A fluorescence-based AhR regulator screening method and its application

Abstract

The present invention provides a fluorescence-based screening method for aryl hydrocarbon receptor (AhR) modulators and its application. The detection principle is to quantitatively evaluate the regulatory effect of small molecules on AhR by biochemically detecting the activity of cytochrome P450 1A1 (CYP1A1) downstream of AhR. To this end, the present invention first developed a class of specific fluorescent substrates suitable for CYP1A1 functional imaging analysis in living cells. Its general structural formula is shown in formula (1): This type of substrate can be specifically catalyzed by intracellular CYP1A1 under physiological conditions 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, thereby indirectly evaluating the agonist activity of AhR. Based on this type of fluorescent substrate, a living cell-based CYP1A1 functional detection method is constructed, which can be used for high-throughput screening and evaluation of the regulatory effect of compounds on the AhR-CYP1A1 axis, and further evaluate the regulatory effect of compounds on the 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 the aryl hydrocarbon receptor (AhR) and its application in drug discovery. Background Art

[0002] The aryl hydrocarbon receptor (AhR) is a prototypical ligand-activated transcription factor (LACT) widely expressed in immune, epithelial, endothelial, and stromal cells of the mammalian barrier system. AhR plays crucial roles in diverse physiological processes, including xenobiotic metabolism, cell development and differentiation, and stem cell maintenance. AhR is closely associated with the development and progression of numerous diseases, including inflammation, metabolic disorders, cancer, and autoimmune diseases. As a typical LACT, AhR can be activated by a diverse array of chemical substances, including endogenous metabolites (such as arachidonic acid metabolites), phytochemicals (such as flavonoids), therapeutic drugs, and environmental pollutants. As one of the best-characterized downstream gene products of the AhR signaling cascade, CYP1A1 has attracted significant attention in the fields of inflammation, immunity, cancer, and other metabolic diseases. A growing number of studies have demonstrated that modulating the AhR-CYP1A axis has emerged as an effective therapeutic strategy for treating autoimmune inflammatory diseases. Activating the AhR-CYP1A axis in specific cellular contexts holds promise for treating 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 commonly used for rapid screening of AhR agonists, they are unable to accurately characterize the interaction network between the 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 would provide an alternative approach for accurately assessing endpoints of test compounds. However, most previously reported fluorogenic substrates suffer from poor isoform specificity, low sensitivity, poor solubility, and very poor cell membrane permeability. Furthermore, most phenolic metabolites of CYP1A1 substrates are readily metabolized by phase II enzymes such as uridine 5'-diphosphate glucuronosyltransferase and sulfonyltransferase, and are also readily transported into the extracellular matrix by efflux pumps. This severely limits their application in in situ visualization of CYP1A1 in complex living systems.

[0004] To address these shortcomings, the present invention employs a computer-aided molecular design strategy combined with a live cell-based screening strategy to design a class of highly specific and cell membrane-permeable fluorescent substrates for in situ functional imaging of CYP1A1. These substrates exhibit subtype specificity, rapid response, excellent cell membrane permeability, and superior optical properties, and can be used for the quantitative detection of CYP1A1 in living cells and living tissues. They offer advantages such as ease of operation, high sensitivity, strong anti-interference properties, and good specificity. Based on these fluorescent substrates, the present invention also developed a method for screening and evaluating AhR modulators based on living cells and living tissues, which can be used for high-throughput screening of compounds or plant extracts for their regulatory effects on AhR. Summary of the Invention

[0005] The present invention provides a living cell-based method for screening and evaluating AhR regulators. 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 use it for drug discovery of AhR regulators.

[0006] The present invention first provides a fluorescent substrate for cytochrome P450 1A1, which is (E)-2-(4-(2-chloroethoxy / isopropyl)phenylvinyl)-3-ethyl-1,1-dimethyl-1 H -Benzo[E]indol-3-ium compounds, the general structural formula of which 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 cytochrome P450 1A1, comprising: H -Indole-1-ium (1.0 mmol) and different benzaldehyde derivatives (1.5 mmol) were dissolved in ethanol, and then catalytic amounts of piperidine and acetic acid were added. The mixture was refluxed at 80 °C for 6 hours. The crude product was further purified by silica gel chromatography (dichloromethane / methanol) to obtain the final product.

[0009] The present invention also provides use of the fluorescent substrate in detecting cytochrome P450 1A1 activity 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. By detecting the amount of metabolite HDBI generated per unit time, the activity of cytochrome P450 1A1 in the biological sample can be quantitatively detected, thereby indirectly evaluating the agonist activity of AhR.

[0011]

[0012] The fluorescent product HDBI, produced by cytochrome P450 1A1, is catalyzed by these fluorescent substrates and has an excitation wavelength of 500-570 nm and an emission wavelength of 550-610 nm. Users can quantitatively detect the formation of the fluorescent product HDBI using any device equipped with a fluorescence detector, including fluorescence microscopes, liquid chromatography-fluorescence detectors, and microplate readers or microplates equipped with fluorescence detection bands.

[0013] The present invention also provides a method for screening and evaluating AhR modulators based on living cells / living tissues, which uses the above-mentioned fluorescent substrate of cytochrome P450 1A1 to measure the regulatory effect of any test compound or animal or plant extract on cytochrome P450 1A1 downstream of AhR 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 existing technologies, the present invention has significant technical advancements. 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 produce a single fluorescent product.

[0017] (2) High affinity: This type of substrate has 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 living cell and living tissue levels: 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 living cell and living tissue levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 . 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 chromatogram of HDB-EtCl 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 are not intended to limit the present invention.

[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-3 H -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%). 1 H NMR (400 MHz, DMSO- d 6) δ 8.48 (d, J = 16.1 Hz, 1H), 8.42 – 8.38 (m, 1H), 8.28 (d, J = 8.9 Hz, 1H), 8.21(dd, J = 8.4, 1.3 Hz, 1H), 8.19 – 8.14 (m, 2H), 8.08 (d, J = 8.9 Hz, 1H), 7.80(ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.70 (ddd, J = 8.0, 6.8, 1.1 Hz, 1H), 7.47 (d, J =16.1 Hz, 1H), 6.99 – 6.94 (m, 2H), 4.77 (q, J = 7.1 Hz, 2H), 2.02 (s, 6H), 1.49(t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 181.57, 164.98, 153.61, 138.69,138.05, 134.32, 133.33, 131.42, 130.49, 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, pure HDB-ipr was obtained as a brown solid powder (yield 36.9%). 1 H NMR (400 MHz, DMSO- d 6) δ 8.54 (d, J = 16.2 Hz, 1H), 8.45 – 8.40 (m, 1H), 8.32 – 8.24 (m, 3H), 8.22 (dd, J = 8.3, 1.3 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.81 (ddd, J= 8.4, 6.9,1.4 Hz, 1H), 7.72 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 7.59 (d, J = 16.3 Hz, 1H),7.16 (d, J = 8.9 Hz, 2H), 4.92 – 4.79 (m, 3H), 2.03 (s, 6H), 1.51 (t, J = 7.1 Hz,3H), 1.34 (d, J = 6.0 Hz, 6H). 13 C NMR (101 MHz, DMSO- d 6) δ 182.27, 162.78, 153.47,138.59, 133.81, 133.54, 131.51, 130.51, 128.88, 127.53, 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 (600 MHz, DMSO- d 6) δ 8.55 (d, J = 16.3 Hz, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.30 (dd, J = 8.9,7.3 Hz, 3H), 8.23 ​​(d, J = 8.2 Hz, 1H), 8.12 (d, J = 8.9 Hz, 1H), 7.84 – 7.80 (m,1H), 7.73 (t, J = 7.4 Hz, 1H), 7.62 (d, J = 16.3 Hz, 1H), 7.22 (d, J = 8.7 Hz, 2H),4.84 (q, J = 7.2 Hz, 2H), 4.45 (t,J = 5.1 Hz, 2H), 4.03 (dd, J = 5.9, 4.1 Hz, 2H),2.04 (s, 6H), 1.52 (t, J = 7.2 Hz, 3H). 13 C NMR (151 MHz, DMSO- d 6) δ 182.35,162.96, 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 analysis of HDB-EtCl and HDB-ipr substrate specificity

[0038] (1) Prepare a 180 μl reaction system, including PBS buffer (100 mM) at pH 7.4, 17 commercially available 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). Preincubate at 37°C with shaking for 3 min, and then initiate the reaction with 20 μl of NADPH (final concentration of 1 mM).

[0039] (2) After 30 minutes, 100 μl of glacial acetonitrile was added and the mixture was shaken vigorously to terminate the reaction. The mixture was centrifuged at 20,000 g for 30 minutes and the supernatant was collected for analysis. 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 a 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 a 180 μL reaction system containing 100 mM PBS buffer (pH 7.4), 2.5 nM recombinant CYP1A1 enzyme (CYP1A1 isoform enzyme, from Cypex), and different concentrations of HDB-EtCl / HDB-ipr. Pre-incubate at 37°C with shaking for 3 min. Then, add 20 μL of NADPH (final concentration of 1 mM) to the reaction system to initiate the reaction.

[0043] (2) Add 20 μl of NADPH (final concentration: 1 mM) to the reaction system to initiate the reaction. After 30 minutes, add 100 μl of glacial acetonitrile and shake vigorously to terminate the reaction. Centrifuge at 20,000 g for 30 minutes and collect the supernatant for analysis. Detect the fluorescence signal of the product (Ex = 545 nm, Em = 585 nm). Calculate the fluorescence intensity of the product in each sample and fit the enzymatic reaction kinetic curve.

[0044] (3) Fitting the Michaelis constant based on the Michaelis equation K m and maximum reaction rate V max ( Figure 4 ).

[0045] V=(V max × [S]) / (K m +[S])

[0046] Where 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 the enzyme kinetic parameters.

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

[0049] (1) Prepare 180 μ The reaction system, including pH 7.4 PBS buffer (100 mM), CYP1A1 recombinant enzyme (2.5 nM), and HDB-EtCl (10 μM), was pre-incubated at 37°C with shaking for 3 min, and then 20 μl of NADPH (final concentration of 1 mM) was added to the reaction system to initiate 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 collect the supernatant for analysis; take the substrate standard, product standard, and metabolic reaction sample 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 was performed using BEAS-2B cells. BEAS-2B cells were cultured in DMEM medium containing 10% FBS. The culture conditions were a humidified atmosphere with 5% CO2 and a temperature of 37°C. When the cells reached a density of about 80%, the cells were plated at a density of 1×10 cells per dish. 5 The cells were seeded at a density of 100 μg / mL in a 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 Hoechst 33342 (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. The cells were then 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 wavelengths of the blue channel are λex = 405 nm, λem = 415-485 nm; the wavelengths of the red channel are λex = 552 nm, λem = 580-640 nm.

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

[0056] Example 6 Construction of a 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 cells per well. 4 The cells were seeded at a density of 100 cells / well in a 96-well black-bottom permeable cell culture plate.

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

[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 modulators.

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 chloroethyl.

2. The method for preparing the fluorescent substrate according to claim 1, wherein: 1-ethyl-2,3,3-trimethyl-3 H -Indol-1-ium and benzaldehyde derivatives 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; The structural formula of the benzaldehyde derivative is shown below: .

3. Use of the fluorescent substrate according to claim 1 in the preparation of a reagent for real-time quantitative detection of the activity of cytochrome P450 1A1 downstream of AhR in living cells or living tissues.

4. The use according to claim 3, characterized in that: The fluorescent substrate is specifically catalyzed by intracellular cytochrome P450 1A1 to undergo a dechloroethylation reaction and produce a single fluorescent product, HDBI. By detecting the amount of fluorescent product HDBI generated per unit time, the activity of CYP1A1 in living cells / tissues can be quantitatively detected. The structural formula of the HDBI is shown below: .

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

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