PubMed · 6243421
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G B Kolata. 1980-02-29. Testing for cancer risk.. https://doi.org/10.1126/science.6243421
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Molecular structural and molecular orbital calculations (AM1 method) are reported on a series of 10 para-substituted toluene derivatives and this structural information has been used to rationalize the differences between both rates of hydroxylation catalysed by cytochrome P4502B4 and binding to the same cytochrome P450, via the generation of quantitative structure-activity relationships (QSARs). It was found that the rate constant for hydroxylation can be described by a two-variable expression involving the dipole moment and volume of the solvent-accessible molecular surface (r = 0.98), whereas binding free energies are well characterized by combinations of molecular volume and various electronic frontier orbital parameters (r = 0.98 and 0.99). This study represents an advance on a previous evaluation by White and McCarthy (Arch Biochem Biophys 246: 19-32, 1986) who used empirical physico-chemical parameters to obtain similar results which were generally of lower statistical significance to those of the present work. The QSAR expressions suggest that both binding to P450 and metabolism for this series of compounds are dependent on the relative ability of the molecules to desolvate and occupy the heme binding site, together with electronic properties of the whole molecule and of the methyl group which undergoes hydroxylation.
Biochemical experiments, using the well-defined human liver CYP2C9 expressed in yeast, and molecular modeling techniques were used to derive a predictive model for substrates of CYP2C9. The ability of 10 2-aroylthiophenes related to tienilic acid to act as substrates for CYP2C9 was studied. Four of them were original compounds that were synthesized and completely characterized by several spectroscopic techniques. In these 10 compounds various chemical functions, such as ester, amide, alcohol, phenol, ether or tetrazole functions, replaced the OCH2COOH function of tienilic acid. Among them, only the derivatives containing an acidic function (carboxylic acids, phenol, and tetrazole whose pKaS are 4.8, 6.3, and 3.8, respectively) underwent a 5-hydroxylation of their thiophene ring like tienilic acid. Despite their close structural analogy with tienilic acid, all of the other compounds not only did not undergo any 5-hydroxylation of their thiophene ring but also failed to act as inhibitors of CYP2C9. These results strongly suggested that the presence, at pH 7.4, of a negative charge on the substrate is a very important feature in its recognition by CYP2C9. In fact, the four new substrates of CYP2C9 described in this study, a carboxylic acid, phenol, and tetrazole derivative, each of which is related to tienilic acid, and the antiinflammatory drug, suprofen (with Km between 12 and 130 microM and kcat between 0.2 and 1.3 min-1), as well as almost all CYP2C9 substrates reported in the literature, exhibit a pKa below 7 (except phenytoin whose pKa is 8.1). They mainly exist as anions at physiological pH. By using molecular modeling techniques, 12 CYP2C9 substrates were superimposed with respect to their hydroxylation site and fitted onto templates, which were rigid molecules such as (S)-warfarin and phenytoin. It was thus possible to arrange them in order that all their anionic sites were at a distance around 4 A from a common point (a putative cationic site of the protein) in space. These results provide a model of the substrate binding site of CYP2C9, in which substrates interact through their anionic site A- with a cationic residue of the CYP2C9 protein C+. In that model, the distance between the hydroxylation site (Hy) and the anionic site (A-) is 7.8 +/- 1.6 A, and the <HyA-C+ angle is 82 +/- 15 degrees.
The preferential hydroxylation of (S)-mephenytoin to 4'-hydroxymephenytoin (4'-OH-M) displays a genetic polymorphism of drug metabolism in humans. Thus the excreted 4'-OH-M is considered to be an important marker for the hepatic (S)-mephenytoin 4'-hydroxylase. Accordingly, a mixture of urine containing total 4'-OH-M after enzymatic deconjugation and phenobarbital as internal standard (I.S.) was extracted with absolute diethyl ether. The residue remaining after evaporation was dissolved in 50 microliters of eluate and 20 microliters were injected into the chromatographic system. All components were separated isocratically on a reversed-phase column using acetonitrile-water (24:76, v/v) as the mobile phase at a flow-rate of 1.2 ml/min. The effluent was monitored at 204 nm. The retention times for 4'-OH-M and the I.S. were within 6 min. The absolute recovery was in the range 84-89% for 4'-OH-M and that of the I.S. was 75.9 +/- 4.2%. Quantification was performed by measuring the peak-height ratio compared with the ratio of the amount of 4'-OH-M divided by that of the I.S. The intra- and inter-day variations were less than 8% and 10%, respectively. The proposed method is simpler and more convenient than those reported previously. Its practical applicability was assessed by phenotyping the efficient and deficient hydroxylators among the Chinese minorities and Han Chinese populations.