Structure and dynamics of clinically-relevant cytochrome P450 enzymes

摘要 人细胞色素P450酶是动态的，通常是混杂的，单加氧酶。中的一些函数 关键内源性化合物的生物合成，是常见的药物靶标。其他是主导因素， 药物代谢，指示药物清除和/或前药活化。对于两者来说，理解P450的相互作用 与底物、抑制剂及其催化伴侣蛋白质的相互作用为药物治疗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酶的结合提供了许多见解， 这一办法无法填补许多空白。P450必须经历的构象变化 将配体引导到活性位点，并使单个P450容纳许多不同的小分子 在比较可实现的X射线结构时，支架仅随机变得明显。尽管作出了重大的 尽管经过努力，一些关键的人类P450酶尚未产生结晶。许多药物底物具有较低的 活性位点亲和性和/或多个取向不适合于确定清晰的X射线结构。没有 具有还原酶或细胞色素b5催化配偶体的人P450酶的结构。因此，药物设计 受到可用结构信息的限制。 我们建议采用溶液NMR作为一种新的可行的正交方法，以获得所需的氨基酸- 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 大小，稳定性，以及缺乏有关个别NMR共振相应的氨基 酸迄今为止都阻止了通过NMR确定任何人类P450结构， 专业知识和初步数据，以证明这一壮举现在在技术上是可能的。Pochapsky实验室 以前开发的专业知识，以确定溶液核磁共振结构略小，可溶性细菌 P450酶。斯科特实验室开发了在细胞中产生人类膜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 基于大量的初步数据，我们建议：1）通过以下方法确定人类P450结构的发展策略： 溶液NMR同时测定人类固醇生成CYP 17 A1结构和2）应用溶液NMR 这两种最重要的人类药物代谢P450酶，CYP 3A 4和CYP 2D 6的策略。 目标1的成功完成不仅将进一步确立核磁共振结构用于人体的可行性， 膜P450酶，但将这样做的一个重要的前列腺癌药物的目标，其中额外的 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套参考资料， 氨基酸分配，然后可以很容易地使用广泛的非NMR专家，在更简单的 实验，以快速确定药物和其他蛋白质在哪里以及如何结合到三个临床重要的 P450酶，以及破译这些事件所需的蛋白质构象的变化。

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.