title: Identification and mechanism of action of the acylguanidine MRT-83, a novel potent Smoothened antagonist.
abstract: There is a clear need to develop novel pharmacological tools to improve our understanding of Smoothened (Smo) function in normal and pathological states. Here, we report the discovery, the mechanism of action, and the in vivo activity of N-(2-methyl-5-(3-(3,4,5-trimethoxybenzoyl)guanidino)phenyl)biphenyl-4-carboxamide (MRT-83), a novel potent antagonist of Smo that belongs to the acylguanidine family of molecules. MRT-83 fits to a proposed pharmacophoric model for Smo antagonists with three hydrogen bond acceptor groups and three hydrophobic regions. MRT-83 blocks Hedgehog (Hh) signaling in various assays with an IC50 in the nanomolar range, showing greater potency than the reference Smo antagonist cyclopamine. MRT-83 inhibits Bodipy-cyclopamine binding to human and mouse Smo but does not modify Wnt signaling in human embryonic kidney 293 transiently transfected with a Tcf/Lef-dependent Firefly luciferase reporter together with a Renilla reniformis luciferase control reporter. MRT-83 abrogates the agonist-induced trafficking of endogenous mouse or human Smo to the primary cilium of C3H10T1/2 or NT2 cells that derive from a pluripotent testicular carcinoma. Stereotaxic injection into the lateral ventricle of adult mice of MRT-83 but not of a structurally related compound inactive at Smo abolished up-regulation of Patched transcription induced by Sonic Hedgehog in the neighboring subventricular zone. These data demonstrate that MRT-83 efficiently antagonizes Hh signaling in vivo. All together, these molecular, functional and biochemical studies provide evidence that MRT-83 interacts with Smo. Thus, this novel Smo antagonist will be useful for manipulating Hh signaling and may help develop new therapies against Hh-pathway related diseases.
authors.affiliation: Laboratoire de Neurobiologie et Développement, Institut de Neurobiologie Alfred Fessard IFR2118, Centre National de la Recherche Scientifique, UPR-3294, Gif-sur-Yvette, France.
authors.forename: Hermine
authors.initials: H
authors.lastname: Roudaut
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Traiffort
authors.forename: Tatiana
authors.initials: T
authors.lastname: Gorojankina
authors.forename: Ludwig
authors.initials: L
authors.lastname: Vincent
authors.forename: Helene
authors.initials: H
authors.lastname: Faure
authors.forename: Angele
authors.initials: A
authors.lastname: Schoenfelder
authors.forename: Andre
authors.initials: A
authors.lastname: Mann
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Manetti
authors.forename: Antonio
authors.initials: A
authors.lastname: Solinas
authors.forename: Maurizio
authors.initials: M
authors.lastname: Taddei
authors.forename: Martial
authors.initials: M
authors.lastname: Ruat
mesh_terms: D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D002460:Cell Line; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000187:drug effects; D006146:Guanidines / Q000494:pharmacology*; D057809:HEK293 Cells; D053823:Hedgehog Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D000072079:Patched Receptors; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects; D000072086:Smoothened Receptor; D014704:Veratrum Alkaloids / Q000494:pharmacology; D051153:Wnt Proteins / Q000187:drug effects / Q000502:physiology
chemical_list: D001549:Benzamides; D006146:Guanidines; D053823:Hedgehog Proteins; C557503:N-(2-methyl-5-(3-(3,4,5-trimethoxybenzoyl)guanidino)phenyl)biphenyl-4-carboxamide; D000072079:Patched Receptors; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; C491022:Smo protein, mouse; D000072086:Smoothened Receptor; D014704:Veratrum Alkaloids; D051153:Wnt Proteins; C000541:cyclopamine