Abstract
The abilities of 7-coumarin propargyl ether (CPE) and 7-(4-trifluoromethyl)coumarin propargyl ether (TFCPE) to act as mechanism-based inactivators of P450 3A4 and 3A5 in the reconstituted system have been investigated using 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and testosterone as probes. CPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner characteristic of a mechanism-based inactivator with a half-maximal inactivation (KI) of 112 μM, a maximal rate of inactivation (kinact) of 0.05 min-1, and a t½ of 13.9 min. Similarly, TFCPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner with a KI of 14 μM, a kinact of 0.04 min-1, and a t½ of 16.5 min. Parallel losses of P450 3A4 enzymatic activity and heme were observed with both compounds as measured by high-performance liquid chromatography and reduced CO spectra. Interestingly, neither compound inhibited the BFC O-debenzylation activity of P450 3A5. Reactive intermediates of CPE and TFCPE formed by P450 3A4 were trapped with glutathione, and the resulting adducts were identified using tandem mass spectral analysis. Metabolism studies using TFCPE resulted in the identification of a single metabolite that is formed by P450 3A4 but not by P450 3A5 and that may play a role in the mechanism-based inactivation.
Footnotes
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The study was supported in part by an MBRS/SCORE Grant (to M.F.) from the National Institutes of Health (NIH) NIGMS Institute (S06 GM 08008) and by NIH Grant CA 16954 (to P.F.H.).
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doi:10.1124/dmd.108.021493.
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ABBREVIATIONS: P450, cytochrome P450; CPE, 7-coumarin propargyl ether; TFCPE, 7-(4-trifluoromethyl)coumarin propargyl ether; BFC, 7-benzyloxy-4-(trifluoromethyl)coumarin; GC, gas chromatography; MS, mass spectrometry; NMR, nuclear magnetic resonance; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS/MS, tandem mass spectrometry; APCI, atmospheric pressure chemical ionization; SRS, substrate recognition site; CPE-GS, CPE-glutathione conjugate.
- Received March 14, 2008.
- Accepted July 23, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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