PMID:21177415

Citation	Roudaut, H, Traiffort, E, Gorojankina, T, Vincent, L, Faure, H, Schoenfelder, A, Mann, A, Manetti, F, Solinas, A, Taddei, M and Ruat, M (2011) Identification and mechanism of action of the acylguanidine MRT-83, a novel potent Smoothened antagonist. Mol. Pharmacol. 79:453-60
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.
Links	PubMed Online version:10.1124/mol.110.069708
Keywords	Animals; Benzamides/pharmacology; Cell Line; Cerebellum/cytology; Cerebellum/drug effects; Cerebellum/metabolism; Dose-Response Relationship, Drug; Gene Expression/drug effects; Guanidines/pharmacology; HEK293 Cells; Hedgehog Proteins/metabolism; Humans; Mice; Protein Binding; Receptors, Cell Surface/antagonists & inhibitors; Receptors, Cell Surface/biosynthesis; Receptors, G-Protein-Coupled/antagonists & inhibitors; Receptors, G-Protein-Coupled/biosynthesis; Signal Transduction/drug effects; Veratrum Alkaloids/pharmacology; Wnt Proteins/drug effects; Wnt Proteins/physiology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HUMAN:SMO	enables	GO:0008144: drug binding	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
HUMAN:SMO	involved_in	GO:0007224: smoothened signaling pathway	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
MOUSE:SMO	enables	GO:0008144: drug binding	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
MOUSE:SMO	involved_in	GO:0007224: smoothened signaling pathway	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete

References

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