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/

The rapid loss of human CYP1A2 (cytochrome P450 1A2) activity caused b y the 8-methylxanthine furafylline is investigated with the aim of det ermining whether a stable covalent adduct of the xanthine to the enzym e could be identified. Metabolic studies employing expressed CYP1A2 wi th radiolabeled furafylline and a close analogue, cyclohexylline, wher e the furan ring is replaced with cyclohexane, indicate that these xan thines are bound in a 1:1 ratio to CYP1A2 protein. This result, combin ed with earlier kinetic studies, verifies that these compounds are mec hanism-based inhibitors of the enzyme. The 8'-methyl carbinols are the only metabolites formed by CYP1A2, and substantial (70-80%) incorpora tion of oxygen from the medium into the carbinols is observed, Carbino l formation is further characterized by high intramolecular isotope ef fects (k(H)/k(D) > 9) and low intermolecular isotope effects (V-D/K < 2). Overall partition ratios are low (5.0 and 7.6, respectively), conf irming our previous conclusion that furafylline is an efficient inacti vator. By contrast, the N-7-methyl-8-methylxanthines are good substrat es for CYP1A2 but are not themselves inactivating agents. In addition to other metabolic products, the 8'-methyl carbinols of these N-7-meth yl-8-methylxanthines are formed in substantial amounts with equally hi gh intramolecular isotope effects; however, the carbinol oxygen is der ived exclusively from molecular oxygen. We conclude that oxidation of the 8-methyl group of furafylline and cyclohexylline, but not their N- 7-methyl analogues, by CYP1A2 promotes a major fraction of the inactiv ating xanthines to a two electron oxidized intermediate which either t erminates enzyme activity by reaction with an active site amino acid o r is decomposed by reaction with the medium to give carbinol.