A fluorescent probe substrate specific for glucuronosyltransferase ugta1a1 and use thereof
By designing specific fluorescent probe substrates, the problem of UGT1A1 activity detection in existing technologies has been solved, achieving efficient and sensitive detection and screening of UGT1A1 enzyme activity, which is applicable to a variety of biological systems.
Patent Information
- Application Number
- CN202411951326.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-11-11
- Estimated Expiration
- 2044-12-27
AI Technical Summary
Existing technologies make it difficult to develop efficient and sensitive glucuronyl transferase UGT1A1-specific probe substrates for screening UGT1A1 activity regulators and quantitatively determining UGT1A1 activity in biological systems.
A specific fluorescent probe substrate for glucuronyl transferase UGT1A1 was designed. This substrate has a significant difference in fluorescence emission wavelength from the N-glucuronidation product and has a high fluorescence quantum yield, making it easy to detect.
It enables rapid and sensitive detection of UGT1A1 activity, and can specifically identify UGT1A1 enzyme in various biological systems. It is suitable for quantitative determination of UGT1A1 enzyme activity in recombinant single enzymes, human or animal tissue preparations and various tissue cells, and is not affected by biological matrix and impurities.
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Figure CN119751352B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and specifically relates to a specific fluorescent probe substrate for glucuronyl transferase UGT1A1 and its application. Background Technology
[0002] The uridine diphosphate-glucuronosyltransferase (UGT) superfamily constitutes a crucial phase II drug metabolism system in organisms. Through an SN2 reaction mechanism, they link lipophilic compounds to the glucuronic acid (GA) moiety of uridine diphosphate glucuronide (UDPGA), thereby enhancing the water solubility of these compounds and facilitating their excretion in urine or bile. This process is often a key step in the body's detoxification process, involving the transformation of numerous endogenous substances (such as bilirubin and estradiol), mutagens, drugs, and their metabolites.
[0003] Human UGT enzymes are classified into four main families: UGT1, UGT2, UGT3, and UGT8, with the UGT1 and UGT2 families playing a central role in the metabolism of endogenous and exogenous substances. To date, scientists have identified 18 human UGT subtypes, including nine of the UGT1A subtypes (UGT1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, and 1A10) and seven of the UGT2B subtypes (UGT2B4, 2B7, 2B10, 2B15, and 2B17).
[0004] Of particular note is uridine diphosphate glucuronide transferase 1A1 (UGT1A1), a key phase I metabolic enzyme crucial for clearing the endogenous toxic metabolite bilirubin and various exogenous substrates. Bilirubin, a product of heme breakdown, can accumulate in the body and lead to cholestasis and nervous system damage, thus requiring timely excretion. Adults produce approximately 250 to 350 mg of bilirubin daily, and UGT1A1 is the only enzyme in the body responsible for bilirubin metabolism. It converts bilirubin into glucuronide conjugates, enabling its excretion via bile. Furthermore, UGT1A1 is widely involved in the metabolism of other endogenous compounds, such as estrogens, androgens, and thyroxines. These hormones play a vital role in regulating cell proliferation and differentiation, and their levels require precise control in target tissues.
[0005] UGT1A1 enzymes are widely distributed in the body and can catalyze the glucuronidation metabolism of these hormones in target tissues, thereby rapidly inactivating them when the body does not need them. However, when UGT1A1 enzyme function is abnormal, it can lead to hormonal imbalances in the body, thereby increasing the risk of cancer. Therefore, developing efficient and sensitive specific UGT1A1 probe substrates is of great significance for screening UGT1A1 activity regulators and quantifying UGT1A1 activity in biological systems. Summary of the Invention
[0006] The purpose of this invention is to provide a specific fluorescent probe substrate for glucuronyl transferase UGT1A1. This specific fluorescent probe substrate has a significantly different fluorescence emission wavelength from the N-glucuronidation product, and the product has a higher fluorescence quantum yield and is easier to detect.
[0007] This scheme includes a specific fluorescent probe substrate for the glucuronyl transferase UGT1A1, which 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:
[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 application of the specific fluorescent probe substrate of the glucuronyl transferase UGT1A1 in determining the activity of UGT1A1 in different biological systems.
[0013] Furthermore, the biological system is any one of the following: 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 into a buffer solution for 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 from 0.1 to 10 μm. For single-point assays, the concentration of the specific fluorescent probe substrate is preferably 5 μm.
[0016] Furthermore, the buffer solution is a Tris-HCl buffer solution with a pH value of 5.5–10.5. Preferably, pH 8.4 is the optimal reaction pH.
[0017] Furthermore, the reaction temperature is 20–60°C, and the reaction time is 0.05–4 h. The reaction is terminated when the corresponding glucuronidation product of the specific fluorescent probe substrate reaches the limit of quantitation and the conversion rate of the specific fluorescent probe substrate does not exceed 20%. Preferably, 37°C is the optimal reaction temperature.
[0018] Furthermore, the elimination rate of the specific fluorescent probe substrate or the generation rate of its glucuronidation product is detected by a fluorescent liquid phase detector. The fluorescence detection conditions for the specific fluorescent probe substrate are: excitation wavelength 455 nm and maximum emission wavelength 535 nm; the fluorescence detection conditions for the glucuronidation product are: excitation wavelength 455 nm and maximum emission wavelength 535 nm.
[0019] The present invention provides the application of a specific fluorescent probe substrate for glucuronyl transferase UGT1A1. Both the probe substrate and its glucuronidation product have fluorescent properties, but they have different optical properties. A fluorescent liquid phase detector can be used to achieve rapid and sensitive detection of both the substrate and the product simultaneously.
[0020] This specific fluorescent probe substrate is not easily affected by biological matrix and impurities during the UGT1A1 activity detection process. It can be used for the quantitative determination of UGT1A1 enzyme activity in various recombinant UGT1A1, human and animal tissue preparations, and various tissue cells. The fluorescence detection method of this specific fluorescent probe substrate and glucuronidation metabolites can also be used for the rapid screening of UGT1A1 activity regulators and the quantitative evaluation of their activity regulation ability.
[0021] The study employed a recombinant glucuronide UGT1A1 single enzyme and a liver microsomal incubation system. Through correlation analysis, specific inhibition experiments, evidence from the recombinant single enzyme metabolic reaction, and enzyme kinetics, it was demonstrated that the specific fluorescent probe substrate can be specifically metabolized by glucuronide UGT1A1 to generate an aminoglucuronidated product. Further investigation using various metabolic evaluation systems, including freshly extracted hepatocytes from various mammals, primary cultured hepatocytes, liver sections, and liver perfusion, revealed that this metabolic reaction exhibits very good specificity.
[0022] As a highly specific fluorescent probe substrate for the glucuronic acid UGT1A1 monoenzyme, this specific fluorescent probe substrate can be used to detect the activity of UGT1A1. It is particularly suitable for the determination of the enzyme activity of UGT1A1 produced by bacterial, insect cell, mammalian cell and yeast clonal expression systems, as well as the labeling of the activity of UGT1A1 in microsomes, S9 and other preparations from various mammalian tissues and organs.
[0023] Using the specific fluorescent probe substrate of the glucuronic acid UGT1A1 single enzyme described in this invention to detect the in vitro activity of UGT1A1 single enzyme has the following outstanding advantages:
[0024] (1) High specificity: The specific fluorescent probe substrate can be metabolized by the glucuronic acid UGT1A1 single enzyme into a metabolite, namely the glucuronidation product of amino group.
[0025] (2) Simple and easy to obtain: Specific fluorescent probe substrates can be obtained by chemical synthesis. The synthesis process is simple and easy to carry out, and the detection cost of fluorescence method is low.
[0026] (3) Easy high-throughput detection: It can be measured on various common laboratory fluorescent microplate readers and biochemical analyzers, and batch detection can be performed using 96 or 386 microplates.
[0027] (4) High sensitivity: The specific fluorescent probe substrates all have good fluorescence emission spectral characteristics (450-700nm), and the specific fluorescent probe substrates and their glucuronidated metabolites have different fluorescence emission spectral characteristics, which can be distinguished and detected well. At the same time, quantitative determination can be performed by establishing a ratio-type standard curve. Attached Figure Description
[0028] Figure 1 N-morpholino-4-amino-1,8-naphthylimide 1 H-NMR spectrum;
[0029] Figure 2 N-morpholino-4-amino-1,8-naphthylimide 13 C-NMR spectrum;
[0030] Figure 3 The liquid-phase metabolic profile of HLM for N-morpholino-4-amino-1,8-naphthalimide;
[0031] Figure 4 Screening assay for recombinant human UGT single enzyme for N-morpholine-4-amino-1,8-naphthylimide;
[0032] Figure 5 Specific inhibition experiments for N-morpholino-4-amino-1,8-naphthylimide;
[0033] Figure 6 Enzyme kinetics experiments for N-morpholine-4-amino-1,8-naphthylimide;
[0034] Figure 7 This is a schematic diagram of the metabolic pathway of N-morpholino-4-amino-1,8-naphthylimide being glucuronidated by UGT1A1. Detailed Implementation
[0035] The following embodiments will further illustrate the present invention, but are not intended to limit the invention.
[0036] The equipment and its model used in this invention are as follows: the fluorescence emission / excitation spectrum was detected by the SynergyH1 full-function microplate detector; 1 The H-NMR spectrum was obtained by nuclear magnetic resonance spectrometer (Avance II 600MHz).
[0037] Example 1
[0038] The synthetic route for N-morpholine-4-amino-1,8-naphthalimide is shown below:
[0039]
[0040] 4.2 mmol of 4-aminomorpholine was added to 50 mL of ethanol solution containing 1 g (3.61 mmol) of 4-amino-1,8-naphthalene anhydride. The mixture was reacted overnight at 70-80 °C. Then, 200 mL of water was added, resulting in the precipitation of a large amount of solid. The solid was filtered and dried under vacuum to obtain a pale yellow solid, N-morpholine-4-amino-1,8-naphthaleneimide, with a yield of 80-90%.
[0041] N-morpholine-4-amino-1,8-naphthylimide 1 The H-NMR spectrum is shown below. Figure 1 , 13 The C-NMR spectrum is shown below. Figure 2 .
[0042] Example 2
[0043] In vitro determination of the metabolism of fluorescent substrates
[0044] (1) Prepare a 95 μL UGT metabolic reaction system, including Tris-Hc1 buffer (100 mM) at pH 8.4, human liver microsomes (0.05 mg / mL), N-morpholino-4-amino-1,8-naphthalimide at final concentrations of 0.5 and 50 μM, and incubate with shaking at 37 °C for 3 minutes.
[0045] (2) Add 5 μL of UDPGA at a concentration of 40 mM (final concentration 2 mM) to the reaction system to initiate the reaction.
[0046] (3) After 60 minutes, add 100 μL of ice-cold acetonitrile, shake vigorously, and then terminate the reaction.
[0047] (4) After centrifuging at 4°C and 20,000 × g for 20 minutes using a high-speed refrigerated centrifuge, the supernatant was collected for fluorescence liquid chromatography detection (Ex = 455 nm, Em = 535 nm). Figure 3 ).
[0048] Example 3
[0049] In vitro determination of the selectivity of human recombinant UGT single enzyme
[0050] (1) Prepare a 95 μL UGT metabolic reaction system, including Tris-Hc1 buffer (100 mM) at pH 8.4, each single enzyme of recombinant human UGT (0.05 mg / mL), and N-morpholine-4-amino-1,8-naphthalimide at final concentrations of 0.5 and 50 μM. Incubate at 37°C with shaking for 3 minutes.
[0051] (2) Add 5 μL of UDPGA at a concentration of 40 mM (final concentration 2 mM) to the reaction system to initiate the reaction.
[0052] (3) After 60 minutes, add 100 μL of ice-cold acetonitrile, shake vigorously, and then terminate the reaction.
[0053] (4) After centrifuging at 4°C and 20,000×g for 20 minutes using a high-speed refrigerated centrifuge, the supernatant was collected for fluorescence liquid chromatography detection (Ex = 455nm, Em = 535nm); the selectivity of recombinant human UGT1A1 enzyme is about 100 times that of other single enzymes. Figure 4 ).
[0054] Example 4
[0055] In vitro specific inhibition test
[0056] (1) Prepare a 190 μL human liver microsome 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-naphthylimide with final concentrations of 0.5 and 50 μM, and pre-incubate different concentrations of hecoginin at 37 °C with shaking for 3 minutes.
[0057] (2) Add 10 μL of UDPGA with a concentration of 40 mM to the reaction system to start the reaction.
[0058] (3) After 60 minutes, add 200 μL of ice-cold acetonitrile, shake vigorously, and then terminate the reaction.
[0059] (4) After centrifuging at 4°C and 20,000 × g for 20 minutes using a high-speed refrigerated centrifuge, the supernatant was collected for fluorescence detection (Ex = 455 nm, Em = 535 nm). The results are shown in the attached figure. Figure 5 As shown.
[0060] Example 5
[0061] Enzyme kinetics experiment
[0062] (1) Prepare a 190 μL human liver microsome and UGT1A1 metabolic reaction system in advance, including Tris-Hc1 buffer (100 mM) at pH 8.4, different concentrations of human liver microsomes, UGT1A1 (0.05 mg / mL), N-n-butyl-4-amino-1,8-naphthalimide with a final concentration of 50 μM, and pre-incubate with shaking at 37 °C for 3 minutes.
[0063] (2) Add 10 μL of UDPGA with a concentration of 40 mM to the reaction system to start the reaction.
[0064] (3) After 60 minutes, add 200 μL of ice-cold acetonitrile, shake vigorously, and then terminate the reaction;
[0065] (4) After centrifuging at 4°C and 20,000 × g for 20 minutes using a high-speed refrigerated centrifuge, the supernatant was collected for fluorescence detection (Ex = 455 nm, Em = 535 nm). The results are shown in the attached figure. Figure 6 As shown.
[0066] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions conceived without inventive effort should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.
Claims
1. A specific fluorescent probe substrate for glucuronyl transferase UGT1A1, characterized in that: The specific fluorescent probe substrate is specifically catalyzed by UGT1A1 to generate the corresponding N-glucuronidation product, and the general structural formula of the specific fluorescent probe substrate is shown below: 。 2. The application of the specific fluorescent probe substrate of glucuronyl transferase UGT1A1 as described in claim 1 for non-disease diagnostic and therapeutic purposes in determining the activity of UGT1A1 in different biological systems.
3. The application of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 2 in determining the activity of UGT1A1 in different biological systems for non-disease diagnostic and therapeutic purposes, characterized in that: The biological system is any one of the following: a recombinant single enzyme containing UGT1A1, a human or animal tissue preparation solution, various mammalian tissue cells and their preparations.
4. The application of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 3 in determining the activity of UGT1A1 in different biological systems for non-disease diagnostic and therapeutic purposes, characterized in that: The determination of UGT1A1 activity in different biological systems includes the following steps: placing a specific fluorescent probe substrate and a biological system containing UGT1A1 into a buffer solution for 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.
5. The application of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 4 for non-disease diagnostic and therapeutic purposes in determining the activity of UGT1A1 in different biological systems, characterized in that: The concentration of the specific fluorescent probe substrate is selected from 0.1 to 10 μm.
6. The application of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 5 for non-disease diagnostic and therapeutic purposes in determining the activity of UGT1A1 in different biological systems, characterized in that: The buffer solution is a Tris-HCl buffer solution with a pH value of 5.5–10.
5.
7. The application of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 6 for non-disease diagnostic and therapeutic purposes in determining the activity of UGT1A1 in different biological systems, characterized in that: The reaction temperature is 20–60°C. The reaction is terminated when the corresponding glucuronidation product of the specific fluorescent probe substrate reaches the limit of quantitation and the conversion rate of the specific fluorescent probe substrate does not exceed 20%.
8. The application of the specific fluorescent probe substrate for glucuronyl transferase UGT1A1 according to claim 7 for non-disease diagnostic and therapeutic purposes 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 fluorescence liquid phase detector is used. The fluorescence detection conditions for the specific fluorescent probe substrate are: excitation wavelength 455 nm and maximum emission wavelength 535 nm; the fluorescence detection conditions for the glucuronidation product are: excitation wavelength 455 nm and maximum emission wavelength 535 nm.
9. The application of the specific fluorescent probe substrate of glucuronyl transferase UGT1A1 as described in claim 1 for rapid screening of UGT1A1 activity regulators and quantitative evaluation of its activity regulation ability, for purposes other than disease diagnosis and treatment.