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: 7169712
• 负责人: BILLY W DAY
• 金额: $ 7.43万
• 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/7169712
• 关键词: NIH Roadmap Initiative tag; chemical registry /resource; cytoplasm; cytoskeleton; drug discovery /isolation; dynein ATPase; enzyme activity; fluorescent dye /probe; high throughput technology; inhibitor /antagonist; pharmacology; protein binding; protein transport; technology /technique development

DESCRIPTION (provided by applicant): 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 DNA damage; (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.

描述（由申请人提供）：细胞质动力蛋白是负责运输具有核定位信号的膜细胞器、病毒、mRNA和蛋白质的主要逆行分子马达。它还参与染色体运动、有丝分裂检查点失活和纺锤体定向以及许多其他基本细胞过程。细胞质动力蛋白是一种大型（约 2 MDa）蛋白质复合物。它沿着微管的运动由 380 kDA 运动域的 ATP 水解提供动力，该运动域与 >500 kDa 的重链亚基相关。除了弱的、非特异性的氧化还原干扰物和 ATP 或磷酸阴离子的模拟物之外，只有一种小分子动力蛋白抑制剂，即我们最近合成和检查的天然产物 Purealin 是已知的。然而，它在细胞中的活性很弱。对动力蛋白和动力蛋白功能子集具有效力和特异性的新型抑制剂将是宝贵的细胞生物学工具。目标是开发一套完善的高通量细胞和生化检测方法，用于化学文库筛选，寻找细胞质动力蛋白的抑制剂。目标 1 是开发基于细胞的表型多参数荧光筛选，以检测间期细胞中的动力蛋白抑制，以及检测由于动力蛋白抑制而可能出现的有丝分裂阻断。初步数据支持三个细胞事件，即从细胞质转运至细胞核：(i)轻度 DNA 损伤后的 p53； (ii)与激动剂结合后稳定表达的绿色荧光蛋白标记的糖皮质激素受体； (iii) 荧光腺病毒。这些将被检查并选择最有用的。由于一些表型可能是由于小分子与脱靶蛋白质的相互作用造成的，目标 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。这些包括基于微量滴定板的比色、比浊和荧光偏振分析，分析文库化学品对重组动力蛋白重链、糖皮质激素受体配体结合结构域、HSP70 和 HSP90 以及分离的肌球蛋白和微管蛋白的直接作用。研究人员是这些领域的专家团队，他们将设计和执行检测以及解释结果，并且之前对大多数检测具有直接或相关的经验。这项工作的产品将是一套精炼、精简的动力蛋白抑制剂快速细胞和生化筛选方法，可在分子库筛选中心实施。