Selective Agonists and Antagonists of Formylpeptide Receptors: Duplex Flow Cytometry and Mixture-Based Positional Scanning Libraries

Abstract

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. Although several Nformyl peptides are known to bind to these receptors, more selective small-molecule, high-affinity ligands are needed for a better understanding of the physiologic roles played by these receptors. High-throughput assays using 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 colorcoded cell lines. Screening 37 different mixture-based combinatorial libraries totaling more than five million small molecules (contained in 5,261 mixture samples) resulted in seven libraries that significantly inhibited activity at the receptors. Using positional scanning deconvolution, selective high-affinity (low nM K i ) 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 EC 50 of 131 nM (4 nM K i ) and an IC 50 of 81 nM (1 nM K i ), respectively, in intracellular Ca 21 response determinations. Comparative analyses of other previous screening approaches clearly illustrate the efficiency of identifying receptor selective, individual compounds from mixture-based combinatorial libraries.

Scaffold Ranking and Positional Scanning Utilized in the Discovery of nAChR-Selective Compounds Suitable for Optimization Studies

“…26,37 Of the compounds described here, 301 is a novel compound, never previously demonstrated to bind to nAChR. In FlexStation assays, 301 appears to have no agonist activity at α4β2 nAChR up to 10 µM.…”

Antagonism of human formyl peptide receptor 1 (FPR1) by chromones and related isoflavones

“…Although there are several examples in which compounds derived from similar chemical scaffolds have opposite functional activities (agonist vs . antagonist) at FPR1 and FPR2 (for example, see [31, 67]), no agonists of human FPR1 with a 4 H -chromen-4-one scaffold have been reported. Thus, 4 H -chromen-4-one may represent a unique chemical scaffold for development of specific FPR1 antagonists that do not have undesirable off-target effects.…”

SAR Studies of Exosite-Binding Substrate-Selective Inhibitors of A Disintegrin And Metalloprotease 17 (ADAM17) and Application as Selective in Vitro Probes

“…56 Boc-amino acids were coupled utilizing standard coupling procedures (6 equiv) with hydroxybenzotriazole hydrate (HOBt, 6 equiv) and N , N ′-diisopropylcarbodiimide (DIC, 6 equiv) in dimethylformamide (DMF, 0.1 M) for 120 min. Boc protecting groups were removed with 55% trifluoroacetic acid (TFA)/45% dichloromethane (DCM) (1×, 30 min) and subsequently neutralized with 5% diisopropylethylamine (DIEA)/95% DCM (3×, 2 min).…”