Microtubule regulation by small molecules.

天然产物历来是大多数微管（MT）靶向小分子的来源，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MT稳定化合物peloruside，是天然产物，临床上建立的MT稳定剂紫杉醇。其他的，如MT-稳定性埃博霉素的类似物，是基于已知天然化合物的半合成衍生物。其他的仍然是完全合成的化合物。我们研究了微管蛋白上两个新的抗MT药物结合位点，以及这些或更早已知的位点上的药物结合对MT性质和对细胞的影响的结果。新的结合位点是合成MT去稳定剂安磺灵和天然产物MT稳定剂Peloruside。对细胞的影响涉及这些药物以及更成熟的药物，特别是临床药物。 由于对植物微管蛋白的高选择性超过哺乳动物微管蛋白，稻磺灵和其它二硝基苯胺是有效的除草剂。我们已经表明，这些化合物，不稳定MT，也显示出选择性原生动物寄生虫微管蛋白相比，哺乳动物微管蛋白。我们继续我们的努力，了解这种选择性，通过映射的结合位点安磺灵微管蛋白使用的突变，赋予这种化合物的抗性寄生虫微管蛋白的详细分析。我们希望利用这些详细的知识来设计更好地结合寄生虫微管蛋白的化合物，从而提供临床上有用的抗寄生虫药物。 我们已经使用了类似的方法来定义的结合位点和模式的peloruside的作用。我们已经通过质谱研究和分子模拟表明，这种化合物与β微管蛋白上的一个位点结合，该位点与临床上重要的MT稳定药物紫杉醇的位点完全不同。选择和映射人类微管蛋白中赋予对peloruside耐药性的突变已经证实了我们的质谱研究，并允许更好地理解结合位点，占据如何改变MT稳定性，以及这与紫杉醇作用的不同。我们希望利用这些知识来了解Peloruside和紫杉醇的不同作用机制，并为临床上这两种药物的联合用药提供依据。 从结合位点定位和临床前研究中已经清楚，紫杉醇和Peloruside通过不同的机制稳定MT。两个结合位点的结构研究表明MT聚合物中纵向和横向稳定性的不同平衡，表明MT的机械性质可能与两种药物不同。未受干扰的MT是已知的最刚性的细胞内蛋白质聚合物，紫杉醇使其柔性增加10倍。我们正在测量的刚度后，结合紫杉醇或peloruside个别荧光MT，以联系在MT属性的差异结合位点结构的差异。这种理解可以为在临床前细胞模型中观察到的这些药物组合的协同效应提供解释。 在细胞内部，MT呈阵列状发生。单个MT的固有极性在整个阵列中共享，并且这种不对称性是几乎所有细胞内转运和信号传导的基础。我们以前的MT阵列极性的作用，在细胞内信号通过p53，我们已经扩展到证明MT的基础上的G-蛋白偶联受体从ER的细胞表面的定向运输。在更大的范围内，我们研究了MT在整体细胞极性和定向运动中的作用。细胞对许多环境信号的反应是通过向上或向下移动分子梯度或其他信号，如光和底物硬度。使用我们实验室开发的方法来研究durotaxis，细胞从较软到较硬表面的优先运动，我们将使用MT靶向药物来定义MT极性在这种定向细胞运动中的作用。 MT的作用在细胞的整个生命周期中延伸，不仅在有丝分裂中，而且在98+%的细胞周期中不是有丝分裂。这些重要的作用包括从上面建立细胞极性，支持细胞内运输和信号，并允许细胞运动的方向性。MT靶向药物不仅在有丝分裂细胞中，而且在所有细胞中都有活性，实际上，临床使用的抗MT药物的一些靶点是有丝分裂后细胞。我们认为，即使在直觉认为有丝分裂是靶点的临床环境中，例如在患者肿瘤中，数据表明MT靶向药物由于干扰非有丝分裂过程而有效，例如上述过程。我们计划联合收割机描述的实验方法，以获得一个更好的理解的非有丝分裂的过程中，有针对性的抗MT药物的作用，以提高这些药物的临床实用性。

Natural products have historically been the source of most of the microtubule (MT)-targeting 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 MT-stabilizing compound peloruside, are natural products, as is the clinically established MT-stabilizer taxol. Others, such as analogs of the MT-stabilizing epothilones, are semisynthetic derivatives based on known natural compounds. Others still are totally synthetic compounds. We have investigated two new binding sites on tubulin for anti-MT drugs, as well as the results of drug binding at these, or the longer-known sites, on the properties of MT and the effects on cells. The new binding sites are for the synthetic MT destabilizer, oryzalin, and the natural product MT stabilizer, peloruside. The effects on cells involve these drugs as well as more established drugs, especially clinical agents. Oryzalin and other dinitroanilines are effective herbicides due to a high selectivity for plant tubulin over mammalian tubulin. We have shown that these compounds, which destabilize MT, also show selectivity for protozoal parasite tubulin compared to mammalian tubulin. We have continued our effort to understand this selectivity by mapping the binding site for oryzalin on tubulin using detailed analysis of mutations in parasite tubulin that confer resistance to this compound. We hope to use this detailed knowledge to design compounds that bind better and with improved selectivity to parasite tubulin, thereby affording clinically useful antiparasite drugs. We have used a similar approach to define the binding site and mode of action of peloruside. We have already shown by mass spectrometric studies and molecular modeling that this compound binds to a site on beta tubulin quite distinct from that of taxol, a clinically important MT-stabilizing drug. Selecting and mapping mutations in human tubulin that confer resistance to peloruside have confirmed our mass spectrometry studies, and allowed an improved understanding of the binding site, how occupancy alters MT stability, and how this differs from taxol action. We hope to use this knowledge to understand the differing mechanisms of peloruside and taxol, and provide a basis for combination of these drugs clinically. It is already clear from the binding site mapping and from preclinical studies that taxol and peloruside stabilize MT by different mechanisms. Structural study of the two binding sites suggests a differing balance of longitudinal and lateral stabilization in the MT polymer, suggesting that the mechanical properties of the MT may differ with the two drugs. Unperturbed MT are the most rigid intracellular protein polymers known, and taxol increases their flexibility 10-fold. We are measuring the rigidity of individual fluorescent MT after binding of taxol or peloruside in order to relate differences in binding site structures to differences in MT properties. This understanding could provide an explanation for the synergistic effect observed for combinations of these drugs in preclinical cellular models. Inside the cell, MT occur in arrays. The intrinsic polarity of individual MT is shared throughout the arrays, and this asymmetry underlies nearly all intracellular transport and signaling. We previously showed the role of MT array polarity in intracellular signaling by p53, and we have extended this to demonstrate the MT basis of the directional transport of G-protein-coupled receptors from the ER to the cell surface. On a larger scale, we have investigated the role of MT in overall cell polarity and directed movement. Cells respond to many environmental signals by moving up or down gradients of molecules or other signals such as light and substrate rigidity. Using the methods developed in our lab to study durotaxis, the preferential movement of cells from softer to more rigid surfaces, we will use MT-targeting drugs to define the role of MT polarity in this directed cell movement. The roles of MT extend throughout the life of the cell, not only in mitosis, but also in the 98+% of the cell cycle that is not mitosis. These vital roles include those from above establishing cellular polarity, supporting intracellular transport and signaling, and allowing directionality in cell movements. MT-targeting drugs are active in all cells, not only in mitotic ones, and indeed some targets of clinical use of anti-MT drugs are post-mitotic cells. We have argued that even in clinical settings where intuition says that mitosis is the target, such as in patient tumors, data indicate that MT-targeting drugs are effective due to interference with non-mitotic processes, such as those mentioned above. We plan to combine the experimental approaches described to obtain a better understanding of the non-mitotic processes that are targeted by the action of anti-MT drugs in order to improve the clinical usefulness of these agents.