The molecular basis of dapsone activation of CYP2C9-catalyzed non-steroidal anti-inflammatory drug (NSAID) oxidation
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(Published version)
Date
2023
Authors
Nair, P.C.
Burns, K.
Chau, N.
McKinnon, R.A.
Miners, J.O.
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Journal article
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Journal of Biological Chemistry, 2023; 199(12):105368-1-105368-17
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Pramod C. Nair, Kushari Burns, Nuy Chau, Ross A. McKinnon, John O. Miners
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Abstract
Positive heterotropic cooperativity, or ‘activation’, results in an instantaneous increase in enzyme activity in the absence of an increase in protein expression. Thus, cytochrome P450 (CYP) enzyme activation presents as a potential drug-drug interaction mechanism. It has been demonstrated previously that dapsone activates the CYP2C9-catalyzed oxidation of a number of NSAIDs in vitro. Here, we conducted molecular dynamics simulations (MDS) together with enzyme kinetic investigations and site-directed mutagenesis to elucidate the molecular basis of the activation of CYP2C9-catalyzed S-flurbiprofen 4’-hydroxylation and S-naproxen Odemethylation by dapsone. Supplementation of incubations of recombinant CYP2C9 with dapsone increased the catalytic efficiency of flurbiprofen and naproxen oxidation by 2.3- and 16.5-fold, respectively. MDS demonstrated that activation arises predominantly from aromatic interactions between the substrate, dapsone, and the phenyl rings of Phe114 and Phe476 within a common binding domain of the CYP2C9 active site, rather than involvement of a distinct effector site. Mutagenesis of Phe114 and Phe476 abrogated flurbiprofen and naproxen oxidation, and MDS and kinetic studies with the CYP2C9 mutants further identified a pivotal role of Phe476 in dapsone activation. MD simulations additionally showed that aromatic stacking interactions between two molecules of naproxen are necessary for binding in a catalytically favorable orientation. In contrast to flurbiprofen and naproxen, dapsone did not activate the 4’-hydroxylation of diclofenac, suggesting that the CYP2C9 active site favors cooperative binding of NSAIDs with a planar or near planar geometry. More generally, the work confirms the utility of MDS for investigating ligand binding in CYP enzymes.
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Published, Papers in Press, October 20, 2023
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© 2023 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article distributed under the terms of the Creative Commons CC-BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.