MOLECULAR BASIS OF HUMAN CYTOCHROME P450 3A FUNCTION

描述（申请人摘要的逐字记录）：长期目标 本提案中描述的研究的一个重要方面是阐明 人细胞色素P450 3A的底物特异性和协同性。 这些酶是非常通用的催化剂，在生物合成中起着至关重要的作用。 多种药理学和毒理学化合物的代谢 兴趣CYP 3A 4是大多数人肝脏中表达最高的P450， 负责代谢的临床使用的药物比任何其他 P450，是许多严重的药物相互作用的位点。CYP 3A 5是 在大约四分之一的个体的肝脏中表达。3a 4和3a 5 表现出84%的氨基酸序列同一性，并代谢许多相同的 印刷受体.然而，每种酶都产生不同的代谢物模式， 某些药物如环孢菌素A和咪达唑仑。一个有趣的问题是 这些酶是如何接受如此多结构多样的底物的 对单一化合物表现出显著的区域和立体选择性。 CYP 3A 4和3A 5也表现出与某些底物的正协同性， 其自身表现为自激活（同向协同性）或 通过第二种化合物，如α-萘酮（异向性）活化 协同性）。在其他情况下，CYP 3A 4可容纳两种底物 对彼此的新陈代谢没有明显影响期间生成的结果 目前的奖励期使我们能够识别许多氨基酸， 负责底物特异性和CYP 3A 4协同性的残基。的 拟议研究的中心假设是， （活化、部分抑制、无抑制） 反映同时占用两个或两个以上的首选位置内， 单个大装订口袋。这将通过以下组合进行测试： 用多种底物进行定点诱变功能分析， 效应器，核磁共振（NMR）光谱，和3-D分子 建模具体目标是：1）确定以下结构基础： CYP 3A 4的同向和异向协同性; 2）确定 人CYP 3A氧化原型药物底物的结构基础 3）通过NMR确定CYP 3A 4活性位点中的底物取向; 4)确定所选化合物抑制CYP 3A的结构基础。 了解人P450 3A功能的分子基础， 预测这些酶的底物、激活剂和抑制剂， 有可能使药物间相互作用和个体间差异最小化， 在药物代谢中。

DESCRIPTION (Verbatim from the Applicant's Abstract): The long-term objective of the research described in this proposal is to elucidate the structural basis for the substrate specificity and cooperativity of human cytochromes P450 3A. These enzymes are very versatile catalysts and play a crucial role in the metabolism of a wide variety of compounds of pharmacological and toxicological interest. CYP3A4 is the most highly expressed P450 in the liver of most humans, is responsible for the metabolism of more clinically used drugs than any other P450, and is the locus of numerous serious drug-drug interactions. CYP3A5 is expressed in the liver of approximately one in four individuals. 3A4 and 3A5 exhibit 84 percent amino acid sequence identity and metabolize many of the same substrates. However, each enzyme produces a distinct pattern of metabolites of certain drugs such as cyclosporin A and midazolam. An intriguing question is how these enzymes can accept so many structurally diverse substrates yet exhibit remarkable regio- and stereoselectivity towards a single compound. CYP3A4 and 3A5 also exhibit positive cooperativity with certain substrates, which manifests itself as autoactivation (homotropic cooperativity) or activation by a second compound, such as alpha-naphthoflavone (heterotropic cooperativity). In other cases, two substrates can be accommodated by CYP3A4 with no apparent effect on each others' metabolism. Results generated during the current award period have allowed us to identify many of the amino acid residues responsible for substrate specificity and cooperativity of CYP3A4. The central hypothesis of the proposed studies is that atypical interactions (activation, partial inhibition, no inhibition) between two CYP3A4 substrates reflect simultaneous occupancy of two or more preferred locations within a single large binding pocket. This will be tested by a combination of site-directed mutagenesis functional analysis with a variety of substrates and effectors, nuclear magnetic resonance (NMR) spectroscopy, and 3-D molecular modeling. The Specific Aims are to: 1) determine the structural basis for homotropic and heterotropic cooperativity of CYP3A4; 2) determine the structural basis for oxidation of prototypical drug substrates by human CYP3A enzymes; 3) determine substrate orientation in the CYP3A4 active site by NMR; 4) determine the structural basis for CYP3A inhibition by selected compounds. Knowledge of the molecular basis of human P450 3A function should allow the prediction of substrates, activators, and inhibitors of these enzymes, making it possible to minimize drug-drug interactions and interindividual differences in drug metabolism.