Target Identification and Validation of (+)-2-(1-Hydroxyl-4-Oxocyclohexyl) Ethyl Caffeate, an Anti-Inflammatory Natural Product

(+)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeate (HOEC) was isolated from Incarvillea mairei var. granditlora (Wehrhahn) Grierson. The plants of the Incarvillea genus have long been used as folk medicines for the treatment of inflammation-related diseases in China. 5-Lipoxygenase (5-LOX), a key enzyme in the arachidonic acid (AA) cascade, was identified as a potential target of HOEC by a pulldown assay, and then extensively validated by biosensor-based affinity detection, enzyme-based activity assays, cell-based AA metabolite analysis and computer-aided AA network simulation. Further in vivo studies of AA-induced ear oedema, ovalbumin (OVA)-induced lung inflammation and collagen-induced arthritis demonstrated the anti-inflammatory potency and validated the therapeutic target of HOEC. This work revealed that HOEC acted as an anti-inflammatory agent targeting 5-LOX, which not only confirmed the key role of 5-LOX in inflammation but also provided a paradigm for the exploration of natural product mechanisms of action.