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: 7615339
• 负责人: NICHOLAS COWAN
• 金额: $ 9.48万
• 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/7615339
• 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; 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

Microtubules are dynamic polymers that play an important role in many vital cellular functions. They are assembled from heterodimers consisting of one a- and one (3-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 (3-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.

微管是动态聚合物，在许多重要的细胞功能中发挥着重要作用。他们 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有吸引力的靶点：几种药物， 正常的微管动力学目前在临床上使用，并且许多其它这样的化合物， 正在进行临床试验因此，微管被公认为是一种有效的、高度 成功的抗癌靶点。目前已知的所有干扰微管的化合物 动力学通过与微管蛋白结合来这样做，但没有已知的干扰导致 微管蛋白异源二聚体的从头组装。这条途径涉及新的 合成微管蛋白多肽与一系列伴侣蛋白，开始与胞质 伴侣蛋白CCT。从CCT释放的准天然亚基与几种微管蛋白特异性 分子伴侣（称为辅因子A-E）的反应，导致释放新产生的 通过辅因子结合的β-微管蛋白水解GTP后的异二聚体。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GTP 伴随微管聚合的水解，发生在微管蛋白含量低得多的地方 浓度.因为辅因子C、D和E对于微管蛋白异源二聚体的形成是必需的，所以它们 代表了独特的和新的潜在的目标，干扰生产性折叠的产生， 微管蛋白异源二聚体。使用系统性siRNA敲除的实验和我们最近的分析 涉及辅因子E的人类遗传性疾病（HRD）的研究提供了概念验证， 对这种方法进行功能验证。我们提出的实验旨在奠定 为寻找干扰从头微管蛋白异二聚体形成的化合物奠定了基础。 我们将1）开发微管蛋白GAP检测，以应用于高通量格式; 2）设计 用于高通量测定的辅因子生产的优化方法; 3）开发 用于阐明微管蛋白GAP测定中抑制机制的方法， 消除伪影并对化合物进行优先级排序以供进一步研究;以及4）进行中试高通量 筛选，以建立适当的条件，优化我们的测定，并定义阈值， 点击率了