Identification of Specific Antagonists that Bind Hedgehog and Block its Activity

鉴定结合 Hedgehog 并阻断其活性的特定拮抗剂

• 批准号: 8100944
• 负责人: Kevin Peter Williams
• 金额: $ 36.29万
• 依托单位: NORTH CAROLINA CENTRAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8100944
• 关键词: Affect; Affinity; Affinity Chromatography; Apoptosis; Binding; Binding Sites; Biological Assay; Biological Factors; Cancer Model; Cancer cell line; Cells; Chemicals; Clinic; Collection; Complex; DNA; DNA Sequence; Development; Diffusion; Disease; Dose; Drug resistance; Erinaceidae; Extracellular Protein; FDA approved; Family; Fluorescence Polarization; Future; Genes; Goals; Growth; Heparan Sulfate Proteoglycan; Heparin; Heparin Antagonists; Heparin Binding; Heparitin Sulfate; Human; Inorganic Sulfates; Length; Libraries; Ligands; Light; Liver; Lung; Malignant Neoplasms; Malignant neoplasm of prostate; Maps; Measures; Mediating; Oligonucleotides; Pancreas; Pathway interactions; Pattern; Peptide aptamers; Peptides; Phase; Play; Proteins; Research; Resistance; Role; Screening procedure; Series; Signal Transduction; Signaling Protein; Site; Solid; Staging; Suramin; Testing; Tissues; Unspecified or Sulfate Ion Sulfates; Western Blotting; Work; anti-cancer therapeutic; aptamer; base; biochip; cancer cell; cancer type; counterscreen; effective therapy; heparin proteoglycan; human SMO protein; in vivo Model; inhibitor/antagonist; interest; migration; novel; novel strategies; novel therapeutics; peptide analog; perlecan; protein protein interaction; receptor; response; small molecule; small molecule libraries; smoothened signaling pathway; synthetic peptide; therapeutic target; tool; trafficking; transcription factor; tumor

DESCRIPTION (provided by applicant): The long term goal of this initiative is to identify novel antagonists that target hedgehog (Hh) binding interactions important for Hh activity as the basis for the development of novel anti-cancer therapeutics. Aberrant expression of the Hh pathway has been implicated in the growth of approximately 20-25% of all cancers, with many driven by the over-expression of Hh ligand. Several natural product and chemical inhibitors that block Hh signaling have anti-tumor effects in models of cancer. These compounds work predominantly by targeting the Hh co-receptor Smoothened (Smo) or Gli transcription factor. Resistance to Smo inhibitors has been observed in the clinic and hence inhibitors targeting other components of the pathway are greatly needed. There is compelling evidence that heparin sulfate proteoglycans (HSPGs) are critically involved in Hh ligand trafficking and signaling activity with a role for HSPGs implicated by restriction in the range of Hh signaling in cells lacking the function of the tout velu gene, which encodes a heparin sulfate copolymerase. The goal of this proposal is to identify novel inhibitors of the Hh pathway by targeting the interaction of Hh ligand with HSPGs. We propose to do this using a novel Hh/heparin binding assay we have developed to identify molecules that can modulate heparin-Hh interactions. In this assay, unlabeled heparin molecules and the small molecule suramin inhibit this interaction and also blocked Hh activity in a cell based assay of Hh function. This assay will be used in high throughput mode to screen a library of 60,000 small molecules to identify small molecule antagonists of Hh/heparin binding. Small molecule mediated disruption of protein- protein interactions involving extracellular proteins such as Hh and their binding partners is challenging and we expect aptamer selection to provide an alternative and complementary approach to identifying antagonists of Hh protein mediated activity whether at the heparin binding site on Hh or elsewhere on the protein. Hence, the goals of the current proposal are to use the Hh/heparin binding assay as a primary screen to identify compounds from large chemical libraries that block Hh binding to heparin and to map the heparin binding site of Hh using synthetic peptides and (Specific Aim 1). To isolate and characterize Hh binding aptamers (Specific Aim 2). Use a series of secondary/orthogonal assays to first confirm and verify hits from primary screening and aptamers from the selection, and secondly identify those that selectively block Hh/HSPG association and inhibit Hh signaling in bioassays and affect proliferation, survival and migration of Hh- dependent cancer cell lines (Specific Aim 3). The various secondary and counter screens will be employed to further characterize the selectivity and activity of the identified molecules, setting the stage for future chemical optimization and subsequent in vivo models of cancer. We believe the resulting molecules will be especially valuable as research tools to better understand HSPG contributions to Hh activity and as novel inhibitors to probe diseases associated with Hh-ligand driven cancers. PUBLIC HEALTH RELEVANCE: The Hedgehog (Hh) pathway is a compelling therapeutic target as it plays a central role in the growth of a vast array of human cancer types including those such as pancreatic, liver and lung with few effective treatment options. The few current inhibitors identified to date for the Hh pathway almost exclusively bind to the smoothened co-receptor or target the Gli transcription factors and hence inhibitors targeting the Hh pathway by alternative mechanisms are of great interest. There is accumulating evidence that the ability of Hh protein to function is modulated by heparin sulfate proteoglycans (HSPG), and so we propose to identify small molecule and DNA-based compounds that selectively block Hh/HSPG interactions, test them as inhibitors in cancers models and so potentially identify a novel therapeutic avenue for hedgehog- dependent cancers.

描述（由申请人提供）：该计划的长期目标是鉴定针对对 Hh 活性至关重要的刺猬蛋白 (Hh) 结合相互作用的新型拮抗剂，作为开发新型抗癌疗法的基础。 Hh 通路的异常表达与大约 20-25% 的所有癌症的生长有关，其中许多癌症是由 Hh 配体的过度表达驱动的。几种阻断 Hh 信号传导的天然产物和化学抑制剂在癌症模型中具有抗肿瘤作用。这些化合物主要通过靶向 Hh 辅助受体 Smoothened (Smo) 或 Gli 转录因子发挥作用。临床上已经观察到对 Smo 抑制剂的耐药性，因此非常需要针对该途径其他成分的抑制剂。有令人信服的证据表明，硫酸肝素蛋白聚糖 (HSPG) 关键参与 Hh 配体运输和信号传导活动，HSPG 的作用与缺乏 Tout velu 基因（编码肝素）功能的细胞中 Hh 信号传导范围的限制有关。硫酸盐共聚酶。该提案的目标是通过针对 Hh 配体与 HSPG 的相互作用来鉴定 Hh 途径的新型抑制剂。我们建议使用我们开发的新型 Hh/肝素结合测定法来识别可以调节肝素-Hh 相互作用的分子。在此测定中，未标记的肝素分子和小分子苏拉明抑制这种相互作用，并且还在基于细胞的 Hh 功能测定中阻断 Hh 活性。该测定将以高通量模式用于筛选 60,000 个小分子文库，以鉴定 Hh/肝素结合的小分子拮抗剂。小分子介导的涉及细胞外蛋白（如 Hh 及其结合伴侣）的蛋白质-蛋白质相互作用的破坏具有挑战性，我们期望适体选择提供一种替代和补充方法来鉴定 Hh 蛋白介导活性的拮抗剂，无论是在 Hh 上的肝素结合位点还是在 Hh 上的肝素结合位点蛋白质的其他地方。因此，当前提案的目标是使用 Hh/肝素结合测定作为主要筛选，以从大型化学库中识别阻断 Hh 与肝素结合的化合物，并使用合成肽和（具体目标）绘制 Hh 的肝素结合位点图谱。 1）。分离和表征 Hh 结合适体（具体目标 2）。使用一系列二次/正交测定首先确认和验证初级筛选的命中和选择的适体，其次鉴定那些选择性阻断 Hh/HSPG 关联并抑制生物测定中的 Hh 信号传导并影响 Hh- 的增殖、存活和迁移的适体。依赖性癌细胞系（具体目标 3）。各种二次和反筛选将用于进一步表征已识别分子的选择性和活性，为未来的化学优化和随后的体内癌症模型奠定基础。我们相信，所得分子作为研究工具特别有价值，可以更好地了解 HSPG 对 Hh 活性的贡献，也可以作为新型抑制剂来探测与 Hh 配体驱动的癌症相关的疾病。 公共健康相关性：Hedgehog (Hh) 通路是一个引人注目的治疗靶点，因为它在多种人类癌症类型的生长中发挥着核心作用，包括胰腺癌、肝癌和肺癌等几乎没有有效治疗选择的癌症。迄今为止发现的少数 Hh 通路抑制剂几乎完全与平滑共受体结合或靶向 Gli 转录因子，因此通过替代机制靶向 Hh 通路的抑制剂引起了人们的极大兴趣。越来越多的证据表明 Hh 蛋白的功能受到硫酸肝素蛋白聚糖 (HSPG) 的调节，因此我们建议鉴定能够选择性阻断 Hh/HSPG 相互作用的小分子和基于 DNA 的化合物，并在癌症模型中测试它们作为抑制剂因此有可能为刺猬依赖性癌症找到一种新的治疗途径。