Microtubule regulation by small molecules.

小分子的微管调节。

• 批准号: 8553912
• 负责人: Dan L Sackett
• 金额: $ 36.3万
• 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/8553912
• 关键词: Adverse effects; Binding; Binding Sites; Biological Factors; Cell Cycle; Cell Polarity; Cell model; Cells; Cellular Morphology; Clinical; Clinical Trials; Clinical effectiveness; Cytoplasm; Cytoskeleton; Data; Disease; Drug Combinations; Drug Delivery Systems; Effectiveness; Epothilones; Equilibrium; Human; Individual; Intracellular Transport; Intuition; Knowledge; Laboratories; Lateral; Life; Location; Maps; Mass Spectrum Analysis; Measures; Mechanics; Microtubules; Mitosis; Mitosis Inhibition; Mitotic; Molecular; Molecular Models; Movement; Mutation; Neurologic; Paclitaxel; Parasites; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacotherapy; Polymers; Process; Property; Protein Subunits; Proteins; Regulation; Resistance; Resolution; Role; Signal Transduction; Site; Source; Structure; Study models; Tubulin; analog; base; beta Tubulin; cell motility; cytotoxic; flexibility; improved; molecular modeling; novel; oryzalin; physical property; pre-clinical; preclinical study; small molecule; trafficking; trial comparing; tumor

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.

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抗MT药物的新结合位点，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。 为了了解新的微管稳定剂Peloruside的活性，有必要了解结合位点的细节。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。我们现在已经确定了这个结合位点在高分辨率映射的几个独立的突变，赋予耐药性的细胞毒性作用的peloruside的位置。这些结果证实了我们以前使用质谱法获得的低分辨率结果，但提供了重要的新细节。结合位点的详细知识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。这一知识也强烈地表明了一个不同于紫杉醇的详细的分子机制，但它稳定了微管结构，就像紫杉醇一样。由于这两种机制在细节上是不同的，这可能会告知这些药物的合理组合，以最大限度地发挥两者的协同作用，这已经被证明。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。 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。 试图提高抗微管药物的临床有效性是基于这样的假设，即这些药物通过抑制有丝分裂起作用，而它们的神经学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，这些化合物针对仅在有丝分裂期间表达的蛋白质。这些有丝分裂特异性药物是以巨大的代价开发的，并投入临床试验，在临床试验中，它们对患者肿瘤的活性非常小。我们结合了所有临床试验的结果并进行比较，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，这些药物在临床上是有效的，因此，抗微管药物的有效性是由于对一些非有丝分裂过程的活性是微管依赖性的。我们正在积极寻找这些过程可能是什么。