在基于高通量培养的6298个化合物的中试筛选中，我们得到的初始命中率为2.3%(147个化合物)[总体Z‘因子=0.81(平均每板Z’=0.9)，S/B=6，CV=6%]，以低于阴性对照的3SD为界值，抑制活性高于30%。通过反筛选、剂量反应曲线和新鲜化学样品的抑制，验证了五次命中。对于一种化合物，数据与以TonB系统为靶标的抑制数据一致。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止细菌中铁的获取。这一发现将推动对不同铁获取机制的生物学研究，并利用铁受体蛋白靶标补充我们的疫苗接种努力。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，以确定肠外致病性大肠杆菌(ExPEC)铁获取的小分子抑制剂；2)进行计数器筛选，使用新的化学样本验证HITS，并分析ExPEC铁获取小分子抑制剂的构效关系(SAR)；以及3)确定每个已验证的小分子抑制物抑制铁获取途径所使用的共同靶点。这些目标的预期结果将是识别化合物和开发通过防止细菌铁同化来阻止细菌生长的衍生物。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种天然产品提取物中确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，并通过评估衍生品来改进这些产品。这一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剂，以防止复发尿路感染妇女和首次尿路感染易感妇女的这一公共卫生祸害。

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.