Structure-Function Studies of Naphthalene, Phenanthrene, Biphenyl, and Their Derivatives in Interaction with and Oxidation by Cytochromes P450 2A13 and 2A6.

Naphthalene, phenanthrene, biphenyl, and their derivatives having different ethynyl, propynyl, butynyl, and propargyl ether substitutions were examined for their interaction with and oxidation by cytochromes P450 (P450) 2A13 and 2A6. Spectral interaction studies suggested that most of these chemicals interacted with P450 2A13 to induce Type I binding spectra more readily than with P450 2A6. Among the various substituted derivatives examined, 2-ethynylnaphthalene, 2-naphthalene propargyl ether, 3-ethynylphenanthrene, and 4-biphenyl propargyl ether had larger ΔAmax/Ks values in inducing Type I binding spectra with P450 2A13 than their parent compounds. P450 2A13 was found to oxidize naphthalene, phenanthrene, and biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2- and/or 4-hydroxybiphenyl, respectively, at much higher rates than P450 2A6. Other human P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, and 3A4 had lower rates of oxidation of naphthalene, phenanthrene, and biphenyl than P450s 2A13 and 2A6. Those alkynylated derivatives that strongly induced Type I binding spectra with P450s 2A13 and 2A6 were extensively oxidized by these enzymes upon analysis with HPLC. Molecular docking studies supported the hypothesis that ligand-interaction energies (U values) obtained with reported crystal structures of P450 2A13 and 2A6 bound to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, indole, pilocarpine, nicotine, and coumarin are of use in understanding the basis of possible molecular interactions of these xenobiotic chemicals with the active sites of P450 2A13 and 2A6 enzymes. In fact, the ligand-interaction energies with P450 2A13 4EJG bound to these chemicals were found to relate to their induction of Type I binding spectra.

Oxidation of pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene by human cytochrome P450 2A13.

1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2. Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3. 1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4. Molecular docking analysis supported preferred orientations of pyrene and its derivatives in the active site of P450 2A13, with lower interaction energies (U values) than observed for P450 2A6 and that several amino acid residues (including Ala-301, Asn-297 and Ala-117) play important roles in directing the orientation of these PAHs in the P450 2A13 active site. In addition, Phe-231 and Gly-329 were found to interact with pyrene to orient this compound in the active site of P450 1B1. 5. These results suggest that P450 2A13 is one of the important enzymes that oxidizes these PAH compounds and may determine how these chemicals are detoxicated and bioactivated in humans.

Binding of diverse environmental chemicals with human cytochromes P450 2A13, 2A6, and 1B1 and enzyme inhibition.

A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e., the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2',5'-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2'-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2'-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family 1 P450 enzymes, is able to catalyze many detoxication and activation reactions with chemicals of environmental interest.

Structure-function relationships of inhibition of human cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives.

Structure-function relationships for the inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced reverse type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, for example, 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 µM to 0.09, 0.21, 0.25, and 0.27 µM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 µM. The introduction of a 4'-methoxy- or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 µM, respectively. The above hydroxyl and/or methoxy groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always toward P450 1A2, where 3-, 5-, or 7-hydroxyflavone and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-, 5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3- and 5-hydroxyflavone. Flavone and several other flavonoids produced type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities toward P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids toward these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.

Reverse type I binding spectra of human cytochrome P450 1B1 induced by flavonoid, stilbene, pyrene, naphthalene, phenanthrene, and biphenyl derivatives that inhibit catalytic activity: a structure-function relationship study.

Fifty-one chemicals including derivatives of 16 flavonoids, three stilbenes, six pyrenes, seven naphthalenes, seven phenanthrenes, 10 biphenyls, 17beta-estradiol, and estrone were examined for their abilities to induce reverse type I binding spectra with human cytochrome P450 (P450) 1B1 and to inhibit 7-ethoxyresorufin O-deethylation (EROD) activities catalyzed by P450 1B1. Forty-nine chemicals showed reverse type I spectra with P450 1B1, and we found that 3,5,7-trihydroxyflavone, 3',4'-dimethoxy-5,7-dihydroxyflavone, 4'-methoxy-5,7-dihydroxyflavone, alpha- and beta-naphthoflavones, 2,4,3',5'-tetramethoxystilbene, pyrene, and several acetylenic pyrenes and phenanthrenes were strong inducers of the spectra and also potent inhibitors of EROD activities catalyzed by P450 1B1. The spectral dissociation constant (K(s)) and the magnitude of the binding (DeltaA(max)/K(s)) of 49 chemicals were correlated with the inhibition potencies of EROD activities by these chemicals [correlation coefficients (r) of 0.72 and 0.74, respectively]. The K(s) and DeltaA(max)/K(s) values were more correlated with IC(50) values when compared in a group of derivatives of flavonoids, stilbenes, and estrogens (r = 0.81 and 0.88, respectively) or a group of derivatives of pyrenes, naphthalenes, phenanthrenes, and biphenyls (r = 0.88 and 0.91, respectively). Among 14 flavonoids examined, 3,5,7-trihydroxyflavone and 4'-methoxy- and 3',4'-dimethoxy-5,7-dihydroxyflavone were more active than flavone in interacting with P450 1B1, but the respective 7,8-dihydroxyflavones were less active. Pyrene itself was highly active in interacting with P450 1B1, but its binding was slightly decreased when substituted with acetylenic groups. In contrast, substitution of naphthalene with methyl and ethyl propargyl ethers led to more interaction with P450 1B1 than with naphthalene itself. Similarly, substitution on phenanthrene or biphenyl with acetylenic groups and propargyl ethers increased affinities to P450 1B1. These results suggest that the reverse type I binding of chemicals to P450 1B1 may determine how they interact with and inhibit the catalytic activity of the enzyme. Substitutions on the compounds with various acetylenic groups and propargyl ethers cause an increase or decrease of their affinities to P450 1B1, depending on the parent compound used.

Interaction of polycyclic aromatic hydrocarbons with human cytochrome P450 1B1 in inhibiting catalytic activity.

Eleven polycyclic aromatic hydrocarbons (PAHs) and 14 acetylenic PAHs and biphenyls were used to analyze interactions with cytochrome P450 (P450) 1B1 in inhibiting catalytic activity, using 7-ethoxyresorufin O-deethylation (EROD) as a model reaction. Most of the chemicals examined were direct inhibitors of P450 1B1 except for 4-(1-propynyl)biphenyl, a mechanism-based inhibitor. In the case of direct inhibition of EROD activity {15 of 24 chemicals, e.g., benzo[a]pyrene, 1-(1-propynyl)pyrene, and 3-(1-propynyl)phenanthrene}, restoration of the EROD activity occurred with increasing incubation time, and kinetic analysis showed that EROD K(m) values were higher with these inhibitors at initial stages of incubation but became lower with increasing incubation time. With the other nine chemicals, the K(m) values for P450 1B1-mediated EROD increased during the incubations. Acetylenic inhibitors, but not the 11 PAHs, induced reverse type I spectral changes with P450 1B1, and the low dissociation constants (K(s)) suggested a role for such interaction in the inhibition of catalytic activity. Studies of quenching of P450 1B1-derived fluorescence with inhibitors demonstrated that acetylenic inhibitors and PAHs interacted rapidly with P450 1B1, with K(d) values < 10 microM. However, studies of quenching of inhibitor-derived fluorescence with P450 1B1 showed these interactions to be different, that is, B[a]P interacted with P450 1B1 more slowly. Molecular docking of P450 1B1, based on P450 1A2 crystal structure, suggested that there are clear differences in the interaction of PAH inhibitors with P450 1B1 and 1A2 and that these differences may explain why PAH inhibitors inhibit P450 1 enzymes by different mechanisms. The results suggest that P450 1B1 interacts with synthetic polycyclic aromatic acetylenes and PAHs in different ways, depending on the chemicals, and that these differences in interactions may explain how these chemicals inhibit P450 activities by different mechanisms.