Microtubule regulation by small molecules.

小分子的微管调节。

• 批准号: 9150114
• 负责人: Dan L Sackett
• 金额: $ 26.94万
• 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/9150114
• 关键词: Biological Assay; Biological Factors; Cell Culture Techniques; Cell Cycle; Cell Nucleus; Cell Respiration; Cells; Cellular Morphology; Cellular biology; Chemosensitization; Colchicine; Combretastatin; Cytoplasm; Cytoskeleton; DNA Damage; DNA Repair; Development; Disease; Drug Combinations; Drug Targeting; Enzymes; Event; Human; Image; In Vitro; Intracellular Transport; Knowledge; Laboratories; Laboratory culture; Malignant Neoplasms; Metabolism; Microtubules; Mitochondria; Mitosis; Mitotic; Mitotic spindle; Movement; Nature; Paclitaxel; Parasites; Patients; Pharmaceutical Preparations; Pharmacotherapy; Polymers; Process; Production; Property; Protein Subunits; Proteins; Reactive Oxygen Species; Regulation; Reporter; Reporting; Research; Respiratory Burst; Role; Schedule; Source; Tubulin; Vincristine; base; cell behavior; chemotherapy; drug mechanism; insight; macromolecule; novel; novel therapeutics; physical property; polymerization; pre-clinical research; research study; response; small molecule; trafficking; 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 that we have used in this study. These include the clinically established MT-active drugs colchicine, combretastatin, vincristine, taxol, and others. Almost all such agents were developed first in pre-clinical research that included in vitro studies of the effect of the compounds on polymerization of tubulin to microtubules as well as the effect of such compounds on cell behavior, especially examining the ability of the compounds to disrupt mitosis through effects on the MT arrays that comprise the mitotic spindle. Indeed the ability to cause mitotic arrest in rapidly growing cell cultures in the laboratory is often considered to be an assay of the principal mechanism of these drugs. Nonetheless we have argued that mitosis is not the central target of these drugs in patient tumors. Human tumors grow very slowly compared to laboratory cultures and mitosis is rare, and therefore is not an abundant target. Microtubules are abundant in non-mitotic as well as mitotic cells, however, and hence we have explored the nature of the microtubule functions that these drugs do target. One of those is intracellular transport, which occurs constantly, throughout the cell cycle, and which is required for many central parts of cell metabolism. This traffic is directional and requires the array of organized microtubules to coordinate the process. One aspect of this is the requirement of directional transport to move enzymes from the cytoplasm to the nucleus in response to DNA damage. This occurs on microtubules, and is disrupted by anti-microtubule drugs. We discovered that this interference with DNA repair processes underlies the well-verified potentiation by microtubule-targeting drugs of the anti-cancer activity of DNA-damaging chemotherapy. This insight may allow better scheduling of therapy and rational development of new therapeutic drug combinations. Additionally we have found that tubulin and microtubules have important roles in the regulation of cell oxidative metabolism through interaction with mitochondria. It has been often reported that microtubule-targeting drugs cause release of bursts of reactive oxygen species. These oxidative bursts result in production of carbonyl residues on cellular proteins. In order to study thus better, we have developed a new fluorescent reporter molecule which reacts covalently with carbonyl residues in cellular macromolecules. This allows the quantitation of oxidative events, the imaging of the distribution of these events in the cell, and potentially the isolation of the oxidized macromolecules (through pull-down experiments).

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。对于我们在这项研究中使用的大多数但不是所有的化合物来说，这一点仍然是正确的。这些药物包括临床上已确定的MT活性药物秋水仙碱、复方丹参素、长春新碱、紫杉醇等。几乎所有此类药物都是在临床前研究中首先开发出来的，包括体外研究化合物对微管蛋白聚合的影响以及此类化合物对细胞行为的影响，特别是检查化合物通过影响组成有丝分裂纺锤体的MT阵列来破坏有丝分裂的能力。事实上，在实验室中引起快速生长的细胞培养中有丝分裂停止的能力通常被认为是对这些药物的主要机制的分析。 尽管如此，我们认为有丝分裂不是这些药物在患者肿瘤中的中心靶点。与实验室培养相比，人类肿瘤生长非常缓慢，有丝分裂很少见，因此不是一个丰富的靶点。然而，微管在非有丝分裂细胞和有丝分裂细胞中都很丰富，因此我们探索了这些药物确实针对的微管功能的性质。其中之一是细胞内转运，它在整个细胞周期中不断发生，是细胞代谢的许多中心部分所必需的。这种流量是方向性的，需要有组织的微管阵列来协调这一过程。其中一个方面是需要定向运输，将酶从细胞质转移到细胞核，以应对DNA损伤。这发生在微管上，并被抗微管药物破坏。我们发现，这种对DNA修复过程的干扰是微管靶向药物增强DNA损伤化疗的抗癌活性的基础。这种洞察力可能会更好地安排治疗和合理开发新的治疗药物组合。 此外，我们还发现微管蛋白和微管通过与线粒体的相互作用在细胞氧化代谢的调节中发挥重要作用。据报道，微管靶向药物会导致活性氧的释放。这些氧化爆发导致细胞蛋白质上产生羰基残基。为了更好地进行研究，我们开发了一种新的荧光报告分子，它可以与细胞大分子中的羰基残基发生共价反应。这使得可以量化氧化事件，成像这些事件在细胞中的分布，并潜在地分离被氧化的大分子(通过下拉实验)。