Structure and dynamics of clinically-relevant cytochrome P450 enzymes

临床相关细胞色素 P450 酶的结构和动力学

• 批准号: 10061625
• 负责人: Thomas Charles Pochapsky
• 金额: $ 42.91万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10061625
• 关键词: Active Sites; Affinity; Amino Acids; Anabolism; Antineoplastic Agents; Binding; Binding Proteins; Binding Sites; CYP17A1 gene; CYP2D6 gene; CYP3A4 gene; Catalysis; Clinical; Complex; Crystallization; Cytochrome P450; Cytochromes b5; Data; Data Analyses; Data Collection; Disease; Drug Design; Drug Interactions; Drug Targeting; Elements; Enzymes; Evaluation; Event; Fatty Acids; Goals; Health; Human; Image; Individual; Infection; Isotope Labeling; Ligand Binding; Ligands; Malignant neoplasm of prostate; Medical; Membrane; Metabolism; Methods; Mixed Function Oxygenases; Molecular Conformation; Oxides; Oxidoreductase; Pharmaceutical Preparations; Play; Prodrugs; Prostate; Protein Conformation; Proteins; Rationalization; Research; Research Personnel; Resistance; Roentgen Rays; Steroid biosynthesis; Structure; Techniques; Vitamins; Work; base; clinically relevant; design; drug clearance; drug development; drug metabolism; enzyme structure; experience; experimental study; flexibility; high risk; human disease; inhibitor/antagonist; insight; interest; new technology; oxidation; polypeptide; prevent; protein complex; scaffold; small molecule

ABSTRACT Human cytochrome P450 enzymes are dynamic, often promiscuous, monooxygenases. Some function in the biosynthesis of critical endogenous compounds and are frequent drug targets. Others are dominant factors in drug metabolism, dictating drug clearance and/or prodrug activation. For both, understanding P450 interactions with substrates, inhibitors, and their catalytic partner proteins provides substantial useful information in drug design. While X-ray structures have provided numerous insights into drug binding to key human P450 enzymes, there are many gaps that cannot be filled by this approach. Conformational changes that P450s must undergo to channel ligands to the active site and for a single P450 to accommodate many different small molecule scaffolds only randomly become apparent in comparing the X-ray structures achievable. Despite substantial efforts, some key human P450 enzymes have not yielded to crystallization. Many drug substrates have lower active site affinity and/or multiple orientations not suitable to determining clear X-ray structures. There are no structures of human P450 enzymes with reductase or cytochrome b5 catalytic partners. As a result, drug design is limited by available structural information. We propose employing solution NMR as a newly-viable orthogonal method to obtain the requisite amino acid- level structural information needed to understand human P450/ligand and P450/protein interactions. While the size, stability, and the absence of information relating individual NMR resonances to the corresponding amino acid have all thus far prevented the determination of any human P450 structures by NMR, we have the combined expertise and preliminary data to demonstrate that this feat is now technically possible. The Pochapsky lab previously developed the expertise to determine solution NMR structures of slightly smaller, soluble bacterial P450 enzymes. The Scott lab developed the capacity to generate human membrane P450 enzymes in the isotopically-labeled forms, with the amounts and with the stability required for NMR experiments. Thus, based on substantial preliminary data, we propose to 1) advance strategies for determining human P450 structures by solution NMR while determining the human steroidogenic CYP17A1 structure and 2) apply solution NMR strategies to the two most important human drug-metabolizing P450 enzymes, CYP3A4 and CYP2D6. Successful completion of aim 1 will not only further establish the feasibility of NMR structures for human membrane P450 enzymes, but will do so for an important prostate cancer drug target for which additional structural information is essential to further drug design. Results of both aims will provide a reference set of amino acid assignments that can then be readily used by a wide range of non-NMR experts, in much simpler experiments, to quickly determine where and how drugs and other proteins bind to three clinically-important P450 enzymes, as well as deciphering the changes in protein conformation required for these events.

抽象的 人细胞色素P450酶是动态的，通常是混杂的单加氧酶。在 关键内源性化合物的生物合成是常见的药物靶标。其他是主要因素 药物代谢，指示药物清除和/或前药激活。两者都了解P450互动 借助底物，抑制剂及其催化伴侣蛋白在药物中提供了实质性的有用信息 设计。尽管X射线结构为与关键人类P450酶结合的药物结合提供了许多见解，但 这种方法无法填补许多空白。 P450必须进行的构象变化 将配体引导到活动位点和单个P450以容纳许多不同的小分子 脚手架仅在比较可实现的X射线结构时随机变得明显。尽管很大 努力，一些关键的人类P450酶尚未屈服于结晶。许多药物底物的较低 主动站点亲和力和/或多个方向不适合确定清晰的X射线结构。没有 具有还原酶或细胞色素B5催化伙伴的人类P450酶的结构。结果，药物设计 受可用结构信息的限制。 我们建议使用溶液NMR作为新的可行正交方法，以获得必要的氨基酸 - 了解人P450/配体和P450/蛋白质相互作用所需的水平结构信息。而 大小，稳定性以及缺乏与相应氨基相关的单个NMR共振有关的信息 到目前为止，酸都阻止了NMR确定任何人类P450结构 专业知识和初步数据以证明这一壮举在技术上是可能的。 Pochapsky实验室 以前开发了专业知识来确定溶液NMR结构稍小，可溶性细菌 P450酶。斯科特实验室（Scott Lab）开发了产生人膜P450酶的能力 同位素标记的形式，具有NMR实验所需的量和稳定性。因此，基于 关于大量初步数据，我们建议1）提前策略来确定人类P450结构 溶液NMR确定人类类固醇生成CYP17A1结构和2）应用溶液NMR 两种最重要的人类药物代谢P450酶的策略，CYP3A4和CYP2D6。 成功完成AIM 1不仅将进一步确定人类NMR结构的可行性 膜P450酶，但对于重要的前列腺癌药物靶标会这样做 结构信息对于进一步的药物设计至关重要。两个目标的结果将提供一组参考 然后，氨基酸分配很容易被广泛的非NMR专家使用，更简单得多 实验，以快速确定药物和其他蛋白质与三种临床重要的何处以及如何结合 P450酶，以及破译这些事件所需的蛋白质构象变化。