Mechanism-Based Inactivation of Cytochrome P450 2B1 by 9-Ethynylphenanthrene (original) (raw)
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Biochemistry, 2000
The inhibition of CYP2A6 by decursinol angelate, a pyranocoumarin isolated from Angelica gigas roots, was examined in human liver microsomes and recombinant CYP2A6. Decursinol angelate moderately inhibited coumarin 7-hydroxylation, but a 20-min preincubation with microsomes and NADPH significantly increased its inhibitory effect (IC 50 ; >20 versus 4.4 M). A similar inhibition pattern was observed in nicotine C oxidation, which is also one of the prototype reactions of CYP2A6. Inactivation by decursinol angelate was selective for CYP2A6 and characterized by K I values of 0.99 and 2.42 M and the k inact values of 0.136 and 0.053 min ؊1 in microsomes and recombinant CYP2A6, respectively. This inactiva-tion was not protected or restored by nucleophiles, reactive oxygen scavengers, or extensive dialysis but was inhibited by the addition of a competitive CYP2A6 inhibitor, pilocarpine. Furthermore, incubation of CYP2A6 with decursinol angelate in the presence of NADPH resulted in a loss of the spectral CYP2A6 content. An in vitro metabolism study revealed that CYP2A6 oxidized decursinol angelate to the dihydrodiol metabolite, presumably via an epoxide intermediate that might be responsible for the inactivation of CYP2A6. These results collectively demonstrated that decursinol angelate inactivated CYP2A6 in a mechanism-based mode.
Biochemistry, 1998
The inhibition of CYP2A6 by decursinol angelate, a pyranocoumarin isolated from Angelica gigas roots, was examined in human liver microsomes and recombinant CYP2A6. Decursinol angelate moderately inhibited coumarin 7-hydroxylation, but a 20-min preincubation with microsomes and NADPH significantly increased its inhibitory effect (IC 50 ; >20 versus 4.4 M). A similar inhibition pattern was observed in nicotine C oxidation, which is also one of the prototype reactions of CYP2A6. Inactivation by decursinol angelate was selective for CYP2A6 and characterized by K I values of 0.99 and 2.42 M and the k inact values of 0.136 and 0.053 min ؊1 in microsomes and recombinant CYP2A6, respectively. This inactiva-tion was not protected or restored by nucleophiles, reactive oxygen scavengers, or extensive dialysis but was inhibited by the addition of a competitive CYP2A6 inhibitor, pilocarpine. Furthermore, incubation of CYP2A6 with decursinol angelate in the presence of NADPH resulted in a loss of the spectral CYP2A6 content. An in vitro metabolism study revealed that CYP2A6 oxidized decursinol angelate to the dihydrodiol metabolite, presumably via an epoxide intermediate that might be responsible for the inactivation of CYP2A6. These results collectively demonstrated that decursinol angelate inactivated CYP2A6 in a mechanism-based mode.
Mechanism-based inactivation of cytochrome P450 2B6 by isoimperatorin
Chemico-biological interactions, 2015
The inhibition of CYP2A6 by decursinol angelate, a pyranocoumarin isolated from Angelica gigas roots, was examined in human liver microsomes and recombinant CYP2A6. Decursinol angelate moderately inhibited coumarin 7-hydroxylation, but a 20-min preincubation with microsomes and NADPH significantly increased its inhibitory effect (IC 50 ; >20 versus 4.4 M). A similar inhibition pattern was observed in nicotine C oxidation, which is also one of the prototype reactions of CYP2A6. Inactivation by decursinol angelate was selective for CYP2A6 and characterized by K I values of 0.99 and 2.42 M and the k inact values of 0.136 and 0.053 min ؊1 in microsomes and recombinant CYP2A6, respectively. This inactiva-tion was not protected or restored by nucleophiles, reactive oxygen scavengers, or extensive dialysis but was inhibited by the addition of a competitive CYP2A6 inhibitor, pilocarpine. Furthermore, incubation of CYP2A6 with decursinol angelate in the presence of NADPH resulted in a loss of the spectral CYP2A6 content. An in vitro metabolism study revealed that CYP2A6 oxidized decursinol angelate to the dihydrodiol metabolite, presumably via an epoxide intermediate that might be responsible for the inactivation of CYP2A6. These results collectively demonstrated that decursinol angelate inactivated CYP2A6 in a mechanism-based mode.
Drug Metabolism and Disposition, 2007
In vitro experiments were conducted to compare k inact , K I and inactivation efficiency (k inact /K I ) of cytochrome P450 (P450) 2C9 by tienilic acid and (؎)-suprofen using (S)-flurbiprofen, diclofenac, and (S)-warfarin as reporter substrates. Although the inactivation of P450 2C9 by tienilic acid when (S)-flurbiprofen and diclofenac were used as substrates was similar (efficiency of ϳ9 ml/min/ mol), the inactivation kinetics were characterized by a sigmoidal profile. (؎)-Suprofen inactivation of (S)-flurbiprofen and diclofenac hydroxylation was also described by a sigmoidal profile, although inactivation was markedly less efficient (ϳ1 ml/min/mol). In contrast, inactivation of P450 2C9-mediated (S)-warfarin 7-hydroxylation by tienilic acid and (؎)-suprofen was best fit to a hyperbolic equation, where inactivation efficiency was moderately higher (10 ml/min/mol) and ϳ3-fold higher (3 ml/min/mol), respectively, relative to that of the other probe substrates, which argues for careful consideration of reporter substrate when mechanismbased inactivation of P450 2C9 is assessed in vitro. Further investigations into the increased inactivation seen with tienilic acid relative to that with (؎)-suprofen revealed that tienilic acid is a higher affinity substrate with a spectral binding affinity constant (K s ) of 2 M and an in vitro half-life of 5 min compared with a K s of 21 M and a 50 min in vitro half-life for (؎)-suprofen. Lastly, a close analog of tienilic acid with the carboxylate functionality replaced by an oxirane ring was devoid of inactivation properties, which suggests that an ionic binding interaction with a positively charged residue in the P450 2C9 active site is critical for recognition and mechanism-based inactivation by these close structural analogs.
European Journal of Biochemistry, 1991
The regulation of CYP2E1 and 2B1 was studied by following mRNA levels, catalytic activities and the subcellular distribution of the apoproteins in rat liver 0, 6, 12, 24, 48 and 96 h after a single intragastric dose of acetone. N o changes were observed in hepatic CYP2E1 mRNA levels at any time after acetone treatment, whereas rapid rises were observed in the microsomal amount of CYP2E1 protein and CYP2E1 -catalyzed 4-nitrophenol hydroxylase and carbon-tetrachloride-initiated lipid-peroxidation activities. However, CYP2E1 -dependent catalytic activities declined much faster than the immunodetectable CYP2E1 protein, suggesting that this cytochrome P-450 is inactivated prior to degradation. Similar results were seen in primary hepatocyte cultures. By contrast, concomitant changes in levels of CYP2B1 and CYP2B1 -dependent 0-depentylation of pentoxyresorufin were observed in the same microsomal preparations. Investigation of the degradative mechanism of both CYP2E1 and CYP2B1 by immunoquantitation of the proteins in lysosomes and by immunohistochemistry indicated their degradation via an autophagic-lysosomal pathway. The data suggest that CYP2E1 is acutely inactivated in the endoplasmic reticulum and that degradation of this isozyme occurs, at least in part, by the lysosomal route. By contrast, CYP2B1 is principally controlled at the level of synthesis.
Toxicology in Vitro, 2013
CYP450 enzymes are key determinants in drug toxicities, reduced pharmacological effect and adverse 28 drug reactions. Mitragynine, an euphoric compound was evaluated for its effects on the expression of 29 mRNAs encoding CYP1A2, CYP2D6 and CYP3A4 and protein expression and resultant enzymatic activity. 30 The mRNA and protein expression of CYP450 isoforms were carried out using an optimized multiplex 31 qRT-PCR assay and Western blot analysis. CYP1A2 and CYP3A4 enzyme activities were evaluated using 32 P450-Glo™ assays. The effects of mitragynine on human CYP3A4 protein expression were determined 33 using an optimized hCYP3A4-HepG2 cell-based assay. An in silico computational method to predict the 34 binding conformation of mitragynine to the active site of the CYP3A4 enzyme was performed and further 35 validated using in vitro CYP3A4 inhibition assays. Mitragynine was found to induce mRNA and protein 36 expression of CYP1A2. For the highest concentration of 25 lM, induction of mRNA was approximately 37 70% that of the positive control and was consistent with the increased CYP1A2 enzymatic activity. Thus, 38 mitragynine is a significant in vitro CYP1A2 inducer. However, it appeared to be a weak CYP3A4 inducer at 39 the transcriptional level and a weak CYP3A4 enzyme inhibitor. It is therefore, unlikely to have any signif-40 icant clinical effects on CYP3A4 activity.
European Journal of Biochemistry
The regulation of CYP2E1 and 2B1 was studied by following mRNA levels, catalytic activities and the subcellular distribution of the apoproteins in rat liver 0, 6, 12, 24, 48 and 96 h after a single intragastric dose of acetone. N o changes were observed in hepatic CYP2E1 mRNA levels at any time after acetone treatment, whereas rapid rises were observed in the microsomal amount of CYP2E1 protein and CYP2E1 -catalyzed 4-nitrophenol hydroxylase and carbon-tetrachloride-initiated lipid-peroxidation activities. However, CYP2E1 -dependent catalytic activities declined much faster than the immunodetectable CYP2E1 protein, suggesting that this cytochrome P-450 is inactivated prior to degradation. Similar results were seen in primary hepatocyte cultures. By contrast, concomitant changes in levels of CYP2B1 and CYP2B1 -dependent 0-depentylation of pentoxyresorufin were observed in the same microsomal preparations. Investigation of the degradative mechanism of both CYP2E1 and CYP2B1 by immunoquantitation of the proteins in lysosomes and by immunohistochemistry indicated their degradation via an autophagic-lysosomal pathway. The data suggest that CYP2E1 is acutely inactivated in the endoplasmic reticulum and that degradation of this isozyme occurs, at least in part, by the lysosomal route. By contrast, CYP2B1 is principally controlled at the level of synthesis.
British Journal of Pharmacology, 2011
BACKGROUND AND PURPOSE Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo. EXPERIMENTAL APPROACH Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS). KEY RESULTS CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC50 value decreased by 98%). This time-dependent inactivation (kinact 0.044 min-1 ; KI 2.64 mM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo.
Effect of pyrantel and dimethoate administration on rat liver cytochrome P450 system
Bulletin Veterinary Institute in Pulawy, 2006
The cytochrome P450 system in rat liver after administration of dimethoate (5 d, 1/10 DL50), pyrantel embonate (3 d, 1/5 DL50) or both xenobiotics simultaneously was analysed. Both compounds were administered directly to the stomach by the tube and the components of cytochrome P450 system were analysed in the microsomal fraction of the liver up to 14 d after the last applied dose. Intoxication with pyrantel diminished the total content of cytochrome P450 in all analysed time intervals. On the other hand, intoxication with dimethoate resulted in increase in the cytochrome P450 content 2 d after the last applied dose. The changes in activities of NADPH: cyt.P450 and NADH: cyt.b 5 reductases were small and statistically not significant. Both dimethoate and pyrantel affected the expression of CYP1A2, CYP2B1/2 and CYP3A1 proteins. Both compounds had a slight negative effect on CYP2B1/2. In animals receiving dimethoate as well as both xenobiotics simultaneously a significant increase in the level of CYP1A2 protein was observed. However, stimulatory effect of dimethoate on the expression of CYP1A2 was abolished by simultaneous intoxication with pyrantel. The changes in CYP3A1 protein expression corresponded with those observed for the total amount of cytochrome P450.
Amiodarone analog-dependent effects on CYP2C9-mediated metabolism and kinetic profiles
Drug metabolism and disposition: the biological fate of chemicals, 2006
CYP2C9 substrates can exhibit both hyperbolic and atypical kinetic profiles, and their metabolism can be activated or inhibited depending on the effector studied. CYP2C9 genetic variants can also affect both substrate turnover and kinetic profile. The present study assessed whether analogs of the effector amiodarone differentially altered the atypical kinetic profile of the substrate naproxen and whether this effect was genotype-dependent. Amiodarone, desethylamiodarone, benzbromarone, and its dimethyl analog (benz(meth)arone) were incubated with naproxen and either CYP2C9.1 or CYP2C9.3. Amiodarone activated naproxen demethylation at lower concentrations, regardless of the CYP2C9 allele, and inhibited metabolism at higher concentrations without altering the kinetic profile. Desethylamiodarone was a potent inhibitor of naproxen demethylation, irrespective of the CYP2C9 allele. Benzbromarone altered naproxen demethylation kinetics from a biphasic profile to that of a hyperbolic form in CYP2C9.1 and CYP2C9.3, resulting in inhibition and activation, respectively. In contrast, benz(meth)arone activated naproxen demethylation in both CYP2C9.1 and CYP2C9.3. In addition, the kinetic profile of naproxen demethylation became more hyperbolic at lower concentrations of benz(meth)arone and then reverted back to biphasic as the benz(meth)arone was increased further. Equilibrium binding and multiple-ligand docking studies were used to propose how such similar compounds exerted very different effects on naproxen metabolism. In summary, effectors of CYP2C9 metabolism can alter not only the degree of substrate turnover (activation or inhibition) but also the kinetic profile of metabolism of CYP2C9 substrates through effects on substrate binding and orientation. In addition, these kinetics effects are concentration-and genotypedependent.