Small Molecule Tools for Modulating Membrane Rafts

用于调节膜筏的小分子工具

• 批准号: 10029455
• 负责人: Anne K Kenworthy
• 金额: $ 39.71万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10029455
• 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.

总结 质膜组织对细胞功能有着巨大的影响。一个众所周知 这种组织的基本机制是通过不同的脂质和蛋白质的纳米级聚集， 膜筏筏结构域富含胆固醇和具有饱和酰基链的脂质， 在体外观察到的液体有序膜相的性质。在细胞膜中，筏结构域是 被认为与更多的流体、无序区域和血浆中富含胆固醇的环境共存 膜被认为特别有利于筏的形成。单个膜蛋白倾向于 更喜欢筏或非筏环境，这种倾向是一个重要的调节机制， 它们的功能。此外，筏依赖性过程已经涉及许多人类病理学，包括 几种形式的癌症，艾滋病和阿尔茨海默病。然而，这些领域的规模和动态 以及膜蛋白在有序和无序结构域之间的分配如何影响它们的功能 相互作用仍然不确定。我们认为，这些基本的知识差距仍然存在，因为很少有人 存在以受控方式实验性地扰动筏的方法。本提案的目的是 因此，为了发现和验证第一代可用于 操纵木筏。为了解决这个问题，我们开发了一种新的高通量 利用巨质膜囊泡（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，以及用于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，改变 GPMV中有序相和无序相的相对比例，以及 这表明有可能改变选定的膜蛋白的相分配。这里我们 我建议在这些初步成功的基础上，从原理验证阶段过渡到我们已经 手工验证具有这些活性的第一代小分子。蛋白质靶点将包括外周 髓鞘蛋白22，一种与腓骨肌萎缩症相关的蛋白质，以及HIV受体CD4及其 共受体CCR5。最终，这些研究的结果将为制定一项 新型化学品可用于在实验室中操纵木筏， 测试有关木筏性质以及药物发现计划中的就业的长期问题。