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

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α和β微管蛋白多肽组成的异源二聚体组装而成的。微管作为有丝分裂纺锤体的重要组成部分参与细胞分裂，这使得这些结构成为癌症化疗的有吸引力的靶点：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，微管作为一种经过验证且非常成功的抗癌靶点已经得到了很好的证实。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，但没有一种已知的化合物会干扰导致微管蛋白异源二聚体重新组装的途径。该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，首先是细胞质伴侣蛋白CCT。从CCT释放的准天然亚基与几个微管蛋白特异性伴侣（称为辅助因子a- e）相互作用，导致GTP被辅助因子结合的β -微管蛋白水解后释放新生成的异源二聚体。辅助因子C、D和E还可作为微管蛋白的GTPase激活蛋白（GAP）；该反应不同于伴随微管聚合的GTP水解，因为它发生在低得多的微管浓度下。由于辅因子C、D和E对微管蛋白异二聚体的形成至关重要，它们代表了干扰有效折叠微管蛋白异二聚体产生的独特而新颖的潜在靶点。使用系统siRNA敲除的实验和我们最近对涉及辅因子E的人类遗传疾病（HRD）的分析为该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)开发用于高通量格式的微管蛋白GAP测定；2)设计用于高通量分析的辅助因子生产优化方法；3)开发微管蛋白GAP测定中抑制机制的阐明方法，以消除伪影并优先考虑化合物进行进一步研究；4)执行试点高通量筛选，以建立适当的条件，优化我们的分析，并定义阈值和命中值。

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.