HTS for Cytochrome C Synthesis Pathways

描述(申请人提供)：细胞色素是对大多数生物，包括人类和人类病原体的有氧和厌氧生长必不可少的血红素蛋白质。C型细胞色素的组装通过三种途径之一发生，称为系统I、II和III。这些途径中的缺陷是某些人类遗传病的基础(在系统III中)，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)的理想胞外靶点。许多不能被培养和/或基因操纵的生物使用这些途径。特定的抑制分子将有助于理解它们是如何生长和存活的。由于细胞色素c直接参与细胞凋亡，并与衰老和神经系统症状有关(通过线粒体的生物能量功能)，细胞色素c合成的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PIS实验室，该实验室设计了系统I和II在大肠杆菌中发挥作用的完整途径。此外，PIS实验室已经开发出重组的、可诱导的细胞色素c报告，以及非常灵敏的化学发光(ECL)方法，可以检测细胞色素c产物的亚皮摩尔水平。在这里，有人提议推动这些工程化大肠杆菌的发展，最终发现这些途径的特定小分子抑制物。对菌株和培养条件的改进将优化和验证用于细胞色素c产物检测的96和384微滴定板技术。将对两种不同的检测方法进行优化：i)基于ECL的高通量检测整个重组E.Coli细胞中的细胞色素c；ii)免疫学检测全息细胞色素c(His标记的和作为PhoA融合的)与已产生的抗血清。然后，这些筛子将被用于发现三条途径中的一条或全部的小分子抑制剂。已经开发的菌株和方法将建立对这些途径的抑制特异性，并将剖析在这些途径中被抑制的步骤(S)。PI小组最近表明，大分子的血红素类似物可以抑制特定的步骤，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，PIS小组开发了快速确定(九种酶中的)哪种酶在血红素合成中被抑制的工具。

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.