Converse Conformational Control of Smoothened Activity by Structurally Related Small Molecules

Shuo Lin,Jing Xiang,James K. Chen,Zhen Yang,Yun Zhao,Jin Jiang,Hongbo Yang,Nengdong Wang,Song Li,Joel Hyman

doi:10.1074/jbc.m807648200

Abstract

The seven-pass transmembrane protein Smoothened (Smo) is an essential component of the Hedgehog (Hh) signaling pathway that is critically involved in normal animal development as well as pathological malignancies. In studying Hh-related biological processes, it would be highly desirable if Smo activity could be instantly switched between activation and inhibition. Using Gli1-dependent GFP transgenic zebrafish and in vitro biochemical assays, we identified and characterized two potent Smo inhibitors, SANT74 and 75 (Smoothened antagonist 74 and 75), by screening a small molecule library designed based on the scaffold of Smo agonist SAG. These compounds are structural analogs of SAG with the methyl group substituted by a propyl or allyl group in SANTs. We show that SANTs and SAG exert opposite effects on Smo activity by regulating protein conformation. Our study represents the first demonstration of conformational regulation of Smo by small molecule analogs, and the combinational use of these Smo modulators in a temporal controlled fashion should be useful for studying Hh biology.

Highlights

The Hh signaling pathway is essential for embryonic development and adult tissue homeostasis in metazoans
A small scale clinic study has found that topical treatment with cyclopamine was effective in reducing the size of basal cell carcinoma lesions [20]
The GFP expression pattern is consistent with the expression of Gli1 and Ptc1 in zebrafish embryos at the stages analyzed

Summary

Introduction

The Hh signaling pathway is essential for embryonic development and adult tissue homeostasis in metazoans. Binding of secreted Hh proteins to Ptch results in subcellular relocation and conformation changes of Smo that in turn promote the expression of Hh target genes through the Gli transcription factors [1]. Hh signaling regulates both cell proliferation and differentiation during normal embryogenesis. Using cell-based screening platforms with a luciferase reporter under the control of multiple Gli1-binding sites, several additional synthetic Hh inhibitors with Smo binding affinity have been identified [23,24,25,26] Some of these Hh inhibitors are able to suppress basal cell carcinoma and medulloblastomas in animal models [25, 27]

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