HTS for Cytochrome C Synthesis Pathways

用于细胞色素 C 合成途径的 HTS

基本信息

批准号：
8191269
负责人：
Robert G. Kranz
金额：
$ 3.8万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-30 至 2011-08-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：细胞色素是血红素蛋白，对于大多数生物体（包括人类和人类病原体）的需氧和厌氧生长至关重要。 c 型细胞色素的组装通过称为系统 I、II 和 III 的三种途径之一进行。这些途径的缺陷是某些人类遗传疾病（系统 III）的基础，并且因为只有原核生物、植物和原生动物使用系统 I 或 II，所以这些途径是抗菌剂（针对病原体）的理想胞质外靶点。许多无法培养和/或基因操纵的生物体都使用这些途径。特定的抑制剂分子将有助于了解它们如何生长和生存。由于细胞色素 c 直接参与细胞凋亡，并且（通过线粒体生物能量功能）与衰老和神经系统表现有关，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 PI 实验室，他们设计了系统 I 和 II 在大肠杆菌中发挥作用的完整途径。此外，PIs 实验室还开发了重组、诱导型细胞色素 c 报告基因，以及非常灵敏的化学发光 (ECL) 方法，可检测亚皮摩尔水平的细胞色素 c 产物。在此，建议推进这些工程化大肠杆菌，以最终发现该途径的特定小分子抑制剂。对菌株和培养条件的修改将优化和验证用于细胞色素 c 产物检测的 96 和 384 孔微量滴定板技术。将优化两种不同的检测方法：i) 在整个重组大肠杆菌细胞中基于 ECL 的细胞色素 c 高通量检测，以及 ii) 已使用抗血清对全细胞色素 c（his 标记且作为 PhoA 融合体）进行免疫学检测生成的。然后，这些筛选将用于发现针对这三种途径之一或全部的小分子抑制剂。已经开发的菌株和方法将建立针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会抑制特定步骤，但这些类似物需要特定的孔蛋白才能进入。基于细胞的测定也将通过检测这种抑制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 小组已开发出工具来快速确定血红素合成中哪种酶（九种）受到抑制。