Evidence for allosteric interactions of antagonist binding to the smoothened receptor.
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, [(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]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 [(3)H]SAG-1.3. In a functional cell-based beta-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 [(3)H]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.
Rominger
Cynthia M
CM
Oncology Center for Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania, USA. drominger@trevenainc.com
Bee
Wei-Lin Tiger
WL
Copeland
Robert A
RA
Davenport
Elizabeth A
EA
Gilmartin
Aidan
A
Gontarek
Richard
R
Hornberger
Keith R
KR
Kallal
Lorena A
LA
Lai
Zhihong
Z
Lawrie
Kenneth
K
Lu
Quinn
Q
McMillan
Lynette
L
Truong
Maggie
M
Tummino
Peter J
PJ
Turunen
Brandon
B
Will
Matthew
M
Zuercher
William J
WJ
Rominger
David H
DH
D000813:Anilides; D000818:Animals; D001549:Benzamides / Q000737:chemistry / Q000378:metabolism; D001562:Benzimidazoles / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003514:Cyclohexylamines / Q000737:chemistry / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007700:Kinetics; D051379:Mice; D015394:Molecular Structure; D009025:Morpholines / Q000737:chemistry / Q000378:metabolism; D041681:NIH 3T3 Cells; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism; D011687:Purines / Q000737:chemistry / Q000378:metabolism; D011720:Pyrazoles / Q000737:chemistry / Q000378:metabolism; D011725:Pyridines; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D000072086:Smoothened Receptor; D013876:Thiophenes / Q000737:chemistry / Q000378:metabolism; D014053:Tomatine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014704:Veratrum Alkaloids / Q000737:chemistry / Q000378:metabolism; D001618:beta-Lactamases / Q000378:metabolism
D000813:Anilides; D001549:Benzamides; D001562:Benzimidazoles; D003514:Cyclohexylamines; C538724:HhAntag691; D009025:Morpholines; D010879:Piperazines; D011687:Purines; D011720:Pyrazoles; D011725:Pyridines; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C468918:SAG compound; C468919:SANT-1 compound; C468920:SANT-2 compound; C491023:SMO protein, human; D000072086:Smoothened Receptor; D013876:Thiophenes; D014704:Veratrum Alkaloids; C003677:tomatidine; D014053:Tomatine; D001618:beta-Lactamases; C470893:purmorphamine; C000541:cyclopamine