Received for publication December 5, 2006.
Revised January 11, 2007.
Accepted for publication January 17, 2007.

High-throughput Screening for Small-molecule Activators of Neutrophils: Identification of Novel N-Formyl Peptide Receptor Agonists

Abstract

We screened a chemolibrary of drug-like molecules for their ability to activate reactive oxygen species (ROS) production in murine phagocytes and identified 26 novel compounds with potent neutrophil activating properties. We used substructure screening, fragment-focusing, and structure-activity relationship (SAR) analyses to further probe the parent library and defined at least two groups of activators of ROS production in murine neutrophils: t-butyl benzene and thiophene-2-amide-3-carboxylic ester derivatives. Further studies of the active compounds revealed 11 compounds that activated ROS production in human neutrophils, and 6 of these compounds also activated intercellular Ca²⁺ mobilization and chemotaxis in human neutrophils. Of the latter compounds, compound 14 (1,3-benzodioxolane-5-carboxylic acid 4'-benzyloxy-3'-methoxybenzylidene-hydrazide) activated neutrophils at nM concentrations, and Ca²⁺ mobilization was inhibited by pertussis toxin and N-t-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc-2), an antagonist of formyl peptide receptors (FPR/FPRL1). Likewise, activation by compound 14 was desensitized after N-formyl-Met-Leu-Phe (fMLF) pretreatment. Similar biological activities were found for compound 104 (1,3-benzodioxolane-5-carboxylic acid 3'-bromo-5'-ethoxy-4'-hydroxybenzylidene-hydrazide), an analog of compound 14. Furthermore, conformational analysis of the activators of chemotaxis and Ca²⁺ mobilization showed a high degree of similarity in distances between pharmacophore points of compounds 14 and 104 with a model of FPR published by Edwards et al. (Mol. Pharm. 2005, 68: 1301-1310), indicating conformational features of the agonists identified here are structurally-compatible with steric constraints of the ligand-binding pocket of the receptor. Based on these results, we conclude that compounds 14 and 104 represent novel small-molecule agonists of FPR. These studies enhance our understanding of FPR ligand/receptor interactions and structure/activity relationships of phagocyte agonists.

Key words: Chemotactic peptides, Gi family, Structure-activity relationships and modeling

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