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确定针对刺猬（HH）结合相互作用的新型拮抗剂对HH活性很重要，这是开发新型抗癌疗法的基础。 HH途径的异常表达与大约20-25％的所有癌症的生长有关，其中许多癌症由HH配体的过表达驱动。阻断HH信号传导的几种天然产物和化学抑制剂对癌症模型具有抗肿瘤作用。这些化合物主要通过靶向HH共受体平滑（SMO）或GLI转录因子来起作用。在临床上已经观察到了对SMO抑制剂的耐药性，因此非常需要针对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）与HH配体运输和信号传导活性至关重要，具有HSPGS的作用，该作用是由HH HH信号限制涉及的HHS，缺少Tout Velu基因功能的HH信号范围，该基因的功能编码了Heparin suparin sulfate Copolymeryase。该提案的目的是通过靶向HH配体与HSPG的相互作用来鉴定HH途径的新型抑制剂。我们建议使用新型的HH/肝素结合测定法进行此操作，以识别可以调节肝素-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蛋白抑制这种相互作用，并且还阻断了基于细胞的HH功能的HH活性。该测定法将在高通量模式下用于筛选一个60,000个小分子的库，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相互作用的破坏涉及涉及HH及其结合伴侣的细胞外蛋白质的蛋白质相互作用是具有挑战性的，我们希望适体的选择可以为识别HH蛋白介导的活性的替代性和互补方法提供一种替代性和互补方法，无论是否在HH上或其他蛋白质上的HH结合位点上的肝素活性。因此，当前建议的目标是使用HH/肝素结合测定法作为主要筛选，以鉴定大型化学文库中阻断HH与肝素结合的大型化合物，并使用合成肽和（特定AIM 1）绘制HH的HH肝素结合位点。隔离和表征HH结合适体（特定目标2）。使用一系列的二级/正交测定首先确认并验证选择中的主要筛选和适体中的命令，其次，选择性地识别那些选择性地阻断HH/HSPG关联并抑制生物测定中的HH信号传导并影响HH-依赖性癌细胞系的增殖，存活和迁移（特定的AIM 3）。将采用各种二次和计数器筛选，以进一步表征已确定的分子的选择性和活性，为将来的化学优化和随后的癌症模型奠定了基础。我们认为，作为更好地了解HSPG贡献HH活性的研究工具以及作为对与HH-配体驱动癌症相关的探针疾病的新型抑制剂的研究工具，所产生的分子将特别有价值。 公共卫生相关性：刺猬（HH）途径是一个引人注目的治疗靶标，因为它在众多人类癌症类型的增长中起着核心作用，包括胰腺，肝脏和肺等有效治疗方案很少的人。 HH途径迄今为止确定的少数当前抑制剂几乎完全与平滑的共受体结合或靶向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关注。有积极的证据表明，HH蛋白功能的能力是由硫酸肝素蛋白聚糖（HSPG）调节的，因此我们建议鉴定小分子和基于DNA的化合物，这些化合物有选择地阻断HH/HSPG相互作用，并将其作为癌症模型中的抑制剂测试，以识别出一种新型的疾病依赖性cance-vence-deflent cance-gededecten cance-gededendendendectect。