Integration of virtual screening with high-throughput flow cytometry to identify novel small molecule formylpeptide receptor antagonists

The formylpeptide receptor (FPR) family of G-protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. We developed a FPR homology model and pharmacophore ( based on the bovine rhodopsin crystal structure and known FPR ligands, respectively) for in silico screening of similar to 480,000 drug-like small molecules. A subset of 4324 compounds that matched the pharmacophore was then physically screened with the HyperCyt flow cytometry platform in high-throughput, no-wash assays that directly measure human FPR binding, with samples (each similar to 2500 cells in 2 mu l) analyzed at 40/min. From 52 confirmed hits (1.2% hit rate), we identified 30 potential lead compounds ( inhibition constant, K-i = 1 - 32 mu M) representing nine distinct chemical families. Four compounds in one family were weak partial agonists. All others were antagonists. This virtual screening approach improved the physical screening hit rate by 12-fold ( versus 0.1% hit-rate in a random compound collection), providing an efficient process for identifying small molecule antagonists.