Inhibition of CYP2B4 by 2-ethynylnaphthalene: Evidence for the co-binding of substrate and inhibitor within the active site
Section snippets
Materials
7-Ethoxycoumarin (7-EC), 7-hydroxycoumarin (7-HC), 7-pentoxyresorufin (7-PR), 7-benzyloxyresorufin (7-BR), resorufin were purchased from Sigma–Aldrich (St. Louis, MO). Benzphetamine (BZP) was a gift from Upjohn (Kalamazoo, MI). 7-Ethoxy-4-trifluoromethylcoumarin (7-EFC), and 7-hydroxy-4-trifluoromethylcoumarin (7-HFC) were obtained from Molecular Probes (Eugene, OR). p-Nitroanisole (PNA) was provided by Acros Organics (Belgium). Testosterone (TS) and its metabolites were from Steraloids Inc.
Examination of the reversible inhibition of CYP2B4-dependent activities by 2EN
As mentioned in our previous report [14], 2EN can serve as both a reversible and an irreversible inhibitor of CYP2B4-mediated metabolism. Although the irreversible inactivation is similar to that expected for typical mechanism-based inhibitors, the reversible component was unusual. The ability of 1 μM 2EN to reversibly inhibit substrate metabolism could be divided into three groups [14]: (a) little to no inhibition (PNA), (b) a moderate degree of inhibition (13–30% for 7EC, 7EFC, and BZP), and
Acknowledgments
This work was supported by US Public Health Service Research Grants from the National Institute of Environmental Health Sciences (R01 ES004344, W.L.B), and the National Institutes of Health (R43-DC-6925, D.H.), and support from the Stanley S. Scott Cancer Center (J.R.R.). We thank Dr. Maryam Foroozesh (Xavier University of Louisiana, New Orleans) for supplying us with 2-ethynylnaphthalene. We would also like to thank Mr. George Cawley for his assistance in purifying CYP2B4 and NADPH-cytochrome
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