A high-throughput screen for small-molecule antagonists of Gli function

描述(由申请人提供)：Hedgehog(HH)信号通路是胎儿发育和儿童和成人肿瘤形成过程中组织模式不可或缺的组成部分。遗传筛选已经揭示了几种调节HH靶基因表达的信号蛋白，其中包括哺乳动物中的Sonic、India和Desert HH配体(Shh、IHH和DHH)、HH受体Patched1(Ptch1)、跨膜蛋白Smoothed(Smo)、Gli转录因子家族(Gli1、Gli2和Gli3)以及Gli拮抗剂融合抑制因子(Sufu)。这些基因是下一代化疗的潜在靶点，几种抑制Smo的化合物已在HH途径依赖性肿瘤的小鼠模型中证明有效，如基底细胞癌、髓母细胞瘤、胰腺癌和前列腺癌。在人类临床试验中，Smo拮抗剂GDC-0449甚至导致转移性基底细胞癌和髓母细胞瘤的消退。然而，越来越明显的是，依赖于HH途径的癌症可以很容易地获得对Smo拮抗剂的耐药性，并且某些肿瘤是通过非Smo依赖的HH靶基因表达而启动或维持的。由于Smo是HH途径中最“可用药”的靶点，而且几乎所有已知的途径抑制剂都以这种跨膜蛋白为靶点，因此显然迫切需要作用于下游的小分子，最好是在Gli转录因子的水平上。本申请描述了一种高通量筛选Gli功能小分子拮抗剂的方法，使用已稳定转染依赖Gli的萤火虫荧光素酶报告基因的Sufu空细胞系(Sufu-KO-light细胞)。由于Sufu直接抑制Gli功能，Sufu-KO-Light细胞表现出由内源性Gli蛋白介导的结构性萤火虫荧光素酶表达。与以前用来发现HH途径抑制剂的基于细胞的分析不同，Sufu-KO-light细胞对化学文库中通常发现的大量Smo靶向化合物没有反应，它们不需要Gli1或Gli2的过表达来实现Smo非依赖性HH靶基因的表达。因此，这一新的检测方法将快速识别阻止内源性Gli因子功能的化合物，这些化合物受到Gli过度表达可以绕过的调控过程的影响。除了这项新的筛选活动外，还概述了一项全面的HIT进展计划，包括评估先导化合物HH途径选择性的几种二次测试，评估化合物对Gli功能作用的细胞和生化实验，以及确定化合物对HH途径依赖的癌细胞的疗效的研究。总而言之，这些研究将为HH途径中的下游信号事件提供有价值的机械探针，并为新的抗癌疗法的开发提供结构性线索。 公共卫生相关性：Hedgehog(HH)通路的不受控制的激活导致了几种癌症的发生和发展，其药理抑制可以在小鼠模型和人类患者中诱导肿瘤消退。这些研究中出现的耐药肿瘤强调了新的HH途径拮抗剂的必要性，特别是使Gli转录因子家族失活的化合物。这项拟议的研究将通过基于细胞的高通量筛选来识别新的Gli功能抑制剂，表征它们的作用机制，并评估它们阻止HH途径依赖型癌症的增殖的能力。

DESCRIPTION (provided by applicant): The Hedgehog (Hh) signaling pathway is integral to tissue patterning during fetal development and oncogenesis in children and adults. Genetic screens have revealed several of the signaling proteins that regulate Hh target gene expression, which in mammals include the Sonic, Indian, and Desert Hh ligands (Shh, Ihh, and Dhh), the Hh receptor Patched1 (Ptch1), the transmembrane protein Smoothened (Smo), the Gli family of transcription factors (Gli1, Gli2, and Gli3), and the Gli antagonist Suppressor of Fused (Sufu). These genes are potential targets for next-generation chemotherapies, and several compounds that inhibit Smo have demonstrated efficacy in mouse models of Hh pathway-dependent tumors, such as basal cell carcinoma, medulloblastoma, pancreatic adenocarcinoma, and prostate cancer. The Smo antagonist GDC-0449 has even caused the regression of metastatic basal cell carcinoma and medulloblastoma in human clinical trials. It has become increasingly apparent, however, that Hh pathway-dependent cancers can readily gain resistance to Smo antagonists and that certain tumors are initiated or maintained by Smo-independent Hh target gene expression. Since Smo is the most "druggable" target within the Hh pathway and nearly all known pathway inhibitors target this transmembrane protein, there is a clear and urgent need for small molecules that act downstream, preferably at the level of the Gli transcription factors. This application describes a high-throughput screen for small-molecule antagonists of Gli function, using a Sufu null cell line that has been stably transfected with a Gli-dependent firefly luciferase reporter (Sufu-KO- LIGHT cells). Since Sufu directly inhibits Gli function, Sufu-KO-LIGHT cells exhibit constitutive firefly luciferase expression that mediated by endogenous Gli proteins. In contrast to cell-based assays used previously to discover Hh pathway inhibitors, the Sufu-KO-LIGHT cells are unresponsive to the large number of Smo- targeting compounds typically found in chemical libraries and they do not require the overexpression of Gli1 or Gli2 to achieve Smo-independent Hh target gene expression. This novel assay will therefore rapidly identify compounds that block the function of endogenous Gli factors, which are subject to regulatory processes that can be circumvented by Gli overexpression. In addition to this new screening campaign, a comprehensive hit advancement plan is outlined, including several secondary assays for assessing the Hh pathway selectivity of lead compounds, cellular and biochemical experiments for evaluating compound action on Gli function, and studies for determining compound efficacy against Hh pathway-dependent cancer cells. Collectively these investigations will provide valuable mechanistic probes of downstream signaling events within the Hh pathway and structural leads for the development of new anti-cancer therapies. PUBLIC HEALTH RELEVANCE: Uncontrolled activation of the Hedgehog (Hh) pathway contributes to the onset and progression of several cancers and its pharmacological inhibition can induce tumor regression in mouse models and human patients. The emergence of drug-resistant tumors in these studies underscores the need for new Hh pathway antagonists, particularly compounds that inactivate the Gli family of transcription factors. The proposed research will identify novel inhibitors of Gli function through a cell-based high-throughput screen, characterize their mechanisms of action, and assess their ability to block the proliferation of Hh pathway-dependent cancers.