PT - JOURNAL ARTICLE AU - Clemencia Pinilla AU - Bruce S. Edwards AU - Jon R. Appel AU - Tina Yates-Gibbins AU - Marc A. Giulianotti AU - Jose L. Medina-Franco AU - Susan M. Young AU - Radleigh G. Santos AU - Larry A. Sklar AU - Richard A. Houghten TI - Selective Agonists and Antagonists of Formylpeptide Receptors: Duplex Flow Cytometry and Mixture-based Positional Scanning Libraries AID - 10.1124/mol.113.086595 DP - 2013 Jun 20 TA - Molecular Pharmacology PG - mol.113.086595 4099 - http://molpharm.aspetjournals.org/content/early/2013/06/20/mol.113.086595.short 4100 - http://molpharm.aspetjournals.org/content/early/2013/06/20/mol.113.086595.full AB - The formylpeptide receptor (FPR1) and formylpeptide-like 1 receptor (FPR2) are G protein coupled receptors that are linked to acute inflammatory responses, malignant glioma stem cell metastasis and chronic inflammation. While several N-formyl peptides are known to bind to these receptors, more selective small molecule high-affinity ligands are needed for a better understanding of the physiological roles played by these receptors. High throughput assays utilizing mixture-based combinatorial libraries represent a unique, highly efficient approach for rapid data acquisition and ligand identification. We report the superiority of this approach in the context of the simultaneous screening of a diverse set of mixture-based small molecule libraries. We used a single cross-reactive peptide ligand for a duplex flow cytometric screen of FPR1 and FPR2 in color-coded cell lines. Upon screening 37 different mixture-based combinatorial libraries totaling more than 5 million small molecules (contained in 5,261 mixture samples), seven libraries significantly inhibited activity at the receptors. Using positional scanning deconvolution, selective high affinity (low nM Ki) individual compounds were identified from two separate libraries, namely pyrrolidine bis-diketopiperazine and polyphenyl urea. The most active individual compounds were characterized for their functional activities as agonists or antagonists with the most potent FPR1 agonist and FPR2 antagonist identified to date with an EC50 of 131 nM (4 nM Ki) and IC50 of 81 nM (1 nM Ki), respectively, in intracellular Ca2+ response determinations. Comparative analyses of other previous screening approaches clearly illustrate the efficiency of identifying receptor selective, individual compounds from mixture-based combinatorial libraries.