Identification of Novel Small-Molecule Agonists for Human Formyl Peptide Receptors and Pharmacophore Models of Their Recognition

Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA.
Molecular pharmacology (Impact Factor: 4.13). 11/2009; 77(2):159-70. DOI: 10.1124/mol.109.060673
Source: PubMed


N-formyl peptide receptor (FPR1) and N-formyl peptide receptor-like 1 (FPRL1, now known as FPR2) are G protein-coupled receptors involved in host defense and sensing cellular dysfunction. Because of the potential for FPR1/FPR2 as a therapeutic target, our recent high-throughput screening efforts have focused on the identification of unique nonpeptide agonists of FPR1/FPR2. In the present studies, we screened a chemolibrary of drug-like molecules for their ability to induce intracellular calcium mobilization in RBL-2H3 cells transfected with human FPR1 or FPR2. Screening of these compounds resulted in the identification of novel and potent agonists that activated both FPR1 and FPR2, as well as compounds that were specific for either FPR1 or FPR2 with EC(50) values in the low micromolar range. Specificity of the compounds was supported by analysis of calcium mobilization in HL-60 cells transfected with human FPR1 and FPR2. In addition, all but one agonist activated intracellular calcium flux and chemotaxis in human neutrophils, irrespective of agonist specificity for FPR1 or FPR2. Molecular modeling of the group of FPR1 and FPR2 agonists using field point methodology allowed us to create pharmacophore models for ligand binding sites and formulate requirements for these specific N-formyl peptide receptor agonists. These studies further demonstrate that agonists of FPR1/FPR2 include compounds with wide chemical diversity and that analysis of such compounds can enhance our understanding of their ligand/receptor interaction.

Download full-text


Available from: Andrei I Khlebnikov,
  • Source
    • "Compared with existing high-throughput screening (HTS) programs, in which hundreds of thousands of individual compounds are screened against therapeutically important targets, millions of compounds formatted as mixtures can be examined using substantially less material and at much reduced time and labor than if these same mixture-based diversities were made and screened as individual compounds (Houghten et al., 1999, 2008; Pinilla et al., 2003). Formylpeptide receptors (FPRs) are a small family of G protein–coupled receptors known to be important in host defense and inflammation, and studies have been carried out to identify small-molecule ligands to characterize the structure and function of these receptors (Ye et al., 2009; Kirpotina et al., 2010). The two receptors of the FPR family that are studied here are formylpeptide receptor (FPR1), which is linked to antibacterial inflammation (Le et al., 2002) and malignant glioma cell metastasis (Zhou et al., 2005), and formylpeptide-like 1 receptor 1 (FPR2; formerly known as FPRL-1), which is linked to chronic inflammation in systemic amyloidosis, Alzheimer disease, and prion diseases (Le et al., 2001). "
    [Show abstract] [Hide abstract]
    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. While several N-formyl peptides are known to bind to these receptors, more selective small molecule high-affinity ligands are needed for a better understanding of the physiological roles played by these receptors. High throughput assays utilizing 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 color-coded cell lines. Upon screening 37 different mixture-based combinatorial libraries totaling more than 5 million small molecules (contained in 5,261 mixture samples), seven libraries significantly inhibited activity at the receptors. Using positional scanning deconvolution, selective high affinity (low nM Ki) 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 EC50 of 131 nM (4 nM Ki) and IC50 of 81 nM (1 nM Ki), respectively, in intracellular Ca(2+) response determinations. Comparative analyses of other previous screening approaches clearly illustrate the efficiency of identifying receptor selective, individual compounds from mixture-based combinatorial libraries.
    Molecular pharmacology 06/2013; 84(3). DOI:10.1124/mol.113.086595 · 4.13 Impact Factor
  • Source
    • "In previous studies investigating FPR agonists, we observed differential activity between FPR-transfected cells and primary neutrophils [10] [11] [15], although neutrophils still responded to all agonists that activated FPR-expressing HL-60 cells. Thus, the NO 2 -and CN-substituted compounds reported here seem to have properties that affect their ability to stimulate Ca 2+ flux or that interfere with the assay system. "
    [Show abstract] [Hide abstract]
    ABSTRACT: N-formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. Recent studies indicated that FPRs have stereo-selective preference for chiral ligands. Here, we investigated the structure-activity relationship of 24 chiral ureidopropanamides, including previously reported compounds PD168368/PD176252 and their close analogs, and used molecular modeling to define chiral recognition by FPR2. Unlike previously reported 6-methyl-2,4-disubstituted pyridazin-3(2H)-ones, whose R-forms preferentially activated FPR1/FPR2, we found that four S-enantiomers in the seven ureidopropanamide pairs tested preferentially activated intracellular Ca(2+) flux in FPR2-transfected cells, while the R-counterpart was more active in two enantiomer pairs. Thus, active enantiomers of FPR2 agonists can be in either R- or S- configurations, depending on the molecular scaffold and specific substituents at the chiral center. Using molecular modeling approaches, including field point methodology, homology modeling, and docking studies, we propose a model that can explain stereoselective activity of chiral FPR2 agonists. Importantly, our docking studies of FPR2 chiral agonists correlated well with the FPR2 pharmacophore model derived previously. We conclude that the ability of FPR2 to discriminate between the enantiomers is the consequence of the arrangement of the three asymmetric hydrophobic subpockets at the main orthosteric FPR2 binding site with specific orientation of charged regions in the subpockets.
    Biochemical pharmacology 12/2012; 85(3). DOI:10.1016/j.bcp.2012.11.015 · 5.01 Impact Factor
  • Source
    • "Note, however, that modification of the R 2 substituent did affect agonist selectivity and/or potency. For example, comparison of our previously reported N-phenethyl-NЈ-phenylurea FPR2-specific agonists (Kirpotina et al., 2010) with the Trp/Phe-based FPR agonists and their related analogs identified here demonstrated that introduction of additional heterocycle-containing groups to the carbon atom in the ␣-position to the carbamide fragment increased potency at FPR2 but led to loss of specificity. Likewise, introduction of an ethyl acetate group into the meta position of the N-piperidine ring increased agonist activity (compare AG-10/5 and AG-10/9), but shifting of the ethyl acetate group from the meta to the para position resulted in decrease FPR2 activity and loss of FPR1 and FPR3 activity (compare AG-10/9 and AG-10/10). "
    [Show abstract] [Hide abstract]
    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.
    Molecular pharmacology 10/2010; 79(1):77-90. DOI:10.1124/mol.110.068288 · 4.13 Impact Factor
Show more