HTS for Cytochrome C Synthesis Pathways

描述（由申请人提供）：细胞色素是大多数生物（包括人类和人类病原体）需氧和厌氧生长所必需的血红素蛋白。c型细胞色素的组装通过三种途径之一进行，称为系统I、II和III。这些途径的缺陷是某些人类遗传疾病（系统III）的基础，因为只有原核生物、植物和原生动物使用系统I或II，这些途径是抗微生物药物（针对病原体）的理想胞质外靶点。许多不能培养和/或基因操纵的生物使用这些途径。一种特定的抑制剂分子将有助于了解它们是如何生长和生存的。由于细胞色素c直接参与细胞凋亡，并（通过线粒体生物能量功能）参与衰老和神经系统表现，因此细胞色素c合成抑制剂将为研究这些过程提供有价值的工具。该领域的一项重大突破来自于PIs实验室在大肠杆菌中工程系统I和II的完整途径。此外，pi实验室还开发了重组，可诱导的细胞色素c报告，以及非常敏感的化学发光（ECL）方法，可检测亚皮摩尔水平的细胞色素c产物。在这里，建议推进这些工程大肠杆菌，最终发现特异性的小分子抑制剂的途径。菌株和培养条件的修改将优化和验证96孔和384孔微滴板检测细胞色素c产物的技术。将优化两种不同的检测方法：i)基于ecl的全重组大肠杆菌细胞中细胞色素c的高通量检测，ii)与已生成的抗血清进行全细胞色素c （his标记和PhoA融合）的免疫学检测。然后，这些筛选将用于发现针对三种途径中的一种或全部的小分子抑制剂。已经开发的菌株和方法将建立对途径的抑制特异性，并将剖析途径中抑制的步骤。pi小组最近表明，大分子血红素类似物抑制特定的步骤，但这些类似物需要特定的孔蛋白才能进入。基于细胞的分析也将通过检测这种抑制作用和使用已知的血红素生物合成抑制剂来验证。因此，HTS还将发现新的血红素生物合成抑制剂，而PIs小组已经开发出工具来快速确定哪种酶（九种）在血红素合成中被抑制。

DESCRIPTION (provided by applicant): Cytochromes are heme proteins essential for the aerobic and anaerobic growth of most organisms, including humans and human pathogens. The assembly of c-type cytochromes occurs by one of three pathways, called systems I, II, and III. Defects in these pathways are the basis for certain human genetic diseases (in system III) and because only prokaryotes, plants, and protozoa use systems I or II, these pathways are ideal extracytoplasmic targets for antimicrobial agents (against pathogens). Many organisms that can not be cultured and/or genetically manipulated use these pathways. A specific inhibitor molecule would facilitate an understanding of how they grow and survive. Since cytochrome c is directly involved in apoptosis, and implicated (by mitochondrial bioenergetic functions) in aging and neurological manifestations, inhibitors of cytochrome c synthesis would provide valuable tools to study these processes. A major breakthrough in the field has come from the PIs lab in engineering the complete pathways of systems I and II to function in Escherichia coli. Moreover, the PIs lab has developed recombinant, inducible cytochrome c reporters, as well as very sensitive chemiluminescent (ECL) methods that detect subpicomolar levels of the cytochrome c product. Here it is proposed to advance these engineered E.coli for ultimately the discovery of specific small molecule inhibitors of the pathways. Modifications of the strains and the culture conditions will optimize and validate 96 and 384 well microtiter plate technologies for cytochrome c product detection. Two different assays will be optimized: i) the ECL-based high-throughput detection of cytochrome c in whole recombinant E.coli cells and ii) the immunological detection of holo cytochrome c (his-tagged and as a PhoA fusion) with antisera already generated. These screens will then be used for the discovery of small molecule inhibitors towards one or all of the three pathways. Strains and methods already developed will establish inhibitory specificity towards the pathways and will dissect the step(s) inhibited in the pathways. The PIs group has recently shown that large molecule, heme analogs inhibit specific steps, but these analogs require specific porins for access. The cell-based assays will also be validated by detection of this inhibition and by the use of known heme biosynthesis inhibitors. Thus, the HTS will also discover new inhibitors of heme biosynthesis, and the PIs group has developed the tools to quickly determine which enzyme (of nine) is inhibited in heme synthesis.