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.
This work was supported by National Science Foundation of China Grants 20325208, 20225318, 20521202, and 20672004, Ministry of Education of China Program 985, the Shenzhen Science and Technology Key Laboratory Promotion Fund, the Instrumental Analysis Fund of Peking University, and funds from the Sidney Kimmel Foundation for Cancer Research (to J. K. C.), the Astellas USA Foundation (to J. K. C.), and the Brain Tumor Society/Rachel Molly Markoff Foundation (to J. K. C.).
