Combinatorial Selection of Beta-Catenin/T Cell Factor Pathway Inhibitors

描述(申请人提供)：结直肠癌(CRC)是美国第二大癌症死亡原因，如果在早期发现，是可以预防和治愈的。尽管有几种化学预防药物可以在实验动物模型中预防结直肠癌，但还没有药物被批准用于人类。除了手术切除，这在晚期疾病中很少治愈，目前的结肠癌治疗依赖于传统的细胞毒剂，效果有限。我们的长期目标是开发修饰的寡核苷酸(ODN)，为癌症，特别是结直肠癌的治疗提供创新的治疗产品。癌基因β-连环蛋白和T细胞因子(TCF)之间的转录复合体的形成被认为是结直肠癌细胞发育的关键事件。调节这一途径和/或破坏β-连环蛋白和TCF相互作用的药物可能会抑制许多随后导致结直肠癌的靶基因的表达。本项目的目标是确定一个紧密结合的、高度特异的ODN，它将与人TCF4的β-连环蛋白结合域(TCF-CBD)结合，以阻断β-连环蛋白与TCF的结合和/或干扰β-连环蛋白与TCF的相互作用。基于结构的设计和新颖的组合选择将被用来实现这一目标。目的(1)表达、纯化和鉴定TCF-CBD蛋白；(2)鉴定针对TCF-CBD的紧密结合且高度特异的5-氨基烯丙基-2‘-脱氧尿苷适配子；(3)体外检测5-AA-适配子对β-连环蛋白-TCF复合体的干扰作用。这些实验最终应该会引入新的含铅抗结直肠癌化合物。这种先导化合物将在细胞培养和动物模型中进行测试。我们新的抗癌发现和开发策略可以应用于其他癌症。此外，这项研究产生的结果将用于未来NIH RO1拨款的申请。LE摘要：抗癌药物的开发已被证明是具有挑战性的。然而，基于寡核苷酸的抑制药物可以从一个非常大的随机池(10[12]大约10[14]个成员)中根据它们结合特定蛋白质的能力进行选择。这一新的抗癌策略很重要，因为它应该允许我们非常迅速地发现高效的药物化合物。

DESCRIPTION (provided by applicant): Colorectal cancer (CRC), the second leading cause of cancer mortality in the United States, is preventable and curable when found in its early stages. Although several chemopreventive agents prevent CRC in experimental animal models, no agent has been approved for human use. Besides surgical resection, which is rarely curative in advanced disease, current therapy for colon cancer relies on traditional cytotoxic agents with limited effects. Our long-term goal is to develop modified oligonucleotides (ODNs) to provide innovative therapeutic products for the treatment of cancer, in particular CRC. The formation of a transcriptional complex between an oncogenic beta-catenin and a T cell factor (TCF) is believed to be a key event in CRC cell development. Agents that modulate this pathway and/or disrupt the beta-catenin and TCF interaction are likely to inhibit the subsequent expression of many target genes that leads to CRC. The goal of this project is to identify a tight-binding and highly specific ODN that will bind to the beta-catenin binding domain of human TCF4 (TCF-CBD) to block the beta-catenin binding with TCF and/or disrupt the beta-catenin and TCF interaction. Structure-based design and novel combinatorial selection will be used to achieve this goal. Three specific aims will be pursued: Aim (1) express, purify, and characterize the TCF-CBD protein; Aim (2) identify tightly binding and highly specific aptamers containing 5-aminoallyl-2'-deoxyuridine (5-AA-aptamers) targeting TCF-CBD; Aim (3) test the 5-AA-aptamers for disruption of the beta-catenin.TCF complex in vitro. These experiments should ultimately introduce new lead anti-CRC compounds. Such lead compounds will be tested in cell cultures and animal models. Our novel anticancer discovery and development strategy can be applied to other cancers. In addition, the results generated from this research will be used in an application for a future NIH RO1 grant. Lay abstract: Development of anticancer drugs has proven to be challenging. However, oligonucleotide- based inhibition drugs can be selected from a very large random pool (10[12] approximately 10[14] members) by their ability to bind a specific protein. This novel anticancer strategy is important because it should allow us to very rapidly discover highly effective drug compounds.