PT  - JOURNAL ARTICLE
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
TI  - Evidence for Allosteric Interactions of Antagonist Binding to the Smoothened Receptor
AID  - 10.1124/jpet.109.152090
DP  - 2009 Jun 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 995--1005
VI  - 329
IP  - 3
4099  - http://jpet.aspetjournals.org/content/329/3/995.short
4100  - http://jpet.aspetjournals.org/content/329/3/995.full
SO  - J Pharmacol Exp Ther2009 Jun 01; 329
AB  - 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