Specific fluorescent probe substrate of glucuronyl transferase UGT1A1 and application of specific fluorescent probe substrate

Abstract

The invention discloses a specific fluorescent probe substrate of glucuronyl transferase UGT1A1 in the technical field of biological medicine, the specific fluorescent probe substrate can be specifically catalyzed by UGT1A1 to generate a corresponding N-glucuronidated product, the structural general formula of the specific fluorescent probe substrate is as follows: # imgabs0 #, R1 is one of-COOH and-morpholine, n is 2-10, and n is 1-10. R2 is one of amino, dimethylamino, N-methyl piperazine, pyridine, imidazole and 1, 2, 5-triazine. The specific fluorescent probe substrate can be used for determining the activity of UGT1A1 in different biological systems.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a specific fluorescent probe substrate of glucuronyl transferase UGT1A1 and application thereof. Background Art

[0002] The glucuronosyltransferase (UGT) superfamily constitutes a vital phase II drug metabolism system in the body. They connect lipophilic compounds to the glucuronic acid (GA) part of uridine diphosphate glucuronic acid (UDPGA) through the SN2 reaction mechanism, thereby enhancing the water solubility of these compounds and facilitating their excretion through urine or bile. This process is often a key step in the body's detoxification, involving the conversion of many endogenous substances (such as bilirubin and estradiol), mutagens, drugs and their metabolites.

[0003] The UGT enzymes in the human body are divided into four major categories: UGT1, UGT2, UGT3 and UGT8, among which the UGT1 and UGT2 families play a central role in metabolizing exogenous and exogenous substances. So far, scientists have identified 18 human UGT subtypes, including 9 in UGT1A (UGT1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10) and 7 in UGT2B (UGT2B4, 2B7, 2B10, 2B15 and 2B17).

[0004] It is particularly worth mentioning that uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) is a key phase I metabolic enzyme that is essential for the clearance of endogenous toxic metabolites bilirubin and a variety of exogenous substrates. Bilirubin is a product of heme decomposition. If it accumulates in the body, it may cause cholestasis and nervous system damage, so it must be excreted in time. The amount of bilirubin produced by adults per day is about 250 to 350 mg, and UGT1A1 is the only enzyme in the body responsible for bilirubin metabolism. It converts bilirubin into glucuronic acid conjugates so that it can be excreted from the body through bile. In addition, UGT1A1 is also widely involved in the metabolism of other endogenous compounds, such as estrogen, androgen, and thyroxine, which play an important role in regulating cell proliferation and differentiation, and their levels need to be precisely controlled in target tissues.

[0005] The UGT1A1 enzyme is widely distributed in the body and can catalyze the glucuronic acid conjugation metabolism of these hormones in target tissues, thereby rapidly inactivating them when the body does not need them. However, when the UGT1A1 enzyme function is abnormal, it can lead to an imbalance in hormone levels in the body, thereby increasing the risk of cancer. Therefore, the development of efficient and sensitive specific UGT1A1 probe substrates is of great significance for screening UGT1A1 activity regulators and quantitatively measuring the activity of UGT1A1 in biological systems. Summary of the invention

[0006] The object of the present invention is to provide a specific fluorescent probe substrate for glucuronyl transferase UGT1A1, the fluorescence emission wavelength of the specific fluorescent probe substrate and the N-glucuronidation product is significantly different, and the fluorescence quantum yield of the product is higher and easier to detect.

[0007] A specific fluorescent probe substrate of glucuronyl transferase UGT1A1 in this scheme can be specifically catalyzed by UGT1A1 to generate a corresponding N-glucuronidation product. The general structural formula of the specific fluorescent probe substrate is as follows:

[0008]

[0009] Among them, R1 is one of -COOH and -morpholine, n is 2 to 10, and R2 is one of amino, dimethylamino, N-methylpiperazine, pyridine, imidazole, and 1,2,5-triazine.

[0010] Furthermore, the specific fluorescent probe substrate is any one of the following structures:

[0011]

[0012] The present invention also provides the use of the specific fluorescent probe substrate of glucuronyl transferase UGT1A1 in determining the activity of UGT1A1 in different biological systems.

[0013] Furthermore, the biological system is any one of a recombinant single enzyme containing UGT1A1, a human or animal tissue preparation solution, various mammalian tissue cells and their preparations.

[0014] Furthermore, when determining the activity of UGT1A1 in different biological systems, the following steps are included: placing a specific fluorescent probe substrate and a biological system containing UGT1A1 in a buffer solution for a glucuronidation binding reaction, and quantitatively determining the activity of UGT1A1 in different biological systems by quantitatively detecting the elimination rate of the specific fluorescent probe substrate or the generation rate of its glucuronidation product per unit time.

[0015] Furthermore, the concentration of the specific fluorescent probe substrate is selected to be 0.1 to 10 μM. The concentration of the specific fluorescent probe substrate is preferably 5 μM in single-point determination. ;

[0016] Furthermore, the buffer is a Tris-HCl buffer, and the pH value of the buffer is 5.5 to 10.5. Preferably, pH 8.4 is the optimal reaction pH value;

[0017] Furthermore, the reaction temperature is 20-60°C, the reaction time is 0.05-4h, and the reaction is terminated when the glucuronidation product corresponding to the specific fluorescent probe substrate reaches the quantitative limit and the conversion rate of the specific fluorescent probe substrate does not exceed 20%. 37°C is preferably the optimal reaction temperature.

[0018] Furthermore, when detecting the elimination rate of the specific fluorescent probe substrate or the generation rate of its glucuronidation product, a fluorescent liquid phase detector is used, and the fluorescence detection conditions of the specific fluorescent probe substrate are: excitation wavelength 455nm, maximum emission wavelength 535nm; the fluorescence detection conditions of the glucuronidation product are: excitation wavelength 455nm, maximum emission wavelength 535nm.

[0019] The invention provides an application of a specific fluorescent probe substrate for glucuronyl transferase UGT1A1. The probe substrate and its glucuronidation product both have fluorescent properties, but the two have different optical properties. A fluorescent liquid phase detector can be used to simultaneously realize rapid and sensitive detection of the substrate and the product.

[0020] The specific fluorescent probe substrate is not easily interfered by biological system matrix and impurities during the UGT1A1 activity detection process, and can be used for the quantitative determination of UGT1A1 enzyme activity in various recombinant UGT1A1, human and animal tissue preparation fluids, and various tissue cells; the specific fluorescent probe substrate and the fluorescence detection method of glucuronidation metabolites can also be used for the rapid screening of UGT1A1 activity regulators and the quantitative evaluation of activity regulation ability.

[0021] The recombinant glucuronic acid UGT1A1 single enzyme and liver microsome incubation system were used for investigation. Through correlation analysis, specific inhibition experiment, recombinant single enzyme metabolic reaction and enzyme reaction kinetics, it was proved that the specific fluorescent probe substrate can be specifically metabolized by glucuronic acid UGT1A1 to generate amino glucuronidation products. Further investigation was carried out using metabolic evaluation systems such as freshly extracted hepatocytes, primary cultured hepatocytes, liver slices, and liver perfusion from various mammals, and it was found that the metabolic reaction has very good specificity.

[0022] As a highly specific fluorescent probe substrate for the glucuronidase UGT1A1 single enzyme, the specific fluorescent probe substrate can be used to detect the activity of UGT1A1, and is particularly suitable for the enzyme activity assay of UGT1A1 produced by bacteria, insect cells, mammalian cells and yeast cloning expression systems, as well as the activity calibration of UGT1A1 in preparations such as microsomes and S9 derived from various mammalian tissues and organs.

[0023] The use of the specific fluorescent probe substrate of the glucuronidase UGT1A1 single enzyme of the present invention to detect the in vitro activity of the UGT1A1 single enzyme has the following outstanding advantages:

[0024] (1) High specificity: The specific fluorescent probe substrate can be metabolized by the glucuronidation UGT1A1 single enzyme with high specificity into a metabolite, namely, the glucuronidation product of the amino group.

[0025] (2) Easy to obtain: Specific fluorescent probe substrates can be obtained through chemical synthesis. The synthesis process is simple and easy, and the cost of fluorescence detection is low.

[0026] (3) Easy high-throughput detection: It can be measured on various common fluorescent microplate readers and biochemical analyzers in the laboratory, and can be used for batch testing using 96 or 386 microplates.

[0027] (4) High sensitivity: The specific fluorescent probe substrates all have good fluorescence emission spectral characteristics (450-700 nm), and the specific fluorescent probe substrates and their glucuronidation metabolites have different fluorescence emission spectral characteristics, which can be better distinguished and detected. At the same time, quantitative determination can be performed by establishing a ratio-type standard curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 N-morpholine-4-amino-1,8-naphthalimide 1 H-NMR spectrum;

[0029] Figure 2 N-morpholine-4-amino-1,8-naphthalimide 13 C-NMR spectrum;

[0030] Figure 3 It is the liquid phase metabolic profile of HLM to N-morpholine-4-amino-1,8-naphthalimide;

[0031] Figure 4 Human UGT recombinant single enzyme screening assay for N-morpholino-4-amino-1,8-naphthalimide;

[0032] Figure 5 It is a specific inhibition experiment of N-morpholine-4-amino-1,8-naphthalimide;

[0033] Figure 6 Enzyme kinetics experiment for N-morpholine-4-amino-1,8-naphthalimide;

[0034] Figure 7 Schematic diagram of the metabolic pathway for glucuronidation of N-morpholine-4-amino-1,8-naphthalimide by UGT1A1. DETAILED DESCRIPTION

[0035] The present invention will be further described in the following examples, but the present invention is not limited thereto.

[0036] The equipment and models used in the present invention are as follows: the fluorescence emission / excitation spectrum is detected by a SynergyH1 full-function microplate detector; 1 The H-NMR spectrum was detected by a nuclear magnetic resonance spectrometer (Avance II 600 MHz).

[0037] Example 1

[0038] The synthetic route of N-morpholine-4-amino-1,8-naphthalimide is as follows:

[0039]

[0040] 4.2mmol 4-aminomorpholine was added to 50mL ethanol solution containing 1g (3.61mmol) 4-amino-1,8-naphthalene anhydride, and reacted at 70-80℃ overnight. Then 200ml water was added to precipitate a large amount of solid, which was filtered and vacuum dried to obtain beige solid N-morpholine-4-amino-1,8-naphthalene imide with a yield of 80-90%.

[0041] N-morpholine-4-amino-1,8-naphthalimide 1 H-NMR spectrum is shown in Figure 1 , 13 C-NMR spectrum is shown in Figure 2 .

[0042] Example 2

[0043] In vitro determination of the metabolism of fluorescent substrates

[0044] (1) Prepare 95 μL of UGT metabolic reaction system in advance, including Tris-Hc1 buffer (100 mM) at pH 8.4, human liver microsomes (0.05 mg / mL), and N-morpholine-4-amino-1,8-naphthalimide at final concentrations of 0.5 and 50 μM, and pre-incubate at 37°C with shaking for 3 min.

[0045] (2) Add 5 μL of 40 mM UDPGA (final concentration 2 mM) to the reaction system to initiate the reaction.

[0046] (3) After 60 minutes, add 100 μL of glacial acetonitrile and shake vigorously to terminate the reaction.

[0047] (4) Centrifuge at 4°C, 20,000×g for 20 minutes in a high-speed refrigerated centrifuge, and take the supernatant for fluorescent liquid phase detection (Ex=455nm, Em=535nm) ( Figure 3 ).

[0048] Example 3

[0049] In vitro determination of selectivity of human recombinant UGT enzymes

[0050] (1) Prepare 95 μL of UGT metabolic reaction system in advance, including Tris-Hc1 buffer (100 mM) at pH 8.4, recombinant human UGT enzymes (0.05 mg / mL), and N-morpholino-4-amino-1,8-naphthalimide at final concentrations of 0.5 and 50 μM. Pre-incubate at 37°C with shaking for 3 min.

[0051] (2) Add 5 μL of 40 mM UDPGA (final concentration 2 mM) to the reaction system to initiate the reaction.

[0052] (3) After 60 minutes, add 100 μL of glacial acetonitrile and shake vigorously to terminate the reaction.

[0053] (4) After centrifugation at 4°C, 20,000×g for 20 minutes, the supernatant was collected for fluorescent liquid phase detection (Ex=455nm, Em=535nm); the selectivity of the recombinant human UGT1A1 enzyme was about 100 times that of other single enzymes ( Figure 4 ).

[0054] Example 4

[0055] In vitro specific inhibition assay

[0056] (1) Prepare 190 μL of human liver microsomes and UGT1A1 metabolic reaction system in advance, including Tris-Hc1 buffer (100 mM) at pH 8.4, human liver microsomes (0.25 mg / m1), UGT1A1 (0.05 mg / m1), N-morpholino-4-amino-1,8-naphthalimide at final concentrations of 0.5 and 50 μM, and pre-incubate with different concentrations of hecogenin at 37°C for 3 min with shaking.

[0057] (2) Add 10 μL of 40 mM UDPGA to the reaction system to start the reaction.

[0058] (3) After 60 minutes, add 200 μL of glacial acetonitrile and shake vigorously to terminate the reaction.

[0059] (4) Centrifuge at 4°C, 20,000 × g for 20 minutes in a high-speed refrigerated centrifuge, and take the supernatant for fluorescence detection (Ex = 455 nm, Em = 535 nm). The results are shown in the attached figure. Figure 5 shown.

[0060] Example 5

[0061] Enzyme kinetics experiments

[0062] (1) Prepare 190 μL of human liver microsomes and UGT1A1 metabolic reaction system in advance, including Tris-Hc1 buffer (100 mM) at pH 8.4, human liver microsomes at different concentrations, UGT1A1 (0.05 mg / ml), and N-butyl-4-amino-1,8-naphthalimide at a final concentration of 50 μM, and pre-incubate at 37°C with shaking for 3 min.

[0063] (2) Add 10 μL of 40 mM UDPGA to the reaction system to start the reaction.

[0064] (3) After 60 minutes, add 200 μL of glacial acetonitrile and shake vigorously to terminate the reaction;

[0065] (4) Centrifuge at 4°C, 20,000 × g for 20 minutes in a high-speed refrigerated centrifuge, and take the supernatant for fluorescence detection (Ex = 455 nm, Em = 535 nm). The results are shown in the attached figure. Figure 6 shown.

[0066] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined in the claims.

Claims

1. A specific fluorescent probe substrate for glucuronyl transferase UGT1A1, characterized in that: The specific fluorescent probe substrate can be specifically catalyzed by UGT1A1 to generate the corresponding N-glucuronidation product. The general structural formula of the specific fluorescent probe substrate is shown below: Among them, R1 is one of -COOH and -morpholine, n is 2 to 10, and R2 is one of amino, dimethylamino, N-methylpiperazine, pyridine, imidazole, and 1,2,5-triazine.

2. The specific fluorescent probe substrate of glucuronyl transferase UGT1A1 according to claim 1, characterized in that: The specific fluorescent probe substrate is any one of the following structures:

3. Use of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 1 or 2 in determining the activity of UGT1A1 in different biological systems.

4. Use of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 3 in determining the activity of UGT1A1 in different biological systems, characterized in that: The biological system is any one of a recombinant single enzyme containing UGT1A1, a human or animal tissue preparation solution, various mammalian tissue cells and preparations thereof.

5. Use of the specific fluorescent probe substrate of glucuronyl transferase UGT1A1 according to claim 4 in determining the activity of UGT1A1 in different biological systems, characterized in that: When determining the activity of UGT1A1 in different biological systems, the method includes the following steps: placing a specific fluorescent probe substrate and a biological system containing UGT1A1 in a buffer solution for a glucuronidation binding reaction, and quantitatively determining the activity of UGT1A1 in different biological systems by quantitatively detecting the elimination rate of the specific fluorescent probe substrate or the generation rate of its glucuronidation product per unit time.

6. Use of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 5 in determining the activity of UGT1A1 in different biological systems, characterized in that: The concentration of the specific fluorescent probe substrate is selected to be 0.1 to 10 μm;.

7. Use of the specific fluorescent probe substrate of glucuronyl transferase UGT1A1 according to claim 6 in determining the activity of UGT1A1 in different biological systems, characterized in that: The buffer is Tris-Hcl buffer, and the pH value of the buffer is 5.5-10.

5.

8. Use of the specific fluorescent probe substrate of glucuronyl transferase UGT1A1 according to claim 7 in determining the activity of UGT1A1 in different biological systems, characterized in that: The reaction temperature is 20-60° C., and the reaction is terminated when the glucuronidation product corresponding to the specific fluorescent probe substrate reaches the quantitative limit and the conversion rate of the specific fluorescent probe substrate does not exceed 20%.

9. Use of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 8 in determining the activity of UGT1A1 in different biological systems, characterized in that: When detecting the elimination rate of the specific fluorescent probe substrate or the generation rate of its glucuronidation product, a fluorescent liquid phase detector is used, and the fluorescence detection conditions of the specific fluorescent probe substrate are: excitation wavelength 455nm, maximum emission wavelength 535nm; the fluorescence detection conditions of the glucuronidation product are: excitation wavelength 455nm, maximum emission wavelength 535nm.

10. Use of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 1 or 2 in the rapid screening of UGT1A1 activity regulators and the quantitative evaluation of activity regulation ability.

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