ABSTRACT
In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-µM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.
Subject(s)
Benzamides/metabolism , Benzimidazoles/metabolism , Cyclohexylamines/metabolism , Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Thiophenes/metabolism , Algorithms , Baculoviridae/genetics , Benzamides/chemistry , Benzamides/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Cell Line , Cyclohexylamines/chemistry , Cyclohexylamines/pharmacology , Fluorescent Dyes , Genes, Reporter , HEK293 Cells , Humans , Morpholines/chemistry , Morpholines/metabolism , Morpholines/pharmacology , Piperazines/chemistry , Piperazines/metabolism , Piperazines/pharmacology , Protein Binding , Purines/chemistry , Purines/metabolism , Purines/pharmacology , Pyrazoles/chemistry , Pyrazoles/metabolism , Pyrazoles/pharmacology , Smoothened Receptor , Thiophenes/chemistry , Thiophenes/pharmacologyABSTRACT
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
A series of 2-aminomethyl piperidines has been discovered as novel urotensin-II receptor antagonists. The synthesis, initial structure-activity relationships, and optimization of the initial hit that resulted in the identification of potent, cross-species active, and functional urotensin-II receptor antagonists such as 1a and 11a are described.