Microtubule regulation by small molecules.

小分子的微管调节。

• 批准号: 8736876
• 负责人: Dan L Sackett
• 金额: $ 35.47万
• 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/8736876
• 关键词: Aneuploidy; Biological Factors; Cells; Cellular Morphology; Cellular biology; Chromosomes; Colchicine; Combretastatin; Cytoplasm; Cytoskeleton; Disease; Error Sources; Failure; Fluorescence; Functional disorder; Goals; Human; In Vitro; Knowledge; Lead; Life; Malignant Neoplasms; Methods; Microtubules; Mitosis; Mitotic; Mitotic spindle; Movement; Optical Methods; Optics; Paclitaxel; Parasites; Pharmaceutical Preparations; Pharmacotherapy; Polymers; Population; Process; Property; Protein Subunits; Proteins; Regulation; Role; Source; System; Tubulin; Vincristine; base; cell behavior; daughter cell; density; novel; physical property; polymerization; pre-clinical research; screening; small molecule; trafficking

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. The in vitro studies with purified tubulin are a critical way in which compounds are evaluated, and indeed this is often used as a screening method to discover new active compounds as well as to quantitatively evaluate established ones. To further this, we have reviewed approaches to quantitating MT assembly by optical methods, with the goal of maximizing the information that may be obtained and minimizing the errors that the system can produce. We used both optical density-based and fluorescence-based approaches, and evaluated sources of error as well as underappreciated sources of extra information on the polymerization process. While the roles of MT extend throughout the life of the cell, it is nonetheless true that mitosis is a central role for MTs. Dysfunction of the MT-based mitotic spindle leads cells to abnormal separation of chromosomes, resulting in abnormal chromosome numbers in daughter cells. This aneuploidy is commonly found in human cancers. Cells have multiple mechanisms to avoid aneuploidy, focusing on maintaining proper functioning of the mitotic spindle. Some of these mechanisms involve proteins that are only expressed and active during mitosis. One of these proteins is known as CKAP2, whose expression is altered in some cancers. We studied this protein to understand how it helps to stabilize mitotic spindle function, and how alterations in its expression in cells can lead to aneuploidy in the daughter cells. We discovered that CKAP2 regulates the functioning of the two poles of the MT arrays in the spindle. Abnormal amounts of CKAP2 reduce the ability of cells to form two and only two spindle poles. When there are multiple poles, it is impossible to divide the chromosomes evenly. Thus failure, or deficit, of CKAP2 function leads to attachment failure in some fraction of the chromosomes and to accumulation of aneuploidy in the cell population.

天然产物历来是大多数微管（MT）靶向小分子的来源，其特性使它们成为有用的药物。我们在这项研究中使用的大部分化合物都是如此，但不是全部。这些包括临床建立的mt活性药物秋水仙碱、康布他汀、长春新碱、紫杉醇等。几乎所有这些药物都是首先在临床前研究中开发出来的，包括化合物对微管蛋白聚合的影响的体外研究，以及这些化合物对细胞行为的影响，特别是通过对构成有丝分裂纺锤体的MT阵列的影响来检测化合物破坏有丝分裂的能力。纯化微管蛋白的体外研究是评价化合物的一种重要方法，它经常被用作发现新的活性化合物和定量评价已建立的活性化合物的筛选方法。为了进一步做到这一点，我们回顾了通过光学方法定量MT装配的方法，目标是最大化可能获得的信息并最小化系统可能产生的误差。我们使用了基于光密度和基于荧光的方法，并评估了误差来源以及聚合过程中未被充分认识的额外信息来源。