Inhibition of cytochrome P450 monooxygenase-catalyzed oxylipin formation by flavonoids: Evaluation of structure-activity relationship towards CYP4F2-selective inhibitors

Epoxy- and hydroxy-fatty acids are physiologically active lipid mediators which are formed from arachidonic acid and other fatty acids by cytochrome P450 monooxygenase (CYP) catalytic activity. In this study, we investigated the structure-activity relationship of the inhibition of fatty acid-oxidizing CYP by flavonoids. A sum of 65 naturally occurring as well as new flavonoids were synthesized and tested in a multi-enzyme assay. Substituents at C2' and C7-position of the flavone structure caused epoxygenase blockade, while electronegative substituents at C4'-position led to ω-hydroxylase-selective inhibition. We identified 4'-trifluoromethylflavone as a potent and selective compound, inhibiting 20-HETE formation with an IC50 of 2.8 μM (1.3 μM-6.1 μM) in human liver microsomes. This inhibition is achieved by selective inhibition of CYP4F2 [IC50: 0.76 μM (0.42 μM-1.4 μM)], while the other human ω-hydroxylating CYP, CYP4A11, is not affected. The compound is also active in microsomes from rat and mouse liver [IC50: 1.4 μM (0.77 μM-2.7 μM) and 0.71 μM (0.24 μM-2.2 μM), respectively]. Moreover, it exhibits moderate permeability properties in PAMPA and CaCo-2 transwell systems (papp: 4.6 ± 0.6 × 10-6 cm/s and 4.1 ± 0.4 × 10-6 cm/s, respectively) and is stable to metabolic conversion in vitro. With this inhibitor, we provide a novel tool to selectively investigate the CYP4F2-catalyzed 20-HETE formation and its role in physiology.