title: MRT-92 inhibits Hedgehog signaling by blocking overlapping binding sites in the transmembrane domain of the Smoothened receptor.
abstract: The Smoothened (Smo) receptor, a member of class F G protein-coupled receptors, is the main transducer of the Hedgehog (Hh) signaling pathway implicated in a wide range of developmental and adult processes. Smo is the target of anticancer drugs that bind to a long and narrow cavity in the 7-transmembrane (7TM) domain. X-ray structures of human Smo (hSmo) bound to several ligands have revealed 2 types of 7TM-directed antagonists: those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7TM cavity (site 2, e.g., SANT-1). Here we report the development of the acylguanidine MRT-92, which displays subnanomolar antagonist activity against Smo in various Hh cell-based assays. MRT-92 inhibits rodent cerebellar granule cell proliferation induced by Hh pathway activation through pharmacologic (half maximal inhibitory concentration [IC50] = 0.4 nM) or genetic manipulation. Using [(3)H]MRT-92 (Kd = 0.3 nM for hSmo), we created a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. Guided by molecular docking and site-directed mutagenesis data, our work convincingly confirms that MRT-92 simultaneously recognized and occupied both sites 1 and 2. Our data demonstrate the existence of a third type of Smo antagonists, those entirely filling the Smo binding cavity from the upper extracellular part to the lower cytoplasmic-proximal subpocket. Our studies should help design novel potent Smo antagonists and more effective therapeutic strategies for treating Hh-linked cancers and associated chemoresistance.
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Lucile
authors: L
authors: Hoch
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Helene
authors: H
authors: Faure
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Hermine
authors: H
authors: Roudaut
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Angele
authors: A
authors: Schoenfelder
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Andre
authors: A
authors: Mann
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Nicolas
authors: N
authors: Girard
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Laure
authors: L
authors: Bihannic
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Olivier
authors: O
authors: Ayrault
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Elena
authors: E
authors: Petricci
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Maurizio
authors: M
authors: Taddei
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
authors: Didier
authors: D
authors: Rognan
authors: *Centre National de la Recherche Scientifique, Unité Mixte de Recherche-9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department, Signal Transduction and Developmental Neuropharmacology Team, Gif-sur-Yvette, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-7200, Laboratoire d'Innovation Thérapeutique, Université de Strasbourg, Illkirch, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche-3306, Institut National de la Santé et de la Recherche Médicale U1005, Institut Curie, Centre Universitaire, Orsay, France; and Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy ruat@inaf.cnrs-gif.fr.
authors: Martial
authors: M
authors: Ruat
mesh_terms: D000328:Adult; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002528:Cerebellar Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D006146:Guanidines / Q000494:pharmacology*; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D008527:Medulloblastoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology*; D000072086:Smoothened Receptor
chemical_list: D000970:Antineoplastic Agents; D006146:Guanidines; D053823:Hedgehog Proteins; C000599830:MRT-92; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D054852:Small Molecule Libraries; D000072086:Smoothened Receptor
keywords: antagonist; medulloblastoma; molecular modeling; stem cell
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