Microtubule regulation by small molecules.

我们一直专注于抗有丝分裂肽，通常是海洋来源，因为这些是已知的最有效的抗MT剂，他们已经合成和类似物是可用的，因为他们诱导MT亚基采取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。我们也在研究沙利度胺和考布他汀A的合成类似物对微管聚合的影响。我们还证明了脱乙酰酶的翻译后修饰的作用，以及蛋白酶体抑制剂对微管稳定性的影响。 我们已经鉴定了一些新的修饰肽衍生自天然肽微管溶素。这些新的肽显示出一系列的抗微管活性的测定与纯化的蛋白质，以及在细胞中。此外，我们还研究了一种新的“第二代”微管稳定剂，重点是天然化合物peloruside。我们还发现，一种古老的化疗药物，亚硝基脲，可能通过改变蛋白质stathmin的活性间接影响微管稳定性。这导致恶性胶质瘤细胞的迁移和侵袭减少。 我们正在寻求识别不能很好地与哺乳动物微管蛋白结合但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。

We have focused on antimitotic peptides, often of marine origin, 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 also demonstrated the role of posttranslational modifications by deacetylases, and the effects of proteasome inhibitors on microtubule stability. We have identified a number of new modified peptides derived from the natural peptide tubulysin. Thes new peptides show a range of antimicrotubule activity in assays with purified proteins as well as in cells. In addition we have examined a new "second generation" of microtubule stabilizers, focusing on the natural compound peloruside. We have also shown that an old chemotherapy agent, a nitrosourea, may affect microtubule stability indirectly by altering the activity of the protein stathmin. This results in reduced migration and invasion by malignant glioma cells. 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.