Inhibition of CYP2B4 by 2-ethynylnaphthalene: Evidence for the co-binding of substrate and inhibitor within the active site

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Abstract

2-Ethynylnaphthalene (2EN) is an effective mechanism-based inhibitor of CYP2B4. There are two inhibitory components: (1) irreversible inactivation of CYP2B4 (a typical time-dependent inactivation), and (2) a reversible component. The reversible component was unusual in that the degree of inhibition was not simply a characteristic of the enzyme-inhibitor interaction, but dependent on the size of the substrate molecule used to monitor residual activity. The effect of 2EN on the metabolism of seven CYP2B4 substrates showed that it was not an effective reversible inhibitor of substrates containing a single aromatic ring; substrates with two fused rings were competitively inhibited by 2EN; and larger substrates were non-competitively inhibited. Energy-based docking studies demonstrated that, with increasing substrate size, the energy of 2EN and substrate co-binding in the active site became unfavorable precisely at the point where 2EN became a competitive inhibitor. Hierarchical docking revealed potential allosteric inhibition sites separate from the substrate binding site.

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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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