CN112725408B - UGT enzyme activity detection method and application thereof - Google Patents
UGT enzyme activity detection method and application thereof Download PDFInfo
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- CN112725408B CN112725408B CN202110082924.0A CN202110082924A CN112725408B CN 112725408 B CN112725408 B CN 112725408B CN 202110082924 A CN202110082924 A CN 202110082924A CN 112725408 B CN112725408 B CN 112725408B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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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- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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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- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/91091—Glycosyltransferases (2.4)
- G01N2333/91097—Hexosyltransferases (general) (2.4.1)
- G01N2333/91102—Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a detection method of UGT enzyme activity which is not used for disease diagnosis and treatment and application thereof in screening and evaluating UGT enzyme modulators. The detection method uses ophiopogon isoflavone A as a substrate, and quantitatively detects the conversion rate of the ophiopogon isoflavone A or the generation rate of 7-O-glucuronic acid products thereof by a liquid phase-ultraviolet method or a liquid phase-mass spectrometry method to detect the activity of UGT enzyme. 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 modulators, 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 which is not used for disease diagnosis and treatment, and application of the detection method in screening and evaluating UGT enzyme modulators.
Background
The uridine diphosphate glucuronyltransferase (UGT) superfamily is an important phase ii metabolic enzyme in the body, which increases the hydrophilicity of a substrate by catalyzing the binding of a compound to the cofactor uridine diphosphate glucuronic acid (UDPGA), enabling the compound to be more effectively excreted from the body from urine or bile, which is an important detoxification process in the body. Mammalian UGTs can be divided into 4 families: UGT1, UGT2, UGT3, and UGT8. Wherein the members involved in drug binding metabolism are most of the UGT1A and UGT2B subfamilies.
As the most important phase II metabolizing enzyme in the body, UGT enzymes play a key role in the metabolic clearance and detoxification of various heterologous organisms (e.g., therapeutic drugs and their phase i metabolites, food chemicals or environmental toxins). In addition to xenobiotic metabolism, UGT enzymes are involved in glucuronidation metabolism of many endogenous substances (e.g., bilirubin, steroids, and bile acids). Endogenous metabolic disorders and drug/herb interactions (DDI/HDI) may be triggered when UGT enzyme is dysfunctional or inhibited with high potency, which can have adverse effects on human health. On the contrary, the agonist of partial UGT enzyme (such as UGT1A 1) can accelerate the metabolism and elimination of toxicants such as bilirubin, thereby relieving the occurrence and development processes of diseases. Accordingly, there is a continuing need in the industry to develop efficient screening and evaluation methods for modulators (inhibitors or agonists) of UGT enzymes.
Since the high identity of the amino acid sequences of UGT enzymes results in a high overlap of their substrate spectra, few specific probe substrates for UGT enzymes are reported. To date, only a few UGT subtype enzymes (including UGT1A1, UGT1A4, UGT1A9 and UGT2B 7) have recognized specific substrates. For the activity detection of other UGT subtype enzymes, the traditional non-fluorogenic substrate 4-methylumbelliferone (4-MU) can be only used, the detection flux is low, the sample pretreatment and detection process are complex and complex in operation, and the large-scale screening and evaluation of the target object on the inhibition capability of various UGT subtype enzymes can not be synchronously carried out.
Therefore, the detection method for developing the non-disease diagnosis treatment of UGT enzyme activity and the application of the detection method in screening and evaluating UGT enzyme modulators have important significance for evaluating UGT mediated drug interactions.
Disclosure of Invention
The invention belongs to the technical field of medicines, and particularly relates to a detection method of UGT enzyme activity which is not used for disease diagnosis and treatment, and application of the detection method in screening and evaluating UGT enzyme modulators. The method takes ophiopogon japonicus high isoflavone A (MOA) as a substrate, under physiological conditions, the ophiopogon japonicus high isoflavone A can be catalyzed by 13 humanized UGT enzymes to generate 7-O-glucuronic acid (MOAG) of the ophiopogon japonicus high isoflavone A, and the product can be detected by liquid phase-ultraviolet or liquid phase-mass spectrum. By means of the probe reaction, the recombinant expressed mammalian UGT enzyme is taken as an enzyme source, and the screening and evaluation of the regulatory capability of the compound to be tested on 13 humanized UGT enzymes can be synchronously realized. The screening and evaluating method is developed by means of a liquid phase-ultraviolet detection system (LC-UV) or a liquid phase-mass spectrum detection system (LC-MS), and has the advantages of sensitive detection, simplicity and convenience in operation, high detection precision and stability, strong anti-interference capability, low cost, high efficiency and the like, and has a good application prospect.
The invention provides a method for detecting UGT enzyme activity which is not used for disease diagnosis and treatment, which adopts ophiopogon isoflavone A as a substrate and detects UGT enzyme activity by quantitatively detecting the conversion rate of ophiopogon isoflavone A or the generation rate of 7-O-glucuronic acid product thereof.
The method of the invention comprises the following steps:
(1) The tris-hydrochloric acid buffer solution is mixed with MgCl 2 Mixing radix Ophiopogonis high isoflavone A, UGT enzyme, pre-incubating for 3-5 min at 20-60deg.C, and pH of the reaction solution at 5-10;
(2) Adding uridine diphosphate glucuronic acid into the reaction solution obtained in the step (1), and reacting for 10-60 minutes to obtain a 7-O-glucuronic acid product of ophiopogon japonicus homoisoflavone A;
(3) Adding glacial acetonitrile or glacial methanol into the reaction solution obtained in the step (2), precipitating proteins, and centrifuging to obtain a supernatant;
(4) Detecting the conversion rate of ophiopogon japonicus high isoflavone A or the generation rate of 7-O-glucuronic acid products of ophiopogon japonicus high isoflavone A in the supernatant obtained in the step (3) by using a liquid phase-ultraviolet system or a liquid phase-mass spectrum system.
The reaction equation is shown in the formula 1:
in the invention, 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 the liquid phase-ultraviolet system is used in the step (4), the detection condition is that octadecylsilane as stationary phase, 0.2% formic acid water (B) and acetonitrile (A) as mobile phase are separated by gradient elution, and the ultraviolet detection wavelength is 300nm.
In the present invention, when the liquid phase-mass spectrometry system is used in the step (4), it is necessary to add an internal standard 4-methyl umbrella-type ketone- β -D-glucuronide in the step (3).
In the invention, when the liquid phase-mass spectrum system is used in the step (4), the detection condition is that octadecylsilane chemically amplified by taking an octadecylsilane column as a stationary phase, taking 0.2% formic acid water (B) and acetonitrile (A) as mobile phases, setting the flow rate to be 0.4ml/min, and separating by adopting a gradient elution mode, wherein the sample injection amount is 2 mu L: 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; adopting an electrospray ion source, negative ions and multiple reaction monitoring mode; nitrogen is atomized gas and dry gas; the high-purity nitrogen is collision gas, and the pressure is 0.1MPa; the mass spectrometer parameters were set as follows: ionization voltage (Is): -4500V; impingement gas (CAD): a Medium; curtain gas (CUR): 20psi, spray gas (GS 1): ion source Temperature (TEM) at 20 psi: de-clustering voltage (DP) at 450 ℃ is: -80V, collision voltage (CE): 15V; the detection ions used for quantitative analysis were: MOAG mass-to-charge ratio 517.138 → 341.105; internal standard 4-methyl umbrella type ketone-beta-D-glucuronide charge ratio 351.0- & gt 175.0.
The second object of the invention is to provide the application of the detection method in screening and evaluating UGT enzyme modulators, wherein the method further comprises the steps of adding a compound to be detected in the step (1) and evaluating the modulation effect of the compound to be detected in the step (4).
In the present invention, the test compound is an agonist when it increases UTG enzyme activity; the test compound is an inhibitor when it decreases UTG enzyme activity.
The invention has the following advantages:
(1) Detection sensitivity: the glucuronidation reaction of the broad-spectrum substrate ophiopogon high isoflavone A can be catalyzed and metabolized by a plurality of UGT enzymes to generate a single metabolite, the substrate has better affinity with the UGT enzymes, an LC-UV or LC-MS detector used in the detection method is also more sensitive, the required sample amount is very small, the consumption of experimental materials can be reduced in actual operation, and the experimental cost is reduced.
(2) The stability is strong: in the invention, the products and the substrates do not react with each other and do not interfere with each other, and the established method uses LC-UV or LC-MS detection, so that good separation of various substances existing in the sample can be realized, interference of impurities on the sample is avoided, and the experimental sample is processed and measured by using the method at the temperature of 4 ℃, so that no obvious response signal intensity change exists for at least two days, and the glucuronidation reaction process of the sample is conveniently and quantitatively monitored in real time.
(3) The operation is simple, the repeatability is good: the invention is constructed by utilizing natural product ophiopogon japonicus high isoflavone A, the raw materials can be directly purchased, and the detection of UGT enzyme activity and the screening method of UGT enzyme regulator also have the characteristics of convenient operation, low consumption and the like, and have better economic applicability. And the broad-spectrum substrate reaction is stable and the experimental repeatability is good.
(4) The UGT enzyme types detected are multiple: the method can realize detection of 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 O-glucuronidation activity of UGT1A4 and UGT2B10, and screen and evaluate modulators of thirteen enzymes under the same condition, so that experimental errors caused by screening and evaluating using different substrates are avoided.
The invention is constructed based on the broad-spectrum substrate ophiopogon japonicus homoisoflavone A found by natural product screening, and the substrate only generates a single glucuronidation product metabolite and can detect the activity of 13 UGT subtype enzymes. The invention has the characteristics of high sensitivity, strong stability, good repeatability, simplicity, easiness in operation and the like. Screening and reevaluation are carried out on various reported UGT enzyme selective inhibitors, and the screening method is proved to have extremely high accuracy and very good application prospect.
Drawings
FIG. 1 is a graph showing the rate at which glucuronidation of MOA occurs in example 1.
FIG. 2 is a mass spectrum of MOAG in example 2.
FIG. 3 stability study in example 3
FIG. 4 is a diagram showing the procedure for detecting MOA glucuronidation by the liquid phase-ultraviolet method in example 4.
FIG. 5A is a diagram showing the procedure for detecting MOA glucuronidation by liquid phase-mass spectrometry in example 5.
FIG. 6 shows a nuclear magnetic resonance identification chart of MOAG in example 2, wherein, the chart (a) is a hydrogen spectrogram and the chart (b) is a carbon spectrogram.
FIG. 7 IC for screening and evaluation of UGTase inhibitors based on UV detection System in example 6 50 The graph, wherein- -indicates no or very weak inhibition, of IC 50 >100μM。
FIG. 8 mass spectrum of the detection using LC-MS in example 7.
FIG. 9 is a graph of a screening evaluation of the effect of glycitein on activating UGT enzyme as a compound using an LC-UV detection system and established methods in example 8.
Detailed Description
The following examples further illustrate the invention, but are not intended to limit it.
Example 1 broad-Spectrum study of the metabolism of ophiopogon japonicus homoisoflavonoid A by human UGT enzyme
Participate in UGT enzyme phenotype research of glucose binding metabolism of a broad-spectrum substrate ophiopogon japonicus homoisoflavone A. The substrate MOA was metabolically characterized using 13 commercial recombinant human UGT singleases (UGT 1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1a10, UGT2B4, UGT2B7, UGT2B10, UGT2B15, UGT2B 17) as target UGT enzyme sources. The method comprises the following steps:
(1) Tris-HCl buffer (50 mM, pH=7.4) and magnesium chloride (5 mM) are mixed in an EP tube, one of 13 UGT singleases is used as a target UGT enzyme (0.05 mg/ml recombinant humanized UGT singlease), ophiopogon root homoisoflavone A (the concentration is unified to be 10 mu M and 100 mu M) is pre-incubated for 3-5 minutes at 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 (DEG C) | 20 | 50 | 60 | 20 | 37 | 60 | 37 |
| pH value of | 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 (temperature) | 37 | 37 | 37 | 37 | 37 | 37 | |
| pH value of | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 |
(2) The UDPGA was added separately 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 |
And detecting the generation amount of the glucuronic acid product by using a liquid phase-ultraviolet system, and obtaining the corresponding substrate reaction rate through data processing. As a result, the substrate ophiopogon japonicus homoisoflavone A can be metabolized by 13 UGT enzymes. (as shown in FIG. 1)
The results show that: compared with the traditional substrate 4-methylumbelliferone, ophiopogon root homoisoflavone A can be metabolized by all 13 UGT subtype enzymes participating in human drug metabolism (comprising UGT1A4 and UGT2B10 which cannot be detected by 4-methylumbelliferone). Based on the discovery of the glucuronidation reaction of ophiopogon japonicus homoisoflavone A and the large number of enzymes covered by the established detection method, the detection of the activity of all 13 UGT enzymes participating in the metabolism of medicaments in a human body can be realized under the same condition, and the broad spectrum is even more excellent than that of the traditional method which takes 4-methylumbelliferone as a substrate.
Example 2 preparation and identification of ophiopogon japonicus homoisoflavone A by UGT enzyme metabolite
After analysis and identification of UGT catalyzed ophiopogon japonicus homoisoflavone A metabolites by high resolution mass spectrometry (as shown in figure 2), a large amount of preparation and structural characterization of the products MOAG are respectively carried out by using a biological preparation method and nuclear magnetic resonance technology. (as shown in fig. 6). The method comprises the following specific steps:
(1) Radix Ophiopogonis homoisoflavone A (8.52 mg), tris-HCl buffer (50 mM, pH 7.4), mgCl 2 (5 mM), polyoxyethylene cetyl ether (0.1 mg/mg protein) and 13 UGT enzymes expressed recombinantly (1.85. Mu.L of each enzyme, 0.8mg/ml final protein concentration) were mixed. After pre-incubation for 5min at 37 ℃, the reaction was started by adding UDPGA (2 mM).
(2) After 6 hours incubation at 37 ℃, the reaction was terminated by adding ice-cold methanol (150 ml). After centrifugation at 20,000Xg for 20 minutes at 4 ℃, the supernatant was collected, then evaporated, and the precipitate was purified by a preparative reverse phase liquid chromatography system using a reverse phase column.
(3) Finally, the purified MOAG (8.0 mg, purity greater than 98% as determined by liquid phase-uv system) obtained was dissolved in deuterated methanol and structurally characterized using a Bruker 400 Nuclear Magnetic Resonance (NMR) spectrometer.
(4) The obtained product is applied to standard curve establishment and related method evaluation experiments in method construction.
The results show that: glucuronidation of the broad-spectrum substrate ophiopogon homoisoflavone A can be metabolized by multiple UGT enzymes to generate a single metabolite mono-glucuronidation product: MOA-7-O-glucuronidation product (MOAG) reacted as follows:
EXAMPLE 3 stability investigation
(1) Tris-HCl buffer (50 mM, pH=7.4) was incubated with MgCl under physiological conditions 2 (5 mM) using 13 recombinant UGT enzymes (0.31 mu L of each enzyme and 0.1mg/ml of protein final concentration) as enzyme source, and using radix Ophiopogonis high isoflavone A solution as substrate (1/10-1K concentration is selected) m ) Mix in centrifuge tube, reaction temperature is 37 ℃, incubation system pH is 7.4.
(2) Pre-incubating, wherein the mixed solution in the step (1) is incubated for 3-5 min at 37 ℃ to ensure that the enzyme is fully contacted with the substrate ophiopogon japonicus homoisoflavone A.
(3) The reaction time for adding UDPGA was 30 minutes.
(4) Sample analysis was performed after 0 hours, 24 hours and 48 hours at 4℃respectively, and stability under 4℃refrigerator storage conditions was examined. The results are shown in FIG. 3.
The results illustrate: the stability is strong, in the probe reaction, the product and the substrate can not react with each other and interfere with each other, and the method is used for treating and measuring experimental samples at the temperature of 4 ℃ without obvious response signal intensity change for at least two days, so that the glucuronidation reaction process can be conveniently and quantitatively monitored in real time.
EXAMPLE 4 LC-UV detection of MOA-7-O-glucuronidation reaction Rate
(1) Tris-HCl buffer (50 mM, pH=7.4) was incubated with MgCl under physiological conditions 2 (5 mM) 13 UGT enzymes (0.31 mu L of each enzyme and 0.1mg/ml of final protein concentration) expressed by recombination are taken as enzyme sources, and a substrate ophiopogon japonicus high isoflavone A solution (the concentration is 1/10-1K m ) Mixing in a centrifuge tube, wherein the total volume is 200 microliters, and the reaction temperature is 37 ℃; the pH value of the incubation system is 7.4.
(2) Pre-incubation. The above mixed solution (blank group provided with substrate-free ophiopogon japonicus homoisoflavone A) was incubated at 37℃for 5min.
(3) The reaction time for adding UDPGA was 60 minutes, ensuring that the product yield or substrate conversion was below 20%.
(4) An equal volume of 200. Mu.l of glacial acetonitrile was added to terminate the reaction and centrifuged at 20,000Xg for 20 minutes at 4℃on a centrifuge. After the reaction solution is centrifuged to precipitate the protein, the generated substrate and product are quantitatively analyzed by a liquid phase-ultraviolet detection system, and the detection is carried out under the ultraviolet wavelength of 300nm. The detection condition uses ODS column as stationary phase, uses 0.2% formic acid water (B) and acetonitrile (A) as mobile phase, 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, and separating the substrate and the product by using a gradient elution mode. The substrates and products were analyzed and detected at ultraviolet wavelength 300nm (FIG. 4).
(5) The rate of formation of the MOAG, a glucuronic acid product of ophiopogon japonicus, was measured over the reaction time and the percentage of the rate of reaction to the inhibitor group relative to the rate of reaction to the control group was calculated as an inhibitory activity evaluation criterion.
After detection, the LC-UV method was found to be useful for detecting MOA-7-O-glucuronidation reaction rates.
EXAMPLE 5 LC-MS method for detecting MOA-7-O-glucuronidation reaction Rate
(1) Tris-HCl buffer (50 mM, pH=7.4) was incubated with MgCl under physiological conditions 2 (5 mM) 13 UGT enzymes (0.31 mu L of each enzyme and 0.1mg/ml of final protein concentration) expressed by recombination are taken as enzyme sources, and a substrate ophiopogon japonicus high isoflavone A solution (the concentration is selected to be 1/10-1K) m ) Mixing in a centrifuge tube, wherein the total volume is 100 microlitres, and the reaction temperature is 37 ℃; the pH value of the incubation system is 7.4.
(2) Pre-incubation. The mixed solution (blank control group with substrate-free ophiopogon japonicus high isoflavone A) is incubated for 3-5 min at 37 ℃.
(3) The reaction time for adding UDPGA was 60 minutes, ensuring that the product yield or substrate conversion was below 20%.
(4) The reaction was terminated by adding ice-equivalent acetonitrile containing the internal standard 4-methyl umbrella ketone-beta-D-glucuronide, and centrifuged at 20,000Xg for 20 minutes at 4 ℃. And (3) carrying out quantitative analysis on the generated product by means of a liquid phase-mass spectrum detection system after centrifuging the reaction liquid to precipitate the protein.
(5) The detection condition uses ODS column as stationary phase, uses 0.2% formic acid water (B) and acetonitrile (A) as mobile phase, the flow rate is set to 0.4ml/min, the sample injection amount is set to 2 mu L, and the substrate and the product are separated by adopting gradient elution mode: 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 (multiple reaction monitoring, MRM) mode was used. Nitrogen is atomized gas and dry gas; the high-purity nitrogen is collision gas, and the pressure is 0.1MPa; the mass spectrometer parameters were set as follows: ionization voltage (Is): -4500V; impingement gas (CAD): a Medium; curtain gas (CUR): 20psi, spray gas (GS 1): ion source Temperature (TEM) at 20 psi: de-clustering voltage (DP) at 450 ℃ is: -80V, collision voltage (CE): 15V. Substrate parent ion mass to charge ratio (m/z): 341.105 and substrate child ion mass to charge ratio (m/z): 206.060. Product parent ion mass to charge ratio (m/z): 517.138, product ion mass to charge ratio (m/z): 341.105, 175.027. The detection results are shown in FIG. 5.
(6) The rate of glucuronic acid product formation during the reaction time was measured and the percentage of the rate of reaction to the inhibitor group relative to the rate of reaction to the blank group was calculated as an inhibitory activity evaluation criterion.
After detection, the LC-MS method is found to be useful for detecting MOA-7-O-glucuronidation reaction rates.
EXAMPLE 6 screening and evaluation of UGTase inhibitors Using LC-UV method
The application of the detection method of UGT enzyme activity which is 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) 200. Mu.L of a reaction system is set, tris-HCl buffer (50 mM, pH=7.4), magnesium chloride (5 mM) and 13 recombinant human UGT enzymes (final concentration 0.05 mg/ml) and ophiopogon high isoflavone A solution (10. Mu.M) are respectively added into a centrifuge tube, and the mixture is uniformly mixed and pre-incubated for 3 minutes at 37 ℃ together with one of the compounds to be tested, namely amentoflavone, nilotinib, magnolol and fluconazole (concentration 1. Mu.M-100. Mu.M).
(2) After 10. Mu.L of UDPGA (final concentration 2 mM) was added to the reaction system to initiate the reaction, the centrifuge tube was placed in an incubator to continue the reaction for 60 minutes, ensuring that the product yield or substrate conversion was less than 20%.
(3) The reaction was stopped by adding an equal volume of 200. Mu.L of glacial acetonitrile and centrifuged at 20,000Xg for 20 minutes at 4℃on a centrifuge. After the reaction solution was centrifuged to precipitate the protein, the resultant was quantitatively analyzed by means of a liquid phase-ultraviolet detection system, and the detection was performed under the above-mentioned detection conditions (example 3) at an ultraviolet wavelength of 300nm. The rate of glucuronic acid production during the reaction time was quantitatively determined and the percentage of the rate of reaction to which the inhibitor group was added relative to the rate of production of the control group product was calculated as an inhibitory activity evaluation criterion, and four compounds were found to have various degrees of inhibitory effects 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 after the addition of amentoflavone, nilotinib, magnolol and fluconazole, the reaction rate became 1.81, 2.63, 13.74, 21.06. Mu. Mol/min/mg protein.
EXAMPLE 7 screening and evaluation of UGTase inhibitors Using LC-MS method
The application of the detection method of UGT enzyme activity which is 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) 100. Mu.L of a reaction system was set, tris-HCl buffer (50 mM, pH=7.4), magnesium chloride (5 mM), 13 recombinant human UGT enzymes (final concentration 0.05 mg/ml) and ophiopogon high isoflavone A solution (10. Mu.M) were added to a centrifuge tube, and mixed uniformly, and pre-incubated for 3 minutes at 37℃with one of the test compounds amentoflavone, nilotinib, magnolol and fluconazole (concentration 1. Mu.M-100. Mu.M).
(2) After adding 5. Mu.L UDPGA (final concentration 2 mM) to the reaction system to initiate the reaction, the centrifuge tube was placed in an incubator to continue the reaction for 60 minutes, ensuring that the product yield or substrate conversion was less than 20%.
(3) The reaction was stopped by adding an equal volume of 100. Mu.L of glacial acetonitrile containing the internal standard 4-methyl umbrella-type ketone-beta-D-glucuronide and centrifuged at 20,000Xg for 20 minutes at 4℃on a centrifuge. After the reaction solution is centrifuged to precipitate the protein, the generated product is quantitatively detected by a liquid phase-mass spectrum detection system under a multi-reaction monitoring (MRM) mode. (FIG. 8)
(4) The detection condition uses ODS column as stationary phase, uses 0.2% formic acid water (B) and acetonitrile (A) as mobile phase, the flow rate is set to 0.4ml/min, the sample injection amount is set to 2 mu L, and the substrate and the product are separated by adopting gradient elution mode: 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 (multiple reaction monitoring, MRM) mode was used. Nitrogen is atomized gas and dry gas; the high-purity nitrogen is collision gas, and the pressure is 0.1MPa; the mass spectrometer parameters were set as follows: ionization voltage (Is): -4500V; impingement gas (CAD): a Medium; curtain gas (CUR): 20psi, spray gas (GS 1): ion source Temperature (TEM) at 20 psi: de-clustering voltage (DP) at 450 ℃ is: -80V, collision voltage (CE): 15V. The detection ions used for quantitative analysis were: MOAG mass to charge ratio 517.138 → 341.105. Internal standard 4-methyl umbrella type ketone-beta-D-glucuronide charge ratio 351.0- & gt 175.0.
(5) The inhibition activity evaluation standard is obtained by quantitatively measuring the production rate of glucuronic acid products in the reaction time and calculating the percentage of the production rate of the products of the inhibitor group relative to the production rate of the blank control group, and the results are similar to the UV detection, so that four compounds are found to have inhibition effects on UGT enzymes to different degrees. Taking UGT1A1 as an example, the reaction rate was 22.88. Mu. Mol/min/mg protein before adding the inhibitor, whereas after adding amentoflavone, nilotinib, magnolol and fluconazole, the reaction rate became 1.62, 2.31, 14.20, 21.97. Mu. Mol/min/mg protein.
(6) The detection method can accurately quantify the target product MOAG, avoids the interference of impurity peaks to the target peaks, which is generated by poor separation effect when a complex sample is detected by using LC-UV, and has high efficiency, and the analysis time is only 3 min.
Example 8 screening and evaluation of UGT enzyme activators Using LC-UV method
The use of a method for detecting UGT enzyme activity that is not used for diagnostic treatment of a disease in screening and evaluating UGT enzyme modulators. The specific operation is as follows:
(1) 200. Mu.L of the reaction system was set, tris-HCl buffer (50 mM, pH=7.4), magnesium chloride (5 mM), recombinant human UGT1A1 enzyme (final concentration 0.05 mg/ml) and ophiopogon root homoisoflavone A solution (10. Mu.M), and glycitein (concentration 0. Mu.M-10. Mu.M) solution as a test compound were added to a centrifuge tube, mixed well, and pre-incubated for 3 minutes at 37 ℃.
(2) After 10. Mu.L of UDPGA (final concentration 2 mM) was added to the reaction system to initiate the reaction, the centrifuge tube was placed in an incubator to continue the reaction for 60 minutes, ensuring that the product yield or substrate conversion was less than 20%.
(3) The reaction was stopped by adding an equal volume of 200. Mu.L of glacial acetonitrile and centrifuged at 20,000Xg for 20 minutes at 4℃on a centrifuge. After the reaction solution was centrifuged to precipitate the protein, the resultant was quantitatively analyzed by a liquid phase-ultraviolet detection system, and the detection was performed under the above-mentioned detection conditions (as in example 4) at an ultraviolet wavelength of 300nm. The rate of glucuronic acid product generation in the reaction time is quantitatively measured, the percentage of the rate of the reaction of the added activator group relative to the rate of the product generation of the blank group is calculated as an activating activity evaluation standard, and the ideal activating effect (more than 2 times) of the glycitein serving as the compound to be tested on UGT enzyme at the concentration of 10 mu M is found. (FIG. 9).
Claims (6)
1. A method for detecting UGT enzyme activity not used for disease diagnosis and treatment is characterized in that the UGT enzyme activity is detected by quantitatively detecting the conversion rate of the ophiopogon isoflavone A or the generation rate of 7-O-glucuronic acid product of the ophiopogon isoflavone A by adopting the ophiopogon isoflavone A as a substrate;
the method comprises the following steps:
(1) The tris-hydrochloric acid buffer solution is mixed with MgCl 2 Mixing radix Ophiopogonis high isoflavone A, UGT enzyme, pre-incubating for 3-5 min at 20-60deg.C, and pH of the reaction solution at 5-10;
(2) Adding uridine diphosphate glucuronic acid into the reaction solution obtained in the step (1), and reacting for 10-60 minutes to obtain a 7-O-glucuronic acid product of ophiopogon japonicus homoisoflavone A;
(3) Adding glacial acetonitrile or glacial methanol into the reaction solution obtained in the step (2), precipitating proteins, and centrifuging to obtain a supernatant;
(4) Detecting the conversion rate of ophiopogon japonicus high isoflavone A or the generation rate of 7-O-glucuronic acid products of ophiopogon japonicus high isoflavone A in the supernatant obtained in the step (3) by using a liquid phase-ultraviolet system or a liquid phase-mass spectrum system;
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.
2. The method according to claim 1, characterized in that: when the liquid phase-ultraviolet system is used in the step (4), the detection condition is that octadecylsilane chemically amplified by taking octadecylsilane as a stationary phase, 0.2% formic acid water (B) and acetonitrile (A) as mobile phases, and a gradient elution mode is adopted for separation, wherein the ultraviolet detection wavelength is 300nm.
3. The method according to claim 1, characterized in that: when step (4) uses a liquid-mass spectrometry system, the addition of the internal standard 4-methyl-umbelliferone- β -D-glucuronide is required in step (3).
4. A method according to claim 3, characterized in that: when the liquid phase-mass spectrum system is used in the step (4), the detection condition is that octadecylsilane chemically amplified by taking an octadecylsilane chemically amplified column as a stationary phase, taking 0.2% formic acid water (B) and acetonitrile (A) as mobile phases, setting the flow rate to be 0.4ml/min, and the sample injection amount to be 2 mu L, and separating by adopting a gradient elution mode: 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; adopting an electrospray ion source, negative ions and multiple reaction monitoring mode; nitrogen is atomized gas and dry gas; the high-purity nitrogen is collision gas, and the pressure is 0.1MPa; the mass spectrometer parameters were set as follows: ionization voltage: -4500V; spraying collision gas: a Medium; air curtain gas: 20psi, spray gas: 20psi, ion source temperature: cluster removal voltage at 450 ℃): -80V, collision voltage: 15V; the detection ions used for quantitative analysis were: MOAG mass-to-charge ratio 517.138 → 341.105; internal standard 4-methyl umbrella type ketone-beta-D-glucuronide charge ratio 351.0- & gt 175.0.
5. Use of the method of claim 1 for screening and evaluating modulators of UGT enzymes, said use further comprising adding a test compound in step (1) and evaluating the modulation of the test compound in step (4).
6. The use according to claim 5, wherein the test compound is an agonist when it increases UTG enzyme activity; the test compound is an inhibitor when it decreases UTG enzyme activity.
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| CN106389405A (en) * | 2015-07-30 | 2017-02-15 | 中国科学院大连化学物理研究所 | A class of glucuronosyltransferase UGT1A1 inducers and applications thereof |
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