Structure and dynamics of clinically-relevant cytochrome P450 enzymes - Summer undergraduate research experience supplement

项目摘要/摘要： 人细胞色素P450酶是动态的，通常是混杂的单加氧酶。一些人 在关键内源性化合物的生物合成中发挥作用，是经常使用的药物靶标。其他 是药物代谢的主导因素，决定药物清除和/或前药物激活。对于两个人来说， 了解P450与底物、抑制剂及其催化伙伴蛋白的相互作用 在药物设计中提供大量有用的信息。P450必须经历的构象变化 通道配体到活性部位，并针对单个P450来容纳许多不同的小分子 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 经过大量的努力，一些关键的人类P450酶还没有屈服于结晶。多种药物 底物具有较低的活性中心亲和力和/或不适合测定透明X- 射线结构。没有含有还原酶或细胞色素b5的人p450酶的结构。 催化剂伙伴结合在一起。因此，药物设计受到现有结构信息的限制。我们是 使用溶液核磁共振作为一种可行的正交法来获得所需的原子级结构 了解人类P450/配体和P450/蛋白质相互作用所需的信息。而大小， 稳定性，以及缺乏将单个核磁共振共振与相应的 到目前为止，氨基酸都阻止了核磁共振对任何人类P450结构的确定，我们 拥有综合的专业知识和初步数据来证明这一壮举现在在技术上是 有可能。波查普斯基实验室已经开发出了确定溶液核磁共振结构的专业知识 稍小的、可溶的细菌P450酶。斯科特实验室开发出了发电能力 同位素标记形式的人膜P450酶，及其数量和 核磁共振实验所需的稳定性。因此，根据大量的初步数据，我们建议 利用溶液核磁共振确定人P450结构的新策略 人类类固醇生成细胞色素P17A1。成功完成这一目标不仅将进一步确立 核磁共振结构用于人膜P450酶的可行性，但将对这样做的一个重要 前列腺癌药物靶点，对于进一步的药物设计来说，额外的结构信息是必不可少的。 目前的要求是为阿里尤·阿尔加利的夏季研究提供补充资料，‘22 a Brandeis 来自塞拉利昂的维恩大学奖学金学生。阿里鱼将在表达和表达方面进行训练 以适合多维核磁共振实验的同位素标记形式纯化细胞色素P17A1。如果 在时间允许的情况下，他还将接受数据获取、处理和分析方面的培训。

Project Summary/Abstract: Human cytochrome P450 enzymes are dynamic and 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. 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 bound. As a result, drug design is limited by available structural information. We are employing solution NMR as a viable orthogonal method to obtain the requisite atomic 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 has 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 advance strategies for determining human P450 structures by solution NMR while determining the human steroidogenic CYP17A1. Successful completion of this aim 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. The current request is for a supplement supporting summer research for Aliyu Alghali, '22 a Brandeis University Wien Scholar student from Sierra Leone. Aliyu will be trained in the expression and purification of CYP17A1 in isotopically labeled form suitable for multidimensional NMR experiments. If time permits, he will also be trained in the acquisition, processing and analysis of the data.