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

描述（由申请人提供）：Hedgehog（Hh）信号通路是儿童和成人胎儿发育和肿瘤发生期间组织模式的组成部分。基因筛选揭示了几种调节Hh靶基因表达的信号蛋白，在哺乳动物中包括Sonic、Indian和Desert Hh配体（Shh、Ihh和Dhh）、Hh受体Patched 1（Ptch 1）、跨膜蛋白Smoothened（Smo）、Gli家族转录因子（Gli1、Gli2和Gli3）和Gli拮抗剂Suppressor of Fused（Sufu）。这些基因是下一代化疗的潜在靶点，并且抑制Smo的几种化合物已经在Hh通路依赖性肿瘤的小鼠模型中证明了疗效，如基底细胞癌、成神经管细胞瘤、胰腺癌和前列腺癌。Smo拮抗剂GDC-0449甚至在人类临床试验中引起转移性基底细胞癌和成神经管细胞瘤的消退。然而，越来越明显的是，Hh途径依赖性癌症可以容易地获得对Smo拮抗剂的抗性，并且某些肿瘤由Smo非依赖性Hh靶基因表达启动或维持。由于Smo是Hh通路中最"可用药"的靶标，并且几乎所有已知的通路抑制剂都靶向这种跨膜蛋白，因此明确且迫切需要在下游作用的小分子，优选在Gli转录因子水平作用。 本申请描述了一种高通量筛选Gli功能的小分子拮抗剂的方法，该方法使用已用Gli依赖性萤火虫荧光素酶报告基因稳定转染的Sufu无效细胞系（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蛋白介导的组成型萤火虫荧光素酶表达。与先前用于发现Hh通路抑制剂的基于细胞的测定相比，Sufu-KO-LIGHT细胞对通常在化学文库中发现的大量Smo靶向化合物无反应，并且它们不需要Gli1或Gli2的过表达来实现Smo非依赖性Hh靶基因表达。因此，这种新的测定将快速鉴定阻断内源性Gli因子功能的化合物，所述内源性Gli因子受到可以通过Gli过表达规避的调节过程的影响。除了这一新的筛选活动，一个全面的命中进步计划的概述，包括几个二级检测评估的Hh通路选择性的先导化合物，细胞和生化实验，以评估化合物的行动Gli功能，并确定化合物对Hh通路依赖性癌细胞的疗效研究。总的来说，这些调查将提供有价值的Hh通路内的下游信号传导事件的机制探针和结构线索，用于开发新的抗癌疗法。 公共卫生相关性：Hedgehog（Hh）通路的不受控制的激活有助于几种癌症的发作和进展，并且其药理学抑制可以在小鼠模型和人类患者中诱导肿瘤消退。这些研究中耐药肿瘤的出现强调了对新的Hh通路拮抗剂的需求，特别是抑制Gli家族转录因子的化合物。拟议的研究将通过基于细胞的高通量筛选来识别Gli功能的新型抑制剂，表征其作用机制，并评估其阻断Hh通路依赖性癌症增殖的能力。

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.