Team:Patras/Results
Results
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Western Blotting
Enzymatic Properties
Western Blotting
Western blotting was performed to determine the protein expression levels of CYP2D6 and CYP2C19 wild type and variant proteins. The analysis was performed according to the standard protocol with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis; for performing Western using a software to evaluate and visualize the protein expression levels.
For CYP2D6, each well was loaded with 30 μg/mL microsomes, and CYP2D6 protein was detected using a polyclonal anti-human CYP2D6 antibody (diluted 1:250). Secondary antibody detection was carried out with horseradish peroxidase-conjugated goat anti-rabbit IgGcat. Additionally, a total protein assay was performed to normalize each signal using 30 μg/mL microsomes.
For CYP2C19, each well was loaded with 100 μg/mL microsomes, and CYP2C19 protein was detected using a polyclonal anti-human CYP2C19 antibody (diluted 1:100). Secondary antibody detection was carried out with horseradish peroxidase-conjugated goat anti-rabbit IgGcat. Additionally, a total protein assay was performed to normalize each signal using 100 μg/mL microsomes.
Enzymatic Properties
The enzymatic properties of wild-type and CYP2D6 and CYP2C19 variants were measured through pharmacokinetic parameters from in vitro drug metabolism data. Enzyme kinetics deals with a quantitative description of the rates of enzyme-catalyzed chemical reactions, mainly how experimental variables affect reaction rates. Enzyme kinetics combined with related approaches can show how the functional properties of a variant or engineered enzyme compare to those of its wild-type.
To display information about enzyme kinetics, Michaelis-Menten graphs (graphing reaction rate as a function of substrate concentration) are used to provide information, such as kinetic parameters: Km, Vmax, and CLint
According to the graph, at a low substrate concentration, there is an abrupt increase in the rate of reaction (V0) as the substrate's concentration increases, then level off to a flat plateau at high substrate concentrations. This plateau occurs because the enzyme is saturated, and any additional substrate molecules will not have an effect due to the saturation and will wait until another enzyme becomes available and so the rate of reaction and product formation is limited and is only dependable on the enzyme’s activity. This maximum rate of reaction is called maximum velocity Vmax.
Km is the substrate concentration that gives the rate that is halfway to Vmax. It measures how quickly reaction rate (V0) increases with substrate concentration, how easily the enzyme can be saturated by the substrate, and also a measure of the affinity of the enzyme for its substrate. Lower Km represents a lower affinity for the substrate.
CLint (CLint = Vmax/Km) is a measure of enzyme activity measured by measuring the increase in the metabolite.
To display information about enzyme kinetics, Michaelis-Menten graphs (graphing reaction rate as a function of substrate concentration) are used to provide information, such as kinetic parameters: Km, Vmax, and CLint
For CYP2D6, the microsomal fraction obtained from 293FT cells were incubated in a mixture containing reagents and as substrate dextromethorphan in variable concentrations (1, 2, 4, 8, 10, 20, 40, 80 μM).
The determination of dextromethorphan O-demethylation in 30 μg of the microsomal protein fractions (containing wild-type CYP2D6 or CYP2D6 variants) showed that dextromethorphan formation was linear for incubations of up to 30 min. Following incubation and protein removal by centrifugation at 14,000 g for 5 min, 50 μL of the resultant supernatant was injected into an ultra-performance liquid chromatography (UPLC)-fluorescence (FLR) detector system.
The Michaelis-Menten kinetic parameters were measured for the dextromethorphan O-demethylation for the wild type and the 3 CYP2D6 variant proteins. Below are represented the means ± standard deviation (SD) of as derived from the three independently performed catalytic assays compared with wild-type CYP2D6.
Differences with *p < 0.05 were considered significant.
| # | CYP2D6 Variants | Km (μM) | V max (nmol/min/mg protein) | CLint (mL/min/mg protein) (% of Wild-type) |
|---|---|---|---|---|
| 1 | Wild-type | 2.01 ± 0.24 | 25.31 ± 0.51 | 12.72 ± 1.35 (100.00) |
| 2 | p.L61S | 1.53 ± 0.45 | 2.26 ± 0.15* | 1.54 ± 0.31* (12.11) |
| 3 | p.F112S | 3.09 ± 0.84 | 15.60 ± 1.86 | 5.19 ± 0.76* (40.80) |
| 4 | p.S476I | N.D. | N.D. | N.D. |
Among the three types of CYP2D6 variants tested, the kinetic parameters of 1 (p.S476I) could not be determined because the amount of the metabolite produced was at or below the detection limit.
The CLint of the wild type CYP2D6 represents 100% of the enzymatic activity, resulting in a diagram representing the enzymatic activity of variant CYP2D6 as a % of the wild type.
Compared to wild type, p.L61S variant CYP2D6 exhibited significantly decreased activity with 12.1 % of the activity of wild type. This hint indicates that maybe in clinical studies, such variant could lead to a Poor Metabolizer.
Additionally, the p.F112S variant showed a decreased activity, 40.7% of CYP2D6. This substitution may be located far from the site that interacts with the substrate and thus does not affect its metabolism as on p.L61S.
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More details about UPLC for CYP2D6
On CYP2D6, dextromethorphan O-demethylation activity was measured as described below.
1) An incubation mixture prepared containing:
- • microsomal fraction (30 μg) obtained from 293FT cells
- • dextromethorphan in concentrations: 1, 2, 4, 8, 10, 20, 40, 80 μM
- • 3.3 mM MgCl2
- • and 100 mM potassium phosphate buffer (pH 7.4) in a final volume of 150 μL
2) Incubation at 37°C for 3 min
3) Initiation of reactions by adding 15 μL of a mixture containing:
- • 1.3 mM NADP+
- • 3.3 mM glucose-6-phosphate
- • and 0.4 U/mL glucose-6-phosphate dehydrogenase
4) Incubation at 37°C for 30 min
5) Termination of reactions by adding 150 μL methanol containing 200 nM levallorphan, as an internal standard
6) Incubation 30 min
7) Protein removed with centrifugation at 14,000 g for 5 min
8) 50 μL of the resultant supernatant was injected into an ultra-performance liquid chromatography (UPLC)-fluorescence (FLR) detector system
Conditions of LC-MS/MS performance
LC-MS/MS system was used to measure the (S)-Mephenytoin 4'-hydroxylation activity on CYP2C19.
4'-Hydroxy mephenytoin was measured using the LC-MS/MS system in the positive ion detection mode at the electrospray ionization interface. Separation by LC was conducted using NANOSPACE SI-2. Chromatographic separation was performed using a Luna C18 100A column (2×150 mm, 5.0-μm particle size) maintained at 40°C. The flow rate was set at 300 μL/min, and the mobile phases were formed using water containing 0.1% formic acid as eluent A and acetonitrile containing 0.1% formic acid as eluent B.The gradient program was as follows: initial elution with 100% A, followed by a linear gradient to 50% B from 3.0 to 4.0 min to 90% B from 4.0 to 6.0 min, and to 100% B from 6.0 to 8.0 min, held at 100% B for 12.0 min, and then immediately returned to initial conditions and maintained for 3.0 min until the end of the run.
LC effluent was introduced into the mass spectrometer between 3.0 and 12.0 min after injection. Quantification analyses were performed in the selected reaction monitoring mode, in which transitions from the precursor into the product ion were monitored: m/z 219.1→134.2 for (S)-mephenytoin (collision energy, 10 V), m/z 235.1→150.2 for 4'-hydroxy mephenytoin (collision energy, 17 V), and m/z 238.1→150.2 for 4'-hydroxy mephenytoin-d3 (collision energy, 19 V).
The optimized parameters for MS were as follows: spray voltage, 3.0 kV; sheath gas pressure, 50 psi; vaporizer temperature, 450°C; capillary temperature, 300°C; and collision pressure, 1.5 mTorr. The sheath gas was nitrogen, and the collision gas was argon. The LC-MS/MS system was controlled by Xcalibur software, which was also used to analyze the attained data. Standard curves for 4'-hydroxy mephenytoin were constructed in the 0.25-5.0 μM range using authentic metabolite standards.
For CYP2C19, the microsomal fraction obtained from 293FT cells was incubated in a mixture containing reagents and as substrate (S)-mephenytoin in variable concentrations (5, 10, 25, 50, 100, 250, 500, 1000 μM).
The determination of (S)-mephenytoin 4'-hydroxylation in 50 μg of the microsomal protein fractions (containing wild-type CYP2C19 or CYP2C19 variants) showed that 4'-hydroxy mephenytoin formation was linear for incubations of up to 30 min. After protein removal by centrifugation at 14,000 g for 5 min, 10 μL of the supernatant was injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system.
The kinetic parameters (Michaelis constant (Km), maximum velocity (Vmax), and intrinsic clearance (CLint = Vmax/Km) were determined using the Enzyme Kinetics Module of SigmaPlot 12.5.
The Michaelis-Menten kinetic parameters were measured for the mephenytoin 4'-hydroxylation for the wild type and the 9 CYP2C19 variant proteins, excepted the variant p.R26* because it results in a stop codon and no protein can be synthesized. Below are the means ± standard deviation (SD) of as derived from the three independently performed catalytic assays compared with wild-type CYP2C19. Differences with *p < 0.05 and ***p <0.005 were considered significant
| # | CYP2C19 Variants | Km (μM) | V max (nmol/min/mg protein) | CLint (mL/min/mg protein) (% of Wild-type) |
|---|---|---|---|---|
| 1 | Wild-type | 0.118 ± 0.013 | 0.278 ± 0.005 | 2.368 ± 0.230 (100.00) |
| 2 | p.L15P | 0.119 ± 0.002 | 0.152 ± 0.001*** | 1.274 ± 0.029 (53.80) |
| 3 | p.E274G | 0.136 ± 0.010 | 0.041 ± 0.001*** | 0.306 ± 0.023* (12.92) |
| 4 | p.N286D | 0.177 ± 0.013 | 0.027 ± 0.001*** | 0.152 ± 0.009* (6.42) |
| 5 | p.D293G | N.D. | N.D. | N.D. |
| 6 | p.T304A | 0.219 ± 0.007* | 0.114 ± 0.002*** | 0.521 ± 0.011* (22.00) |
| 7 | p.I327V | 0.132 ± 0.005 | 0.073 ± 0.001*** | 0.550 ± 0.018* (23.22) |
| 8 | p.L380P | N.D. | N.D. | N.D. |
| 9 | p.L413M | 0.133 ± 0.013 | 0.080 ± 0.003*** | 0.605 ± 0.047* (25.55) |
| 10 | p.F487S | N.D. | N.D. | N.D. |
From the measurement of 9 types of CYPC19 variants, the kinetic parameters of p.D293G, p.L380P, and p.F487S could not be determined because the amount of the metabolite produced was at or below the detection limit. Proceeding to the analysis of the enzymatic activity of each variant compared against the wild type CYP2C19, the following charts are obtained.
It is observed that all the variant CYP2C19 exhibited a decreased enzyme activity, with most of them appearing a 20% activity of the wild type CYP2C19. Precisely, for p.T304A, p.I327V, and p.L413M the enzymatic activity of variant CYP2C19 was 22.02%, 23.25%, and 25.84%, respectively.
Among the variant, two showed significantly decreased activity, the p.E274G with 12.9 % of the activity of wild type and p.N286D with even more significant reduction of the activity, presenting a 6.42% of the activity of wild type.
The variant, p.L15P appears a 53.80% of the activity of wild type, indicating that the substitution may occur at a site that does not affect the interaction with the substrate as on the other variants.
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More details about LC/MS-MS for CYP2C19
On CYP2C19, dextromethorphan O-demethylation activity was measured as described below.
1) An incubation mixture prepared containing:
- • microsomal fraction (50 μg) obtained from 293FT cells
- • (S)-mephenytoin in concentrations 5, 10, 25, 50, 100, 250, 500, 1000 μ
- • 3.3 mM MgCl2
- • and 100 mM potassium phosphate buffer (pH 7.4) in a final volume of 150 μL
2) Incubation at 37°C for 3 min
3) Initiation of reactions by adding 15 μL of a mixture containing:
- • 1.3 mM NADP+
- • 3.3 mM glucose-6-phosphate
- • and 0.4 U/mL glucose-6-phosphate dehydrogenase
4) Incubation at 37°C for 30 min
5) Termination of reactions by adding 150 μL acetonitrile containing 10 μM 4'-hydroxy mephenytoin-d3, as an internal standard
6) Incubation 30 min
7) Protein removed with centrifugation at 14,000 g for 5 min
8) 10 μL of the resultant supernatant was injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system
Conditions of UPLC performance
UPLC was performed to measure the dextromethorphan O-demethylation activity on CYP2D6.
The UPLC-FLR system was comprised of an ACQUITY UPLC H-Class, connected to an ACQUITY UPLC FLR Detector. Separation was performed on an analytical column (ACQUITY UPLC CSH Fluoro-Phenyl, 2.1⋅75 mm, 1.7-μm particle size; Waters Corporation) maintained at 40°C. The mobile phase was composed of 75% buffer (20 mM potassium phosphate and 20 mM hexane sulfonic acid, pH 4.0) and 25% acetonitrile. The flow rate was set at 0.5 mL/min with fluorescence detection at excitation and emission wavelengths of 280 nm and 310 nm, respectively. Standard curves for dextrorphan were constructed in the 0.5-100 μM range using authentic metabolite standards.
From the in-vitro assays, we have evidence about how every variant, alongside its consequent substitution on the amino acid sequence, affects the enzyme’s activity and determines the variant CYP2D6/CYP2C19 enzyme activity compared against the wild type’s activity. However, it would be difficult to assess the clinical outcome in subjects who express those CYP2D6 and CYP2C19 variants without in vivo data. To better understand our findings, it would be great to examine the clinical relationships among CYP2D6 and CYP2C19 variants and the plasma concentrations of the given drug-substrate and its metabolite. Thus, further studies will be needed to define the importance of the above novel variants in clinical settings.
*The results presented in this section are a part of the collaboration between our Laboratory of Pharmacogenomics and Individualized Therapy, School of Pharmacy, University of Patras with the Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences at Tohoku University, Japan.