br Discussion and concluding remarks FPR
Discussion and concluding remarks FPR1 is considered to be a promising drug target for treating inflammatory and immunological diseases. Thus, natural compounds that could block and/or regulate FPR1 activity may be an important source of novel therapeutics for modulating inflammatory processes. Here, we reviewed the literature and conclude that only a few natural products and their synthetic derivatives are characterized well enough to be defined as FPR1 antagonists. CsH, CHIPS, cyclic peptide SRSRY, Boc-1, several synthetic isoflavones, PP-6, and SMG-1 are FPR1 antagonists with quantified FPR1 binding affinity. Conversely, numerous other natural compounds with the ability to suppress O2 production and HNE release and/or other functional activities in fMLF-stimulated neutrophils that have been reported have not been evaluated for direct FPR1 binding activity and/or had off-target effects, suggesting that they could not be bona fide receptor antagonists. In an effort to uncover additional natural products that may represent potential FPR1 antagonists, we used this review of the literature and additional screening of selected compounds for their ability to inhibit Ca flux in fMLF-stimulated human neutrophils to define a set of potential FPR1 antagonists. For a compound to be considered, it had to: (a) inhibit all fMLF-induced functional responses studied; (b) not directly inhibit known processes downstream of FPR1 that could interfere with the functional responses tested, including inhibition of ion Z-LEHD-FMK and eicosanoid biosynthesis; and (c) not directly activate functional responses in neutrophils. Based on all of these restrictions, we selected a prospective set of 24 natural products from the literature that were all relatively potent inhibitors of fMLF-induced signaling (IC50<30μM) and conducted molecular modeling to see if these compounds fit the structural requirements of an FPR1 antagonist. Four natural compounds (cnidimol A, PP-6, PL3S, and garcimultiflorone B) met this additional requirement, suggesting they may be FPR1 antagonists. Indeed, one of these compounds (PP-6) has already been shown to compete with fMLF for binding to FPR1 . Thus, further investigation of the binding of cnidimol A, PL3S, and garcimultiflorone B to the FPR1 ligand binding site will be important to evaluate. Cnidimol A has a 4H-chromen-4-one scaffold, which is similar to recently reported isoflavone FPR1 antagonists . Thus, the high similarity of cnidimol A to the FPR1 pharmacophore model suggests 4H-chromen-4-one may represent an important scaffold for developing FPR1 antagonists. Although we predict garcimultiflorone B could be an FPR1 antagonist, it is also possible that this natural product could inhibit fMLF-induced functional activity via downstream pathways, as some natural compounds related to garcimultiflorone B, such as garcinol and hyperforin, inhibited 5-lipoxygenase, a key enzyme in leukotriene biosynthesis , . Our docking study showed that PP-6 formed three H-bonds with FPR1. This lignan is structurally related to the mammalian lignans enterolactone and prestegane B. Various mammalian-type lignan derivatives are now commercially available, and study of their FPR1-regulatory activity would be desirable. Importantly, key chemical moieties of these natural compounds could provide leads for the development of effective natural compound-inspired small molecule FPR1 antagonists. Since our molecular modeling only evaluated orthosteric interaction of a ligand with FPR1, possible allosteric mechanisms for various compounds cannot be excluded. It is now recognized that GPCRs possess spatially distinct allosteric sites, and G protein-GPCR interactions, including β-arrestin-GPCR interactions, can involve allosteric interactions . Thus, compounds that are able to interfere with these FPR1 interactions or directly insert into the FPR1 transmembrane-extracellular interface could modify FPR1-dependent signal transduction pathways. Finally, FPR1 molecules can interact laterally in the plasma membrane  and, for example, bile acids, plant steroids, and saponins may alter lateral allosteric regulation, modifying the membrane environment responsible for the receptor dimerization, FPR1 coupling with its G proteins, and other scaffolding/accessory proteins.