Redox-Active and Luminescent Probes for Heme Enzymes

用于血红素酶的氧化还原活性和发光探针

• 批准号: 6940828
• 负责人: DAVID B. GOODIN
• 金额: $ 29.54万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6940828
• 关键词: active sites; binding sites; cytochrome P450; cytochrome oxidase; electron transport; enzyme activity; enzyme inhibitors; enzyme mechanism; heme; high performance liquid chromatography; luminescence; metalloproteins; nitric oxide synthase; oxidation reduction reaction; reducing agents

DESCRIPTION (provided by applicant): Integrated efforts of the Gray and Goodin laboratories will focus on a series of designed sensitizer-linked substrates (SLS) to be used as probes of enzyme function, for the discovery of inhibitors, and as imaging agents for biomedically important metalloproteins. Two primary hypotheses drive these studies: 1) rational design of structural elements into a protein is feasible when coupled to a closed design-feedback cycle, and 2) questions about the function of important enzymes may be addressed using such models. The Gray laboratory has pioneered the use of SLS probes, allowing electron transfer (ET) to be directed into deeply buried protein active sites. Results in the Goodin laboratory have shown that proteins may be manipulated by iterative design to create binding sites for small molecules. In this proposal, a combined molecular design approach will be used in which both the protein and the SLS are designed to be self complementary. The proposed electron transfer pathway in cytochrome c peroxidase will be excised and replaced with redox active SLS probes to address questions about the chemical nature and context of specific ET pathways. SLS probes will be coupled to a protein-based model for the heme a3/CUB center of cytochrome c oxidase, providing new ways to test emerging ideas about how electron donors participate in O2 reduction. Novel SLS probes will be developed for identification of enzyme inhibitors and for imaging specific biological targets. One class will be designed to switch from a luminescent to a dark state upon binding to the protein target, and to recover luminescence upon displacement by a competitive inhibitor. The long-term goal is to develop isoform specific probes for screening inhibitors of important enzymes such as cytochrome P450 and nitric oxide synthase (NOS). A second class of probes will be designed to switch from a dark to a luminescent state upon binding to a target. These probes will be designed to detect NOS isoforms; their study will represent a critical first step toward our long-term goal of developing new tools for imaging and spatial localization of proteins in vivo. Overall, this proposal will contribute a completely novel approach to the design of structural elements into protein frameworks for the purpose of detecting, controlling and understanding the behavior of metalloproteins.

描述（由申请人提供）：灰色和良好实验室的综合努力将集中在一系列设计的敏化剂链接底物（SLS）上，以用作酶功能的探针，用于发现抑制剂，并作为生物医学上的成像剂重要的金属蛋白。两个主要的假设推动了这些研究：1）当耦合到封闭的设计回馈周期时，将结构元素的合理设计可行，以及2）有关重要酶功能的问题，可以使用此类模型来解决重要酶的功能。灰色实验室已经开创了SLS探针的使用，使电子转移（ET）可以被引导到深埋的蛋白质活性位点中。 Goodin实验室的结果表明，蛋白质可以通过迭代设计来操纵以创建小分子的结合位点。在此提案中，将使用一种组合的分子设计方法，其中蛋白质和SLS均设计为自我补充。细胞色素C过氧化物酶中提出的电子转移途径将被切除并用氧化还原活性SLS探针取代，以解决有关特定ET途径的化学性质和背景的问题。 SLS探针将与细胞色素C氧化酶的血红素A3/CUB中心的基于蛋白质的模型耦合，从而提供了新的方法来测试有关电子供体如何参与O2减少的新想法。新型SLS探针将开发用于鉴定酶抑制剂和成像特定的生物学靶标。一个类将设计为在与蛋白质靶标结合时从发光变为黑暗状态，并在竞争性抑制剂移位后恢复发光。长期目标是开发同工型特异性探针，以筛选重要酶的抑制剂，例如细胞色素P450和一氧化氮合酶（NOS）。第二类探针将被设计为与目标结合后，将从黑暗切换到发光状态。这些探针将被设计为检测NOS同工型。他们的研究将是朝着我们的长期目标迈出的关键第一步，即开发新的工具，用于在体内成像和空间定位。总体而言，该提案将为将结构元素设计成蛋白质框架的设计做出一种新颖的方法，以检测，控制和理解金属蛋白的行为。