Fluorescence-based AhR regulating agent screening method and application thereof

Abstract

The invention provides a screening method of an aromatic hydrocarbon receptor (AhR) regulating agent based on fluorescence and application of the screening method. The detection principle is that the regulation effect of the small molecule on the AhR is quantitatively evaluated by detecting the activity of the AhR downstream cytochrome P450 1A1 (CYP1A1) through a biochemical method. Therefore, the invention firstly develops a specific fluorescent substrate suitable for functional imaging analysis of CYP1A1 in living cells. The structural general formula is as shown in formula (1): # imgabs0, the substrate can be specifically catalyzed by intracellular CYP1A1 under physiological conditions to perform dechloroethyl reaction and generate a single fluorescent product HDBI, the activity of CYP1A1 in living cells / living tissues can be quantitatively detected by detecting the generation amount of the fluorescent product HDBI in unit time, and then the agonistic activity of AhR is indirectly evaluated. A living cell-based CYP1A1 function detection method is constructed on the basis of the fluorescent substrate, and the fluorescent substrate can be used for high-throughput screening and evaluation of the regulation effect of a compound on an AhR-CYP1A1 axis so as to evaluate the regulation effect of the compound on a CYP1A1 upstream gene AhR.

Description

Technical Field

[0001] The present invention belongs to the field of drug screening and pharmacology, and specifically relates to a fluorescence detection method for detecting cytochrome P450 1A1 (CYP1A1) downstream of aryl hydrocarbon receptor (AhR) and its application in drug discovery. Background Art

[0002] The aryl hydrocarbon receptor (AhR) is a typical ligand-activated transcription factor that is widely expressed in immune, epithelial, endothelial, and stromal cells of the mammalian barrier system. AhR plays a vital role in various physiological processes, such as xenobiotic metabolism, cell development and differentiation, and stem cell maintenance. AhR is closely associated with the occurrence and development of a variety of diseases, including inflammation, metabolic disorders, cancer, and autoimmune diseases. As a typical ligand-activated transcription factor, AhR can be activated by a variety of structurally diverse chemicals, including endogenous metabolites (such as arachidonic acid metabolites), phytochemicals (such as flavonoids), therapeutic drugs, and environmental pollutants. As one of the most characterized downstream gene products of the AhR signaling cascade, CYP1A1 has attracted widespread attention in the fields of inflammation, immunity, cancer, and other metabolic diseases. An increasing number of studies have shown that regulating the AhR-CYP1A axis has become an effective therapeutic strategy for the treatment of autoimmune inflammatory diseases. Activating the AhR-CYP1A axis in a specific cellular environment is expected to treat a variety of inflammatory diseases, including intestinal inflammation, skin inflammation, acute lung injury, and acute kidney injury. Therefore, there is an urgent need to develop reliable and practical drug screening methods to discover and evaluate regulators of the AhR-CYP1A1 axis.

[0003] Although luciferase reporter gene assays are often used to rapidly screen AhR agonists, this method cannot accurately characterize the interaction network between AhR, CYP1As, and small molecule ligands. Given that CYP1A1 is a key downstream gene product, an important phenotypic marker of AhR activation, and a key mediator of various inflammatory diseases, in situ visualization of the dynamic changes in CYP1A1 function in living systems will provide an alternative approach to accurately assess the endpoint results of test substances. However, most of the previously reported fluorescent substrates have problems with poor subtype specificity, low sensitivity, poor solubility, and very poor cell membrane permeability. In addition, most phenolic metabolites of CYP1A1 substrates are easily metabolized by phase II metabolic enzymes such as uridine 5'-diphospho-glucuronosyltransferase and sulfonyltransferase, and are also easily transported to the extracellular matrix by efflux pumps. This severely limits their in situ visualization of CYP1A1 in complex living systems.

[0004] In order to solve the above shortcomings, the present invention adopts a computer-aided molecular design strategy and combines it with a screening strategy based on living cells to design a class of fluorescent substrates with high specificity and good cell membrane permeability for in situ functional imaging of CYP1A1. This type of substrate shows subtype specificity, rapid response, excellent cell membrane permeability and excellent optical properties, and can be used for quantitative detection of CYP1A1 in living cells and living tissues, with the advantages of simple operation, high sensitivity, strong anti-interference, good specificity, etc. Based on the above fluorescent substrate, the present invention also developed a method for screening and evaluating AhR regulators based on living cells and living tissues, which can be used for high-throughput screening of the regulatory effects of compounds or plant extracts on AhR. Summary of the invention

[0005] The present invention provides a method for screening and evaluating AhR regulators based on living cells. The detection principle is to quantitatively evaluate the regulatory effect of small molecules on AhR by detecting the enzyme activity of cytochrome P450 1A1 (CYP1A1) downstream of AhR, and then to use it for drug discovery of AhR regulators.

[0006] The present invention first provides a type of fluorescent substrate for cytochrome P450 1A1, which is a (E)-2-(4-(2-chloroethoxy / isopropyl)phenylvinyl)-3-ethyl-1,1-dimethyl-1H-benzo[E]indol-3-ium compound, and its general structural formula is shown in formula (1):

[0007] Wherein R is isopropyl or chloroethyl.

[0008] The present invention also provides a method for preparing the fluorescent substrate of the above-mentioned cytochrome P450 1A1, wherein 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium (1.0 mmol) and different benzaldehyde derivatives (1.5 mmol) are dissolved in ethanol, and then a catalytic amount of piperidine and acetic acid are added, and the mixture is refluxed at 80° C. for 6 hours. The crude product is further purified by silica gel chromatography (dichloromethane / methanol) to obtain the final product.

[0009] The present invention also provides the use of the fluorescent substrate in detecting the activity of cytochrome P450 1A1 in biological samples.

[0010] Furthermore, the fluorescent substrate can be specifically catalyzed by cytochrome P450 1A1 to undergo a dechloroethylation reaction and produce a single fluorescent product HDBI. The activity of cytochrome P450 1A1 in a biological sample can be quantitatively detected by detecting the amount of metabolite HDBI generated per unit time, thereby indirectly evaluating the agonist activity of AhR.

[0011]

[0012] The fluorescent product HDBI produced by this type of fluorescent substrate after being catalyzed by cytochrome P450 1A1 has an excitation wavelength of 500-570nm and an emission wavelength of 550-610nm. Users can use any device equipped with a fluorescence detector to quantitatively detect the generation of the fluorescent product HDBI, including various types of fluorescence microscopes, liquid chromatography-fluorescence detection instruments, and microplate readers or microplates equipped with fluorescence detection bands.

[0013] The present invention also provides a method for screening and evaluating AhR regulators based on living cells / living tissues, using the above-mentioned fluorescent substrate of cytochrome P450 1A1 to measure the regulatory effect of any test compound or animal or plant extract on AhR downstream cytochrome P450 1A1 in living cells, living tissues or living bodies, thereby indirectly reflecting the agonist activity of the test compound or animal or plant extract on AhR.

[0014] The samples to be tested include, but are not limited to, various cells or cell preparations expressing AhR and cytochrome P450 1A1, engineered cells or their preparations overexpressing AhR and cytochrome P450 1A1, commercially available tissue preparations or other biological samples containing AhR and cytochrome P450 1A1.

[0015] Compared with the prior art, the present invention has significant technical progress. The use of the fluorescence detection method of the present invention to screen agonists of the AhR-CYP1A1 axis has the following advantages:

[0016] (1) Good specificity and single metabolite: This type of substrate can be metabolized by CYP1A1 with high specificity to generate a single fluorescent product.

[0017] (2) High affinity: This type of substrate has a high affinity for CYP1A1, K m Less than 1 micromole.

[0018] (3) High detection throughput: The AhR regulator screening method established using this type of fluorescent substrate can achieve efficient and rapid real-time detection in 96- and 384-well plates, and can achieve high-throughput detection of 20,000 samples per day, with a low cost per test (<0.5 yuan).

[0019] (4) Both substrates and products are easy to synthesize: The above-mentioned fluorescent substrates and their metabolites can be obtained by chemical synthesis, and the synthesis process is simple and easy.

[0020] (5) Screening of AhR-CYP1A1 axis regulators can be carried out at the level of living cells and living tissues: The above-mentioned fluorescent substrates have good cell membrane permeability and can be used for visual screening and evaluation of AhR regulators and CYP1A1 inducers at the level of living cells and living tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 .The general structural formula of CYP1A1-specific fluorescent substrate.

[0022] Figure 2 .Synthetic routes of HDB-EtCl, HDB-ipr and their products.

[0023] Figure 3 . Specificity characterization of HDB-EtCl and HDB-ipr.

[0024] Figure 4 .Enzyme kinetic analysis of HDB-EtCl and HDB-ipr.

[0025] Figure 5 .Liquid chromatograms of HDB-EtCl and HDB-ipr in CYP1A1.

[0026] Figure 6 .Long-term imaging of HDB-EtCl and HDB-ipr in living cells.

[0027] Figure 7 .Construction of a screening method for AhR regulators in living cells. DETAILED DESCRIPTION

[0028] The present invention will be further described in the following examples, but the present invention is not limited thereto.

[0029] Preparation Example 1 Chemical synthesis of the above fluorescent substrate and its metabolites (such as Figure 2 (shown)

[0030] (1) In a 50 mL flask, 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium (1.0 mmol) and different benzaldehyde derivatives (1.5 mmol) were dissolved in anhydrous ethanol, and then catalytic amounts of piperidine and acetic acid were added. The mixture was refluxed at 80° C. for 6 hours, and the crude product was further purified by silica gel chromatography (dichloromethane / methanol).

[0031]

[0032] (2) As described above, pure HDBI was obtained as a brown solid powder (yield 49%). 1H NMR(400MHz, DMSO-d6)δ8.48(d,J=16.1Hz,1H),8.42-8.38(m,1H),8.28(d,J=8.9H z,1H),8.21(dd,J=8.4,1.3Hz,1H),8.19-8.14(m,2H),8.08(d,J=8.9Hz,1H),7.80 (ddd,J=8.4,6.9,1.4Hz,1H),7.70(ddd,J=8.0,6.8,1.1Hz,1H),7.47(d,J=16.1Hz ,1H),6.99-6.94(m,2H),4.77(q,J=7.1Hz,2H),2.02(s,6H),1.49(t,J=7.2Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ181.57,164.98,153.61,138.69,138.05,134.32,133.33,131.42,130.4 9,128.80,127.35,127.26,126.16,123.40,117.36,113.33,107.85,53.72,42.11,26.27,14.16.

[0033]

[0034] (3) As described above, the pure HDB-ipr was obtained as a brown solid powder (yield 36.9%). 1 H NMR(400MHz,DMSO-d6)δ8.54(d,J=16.2Hz,1H),8.45-8.40(m,1H),8.32-8.24(m,3 H),8.22(dd,J=8.3,1.3Hz,1H),8.12(d,J=9.0Hz,1H),7.81(ddd,J=8.4,6.9,1.4H z,1H),7.72(ddd,J=8.1,6.9,1.1Hz,1H),7.59(d,J=16.3Hz,1H),7.16(d,J=8.9Hz ,2H),4.92-4.79(m,3H),2.03(s,6H),1.51(t,J=7.1Hz,3H),1.34(d,J=6.0Hz,6H). 13C NMR(101MHz,DMSO-d6)δ182.27,162.78,153.47,138.59,133.81,133.54,131.51,130.51,128.88,1 27.53,127.49,127.28,123.52,116.55,113.52,109.53,70.53,54.06,42.52,26.12,22.17,14.31.

[0035]

[0036] (4) As described above, pure HDB-EtCl was obtained as a brown solid powder (yield 27.9%). 1 H NMR (600MHz, DMSO-d6) δ8.55(d,J=16.3Hz,1H),8.43(d,J=8.4Hz,1H),8.30(dd,J=8. 9,7.3Hz,3H),8.23(d,J=8.2Hz,1H),8.12(d,J=8.9Hz,1H),7.84-7.80(m,1H),7.73(t ,J=7.4Hz,1H),7.62(d,J=16.3Hz,1H),7.22(d,J=8.7Hz,2H),4.84(q,J=7.2Hz,2H),4 .45(t,J=5.1Hz,2H),4.03(dd,J=5.9,4.1Hz,2H),2.04(s,6H),1.52(t,J=7.2Hz,3H). 13 CNMR(151MHz,DMSO-d6)δ182.35,162.96,153.21,138.77,138.58,133.66,133.59,131.53,130.52,128. 90,128.26,127.59,127.27,123.55,115.91,113.55,110.05,68.92,54.14,43.41,42.63,26.07,14.35.

[0037] Example 2 Metabolic phenotyping of HDB-EtCl and HDB-ipr substrate specificity

[0038] (1) Prepare 180 μl of reaction system, including pH 7.4 PBS buffer (100 mM), 17 commercial CYP recombinant enzymes (2.5 nM) (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP1A1, CYP3A5, CYP4A11, CYP4F2, all from Cypex), and HDB-EtCl / HDB-ipr (5 μM). Pre-incubate at 37°C with shaking for 3 min, and then start the reaction with 20 μl of NADPH with a final concentration of 1 mM.

[0039] (2) After 30 minutes, 100 μl of acetonitrile was added, and the reaction was terminated after vigorous shaking. The supernatant was collected for testing after centrifugation at 20,000 g for 30 minutes. The fluorescence signal of the product was detected (Ex = 545 nm, Em = 585 nm); the fluorescence intensity of the product in each sample was obtained. The selectivity of HDB-EtCl and HDB-ipr for the CYP1A1 family isoform (CYP1B1) was 46.34 and 35.1 times, respectively ( Figure 3 ).

[0040] Conclusion: Both HDB-EtCl and HDB-ipr can be specifically metabolized by CYP1A1, with fast product formation rate and good specificity.

[0041] Example 3 Kinetic Analysis of Enzymatic Reaction of HDB-EtCl and HDB-ipr Substrates

[0042] (1) Prepare 180 μL of reaction system, including pH 7.4 PBS buffer (100 mM), CYP1A1 recombinant single enzyme (2.5 nM) (CYP1A1 isoform enzyme, from Cypex), and different concentrations of HDB-EtCl / HDB-ipr, pre-incubate at 37°C with shaking for 3 min, and then add 20 μL of NADPH with a final concentration of 1 mM to the reaction system to start the reaction;

[0043] (2) Add 20 μl of NADPH with a final concentration of 1 mM to the reaction system to start the reaction; after 30 minutes, add 100 μl of glacial acetonitrile, shake vigorously, terminate the reaction, centrifuge at 20,000 g for 30 minutes, and take the supernatant for testing. Detect the fluorescence signal of the product (Ex = 545 nm, Em = 585 nm); obtain the fluorescence intensity of the product in each sample, and fit the enzyme reaction kinetic curve;

[0044] (3) Fit the Michaelis constant K according to the Michaelis equation m With the maximum reaction rate V max ( Figure 4 ).

[0045] V=(V max ×[S]) / (K m +[S])

[0046] Wherein, V is the reaction rate, and [S] is the concentration of HDB-EtCl / HDB-ipr.

[0047] Conclusion: The metabolism of HDB-EtCl / HDB-ipr substrates conforms to Michaelis-Menten kinetics, and the Michaelis-Menten equation can be used to characterize its enzyme kinetic parameters.

[0048] Example 4 Uniqueness Analysis of HDB-EtCl Metabolites

[0049] (1) Prepare 180 μL of reaction system, including pH 7.4 PBS buffer (100 mM), CYP1A1 recombinant enzyme (2.5 nM) and HDB-EtCl (10 μM), pre-incubate at 37°C for 3 min with shaking, and then add 20 μL of NADPH with a final concentration of 1 mM to the reaction system to start the reaction;

[0050] (2) After 30 minutes, add 100 μl of glacial acetonitrile, shake vigorously, terminate the reaction, centrifuge at 20,000 g for 30 minutes, and take the supernatant for analysis; take substrate standards, product standards, and metabolic reaction samples for liquid chromatography analysis (see Figure 5 ).

[0051] Conclusion: HDB-EtCl and HDB-ipr can generate a single metabolite after CYP1A1.

[0052] Example 5 Functional imaging of CYP1A1 in living cells

[0053] (1) Functional imaging of CYP1A1 in living cells using BEAS-2B cells. BEAS-2B cells were cultured in DMEM medium containing 10% FBS. The culture conditions were a humidified environment with 5% CO2 and a temperature of 37°C. When the cells reached a density of about 80%, the cells were plated at 1×10 5 The density of each was seeded in the confocal culture dish.

[0054] (2) After 24 hours, when the cells were completely adhered to the bottom of the culture dish, HDB-EtCl (5 μM) and Hoechst33342 (5 μg / ml) were added and cultured for 0-120 minutes. The culture medium was discarded, and the cells were rinsed 2-3 times with PBS. Then, the cells were fixed with 4% paraformaldehyde for 10-15 minutes, the culture medium was discarded, and the cells were rinsed 2-3 times with PBS. Finally, the cells were immersed in PBS. Functional imaging of CYP1A1 was performed using a laser confocal scanning microscope (Leica SP8, Wetzlar, Germany). Figure 6 ). The wavelength of the blue channel is λex=405nm, λem=415-485nm; the wavelength of the red channel is λex=552nm, λem=580-640nm.

[0055] Conclusion: HDB-EtCl can be used for long-term imaging of CYP1A1 in living cells.

[0056] Example 6 Construction of screening and evaluation method for CYP1A1 regulators

[0057] (1) BEAS-2B cells were cultured in DMEM medium containing 10% FBS. The culture conditions were a humidified environment with 5% CO2 and a temperature of 37°C. When the cells reached a density of about 80%, BEAS-2B cells were plated at 1×10 4 The cells were seeded at a density of 1000 cells / mL in a 96-well black-bottom permeable cell culture plate.

[0058] (2) When the cells reached a density of about 60%, benzo[a]pyrene (a reported CYP1A1 inducer) was added and cultured for 24 hours, and then 100 μL HDB-EtCl (5 μM) and Hoechst 33342 (5 μg / ml) were added to initiate the reaction. After 30 minutes of culture, fluorescence imaging of CYP1A1 was performed using a high-content cell imaging analysis system (Operetta CLS, PE). Figure 7 ). The blue channel is λex=355-385nm, λem=430-500nm; the red channel is λex=530-560nm, λem=570-650nm.

[0059] Conclusion: Benzo[a]pyrene can induce CYP1A1 activity in BEAS-2B cells in a concentration-dependent manner, indicating that HDB-EtCl can be used as a fluorescent substrate for the screening and evaluation of AhR regulators.

Claims

1. A fluorescent substrate for detecting the activity of CYP1A1 enzyme downstream of AhR, characterized in that: Its general structural formula is shown in formula (1): Wherein R is isopropyl or chloroethyl.

2. The method for preparing a fluorescent substrate for detecting cytochrome P450 1A1 enzyme activity according to claim 1, characterized in that: 1-Ethyl-2,3,3-trimethyl-3H-indol-1-ium and a benzaldehyde derivative were dissolved in ethanol, and then catalytic amounts of piperidine and acetic acid were added, and the mixture was refluxed at 80°C for 6 hours. The crude product was quickly purified by normal phase column chromatography to obtain a pure product.

3. A use of the fluorescent substrate according to claim 1, characterized in that: This type of substrate can be used for real-time quantitative detection of the activity of cytochrome P450 1A1 downstream of AhR in biological samples such as living cells or living tissues, thereby indirectly evaluating the agonist activity of AhR.

4. The use according to claim 3, characterized in that: This type of fluorescent substrate is specifically catalyzed by intracellular cytochrome P4501A1 to undergo a dechloroethylation reaction and produce a single fluorescent product HDBI. The activity of CYP1A1 in living cells / living tissues can be quantitatively detected by detecting the amount of fluorescent product HDBI generated per unit time.

5. The use according to claim 3, characterized in that: The fluorescent substrate of cytochrome P450 1A1 is catalyzed by cytochrome P450 1A1 to release the fluorescent product HDBI, with an excitation wavelength of 500-570nm and an emission wavelength of 550-610nm.

6. A method for screening and evaluating AhR modulators, characterized in that: Using any fluorescent substrate of cytochrome P4501A1 described in claim 1 as a probe substrate, the regulatory effect of any test compound or animal or plant extract on AhR downstream cytochrome P450 1A1 is measured in living cells, living tissues or living bodies, thereby indirectly reflecting the agonist activity of the test compound or animal or plant extract on AhR.

7. The method for screening and evaluating AhR modulators according to claim 6, characterized in that: The formation of the fluorescent product HDBI can be quantitatively detected by any device equipped with a fluorescence detector, including various types of fluorescence microscopes, liquid chromatography-fluorescence detection instruments, and microplate readers or microtiter plates equipped with fluorescence detection bands.

8. The method for screening and evaluating AhR modulators according to claim 6, characterized in that: Biological systems for screening and evaluating AhR agonists include, but are not limited to, various types of tool cells or immortalized cells expressing AhR, engineered cells transfected or overexpressing AhR or their preparations, animal tissues expressing AhR and their preparations, or other commercially available cells or tissues that highly express AhR.

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