Cyclopropylamine inactivation of cytochromes P450: Role of metabolic intermediate complexes

https://doi.org/10.1016/j.abb.2005.02.020Get rights and content

Abstract

The inactivation of cytochrome P450 enzymes by cyclopropylamines has been attributed to a mechanism involving initial one-electron oxidation at nitrogen followed by scission of the cyclopropane ring leading to covalent modification of the enzyme. Herein, we report that in liver microsomes N-cyclopropylbenzylamine (1) and related compounds inactivate P450 to a large extent via formation of metabolic intermediate complexes (MICs) in which a nitroso metabolite coordinates tightly to the heme iron, thereby preventing turnover. MIC formation from 1 does not occur in reconstituted P450 systems with CYP2B1/2, 2C11 or 2E1, or in microsomes exposed to gentle heating to inactivate the flavin-containing monooxygenase (FMO). In contrast, N-hydroxy-N-cyclopropylbenzylamine (3) and N-benzylhydroxylamine (4) generate MICs much faster than 1 in both reconstituted and microsomal systems. MIC formation from nitrone 5 (PhCH = N(O)cPr) is somewhat faster than from 1, but very much faster than the hydrolysis of 5 to a primary hydroxylamine. Thus the major overall route from 1 to a P450 MIC complex would appear to involve FMO oxidation to 3, further oxidation by P450 and/or FMO to nitrone 5′ (C2H4C = N(O)CH2Ph), hydrolysis to 4, and P450 oxidation to α-nitrosotoluene as the precursor to oxime 2 and the major MIC from 1.

Section snippets

Reagents and general procedures

[7-14C]Benzoic acid and sodium [14C]formate were purchased from Moravek Biochemicals (Brea, CA) with specific activities of 53 and 56 Ci/mol, respectively. [7-13C]Benzoic acid and sodium [13C]formate were purchased from Cambridge Isotopes Laboratories (Andover, MA). Compounds 2, 4, 6–9 and cyclopropylamine were obtained from Aldrich (Milwaukee, WI). NADPH, glucose-6-phosphate and glucose-6-phosphate dehydrogenase were purchased from Sigma (St. Louis, MO). Aminopyrine was from Mallinckrodt (St.

Results

Incubation of varying concentrations of 1 with rat liver microsomes and NADPH leads to a time-, concentration-, and cofactor-dependent loss of cytochrome P450 activity as assessed by the aminopyrine N-demethylase (APD) assay. A replot of the reciprocals of the slopes of Fig. 3 vs. the reciprocals of inhibitor concentration [34] leads to the kinetic constants reported in Table 1. As described in earlier literature, the loss of APD activity in microsomes incubated with 1 does not proceed to

Discussion

The discovery of 1 as a suicide substrate for cytochrome P450 enzymes was based on the observation of time- and cofactor-dependent loss of the aminopyrine N-demethylase and p-nitroanisole O-demethylase activities of PB rat liver microsomes [35], [42]. Results presented in Fig. 3 confirm the time-dependence and also indicate the concentration dependence of the inactivation process. They also confirm that as originally described, the inactivation of APD activity does not go to completion but

Acknowledgments

We thank Drs. Yakov Koen and Emily Scott and Ms. Xin Wang for assistance and advice concerning enzyme expression and purification. We also thank Dr. M.A. Correia for providing (with kind permission from Dr. T. Omura) plasmid EL2 for CYP2C11 expression, Dr. F.P. Guengerich for providing an E. coli strain expressing CYP2E1, and Dr. C.B. Kasper for providing an E. coli strain expressing P450 oxidoreductase. We also thank Pfizer Global Research and Development for generous financial support of this

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