Identification of inhibitors of hedgehog autoprocessing

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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.