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Integration of Virtual Screening with High-Throughput Flow Cytometry to Identify Novel Small Molecule Formylpeptide Receptor Antagonists

Departments of Cytometry (B.S.E., S.M.Y., L.A.S.), Biocomputing (C.B., T.I.O.), Pathology (B.S.E., L.A.S.), Cell Biology and Physiology (E.R.P.), University of New Mexico Health Sciences Center, Albuquerque, New Mexico; and Chemical Diversity Laboratories, Inc., San Diego, California (K.V.B., N.P.S.)

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 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 2500 cells in 2 µl) analyzed at 40/min. From 52 confirmed hits (1.2% hit rate), we identified 30 potential lead compounds (inhibition constant, K_i = 1-32 µM) 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.

Address correspondence to: Tudor I. Oprea, Division of Biocomputing, MSC11 6145, Research Incubator Building S-te 190, UNM HSC, 1 University of New Mexico, Albuquerque, NM 87131-0001. E-mail: toprea{at}salud.unm.edu

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