Identification of inhibitors of hedgehog autoprocessing

描述(申请人提供)：Hedgehog(HH)信号在胚胎模式形成和成人干细胞更新中发挥重要作用，但最近发现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，所有这些癌症都是出了名的难以治疗。HH信号的第一步是HH蛋白的自动处理，其中C-末端结构域(HH-C)催化胆固醇依赖的自裂解反应，导致N-末端HH结构域(HH-N)的胆固醇酯的产生，从而产生信号分子。分泌的HH配体结合到修补的受体上，导致Smoothens(Smo)的激活和一系列信号转导，最终激活Gli转录因子，转录Gli和其他基因。关于HH信号在干细胞癌中作用的研究大多集中在Smo激活和Gli激活之间的一系列事件上，目的是为了了解这一复杂的信号通路，并希望开发癌症治疗药物。相反，尽管HH的自动处理和修饰是HH信号通路中的第一步，但很少有人关注它的作用。由于其研究缺乏药理学工具，对于HH自切和与胆固醇的酯化在HH信号中的作用，无论是在发育中的胚胎中的HH信号中，还是在HH配体依赖的癌症的生长中，都存在相当大的不确定性。这项建议旨在识别减弱HH自动处理的化合物，作为一种研究工具来剖析自动处理和伴随的胆固醇修饰在HH功能中的作用，无论是在许多不同的胚胎发育背景下，还是在HH配体依赖的干细胞癌中。这种方法是基于体外均相检测系统，该系统测量伴随着胆固醇依赖的HH蛋白自裂解的荧光偏振的变化。特别令人感兴趣的将是研究这项研究确定的抑制物对HH依赖的内皮肿瘤细胞系生长的影响。如果真的观察到肿瘤细胞系的生长抑制，这将表明作为HH信号通路的第一步，HH自我处理可能是一个可能的治疗靶点。 公共卫生相关性：Hedgehog(HH)信号在胚胎模式形成和成人干细胞更新中发挥重要作用，但最近发现也与某些干细胞癌有关，包括胰腺癌、小细胞肺癌、肠道和前列腺癌，所有这些癌症都是出了名的难以治疗。HH信号的第一步是自动处理反应，但目前还没有可用的化学生物学工具来研究这一重要步骤。这项提议寻求确定HH自动处理的抑制剂，这些抑制剂可以作为工具来研究其在正常发育和癌症中的作用，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.