Document Detail

Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes.
MedLine Citation:
PMID:  18785269     Owner:  NLM     Status:  MEDLINE    
Of recent, clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to antibacterial inflammation and malignant glioma cell metastasis; and FPR like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease, and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow-cytometry-based high-throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein- labeled peptide ligand from FPR, FPRL1, or both. U937 cells expressing FPR and rat basophil leukemia (RBL) cells expressing FPRL1 were tested together in a "duplex" format. The U937 cells were color coded with red-fluorescent dye allowing their distinction during analysis. Compounds, cells, and fluorescent ligand were sequentially combined (no wash) in 15 microl assay volumes in 384-well plates. Throughput averaged approximately 11 min per plate to analyze approximately 4,000 cells ( approximately 2,000/receptor) in a 2 microl aspirate from each well. In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (K(i)) < or = 4 microM (34 for FPR and 6 for FPRL1). An additional 1,446 candidate compounds were selected by structure-activity-relationship analysis of the hits and screened to identify novel ligands for FPR (3570-0208, K(i) = 95 +/- 10 nM) and FPRL1 (BB-V-115, K(i) = 270 +/- 51 nM). Each was a selective antagonist in calcium response assays and the most potent small molecule antagonist reported for its respective receptor to date. The duplex assay format reduced assay time, minimized reagent requirements, and provided selectivity information at every screening stage, thus proving to be an efficient means to screen for selective receptor ligand probes.
Susan M Young; Cristian M Bologa; Dan Fara; Bj K Bryant; Juan Jacob Strouse; Jeffrey B Arterburn; Richard D Ye; Tudor I Oprea; Eric R Prossnitz; Larry A Sklar; Bruce S Edwards
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Cytometry. Part A : the journal of the International Society for Analytical Cytology     Volume:  75     ISSN:  1552-4930     ISO Abbreviation:  Cytometry A     Publication Date:  2009 Mar 
Date Detail:
Created Date:  2009-02-19     Completed Date:  2009-06-22     Revised Date:  2014-09-21    
Medline Journal Info:
Nlm Unique ID:  101235694     Medline TA:  Cytometry A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  253-63     Citation Subset:  IM    
Copyright Information:
2008 International Society for Advancement of Cytometry.
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MeSH Terms
Cells, Cultured
Chemotactic Factors / metabolism
Flow Cytometry / methods*
Fluorescent Dyes
Molecular Probes / analysis
Receptors, Formyl Peptide / antagonists & inhibitors*,  metabolism
Receptors, Lipoxin / antagonists & inhibitors*,  metabolism
Sensitivity and Specificity
U937 Cells
Grant Support
R03 MH076381/MH/NIMH NIH HHS; R03 MH076381-01/MH/NIMH NIH HHS; R03 MH076381-01/MH/NIMH NIH HHS; U54 MH074425/MH/NIMH NIH HHS; U54 MH074425-01/MH/NIMH NIH HHS
Reg. No./Substance:
0/Chemotactic Factors; 0/FPR2 protein, human; 0/Fluorescent Dyes; 0/Ligands; 0/Molecular Probes; 0/Receptors, Formyl Peptide; 0/Receptors, Lipoxin

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