Small Molecule Tools for Modulating Membrane Rafts

用于调节膜筏的小分子工具

基本信息

批准号：
10474445
负责人：
Anne K Kenworthy
金额：
$ 38.22万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10474445
关键词：
Acquired Immunodeficiency Syndrome Affect Alzheimer&apos s Disease Behavior Benchmarking Biological Biological Assay Biological Models Biophysics CCR5 gene CD4 Antigens Cell membrane Cell physiology Cellular biology Charcot-Marie-Tooth Disease Chemicals Cholesterol Communities Computer software Custom Data Set Development Disease Employment Environment FDA approved Fluorescence Microscopy Generations HIV HIV Receptors Hand Human Human Pathology Image In Vitro Individual Integral Membrane Protein Knowledge Laboratories Lead Lipids Liquid substance Malignant Neoplasms Membrane Membrane Microdomains Membrane Proteins Methods Microscopy Myelin Proteins Nature PMP22 gene Peripheral Pharmaceutical Preparations Pharmacology Phase Population Process Property Proteins Syndrome Technology Testing United States National Institutes of Health Vesicle base chemical property drug discovery druggable target high throughput screening microscopic imaging novel preference programs research and development small molecule small molecule libraries success tool

项目摘要

Summary Plasma membrane organization exerts tremendous influence on cellular functionality. A well known mechanism underlying this organization is through nanoscopic clustering of distinct lipids and proteins in membrane rafts. Raft domains are enriched in cholesterol and lipids with saturated acyl chains and share properties with liquid-ordered membrane phases observed in vitro. In cell membranes, raft domains are thought to co-exist with more fluid, disordered domains, and the cholesterol-rich environment of the plasma membrane is thought to be especially conducive for raft formation. Individual membrane proteins tend to prefer either raft or non-raft environments, and this propensity serves as an important regulatory mechanism of their function. Further, raft-dependent processes have been implicated in many human pathologies including several forms of cancer, AIDS and Alzheimer's disease. However, the size and dynamics of these domains and how the partitioning of membrane proteins between ordered and disordered domains affect their functions and interactions remain uncertain. We argue that these fundamental gaps in knowledge remain because few methods exist to experimentally perturb rafts in a controlled manner. The objective of this proposal is therefore to discover and validate first generation small molecules that can be used to pharmacologically manipulate rafts. To tackle this problem, we have developed a novel high throughput screen (HTS) assay exploiting giant plasma membrane vesicles (GPMVs) as a model system to visualize rafts and their associated proteins, as well as custom software to quantitate these datasets. Using these approaches, we have now identified several examples of bioactive lipids and FDA-approved drugs that alter the relative proportions of ordered and disordered phases in GPMVs, as well as have preliminary evidence in hand that suggests it is possible to alter the phase partitioning of selected membrane proteins. Here, we propose to build on these initial successes to move from proof-of-principle stage to the point where we have in hand validated first generation small molecules with these activities. Protein targets will include peripheral myelin protein 22, a protein associated with Charcot-Marie-Tooth syndrome, and the HIV receptor CD4 and its co-receptor CCR5. Ultimately, the results of these studies will lay the groundwork for the development of a new class of chemicals that can be used to pharmacologically manipulate rafts in the laboratory and definitively test long-standing questions about the nature of rafts as well as for employment in drug discovery programs.

概括质膜组织对细胞功能产生巨大影响。一个众所周知的这种组织的机制是通过不同脂质和蛋白质的纳米级聚集膜筏。筏结构域富含具有饱和酰基链的胆固醇和脂质，并共享体外观察到的液体有序膜相的特性。在细胞膜中，筏结构域是被认为与更多流体、无序域和富含胆固醇的血浆环境共存膜被认为特别有利于筏的形成。个别膜蛋白倾向于喜欢筏或非筏环境，这种倾向是重要的调节机制他们的功能。此外，筏依赖性过程与许多人类病理有关，包括多种癌症、艾滋病和阿尔茨海默病。然而，这些领域的规模和动态以及膜蛋白在有序结构域和无序结构域之间的划分如何影响其功能和相互作用仍然不确定。我们认为，这些基本知识差距仍然存在，因为很少有人存在以受控方式对筏进行实验扰动的方法。该提案的目标是因此，发现并验证可用于通过药物操纵筏。为了解决这个问题，我们开发了一种新型的高通量利用巨型质膜囊泡（GPMV）作为模型系统来可视化筏的筛选（HTS）测定及其相关蛋白质，以及定量这些数据集的定制软件。使用这些方法，我们现在已经确定了几个生物活性脂质和 FDA 批准的药物的例子，这些药物可以改变 GPMV 中有序相和无序相的相对比例，并有初步证据这表明可以改变选定膜蛋白的相分配。在这里，我们建议在这些初步成功的基础上，从原理验证阶段转向我们所拥有的阶段手工验证第一代小分子具有这些活性。蛋白质靶标将包括外周血髓磷脂蛋白 22（一种与夏科-玛丽-图斯综合征相关的蛋白）以及 HIV 受体 CD4 及其辅助受体CCR5。最终，这些研究结果将为开发新型化学物质，可用于在实验室中对筏进行药理学操纵，并且最终测试有关筏性质以及药物发现计划中的就业的长期存在的问题。