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

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。他们 由一个α-和一个(3-微管蛋白多肽)组成的杂二聚体组装而成。这个 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诱人靶点：几种干扰药物 正常的微管动力学目前在临床上使用，许多其他类似的化合物是 目前正在进行临床试验。因此，微管作为一种有效的和高度有效的 成功的抗癌目标。所有目前已知的干扰微管的化合物 动力学通过与微管蛋白结合来实现这一点，但目前还没有人知道干扰导致 微管蛋白异源二聚体的从头组装。这条途径涉及到新生的 合成含有一系列伴侣蛋白的微管蛋白多肽，从胞浆开始 监护人CCT。CCT释放的准天然亚基与几种微管蛋白特异性相互作用 伴侣(称为辅因子A-E)在导致释放新生成的 辅因子结合的GTP水解后的杂二聚体(3-微管蛋白。辅因子C、D和E也 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，因为它发生在低得多的微管蛋白 集中精神。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不可少的，所以它们 代表独特的和新的潜在目标，以干扰生产性折叠的产生 微管蛋白杂二聚体。使用系统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)提供了概念证明，并进一步 此方法的功能验证。我们提出的这些实验旨在为 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)开发应用于高通量形式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 在微管蛋白间隙分析中阐明抑制机制的方法 消除伪影并为进一步研究确定化合物的优先顺序；以及4)进行高吞吐量试验 筛选，以建立适当的条件，优化我们的分析，并定义阈值和 点击率。

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.