Abstract

A series of fluoroquinolone compounds (compounds 1–9), which contain a common quinolone scaffold, inactivated the metabolic activity of CYP3A. The purpose of this study was to identify mechanism-based inhibition (MBI) among these fluoroquinolone compounds by metabolite profiling to elucidate the association of the substructure and MBI potential. Reversibility of MBI after incubation with potassium ferricyanide differed among the test compounds. Representative quasi-irreversible inhibitors form a metabolite-intermediate (MI) complex with the heme of CYP3A4 according to absorption analysis. Metabolite profiling identified the cyclopropane ring-opened metabolites from representative irreversible inhibitors, suggesting irreversible binding of the carbon-centered radical species with CYP3A4. On the other hand, the oxime form of representative quasi-irreversible inhibitors was identified, suggesting generation of a nitroso intermediate that could form the MI complex. Metabolites of compound 10 with a methyl group at the carbon atom at the root of the amine moiety of compound 8 include the oxime form, but compound 10 did not show quasi-irreversible inhibition. The docking study with CYP3A4 suggested that a methyl moiety introduced at the carbon atom at the root of the primary amine disrupts formation of the MI complex between the heme and the nitroso intermediate because of steric hindrance. This study identified substructures of fluoroquinolone compounds associated with the MBI mechanism; introduction of substituted groups inducing steric hindrance with the heme of P450 can prevent formation of an MI complex. Our series of experiments may be broadly applicable to prevention of MBI at the drug discovery stage.