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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.

摘要外周膜蛋白（PMPs）是生物学和疾病过程的重要介质。这一类蛋白质以水溶性状态存在，直到靶向粘附在膜上，使它们能够履行其职能。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没有解决。此外，利用由于目前的方法是针对水溶性蛋白质设计的，因此很难将PMP作为药物靶标对膜相关蛋白的表达也很好。因此，非常需要新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的PMP抑制剂。的我们研究的目标是实现PMP相互作用的高分辨率定量研究，并允许抑制剂设计这种难以捉摸的蛋白质。我们将首先集中精力在三个特别的PMP上，生物医学兴趣：NADPH氧化酶中的谷胱甘肽过氧化物酶4（GPx 4）和Phox同源（PX）结构域 p47 phox-PX和NOXO 1-PX。我们已经开始开发一种新的膜模型，膜- 模拟反胶束（mmRM），其基于细胞膜的化学性质。PMP嵌入像处理细胞膜一样，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与当前模型相比具有许多优点，包括大大增强了稳定性，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，允许根据自然PMP环境来调谐系统。我们的重点将是利用我们的mmRM具有独特的特性，能够在PMP相互作用的研究和量化中提供无与伦比的细节与膜和脂质基质。为了促进抑制剂设计，我们将开发一种新方法，膜包埋的PMP的片段筛选，目前的方法不适合。使用这些工具，我们将启动一个抑制剂的设计活动，我们的重要PMP目标。总的来说，我们的目标是发展这些工具将使PMP生物学的详细研究以及抑制剂和药物开发取得突破对于这类尚未开发的蛋白质。