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.

概括 质膜组织对细胞功能产生巨大影响。一个众所周知的 该组织的基础机制是通过纳米镜簇的不同脂质和蛋白质的纳米群聚类 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 在体外观察到的具有液态膜相的性质。在细胞膜中，筏结构域是 被认为与更多的流体，无序结构域和等离子体的胆固醇环境共存 膜被认为特别有利于筏形成。单个膜蛋白倾向于 更喜欢筏或非收割环境，并且这种倾向是 他们的功能。此外，与筏相关的过程已经与许多人类病理有关 几种形式的癌症，艾滋病和阿尔茨海默氏病。但是，这些域的大小和动态 以及膜蛋白在有序域和无序域之间的分配如何影响其功能 相互作用仍然不确定。我们认为，这些知识中的这些基本差距仍然是因为很少 存在以受控方式进行实验扰动筏的方法。该提议的目的是 因此，发现和验证一代小分子 药理学操纵筏。为了解决这个问题，我们已经开发了一个新颖的高通量 屏幕（HTS）测定利用巨型质膜囊泡（GPMV）作为模型系统，可视化筏 及其相关的蛋白质以及定制这些数据集的自定义软件。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 GPMV中有序相和无序相的相对比例，并且具有初步证据 表明可以改变选定膜蛋白的相分配的手。在这里，我们 建议以这些最初的成功为基础，以从原理证明阶段转变为我们所拥有的 手工验证的第一代小分子使用这些活动。蛋白质靶标将包括周围 髓磷脂蛋白22，一种与Charcot-Marie-Tooth综合征相关的蛋白质，HIV受体CD4及其ITS蛋白 共受体CCR5。最终，这些研究的结果将为开发 新的化学物质可用于在实验室操纵筏子的药理学，并确定 测试有关筏子性质以及在药物发现计划中的工作的长期问题。