TY - JOUR T1 - Evidence for Allosteric Interactions of Antagonist Binding to the Smoothened Receptor JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 995 LP - 1005 DO - 10.1124/jpet.109.152090 VL - 329 IS - 3 AU - Cynthia M. Rominger AU - Wei-Lin Tiger Bee AU - Robert A. Copeland AU - Elizabeth A. Davenport AU - Aidan Gilmartin AU - Richard Gontarek AU - Keith R. Hornberger AU - Lorena A. Kallal AU - Zhihong Lai AU - Kenneth Lawrie AU - Quinn Lu AU - Lynette McMillan AU - Maggie Truong AU - Peter J. Tummino AU - Brandon Turunen AU - Matthew Will AU - William J. Zuercher AU - David H. Rominger Y1 - 2009/06/01 UR - http://jpet.aspetjournals.org/content/329/3/995.abstract N2 - The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [3H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [3H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [3H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [3H]SAG-1.3. In a functional cell-based β-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed “Schild-type” radioligand binding analysis with [3H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway. The American Society for Pharmacology and Experimental Therapeutics ER -