Mechanism, Specificity, and Inhibition of Cytochrome P450

细胞色素 P450 的机制、特异性和抑制

• 批准号: 8107702
• 负责人: Paul R Ortiz De Montellano
• 金额: $ 42.26万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8107702
• 关键词: Active Sites; Algorithms; Amino Acids; Anabolism; Antifungal Agents; Antineoplastic Agents; Antiparasitic Agents; Area; Binding; Biochemistry; Biology; CYP2C9 gene; Camphor 5-Monooxygenase; Catalysis; Computational algorithm; Computers; Coupled; Cysteine; Cytochrome P450; Development; Disease; Distant; Docking; Drug Industry; Drug Interactions; Drug Kinetics; Drug effect disorder; Drug toxicity; Electronics; Endogenous Factors; Environment; Enzymes; Fluorescent Probes; Goals; Grant; Hormones; Human; Individual; Iron; Label; Ligands; Ligation; Link; Malignant Neoplasms; Membrane; Metabolism; Methodology; Methods; Molecular; Molecular Conformation; Molecular Evolution; Motion; Mutation; Nature; Outcome; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Positioning Attribute; Process; Property; Protein Analysis; Protein Isoforms; Proteins; Protocols documentation; Rationalization; Research; Rest; Role; Site; Solutions; Specificity; Speed; Sterols; Structure; Substrate Specificity; System; Techniques; Technology; Testing; Tretinoin; Work; base; catalyst; computer studies; cytochrome P-450 CYP119 (Sulfolobus solfataricus); design; drug candidate; drug development; drug metabolism; environmental chemical; enzyme structure; flexibility; improved; inhibitor/antagonist; interest; molecular dynamics; novel strategies; physical property; tool

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the interrelationships between the structure and conformational dynamics of cytochrome P450 enzymes, their catalytic mechanisms, and the consequences of these interactions in terms of catalytic outcome and substrate specificity. The phase of the work proposed here has three major goals: (a) To utilize NMR and fluorescent probes to elucidate the conformational range of bacterial P450 enzymes and the nature of the environment in the vicinity of the thiolate-iron ligand; (b) to investigate the mechanisms by which mutations distant from the active site alter the catalytic process, focusing on the possible role of coupled protein motions and conformational links; (c) to further stabilize the monomeric state of solubilized human P450 enzymes and to extend the NMR and fluorescent conformational probe studies to these proteins; and (d) to apply cutting edge computational docking techniques to the conformational ensembles emerging from the combined use of molecular dynamics and NMR to construct refined protocols for the prediction and rationalization of cytochrome P450 substrate and inhibitor specificity, focusing initially on CYP2C9 The work proposed in this application rests on a number of technical advances. First, efforts to determine the structures of membrane-bound human P450 enzymes have succeeded in the past decade and more than a dozen P450 enzyme structures in various ligation states are now available. Second, the rapid progress in NMR techniques has allowed us to demonstrate the utility of site-specific NMR probes in conformational analysis of proteins as large as the P450 enzymes. Third, methods for the site-specific incorporation of unnatural and labeled amino acids now make it possible to tag individual positions in a protein with NMR or fluorescent probes. Fourth, advances in computational algorithms and the increase in computer speed now allow long-term molecular dynamics studies and computational docking of flexible ligands in flexible active sites. This is a broad study of the interplay of conformational dynamics and its role in modulating catalysis and determining substrate specificity in a set of enzymes that are notoriously malleable and challenging. The results should have a major impact in the pharmaceutical industry, for which the P450 specificity for a given drug candidate can determine whether it progresses or is discarded. They will also impact the design of inhibitors for P450 enzyme targeted in cancer and other diseases, and the design of P450 catalysts for biotechnological applications. Beyond P450, the conformational issues dealt with in this project and the techniques being explored have broader applicability and will have an impact in completely different areas of biochemistry and biology. PUBLIC HEALTH RELEVANCE: Cytochrome P450 enzymes fulfill critical roles in the metabolism of drugs and environmental chemicals, the biosynthesis of endogenous factors such as the sterol hormones, and the termination of the action of endogenous factors such as retinoic acid. They are of major practical interest because: (a) they are important targets for clinically used antifungal, antiparasitic, and anticancer drugs, (b) they are major determinants of drug pharmacokinetics and drug action, (c) they are responsible for a broad range of metabolism-dependent drug toxicities, and (d) they have high potential as biotechnological catalysts. The ability to predict P450 substrate and inhibitor specificity, and the effect of mutations on catalytic function, is central to all of these areas.

描述（由申请人提供）：该项目的长期目标是阐明细胞色素P450酶的结构和构象动力学之间的相互关系，它们的催化机制以及这些相互作用在催化结果和底物特异性方面的相互作用。这里提出的工作的阶段具有三个主要目标：（a）利用NMR和荧光探针阐明细菌P450酶的构象范围以及在硫醇酸盐配体附近的环境性质； （b）研究与活性位点突变距离的机制改变了催化过程，重点是耦合蛋白质运动和构象联系的可能作用； （c）进一步稳定溶解的人P450酶的单体状态，并将NMR和荧光构象探针研究扩展到这些蛋白质； （d）将尖端计算对接技术应用于从分子动力学和NMR的综合使用中出现的构象合奏，以构建精制方案，以预测和合理化细胞色素p450底物和抑制剂特异性，最初将其集中在CYP2C9上，以CYP2C9的要求在本应用程序上的工作质量上的启用。首先，在过去的十年中，确定膜结合的人P450酶结构的努力已经成功，现在有多种在各种连接状态下的P450酶结构。其次，NMR技术的快速进步使我们能够证明位点特异性NMR探针在与P450酶一样大的蛋白质构象分析中的实用性。第三，现在，非自然和标记的氨基酸的位点特异性掺入方法使得可以在具有NMR或荧光探针的蛋白质中标记单个位置。第四，计算算法的进步和计算机速度的提高现在允许长期的分子动力学研究和柔性活性位点柔性配体的计算对接。 这是对构象动力学相互作用的广泛研究及其在调节催化和确定一组酶的底物特异性中的作用，这些酶是众所周知的可延展和具有挑战性的酶。结果应该对制药行业产生重大影响，对于给定药物的P450特异性可以确定其进展还是被丢弃。它们还将影响针对癌症和其他疾病的P450酶的抑制剂的设计，以及用于生物技术应用的P450催化剂的设计。除P450外，该项目中处理的构象问题和所探索的技术具有更广泛的适用性，并且会影响完全不同的生物化学和生物学领域。 公共卫生相关性：细胞色素P450酶在药物和环境化学物质的代谢中扮演关键作用，内源性因子（例如固醇激素）的生物合成以及诸如视市酸等内源性因素的作用终止。 They are of major practical interest because: (a) they are important targets for clinically used antifungal, antiparasitic, and anticancer drugs, (b) they are major determinants of drug pharmacokinetics and drug action, (c) they are responsible for a broad range of metabolism-dependent drug toxicities, and (d) they have high potential as biotechnological catalysts.预测P450底物和抑制剂特异性以及突变对催化功能的影响的能力至关重要。