Synthesis of substituted phenyl diaziridines and characterization as mechanism-based inactivators of human cytochrome P450 2B6.
The metabolism of arylhydrazines by cytochromes P450 (P450s) has previously been shown to yield aryl-iron complexes that inhibit P450 enzymes as a result of heme modification. These modifications of the heme have been used to probe the topology of the active site of several P450s. Therefore, diaziridines containing one or more substitutions on the phenyl ring were synthesized and evaluated as potential mechanism-based inactivators of P450 2B enzymes that could be used to elucidate the active site topology. Five of the six trifluoroaryldiaziridines tested selectively inactivated P450 2B6 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner as measured using the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation assay. The kinetic parameters for P450 2B6 inactivation by the five compounds were calculated. Analysis of the P450 heme from P450s inactivated by the five substituted diaziridines suggested that the activity loss was not due to heme destruction as measured by the reduced-CO spectrum or high-performance liquid chromatography of the P450 heme. Dialysis experiments indicated the irreversible nature of the inactivation and the reaction between the diaziridine compounds and the P450 enzyme. Interestingly, a thiomethyl-substituted phenyl diaziridine had no effect on the activity of P450 2B6 in the reconstituted system, but competitively inhibited the O-debenzylation activity of P450 3A4 with 7-benzyloxy-4-(trifluoromethyl)coumarin as substrate. Binding spectra suggest that this compound bound reversibly to P450 2B6, and preliminary results indicate that 3-(4-methylthiophenyl)-3-(trifluoromethyl)diaziridine is metabolized by P450 2B6.
Sridar
Chitra
C
Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109-0632, USA.
Kobayashi
Yoshimasa
Y
Brevig
Holly
H
Kent
Ute M
UM
Puppali
Satish G
SG
Rimoldi
John M
JM
Hollenberg
Paul F
PF
D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors*; D001388:Aziridines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D065702:Cytochrome P-450 CYP2B6; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D015394:Molecular Structure; D009249:NADP / Q000737:chemistry; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D013997:Time Factors
D001388:Aziridines; D004791:Enzyme Inhibitors; D009249:NADP; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating