Identification and target-pathway deconvolution of FFA4 agonists with anti-diabetic activity from Arnebia euchroma (Royle) Johnst

FFA4 is a novel therapeutic target for the treatment of metabolic diseases, such as type II diabetes. However, there are still few ligands with structural diversity, selectivity and high potency, and the signaling pathway downstream of FFA4 remains to be poorly characterized. In this study, a high performance liquid chromatography-corona charged aerosol detector (HPLC-CAD) combined with label-free dynamic mass redistribution (DMR) method was introduced to guide the discovery of FFA4 agonists from Arnebia euchroma (Royle) Johnst. Ten compounds were identified as FFA4 agonists and structure-activity relationship was obtained. Among them, shikonin displayed the most potent activity with pEC(50) value of 6.02 +/- 0.19. The activity of shikonin was confirmed by FLIPR (fluommetric imaging plate reader) assay. Signaling pathways of FFA4 were explored in HT-29 cells endogenously expressing FFA4 using shikonin and known FFA4 agonists a-linolenic acid (ALA) and TUG891. Multiple pathways included Gq/11-PLC-Ca2+-PKC, RohA, JNK, p38 MAPK, Gi/o and PI3K signaling but may not involve Gs signaling triggered by shikonin, ALA and TUG891. Besides, shikonin, TUG891 and ALA could induce ERK1/2 and AKT phosphorylation in HT-29 cells. Moreover, anti-diabetes effects of shikonin were evaluated on the glucose intolerance in diabetic db/db mice. Shikonin reduced plasma glucose level, suggesting that it had the potential in treatment of type II diabetes. The agonists identified in this study provided structure guidance for FFA4 drug design. This study was also useful for understanding FFA4 pharmacology and its biological function.