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

临床相关细胞色素 P450 酶的结构和动力学 - 暑期本科生研究经验补充

基本信息

批准号：
10392567
负责人：
Thomas Charles Pochapsky
金额：
$ 0.93万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
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

项目摘要

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.

项目概要/摘要：人细胞色素P450酶是动态的并且通常是混杂的单加氧酶。一些在关键内源性化合物的生物合成中起作用，并且是常见的药物靶标。别人是药物代谢中的主导因素，决定药物清除和/或前药活化。对于两者来说，了解P450与底物、抑制剂及其催化伴侣蛋白的相互作用，药物设计中的大量有用信息。P450必须经历的构象变化通道配体的活性位点和一个单一的P450，以适应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。尽管尽管进行了大量的努力，但一些关键的人类P450酶尚未产生结晶。许多药物基底具有较低的活性位点亲和力和/或多个取向，不适合于确定透明的X-射线衍射。射线结构没有人P450酶与还原酶或细胞色素b5的结构催化剂伙伴绑定。因此，药物设计受到可用结构信息的限制。我们采用溶液NMR作为可行的正交方法来获得所需的原子水平结构，了解人类P450/配体和P450/蛋白质相互作用所需的信息。虽然大小，稳定性，以及缺乏有关个别NMR共振的信息，以相应的氨基酸迄今为止都阻止了通过NMR确定任何人P450结构，我们结合专业知识和初步数据，证明这一壮举现在在技术上是可能Pochapsky实验室已经开发了确定溶液NMR结构的专业知识，稍小的可溶性细菌P450酶。斯科特实验室开发了一种同位素标记形式的人膜P450酶，其量和 NMR实验所需的稳定性。因此，根据大量的初步数据，我们建议通过溶液NMR确定人类P450结构的先进策略，同时确定人类固醇生成CYP 17 A1。成功地实现这一目标不仅将进一步建立的可行性NMR结构的人膜P450酶，但将这样做的一个重要的前列腺癌药物靶点，其额外的结构信息对于进一步的药物设计是必不可少的。目前的要求是一个补充支持夏季研究阿利尤阿尔加利，'22一个布兰代斯来自塞拉利昂的维也纳大学奖学金学生。阿利尤将接受表情训练，纯化同位素标记形式的CYP 17 A1，适用于多维NMR实验。如果如果时间允许，他还将接受数据采集、处理和分析方面的培训。