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

• 批准号: 7321819
• 负责人: NICHOLAS COWAN
• 金额: $ 31.51万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7321819
• 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; Compatible; Complex; Condition; 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; Hydrolysis; Inherited; Kinetics; Letters; Libraries; Malignant Neoplasms; Measures; Mediating; Methods; Microtubule Polymerization; Microtubules; Mitotic spindle; Molecular Chaperones; Monitor; Morphologic artifacts; Mutation; Numbers; Pathogenesis; Pathway interactions; Peptide Elongation Factor Tu; Pharmaceutical Preparations; Play; Polymers; Production; Proliferating; Proteins; Reaction; Ribosomes; Role; Screening procedure; Series; Small Interfering RNA; Structure; Temperature; Testing; Tubulin; Validation; base; beta Tubulin; cancer cell; cancer therapy; chaperonin CCT; cofactor; concept; cytosolic chaperonin; experience; high throughput screening; human EEF1A1 protein; human disease; inorganic phosphate; novel; polymerization; polypeptide; research study; scale up; size; tubulin-specific chaperone C

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(申请人提供)：微管是一种动态聚合物，在许多重要的细胞功能中发挥着重要作用。它们是由一个α和一个β-微管蛋白多肽组成的异二聚体组装而成的。微管作为有丝分裂纺锤体的重要组成部分参与细胞分裂，使这些结构成为癌症化疗的靶点：几种干扰正常微管动力学的药物目前正在临床使用，许多其他此类化合物目前正在进行临床试验。因此，微管被公认为是一个有效且非常成功的抗癌靶点。目前已知的所有干扰微管动力学的化合物都是通过与微管蛋白结合来实现的，但没有一种化合物干扰导致微管蛋白异源二聚体从头组装的途径。这一途径涉及新合成的微管蛋白多肽与一系列伴侣蛋白的相互作用，从胞浆伴侣蛋白CCT开始。CCT释放的准天然亚基与几个微管蛋白特异的伴侣蛋白(称为辅因子A-E)相互作用，导致辅因子结合的β-微管蛋白对GTP进行水解后新生成的异源二聚体的释放。辅因子C、D和E还作为微管蛋白的GTP酶激活蛋白(GAP)发挥作用；该反应不同于伴随微管聚合的GTP水解，因为它发生在微管蛋白浓度低得多的情况下。因为辅因子C、D和E是微管蛋白异源二聚体形成所必需的，所以它们是干扰高效折叠微管蛋白异源二聚体产生的独特和新的潜在靶点。使用系统siRNA敲除的实验和我们最近对涉及辅因子E的人类遗传病(HRD)的分析为这一方法提供了概念验证和进一步的功能验证。我们提出的实验旨在为寻找干扰新生微管蛋白异二聚体形成的化合物奠定基础。我们将1)开发应用于高通量分析的微管蛋白间隙分析；2)设计用于高通量分析的优化辅因子生产的方法；3)开发用于阐明微管蛋白间隙分析中抑制机制的方法，以便消除伪影并优先选择化合物进行进一步研究；以及4)进行中试高通量筛选，以便建立合适的条件，优化我们的分析，并定义阈值和命中值。