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 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 (kH/kD > 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.
Racha
J K
JK
Department of Medicinal Chemistry, University of Washington, Seattle 98195, USA.
Rettie
A E
AE
Kunze
K L
KL
D002461:Cell Line, Transformed; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D003903:Deuterium; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D010100:Oxygen / Q000378:metabolism; D013806:Theophylline / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology
D065609:Cytochrome P-450 CYP1A2 Inhibitors; D007093:Imidazoles; D014970:Xanthines; C008514:methylxanthine; D003903:Deuterium; D013806:Theophylline; C050131:furafylline; D019388:Cytochrome P-450 CYP1A2; D010100:Oxygen