Discovery, Molecular and Pharmacological Characterization of GSA-10, a Novel Small-Molecule Positive Modulator of Smoothened

• Published in: Molecular pharmacology 2013-05, Vol.83 (5), p.1020-1029
• Main Authors: Gorojankina, Tatiana, Hoch, Lucile, Faure, Hélène, Roudaut, Hermine, Traiffort, Elisabeth, Schoenfelder, Angèle, Girard, Nicolas, Mann, André, Manetti, Fabrizio, Solinas, Antonio, Petricci, Elena, Taddei, Maurizio, Ruat, Martial
• Format: Article
• Language: English
• Subjects: Benzoates - pharmacology; Binding Sites - drug effects; Bone Morphogenetic Protein Receptors - metabolism; Cell Differentiation - drug effects; Cell Line; Cyclic AMP - metabolism; Cyclohexylamines - pharmacology; Hedgehog Proteins - metabolism; HEK293 Cells; Humans; Life Sciences; Ligands; Neurons and Cognition; Quinolines - pharmacology; Receptors, G-Protein-Coupled - agonists; Receptors, G-Protein-Coupled - antagonists & inhibitors; Receptors, G-Protein-Coupled - metabolism; Small Molecule Libraries; Smoothened Receptor; Thiophenes - pharmacology; Transcription Factors - metabolism; Wnt Proteins - metabolism; Zinc Finger Protein GLI1
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.112.084590

Activation of the Smoothened (Smo) receptor mediates Hedgehog (Hh) signaling. Hh inhibitors are in clinical trials for cancer, and small-molecule Smo agonists may have therapeutic interests in regenerative medicine. Here, we have generated and validated a pharmacophoric model for Smo agonists and used this model for the virtual screening of a library of commercially available compounds. Among the 20 top-scoring ligands, we have identified and characterized a novel quinolinecarboxamide derivative, propyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido) benzoate, (GSA-10), as a Smo agonist. GSA-10 fits to the agonist pharmacophoric model with two hydrogen bond acceptor groups and four hydrophobic regions. Using pharmacological, biochemical, and molecular approaches, we provide compelling evidence that GSA-10 acts at Smo to promote the differentiation of multipotent mesenchymal progenitor cells into osteoblasts. However, this molecule does not display the hallmarks of reference Smo agonists. Remarkably, GSA-10 does not recognize the classic bodipy-cyclopamine binding site. Its effect on cell differentiation is inhibited by Smo antagonists, such as MRT-83, SANT-1, LDE225, and M25 in the nanomolar range, by GDC-0449 in the micromolar range, but not by cyclopamine and CUR61414. Thus, GSA-10 allows the pharmacological characterization of a novel Smo active site, which is notably not targeted to the primary cilium and strongly potentiated by forskolin and cholera toxin. GSA-10 belongs to a new class of Smo agonists and will be helpful for dissecting Hh mechanism of action, with important implications in physiology and in therapy.