ABSTRACT
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.
Subject(s)
Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Anilides , Animals , Benzamides/chemistry , Benzamides/metabolism , Benzimidazoles/chemistry , Benzimidazoles/metabolism , Binding Sites , Binding, Competitive , Cell Line , Cell Membrane/metabolism , Cyclohexylamines/chemistry , Cyclohexylamines/metabolism , Genes, Reporter/genetics , Humans , Kinetics , Mice , Molecular Structure , Morpholines/chemistry , Morpholines/metabolism , NIH 3T3 Cells , Piperazines/chemistry , Piperazines/metabolism , Purines/chemistry , Purines/metabolism , Pyrazoles/chemistry , Pyrazoles/metabolism , Pyridines , Radioligand Assay , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/chemistry , Recombinant Proteins/agonists , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Smoothened Receptor , Thiophenes/chemistry , Thiophenes/metabolism , Tomatine/analogs & derivatives , Tomatine/chemistry , Tomatine/metabolism , Transfection , Veratrum Alkaloids/chemistry , Veratrum Alkaloids/metabolism , beta-Lactamases/metabolismABSTRACT
Apelin peptides have recently been identified to be the endogenous ligands for the G protein-coupled receptor APJ. However, little is known about the physiological roles of this ligand-receptor pairing. In the present study we investigated the pharmacology of several apelin analogues at the human recombinant APJ receptor using radioligand binding and functional assays. This has led to the identification of key residues in the apelin peptide required for functional potency and binding affinity through structure-activity studies. In particular, we have identified that replacement of leucine in position 5, or arginine in position 2 and 4 of the C-terminal apelin peptide, apelin-13, resulted in significant changes in pharmacology. We also investigated the detailed localization of pre-proapelin and APJ receptor mRNA in a wide range of human, rat and mouse tissues using quantitative RT-PCR, and carried out a detailed immunohistochemical study of the distribution of the APJ receptor in rat brain and spinal cord. Interestingly, the APJ receptor was not only co-localized in white matter with GFAP in the spinal cord, but was also clearly localized on neurones in the brain, suggesting that this receptor and its peptide may be involved in a wide range of biological process yet to be determined.
