Small Molecule Tools for Modulating Membrane Rafts

用于调节膜筏的小分子工具

• 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
• 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' 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

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.

摘要 质膜结构对细胞功能有很大的影响。一位著名的 这种组织背后的机制是通过不同的脂质和蛋白质在纳米级聚集在一起 膜筏。RAFT结构域富含胆固醇和脂类，具有饱和的酰基链和 在体外观察到具有液体有序膜相的性质。在细胞膜中，RAFT结构域是 被认为与更多的流体、无序区域和富含胆固醇的血浆环境共存 膜被认为特别有利于筏子的形成。个别膜蛋白倾向于 喜欢木筏或非木筏环境，这种倾向是一种重要的调节机制 它们的功能。此外，RAFT依赖的过程在许多人类病理中都有牵连，包括 几种形式的癌症、艾滋病和阿尔茨海默氏症。然而，这些领域的大小和动态 以及膜蛋白在有序和无序结构域之间的分配如何影响其功能 而且相互作用仍然不确定。我们认为，知识中的这些根本差距仍然存在，因为很少有人 存在以受控方式对木筏进行实验扰动的方法。这项提议的目标是 因此，要发现和验证第一代小分子可以用来 从药理上操纵木筏。为了解决这个问题，我们开发了一种新型的高吞吐量 以巨型质膜囊泡(GPMV)为模型系统的筛查(HTS)试验 及其相关蛋白质，以及对这些数据集进行量化的定制软件。使用这些 方法，我们现在已经确定了几个生物活性脂类和FDA批准的药物改变 GPMV中有序相和无序相的相对比例，以及在 这表明有可能改变选定的膜蛋白的相分配。在这里，我们 建议在这些初步成功的基础上，从原则证明阶段进入我们在 手工验证具有这些活性的第一代小分子。蛋白质靶标将包括外周血细胞 髓鞘蛋白22，一种与夏科-玛丽-图斯综合征相关的蛋白质，以及HIV受体CD4和它的 共同受体CCR5。最终，这些研究的结果将为制定 一种新的化学物质，可用于在实验室和最终药理上操纵木筏 测试长期存在的关于木筏性质的问题，以及药物发现项目中的就业问题。