Discovery of rubiarbonone C as a selective inhibitor of cytochrome P450 4F enzymes

Abstract

Cytochrome P450 (CYP) enzymes, particularly CYP4A/4F, are the major -hydroxylases of arachidonic acid (AA) that can produce 20-hydroxyeicosatetraenoic acid (20-HETE). Although there are dissimilarities in substrate specificity, tissue distribution, and gene regulation between CYP4A and CYP4F, selective CYP4A or 4F inhibitors are currently unavailable. Therefore, this study was designed to develop CYP4F selective inhibitors using a novel inhibitory assay of 20-HETE formation. The assay was established using pooled human kidney microsomes (HKMs) and human recombinant CYP4 enzymes incubated with 1,2,3,4,5-C-13 AA (C-13(5) AA) as a substrate to minimize interference by endogenous AA. The intrinsic clearance (V-max/K-m) values were 9.5 mu L/min/mg for HKMs and 0.02, 0.9, and 10.1 mu L/min/pmol for CYP4A11, CYP4F2, and CYP4F3B, respectively, which suggests a major role for CYP4F in -hydroxylation of AA. To validate the assay, we tested well-known pan-CYP4 inhibitor HET0016 along with 50 compounds derived from natural products. Of the screened compounds, rubiarbonone C showed the most potent inhibitory activity. The 50% inhibitory concentrations of rubiarbonone C against CYP4A11, CYP4F2, and 4F3B were >50, 4.2, and 4.2 mu M, respectively. Moreover, epoxyeicosatrienoic acid formation from C-13(5) AA was not inhibited by up to 30 mu M rubiarbonone C. Meanwhile, in pooled human liver microsomes, CYP1, 2, and 3 family enzymes involved in drug metabolism were not substantially inhibited by rubiarbonone C. Thus, rubiarbonone C is a selective inhibitor of CYP4F and can be used to discriminate among CYP4 family enzymes and evaluate their roles in physiological and pathophysiological conditions.