Compound and several additional FPR antagonists identified h
Compound 10 and several additional FPR1 antagonists identified here specifically blocked fMLF-induced responses mediated via FPR1 in FPR1-HL60 ikk and human neutrophils, but not responses mediated via FPR2 or FPR3 (in human neutrophils and transfected HL60 cells) or Fpr1 (murine neutrophils and transfected RBL cells). These compounds also did not inhibit IL-8-induced Ca2+ mobilization in RBL cells transfected with CXCR1 and human neutrophils. The selected chromone/isoflavone derivatives (Table 2) were also inactive as direct agonists of FPR2 and Fpr1 and did not activate Ca2+ flux in murine and human neutrophils. 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 , ), no agonists of human FPR1 with a 4H-chromen-4-one scaffold have been reported. Thus, 4H-chromen-4-one may represent a unique chemical scaffold for development of specific FPR1 antagonists that do not have undesirable off-target effects. In addition to mobilizing calcium from intracellular stores, fMLF activates the mitogen-activated protein kinases ERK1/2 , . We evaluated this response to test the relative potency of selected FPR1 antagonists. Compounds 10 and 36, which exhibited high potency in the Ca2+ flux assay, also inhibited fMLF-induced ERK phosphorylation. In comparison, antagonists with low potency in the Ca2+ flux assay, such as 15 and 38, or the inactive compound 35 had much less or no effect on ERK1/2 phosphorylation. These results verify the Ca2+ flux data and together established a dose-effect relationship for these FPR1 antagonists. Comprehensive SAR analysis of all 100 analogs was performed mainly at positions 2 and 7 of the chromone scaffold due to the apparent importance of these regions of the molecules. Our analysis suggests the importance of a small hydrophobic group (CH3/CF3) at position 2 of the heterocycle (R3 functionality) for FPR1 antagonist activity. Substitution at position 7 of the chromone scaffold also had effects on antagonist activity, but a wider range of modifications was tolerated. Although several substituents at positions 6 and 2′ of the chromone and isoflavone scaffolds were evaluated, modification of these regions will be necessary for further optimization of chromone-based FPR1 antagonists. Our molecular modeling showed a noticeably higher degree of similarity of the active molecules to the pharmacophore model, which was developed based on the structure of previously reported potent FPR1 antagonists with diverse chemical scaffolds, including chromone 1, methionine-derived benzimidazole 5, and pyrazolo[1,5-a]pyrimidin-7(1H)-one 7. This reflects the main hypothesis of molecular recognition using a field point approach : molecules of different chemical groups have the same biological action if they have similar geometrical arrangement of key pharmacophore features, including hydrophobic, electronegative, or electropositive features. Indeed, a significant statistical difference between groups of FPR1 antagonists with different sub-scaffolds and their inactive derivatives with respect to their similarity scores compared to the 3-molecule FPR1 antagonist pharmacophore was observed. The docking study of the most potent FPR1 antagonist 10 showed that the subpockets of FPR1 binding site could also fully accommodate the FPR1 antagonist pharmacophore. Further studies are clearly required to define the nature of molecular interactions between the chromone/isoflavone based ligands and FPR1 binding site. Although biochanin A (compound 94) inhibited fMLF-induced Ca2+ flux in FPR1-HL60 cells and human neutrophils, this natural occurring isoflavone did not displace WKYMVm-FITC from FPR1-HL60 cells. In addition, biochanin A and its synthetic 2-trifluoromethyl derivative 95 had direct agonist activity in cell lines and primary neutrophils. Thus, compounds 94 and 95 may activate Ca2+ flux via FPR-independent pathways, which leads to cross-desensitization of FPR1 and/or cross-talk with the receptor. Indeed, several other synthetic and natural flavonoids (wogonin, apigenin, genistein, kazinol B, and ugonin U) have been reported to activate intracellular Ca2+ flux in various cell lines ,  or neutrophils , . Clearly, additional research is required to define the mechanisms of biochanin A action on neutrophils.