High-throughput fluorescence screening for inhibitors of TonB-dependent iron transport

 描述（由申请人提供）：微生物获得铁的能力是微生物侵入性和致病性的一个关键方面。革兰氏阴性细菌外膜中的TonB依赖性转运蛋白催化称为铁铁载体的铁络合物的摄取，并且这些金属螯合物的同化是许多物种的发病机制的决定因素，包括大肠杆菌、伤寒沙门氏菌、脑膜炎奈瑟氏菌、霍乱弧菌等。不幸的是，缺乏对这些转运反应的完整分子理解，这削弱了我们构思潜在抑制剂的能力。尽管如此，已知E.大肠杆菌外膜蛋白FepA通过广泛的构象运动完成主动铁转运。拟议的研究利用这一知识与荧光光谱分析，监测铁载体铁肠杆菌素的吸收。实验将包括在堪萨斯大学高通量筛选设施中对化合物的验证和初级文库进行高通量筛选。这三个库包括标准品和测试化学品，总共约17，500个独特的分子。在384孔微量滴定板中进行的这些测试将鉴定抑制革兰氏阴性细菌的TonB依赖性铁转运的化合物，从而产生一组候选物用于进一步测试作为针对细菌发病机制的抗生素。

 DESCRIPTION (provided by applicant): The ability of microbes to acquire iron is a key aspect of microbial invasiveness and pathogenicity. TonB-dependent transporters in the Gram-negative bacterial outer membrane catalyze the uptake of iron complexes called ferric siderophores, and the assimilation of these metal chelates is a determinant of the pathogenesis of many species, including Escherichia coli, Salmonella typhi, Neisseria meningitidis, Vibrio cholerae and others. Unfortunately, the full molecular understanding of these transport reactions is lacking, which impairs our ability to conceive of potential inhibitors. Nevertheless, it's known that the E. coli outer membrane protein FepA accomplishes active iron transport by a process that involves extensive conformational motion. The proposed research capitalizes on this knowledge with a fluorescence spectroscopic assay that monitors uptake of the siderophore ferric enterobactin. The experiments will encompass high-throughput screening of validation and primary libraries of compounds at the University of Kansas High-Throughput Screening facility. The three libraries include standards and test chemicals that total ~17,500 unique molecules. These tests, performed in 384-well microtiter plates, will identify compounds that inhibit TonB-dependent iron transport by Gram- negative bacteria, creating a panel of candidates for further testing as antibiotics against bacterial pathogenesis.