CN112725408A - UGT enzyme activity detection method and application thereof - Google Patents
UGT enzyme activity detection method and application thereof Download PDFInfo
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- CN112725408A CN112725408A CN202110082924.0A CN202110082924A CN112725408A CN 112725408 A CN112725408 A CN 112725408A CN 202110082924 A CN202110082924 A CN 202110082924A CN 112725408 A CN112725408 A CN 112725408A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention provides a detection method of UGT enzyme activity not used for disease diagnosis and treatment and application thereof in screening and evaluating UGT enzyme regulators. The detection method takes ophiopogonin A as a substrate, and detects the UGT enzyme activity by quantitatively detecting the conversion rate of the ophiopogonin A or the generation rate of the 7-O-glucuronic acid product of the ophiopogonin A by a liquid-ultraviolet method or a liquid-mass spectrometry method. The method has the characteristics of high sensitivity, strong stability, good repeatability, simplicity, easiness in operation and the like; the method is used for screening and evaluating various reported UGT enzyme regulators, has extremely high accuracy and has very good application prospect.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a detection method of UGT enzyme activity not used for disease diagnosis and treatment, and application of the detection method in screening and evaluation of a UGT enzyme regulator.
Background
The uridine diphosphate glucuronyl transferase (UGT) superfamily is an important phase ii metabolic enzyme in the body, which increases the hydrophilicity of the substrate by catalyzing the compound to bind with the cofactor uridine diphosphate glucuronate (UDPGA), which enables the compound to be more efficiently excreted from the body in the urine or bile, an important detoxification process of the body. Mammalian UGTs can be divided into 4 families: UGT1, UGT2, UGT3, and UGT 8. Among them, most of them involved in drug-binding metabolism are members of the UGT1A and UGT2B subfamilies.
As the most important II-phase metabolic enzyme of the body, UGT enzyme plays a key role in the metabolic clearance and detoxification of various heterologous organisms (such as therapeutic drugs and I-phase metabolites thereof, food chemical components or environmental toxins). In addition to xenometabolism, UGT enzymes are involved in the glucuronidation metabolism of many endogenous substances (e.g., bilirubin, steroids, and bile acids). When UGT enzymes are dysfunctional or strongly inhibited, endogenous metabolism disorders and drug/herbal interactions (DDI/HDI) may be induced, which can have adverse effects on human health. On the contrary, partial agonists of UGT enzyme (such as UGT1A1) can accelerate the metabolic clearance of toxicants such as bilirubin and the like, thereby relieving the occurrence and development processes of diseases. Therefore, it is desirable to develop efficient screening and evaluation methods for UGT enzyme modulators (inhibitors or agonists).
Because of the high degree of identity of the UGT enzyme amino acid sequence, which results in high overlap of its substrate spectra, there are few reports of probe substrates specific for UGT enzymes. To date, only a few UGT subtype enzymes (including UGT1a1, UGT1a4, UGT1a9, and UGT2B7) have recognized specific substrates. For the activity detection of other UGT subtype enzymes, only the traditional non-fluorescent substrate 4-methylumbelliferone (4-MU) can be relied on, the detection flux is low, the sample pretreatment and detection process are complicated and complicated to operate, and the large-scale screening and evaluation of the inhibition capability of a target object on various UGT subtype enzymes cannot be synchronously carried out.
Therefore, the detection method for designing and developing non-disease diagnosis and treatment of UGT enzyme activity and the application of the detection method in UGT enzyme regulation agent screening and evaluation have important significance for UGT mediated drug interaction evaluation.
Disclosure of Invention
The invention belongs to the technical field of medicines, and particularly relates to a detection method of UGT enzyme activity not used for disease diagnosis and treatment, and application of the detection method in screening and evaluation of a UGT enzyme regulator. The method takes ophiopogonin A (MOA) as a substrate, under physiological conditions, the ophiopogonin A can be catalyzed by 13 human UGT enzymes to generate 7-O-glucuronic acid (MOAG) of the ophiopogonin A, and the product can be detected by liquid phase-ultraviolet or liquid phase-mass spectrometry. By means of the probe reaction, the recombinant mammalian UGT enzyme is used as an enzyme source, and the screening and evaluation of the regulation and control capacity of the compound to be tested on the 13 human UGT enzymes can be synchronously realized. The screening and evaluation method is carried out by means of a liquid phase-ultraviolet detection system (LC-UV) or a liquid phase-mass spectrum detection system (LC-MS), and the method has the advantages of sensitive detection, simplicity and convenience in operation, high detection precision and stability, strong anti-interference capability, low price, high efficiency and the like, and has a good application prospect.
The invention provides a UGT enzyme activity detection method not used for disease diagnosis and treatment, which adopts ophiopogonone A as a substrate and detects the UGT enzyme activity by quantitatively detecting the conversion rate of the ophiopogonone A or the generation rate of 7-O-glucuronic acid product thereof.
The method of the invention comprises the following steps:
(1) mixing tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution with MgCl2Mixing with high isoflavone A, UGT enzyme, pre-incubating for 3-5 min at 20-60 deg.C, and adjusting pH of the reaction solution to 5-10;
(2) adding uridine diphosphate glucuronate into the reaction liquid obtained in the step (1) to react for 10-60 minutes to obtain a 7-O-glucuronate product of ophiopogonone homoisoflavone A;
(3) adding glacial acetonitrile or glacial methanol into the reaction liquid obtained in the step (2), precipitating protein, and centrifuging to obtain supernatant;
(4) detecting the conversion rate of the ophiopogonone A or the generation rate of the 7-O-glucuronic acid product of the ophiopogonone A in the supernate obtained in the step (3) by using a liquid-ultraviolet system or a liquid-mass spectrum system.
The reaction equation is shown in formula 1:
in the invention, the UGT enzyme is selected from one or more of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15 or UGT2B 17.
In the invention, when a liquid phase-ultraviolet system is used in the step (4), the detection conditions are that an octadecylsilane column is used as a stationary phase, 0.2% formic acid (B) and acetonitrile (A) are used as mobile phases, a gradient elution mode is adopted for separation, and the ultraviolet detection wavelength is 300 nm.
In the present invention, when the liquid-mass spectrometry system is used in step (4), the internal standard 4-methylumbelliferyl-beta-D-glucuronide is added in step (3).
In the present invention, when a liquid-mass spectrometry system is used in step (4), the detection conditions are that an octadecylsilane chemically bonded silica column is used as a stationary phase, 0.2% formic acid (B) and acetonitrile (a) are used as mobile phases, the flow rate is set to 0.4ml/min, the sample size is 2 μ L, and a gradient elution method is adopted to separate: 0-0.5min, 98% B, 0.5-0.6min, 98% -15% B; 0.6-1.6min, 15% B; 1.6-1.7min, 98% B; 1.7-3min, 98% B; an electrospray ion source, negative ions and a multi-reaction monitoring mode are adopted; the nitrogen is atomized gas and dry gas; the high-purity nitrogen is used as collision gas, and the pressure is 0.1 MPa; the mass spectrometer parameters were set as follows: ionization voltage (Is): -4500V; jet impingement (CAD): medium; air curtain gas (CUR): 20psi, spray mist (GS 1): 20psi, ion source Temperature (TEM): 450 ℃, declustering voltage (DP): -80V, impact voltage (CE): 15V; the detected ions used for quantitative analysis were: MOAG mass to charge ratio 517.138 → 341.105; internal standard 4-methyl umbelliferone-beta-D-glucuronide mass-to-charge ratio 351.0 → 175.0.
The second purpose of the invention is to provide the application of the detection method in screening and evaluating UGT enzyme modulators, and the method also comprises the steps of adding the test compound in the step (1) and evaluating the modulation effect of the test compound in the step (4).
In the present invention, a test compound is an agonist when it increases UTG enzymatic activity; a test compound is an inhibitor when it decreases UTG enzyme activity.
The invention has the following advantages:
(1) and (3) detection sensitivity: the broad-spectrum substrate, the glucuronidation reaction of the ophiopogonone root homoisoflavone A, can be catalyzed and metabolized by various UGT enzymes to generate a single metabolite, the affinity of the substrate and the UGT enzymes is good, an LC-UV or LC-MS detector used in the detection method is sensitive, the required sample amount is very small, the use amount of experimental materials can be reduced in actual operation, and the experimental cost is reduced.
(2) The stability is strong: in the invention, the product and the substrate do not react with each other and interfere with each other, and the established method uses LC-UV or LC-MS for detection, can realize good separation of various substances existing in the sample, avoids the interference of impurities to the sample, carries out experimental sample treatment and determination by using the method under the condition of 4 ℃, has no obvious response signal intensity change for at least two days, and is convenient for quantitatively monitoring the glucuronidation reaction process in real time.
(3) The operation is simple, the repeatability is good: the natural product ophiopogonin A is used for construction, raw materials can be directly purchased, and the UGT enzyme activity detection and UGT enzyme regulating agent screening method has the characteristics of convenience in operation, low consumption and the like, and is good in economic applicability. And the broad-spectrum substrate reaction is relatively stable, and the experimental repeatability is relatively good.
(4) The UGT enzyme types detected are many: the method can realize the detection of the activity of all 13 UGT enzymes participating in drug metabolism of a human body, and compared with the traditional method using 4-methylumbelliferone as a substrate, the method can detect the O-glucuronic acid activity of UGT1A4 and UGT2B10, and can screen and evaluate regulating agents of thirteen enzymes under the same condition, thereby avoiding experimental errors when different substrates are used for screening and evaluating.
The invention is constructed based on broad-spectrum substrate ophiopogonone A found by natural product screening, the substrate only generates a single metabolic product of a single glucuronic acid product, and the activity of 13 UGT subtype enzymes can be detected. The method has the characteristics of high sensitivity, strong stability, good repeatability, simplicity, easiness in operation and the like. Screening and re-evaluating a plurality of reported UGT enzyme selective inhibitors, and proving that the screening method has extremely high accuracy and very good application prospect.
Drawings
FIG. 1 is a graph of the rate of glucuronidation of MOA in example 1.
FIG. 2 mass spectrum of MOAG in example 2.
FIG. 3 stability test chart in example 3
FIG. 4 is a diagram showing the reaction process of liquid phase-UV assay for MOA glucuronidation in example 4.
FIG. 5 is a diagram showing the reaction process of liquid-mass spectrometry for detecting MOA glucuronidation in example 5.
FIG. 6 is a nuclear magnetic identification chart of MOAG in example 2, wherein the chart (a) is a hydrogen spectrum and the chart (b) is a carbon spectrum.
FIG. 7 IC-screening and evaluation of UGT enzyme inhibitors based on UV detection System in example 650Graph wherein- -indicates no or very weak inhibition, IC50>100μM。
FIG. 8 mass spectrum measured using LC-MS in example 7.
FIG. 9 is a graph showing the screening and evaluation of the effect of glycitein activating UGT enzyme in example 8 by using the LC-UV detection system and the established method.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1 broad-spectrum study of human UGT enzyme metabolism of ophiopogonone root homoisoflavone A
And the UGT enzyme phenotype research which participates in broad-spectrum substrate ophiopogon japonicus homoisoflavone A glucose binding metabolism. 13 commercial recombinant humanized UGT single enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) are adopted as target UGT enzyme sources, and the substrate MOA is subjected to metabolic characterization. The method comprises the following specific steps:
(1) Tris-HCl buffer (50mM, pH 7.4) and magnesium chloride (5mM) are respectively mixed in an EP tube by taking one of 13 UGT single enzymes as target UGT enzyme (0.05mg/ml recombinant humanized UGT single enzyme), ophiopogonin A (the concentration is unified to 10 mu M and 100 mu M) is pre-incubated for 3-5 minutes at the temperature of 20-60 ℃, and the pH value of an incubation system is 5-10. The specific reaction conditions are as follows:
| UGT1A1 | UGT1A3 | UGT1A4 | UGT1A6 | UGT1A7 | UGT1A8 | UGT1A9 | |
| pre-incubation time (minutes) | 3 | 4 | 5 | 3 | 4 | 5 | 3 |
| Temperature of | 20 | 50 | 60 | 20 | 37 | 60 | 37 |
| |
5 | 8 | 10 | 6 | 9 | 7.4 | 7.4 |
| UGT1A10 | UGT2B4 | UGT2B7 | UGT2B10 | UGT2B15 | UGT2B17 | ||
| Pre-incubation time (minutes) | 4 | 5 | 3 | 4 | 5 | 5 | |
| Temperature of | 37 | 37 | 37 | 37 | 37 | 37 | |
| pH value | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 |
(2) Respectively adding UDPGA to react for 10-60 minutes, and concretely, adding the UDPGA to react for 10-60 minutes as follows:
| UGT1A1 | UGT1A3 | UGT1A4 | UGT1A6 | UGT1A7 | UGT1A8 | UGT1A9 | |
| reaction time (minutes) | 10 | 40 | 60 | 20 | 40 | 60 | 30 |
| UGT1A10 | UGT2B4 | UGT2B7 | UGT2B10 | UGT2B15 | UGT2B17 | ||
| Reaction time (minutes) | 60 | 60 | 60 | 60 | 60 | 60 |
Detecting the generation amount of glucuronic acid product by using a liquid-phase ultraviolet system, and processing data to obtain corresponding substrate reaction rate. As a result, the substrate ophiopogonin A can be metabolized by 13 UGT enzymes. (as shown in FIG. 1)
The results show that: compared with the traditional substrate 4-methylumbelliferone, the ophiopogonone A can be metabolized by all 13 UGT subtype enzymes participating in human drug metabolism (including UGT1A4 and UGT2B10 which cannot be detected by 4-methylumbelliferone). Based on the discovery of the ophiopogonone A glucuronic acid reaction and the established detection method, the number of covered enzymes is large, all 13 UGT enzyme activities participating in drug metabolism in a human body can be detected under the same condition, and the broad spectrum property of the method is even superior to that of the traditional method taking 4-methylumbelliferone as a substrate.
Example 2 preparation and identification of A metabolite of ophiopogonin A by UGT enzyme
After UGT-catalyzed ophiopogonone A metabolites are analyzed and identified by using high-resolution mass spectrometry (as shown in figure 2), the product MOAG is prepared and structurally characterized in a large scale by using a biological preparation method and a nuclear magnetic resonance technology respectively. (as shown in fig. 6). The method comprises the following specific steps:
(1) mixing radix Ophiopogonis homoisoflavone A (8.52mg), Tris-HCl buffer (50mM, pH 7.4), and MgCl2(5mM), polyoxyethylene cetyl ether (0.1mg/mg protein) and 13 UGT enzymes expressed recombinantly (1.85. mu.L of each enzyme, final protein concentration 0.8mg/ml) were combined. After preincubation at 37 ℃ for 5 minutes, UDPGA (2mM) was added to start the reaction.
(2) After incubation at 37 ℃ for 6 hours, the reaction was stopped by adding ice-cold methanol (150 ml). After centrifugation at 20,000 Xg for 20 minutes at 4 ℃, the supernatant was collected, then evaporated, and the precipitate was purified by a preparative reverse liquid chromatography system using a reverse phase column.
(3) Finally, the obtained purified MOAG (8.0mg, purity greater than 98% by liquid-uv system) and MOA were dissolved in deuterated methanol for structural characterization using Bruker 400 Nuclear Magnetic Resonance (NMR) spectrometer.
(4) The obtained product is applied to standard curve establishment in method construction and related method evaluation experiments.
The results show that: the glucuronic acid reaction of the broad-spectrum substrate ophiopogon root homoisoflavone A can be catalyzed and metabolized by various UGT enzymes to generate a single metabolite, namely a single glucuronic acid product: MOA-7-O-glucuronic acid product (MOAG), the reaction is as follows:
example 3 stability Studies
(1) Tris-HCl buffer (50mM, pH 7.4) was mixed with MgCl under physiological conditions2(5mM), 13 recombinant UGT enzymes (0.31 mu L of each enzyme, the final protein concentration is 0.1mg/ml) are used as enzyme sources, and the substrate radix ophiopogonis homoisoflavone A solution (the concentration is selected from 1/10-1K)m) Mixing in a centrifuge tube, wherein the reaction temperature is 37 ℃, and the pH of the incubation system is 7.4.
(2) And (3) pre-incubation, namely incubating the mixed solution in the step (1) for 3-5 min at 37 ℃ to ensure that the enzyme is fully contacted with a substrate, namely the ophiopogonone A.
(3) UDPGA was added for 30 minutes.
(4) Samples were taken after 0 hours, 24 hours and 48 hours at 4 ℃ and analyzed to determine the stability under the refrigerator storage conditions at 4 ℃. The results are shown in FIG. 3.
The results show that: the stability is strong, in the probe reaction, the product and the substrate do not react with each other and interfere with each other, the method is used for processing and measuring the experimental sample at 4 ℃, obvious response signal intensity change does not occur for at least two days, and the real-time quantitative monitoring of the glucuronic acid reaction process is convenient.
Example 4 detection of MOA-7-O-glucuronidation reaction Rate by LC-UV method
(1) Tris-HCl buffer (50mM, pH 7.4) was mixed with MgCl under physiological conditions2(5mM), 13 recombinant UGT enzymes (0.31 mu L of each enzyme, the final protein concentration is 0.1mg/ml) are used as enzyme sources, and the substrate radix ophiopogonis homoisoflavone A solution (the concentration is selected from 1/10-1K)m) Mixing in a centrifuge tube, wherein the total volume is 200 microliters, and the reaction temperature is 37 ℃; the pH of the incubation system was 7.4.
(2) And (4) pre-incubation. The mixed solution (blank control group without substrate, ophiopogonin A) is incubated at 37 deg.C for 5 min.
(3) The UDPGA reaction time was 60 minutes to ensure that the product formation or substrate conversion was less than 20%.
(4) The reaction was stopped by adding an equal volume of 200. mu.l of glacial acetonitrile and centrifuged in a centrifuge at 20,000 Xg for 20 minutes at 4 ℃. After the reaction solution centrifugally precipitates protein, the generated substrate and product are quantitatively analyzed by a liquid-phase ultraviolet detection system, and detection is carried out under the ultraviolet wavelength of 300 nm. The detection conditions are that ODS column is used as a stationary phase, 0.2% formic acid (B) and acetonitrile (A) are used as mobile phases, and 0-1min, 80% B, 1-2min and 80% -60% B are used; 2-6min, 60% -20% B; 6-6.5min, 20% B; separating the substrate and the product by a gradient elution mode at 20-80% B for 6.5-7.5 min. The substrates and products were detected analytically at a UV wavelength of 300nm (FIG. 4).
(5) The production rate of the ophiopogonone homoisoflavone A glucuronic acid product MOAG in the reaction time is measured, and the percentage of the reaction rate of the inhibitor-added group relative to the reaction rate of the blank control group is calculated to be used as the evaluation standard of the inhibitory activity.
After detection, the LC-UV method can be used for detecting the MOA-7-O-glucuronic acid reaction rate.
Example 5 detection of MOA-7-O-glucuronidation reaction Rate by LC-MS method
(1) Tris-HCl buffer (50mM, pH 7.4) was mixed with MgCl under physiological conditions2(5mM), 13 recombinant UGT enzymes (0.31 mu L of each enzyme, and final protein concentration of 0.1mg/ml) are used as enzyme sources, and the substrate radix ophiopogonis homoisoflavone A solution (the concentration is selected from 1/10-1K)m) Mixing in a centrifuge tube, wherein the total volume is 100 microliters, and the reaction temperature is 37 ℃; the pH of the incubation system was 7.4.
(2) And (4) pre-incubation. And (3) incubating the mixed solution (a blank control group without the substrate, namely the ophiopogonone A) at 37 ℃ for 3-5 min.
(3) The UDPGA reaction time was 60 minutes to ensure that the product formation or substrate conversion was less than 20%.
(4) The reaction was stopped by adding an equal volume of glacial acetonitrile containing internal standard 4-methylumbelliferone-beta-D-glucuronide and centrifuged at 20,000 Xg for 20 minutes at 4 ℃ in a centrifuge. After the reaction solution centrifugally precipitates the protein, the generated product is quantitatively analyzed by a liquid phase-mass spectrum detection system.
(5) The detection conditions are that ODS column is used as a stationary phase, 0.2% formic acid (B) and acetonitrile (A) are used as mobile phases, the flow rate is set to be 0.4ml/min, the sample injection amount is set to be 2 mu L, and a gradient elution mode is adopted to separate a substrate from a product: 0-1min, 80% B, 1-2min, 80% -60% B; 2-6min, 60% -20% B; 6-6.5min, 20% B; 6.5-7.5min, 20% -80% B. Electrospray ion source, negative ion, Multiple Reaction Monitoring (MRM) mode was used. The nitrogen is atomized gas and dry gas; the high-purity nitrogen is used as collision gas, and the pressure is 0.1 MPa; the mass spectrometer parameters were set as follows: ionization voltage (Is): -4500V; jet impingement (CAD): medium; air curtain gas (CUR): 20psi, spray mist (GS 1): 20psi, ion source Temperature (TEM): 450 ℃, declustering voltage (DP): -80V, impact voltage (CE): 15V. The mass-to-charge ratio (m/z) of parent substrate ions is 341.105, and the mass-to-charge ratio (m/z) of child substrate ions is 206.060. Mass-to-charge ratio (m/z) of product parent ion: 517.138, mass-to-charge ratio (m/z) of product ion 341.105, 175.027. The results of the detection are shown in FIG. 5.
(6) The production rate of glucuronic acid product over the reaction time was measured and the percentage of the reaction rate of the inhibitor-added group relative to the reaction rate of the blank group was calculated as an evaluation criterion of inhibitory activity.
After detection, the LC-MS method can be used for detecting the MOA-7-O-glucuronic acid reaction rate.
Example 6 screening and evaluation of UGT enzyme inhibitors Using LC-UV method
The application of the detection method of UGT enzyme activity not used for disease diagnosis and treatment in screening and evaluating UGT enzyme modulators can screen and evaluate the inhibition effect of unknown compounds on 13 UGT enzymes. The specific operation is as follows:
(1) setting a reaction system of 200 mu L, adding Tris-HCl buffer solution (50mM, pH 7.4) and magnesium chloride (5mM) into a centrifuge tube by using 13 recombinant humanized UGT enzymes (final concentration is 0.05mg/ml) and a ophiopogonone A solution (10 mu M) and one of the compounds to be tested, namely amentoflavone, nilotinib, magnolol and fluconazole (concentration is 1 mu M-100 mu M) and the like respectively, mixing uniformly, and pre-incubating for 3 minutes at 37 ℃.
(2) After the reaction was initiated by adding 10. mu.L of UDPGA (final concentration: 2mM) to the reaction system, the centrifuge tube was placed in an incubator and continuously reacted for 60 minutes, ensuring that the product formation rate or substrate conversion rate was less than 20%.
(3) The reaction was stopped by adding an equal volume of 200. mu.L of glacial acetonitrile and centrifuged in a centrifuge at 20,000 Xg for 20 minutes at 4 ℃. After the protein was precipitated by centrifugation of the reaction solution, the resultant product was quantitatively analyzed by means of a liquid-UV detection system, and detection was carried out under the above-mentioned detection conditions (example 3) at a UV wavelength of 300 nm. The production rate of glucuronic acid product in the reaction time was quantitatively measured and the percentage of the reaction rate of the inhibitor-added group relative to the production rate of the blank group product was calculated as an evaluation criterion of inhibitory activity, and it was found that each of the four compounds had different degrees of inhibitory effect on UGT enzyme (fig. 7). Taking UGT1A1 as an example, the reaction rate was 23.97. mu. mol/min/mg protein before the addition of the inhibitor, whereas the reaction rates became 1.81, 2.63, 13.74, 21.06. mu. mol/min/mg protein after the addition of amentoflavone, nilotinib, magnolol and fluconazole.
Example 7 screening and evaluation of UGT enzyme inhibitors Using LC-MS
The application of the detection method of UGT enzyme activity not used for disease diagnosis and treatment in screening and evaluating UGT enzyme modulators can screen and evaluate the inhibition effect of unknown compounds on 13 UGT enzymes. The specific operation is as follows:
(1) setting a reaction system of 100 mu L, adding Tris-HCl buffer solution (50mM, pH 7.4) and magnesium chloride (5mM) into a centrifuge tube by using 13 recombinant humanized UGT enzymes (final concentration is 0.05mg/ml) and a ophiopogonone A solution (10 mu M) and one of the compounds to be tested, namely amentoflavone, nilotinib, magnolol and fluconazole (concentration is 1 mu M-100 mu M) and the like respectively, mixing uniformly, and pre-incubating for 3 minutes at 37 ℃.
(2) After the reaction was initiated by adding 5. mu.L of UDPGA (final concentration: 2mM) to the reaction system, the centrifuge tube was placed in an incubator and continuously reacted for 60 minutes, ensuring that the product formation rate or substrate conversion rate was less than 20%.
(3) The reaction was stopped by adding an equal volume of 100. mu.L of glacial acetonitrile containing internal standard 4-methylumbelliferone-beta-D-glucuronide and centrifuged at 20,000 Xg for 20 minutes at 4 ℃ in a centrifuge. After the reaction liquid is centrifuged to precipitate the protein, the generated product is quantitatively detected in a Multiple Reaction Monitoring (MRM) mode by means of a liquid phase-mass spectrum detection system. (FIG. 8)
(4) The detection conditions are that ODS column is used as a stationary phase, 0.2% formic acid (B) and acetonitrile (A) are used as mobile phases, the flow rate is set to be 0.4ml/min, the sample injection amount is set to be 2 mu L, and a gradient elution mode is adopted to separate a substrate from a product: 0-0.5min, 98% B, 0.5-0.6min, 98% -15% B; 0.6-1.6min, 15% B; 1.6-1.7min, 98% B; 1.7-3min, 98% B. Electrospray ion source, negative ion, Multiple Reaction Monitoring (MRM) mode was used. The nitrogen is atomized gas and dry gas; the high-purity nitrogen is used as collision gas, and the pressure is 0.1 MPa; the mass spectrometer parameters were set as follows: ionization voltage (Is): -4500V; jet impingement (CAD): medium; air curtain gas (CUR): 20psi, spray mist (GS 1): 20psi, ion source Temperature (TEM): 450 ℃, declustering voltage (DP): -80V, impact voltage (CE): 15V. The detected ions used for quantitative analysis were: MOAG mass to charge ratio 517.138 → 341.105. Internal standard 4-methyl umbelliferone-beta-D-glucuronide mass-to-charge ratio 351.0 → 175.0.
(5) The quantitative determination of the production rate of glucuronic acid product in the reaction time and the calculation of the percentage of the reaction rate of the inhibitor-added group relative to the production rate of the blank control group product were used as the evaluation criteria of inhibitory activity, and the results were similar to those of UV detection, and it was found that the four compounds all had different degrees of inhibitory effect on UGT enzyme. Taking UGT1A1 as an example, the reaction rate was 22.88. mu. mol/min/mg protein before the addition of the inhibitor, whereas the reaction rates became 1.62, 2.31, 14.20, 21.97. mu. mol/min/mg protein after the addition of amentoflavone, nilotinib, magnolol and fluconazole.
(6) The detection method can accurately quantify the target product MOAG, avoids the interference of impurity peaks to target peaks caused by poor separation effect when a complex sample is detected by LC-UV, only needs 3min for analysis, and is relatively efficient.
Example 8 screening and evaluation of UGT enzyme activators Using LC-UV method
The use of assay methods for UGT enzyme activity other than for the diagnostic treatment of disease for screening and evaluating UGT enzyme modulators. The specific operation is as follows:
(1) a reaction system of 200. mu.L is set, and Tris-HCl buffer (50mM, pH 7.4), magnesium chloride (5mM), recombinant humanized UGT1A1 enzyme (final concentration is 0.05mg/ml), ophiopogonin A solution (10. mu.M), and glycitein solution (concentration is 0. mu.M-10. mu.M) of a compound to be detected are added into a centrifuge tube, mixed uniformly and pre-incubated for 3 minutes at 37 ℃.
(2) After the reaction was initiated by adding 10. mu.L of UDPGA (final concentration: 2mM) to the reaction system, the centrifuge tube was placed in an incubator and continuously reacted for 60 minutes, ensuring that the product formation rate or substrate conversion rate was less than 20%.
(3) The reaction was stopped by adding an equal volume of 200. mu.L of glacial acetonitrile and centrifuged in a centrifuge at 20,000 Xg for 20 minutes at 4 ℃. After the reaction solution was centrifuged to precipitate proteins, the resultant product was quantitatively analyzed by means of a liquid-UV detection system, and detection was carried out under the above-mentioned detection conditions (the same as in example 4) at a UV wavelength of 300 nm. The production rate of glucuronic acid product in the reaction time is quantitatively measured and the percentage of the reaction rate of the activator-added group relative to the production rate of the blank control group is calculated as an activation activity evaluation criterion, and the ideal activation effect (more than 2 times) of glycitein of the compound to be tested on UGT enzyme at the concentration of 10 mu M is found. (FIG. 9).
Claims (8)
1. A UGT enzyme activity detection method not used for disease diagnosis and treatment is characterized in that ophiopogonin A is used as a substrate, and UGT enzyme activity is detected by quantitatively detecting the conversion rate of ophiopogonin A or the generation rate of 7-O-glucuronic acid product thereof.
2. Method according to claim 1, characterized in that it comprises the following steps:
(1) mixing tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution with MgCl2Mixing with high isoflavone A, UGT enzyme, pre-incubating for 3-5 min at 20-60 deg.C, and adjusting pH of the reaction solution to 5-10;
(2) adding uridine diphosphate glucuronate into the reaction liquid obtained in the step (1) to react for 10-60 minutes to obtain a 7-O-glucuronate product of ophiopogonone homoisoflavone A;
(3) adding glacial acetonitrile or glacial methanol into the reaction liquid obtained in the step (2), precipitating protein, and centrifuging to obtain supernatant;
(4) detecting the conversion rate of the ophiopogonone A or the generation rate of the 7-O-glucuronic acid product of the ophiopogonone A in the supernate obtained in the step (3) by using a liquid-ultraviolet system or a liquid-mass spectrum system.
3. The method of claim 2, wherein: the UGT enzyme is selected from one or more of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B15 or UGT2B 17.
4. The method of claim 2, wherein: when a liquid phase-ultraviolet system is used in the step (4), the detection conditions are that an octadecylsilane column is used as a stationary phase, 0.2% formic acid (B) and acetonitrile (A) are used as mobile phases, a gradient elution mode is adopted for separation, and the ultraviolet detection wavelength is 300 nm.
5. The method of claim 2, wherein: when a liquid phase-mass spectrometry system is used in step (4), an internal standard of 4-methylumbelliferyl-beta-D-glucuronide is added in step (3).
6. The method of claim 5, wherein: when a liquid phase-mass spectrum system is used in the step (4), the detection conditions are that an octadecylsilane chemically bonded silica column is used as a stationary phase, 0.2% formic acid (B) and acetonitrile (A) are used as mobile phases, the flow rate is set to be 0.4ml/min, the sample injection amount is 2 mu L, and a gradient elution mode is adopted for separation: 0-0.5min, 98% B, 0.5-0.6min, 98% -15% B; 0.6-1.6min, 15% B; 1.6-1.7min, 98% B; 1.7-3min, 98% B; an electrospray ion source, negative ions and a multi-reaction monitoring mode are adopted; the nitrogen is atomized gas and dry gas; the high-purity nitrogen is used as collision gas, and the pressure is 0.1 MPa; the mass spectrometer parameters were set as follows: ionization voltage (Is): -4500V; jet impingement (CAD): medium; air curtain gas (CUR): 20psi, spray mist (GS 1): 20psi, ion source Temperature (TEM): 450 ℃, declustering voltage (DP): -80V, impact voltage (CE): 15V; the detected ions used for quantitative analysis were: MOAG mass to charge ratio 517.138 → 341.105; internal standard 4-methyl umbelliferone-beta-D-glucuronide mass-to-charge ratio 351.0 → 175.0.
7. Use of the method of claim 2 for screening and evaluating UGT enzyme modulators, further comprising adding a test compound in step (1) and evaluating the modulating effect of the test compound in step (4).
8. The use of claim 7, wherein the test compound is an agonist when it increases UTG enzymatic activity; a test compound is an inhibitor when it decreases UTG enzyme activity.
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