Microtubule regulation by small molecules.

历史上，天然产品一直是大多数微管(MT)的来源-以小分子为靶标，这些小分子的性质使它们能够成为有用的药物。对于这项研究中的大多数但不是所有的化合物来说，这仍然是正确的。有些是天然产物，如新的MT稳定剂Peloruside，以及临床上已建立的MT稳定剂紫杉醇。其他的类似物，如稳定MT的Epothilone，是基于已知的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。我们已经研究了抗MT药物在微管蛋白上的新结合部位，以及药物在这些或较长已知部位结合的结果，对MT的性质和对细胞的影响。新的结合部位是合成的MT稳定剂oryzalin和天然产物MT稳定剂Peloruside。对细胞的影响涉及到这些药物以及更成熟的药物，特别是临床药物。 为了了解新型微管稳定剂Peloruside的活性，有必要了解结合部位的细节。我们已经通过质谱学研究和分子模拟表明，这种化合物结合在β-微管蛋白上的位置与紫杉醇非常不同，紫杉醇是一种临床上重要的MT稳定药物。选择和定位人类微管蛋白中对绿豆苷具有耐药性的突变，证实了我们的质谱学研究，并使我们能够更好地理解结合部位，占有率如何改变MT的稳定性，以及这与紫杉醇的作用有何不同。我们现在已经通过定位几个独立的突变的位置以高分辨率定义了这个结合位点，这些突变赋予了对Peloruside的细胞毒作用的抗性。这些结果证实了我们之前使用质谱学获得的较低分辨率的结果，但提供了重要的新细节。对结合位点的详细了解加强了该结合位点与紫杉醇结合位点的独立性，尽管它们的作用相似。这一知识也有力地表明了一种与紫杉醇不同的详细分子机制，但它像紫杉醇一样稳定微管结构。由于这两种机制在细节上不同，这可能会使这两种药物的合理组合，以最大限度地发挥两种药物的协同作用，这一点已经得到证明。我们希望利用这些知识来了解骨肉苷和紫杉醇的不同作用机制，并为这两种药物的临床联合应用提供依据。 结合部位图和临床前研究已经清楚地表明，紫杉醇和紫杉醇通过不同的机制稳定MT。对这两个结合部位的结构研究表明，MT聚合物中纵向和横向稳定性的平衡不同，这表明MT的机械性能可能因这两种药物而不同。无拘无束的MT是已知的最坚硬的细胞内蛋白质聚合物，紫杉醇使它们的灵活性增加了10倍。我们正在测量紫杉醇或紫杉苷结合后单个荧光MT的刚性，以便将结合部位结构的差异与MT性质的差异联系起来。这一理解可以为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。 MT的作用贯穿于细胞的整个生命周期，不仅在有丝分裂中，而且在98+%的非有丝分裂的细胞周期中也是如此。这些重要的作用包括从上面建立细胞极性，支持细胞内的运输和信号，并允许细胞运动的方向性。MT靶向药物在所有细胞中都是有效的，不仅在有丝分裂细胞中，而且确实有一些抗MT药物的临床使用靶点是有丝分裂后的细胞。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靶向药物由于干扰了非有丝分裂过程而有效，如上面提到的那些。我们计划结合所描述的实验方法，以更好地了解抗MT药物作用的非有丝分裂过程，以提高这些药物的临床实用性。 一项旨在提高抗微管药物临床疗效的尝试是基于这样一种假设，即这些药物通过抑制有丝分裂发挥作用，而它们的神经副作用与有丝分裂无关。基于这一观点，多个实验室和制药公司已经开发了许多化合物，这些化合物针对的是只在有丝分裂期间表达的蛋白质。这些针对有丝分裂的药物是以高昂的费用开发出来的，并投入了临床试验，在这些试验中，它们对患者肿瘤的活性很小。我们综合了所有临床试验的结果，并将它们进行比较，以了解它们失败的原因。我们的结论是，问题是有丝分裂在肿瘤中太罕见，这些药物无法在临床上有效，因此抗微管药物的有效性是由于对一些微管依赖的非有丝分裂过程(ES)的活性。我们正在积极寻找这些过程可能是什么。

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 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. In order to understand the activity of the new microtubule stabilizer, peloruside, it is necessary to know the details of the binding site. 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 have now defined this binding site at high resolution by mapping the location of several independent mutations that confer resistance to the cytotoxic action of peloruside. These results confirm the lower-resolution results we previously obtained using mass spectrometry, but provide significant new details. The detailed knowledge of the binding site reinforces the independence of this binding site from that for taxol, despite the similarity of their actions. This knowledge also strongly suggests a detailed molecular mechanism different from that of taxol, but which stabilizes the microtubule structure just as taxol does. Since these two mechanisms are different in detail, this may inform a rational combination of these drugs to maximize the synergistic action of the two together which has already been demonstrated. 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. 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. An attempt to improve on the clinical effectiveness of anti-microtubule drugs was based on the assumption that these agents act via inhibition of mitosis, while their neurological side effects were unrelated to mitosis. Based on this view, a number of compounds have been developed by multiple laboratories and pharmaceutical companies that target proteins that are only expressed during mitosis. These mitosis-specific drugs were developed, at great expense, and put in clinical trials where they have shown very little activity against patient tumors. We combined results of all of the clinical trials and compared them to understand why they failed. We concluded that the problem is that mitosis is too rare in tumors for these drugs to be clinically effective, and that therefore the effectiveness seen with anti-microtubule agents is due to activity against some non-mitotic process(es) that are microtubule-dependent. We are actively seeking what these process(es) might be.