Inhibition of cytochrome P450 2B6 by Astragalus extract mixture HT042

Kim Harim,Lee Yejin,Kim Vitchan,Lee Rowoon,Bae Soo Kyung,Kwak Mi-Kyoung,Lee Sung Hoon,Kim Donghak 한국독성학회 2020 Toxicological Research Vol.36 No.3

Astragalus extract mixture (AEM) HT042 is a functional food approved by the MFDS (Korean FDA) for increasing height. It comprises a mixture of three standardized extracts from Astragalus membranaceus root, Eleutherococcus senticosus stem, and Phlomis umbrosa root. In this study, drug–functional food interaction was analyzed using six major human cytochrome P450 enzymes. The inhibitory effect of AEM HT042 on P450 activities was studied using a P450–NADPH P450 reductase reconstitution system. Among the six P450 enzymes (1A2, 2A6, 2B6, 2D6, 2C9, and 3A4), only P450 2B6 activity was markedly decreased by AEM HT042 addition. The bupropion hydroxylation activity of P450 2B6 was analyzed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS). A calculated IC50 value of 10.62 μg/ml was obtained. To identify the inhibitory compounds in the mixture, four active compounds in AEM HT042 were analyzed. Shanzhiside methylester exhibited inhibitory effects on P450 2B6, whereas formononetin, eleutheroside E, and sesamoside did not affect P450 2B6 activity at all. Our results suggest that shanzhiside methylester in AEM HT042 is responsible for the inhibitory effect on P450 2B6 metabolism. Characterization of the inhibitory effect on P450 can help determine the safe administration of functional foods along with many clinical drugs that are metabolized by P450.

Catalytic enhancements in cytochrome P450 2C19 by cytochrome b5

Lee Gyu-Hyeong,Kim Vitchan,Lee Sung-Gyu,Jeong Eunseo,Kim Changmin,Lee Yoo-Bin,Kim Donghak 한국독성학회 2024 Toxicological Research Vol.40 No.2

Human cytochrome P450 2C19 catalyzes P450 enzyme reactions of various substrates, including steroids and clinical drugs. Recombinant P450 2C19 enzyme with histidine tag was successfully expressed in Escherichia coli and purified using affinity column chromatography. Ultra-performance liquid chromatography-tandem mass (UPLC-MS/MS) spectrometry showed that the purified P450 2C19 enzyme catalyzed 5-hydroxylation reaction of omeprazole. The purified enzyme displayed typical type I binding spectra to progesterone with a Kd value of 4.5 ± 0.2 μM, indicating a tight substrate binding. P450 2C19 catalyzed the hydroxylation of progesterone to produce 21-hydroxy (OH) as a major and 17-OH product as a minor product. Steady-state kinetic analysis of progesterone 21-hydroxylation indicated that the addition of cytochrome b5 stimulated a five-times catalytic turnover number of P450 2C19 with a kcat value of 1.07 ± 0.08 min− 1. The molecular docking model of progesterone in the active site of P450 2C19 displayed that the 21-carbon of progesterone was located close to the heme with a distance of 4.7 Å, suggesting 21-hydroxylation of progesterone is the optimal reaction of P450 2C19 enzyme for a productive orientation of the substrate. Our findings will help investigate the extent to which cytochrome b5 affects the metabolism of P450 2C19 to drugs and steroids.

Functional Characterization of Pharmcogenetic Variants of Human Cytochrome P450 2C9 in Korean Populations

( Myung-a Cho ),( Jihoon G Yoon ),( Vitchan Kim ),( Harim Kim ),( Rowoon Lee ),( Min Goo Lee ),( Donghak Kim ) 한국응용약물학회 2019 Biomolecules & Therapeutics(구 응용약물학회지) Vol.27 No.6

Human cytochrome P450 2C9 is a highly polymorphic enzyme that is required for drug and xenobiotic metabolism. Here, we studied eleven P450 2C9 genetic variants―including three novel variants F69S, L310V, and Q324X―that were clinically identified in Korean patients. P450 2C9 variant enzymes were expressed in Escherichia coli and their bicistronic membrane fractions were prepared The CO-binding spectra were obtained for nine enzyme variants, indicating P450 holoenzymes, but not for the M02 (L90P) variant. The M11 (Q324X) variant could not be expressed due to an early nonsense mutation. LC-MS/MS analysis was performed to measure the catalytic activities of the P450 2C9 variants, using diclofenac as a substrate. Steady-state kinetic analysis revealed that the catalytic efficiency of all nine P450 2C9 variants was lower than that of the wild type P450 2C9 enzyme. The M05 (R150L) and M06 (P279T) variants showed high k_cat values; however, their K_m values were also high. As the M01 (F69S), M03 (R124Q), M04 (R125H), M08 (I359L), M09 (I359T), and M10 (A477T) variants exhibited higher K_m and lower k_cat values than that of the wild type enzyme, their catalytic efficiency decreased by approximately 50-fold compared to the wild type enzyme. Furthermore, the novel variant M07 (L310V) showed lower k_cat and K_m values than the wild type enzyme, which resulted in its decreased (80%) catalytic efficiency. The X-ray crystal structure of P450 2C9 revealed the presence of mutations in the residues surrounding the substrate-binding cavity. Functional characterization of these genetic variants can help understand the pharmacogenetic outcomes.

Inhibitory effect of α-terpinyl acetate on cytochrome P450 2B6 enzymatic activity

Lee, Yejin,Park, Hyoung-Goo,Kim, Vitchan,Cho, Myung-A.,Kim, Harim,Ho, Thien-Hoang,Cho, Kyoung Sang,Lee, Im-Soon,Kim, Donghak Elsevier Pub. Co 2018 Chemico-biological interactions Vol.289 No.-

<P><B>Abstract</B></P> <P>Human cytochrome P450 2B6 is an important hepatic enzyme for the metabolism of xenobiotics and clinical drugs. Recently, more attention has been paid to P450 2B6 because of the increasing number of drugs it metabolizes. It has been known to interact with terpenes, the major constituents of the essential oils used for various medicinal purposes. In this study, the effect of monoterpenes on P450 2B6 catalytic activity was investigated. Recombinant P450 2B6 was expressed in <I>Escherichia coli</I> and purified using Ni-affinity chromatography. The purified P450 2B6 enzyme displayed bupropion hydroxylation activity in gas-mass spectrometry (GC-MS) analysis with a <I>k</I> <SUB>cat</SUB> of 0.5 min<SUP>−1</SUP> and a <I>K</I> <SUB>m</SUB> of 47 μM. Many terpenes displayed the type I binding spectra to purified P450 2B6 enzyme and α-terpinyl acetate showed strong binding affinity with a <I>K</I> <SUB>d</SUB> value of 5.4 μM. In GC-MS analysis, P450 2B6 converted α-terpinyl acetate to a putative oxidative product. The bupropion hydroxylation activity of P450 2B6 was inhibited by α-terpinyl acetate and its IC<SUB>50</SUB> value was 10.4 μM α-Terpinyl acetate was determined to be a competitive inhibitor of P450 2B6 with a <I>K</I> <SUB>i</SUB> value of 7.6 μM. The molecular docking model of the binding site of the P450 2B6 complex with α-terpinyl acetate was constructed. It showed the tight binding of α-terpinyl acetate in the active site of P450 2B6, which suggests that it could be a competitive substrate for P450 2B6.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Recombinant P450 2B6 was expressed and purified. </LI> <LI> Purified P450 2B6 displayed the bupropion hydroxylation activity. </LI> <LI> Terpenes displayed the typical type I binding spectra to P450 2B6. </LI> <LI> α-Terpinyl acetate showed strong binding affinity to P450 2B6. </LI> <LI> α-Terpinyl acetate is a competitive inhibitor of P450 2B6 to bupropion. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Functional Characterization of Drosophila melanogaster CYP6A8 Fatty Acid Hydroxylase

Lee Sang-A,Kim Vitchan,Choi Byoungyun,Lee Hyein,Chun Young-Jin,Cho Kyoung Sang,Kim Donghak 한국응용약물학회 2023 Biomolecules & Therapeutics(구 응용약물학회지) Vol.31 No.1

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni2+-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type І spectral changes, with Kd values 28 ± 4 and 144 ± 20 μM, respectively. Ultra-performance liquid chromatography–mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a kcat value of 0.038 ± 0.002 min–1 and a Km value of 10 ± 2 μM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a kcat value of 0.135 ± 0.007 min–1 and a Km value of 21 ± 4 μM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

Cytochrome P450 2A6 and other human P450 enzymes in the oxidation of flavone and flavanone

Kakimoto, Kensaku,Murayama, Norie,Takenaka, Shigeo,Nagayoshi, Haruna,Lim, Young-Ran,Kim, Vitchan,Kim, Donghak,Yamazaki, Hiroshi,Komori, Masayuki,Guengerich, F. Peter,Shimada, Tsutomu Taylor & Francis 2019 Xenobiotica Vol.49 No.2