Active-site characteristics of CYP2C19 and CYP2C9 probed with hydantoin and barbiturate inhibitors

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Abstract

Three series of N-3 alkyl substituted phenytoin, nirvanol, and barbiturate derivatives were synthesized and their inhibitor potencies were tested against recombinant CYP2C19 and CYP2C9 to probe the interaction of these ligands with the active sites of these enzymes. All compounds were found to be competitive inhibitors of both enzymes, although the degree of inhibitory potency was generally much greater towards CYP2C19. Inhibitor stereochemistry did not markedly influence Ki towards CYP2C9, and log P adequately predicted inhibitor potency for this enzyme. In contrast, stereochemistry was an important factor in determining inhibitor potency towards CYP2C19. (S)-(+)-N-3-Benzylnirvanol and (R)-(−)-N-3-benzylphenobarbital emerged as the most potent and selective CYP2C19 inhibitors, with Ki values of <250 nM—at least two orders of magnitude greater inhibitor potency than towards CYP2C9. Both inhibitors were metabolized preferentially at their C-5 phenyl substituents, indicating that CYP2C19 prefers to orient the N-3 substituents away from the active oxygen species. These features were incorporated into expanded CoMFA models for CYP2C9, and a new, validated CoMFA model for CYP2C19.

Section snippets

Materials

(S)-Flurbiprofen, 4-hydroxyflurbiprofen, and 2-fluoro-4-biphenylacetic acid were gifts from Dr. T.S. Tracy (West Virginia University, Morgantown, WV). Mephobarbital, 3-O-methylfluorescein, fluorescein, and 2,7-dichlorofluorescein were purchased from Sigma–Aldrich Chemical (St. Louis, MO). Nirvanol, mephenytoin, and (S)-nirvanol were prepared as reported previously [21]. TLC plates, POLYGRAM SIL G/UV254, were Macherey-Nagel from Alltech Associates (Deerfield, IL). All other chemicals and

Experimental approach

These studies were initiated in an effort to design new high-affinity inhibitors of CYP2C19 with which to begin constructing computational models for this enzyme. We chose to focus on hydantoin and barbiturate structures as templates because (S)-mephenytoin and mephobarbital are well-recognized selective substrates for CYP2C19 [32], and it had been reported that N-3 methylation of phenytoin enhanced affinity for CYP2C19 at the expense of CYP2C9 [33]. In each series, the readily available parent

Acknowledgements

This work was supported by NIH Grant GM32165 (W.F.T., J.P.J., and A.E.R.). The authors acknowledge the excellent technical assistance of Ms. Alison Bickerstaff.

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