Evidence for Allosteric Interactions of Antagonist Binding to the Smoothened Receptor

Rominger, Cynthia M.; Bee, Weilin Tiger; Copeland, Robert A.; Davenport, Elizabeth A.; Gilmartin, Aidan G.; Gontarek, Richard R.; Hornberger, Keith R.; Kallal, Lorena A.; Lai, Zhihong; Lawrie, K. W. M.; Lu, Quinn; McMillan, Lynette J.; Truong, Maggie; Tummino, Peter J.; Turunen, Brandon J.; Will, Matthew; Zuercher, William J.; Rominger, David H.

doi:10.1124/jpet.109.152090

Cited by 61 publications

(65 citation statements)

References 23 publications

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“…Within the family of GPCRs, crosstalk pharmacology between members displaying significant homology has been observed, such as between the calciumsensing receptor and the related GPCRC6A receptor (Faure et al, 2009;Rosenbaum et al, 2009 (Chen et al, 2002b). Radioligand binding analysis with tritiated SAG derivatives also revealed that the antagonist SANT-1 binds in a manner consistent with that of an allosteric modulation (Rominger et al, 2009). Itraconazole, an antifungal compound, has been shown to inhibit Hh signaling and to act on Smo at a site distinct from cyclopamine (Kim et al, 2010).…”

Section: Discussionmentioning

confidence: 90%

Identification and Mechanism of Action of the Acylguanidine MRT-83, a Novel Potent Smoothened Antagonist

et al. 2010

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Section: Discussionmentioning

confidence: 90%

Identification and Mechanism of Action of the Acylguanidine MRT-83, a Novel Potent Smoothened Antagonist

et al. 2010

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“…Various classes of Smo modulators stabilize distinct conformations. For example, cyclopamine drives Smo from the cytoplasm into the cilium in an inactive state, whereas SAG and purmorphamine stabilize an active conformation in the cilium Rominger et al, 2009;Wilson et al, 2009;Yang et al, 2009;Belgacem and Borodinsky, 2011;Roudaut et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2013

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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.

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“…CHO cells containing M1, M3, or M5 mAChR were cultured in Alpha minimum essential medium (MEM) (Gibco, Green Island, NY) with nucleosides, L-glutamine, and 10% fetal calf serum, whereas those expressing M2 and M4 mAChRs were cultured in Dulbecco's modified Eagle's medium nutrient mixture F12 media (Gibco) supplemented with 200 mg/l G418 (Geneticin) and 10% fetal calf serum. Membranes were prepared by centrifugation (1000g for 10 minutes at 4°C) and washing the cell pellet with phosphate-buffered saline followed by rapid freezing using liquid N 2 and storage at 280°C (Rominger et al, 2009). The frozen pellet was thawed, resuspended in cold hypotonic medium (40 mM Tris, pH 7.5; 1 mM MgSO 4 ; 0.5 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; 2.5 mg/l leupeptin; 0.1 mg/ml aprotinin), and incubated on ice for 5 minutes.…”

Section: Methodsmentioning

confidence: 99%

Pharmacological Characterization of GSK573719 (Umeclidinium): A Novel, Long-Acting, Inhaled Antagonist of the Muscarinic Cholinergic Receptors for Treatment of Pulmonary Diseases

Salmon¹,

Luttmann²,

Foley³

et al. 2013

J Pharmacol Exp Ther

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Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl) bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA 2 5 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED 50 value was 0.02 mg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 mg elicited 50% bronchoprotection for .24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.

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Evidence for Allosteric Interactions of Antagonist Binding to the Smoothened Receptor

Abstract: methylidene]-

Cited by 61 publications

References 23 publications

Identification and Mechanism of Action of the Acylguanidine MRT-83, a Novel Potent Smoothened Antagonist

Identification and Mechanism of Action of the Acylguanidine MRT-83, a Novel Potent Smoothened Antagonist

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

Pharmacological Characterization of GSK573719 (Umeclidinium): A Novel, Long-Acting, Inhaled Antagonist of the Muscarinic Cholinergic Receptors for Treatment of Pulmonary Diseases

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