RT Journal Article SR Electronic T1 Identification and Mechanism of Action of the Acylguanidine MRT-83, a Novel Potent Smoothened Antagonist JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.110.069708 DO 10.1124/mol.110.069708 A1 Hermine Roudaut A1 Elisabeth Traiffort A1 Tatiana Gorojankina A1 Ludwig Vincent A1 Helene Faure A1 Angele Schoenfelder A1 Andre Mann A1 Fabrizio Manetti A1 Antonio Solinas A1 Maurizio Taddei A1 Martial Ruat YR 2010 UL http://molpharm.aspetjournals.org/content/early/2010/12/21/mol.110.069708.abstract AB 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 which 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 HEK293 transiently transfected with a Tcf/Lef-dependent Firefly luciferase reporter together with a Renilla 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 upregulation 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.