title: Selective identification of hedgehog pathway antagonists by direct analysis of smoothened ciliary translocation.
abstract: Hedgehog (Hh) signaling promotes tumorigenesis. The accumulation of the membrane protein Smoothened (Smo) within the primary cilium (PC) is a key event in Hh signal transduction, and many pharmacological inhibitors identified to date target Smo's actions. Smo ciliary translocation is inhibited by some pathway antagonists, while others promote ciliary accumulation, an outcome that can lead to a hypersensitive state on renewal of Hh signaling. To identify novel inhibitory compounds acting on the critical mechanistic transition of Smo accumulation, we established a high content screen to directly analyze Smo ciliary translocation. Screening thousands of compounds from annotated libraries of approved drugs and other agents, we identified several new classes of compounds that block Sonic hedgehog-driven Smo localization within the PC. Selective analysis was conducted on two classes of Smo antagonists. One of these, DY131, appears to inhibit Smo signaling through a common binding site shared by previously reported Smo agonists and antagonists. Antagonism by this class of compound is competed by high doses of Smo-binding agonists such as SAG and impaired by a mutation that generates a ligand-independent, oncogenic form of Smo (SmoM2). In contrast, a second antagonist of Smo accumulation within the PC, SMANT, was less sensitive to SAG-mediated competition and inhibited SmoM2 at concentrations similar to those that inhibit wild-type Smo. Our observations identify important differences among Hh antagonists and the potential for development of novel therapeutic approaches against mutant forms of Smo that are resistant to current therapeutic strategies.
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Arvanites
authors.forename: Lance
authors.initials: L
authors.lastname: Davidow
authors.forename: Joel
authors.initials: J
authors.lastname: Blanchard
authors.forename: Kelvin
authors.initials: K
authors.lastname: Lam
authors.forename: Jin Woo
authors.initials: JW
authors.lastname: Yoo
authors.forename: Shannon
authors.initials: S
authors.lastname: Coy
authors.forename: Lee L
authors.initials: LL
authors.lastname: Rubin
authors.forename: Andrew P
authors.initials: AP
authors.lastname: McMahon
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology*; D002460:Cell Line; D002923:Cilia / Q000187:drug effects / Q000378:metabolism; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D000072086:Smoothened Receptor
chemical_list: D000970:Antineoplastic Agents; D053823:Hedgehog Proteins; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor