title: Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes.
abstract: 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.
authors.affiliation: University of New Mexico, Albuquerque, 87131, USA.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Young
authors.forename: Cristian M
authors.initials: CM
authors.lastname: Bologa
authors.forename: Dan
authors.initials: D
authors.lastname: Fara
authors.forename: Bj K
authors.initials: BK
authors.lastname: Bryant
authors.forename: Juan Jacob
authors.initials: JJ
authors.lastname: Strouse
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Arterburn
authors.forename: Richard D
authors.initials: RD
authors.lastname: Ye
authors.forename: Tudor I
authors.initials: TI
authors.lastname: Oprea
authors.forename: Eric R
authors.initials: ER
authors.lastname: Prossnitz
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Edwards
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002630:Chemotactic Factors / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D008024:Ligands; D015335:Molecular Probes / Q000032:analysis; D051381:Rats; D044042:Receptors, Formyl Peptide / Q000037:antagonists & inhibitors* / Q000378:metabolism; D044067:Receptors, Lipoxin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012680:Sensitivity and Specificity; D020298:U937 Cells
chemical_list: D002630:Chemotactic Factors; C105101:FPR2 protein, human; D005456:Fluorescent Dyes; D008024:Ligands; D015335:Molecular Probes; D044042:Receptors, Formyl Peptide; D044067:Receptors, Lipoxin
references.citation: Blood. 2003 Feb 15;101(4):1572-81
references.pmid: 12393391
references.citation: J Exp Med. 1995 Jul 1;182(1):207-17
references.pmid: 7540650
references.citation: J Exp Med. 1982 Jan 1;155(1):264-75
references.pmid: 6274994
references.citation: J Biol Chem. 1992 Apr 15;267(11):7637-43
references.pmid: 1373134
references.citation: J Mol Cell Cardiol. 1997 Mar;29(3):881-94
references.pmid: 9152849
references.citation: Bioorg Med Chem Lett. 2006 Jul 15;16(14):3713-8
references.pmid: 16697190
references.citation: Cytokine Growth Factor Rev. 2001 Mar;12(1):91-105
references.pmid: 11312121
references.citation: J Natl Cancer Inst. 2005 Jun 1;97(11):823-35
references.pmid: 15928303
references.citation: J Exp Med. 2000 Oct 2;192(7):1069-74
references.pmid: 11015447
references.citation: Annu Rev Immunol. 1991;9:617-48
references.pmid: 1910690
references.citation: J Exp Med. 1999 Feb 15;189(4):657-62
references.pmid: 9989980
references.citation: Clin Immunol. 2000 Sep;96(3):236-42
references.pmid: 10964542
references.citation: J Immunol. 2003 Aug 15;171(4):2050-6
references.pmid: 12902510
references.citation: J Immunol. 1975 Jun;114(6):1831-7
references.pmid: 165239
references.citation: Biochem Biophys Res Commun. 1992 Apr 30;184(2):582-9
references.pmid: 1374236
references.citation: Pharmacol Ther. 1997;74(1):73-102
references.pmid: 9336017
references.citation: Mol Cell. 2000 May;5(5):831-40
references.pmid: 10882119
references.citation: J Neuroimmunol. 1995 Aug;61(1):71-8
references.pmid: 7560015
references.citation: Proc Natl Acad Sci U S A. 1998 May 26;95(11):6448-53
references.pmid: 9600986
references.citation: J Immunol. 1999 May 15;162(10):5924-30
references.pmid: 10229829
references.citation: Mol Cancer Ther. 2007 Oct;6(10):2618-25
references.pmid: 17938258
references.citation: Curr Opin Hematol. 1999 Jan;6(1):15-24
references.pmid: 9915549
references.citation: Mol Pharmacol. 2007 Oct;72(4):976-83
references.pmid: 17652444
references.citation: Genomics. 1992 Jun;13(2):437-40
references.pmid: 1612600
references.citation: J Neuroimmunol. 2000 Nov 1;111(1-2):102-8
references.pmid: 11063827
references.citation: Cytometry. 1998 Oct 1;33(2):234-43
references.pmid: 9773885
references.citation: J Exp Med. 1999 Jan 18;189(2):395-402
references.pmid: 9892621
references.citation: Cytometry A. 2003 May;53(1):55-65
references.pmid: 12701132
references.citation: J Exp Med. 2000 Apr 3;191(7):1197-208
references.pmid: 10748237
references.citation: J Neurooncol. 2008 Jan;86(1):47-53
references.pmid: 17611713
references.citation: J Exp Med. 1998 Apr 20;187(8):1285-94
references.pmid: 9547339
references.citation: Nature. 1996 Mar 28;380(6572):345-7
references.pmid: 8598929
references.citation: Cytometry. 2001 May 1;44(1):83-90
references.pmid: 11309812
references.citation: J Immunol. 2001 Feb 1;166(3):1448-51
references.pmid: 11160182
references.citation: J Recept Res. 1988;8(1-4):533-46
references.pmid: 3385692
references.citation: J Biomol Screen. 2005 Jun;10(4):374-82
references.pmid: 15964939
references.citation: J Immunol. 1995 Oct 1;155(7):3292-5
references.pmid: 7561021
references.citation: Annu Rev Immunol. 1994;12:593-633
references.pmid: 8011292
references.citation: Trends Biotechnol. 2002 Jan;20(1):9-12
references.pmid: 11742671
references.citation: Trends Immunol. 2002 Nov;23(11):541-8
references.pmid: 12401407
references.citation: Int Immunopharmacol. 2002 Jan;2(1):1-13
references.pmid: 11789660
references.citation: J Biol Chem. 1984 May 10;259(9):5430-9
references.pmid: 6371005
references.citation: Mol Pharmacol. 2007 Apr;71(4):1061-74
references.pmid: 17229869
references.citation: Mol Pharmacol. 2004 Nov;66(5):1213-22
references.pmid: 15308762
references.citation: Nat Methods. 2006 May;3(5):361-8
references.pmid: 16628206
references.citation: Mol Pharmacol. 2008 Aug;74(2):392-402
references.pmid: 18458054
references.citation: Int Immunol. 1993 Oct;5(10):1239-49
references.pmid: 7505609
references.citation: Proc Natl Acad Sci U S A. 1975 Mar;72(3):1059-62
references.pmid: 1093163
references.citation: J Neurosci. 2001 Jan 15;21(2):RC123
references.pmid: 11160457
references.citation: FASEB J. 2001 Nov;15(13):2454-62
references.pmid: 11689470
references.citation: Nat Biotechnol. 2002 Feb;20(2):155-62
references.pmid: 11821861
references.citation: J Biol Chem. 2001 Jun 29;276(26):23645-52
references.pmid: 11316806
references.citation: Mol Pharmacol. 2005 Nov;68(5):1301-10
references.pmid: 16118363