Microtubule regulation by small molecules

小分子的微管调节

• 批准号: 6828480
• 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/6828480
• 关键词: Leishmania; antimitotics; biological products; cell morphology; cryoelectron microscopy; cytoplasm; fluorescence spectrometry; intracellular parasitism; intracellular transport; microtubules; molecular dynamics; tubulin; ultracentrifugation

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. 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. 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.

该项目的重点是小分子改变细胞骨架，特别是微管（MT）的能力。MT是最刚性的细胞骨架聚合物，并且是建立和维持非球形细胞形态的核心。此外，MT具有内在极性，其胞质排列为定向胞内运动提供了基质。因此，改变MT完整性和/或动力学的小分子可以扰乱细胞内运输并改变细胞质的物理性质。这个项目有两个部分。一个是详细了解MT活性小分子与MT或MT亚基蛋白微管蛋白的相互作用，重点是来自海洋来源的抗有丝分裂肽天然产物。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疗法。 在第一部分中，我们集中于抗有丝分裂肽，因为这些是已知的最有效的抗MT剂之一，它们已经被合成并且类似物是可用的，并且因为它们诱导MT亚基呈现不寻常的和特征的环状。我们正在研究这些环聚合物的结构和动力学性质的分析超离心，冷冻电子显微镜，荧光相关光谱和蛋白酶映射。我们的研究揭示了这些环的高稳定性和均匀性，这使我们尝试结晶这些聚合物以实现其结构的原子分辨率。 在第二部分中，我们正在寻求确定小分子，不能很好地结合哺乳动物微管蛋白，但结合寄生虫微管蛋白。微管蛋白分子在进化上是相当保守的，但确实存在差异，并且已知几种分子可以靶向例如酵母而不是哺乳动物微管蛋白，反之亦然。我们正在寻找针对利什曼原虫的分子，利什曼原虫是一组重要的人类疾病的传染性病因。我们已经确定了几种小分子，显示出作为选择性试剂的前景，优先结合利什曼原虫微管蛋白超过哺乳动物微管蛋白，并防止人体巨噬细胞内的寄生虫繁殖。