Inhibition of CYP2B4 by the mechanism-based inhibitor 2-ethynylnaphthalene: Inhibitory potential of 2EN is dependent on the size of the substrate

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Abstract

2-Ethynylnaphthalene (2EN) is a mechanism-based inhibitor of CYP2B4 with two components to the inhibition, (1) enzyme inactivation, which requires covalent binding of the 2EN metabolite, and (2) reversible inhibition by 2EN itself. Both inhibitory components were examined using several different CYP2B4 substrates. Preincubation of CYP2B4 with 2EN led to a time-dependent inactivation of each of the CYP2B4-dependent activities examined; however, the ability of 2EN to reversibly inhibit CYP2B4 depended on the substrate employed, which is inconsistent with classical inhibition patterns. The degree 2EN’s reversible inhibition was shown not to correlate with the substrate affinity for the active site, but with parameters related to the molecular size of the substrate. The results are consistent with 2EN and the smaller substrates simultaneously fitting in the CYP2B4 active site, leading to very little inhibition. Larger substrates exhibited greater degrees of inhibition because of their inability to co-bind with inhibitor in the active site.

Section snippets

Materials

7-Ethoxycoumarin (7-EC), 7-hydroxycoumarin (7-HC), 7-pentoxyresorufin (7-PR), 7-benzyloxyresorufin (7-BR), resorufin, dilauroylphosphatidylcholine (DLPC), nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt (NADPH), glucose-6-phosphate, glucose-6-phosphate dehydrogenase, formaldehyde, and dimethylsulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO). Benzphetamine (BZP) was a gift from Upjohn (Kalamazoo, MI). 7-Ethoxy-4-trifluoromethylcoumarin (7-EFC) and

Results

2EN has been characterized as a selective mechanism based inhibitor for CYP2B4 [4], [5], [6]. The goal of this study was to examine the ability of 2EN to inhibit CYP2B4-dependent monooxygenase activities. As an initial experiment, the effect of 2EN on 7-pentoxyresorufin-O-dealkylation (PROD) was examined (Fig. 1a). Preincubation with 2EN for 10 min led to an 85% decrease in PROD activity, a response typically observed with mechanism-based inhibitors [2], [7], [8], [25]. However, a significant

Discussion

Previous studies [3], [4], [26] have shown that the aryl acetylene, 2EN, inactivates CYP2B4 in a mechanism-based manner. 2EN covalently modifies an amino acid in the peptide Glu 273-Met 314 with a molecular fragment that is consistent with a 2-naphthylacetyl group. The metabolism of 2EN results in the inactivation and covalent modification of the protein moiety, with the modified protein being unable to oxygenate substrates. Inhibition by 2EN has two components: [1] the inactivation of CYP2B4

Acknowledgments

This work was supported by US Public Health Service Research Grants from the National Institute of Environmental Health Sciences (R01 ES004344 – WLB), and the National Institutes of Health (R43-DC-6925 – DH), and support from the Stanley S. Scott Cancer Center (JRR). We thank Dr. William Alworth (Tulane University, New Orleans) for supplying us with 2-ethynylnaphthalene, and for his suggestions associated with these studies. We also thank Mr. George Cawley for his assistance in purifying CYP2B4

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