Microtubule regulation by small molecules

小分子的微管调节

• 批准号: 7333359
• 负责人: Dan L Sackett
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7333359
• 关键词: Microtubule; regulation; small; molecules

This project focuses on the ability of small molecules to alter the cell's cytoskeleton and, in particular, microtubules (MT). MT are the most rigid of the cytoskeletal polymers, and are central to establishing and maintaining non-spherical cell morphology. In addition, MT have intrinsic polarity, and the cytoplasmic array of MT provides the substrate for directional intracellular movement. Thus, small molecules that alter MT integrity and/or dynamics can scramble intracellular trafficking and alter the physical properties of the cytoplasm. This project has two parts. One is to understand in detail the interaction of MT-active small molecules with MT or the MT subunit protein, tubulin, focusing on anti-mitotic peptide natural products from marine sources, as well as compounds produced from knowledge-directed synthesis. The second part aims to use the knowledge of MT-small molecule interactions to identify drug therapies for parasite diseases. In part one we have focused on antimitotic peptides because these are among the most potent anti-MT agents known, they have been synthesized and analogs are available, and because they induce the MT subunits to assume unusual and characteristic ring shapes. We are studying the structural and dynamic properties of these ring polymers by analytical ultracentrifugation, cryoelectron microscopy, fluorescence correlation spectroscopy, and protease mapping. The high stability and uniformity of these rings that our studies revealed have led us to attempt crystallization of these polymers to achieve atomic resolution of their structure. We are also examining the effects on microtubule polymerization of synthetic analogs of thalidomide and combretastatin A. We have discovered an anlog of thalidomide that stabilizes microtubules. In part two, we are seeking to identify small molecules that do not bind well with mammalian tubulin but do bind to parasite tubulin. The tubulin molecule is quite conserved evolutionarily, but differences do exist, and several molecules are known that can target, for example, yeast rather than mammalian tubulin or vice-versa. We are looking for molecules that will target Leishmania, the infectious cause of an important group of human diseases. We have identified several small molecules that show promise as selective agents, binding to Leishmania tubulin preferentially over mammalian tubulin, and preventing parasite multiplication inside human macrophage cells. In addition, we have produced purified tubulin from cultured Leishmania and evaluated it for use in drug screening.

这个项目的重点是小分子改变细胞细胞骨架的能力，特别是微管(MT)。MT是最刚性的细胞骨架聚合物，是建立和维持非球形细胞形态的核心。此外，MT具有固有的极性，其胞质阵列为细胞内的定向运动提供了底物。因此，改变MT完整性和/或动力学的小分子可以扰乱细胞内的运输并改变细胞质的物理性质。这个项目包括两个部分。一是详细了解MT活性小分子与MT或MT亚单位蛋白微管蛋白的相互作用，重点是海洋来源的抗有丝分裂肽天然产物，以及通过知识定向合成产生的化合物。第二部分旨在利用MT-小分子相互作用的知识来确定寄生虫病的药物治疗方法。 在第一部分中，我们重点介绍了抗有丝分裂多肽，因为它们是已知的最有效的抗MT药物之一，它们已经被合成和类似物可用，而且因为它们诱导MT亚单位呈现不寻常的和特征的环状。我们正在通过分析超速离心法、低温电子显微镜、荧光相关光谱和蛋白酶图谱来研究这些环聚合物的结构和动力学性质。我们的研究揭示了这些环的高度稳定性和一致性，这使得我们试图对这些聚合物进行结晶，以实现其结构的原子分辨。我们还在研究合成的沙利度胺类似物和沙利度胺A对微管聚合的影响。我们发现了一种稳定微管的沙利度胺。 在第二部分，我们正在寻找不能很好地与哺乳动物微管蛋白结合，但确实能与寄生虫微管蛋白结合的小分子。微管蛋白分子在进化上是相当保守的，但确实存在差异，并且已知有几个分子可以靶向例如酵母而不是哺乳动物的微管蛋白，反之亦然。我们正在寻找针对利什曼原虫的分子，利什曼原虫是人类一组重要疾病的感染原因。我们已经确定了几个小分子作为选择性试剂，优先与利什曼原虫微管蛋白结合，而不是哺乳动物微管蛋白，并防止寄生虫在人巨噬细胞内繁殖。此外，我们还从培养的利什曼原虫中制备了纯化的微管蛋白，并对其用于药物筛选进行了评估。