Identification of Specific Antagonists that Bind Hedgehog and Block its Activity

描述（由申请人提供）：该项目的长期目标是确定靶向对Hh活性重要的hedgehog （Hh）结合相互作用的新型拮抗剂，作为开发新型抗癌疗法的基础。Hh通路的异常表达与大约20-25%的癌症的生长有关，其中许多是由Hh配体的过度表达驱动的。几种天然产物和阻断Hh信号的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。临床已经观察到对Smo抑制剂的耐药性，因此迫切需要针对该途径其他组分的抑制剂。有令人信服的证据表明，硫酸肝素蛋白多糖（HSPGs）在Hh配体运输和信号传导活性中起关键作用，在缺乏编码硫酸肝素共聚酶的tout velu基因功能的细胞中，HSPGs的作用涉及Hh信号传导范围的限制。本提案的目标是通过靶向Hh配体与HSPGs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一种新的Hh/肝素结合试验来鉴定可以调节肝素-Hh相互作用的分子。在这项实验中，未标记的肝素分子和小分子苏拉明抑制这种相互作用，并在Hh功能的细胞基础实验中阻断Hh活性。该试验将在高通量模式下用于筛选60000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及其结合伙伴）的蛋白-蛋白相互作用的破坏是具有挑战性的，我们期望适体选择提供一种替代和互补的方法来识别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/肝素结合试验作为主要筛选，从大型化学文库中识别阻断Hh与肝素结合的化合物，并使用合成肽和绘制Hh的肝素结合位点（Specific Aim 1）。分离和表征Hh结合适体（Specific Aim 2）。使用一系列次级/正交试验首先确认和验证初级筛选的命中值和选择的适体，然后确定那些选择性阻断Hh/HSPG关联并抑制生物测定中Hh信号传导并影响Hh依赖性癌细胞系增殖、存活和迁移的适体（Specific Aim 3）。各种二次筛选和反筛选将用于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体内癌症模型奠定基础。我们相信，所得到的分子将特别有价值，可以作为研究工具，更好地了解HSPG对Hh活性的贡献，以及作为探索与Hh配体驱动的癌症相关疾病的新型抑制剂。

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.