High Throughput/Content Screens for Dynein Inhibitors

动力蛋白抑制剂的高通量/含量筛选

• 批准号: 7622278
• 负责人: BILLY W DAY
• 金额: $ 3.78万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7622278
• 关键词: 9-(2-hydroxy-3-nonyl)adenine; ATP Hydrolysis; Adenoviruses; Agonist; Anions; Area; Binding; Biochemical; Biological; Biological Assay; Biological Factors; Biology; Cell Nucleus; Cell physiology; Cells; Cellular biology; Chemicals; Complex; Cytoplasm; DNA Damage; Data; Dynein ATPase; Event; Fluorescence; Fluorescence Polarization; Glucocorticoid Receptor; Goals; Green Fluorescent Proteins; Heat-Shock Proteins 70; Heat-Shock Proteins 90; Interphase Cell; Kinesin; Label; Ligand Binding Domain; Liquid substance; Messenger RNA; Microtubules; Minus End of the Microtubule; Mitosis; Mitotic; Mitotic Checkpoint; Molecular Bank; Molecular Motors; Molecular Target; Motor; Movement; Myosin ATPase; Nuclear Envelope; Nuclear Localization Signal; Organelles; Outcome; Oxidation-Reduction; Phenotype; Plus End of the Microtubule; Proteins; Recombinants; Relative (related person); Research Personnel; Robotics; Screening procedure; Sister Chromatid Exchange; Specificity; System; TP53 gene; Tubulin; Urination; Vanadates; Virus; Work; base; chromosome movement; design; experience; high throughput screening; inhibitor/antagonist; inorganic phosphate; purealin; size; small molecule; small molecule libraries; tool

Cytoplasmic dynein is the major retrograde molecular motor responsible for transport of membranous organelles, viruses, mRNA and proteins with nuclear localization signals. It is also involved in chromosome movement, mitotic checkpoint inactivation and spindle orientation, and many other basic cellular processes. Cytoplasmic dynein is a large (ca. 2 MDa) complex of proteins. Its movement along microtubules is powered by ATP hydrolysis by its 380 kDA motor domains, which are associated with the >500 kDa heavy chain subunits. Other than weak and nonspecific redox perturbers and mimics of ATP or phosphate anion, only one small molecule inhibitor of dynein, the natural product purealin, which we have recently synthesized and examined, is known. It is, however, only weakly active in cells. New inhibitors with potency and specificity for dynein and subsets of dynein function would be invaluable cell biology tools. The goal is to develop a refined suite of high throughput cell and biochemical assays for chemical library screening find inhibitors of cellular cytoplasmic dynein. Aim 1 is to develop a cell-based phenotypic multiparameter fluorescence screen to detect dynein inhibition in interphase cells, as well as to detect possible mitotic block due to inhibition of dynein. Preliminary data provides support of three cellular events, transport from the cytoplasm to the nucleus of: (i) p53 after mild DMAdamage; (ii) stably expressed green fluorescent protein-labeled glucocorticoid receptor after binding with an agonist; and (iii) a fluorescent adenovirus. These will be examined and the most useful selected. As some phenotypes could be due to interaction of the small molecules with off-target proteins, Aim 2 is to implement high throughput biochemical screens to confirm that a small molecule's molecular target is indeed cytoplasmic dynein. These include microtiter plate-based colorimetric, turbidimetric and fluorescence polarization analyses of the direct action of library chemicals on recombinant dynein heavy chain, glucocorticoid receptor ligand binding domain, HSP70 and HSP90, and on isolated myosin and tubulin. The investigators, a team of experts in these areas, will design and perform the assays as well as interpret results, and have previous direct or related experience with most of the assays. The product of this work will be a refined, streamlined set of rapid cell and biochemical screens for dynein inhibitors that can be implemented in molecular library screening centers.

细胞质动力蛋白是负责膜运输的主要逆行分子电动机 带有核定位信号的细胞器，病毒，mRNA和蛋白质。它也参与染色体 运动，有丝分裂检查点的失活和纺锤取向以及许多其他基本的细胞过程。 细胞质动力蛋白是蛋白质的大型（C. 2 MDA）复合物。它沿着微管运动是动力的 通过其380 kDa电动域的ATP水解，与> 500 kDa重链有关 亚基。除了弱和非特异性氧化还原蛋白酶以及ATP或磷酸根阴离子的模仿 一种小分子抑制剂Dynein，天然产物纯素，我们最近合成了，并 已知，已知。但是，它仅在细胞中活跃。具有效力和特异性的新抑制剂 动力蛋白和动力蛋白功能的子集将是宝贵的细胞生物学工具。目标是开发一个精致的 化学库筛选的高吞吐量细胞和生化测定的套件发现细胞的抑制剂 细胞质动力蛋白。目的1是开发基于细胞的表型多参数荧光屏幕 检测相间细胞中的动力蛋白抑制作用，以及由于抑制而检测到可能的有丝分裂阻滞 动力蛋白。初步数据提供了三个细胞事件的支持，从细胞质转运到 核的核：（i）p53轻度dmadamage之后； （ii）稳定表达的绿色荧光蛋白标记 与激动剂结合后，糖皮质激素受体； （iii）荧光腺病毒。这些将是 检查和最有用的选择。因为某些表型可能是由于小的相互作用 具有脱靶蛋白的分子，AIM 2是实施高通量生化筛选，以确认 小分子的分子靶标确实是细胞质动力蛋白。这些包括基于微滴定板的 图书馆化学物质对直接作用的比色，浊度和荧光极化分析 重组动力蛋白重链，糖皮质激素受体配体结合结构域，HSP70和HSP90，以及On 孤立的肌球蛋白和微管蛋白。调查人员是这些领域的专家团队，将设计和执行 测定法和解释结果，并具有以前的直接或相关经验。 这项工作的产物将是一组精简的快速细胞和动力蛋白的生化筛选 可以在分子库筛选中心实施的抑制剂。