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过程中组织模式的组成部分。遗传筛选已经揭示了几种调节Hh靶基因表达的信号蛋白，其中在哺乳动物中包括Sonic、Indian和Desert Hh配体（Shh、Ihh和Dhh）、Hh受体Patched1 （Ptch1）、跨膜蛋白Smoothened （Smo）、Gli转录因子家族（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，一些抑制Smo的化合物已经在Hh通路依赖性肿瘤的小鼠模型中显示出疗效，如基底细胞癌、成神经管细胞瘤、胰腺腺癌和前列腺癌。在人类临床试验中，Smo拮抗剂GDC-0449甚至引起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，Hh通路依赖的癌症可以很容易地获得对Smo拮抗剂的耐药性，并且某些肿瘤是由Smo不依赖的Hh靶基因表达引发或维持的。由于Smo是Hh通路中最“可药物化”的靶点，几乎所有已知的通路抑制剂都以这种跨膜蛋白为靶点，因此迫切需要在下游发挥作用的小分子，最好是在Gli转录因子水平上。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，使用稳定转染Gli依赖性萤火虫荧光素酶报告细胞（Sufu- ko - LIGHT细胞）的Sufu零细胞系。由于Sufu直接抑制Gli功能，Sufu- ko - light细胞表现出内源性Gli蛋白介导的萤火虫荧光素酶的组成性表达。与先前用于发现Hh通路抑制剂的基于细胞的分析相比，Sufu-KO-LIGHT细胞对化学文库中通常发现的大量Smo靶向化合物无反应，并且它们不需要过表达Gli1或Gli2来实现Smo独立的Hh靶基因表达。因此，这种新的检测方法将快速识别阻断内源性Gli因子功能的化合物，这些化合物受Gli过表达的调节过程所影响。除了这项新的筛选活动外，还概述了一项全面的打击进展计划，包括评估先导化合物Hh通路选择性的几项二级分析，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通路依赖的癌细胞的功效的研究。总的来说，这些研究将为Hh通路中的下游信号事件提供有价值的机制探测，并为开发新的抗癌疗法提供结构线索。

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.