New tools to advance biophysical study and inhibition of peripheral membrane proteins.

推进生物物理研究和外周膜蛋白抑制的新工具。

基本信息

批准号：
10795403
负责人：
Brian Fuglestad
金额：
$ 8.76万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2027-07-31
项目状态：
未结题

项目摘要

Abstract Peripheral membrane proteins (PMPs) represent crucial mediators of biological and disease processes. This class of proteins exists in a water-soluble state until targeted to adhere on membranes, enabling them to perform their function. As with any membrane associated protein, PMPs are challenging to study, particularly in their membrane bound state, leaving many basic questions about function unresolved. Additionally, utilizing PMPs as drug targets is difficult since current methods are designed for water-soluble proteins and do not work well for membrane associated proteins. Thus, there is a great need for novel tools and procedures to illuminate details of PMP function and to create PMP inhibitors for use in chemical biology study and as drug leads. The goal of our research is to enable high-resolution, quantitative study of PMP interactions and allow inhibitor design for this elusive type of protein. We will initially focus our efforts on three PMPs of extraordinary biomedical interest: glutathione peroxidase 4 (GPx4) and Phox homology (PX) domains in the NADPH oxidase family (p47phox-PX and NOXO1-PX). We have initiated development of a novel membrane model, membrane- mimicking reverse micelles (mmRMs), which is based on the chemistry of cellular membranes. PMPs embed into mmRMs as they do with cellular membranes, enabling high-resolution study using NMR spectroscopy and other techniques. mmRMs have a number of advantages over current models, including greatly enhanced stability, outstanding spectroscopic properties, and an ability to house high concentrations of analyte along with the protein. We will modify our mmRM system to better reflect a variety of cellular and organelle membranes, allowing the system to be tuned according to the natural PMP environment. Our focus will then be to harness the unique properties of our mmRMs to enable unrivaled detail in study and quantification of PMP interactions with membranes and lipid substrates. To facilitate inhibitor design, we will develop a novel method that allows fragment screening of membrane-embedded PMPs, for which current methods are not suitable. Using these tools, we will initiate an inhibitor design campaign for our important PMP targets. Overall, our goal is to develop tools that will enable breakthroughs in detailed study of PMP biology as well as inhibitor and drug development for this largely untapped class of proteins.

抽象的外周膜蛋白（PMP）代表生物和疾病过程的关键介质。这一类蛋白质以水溶性状态存在，直到被靶向粘附在膜上，使它们能够履行他们的职能。与任何膜相关蛋白一样，PMP 的研究具有挑战性，特别是在它们的膜结合状态，留下了许多关于功能的基本问题尚未解决。此外，利用 PMP 作为药物靶点很困难，因为目前的方法是针对水溶性蛋白质设计的，不起作用对于膜相关蛋白来说效果很好。因此，非常需要新颖的工具和程序来阐明 PMP 功能的详细信息并创建用于化学生物学研究和作为药物先导物的 PMP 抑制剂。这我们研究的目标是实现 PMP 相互作用的高分辨率、定量研究，并允许抑制剂针对这种难以捉摸的蛋白质类型进行设计。我们首先将重点放在三名杰出的 PMP 上生物医学兴趣：NADPH 氧化酶中的谷胱甘肽过氧化物酶 4 (GPx4) 和 Phox 同源 (PX) 结构域家族（p47phox-PX 和 NOXO1-PX）。我们已经开始开发一种新型膜模型，膜- 模仿基于细胞膜化学的反胶束 (mmRM)。 PMP 嵌入就像对细胞膜一样，可以将其转化为 mmRM，从而能够使用 NMR 光谱进行高分辨率研究，其他技术。 mmRM 与当前型号相比具有许多优势，包括大大增强稳定性、出色的光谱特性以及容纳高浓度分析物的能力蛋白质。我们将修改我们的 mmRM 系统，以更好地反映各种细胞和细胞器膜，允许系统根据自然的 PMP 环境进行调整。我们的重点将是利用我们的 mmRM 具有独特的特性，可在 PMP 相互作用的研究和量化中提供无与伦比的细节具有膜和脂质底物。为了促进抑制剂设计，我们将开发一种新方法，允许膜嵌入 PMP 的片段筛选，目前的方法不适合。使用这些工具，我们将为重要的 PMP 目标发起抑制剂设计活动。总体而言，我们的目标是发展这些工具将为 PMP 生物学以及抑制剂和药物开发的详细研究带来突破对于这一类很大程度上尚未开发的蛋白质。