Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes

Abstract

The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2'-hydroxy-, 4'-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2'-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities. Molecular docking analysis showed that the C2'-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2' and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

A Complete Study of Farrerol Metabolites Produced In Vivo and In Vitro

“…Cytochrome P450 enzymes (CYPs) constitute a major enzyme family and are responsible for phase I metabolism of about 80% endogenous and exogenous substances [32]. The subtypes of CYP1A2, CYP2A6, and CYP2B6 play a vital role in the oxidation of flavanone and flavone [33]. Among them, CYP2A6 has high activities in oxidizing flavanone to form 2′-hydroxyflavanones and flavone, such as metabolites M3 and M10.…”

Natural Product Diversification by One‐Step Biocatalysis using Human P450 3A4

“…[57][58][59][60] Hence, only two flavonoids were tested because literature data also indicated a similar effect on P450 3A4. [61][62][63] On the other hand, different compounds of a natural product class can have contrary fates when exposed to this P450 as exemplified by the metabolism and inhibition of the coumarins warfarin [64] and bergamottin, [35] respectively. While the enzyme showed good activity in the 7-methoxy-4-(trifluoromethyl)coumarin (MFC) fluorescent assay, the well-known O-debenzylation catalysis of 7-benzyloxy-4-(trifluoromethyl)coumarin suggests demethylation of MFC.…”

“…The chemical shifts were almost identical to those in the literature. [82,83] (+)-(4R,5S,7R,9R,11S)-11,12-epoxy-9-hydroxynootkatone (13, C 15 H 22 O 3 , yellow oil, 6 mg, 4 %): 1 H NMR (300 MHz, CDCl 3 ): δ = 5.83 ChemCatChem (1H, br, 1-H), 4.40 (1H, dd, J = 2.9, 2.9 Hz, 9-H), 2.69 (1H, dd, J = 4.6, 0.3 Hz, 12-H), 2.59 (1H, d, J = 4.6 Hz, 12-H), 2.35 (1H, d, J = 17.9, 13.6 Hz, 3α-H), 2.27 (1H, dd, J = 17.9, 4.5, 0.7 Hz, 3β-H), 2.17 (1H, ddd, J = 12.8, 3.4, 3.0 Hz, 7-H), 2.09-2.01 (1H, m, 8α-H), 2.00-1.88 (2H, m, 4-, 6α-H), 1.48 (1H, J = 12.8, 12.7, 3.2 Hz, 8β-H), 1.27 (3H, s, 15-H), 1.25 (3H, s, 13-H), 0.97 (1H, J = 12.9, 12.7 Hz, 6β-H), 0.95 (3H, d, J = 6.8 Hz, 14-H); GC-MS (EI + , 70 eV): m/z (%) = 250 ([M] + , 22), 232 (22), 217 (44), 191 (100), 175 (44), 161 (44), 147 (56), 137 (56), 121 (94), 105 (78), 91 (78), 79 (61), 69 (61), 55 (66), 41 (83). The absolute configuration was assigned by comparison to literature data, which were almost identical.…”

“…In addition to P450 3A4, other human liver P450s [32,55,132] or homologous liver P450s of other mammal species [105,133,134] might complement the observed degree of diversification with different regio-and stereoselectivitities or substrate tolerance of other natural product classes such as P450 2D6 with its known activity towards flavonoids. [61,62] It remains to be investigated why substrates like 3 and 6 are accepted or diversified to an extent of di-hydroxylation, while close derivatives such as parthenolide and progesterone are not converted or do not experience the same degree of diversification (peaks observed only implied polarity caused by monohydroxylation; data not shown), respectively.…”

Oxidation of Flavone, 5-Hydroxyflavone, and 5,7-Dihydroxyflavone to Mono-, Di-, and Tri-Hydroxyflavones by Human Cytochrome P450 Enzymes

“…Purified human P450 enzymes used included CYP1A1, 1A2, 1B1.1, 1B1.3, 2A6, 2C9, and 3A4, and we also used recombinant CYP2B6 expressed in microsomes of Trichoplusia ni cells in which human NADPH-P450 reductase was coexpressed. Among liver microsomes prepared from human samples HH2, HH47, and HH54, the HH2 sample was defective in coumarin 7-hydroxylation activity and thus categorized to be a poor metabolizer of CYP2A6 . Molecular docking analysis of the interaction of 57diOHF with different forms of human P450s, particularly CYP1B1.1 and 1B1.3, was done to examine the basis of catalytic differences found in these P450s in oxidizing these flavonoids.…”