The effect of flavonoids on the metabolic activity of cytochromes P450 in vitro

U svrhu ispitivanja inhibicijskog učinka flavonoida na metaboličku aktivnost enzima citokrom P450, primijenjene su reakcije biotransformacije: 6β-hidroksilacija testosterona (CYP3A4), O-demetilacija dekstrometorfana (CYP2D6), O-deetilacija fenacetina (CYP1A2), 7-hidroksilacija kumarina (CYP2A6) i 6α-hidroksilacija paklitaksela (CYP2C8). Ostatna aktivnost enzima citokrom P450 određena je primjenom HPLC-DAD analitičke metode. Sedam flavonoida pokazalo je statistički značajan potencijal za inhibiciju CYP3A4 (P < 0,05), pet za inhibiciju CYP1A2, dok rezultati ispitivanja inhibicije enzima CYP2A6 ukazuju na značajan inhibicijski potencijal ukupno četiri flavonoida. Niti jedan ispitivani flavonoid nije pokazao statistički značajnu inhibiciju CYP2D6. Tri flavonoida inhibirala su aktivnost enzima CYP2C8. Za najpotentnije inhibitore (akacetin, krizin, apigenin), metodom molekularnog sidrenja, procijenila su se potencijalna vezna mjesta te najvjerojatnija konformacija kompleksa flavonoida i enzima CYP3A4. Rezultati molekularnog sidrenja pokazuju kako su energije vezanja molekulskih specija u aktivno mjesto CYP3A4 niže od energija vezanja anionskih specija. Sve najbolje rangirane molekulske specije orijentirane su s prstenom B u blizini hemskog željeza. U konačnici je provedena analiza kvantitativnog odnosa strukture i djelovanja (QSAR) primjenom metode slučajne šume i metodom tvorbe dendrograma. Svi odabrani deskriptori ukazuju da je za značajnu inhibiciju CYP3A4 potreban što hidrofobniji prsten B koji stupa u interakciju s hemskim željezom. Hijerarhijska tvorba klastera pokazala je kako ispitivani flavonoidi podjednako uspješno inhibiraju CYP3A4, CYP1A2, CYP2A6 i CYP2C8. U slučaju CYP2A6 dodatno je utvrđena statistički značajna povezanost ostatne aktivnosti s energijom najviše popunjene molekulske orbitale (EHOMO).The inhibitory effect of flavonoids on the metabolic activity of the cytochrome P450 enzyme was tested using the biotransformation reactions: 6β-hydroxylation of testosterone (CYP3A4), O-demethylation of dextromethorphan (CYP2D6), O-deethylation of phenacetin (CYP1A2), coumarin 7-hydroxylation (CYP2A6) and 6α-hydroxylation of paclitaxel (CYP2C8). The residual activity of cytochrome P450 enzymes was determined using HPLC-DAD analytical method. Seven flavonoids showed statistically significant potential for CYP3A4 inhibition (P < 0.05), five flavonoids showed CYP1A2 inhibition, while the results of the CYP2A6 enzyme inhibition assay showed significant inhibitory potential of four flavonoids. None of the flavonoids tested showed statistically significant CYP2D6 inhibition. Three flavonoids, inhibited CYP2C8 enzyme activity. For the most potent inhibitors (acacetin, chrysin, apigenin), the most likely conformation in the active site of the CYP3A4, as well as the potential binding site, was determined by molecular docking study. Docking results showed that the binding energies of the molecular spices in the CYP3A4 active site are lower than the anion spices. All the top-ranked molecular species are oriented with ring B toward heme iron. Quantitative structure activity relationship (QSAR) analysis was performed using the random forest method and the hierarchical clustering method. All selected descriptors indicate that a significant hydrophobic B ring, interacting with the heme iron, is required for significant inhibition of CYP3A4. The hierarchical cluster formation showed that tested flavonoids equally successfully inhibit CYP3A4, CYP1A2, CYP2A6 and CYP2C8. In the case of CYP2A6, a statistically significant dependence of residual activity and the highest occupied molecular orbital energy (EHOMO) was further established