Metabolism of N,N',N"-triethylenethiophosphoramide by CYP2B1 and CYP2B6 results in the inactivation of both isoforms by two distinct mechanisms.
The anticancer drug N,N,"N"-triethylenethiophosphoramide (tTEPA) inactivated CYP2B6 and CYP2B1 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner indicative of mechanism-based inactivation. The KI value for the inactivation of CYP2B1 was 38 microM, the kinact was 0.3 min(-1), and the t1/2 value was 2.5 min. Spectral carbon monoxide (CO) binding and high-performance liquid chromatography heme studies of the tTEPA-inactivated CYP2B1 suggest that the loss in the enzymatic activity was primarily due to the binding of a reactive tTEPA intermediate to the 2B1 apoprotein. Inactivation by tTEPA in the presence of 7-ethoxycoumarin, an alternate substrate, reduced the rate of inactivation of CYP2B1. Incubations with tTEPA and NADPH resulted in greater than 90% loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation and testosterone hydroxylation activity of CYP2B1. In contrast, benzphetamine metabolism was significantly less inhibited (47%). CYP2B6 was inactivated by tTEPA with a KI value of 50 microM, a k inact value of 0.1 min(-1), and a t1/2 value of 14 min. However, unlike CYP2B1, the tTEPA-inactivated human isoform showed losses in the cytochrome P450 (P450) CO spectrum, the pyridine hemochrome spectrum, and in the amount of native heme that were comparable with the loss in the 7-EFC and benzphetamine activity, suggesting that activity loss was brought about by a tTEPA-reactive intermediate damaging the CYP2B6 heme. CYP2B6 could only be protected from the tTEPA-dependent inactivation by the 2B6-specific substrate bupropion but not by other substrates of CYP2B such as benzphetamine, testosterone, or 7-ethoxycoumarin. The data indicate that tTEPA metabolism by these two 2B isoforms results in inactivation of the P450s by two distinct mechanisms.
Harleton
Erin
E
Department of Pharmacology, Medical Science Research Building III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0632, USA.
Webster
Marie
M
Bumpus
Namandjé N
NN
Kent
Ute M
UM
Rae
James M
JM
Hollenberg
Paul F
PF
D000588:Amines / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001589:Benzphetamine / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D065702:Cytochrome P-450 CYP2B6; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism; D013852:Thiotepa / Q000378:metabolism* / Q000494:pharmacology
D000588:Amines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D001589:Benzphetamine; D013739:Testosterone; D013852:Thiotepa; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D019362:Cytochrome P-450 CYP2B1; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; C008699:octylamine