Microtubule regulation by small molecules.

天然产物历来是大多数抗有丝分裂小分子的来源，这些小分子的特性使它们成为有用的药物。 这仍然适用于本研究中的大多数化合物，但不是所有化合物。一些，如新的微管稳定化合物peloruside，是天然产品。其它的，例如微管稳定化合物埃坡霉素的类似物，和微管去稳定肽微管溶素的类似物，是通过基于已知天然化合物的结构的合成得到的。我们还从合成分子库的高通量筛选中发现了抗有丝分裂化合物，这些合成分子的结构与特定的天然化合物无关。所有这些化合物通过与微管蛋白（微管的亚基蛋白）结合发挥作用。 我们还证明，像亚硝基脲这样的化合物，不与微管蛋白相互作用，可以通过与本身与微管蛋白结合的小蛋白相互作用来影响微管特性。 我们已经确定了一些新的修饰肽来自天然肽微管去稳定剂，微管溶素。这些新的肽显示出一系列的抗微管活性的测定纯化的蛋白质以及在细胞中。我们还设计、合成并测试了一种基于天然化合物埃坡霉素的新型有效微管稳定剂。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。通过氢氘交换质谱法研究其与微管蛋白的结合，对正常微管组装的机制有了新的认识。我们还开发了新的质谱数据分析方法，用于研究组织样品中的微管蛋白。如我们所示，小分子与微管蛋白的结合可以通过荧光相关光谱有效地跟踪。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。 除了研究可能对人类微管蛋白具有有用活性的小分子外，我们还试图鉴定与哺乳动物微管蛋白结合不好但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。

Natural products have historically been the source of most of the anti-mitotic small molecules whose properties have allowed them to become useful drugs. That remains true of most but not all of the compounds in this study. Some, such as the new microtubule-stabilizing compound peloruside, are natural products. Others, such as analogs of the microtubule-stabilizing compound epothilone, and analogs of the microtubule-destabilizing peptide tubulysin, are derived by synthesis based on the structure of known natural compounds. We have also discovered anti-mitotic compounds from a high-throughput screen of libraries of synthetic molecules whose structures are unrelated to particular natural compounds. All of these compounds exert their actions through binding to tubulin, the subunit protein of microtubules. We have also demonstrated that compounds like nitrosoureas, that do not interact with tubulin, can affect microtubule properties by interacting with small proteins that themselves bind to tubulin. We have identified a number of new modified peptides derived from the natural peptide microtubule destabilizer, tubulysin. These new peptides show a range of antimicrotubule activity in assays with purified proteins as well as in cells. We have also designed, synthesized, and tested a new, potent microtubule stabilizer based on the natural compound, epothilone. In addition we have examined a new "second generation" of microtubule stabilizers, focusing on the natural compound peloruside. Study of its binding to tubulin by hydrogen-deuterium exchange mass spectrometric methods has led to new insights into the mechansims of normal microtubule assembly. We also have developed new methods of analysis of mass spectrometric data for studying tubulin from tissues samples. Binding of small molecules to tubulin can be usefully followed by fluorescence correlation spectroscopy, as we have shown. 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. In addition to studying small molecules that may have useful activity against human tubulin, 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.