Identification of a Novel FPR1 Antagonist in Human Neutrophils from Natural Sources

Neutrophils are a crucial component of the innate immune response against invading microorganisms; however there is increasing evidence that extensive or inappropriate activation of neutrophils is a major contributor to tissue damage in inflammatory diseases. Suppression of the extensive or inappropriate activation of neutrophils using drugs has been proposed as a way to ameliorate inflammatory diseases. Despite this, there are only a few currently available agents that directly modulate neutrophil proinflammatory responses in clinical practice. Therefore, the purpose of this study is to research and development of new generation anti-inflammatory agents to treat the neutrophilic inflammatory diseases. In response to endogenous and exogenous chemoattractants, activated neutrophils secrete a series of cytotoxins. One of the most studied and significant chemoattractants is formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), which is a prototype for microbe-derived formylated peptides. Significantly, recent studies have shown that formylated peptides are produced and released by damaged mitochondria. Thus, formylated peptides can also be considered alarmins, which are defined as endogenous molecules that signal tissue and cell damage. The molecular and functional responses for human neutrophils recognition of formylated peptides are their binding to formyl peptide receptor (FPR). Human neutrophils express two members of this family, FPR1 and FPR2. FPR1 is a high-affinity receptor for fMLP. Based on the importance of FPR1 in inflammatory diseases and the need for development of FPR1 antagonists, we screened more than 500 crude extracts from natural products to indentify potent and selective inhibitors for FPR1. Among them, an extract of fruit of Garcinia multiflora (Guttiferae) Champ (GMC) showed the most-potent inhibitory effect on superoxide anion generation and elastase release in fMLP-activated human neutrophils with IC50 values of 3.060.50 and 4.340.95 ng/ml, respectively. In our preliminary experiments, GMC attenuated fMLP-induced increase of cytosolic calcium concentration and phosphorylation of ERK, p38 MAPK and JNK. Significantly, receptor-binding analysis showed that GMC inhibited fMLP binding to its receptor (FPR1) in a concentration-dependent manner. Taken together, our preliminary results demonstrate that GMC is a natural inhibitor of the FPR1, which may have the potential to be developed into a useful new therapeutic agent for treating neutrophilic inflammatory diseases. G. multiflora is a small evergreen tree, distributed in South China, Hong Kong, and Taiwan. Benzophenones, xanthones, flavonoids, isocoumarins, and steroid are widely distributed in this plant. However, the anti-inflammatory effects and active ingredients of G. multiflora are still unknown. In this project, a cellular model in isolated human neutrophils and two animal models of multiple organ dysfunctions will be established to elucidate the anti-inflammatory functions of GMC and its active ingredients. We will explore the novel mechanism of the potential candidates and evaluate their potentials as the lead compounds for the drug development in this field. The study of the pharmacological mechanisms of potential natural products is useful for developing better therapeutic strategies. These studies will provide new insight into the pharmacological functions of G. multiflora and support the hypothesis that GMC and its active ingredients may have the potential to protect against the progression of inflammatory diseases.

Project ID:PC10007-0346
External Project ID:NSC100-2628-B182-001-MY3