Anti-Cancer Drugs that Target Diaphanous-related Formins

描述(由申请人提供)：基本原理。以药物紫杉醇为代表的紫杉烷及其衍生物代表了一种最先进的抗癌疗法。它们与微管蛋白结合并破坏微管细胞骨架的动态重塑，导致细胞运动和增殖受到抑制；细胞通过启动细胞程序性死亡来做出反应。然而，这些药物在临床上受到副作用和肿瘤细胞耐药性的限制。因此，有必要进一步鉴定破坏细胞骨架功能的抗癌化合物。 指导性假设。Rho小GTP酶在生长因子反应和细胞分裂过程中是细胞形状的关键调节因子；它们的活性也是致癌转化所必需的。透明相关福尔曼(Diaphanous Related Forins，DRF)是细胞骨架调节蛋白家族中的一员，在与DRF GTPase结合域(GBD)结合后被Rho蛋白激活。这种相互作用破坏了GBD和Dia-自动调节结构域(DAD)之间的分子内联系。将DAD衍生的多肽引入细胞足以解除对DRF蛋白的调控；DAD模拟Rho结合，从而破坏分子内的自我抑制。其结果是细胞骨架的不适当稳定。与紫杉烷一样，DAD多肽也会导致细胞最终发生凋亡。我们推测，DAD多肽代表了一类新的抗癌药物，它针对癌细胞中的DRf并解除对其的调控。拟议的实验将检验我们的指导性假设。 明确的目标。(1)鉴定DAD多肽靶向的透明相关福尔明(S) 目的：(1)在对紫杉醇敏感和耐药的乳腺和前列腺癌细胞系中(从DRf3蛋白衍生的DAD多肽开始)；(2)确定与其靶标GTPase结合域结合的DAD多肽的结构；以及(3)开发一种新的筛选方法，用于识别破坏DRF自动调节的新分子，该方法适用于药物文库的高通量筛选(HTS)。如果我们的假设是正确的，那么DAD多肽将足以杀死甚至对紫杉醇耐药的癌细胞。不能与细胞DRF的GBD相互作用的DAD变体将是不活跃的，被破坏的位点将对应于定义与GBD结合的DAD结构的分子决定因素。最后，DAD多肽将破坏GBD-DAD在体外的相互作用；然后将使用DAD多肽及其衍生物优化分析，以筛选策略，使我们能够识别更多具有DRF去调节活性的分子。

DESCRIPTION (provided by applicant): Rationale. Taxanes, epitomized by the drug Taxol, and their derivatives represent a state-of-the-art anti-cancer therapy. They bind to tubulin and disrupt the dynamic remodeling of the microtubule cytoskeleton, leading to inhibition of cell motility and proliferation; cells respond by initiating programmed cell death. These drugs are limited clinically, however, by their side effects and tumor cell resistance. Thus, there is a need to further identify anti-cancer compounds that disrupt cytoskeletal function. Guiding Hypothesis. Rho small GTPases act as critical regulators of cellular shape during growth factor responses and cell division; their activity is also required for oncogenic transformation. Diaphanous related formins (Drf), members of the formin family of cytoskeletal regulators, are activated by Rho proteins upon binding to the Drf GTPase binding domains (GBDs). The interaction disrupts an intramolecular association between the GBD and the Dia-autoregulatory domain (DAD). Introduction of a DAD-derived peptide into cells is sufficient to deregulate Drf proteins; DAD mimics Rho binding, thereby disrupting intramolecular autoinhibition. The result is the inappropriate stabilization of the cytoskeleton. Like taxanes, DAD peptide causes cells to eventually undergo apoptosis. We hypothesize that DAD peptide represents a novel class of anti-cancer drug that targets and deregulates Drfs in cancer cells. The proposed experiments will test our guiding hypothesis. Specific Aims. (1) Identify the Diaphanous-related formin(s) that is/are targeted by DAD peptide (beginning with DAD peptide derived from the Drf3 protein) in Taxol-sensitive and -resistant breast and prostate cancer cell lines, (2) determine the structure of DAD peptide bound to its target, the GTPase binding domain, and (3) develop a screening method for identifying novel molecules that disrupt Drf autoregulation that is amenable to high-throughput screening (HTS) of drug libraries. If our hypothesis is correct, then DAD peptide will be sufficient to kill even Taxol-resistant cancer cells. DAD variants, that fail to interact with the GBD of cellular Drfs, will be inactive, and the disrupted sites will correspond to molecular determinants that define the structure of DAD bound to the GBD. Finally, DAD peptides will disrupt the GBD-DAD interaction in vitro; the assays will then be optimized, using DAD peptides and their derivatives, for screening strategies that will allow us to identify additional molecules that harbor Drf-deregulating activity.