Microtubule regulation by isotype expression and by small molecules.

通过同种型表达和小分子调节微管。

• 批准号: 10011339
• 负责人: Danny Sackett
• 金额: $ 52.32万
• 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/10011339
• 关键词: Address; Affinity; Anti-Inflammatory Agents; Antibodies; Binding; Biological; Biological Assay; Biology; Cell Culture Techniques; Cells; Cellular Morphology; Cellular biology; Chemistry; Clinical; Colchicine; Combretastatin; Cytoplasm; Cytoskeleton; Development; Disease; Dissociation; Drug Targeting; Elements; Equilibrium; Fluorescence; Fluorescence Polarization; Genes; Human; In Vitro; Knowledge; Laboratories; Light; Mediating; Methods; Microscope; Microscopy; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular Conformation; Movement; Muscle; Muscle Fibers; Muscle function; Muscular Dystrophies; Names; Natural Product Drug; Natural Products; Neoplasm Metastasis; Organism; Paclitaxel; Parasites; Patients; Peptides; Pharmaceutical Preparations; Pharmacotherapy; Photobleaching; Physiology; Polymers; Process; Property; Protein Subunits; Proteins; Reaction; Regulation; Reporting; Research; Resolution; Role; Skeletal Muscle; Source; Specificity; Spongistatin; Structure; Surface; Tail; Tissues; Tubulin; Vincristine; Work; analog; analytical ultracentrifugation; arm; beta Tubulin; cell behavior; chemotherapy; cytotoxic; dimer; fluorophore; in vitro Assay; light microscopy; mdx mouse; monomer; mouse model; novel therapeutics; overexpression; physical property; polymerization; pre-clinical research; protein protein interaction; side effect; small molecule; spongistatin 1; structural biology; tool; 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. Often study of the regulation of microtubules and microtubule arrays and the effects of small molecules on those arrays depends on tools of microscopy, both for in vitro assays with purified proteins and especially for assays that address microtubule arrays inside cells. For this reason we have collaborated with labs that focus on fluorescence methods and advanced microscope applications. One such application is super-resolution microscopy, which is actually a class of methods for achieving enhanced spatial resolution using light microscopy, that until a few years ago was thought to be not possible. One such method is STED microscopy, Stimulated Emission Depletion Microscopy. This method can require high power of light, resulting in photobleaching of the fluorophores. To minimize this and optimize resolution we have developed a simple method to check power optimization in STED microscopy that can be readily accomplished at the microscopy bench. Our work with small molecules that target tubulin / microtubules has focused on using our knowledge of tubulin structure to identify new molecules that could become useful drugs, and also to take potent existing drugs and modify them to extend their usefulness or better target them in the body in order to minimize side effects on bystander tissues. We have previously developed chemistry to attach linker arms to some known drugs and this year extend that work by demonstrating how to attach linker arms to the potent natural product drug, spongistatin 1, without losing potency. This accomplishment will allow this drug, one of the most potent cytotoxic compounds known, to be attached to antibodies or other affinity-conferring entities that will target the potent drug to the intended target, such as a tumor or metastases. While spongistatin destabilizes microtubules, other drugs stabilize them (such as paclitaxel, commercial name Taxol), and we have also looked for new varients of these. Two of the most promising of these are the structural analogs zampanolide and dactylolide. These bind to the same place on tubulin; zampanolide binds covalently and dactylolide binds non-covalently. We have prepared and examined conformational analogs of these compounds to better understand their stabilizing potency and the significance of the covalent reaction. We have also pursued the basic structural biology of the tubulin dimer. This knowledge is required to understand how small molecules regulate tubulin and how tubulin binding domains regulate interaction with other proteins. The most basic step in tubulin biology is assembly of the heterodimer, and we previously described studies that used analytical ultracentrifugation as well as fluorescence polarization to show that dimer formation is a reversible, mass-action-driven process. We also examined the role of the tubulin carboxyl terminal tail peptides which mediate interaction of many proteins with tubulin and/or microtubules. We have extended those studies, showing that dimer dissociation and monomer exchange are controlled by equilibrium constants that differ by orders of magnitude between tubulins from different biological sources. These include tubulins from human cells compared to tubulins from different organisms, including protozoan parasites, and also between tubules from different tissues, which represent different expression levels of tubule isotypes. This work also demonstrated the importance of non-binding surfaces in protein-protein interactions. Beyond the formation of heterodimers is the regulation of expression of which isotypes of alpha and of beta tubulin contributes to the heterodimers. The tissue specificity of expression strongly indicates that isotypes contribute function that is specific to particular cell- or tissue types but the evidence for this is not abundant. We report the effect of altered expression of beta tubulin isotypes in skeletal muscle on muscle function in Duschenne Muscular Dystrophy (DMD) and the commonly used mdx mouse model of dystrophic muscle. Microtubule arrays are known to be disrupted in DMD and mdx skeletal muscle fibers, and here we show that over expression of the tubulin beta6 isotype (gene = tubb6) results in the microtubule disorganization seen in dystrophic muscle. Overexpression of the beta5 isotype has no such effect.

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。事实上，在实验室中，在快速生长的细胞培养物中引起有丝分裂停止的能力通常被认为是这些药物的主要机制的一种测定方法。