Inhibition of the Tubulin Folding Pathway as a Novel Therapy for Cancer

抑制微管蛋白折叠途径作为癌症的新疗法

基本信息

批准号：
7608641
负责人：
NICHOLAS COWAN
金额：
$ 32.21万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7608641
关键词：
Affect Affinity Antineoplastic Agents Binding Biological Assay Cancerous Cell Cycle Arrest Cell Death Cell division Cell physiology Cells Chemotherapy-Oncologic Procedure Classification Clinical Clinical Trials Collaborations Color Complex Cultured Cells Development Disease Drug Delivery Systems EEF1A1 gene Environment Eukaryotic Cell GTPase-Activating Proteins Generations Genes Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Hereditary Disease Human Genetics Hydrolysis Inherited Kinetics Letters Libraries Malignant Neoplasms Measures Mediating Methods Microtubule Polymerization Microtubules Mitotic spindle Molecular Chaperones Monitor Morphologic artifacts Mutation Pathogenesis Pathway interactions Pharmaceutical Preparations Play Polymers Production Proliferating Proteins Reaction Ribosomes Role Screening procedure Series Small Interfering RNA Structure Temperature Tubulin Validation base beta Tubulin cancer cell cancer therapy chaperonin CCT cofactor cytosolic chaperonin efficacy testing experience high throughput screening human disease inorganic phosphate novel polymerization polypeptide research study scale up tubulin-specific chaperone C

项目摘要

DESCRIPTION (provided by applicant): Microtubules are dynamic polymers that play an important role in many vital cellular functions. They are assembled from heterodimers consisting of one alpha and one beta-tubulin polypeptide. The participation of microtubules in cell division as an essential component of the mitotic spindle has made these structures attractive targets for cancer chemotherapy: several drugs that interfere with normal microtubule dynamics are currently in clinical use and many other such compounds are currently undergoing clinical trials. Microtubules are thus well established as a validated and highly successful anti-cancer target. All of the currently known compounds that interfere with microtubule dynamics do so by binding to tubulin, but none are known that interfere with the pathway leading to the de novo assembly of the tubulin heterodimer. This pathway involves interaction of newly synthesized tubulin polypeptides with a series of chaperone proteins, beginning with the cytosolic chaperonin CCT. Quasi-native subunits released from CCT interact with several tubulin-specific chaperones (known as cofactors A-E) in a reaction that leads to release of newly generated heterodimers following GTP hydrolysis by cofactor-bound beta-tubulin. Cofactors C, D and E also function as a GTPase activating protein (GAP) for tubulin; this reaction is distinct from the GTP hydrolysis that accompanies microtubule polymerization in that it occurs at a much lower tubulin concentration. Because cofactors C, D and E are essential for tubulin heterodimer formation, they represent unique and novel potential targets for interfering with the generation of productively folded tubulin heterodimers. Experiments using systematic siRNA knockdown and our recent analysis of a human genetic disorder (HRD) involving cofactor E provide proof-of-concept and further functional validation for this approach. The experiments we propose are intended to lay the groundwork for a search for compounds that interfere with de novo tubulin heterodimer formation. We will 1) Develop the tubulin GAP assay for application to a high throughput format; 2) Devise methods for the optimization of cofactor production for use in high throughput assays; 3) Develop methods for the elucidation of the mechanism of inhibition in tubulin GAP assays in order to eliminate artifacts and prioritize compounds for further study; and 4) Perform pilot high throughput screens in order to establish appropriate conditions, optimize our assays, and define thresholds and hits.

描述（由申请人提供）：微管是动态聚合物，在许多重要的细胞功能中起重要作用。它们是由一个由一个α和一个β-微管蛋白多肽组成的异二聚体组装的。微管在细胞分裂中的参与是有丝分裂主轴的重要组成部分，使这些结构具有吸引力的癌症化学疗法靶标：目前正在临床使用中，几种干扰正常微管动力学的药物目前正在临床中，目前正在进行临床试验。因此，微管被很好地确定为经过验证且非常成功的抗癌目标。干扰微管动力学的所有当前已知化合物都通过与微管蛋白结合来做到这一点，但众所周知，没有人会干扰导致小管蛋白异二聚体从头组装的途径。该途径涉及新合成的小管蛋白多肽与一系列伴侣蛋白的相互作用，从胞质伴侣CCT开始。从CCT释放的准本地亚基与反应中的几个小管蛋白特异性伴侣（称为辅因子A-E）相互作用，该反应导致通过辅助剂结合β-β-β-β-β-β-β-β-结合β-结合β-蛋白的GTP水解后，新产生的异二聚体释放。辅助因子C，D和E还充当微管蛋白的GTPase激活蛋白（GAP）。该反应与伴随微管聚合的GTP水解不同，因为它发生在小管蛋白浓度下。由于辅因子C，D和E对于微管蛋白异二聚体的形成至关重要，因此它们代表了干扰产生有效折叠的微管蛋白异二聚体的独特而新颖的潜在靶标。使用系统的siRNA敲低的实验以及我们最近对涉及共同作用者E的人类遗传疾病（HRD）的分析提供了这种方法的概念证明和进一步的功能验证。我们提出的实验旨在为搜索干扰从头微管蛋白异二聚体形成的化合物奠定基础。我们将1）开发用于应用高吞吐量格式的小管蛋白间隙测定； 2）设计用于优化辅因子生产的方法，以用于高吞吐量测定； 3）开发方法来阐明微管蛋白间隙测定中抑制机制，以消除伪影并确定化合物以进行进一步研究； 4）执行试点高通量屏幕，以建立适当的条件，优化我们的测定并定义阈值和命中。