Activation of cytochrome P450 2C9-mediated metabolism: mechanistic evidence in support of kinetic observations

doi:10.1016/S0003-9861(02)00665-3

Archives of Biochemistry and Biophysics

Volume 410, Issue 1, 1 February 2003, Pages 16-24

https://doi.org/10.1016/S0003-9861(02)00665-3 Get rights and content

Abstract

Studies were designed to investigate the possible mechanisms associated with the kinetic observation of CYP2C9 activation by dapsone and its phase I metabolite, N-hydroxydapsone. Kinetic studies suggested that dapsone activated CYP2C9-mediated flurbiprofen 4^′-hydroxylation by decreasing the K_m (α=0.2) and increasing the V_max (β=1.9). Interestingly, N-hydroxydapsone also activated flurbiprofen 4^′-hydroxylation by increasing V_max (β=1.5) but had no effect on K_m (α=0.98). To study the effects of these modulators on the binding affinity of flurbiprofen, spectral binding studies were performed. In the presence of dapsone, the spectral binding constant (K_s) for flurbiprofen was reduced from 14.1 to 2.1 μM, while in the presence of N-hydroxydapsone, the K_s remained unchanged (14.0 μM), which suggests that dapsone causes an increase in the affinity of flurbiprofen for CYP2C9, whereas N-hydroxydapsone does not. Additionally, stoichiometry measurements under activation conditions in the presence of dapsone resulted in a doubling of both NADPH and oxygen consumption for flurbiprofen 4^′-hydroxylation, with an overall increase in metabolite formation and a decrease in formation of peroxide and excess water. Interestingly, the presence of N-hydroxydapsone generally caused the same effects on stoichiometry as those of flurbiprofen 4^′-hydroxylation but failed to reduce excess water formation, which suggests that, while N-hydroxydapsone activates CYP2C9, it does so less efficiently and possibly through a mechanism different from that of dapsone.

Section snippets

Materials

Acetonitrile and dibasic potassium phosphate were obtained from Fisher Scientific (Pittsburgh, PA). (S)-flurbiprofen, 4^′-hydroxyflurbiprofen, and 2-fluoro-4-biphenyl acetic acid (internal standard) were gift from Pharmacia (Kalamazoo, MI). Dapsone was purchased from Sigma Chemical (St. Louis, MO) and N-hydroxydapsone was a gift from Dr. Reggie Frye of the University of Pittsburgh. Expressed CYP2C9 microsomes were obtained from ArQule (Redwood City, CA), while purified CYP2C9, NADPH P450

Activation of flurbiprofen 4^′-hydroxylation

Both dapsone and its major Phase I metabolite N-hydroxydapsone (Fig. 1) activated CYP2C9-mediated flurbiprofen 4^′-hydroxylation. 4^′-Hydroxyflurbiprofen formation data from coincubation of flurbiprofen with dapsone were fit to a two-site effector model equation (Eq. (1)) to generate a surface plot (Fig. 2), yielding an α of 0.23 and a β of 1.9 (Table 1), suggestive of a decrease in K_m and an increase in V_max for flurbiprofen 4^′-hydroxylation. N-Hydroxydapsone activated flurbiprofen 4^′

Discussion

Despite the numerous studies describing cytochrome P450 atypical kinetics such as activation [6], [15], homotropic positive cooperativity [7], [16], and substrate inhibition kinetics [8], [17], studies defining the underlying causes of these phenomena are absent. To date, CYP3A4 has been the most extensively studied P450 enzyme with respect to atypical kinetic profiles, as many substrates appear to exhibit these phenomena [6], [5]. The preponderance of evidence suggests that there are at least

Acknowledgements

This work was supported in part by a grant from the Public Health Service (GM-63215; T.S.T). J.M.H. was supported in part by a fellowship from the American Foundation for Pharmaceutical Education. Additionally, we thank Dr. Andrew Shiemke from the Biochemistry Department at West Virginia University for the use of his spectrophotometer for spectral binding studies and Dr. Ken Korzekwa for thoughtful review of the manuscript.

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We report that trace amounts (nano- to picomolar concentrations) of dapsone and amiodarone in reaction mixtures of structurally and functionally distinct heme enzymes gave increased product formation rates from diverse substrates. These enhancements are found to be lowered by mild radical quenchers; a lowering well differentiated from heme active site inhibitions. Based on the non-specific nature of the activations in the diverse heme enzyme reactions studied, a generic mechanism is proposed to explain additive-based activity enhancements in heme enzymology.
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¹: Current address: Pharmacia Corp., Global Drug Metabolism, Kalamazoo, MI 49007, USA.

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Activation of cytochrome P450 2C9-mediated metabolism: mechanistic evidence in support of kinetic observations

Abstract

Section snippets

Materials

Activation of flurbiprofen 4′-hydroxylation

Discussion

Acknowledgements

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

Crit. Rev. Toxicol.

Drug Metab. Dispos.

Drug Metab. Dispos.

Activation of flurbiprofen 4^′-hydroxylation