Mechanism-Based Inactivation of Human Cytochrome P450 1A2 by Furafylline: Detection of a 1:1 Adduct to Protein and Evidence for the Formation of a Novel Imidazomethide Intermediate

Abstract

The rapid loss of human CYP1A2 (cytochrome P450 1A2) activity caused by the 8-methylxanthine furafylline is investigated with the aim of determining whether a stable covalent adduct of the xanthine to the enzyme could be identified. Metabolic studies employing expressed CYP1A2 with radiolabeled furafylline and a close analogue, cyclohexylline, where the furan ring is replaced with cyclohexane, indicate that these xanthines are bound in a 1:1 ratio to CYP1A2 protein. This result, combined with earlier kinetic studies, verifies that these compounds are mechanism-based inhibitors of the enzyme. The 8‘-methyl carbinols are the only metabolites formed by CYP1A2, and substantial (70−80%) incorporation of oxygen from the medium into the carbinols is observed. Carbinol formation is further characterized by high intramolecular isotope effects (k_H/k_D > 9) and low intermolecular isotope effects (^DV/K < 2). Overall partition ratios are low (5.0 and 7.6, respectively), confirming our previous conclusion that furafylline is an efficient inactivator. By contrast, the N⁷-methyl-8-methylxanthines are good substrates for CYP1A2 but are not themselves inactivating agents. In addition to other metabolic products, the 8‘-methyl carbinols of these N⁷-methyl-8-methylxanthines are formed in substantial amounts with equally high intramolecular isotope effects; however, the carbinol oxygen is derived exclusively from molecular oxygen. We conclude that oxidation of the 8-methyl group of furafylline and cyclohexylline, but not their N⁷-methyl analogues, by CYP1A2 promotes a major fraction of the inactivating xanthines to a two electron oxidized intermediate which either terminates enzyme activity by reaction with an active site amino acid or is decomposed by reaction with the medium to give carbinol.