Tetracyclic triterpenes from Chios Mastic Gum act as TGR5 and RORγ modulators.

BACKGROUND: Chios mastic gum (CMG) is a natural resin from Pistacia lentiscus L. var. Chia (Anacardiaceae). It contains high concentrations of triterpenoids, with 24Z-masticadienonic acid (24Z-MNA) and 24Z-isomasticadienonic acid (24Z-IMNA) being the predominant compounds. CMG has been reported to possess anti-inflammatory activity in vitro; however, the molecular targets remain unknown. PURPOSE: Due to the structural similarity of tetracyclic CMG triterpenoids to bile acids, we investigated whether CMG constituents modulate bile acid-regulated receptors. METHODS: Receptor activities and in vitro anti-inflammatory effects of CMG triterpenoids were assessed using cell-based assays and computational modeling. RESULTS: We show that 24Z-IMNA acts as both a potent TGR5 agonist (EC50 = 1.7 µM) and an RORγ inverse agonist (IC50 = 329 nM), whereas 24Z-MNA shows modest activity on both receptors in luciferase reporter gene assays. The semi-synthetic isomer 24E-IMNA acted as a TGR5 agonist (EC50 = 2.2 µM) but lacked RORγ activity. TGR5 agonistic activities were confirmed in cAMP accumulation assays. 24Z-MNA, 24Z-IMNA and 24E-IMNA inhibited NF-κB transactivation TGR5-dependently and reduced NO production and Il1b mRNA expression in LPS-stimulated J774A.1 macrophages. The identified RORγ inverse agonist, 24Z-IMNA, showed concentration-dependent reductions in RORγ target gene expression (Il17a, Il17f and Il23r) by RT-qPCR. Computational modeling, including molecular docking, molecular dynamics (MD) simulations and free energy calculations, suggests plausible binding modes of those three compounds on TGR5 and RORγ, providing a structural basis for the observed activities. CONCLUSION: Taken together, this study shows that CMG triterpenoids target TGR5 and RORγ, potentially contributing to their reported anti-inflammatory activities.