
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
Cytochrome P450 (P450, CYP) is a superfamily of heme-containing enzymes and is evolutionary conserved across species (Danielson, 2002; Cho
Genetic variants of metabolic enzymes can affect drug response in patients by affecting the efficacy or toxicity of the drug. In particular, polymorphisms in human P450 enzymes can affect catalytic activity. Therefore, the metabolic outcomes of P450 genetic variants can have pharmacological and toxicological impacts on the therapeutic drugs (Lee and Kim, 2011; Jeong
The human CYP2C gene cluster is located on chromosome 10q24 with the P450s 2C8 and 2C19 (Gray
P450 2C9 can be regulated on multiple levels. Inducers, such as rifampicin, dexamethasone, and phenobarbital, increase the gene expression of P450 2C9, whereas aminodarone, sulphaphenazole, and fluconazole, have been reported as a selective, competitive inhibitor of P450 2C9 (Miners and Birkett, 1998; Danielson, 2002; Nebert and Russell, 2002; Guengerich, 2015). These drugs can accelerate or delay the clearance of drugs metabolized by P450 2C9. The crystal structure of P450 2C9 published by Williams
The gene encoding P450 2C9 is highly polymorphic, and at least 65 alleles have been reported (https://www.pharmvar.org/). These polymorphisms include functional variants, and pharmacogenetic studies regarding the clinical effect of the alleles are important to decrease adverse drug reactions (Van Booven
In this study, the genetic screening of 433 Korean patients with P450 2C9 polymorphisms was conducted and 11 allelic variants of P450 2C9, including three novel variants (F69S, L310V, and Q324X), were detected (Table 1) (Han
Diclofenac, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and NADP+ were purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were of the highest commercially available grade.
The pBL vector containing the wild type P450 2C9 gene with 6× His-tag was used to construct allelic variant clones. Site-directed mutagenesis (Quick Change Lightning Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara, CA, USA) with PCR was performed and 11 allelic variant clones were obtained using specific primers. The constructed clones with desired mutations were subcloned into the pCW bicistronic expression vector containing the NADPH-P450 reductase (NPR) gene, using
P450 2C9 wild type and its allelic variants were expressed as previously described, with minor modifications (Jeong
The catalytic activity of P450 2C9 variants was determined by measuring diclofenac hydroxylation reaction. The reaction mixture contained 0.1 μM P450 2C9 BC membrane fraction, 0.1 M potassium phosphate buffer (pH 7.4) and various concentrations of diclofenac substrate in total volume of 0.1 ml. After preincubation at 37°C for 3 min, the reaction was initiated by adding the NADPH-generating system [100 mM glucose 6-phosphate, 1 mg/ml glucose-6-phosphate dehydrogenase, and 10 mM NADP+]. Reaction mixtures were incubated for 10 min at 37°C and were then stopped by adding two volumes of pre-chilled CH3CN containing 200 ng/ml chlorpropamide as an internal standard. After vortexing, the mixture was centrifuged at 3,000 rpm for 20 min and then 100 μl of the supernatant was transferred to a glass vial and analyzed by LC-MS/MS.
The concentration of 4-hydoroxydiclofenac was measured by LC-MS/MS using Waters ACQUITY UPLCTM and Waters Quattro PremierTM. ACQUITY UPLCTM BEH C18 (2.1×50 mm, 1.7 μm) was equipped with Waters ACQUITY UPLCTM. The isocratic mobile phase comprising CH3CN and water in the ratio of 75:25 with 0.1% formic acid was pumped at a flow rate of 0.2 ml/min. An electrospray ionization source was used in the positive ion mode. The operation conditions were as follows: capillary voltage 3.9 kV, extractor voltage 3 V, RF lens 0.1 V, source temperature 150°C, dislocation gas flow 600 L/H, cone gas flow 0.0 L/hr, and collision gas flow 0.2 ml/min. The system was operated in the multiple reaction mode (MRM) to measure the peak area of compounds. The transitions of the 4-hydroxydiclofenac-H+ adduct (m/z 314.15>231.15), diclofenac-H+ adduct (m/z 296>214), and chlorpropamide-H+ adduct (m/z 277>111) were monitored at collision energies of 34, 32, and 20 eV, respectively. The QuanLynx software was used to calculate the peak area.
Eleven variants of P450 2C9 were successfully constructed in a pCW bicistronic vector and the recombinant P450 enzymes were expressed in
The enzyme activity of P450 2C9 can be measured from the 4-hydroxylation reaction of diclofenac (Guengerich, 2015). LC-MS/MS analysis showed the diclofenac substrate at 1.77 min with an MRM of 296>214 and 4-hydroxydiclofenac at 1.27 min with an MRM of 314.15>231.15 (Fig. 2). The catalytic activity of the P450 2C9 variant enzymes was determined by measuring the rates of diclofenac 4-hydroxylation using bicistronic membrane fractions. Steady-state kinetic analysis of diclofenac 4-hydroxylation indicated that the catalytic efficiencies (
Previously, the X-ray crystal structure of human P450 2C9 with bound warfarin was determined by Williams
Five human P450 enzymes—CYP1A2, 2C9, 2C19, 2D6, and 3A4—are responsible for the metabolism of more than 90% clinical drugs and P450 2C9 is the second major hepatic P450 enzyme that is responsible for the metabolic clearance of 15% to 20% of drugs (Si
In this study, the recombinant variant enzymes of P450 2C9 found in Korean populations (M01–M11) were heterologously expressed in
The novel variant M01 exhibited similar expression level to that of the wild type enzyme. However, a two-fold decrease in its
The M03 and M04 variants showed similar expression levels compared to the wild type, but their catalytic efficiency was dramatically reduced (Fig. 3, Table 2). According to a previous study, the M04 variant in the P450 2C9*14 allele showed increased
Interestingly, the M05 and M06 variants exhibited higher turn-over numbers than the wild type enzyme (Table 2). The clinical outcomes of 50% to 80% increases in turn-over numbers (albeit increases in
The M08 and M09 variants contain Ile359 mutations in the K-helix and are called P450 2C9*3 and *4 alleles, respectively (Sullivan-Klose
The M10 variant (P450 2C9*30 allele) showed a 3-fold lower expression than the wild type enzyme, a 5-fold decrease in the
In the M11 variant, Glu324 is substituted with a stop codon. Thus, this mutation is incapable of producing proteins. Because Glu324 is downstream of the heme-binding signature sequences of P450 2C9, the holoenzyme containing the heme prosthetic group cannot be obtained for this variant.
In conclusion, 11 allelic variants of P450 2C9 were constructed and their functional attributes were examined. Nine variants and the wild type enzyme were successfully expressed and their functional alterations toward diclofenac were analyzed. Most of these allelic variants reduced catalytic efficiency; in particular, the M03 and M10 variants showed significantly reduced catalytic efficiency. Three novel variants—M01, M07, and M11—demonstrated significant alterations in their expression or catalytic activity. As P450 2C9 is important for the metabolism of various drugs, the study of 11 variants found in Korean populations can provide valuable information on their pharmacogenetic effects.
The authors declared no conflict of interest.
This work was supported by Konkuk University in 2018.
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