Identification of inhibitors of hedgehog autoprocessing

描述（申请人提供）：Hedgehog（Hh）信号传导在胚胎模式和成体干细胞更新中起着重要作用，但最近发现也参与某些干细胞癌症，包括胰腺癌、小细胞肺癌、肠癌和前列腺癌，所有这些都是众所周知的难以治疗的。Hh信号传导的第一步是Hh蛋白的自动加工，其中C-末端结构域（Hh-C）催化胆固醇依赖性自裂解反应，该反应导致产生N-末端Hh结构域（Hh-N）的胆固醇酯，从而产生信号分子。分泌的Hh配体与Patched受体结合，导致Smoothened（Smo）的激活和信号转导级联，最终激活Gli转录因子和Gli及其他基因的转录。大多数关于Hh信号在干细胞癌症中的作用的研究都集中在Smo激活和Gli激活之间的一系列事件上，目的是了解这种复杂的信号通路，并希望开发癌症治疗方法。相反，很少有人注意到Hh的自动加工和修饰的作用，尽管它代表了Hh信号通路的初始步骤。由于缺乏药理学的工具，其研究，有相当大的不确定性的作用Hh的自裂解和酯化与胆固醇在Hh信号，无论是在Hh信号在发育中的胚胎和生长的Hh配体依赖性癌症。该提案旨在确定减弱Hh自动加工的化合物，作为解剖Hh功能中自动加工和伴随的胆固醇修饰的作用的研究工具，无论是在胚胎发育的许多不同背景下，还是在Hh配体依赖性干细胞癌症中。这种方法是基于一个在体外均相测定系统，测量伴随胆固醇依赖性的Hh蛋白的自裂解的荧光偏振的变化。特别感兴趣的将是本研究确定的抑制剂对Hh依赖性内胚层肿瘤细胞系生长的影响的研究。如果确实观察到肿瘤细胞系的生长抑制，这将表明作为Hh信号传导途径的第一步的Hh自动加工可能是一个可能的治疗靶点。 公共卫生相关性：Hedgehog（Hh）信号在胚胎发育和成体干细胞更新中起着重要作用，但最近发现它也与某些干细胞癌有关，包括胰腺癌、小细胞肺癌、肠癌和前列腺癌，所有这些都是众所周知的难以治疗的疾病。Hh信号的第一步是自动加工反应，但目前还没有化学生物学工具来研究这一重要步骤。该提案旨在确定Hh自动加工的抑制剂，可以作为研究其在正常发育和癌症中的作用的工具，并可能导致潜在的癌症治疗。

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling plays an important role in embryonic patterning and adult stem cell renewal, but has recently been found also to be involved in certain stem-cell cancers, including pancreatic, small cell lung, intestinal, and prostate, all of which are notoriously difficult to treat. One of the first steps in Hh signaling is the autoprocessing of Hh protein, in which the C-terminal domain (Hh-C) catalyzes a cholesterol-dependent autocleavage reaction that leads to the production of the cholesterol ester of the N-terminal Hh domain (Hh-N), thereby yielding a signaling molecule. The secreted Hh ligand binds to the Patched receptor, leading to activation of Smoothened (Smo) and a signal transduction cascade that culminates in the activation of Gli transcription factors and the transcription of Gli and other genes. Most research on the role of Hh signaling in stem cell cancers is focused on the series of events that lie between the activation of Smo and the activation of Gli, with the aim of understanding this complex signaling pathway and in the hope to develop cancer therapeutics. In contrast, very little attention has been given to the role of Hh autoprocessing and modification, in spite of the fact that it represents the initial step in the Hh signaling pathway. Due to the absence of pharmacologic tools for its study, there is considerable uncertainty about the role of Hh autocleavage and esterification with cholesterol in Hh signaling, both in Hh signaling in the developing embryo and in the growth of Hh ligand-dependent cancers. This proposal aims to identify compounds that attenuate Hh autoprocessing as a research tool for dissecting the role of autoprocessing and the attendant cholesterol modification in Hh function, both in the many different contexts of embryonic development and in Hh ligand-dependent stem cell cancers. This approach is based on an in vitro homogeneous assay system which measures changes in fluorescence polarization that accompany the cholesterol-depended autocleavage of Hh protein. Of special interest will be the study of the effect of the inhibitors identified by this research on the growth of Hh-dependent endodermal tumor cell lines. If growth inhibition of the tumor cell lines should indeed be observed, this would suggest that Hh autoprocessing, as the first step in the Hh signaling pathway, could be a possible therapeutic target. PUBLIC HEALTH RELEVANCE: Hedgehog (Hh) signaling plays an important role in embryonic patterning and adult stem cell renewal, but has recently been found also to be responsible for certain stem-cell cancers, including pancreatic, small cell lung, intestinal, and prostate, all of which are notoriously difficult to treat. The first step in Hh signaling is an autoprocessing reaction, but there are no chemical biology tools available for studying this important step. This proposal seeks to identify inhibitors of Hh autoprocessing that can serve as tools for studying its role in normal development and in cancer and which may lead to potential cancer therapeutics.