The development of a high-content screening binding assay for the smoothened receptor.
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.
Department of Biological Reagents and Assay Development, Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA 19426-0989, USA.
Weilin Tiger
WT
Bee
Wensheng
W
Xie
Maggie
M
Truong
Matthew
M
Will
Brandon
B
Turunen
William J
WJ
Zuercher
Lynette
L
McMillan
Hu
H
Li
Keith R
KR
Hornberger
Elizabeth A
EA
Davenport
Robert S
RS
Ames
Lorena A
LA
Kallal
D000465:Algorithms; D016367:Baculoviridae / Q000235:genetics; D001549:Benzamides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001562:Benzimidazoles / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D003514:Cyclohexylamines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009025:Morpholines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011687:Purines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011720:Pyrazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000072086:Smoothened Receptor; D013876:Thiophenes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D001549:Benzamides; D001562:Benzimidazoles; D003514:Cyclohexylamines; D005456:Fluorescent Dyes; D009025:Morpholines; D010879:Piperazines; D011687:Purines; D011720:Pyrazoles; D043562:Receptors, G-Protein-Coupled; C468918:SAG compound; C468919:SANT-1 compound; C468920:SANT-2 compound; C491023:SMO protein, human; D000072086:Smoothened Receptor; D013876:Thiophenes; C470893:purmorphamine