Gastrin-releasing peptide/neuromedin B receptor antagonists PD176252, PD168368, and related analogs are potent agonists of human formyl-peptide receptors

Abstract

N-Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) involved in host defense and sensing cellular dysfunction. Thus, FPRs represent important therapeutic targets. In the present studies, we screened 32 ligands (agonists and antagonists) of unrelated GPCRs for their ability to induce intracellular Ca²+ mobilization in human neutrophils and HL-60 cells transfected with human FPR1, FPR2, or FPR3. Screening of these compounds demonstrated that antagonists of gastrin-releasing peptide/neuromedin B receptors (BB₁/BB₂) PD168368 [(S)-a-methyl-a-[[[(4-nitrophenyl)amino]carbonyl]amino]-N-[[1-(2-pyridinyl) cyclohexyl]methyl]-1H-indole-3-propanamide] and PD176252 [(S)-N-[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]-a-methyl-a-[[-(4-nitrophenyl)amino]carbonyl]amino-1H-indole-3-propanamide] were potent mixed FPR1/FPR2 agonists, with nanomolar EC₅₀ values. Cholecystokinin-1 receptor agonist A-71623 [Boc-Trp-Lys(ε-N-2-methylphenylaminocarbonyl)-Asp-(N-methyl)-Phe-NH₂] was also a mixed FPR1/FPR2 agonist, but with a micromolar EC₅₀. Screening of 56 Trp- and Phe-based PD176252/PD168368 analogs and 41 related nonpeptide/nonpeptoid analogs revealed 22 additional FPR agonists. Most were potent mixed FPR1/FPR2/FPR3 agonists with nanomolar EC₅₀ values for FPR2, making them among the most potent nonpeptide FPR2 agonists reported to date. In addition, these agonists were also potent chemoattractants for murine and human neutrophils and activated reactive oxygen species production in human neutrophils. Molecular modeling of the selected agonists using field point methods allowed us to modify our previously reported pharmacophore model for the FPR2 ligand binding site. This model suggests the existence of three hydrophobic/aromatic subpockets and several binding poses of FPR2 agonists in the transmembrane region of this receptor. These studies demonstrate that FPR agonists could include ligands of unrelated GPCR and that analysis of such compounds can enhance our understanding of pharmacological effects of these ligands.

Fig. 1.

Structure and activity of selected GPCR agonists. A, chemical structures of CCK-1 receptor agonist A-71623 and bombesin-related receptor BB₁ and BB₂ antagonists PD176252 and PD168368. B, human neutrophils were treated with 300 nM PD176252 or PD168368, 20 μM A-716235, 5 nM fMLF (positive control), or 1% DMSO (negative control), and Ca²⁺ mobilization was monitored for the indicated times (arrow indicates when treatment was added). Arrow indicates time of treatment addition. C, human neutrophils were treated with the indicated concentrations of PD168368, PD176252, A-716235, and fMLF (all in micromolar), and MPO release was determined as described under Materials and Methods. The data are presented as mean ± S.D. of triplicate samples. In B and C, the data are from one experiment that is representative of three independent experiments.

Fig. 2.

Stimulation of human neutrophil migration by selected compounds. Human neutrophil chemotaxis toward the indicated concentrations of AG-10/1 and AG-10/2 was determined, as described under Materials and Methods. The data are presented as the mean ± S.D. of triplicate samples from one experiment that is representative of three independent experiments.

Fig. 3.

ROS production by human neutrophils treated with WKYMVm or AG-10/22. A, kinetic curves of ROS production induced by 100 nM WKYMVm or 100 nM AG-10/22. Arrow indicates time of treatment addition. B, integrated luminescence (120 s) induced in human neutrophils plotted against the compound concentration. The data are presented as the mean ± S.D. of triplicate samples. A representative experiment from three independent experiments is shown in each panel.

Fig. 4.

Desensitization of Ca²⁺ mobilization in human neutrophils by selected FPR agonists. Human neutrophils were loaded with Fluo-4AM dye and pretreated with vehicle (DMSO), PD168368 (10 μM), AG-10/16 (100 nM), 5 nM fMLF (A), 5 nM WKYMVm (B), or 5 nM WKYMVM (C), and Ca²⁺ mobilization was monitored. The same wells were then treated with one of peptides (in 5 nM concentrations) as indicated, and Ca²⁺ mobilization was monitored after this second treatment. In each panel, the data are from representative experiments from three independent experiments.

Fig. 5.

Multimolecule template for FPR2 and alignments of two molecules on the template. A, the multimolecule template was created using the best conformations of the following five molecules: AG-10/5, AG-10/8, AG-10/17, PD168368, and Frohn-11. Field points are colored as follows: blue, electron-rich (negative); red, electron-deficient (positive); yellow, van der Waals attractive (steric); and orange, hydrophobic. B, alignments for Cilibrizzi-14x and AG-09/42 in the template represent examples of two different modes of ligand-receptor interaction with the three hypothetical receptor subpockets I, II, and III. Arrows indicate directions of alignments for AG-09/42 in subpockets I/II and for Cilibrizzi-14x in subpockets I/III. Negative field points (blue spheres A and B) correspond to the receptor's positively charged regions (e.g., amino and hydroxyl groups in the active site that are capable of forming hydrogen bonds with electronegative atoms of the agonist). Positive field points (red sphere C) correspond to the receptor's negatively charged regions or to hydrogen bond acceptors in the FPR2 active site. Spheres H₁, H₂, and H₃ correspond to hydrophobic centers. Substituents R₁, R₂, and R₃ may influence lipophilicity, molar refraction, and atomic charges for respective groups of particular FPR2 agonists. Dashed lines show correspondences between centers of the main field points on the multimolecule template (A) and their schematic representations in B.