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.

抽象的周围膜蛋白（PMP）代表生物学和疾病过程的关键介质。这蛋白质类别存在于水溶性状态下，直到有针对性地粘附在膜上，使它们能够执行其功能。与任何膜相关蛋白一样，PMP在研究中都具有挑战性，特别是他们的膜约束状态，留下了有关功能未解决的许多基本问题。另外，利用 PMP作为药物靶标很困难，因为当前方法是为水溶性蛋白设计的，并且不起作用很好地适用于膜相关蛋白。因此，非常需要新颖的工具和程序来照亮 PMP功能的详细信息并创建用于化学生物学研究的PMP抑制剂和作为药物铅。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这种难以捉摸的蛋白质的设计。我们最初将把精力集中在三个特殊的PMP上生物医学的兴趣：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）。我们已经开发了一种新型的膜模型，膜 - 模仿反向胶束（MMRMS），基于细胞膜的化学。嵌入PMP 与细胞膜一样，进入MMRM，可以使用NMR光谱和高分辨率研究其他技术。 MMRM比当前模型具有许多优势，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以及蛋白质。我们将修改MMRM系统，以更好地反映各种细胞和细胞器膜，允许根据天然PMP环境对系统进行调整。然后，我们的重点是利用 MMRMS的独特属性可以在PMP相互作用的研究和量化中实现无与伦比的细节膜和脂质底物。为了促进抑制剂设计，我们将开发一种新颖的方法，允许膜上包裹的PMP的片段筛选，目前的方法不合适。使用这些工具，我们将针对重要的PMP目标启动抑制剂设计活动。总体而言，我们的目标是发展可以在PMP生物学以及抑制剂和药物开发的详细研究中取得突破的工具对于这种很大程度上未开发的蛋白质。