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靶向对Hh活性重要的Hedgehog（Hh）结合相互作用的新型拮抗剂，作为开发新型抗癌治疗剂的基础。Hh途径的异常表达与大约20-25%的所有癌症的生长有关，其中许多是由Hh配体的过表达驱动的。阻断Hh信号传导的几种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共受体Smoothened（Smo）或Gli转录因子起作用。在临床中已经观察到对Smo抑制剂的抗性，因此非常需要靶向该途径的其他组分的抑制剂。有令人信服的证据表明，硫酸肝素蛋白聚糖（HSPG）是关键参与Hh配体运输和信号传导活性的作用，HSPG的牵连限制在细胞中的Hh信号传导的范围内缺乏tout velu基因的功能，它编码的硫酸肝素共聚酶。该提案的目标是通过靶向Hh配体与HSPG的相互作用来识别Hh途径的新型抑制剂。我们建议这样做，我们已经开发了一种新的Hh/肝素结合试验，以确定分子，可以调节肝素Hh相互作用。在该测定中，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个小分子的文库，以鉴定Hh/肝素结合的小分子拮抗剂。小分子介导的蛋白质-蛋白质相互作用的破坏涉及细胞外蛋白质如Hh及其结合配偶体是具有挑战性的，我们期望适体选择提供一种替代和互补的方法来鉴定Hh蛋白质介导的活性的拮抗剂，无论是在Hh上的肝素结合位点还是在蛋白质上的其他地方。因此，当前提案的目标是使用Hh/肝素结合试验作为初步筛选，以从大型化学文库中鉴定阻断Hh与肝素结合的化合物，并使用合成肽和（具体目标1）绘制Hh的肝素结合位点。分离和表征Hh结合适体（具体目标2）。使用一系列二次/正交试验，首先确认和验证来自初步筛选的命中物和来自选择的适体，其次鉴定在生物试验中选择性阻断Hh/HSPG缔合和抑制Hh信号传导并影响Hh依赖性癌细胞系的增殖、存活和迁移的那些适体（具体目标3）。将采用各种次级和反向筛选来进一步表征所鉴定分子的选择性和活性，为未来的化学优化和随后的癌症体内模型奠定基础。我们相信，所得到的分子将是特别有价值的研究工具，以更好地了解HSPG的贡献Hh活性和作为新的抑制剂，探针与Hh-配体驱动的癌症相关的疾病。 公共卫生关系：Hedgehog（Hh）通路是一个引人注目的治疗靶点，因为它在包括胰腺癌、肝癌和肺癌在内的多种人类癌症类型的生长中起着核心作用，而有效的治疗方案却很少。迄今为止鉴定的用于Hh途径的几种当前抑制剂几乎专门结合平滑的共受体或靶向Gli转录因子，因此通过替代机制靶向Hh途径的抑制剂引起了极大的兴趣。越来越多的证据表明，Hh蛋白发挥功能的能力受到硫酸肝素蛋白聚糖（HSPG）的调节，因此我们提出鉴定选择性阻断Hh/HSPG相互作用的小分子和基于DNA的化合物，在癌症模型中测试它们作为抑制剂，从而潜在地鉴定刺猬依赖性癌症的新治疗途径。