HUMAN P450S--HIGH LEVEL EXPRESSION AND IMMUNOREAGENTS

人 P450S--高水平表达和免疫试剂

• 批准号: 2672340
• 负责人: HENRY J BARNES
• 金额: $ 33.14万
• 依托单位: IMMUNE COMPLEX CORPORATION
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672340
• 关键词: Escherichia coli; biological products; biosensor device; biotechnology; cytochrome P450; drug metabolism; drug screening /evaluation; enzyme activity; method development; microcalorimetry; molecular cloning; protein purification; recombinant proteins

Cytochrome P450 enzymes play a critical role in the metabolism of a wide variety of drugs and other ingested chemical compounds. These enzymes represent the first step in the metabolism of these chemicals; a process that yields products which have very different biological effects from the original compound. A key parameter in the profiling of a candidate drug is the identification of a particular cytochrome P450 enzyme(s) responsible for its metabolism. The objective of this project is to generate simple as well as more sophisticated high throughput screens that will enable individual researchers and large pharmaceutical companies to rapidly identify, in vitro, significant specific P450 drug interactions. Expanding o the success of the Phase I data which demonstrates that human P450s and NADPH reductase can be co-expressed in E coli at high levels, devoid of the intrinsic P450 activities plaguing systems based on eukaryotic expression, Phase II will: (1) generate cytochrome P450 isoforms by their expression in E coli; (2) forma the P450s into simple unambiguous assays that will permit pharmacological and kinetic profiles to be determined in vitro; (3) generate methods based on microcalorimetry and surface plasmon resonance technologies that will determine P450-substrate binding constants; and, (4) adapt the recombinant human P450s into high throughput systems that will form the basis on the Company's drug screening service. PROPOSED COMMERCIAL APPLICATIONS: A comprehensive panel of recombinant, human P450s and human NADPH P450 reductase, in the form of individual reagents, and well as the more "user-friendly" P450-reductase co-expressed membranes will be made available to research and development labs for in vitro drug metabolism studies. These reagents will also be incorporated into an "in-house", custom screening service which will provide high- throughput analysis of P450-drug interactions on a contractual basis.

细胞色素P450酶在广泛的代谢中起关键作用。 各种药物和其他摄入的化合物。 这些酶 是这些化学物质新陈代谢的第一步;过程 它产生的产物具有非常不同的生物效应， 原始化合物。 候选药物特征分析的一个关键参数 是一种特定的细胞色素P450酶的鉴定 负责新陈代谢。 这个项目的目标是产生简单的，以及更多的 先进的高通量筛选， 研究人员和大型制药公司迅速确定， 体外，显著的特异性P450药物相互作用。 扩展O I期数据的成功，证明了人类P450和NADPH 还原酶可以在大肠杆菌中以高水平共表达，而不含 基于真核表达的内源性P450活性检测系统， 第二阶段将：（1）通过表达细胞色素P450亚型， 在大肠杆菌中;（2）将P450形成简单明确的测定， 允许在体外确定药理学和动力学特征;（3） 基于微量热和表面等离子体共振的产生方法 确定P450-底物结合常数的技术;以及， (4)使重组人P450适应高通量系统， 将构成公司药物筛选服务的基础。 拟议的商业应用：一个全面的重组， 人P450和人NADPH P450还原酶，以个体形式 试剂，以及更"用户友好"的P450-还原酶共表达 膜将提供给研究和开发实验室， 体外药物代谢研究。 这些试剂也将被纳入 成为一个“内部”，客户筛选服务，将提供高- 在合同基础上进行P450-药物相互作用的通量分析。