Integrating High-Content Analysis into a Multiplexed Screening Approach to Identify and Characterize GPCR Agonists.
G protein-coupled receptors (GPCRs) are one of the most popular and proven target classes for therapeutic intervention. The increased appreciation for allosteric modulation, receptor oligomerization, and biased agonism has led to the development of new assay platforms that seek to capitalize on these aspects of GPCR biology. High-content screening is particularly well suited for GPCR drug discovery given the ability to image and quantify changes in multiple cellular parameters, to resolve subcellular structures, and to monitor events within a physiologically relevant environment. Focusing on the sphingosine-1-phosphate (S1P1) receptor, we evaluated the utility of high-content approaches in hit identification efforts by developing and applying assays to monitor β-arrestin translocation, GPCR internalization, and GPCR recycling kinetics. Using these approaches in combination with more traditional GPCR screening assays, we identified compounds whose unique pharmacological profiles would have gone unnoticed if using a single platform. In addition, we identified a compound that induces an atypical pattern of β-arrestin translocation and GPCR recycling kinetics. Our results highlight the value of high-content imaging in GPCR drug discovery efforts and emphasize the value of a multiassay approach to study pharmacological properties of compounds of interest.
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
Yingjie
Y
Zhu
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
John
J
Watson
Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA.
Mengjie
M
Chen
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
Ding Ren
DR
Shen
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
Melissa
M
Yarde
Medicinal Chemistry (High-Throughput Biology), Boehringer Ingelheim, Ridgefield, CT, USA.
Michele
M
Agler
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
Neil
N
Burford
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
Andrew
A
Alt
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
Sukhanya
S
Jayachandra
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
Mary Ellen
ME
Cvijic
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
Litao
L
Zhang
Discovery Chemistry, Bristol-Myers Squibb, Wallingford, CT, USA.
Alaric
A
Dyckman
Discovery Biology, Bristol-Myers Squibb, Wallingford, CT, USA.
Jenny
J
Xie
High Throughput Screening, Forma Therapeutics, Watertown, MA, USA.
Jonathan
J
O'Connell
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA.
Martyn
M
Banks
Lead Discovery and Optimization, Bristol-Myers Squibb, Wallingford, CT, USA andrea.weston@bms.com.
Andrea
A
Weston
D000495:Allosteric Site; D000818:Animals; D001681:Biological Assay / Q000379:methods; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000737:chemistry; D055808:Drug Discovery; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000819:agonists*; D049349:Receptors, Lysosphingolipid / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D015203:Reproducibility of Results; D000071557:beta-Arrestins / Q000378:metabolism
D043562:Receptors, G-Protein-Coupled; D049349:Receptors, Lysosphingolipid; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP
G protein–coupled receptors (GPCR); cell-based assays; high-content screening; image analysis