Structure and dynamics of clinically-relevant cytochrome P450 enzymes

摘要 人类细胞色素P450酶是动态的，通常是混杂的单加氧酶。中的一些函数 是关键的内源性化合物的生物合成，是经常使用的药物靶标。其他因素是主要因素 药物代谢，决定药物清除和/或前药物激活。对于两者来说，了解P450相互作用 底物、抑制剂及其催化伙伴蛋白在药物方面提供了大量有用的信息。 设计。虽然X射线结构为药物与关键的人类P450酶的结合提供了大量的见解， 有许多空白是这种方法无法填补的。P450必须经历的构象变化 将配体引导到活性部位，并使单个P450适应许多不同的小分子 支架只有在比较可实现的X射线结构时才会随机变得明显。尽管有大量的 经过努力，一些关键的人类P450酶还没有屈服于结晶。许多药物底物具有较低的 活性中心亲和力和/或多重取向不适合确定清晰的X射线结构。没有 人p450酶与还原酶或细胞色素b5催化配对的结构。因此，药物设计 受到现有结构信息的限制。 我们建议使用溶液核磁共振作为一种新的可行的正交法来获得所需的氨基酸- 了解人类P450/配体和P450/蛋白质相互作用所需的水平结构信息。而当 大小、稳定性和缺乏将单个核磁共振共振与相应氨基相关联的信息 到目前为止，所有酸都阻止了用核磁共振确定任何人类P450结构，我们结合了 专业知识和初步数据表明，这一壮举现在在技术上是可能的。波查普斯基实验室 以前开发了确定稍小的可溶细菌的溶液核磁共振结构的专业知识 P450酶。斯科特实验室开发了在体内产生人膜P450酶的能力 同位素标记形式，具有核磁共振实验所需的数量和稳定性。因此，基于 根据大量的初步数据，我们建议1)通过以下方式提高确定人类P450结构的策略 溶液核磁共振在测定人类固醇合成细胞色素P17A1结构中的应用 人类两种最重要的药物代谢P450酶--细胞色素P3A4和细胞色素P2D6的研究策略。 目标1的成功完成不仅将进一步确立用于人类的核磁共振结构的可行性 膜上的P450酶，但会为前列腺癌药物的一个重要靶点，额外 结构信息对于进一步的药物设计是必不可少的。这两个目标的结果将提供一个参考集 氨基酸分配，然后可以很容易地被广泛的非核磁共振专家使用，要简单得多 实验，以快速确定药物和其他蛋白质在哪里以及如何与三种临床重要的 P450酶，以及破译这些事件所需的蛋白质构象变化。

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.