Methods for screening acetylcholinesterase inhibitors - Patents.com

Abstract

The present disclosure relates to a method for screening acetylcholinesterase inhibitors, and further to a kit and a system for screening acetylcholinesterase inhibitors.

Description

[Technical field]

[0001] The present disclosure is in the fields of food, environment, pharmacology and medicine, and particularly relates to a method for screening acetylcholinesterase inhibitors, and also to a kit and system for screening acetylcholinesterase inhibitors. [Background technology]

[0002] Acetylcholinesterase (AChE), also known as true cholinesterase, is a key enzyme in biological nerve conduction processes. AChE can degrade acetylcholine (ACh) at cholinergic synapses, halting the excitatory action of neurotransmitters in the postsynaptic membrane and ensuring the normal transmission of nerve signals in the body. All currently known organophosphate (OP) neurotoxicants, carbamate (CM) pesticides, and Alzheimer's disease (AD) therapeutics have AChE as their key target, and induce neurotoxic effects or improve cognitive function in AD by inhibiting normal or pathologically elevated AChE activity. OPs are the largest class of AChE inhibitors (AChEIs), and related compounds include pesticides, defoliants, flame retardants, industrial solvents, lubricants, plasticizers, fuel additives, and neurotoxic chemical warfare agents, as well as the AD prescription drug metrifonate, which are found in hundreds of products currently used worldwide. Due to the great benefits of pesticides in improving agricultural productivity and controlling deadly vector-borne diseases, and the rapid development of industrialization, OPs have been widely used. Statistics show that OPs have become one of the most common synthetic chemicals found in the environment, animal tissues, and human tissues, with approximately one billion pounds of OPs being released into the environment, food, and water supplies worldwide every year. This has led to increasingly severe environmental and ecological problems and has become a major threat to human health and public safety. Furthermore, faced with the increasingly severe problem of an aging world population and a completely unmet need for AD treatment, new types of (non-organophosphorus) AChEIs that are recognized as AD treatment drugs remain the focus of AD drug research and development. Therefore, screening and accurate characterization of known and potential AChEIs (including organophosphorus nerve agents) are important for early warning of environmental and food safety, understanding of potentially toxic substances, precise treatment of poisoning, and research of new and more effective AD drugs.

[0003] To date, there are three major types of commonly used AChEI screening methods, namely, determination based on known OP structures, analysis based on inhibition of AChE activity, and immunoassay based on OP immunogenicity. Due to limitations in preparing antibodies with strong affinity and high specificity, immunoassay methods not only have challenges in successfully detecting low levels of OPs, but also cannot distinguish between OPs with different structures. Analysis based on chemical structures mainly uses chromatographic methods, such as liquid chromatography (LC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS). These methods provide high sensitivity with detection limits in the nanomolar concentration range and are the preferred methods for trace component quantitative analysis. However, these methods are only suitable for known molecules, have low throughput, and are not suitable for large-scale screening. In addition, these methods are time-consuming, use expensive equipment, and must be performed by well-trained technicians. Enzyme activity inhibition methods are based on the inhibition of AChE, and AChEI can be detected by measuring the enzyme activity before and after exposure to the test sample. The enzyme activity is usually measured by Ellman colorimetry, radioactivity analysis, fluorescence analysis, electrochemical analysis, or chemiluminescence analysis. These methods have low cost, high throughput, and fast analysis speed, and are the most promising alternatives to classical methods (GC / MS, LC / MS). However, these methods have low sensitivity to the enzyme activity changes after exposure to some OP compounds, which significantly limits the detection sensitivity of these methods. Moreover, from the perspective of efficient screening of AChEI, the above methods can only provide single characterization of the test chemicals at the level of known structure or enzyme molecules, but cannot provide biological characterization of potentially toxic molecules, and cannot provide multidimensional information for accurate early warning and effective treatment guidance. Summary of the Invention

[0004] The present disclosure provides a method for screening for acetylcholinesterase inhibitors, comprising: 1) mixing a sample to be screened with acetylcholinesterase; 2) mixing the mixture obtained in step 1) with acetylcholine; 3) contacting the reporter cell M3:NFATc1-EGFP U2OS with the mixture obtained in step 2); 4) The method includes a step of detecting the degree of activation of reporter cell M3:NFATc1-EGFP U2OS.

[0005] In some embodiments, the activation degree of reporter cell M3:NFATc1-EGFP U2OS is expressed as the degree of NFATc1-EGFP translocation from the cytoplasm to the nucleus of reporter cell. When NFATc1-EGFP in reporter cell translocates from the cytoplasm to the nucleus, it is determined that the screening sample contains an acetylcholinesterase inhibitor. The inhibitory activity of acetylcholinesterase inhibitor against acetylcholinesterase can also be quantified according to the number of intracellular NFATc1-EGFP translocated from the cytoplasm to the nucleus.

[0006] In some embodiments, samples to be screened according to the present disclosure may be chemicals, foods (eg, various meats, vegetables, teas, etc.), environmental organophosphorus pesticides, AD medications, nerve agents, and the like.

[0007] In some embodiments, the acetylcholine is iodinated acetylcholinesterase and the acetylcholinesterase is recombinant human acetylcholinesterase.

[0008] In some embodiments, in step 1) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, the sample to be screened is mixed with acetylcholinesterase in a first solvent.

[0009] In some embodiments, in step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, the mixture obtained in step 1) is mixed with acetylcholine in a second solvent.

[0010] In some embodiments, the first solvent and the second solvent are the same or different and each independently is a medium suitable for normal growth of the reporter cells M3:NFATc1-EGFP U2OS, for example, DMEM high glucose medium.

[0011] In some embodiments, in step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, after the mixed system obtained in step 1) is mixed with acetylcholine, the initial concentration of acetylcholinesterase in the resulting mixed system (the concentration when not reacting with acetylcholine) is 0.005 to 0.018 U / mL, preferably 0.007 to 0.015 U / mL, and even more preferably 0.01 U / mL.

[0012] In some embodiments, in step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, after the mixed system obtained in step 1) is mixed with acetylcholine, the initial concentration of acetylcholine in the obtained mixed system (concentration when not reacting with acetylcholinesterase) is 10 to 1000 nM, preferably 30 to 800 nM, more preferably 30 to 500 nM, more preferably 30 to 400 nM, even more preferably 30 to 300 nM, even more preferably 30 to 200 nM, and even more preferably 30 to 100 nM.

[0013] In some embodiments, in step 1) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, the sample to be screened is mixed with acetylcholinesterase at 25 to 40°C (e.g., 30°C, 32°C, 35°C, 37°C) for 10 to 60 minutes, e.g., 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 50 minutes.

[0014] In some embodiments, in step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, the mixture obtained in step 1) is mixed with acetylcholine at 25 to 40°C (e.g., 30°C, 32°C, 35°C, 37°C) for 10 to 60 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 50 minutes.

[0015] In some embodiments, in step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, after the mixed system obtained in step 1) is mixed with acetylcholine, the initial concentration of acetylcholinesterase in the obtained mixed system (the concentration when not reacting with acetylcholine) is 0.005 to 0.018 U / mL, and the initial concentration of acetylcholine in the obtained mixed system is 10 to 1000 nM (e.g., 30 to 800 nM, 30 to 500 nM, 30 to 400 nM, 30 to 300 nM, 30 to 200 nM, or 30 to 100 nM).

[0016] In some embodiments, in step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, after the mixed system obtained in step 1) is mixed with acetylcholine, the initial concentration of acetylcholinesterase in the obtained mixed system (the concentration when not reacting with acetylcholine) is 0.007 to 0.015 U / mL, and the initial concentration of acetylcholine in the obtained mixed system is 10 to 1000 nM (e.g., 30 to 800 nM, 30 to 500 nM, 30 to 400 nM, 30 to 300 nM, 30 to 200 nM, or 30 to 100 nM).

[0017] In some embodiments, in step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, after the mixed system obtained in step 1) is mixed with acetylcholine, the initial concentration of acetylcholinesterase in the obtained mixed system (the concentration when not reacting with acetylcholine) is 0.01 U / mL, and the initial concentration of acetylcholine in the obtained mixed system is 10 to 1000 nM (e.g., 30 to 800 nM, 30 to 500 nM, 30 to 400 nM, 30 to 300 nM, 30 to 200 nM, or 30 to 100 nM).

[0018] In some embodiments, in step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, after the mixed system obtained in step 1) is mixed with acetylcholine, the initial concentration of acetylcholinesterase in the obtained mixed system is 0.01 U / mL, the initial concentration of acetylcholine in the obtained mixed system is 30 nM, and preferably, the mixed system obtained in step 1) is mixed with acetylcholine for 10 minutes.

[0019] In some embodiments, in step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, after the mixed system obtained in step 1) is mixed with acetylcholine, the initial concentration of acetylcholinesterase in the obtained mixed system is 0.01 U / mL, the initial concentration of acetylcholine in the obtained mixed system is 100 nM, and preferably, the mixed system obtained in step 1) is mixed with acetylcholine for 20 minutes.

[0020] In some embodiments, in step 3) of the method for screening acetylcholinesterase inhibitors described in the present disclosure, the reporter cells M3:NFATc1-EGFP U2OS are contacted with the mixed system obtained in step 2) for 10 to 60 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or 50 minutes.

[0021] In some embodiments, the method further comprises, prior to step 4) of the method for screening acetylcholinesterase inhibitors described herein, Fixing the reporter cells with a formaldehyde solution diluted in phosphate buffered saline (PBS); The method further includes the step of staining (eg, fluorescently staining) the nuclei and / or cytoskeleton (actin, tubulin) of the reporter cells.

[0022] In some embodiments, the method for screening acetylcholinesterase inhibitors described in the present disclosure uses a high content cell imaging and analysis system to detect the activation degree of reporter cells M3:NFATc1-EGFP U2OS.

[0023] The present disclosure also relates to a kit comprising reporter cells M3:NFATc1-EGFP U2OS, acetylcholine (eg, acetylcholine iodide), acetylcholinesterase (eg, recombinant human acetylcholinesterase), and a fluorescent dye (eg, Hoechst 33342, phalloidin).

[0024] In some embodiments, the kit further comprises a medium suitable for growing the reporter cells M3:NFATc1-EGFP U2OS, for example, DMEM high glucose medium.

[0025] In some embodiments, the kit further comprises phosphate (PBS) buffer (either in aqueous or anhydrous form), and formaldehyde.

[0026] In some embodiments, the kit further comprises instructions describing methods for screening for acetylcholinesterase inhibitors described in this disclosure.

[0027] The present disclosure also relates to the use of the kit for screening for acetylcholinesterase inhibitors.

[0028] The present disclosure also relates to a system for screening acetylcholinesterase inhibitors, including a kit and a high-content cellular imaging and analysis system.

[0029] The reporter cell M3:NFATc1-EGFP U2OS described in this disclosure is a U2OS cell that stably expresses human muscarinic M3 receptor (muscarinic receptor 3, M3) and human NFATc1 (Nuclear Factor Of Activated T Cells 1) fused to the C-terminus of EGFP (enhanced green fluorescent protein). When M3 is activated by the agonist ACh, it increases intracellular Ca 2+ is released and upon increase in calcium levels, NFATc1 is dephosphorylated and rapidly translocates from the cytoplasm to the nucleus. Cells can be commercially available or constructed according to existing techniques.

[0030] In this disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory operation steps of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are common operation steps widely used in the corresponding fields.

[0031] Beneficial Effects of the Present Disclosure In this disclosure, a high-throughput screening method for AChE inhibitors based on ACh activity is established by using M3 receptor-activated reporter cells as a detection device and optimizing the rhAChE reaction system. This method has been successfully used to screen cholinesterase inhibitors of OP pesticides, nerve agents, and AD drugs, with a detection limit of 1 nM for paraoxon, 1 nM for donepezil, 0.1 nM for GD (soman), and 30 pM for VX. Compared with previous methods, this method can achieve high throughput, high sensitivity, and low consumption costs of time and reagents. In particular, this method can obtain phenotypic spectrum information on the cytotoxicity and cellular effects of the test substance in a single experiment, resulting in richer characteristic information for identifying potential drugs or toxic substances, and can provide a new and effective tool for screening a large number of neurotoxic compounds. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 shows a detection system of a method for screening AChEI established in an embodiment of the present disclosure. [Figure 2A] FIG. 1 shows that AChI concentration-dependently activates reporter cell lines and induces nuclear translocation of EGFP-NFATc1 in M3:NFATc1-EGFP U2OS human osteosarcoma cells. [Figure 2B] FIG. 1 shows that AChI concentration-dependently activates reporter cell lines and induces nuclear translocation of EGFP-NFATc1 in M3:NFATc1-EGFP U2OS human osteosarcoma cells. [Figure 3A] FIG. 1 shows the effect of incubation of different concentrations of rhAChE with AChI for 10 min on the nuclear translocation of EGFP-NFATc1. [Figure 3B] FIG. 1 shows the effect of incubation of 0.01 U / mL rhAChE with different concentrations of AChI for different times on the nuclear translocation of EGFP-NFATc1. [Figure 3C]FIG. 1 shows the effect of VX on the nuclear translocation of EGFP-NFATc1 in two reaction systems. [Figure 4A] FIG. 1 shows the detection results of organophosphorus pesticide AChEI using the method for screening AChEI established in an embodiment of the present disclosure, where A shows the detection result of paraoxon; B shows the detection result of dichlorvos; and C shows the detection result of chlorpyrifos. [Figure 4B] FIG. 1 shows the detection results of organophosphorus pesticide AChEI using the method for screening AChEI established in an embodiment of the present disclosure, where A shows the detection result of paraoxon; B shows the detection result of dichlorvos; and C shows the detection result of chlorpyrifos. [Figure 4C] FIG. 1 shows the detection results of organophosphorus pesticide AChEI using the method for screening AChEI established in an embodiment of the present disclosure, where A shows the detection result of paraoxon; B shows the detection result of dichlorvos; and C shows the detection result of chlorpyrifos. [Figure 5A] FIG. 1 shows the detection results of AD therapeutic drug AChEIs using the method for screening AChEIs established in an embodiment of the present disclosure, where A shows the detection results of donepezil; B shows the detection results of rivastigmine. [Figure 5B] FIG. 1 shows the detection results of AD therapeutic drug AChEIs using the method for screening AChEIs established in an embodiment of the present disclosure, where A shows the detection results of donepezil; B shows the detection results of rivastigmine. [Figure 6A] FIG. 1 shows the detection results of the nerve agent AChEI using the method for screening AChEI established in an embodiment of the present disclosure, where A represents the detection result of tabun; B represents the detection result of sarin; and C represents the detection result of soman. [Figure 6B] FIG. 1 shows the detection results of the nerve agent AChEI using the method for screening AChEI established in an embodiment of the present disclosure, where A represents the detection result of tabun; B represents the detection result of sarin; and C represents the detection result of soman. [Figure 6C]FIG. 1 shows the detection results of the nerve agent AChEI using the method for screening AChEI established in an embodiment of the present disclosure, where A represents the detection result of tabun; B represents the detection result of sarin; and C represents the detection result of soman. [Figure 7A] FIG. 1 shows the results of characterization and analysis of the cellular phenotype spectrum of AChEI using the method for screening AChEI established in an embodiment of the present disclosure. [Figure 7B] FIG. 1 shows the results of characterization and analysis of the cellular phenotype spectrum of AChEI using the method for screening AChEI established in an embodiment of the present disclosure. [Figure 7C] FIG. 1 shows the results of characterization and analysis of the cellular phenotype spectrum of AChEI using the method for screening AChEI established in an embodiment of the present disclosure. [Figure 7D] FIG. 1 shows the results of characterization and analysis of the cellular phenotype spectrum of AChEI using the method for screening AChEI established in an embodiment of the present disclosure. [Figure 7E] FIG. 1 shows the results of characterization and analysis of the cellular phenotype spectrum of AChEI using the method for screening AChEI established in an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] [Specific Model for Carrying Out the Invention] The technical solutions in some embodiments of the present disclosure are clearly and completely described below with reference to the drawings in the examples of the present disclosure. Of course, the described examples are only some of the examples of the present disclosure, not all of the examples. The following description of at least one exemplary embodiment is merely exemplary in nature and is not intended to limit the present disclosure, its application or use. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative efforts are included in the scope of protection of the present disclosure.

[0034] In all examples shown and discussed herein, any particular values ​​should be understood as exemplary only and not limiting, and thus other examples of the example embodiments may have different values. EXAMPLES

[0035] The present disclosure is described in detail below through specific examples.

[0036] Unless otherwise indicated, the molecular biological experimental methods and immunoassays used in this disclosure are carried out substantially according to the methods described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. If no specific conditions are shown in the examples, the conditions should be carried out according to conventional conditions or conditions recommended by the manufacturer. If no specific conditions are shown in the examples, the manufacturers of the reagents or instruments used were all conventional products that can be purchased commercially. Those skilled in the art will understand that the examples are for illustrative purposes only and are not intended to limit the scope of protection of the present disclosure.

[0037] The main drugs and reagents used in the examples of this disclosure include: DMEM high glucose culture medium, fetal bovine serum (FBS), G418 (geneticin, 50 mg / mL), penicillin / streptomycin mixed solution (100x), 0.05% trypsin-EDTA solution, 1x PBS phosphate buffer solution (pH=7.4), and 10x PBS phosphate buffer solution (pH=7.4), all purchased from Gibco; bovine serum albumin (BSA) purchased from Amresco; acetylcholine iodide (AChI) purchased from Selleck; recombinant human acetylcholinesterase (rhAChE) purchased from Sigma-Aldrich; Hoechst 33342 fluorescent dye purchased from Invitrogen; 633-labeled phalloidin purchased from Solarbio; mouse anti-human α-tubulin primary antibody purchased from Thermo; Alexa Flour purchased from Invitrogen. 546-labeled donkey anti-mouse secondary antibody; paraoxon, dichlorvos, and chlorpyrifos purchased from Merck; donepezil and rivastigmine purchased from Selleck; sarin, tabun, and soman and VX purchased from the Institute of Chemical Defense; and other reagents that were of analytical grade reagents.

[0038] The primary instrument used in some embodiments of the present disclosure is Perkin Elmer's Opera Phenix™ high content cellular imaging and analysis system.

[0039] Cell lines and culture conditions used in the examples of the present disclosure: M3:NFATc1-EGFP U2OS human osteosarcoma cell line (Thermo) was cultured in DMEM high glucose medium containing 10% FBS in a 5% CO2 incubator at 37° C. In addition, 0.5 mg / mL G418, 100 U / mL penicillin, and 100 μg / mL streptomycin were added to the culture medium.

[0040] Preparation of solutions used in the examples of this disclosure: 1. Recombinant human acetylcholinesterase (rhAChE) was prepared as a 1 U / mL stock solution in 10x PBS buffer (pH = 7.4) containing 0.1% BSA, and the stock solution was diluted to the desired working solution with serum-free culture medium during the experiment.

[0041] 2. Acetylcholine iodide (AChI) was prepared to a 5 mM stock solution in sterile deionized water, and the stock solution was diluted to the desired working solution with serum-free culture medium during the experiment.

[0042] 3. Paraoxon, dichlorvos, and chlorpyrifos were prepared in 500 μM stock solutions in methanol, and the stock solutions were diluted to the intended working solutions in serum-free medium during the experiments.

[0043] 4. Donepezil hydrochloride (donepezil) and rivastigmine tartrate (rivastigmine) were prepared into 5 mM stock solutions using sterile deionized water, and the stock solutions were diluted to the intended working solutions using serum-free medium during the experiment.

[0044] 5. Tabun (GA), sarin (GB), soman (GD), and VX were prepared in 0.1 M stock solutions in acetonitrile, and the stock solutions were diluted to the desired working solutions in serum-free medium during the experiments.

[0045] The detection system of the method for screening AChEI established in the embodiment of the present disclosure is shown in FIG. 1 and includes (1) a cholinergic receptor-based reporter cell line, (2) an AChE reaction system, and (3) a high content screening (HCS) detection system composed of (1) and (2); (1) is a cholinergic receptor cell line M3:NFATc1-EGFP U2OS, which is a U2OS cell stably expressing human muscarinic cholinergic receptor 3 (M3) and human NFATc1 fused to the C-terminus of EGFP (enhanced green fluorescent protein), and when M3 is activated by the agonist ACh, it can detect intracellular Ca 2+In the present disclosure, an optimized concentration of acetylcholine iodide (ACh) was used as an agonist; AChE was pretreated with AChEI, then ACh was added and incubated for a certain period of time, and the reporter cell line was treated with the resulting mixture, and the HCS system was used to quantitatively detect the activity of reporter cells after adding AChEI to the AChE reaction system, thereby determining the inhibitory activity of AChEI against AChE and its inherent cellular response characteristics.

[0046] Example 1: High-content analysis of activation of the M3:NFATc1-EGFP U2OS reporter cell line by ACh In this example, we investigated whether ACh could stably activate M3:EGFP-NFATc1 cells. AChI was selected as the ACh, and after ACh treatment of the reporter cell line, a high-content cell imaging analysis system was used to collect cell images, and the degree of nuclear translocation of EGFP-NFATc1 was quantitatively analyzed to determine the extent and concentration range of ACh-activated M3 receptor. The specific steps are as follows: 1×10 5A cell suspension of 100 cells / mL was prepared and seeded in a black clear-bottom 96-well culture plate at 100 μL / well, then cultured for 24 hours, the culture supernatant was discarded, the cells were washed twice with serum-free medium, and then AChI gradient solutions (diluted with serum-free culture medium, final concentrations were 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000, 30000, 100000nmol / L, respectively, 3 wells for each concentration) were added at 100 μL / well, and the cells were then placed in a 37°C, 5% CO2 incubator for 20 minutes. Then 12% formaldehyde in 1×PBS solution was added at 50 μL / well (final concentration of formaldehyde was 4%) to fix the cells in the dark at room temperature for 20 minutes. The cells were washed twice with 1x PBS solution, 200 μL / well of 1x PBS solution containing 1 μM Hoechst 33342 was added, and then the cells were incubated at 37°C in the dark for 30 min. The cells were washed twice with 1x PBS solution, and then 100 μL / well of 1x PBS was added. The cells were loaded into the instrument, and Alexa 488 (absorption wavelength: 495 nm, emission wavelength: 519 nm) and DAPI (absorption wavelength: 340 nm, emission wavelength: 488 nm) channels were selected, and images of the cells were collected using a 20x objective lens, with 9 fields per well set for detection. Harmony 4.9 software was used to quantitatively analyze the number of cell nuclei and the intracellular NFATc1-EGFP translocation from the cytoplasm to the nucleus, and the activation effect of ACh was expressed as the average nuclear translocation degree of NFATc1-EGFP in each cell in triplicate wells, more than 1,000 cells per well, and was calculated as EC 50 Values ​​were calculated using Prism 8.

[0047] The results of high-content analysis of intracellular EGFP-NFATc1 translocation after treating M3:NFATc1-EGFP U2OS with different concentrations of AChI are shown in Figure 2. The reporter cell line was activated in an AChI concentration-dependent manner in the range of 0.1-100 nM. When the concentration of AChI was 0.3 nM, the cells showed an activation response; when the concentration of AChI reached 1 nM, the nuclear translocation degree of EGFP-NFATc1 was about 40% of the maximum nuclear translocation degree; when the concentration of AChI reached 10 nM, the nuclear translocation degree of EGFP-NFATc1 approached the maximum nuclear translocation degree; when the concentration of AChI was 30 nM, the maximum nuclear translocation degree of EGFP-NFATc1 was reached. After using Prism 8.0 software to nonlinearly fit the log(agonist) vs. response to obtain a S-shaped curve, the analysis showed that the EC 50 The value was 1.76 ± 0.34 nM with a Z' factor of 0.685, demonstrating that the cholinergic receptor reporter cell assay model using AChI as an agonist is a sensitive and stable HTS method.

[0048] Example 2: Establishment of the AChE reaction system In this example, an AChE reaction system suitable for a reporter cell line was established. The effects of different substrate (AChI) concentrations, different enzyme (rhAChE) concentrations, and different enzyme-substrate reaction times on the reaction system were examined according to the quantitative analysis results of the activation of the reporter cell line. The specific steps are as follows: First, the gradient rhAChE working solution and the AChI working solution were combined with each other and mixed in a 96-well plate at a ratio of 1:1 (v / v), and reacted for 5, 10, 15, and 20 minutes, respectively, at 37° C. Then, the mixed solution was added to a black clear-bottom 96-well plate pre-seeded with the reporter cell line at 100 μL / well, and the cells were incubated for 20 minutes, and then the activation degree of the reporter cell line was quantitatively analyzed according to the method described in Example 1, and the Z' factor was calculated to obtain the optimal experimental system. The formula for calculating the Z' factor is:

[0049]

number

[0050] The value of the Z' factor should be between 0 and 1. When the Z' factor is 0, it indicates that the experimental system has not been established; when 0 < Z' factor < 0.5, it indicates that the experimental system has poor stability and low reliability; when 0.5 ≤ Z' factor < 1, it indicates that the experimental system has good stability and high reliability; when the Z' factor is 1, the standard deviations of the positive control and the negative control are 0, indicating that the experimental system is an ideal experimental system (Zhang, Chung, Oldenburg. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal of biomolecular screening, 1999, 4(2):67~73).

[0051] According to the concentration range of AChI that activates the reporter cell line, five points of AChI concentration were selected in the range of 10 to 1×10 5 nM, and the optimal content of AChE and reaction time in the AChE reaction system were examined under these conditions.

[0052] The results of reporter cell line activation by each reaction system when 0.001, 0.003, 0.01, and 0.02 U / mL rhAChE (these concentrations are the initial concentration of rhAChE in the AChE reaction system, i.e., the enzyme concentration in the system before reaction with AChI) was incubated with different concentrations of AChI for 10 min are shown in Figure 3A. When the enzyme content was too low (≦0.003 U / mL), AChI hydrolysis could not be carried out effectively; when the enzyme content was too high (0.02 U / mL), AChI hydrolysis was carried out excessively, and the difference between the two conditions in the reporter cell analysis was limited; under the condition of 0.01 U / mL rhAChE, AChI induced the nuclear translocation of EGFP-NFATc1 in a concentration-dependent manner in the range of 10 to 1000 nM. AChI in the above concentration range has almost 100% cell activation effect in an enzyme-free system, which suggests that the amount of substrate AChI hydrolyzed by 0.01U / mL rhAChE within 10 minutes can be quantified using a reporter cell line. Therefore, the appropriate initial concentration of rhAChE in the reaction system may be 0.005-0.018U / mL, preferably 0.007-0.015U / mL, and more preferably 0.01U / mL.

[0053] Considering the feasibility of antagonist screening experiments, the initial concentration of the substrate AChI in the AChE reaction system was set to 10, 30, 100, and 300 nM within the range showing concentration dependency, and the influence of the difference in incubation time of 0.01 U / mL enzyme and substrate on the results was examined. The results are shown in Figure 3B. At the lowest substrate concentration (10 nM), hydrolysis was completed within 5 minutes, and the feasibility of subsequent experiments was low. At the highest substrate concentration (300 nM), the remaining amount of substrate at each time point was too high and the difference was significant, so it lacked sufficient window and stability for subsequent experiments. On the other hand, in the following two cases, i.e., when the substrate at a concentration of 30 nM was incubated with the enzyme for 10 minutes and when the substrate at a concentration of 100 nM was incubated with the enzyme for 20 minutes, the hydrolysis of AChI was more than 90%, and the system basically reached stability. This condition not only met the requirement of the maximum window value for inhibitor screening, but also benefited the stability of the system and had high operability. Therefore, two reaction systems were previously determined to be preferable: 0.01 U / mL rhAChE and 30 nM ACh incubated for 10 min, and 0.01 U / mL rhAChE and 100 nM ACh incubated for 20 min.

[0054] Based on this, the above two screening systems were further evaluated using a known acetyl esterase inhibitor VX. The results are shown in Figure 3C, and in both reaction systems, VX can increase the activity of cholinergic receptors in reporter cells in a concentration-dependent manner, i.e., induce the nuclear translocation of EGFP-NFATc1 in reporter cells, with the lowest detection concentration being 0.1 nM; the Z' factors of the two methods composed of the two reaction systems were 0.716 and 0.614, respectively, i.e., both of the two reaction systems could sensitively and reliably reflect the inhibitory activity of VX against AChE when combined with a reporter cell line, where the Z' factor under the condition of 0.01 U / mL rhAChE incubated with 30 nM ACh for 10 minutes was higher, and therefore this system was selected as the optimal reaction system. The method for screening cholinesterase inhibitors (AChEI) composed of this system and a reporter cell line had high-throughput detection capability and relatively good stability and sensitivity.

[0055] Example 3: Evaluation of the inhibitory activity of different types of AChEI against AChE using the method for screening AChEI established in the present disclosure In this example, the method established in the present disclosure example was used to detect the inhibitory activity against AChE of organophosphorus pesticides commonly used in agricultural production, such as paraoxon, dichlorvos (DDVP) and chlorpyrifos, AD treatment drugs, such as donepezil and rivastigmine, and nerve agents, such as sarin (GB), tabun (GA), soman (GD) and VX. The specific steps are as follows: To 30 μL of rhAChE working solution in each well, 30 μL of AChEI with corresponding gradient concentration was added, and the mixed solution was incubated at 37° C. for 30 min to allow AChEI and rhAChE to fully interact with each other, and then 60 μL of AChI working solution was added and reacted at 37° C. for 10 min. Then, 100 μL of the reaction solution was pipetted and added to a black clear-bottom 96-well plate in which the reporter cell line was pre-seeded. After 20 min of incubation, the activation degree of the reporter cell line was quantitatively analyzed according to the method described in Example 1 to determine the inhibitory activity of AChEI against AChE.

[0056] The results of the inhibitory effects of organophosphorus pesticides on rhAChE activity are shown in Figure 4, where paraoxon, dichlorvos, and chlorpyrifos could all inhibit rhAChE in the cell detection system, reduce the hydrolysis of AChI in the system, and then activate the M3 receptor in the reporter cells to promote the nuclear translocation of EGFP-NFATc1. The lowest detectable concentrations of the three organophosphorus pesticides were 1 nM, 0.3 μM, and 3 μM, respectively, with IC 50 The values ​​were 4.75±0.05 nM, 0.93±0.04 μM, and 13.28±0.87 μM, respectively. The order of the three inhibitory activities was suggested to be paraoxon > dichlorvos > chlorpyrifos, which is consistent with the reported results, indicating that the method established in the examples of the present disclosure can be used to evaluate the acetylcholinesterase inhibitory activities of different OP pesticides.

[0057] The results of the inhibitory effect of donepezil and rivastigmine on rhAChE are shown in Figure 5. Both donepezil and rivastigmine can promote the nuclear translocation of EGFP-NFATc1 by inhibiting rhAChE in the cell detection system, and IC 50 The values ​​were 26.21±1.45 nM and 4.05±1.26 μM, respectively, and were essentially consistent with those reported in the literature.

[0058] The results of the inhibitory effects of OP nerve agents on rhAChE are shown in Figure 6 and Figure 3C. Tabun, sarin, soman and VX all significantly inhibited rhAChE in the cell detection system, promoted the nuclear translocation of EGFP-NFATc1 and increased the IC 50 The values ​​were 3.75±0.03nM (GA); 0.62±0.05nM (GB); 0.19±0.05nM (GD), and 0.19±0.03nM (VX). The minimum detectable concentration of VX reached 30pM. This result was consistent with the results from our laboratory using the Ellman method, where tabun was partially degraded.

[0059] Based on the evaluation results of the above three major categories of AChEIs, the method established in this disclosure is capable of quantitatively screening different types of AChEIs with high throughput, and the method has the characteristics of high sensitivity, high stability, simple operation, and low consumption.

[0060] Example 4: Application of the method for screening AChEI established in this disclosure in characterizing the phenotypic properties of AChEI cells In addition to screening AChEI and detecting the enzyme inhibition effect of AChEI, the method for screening AChEI established in the present disclosure can also characterize the phenotypic characteristics of cells used to detect AChEI, in combination with fluorescent staining of nuclei and cytoskeleton (actin, tubulin), which shows the information of the inherent cellular activity and toxicity of the test substance. The specific method is as follows: The treated cells were stained with Hoechst 33342 according to the method described in Example 1 and permeabilized with 200 μL of 1×PBS solution containing 0.3% Triton X-100 at 37° C. in the dark for 30 min; then, 50 μL / well of 633-labeled phalloidin was added and stained at 37° C. in the dark for 30 min. The supernatant was discarded. The cells were washed three times with 1×PBS solution, then, 200 μL of blocking solution (PBS solution containing 5% BSA) was added to each well, and the cells were blocked at room temperature for 1 h. The blocking solution was discarded, and 50 μL of mouse anti-human α-tubulin monoclonal antibody was added to each well as the primary antibody, and then, the cells were incubated at 37° C. in the dark for 1 h. The primary antibody was removed, the cells were washed three times with blocking solution, 50 μL of Alexa Flour 546-conjugated donkey anti-mouse IgG was added to each well as the secondary antibody, and the cells were incubated at room temperature in the dark for 30 min. The cells were washed twice with 1× PBS solution, and 100 μL of 1× PBS solution was added to the well. A high content cell imaging and analysis system was used to capture images of the cells and analyze the nuclear morphology, actin, and tubulin status.

[0061] Different exogenous substances have different targets, induce different cellular changes, and have different cellular phenotypic characteristics. There are many types of AChEIs with diverse structures and different biological effects. Research has shown that in addition to the specific target AChE, OPs also affect other targets, such as hundreds of enzymes, receptors, and other proteins, some of which may be particularly relevant to long-term CNS effects and developmental neurotoxicity due to chronic OP exposure, and AChE's interaction with these targets is becoming part of the cumulative risk assessment of OPs. The method for screening AChEIs established in this disclosure not only identifies AChEIs by detecting the nuclear translocation degree of EGFP-NFATc1, but also quantitatively detects the changes in cell and subcellular morphology, signal transduction pathways, and target proteins induced by the test molecule through matching fluorescent labeling in the same experiment, thereby obtaining the unique cellular phenotypic spectrum characteristics of the test molecule. This provides more abundant information for screening and identifying new AChEIs.

[0062] In this example, different types of AChEI (nerve agent VX, OP pesticide paraoxon, and AD drug donepezil) were used as representatives to detect the nuclear translocation of EGFP-NFATc1 to determine target-specific activity, and in the same analysis process, the changes in cell nucleus, actin, and tubulin were exemplarily and quantitatively analyzed through different fluorescent labels of channels. These not only reflect the rapid response characteristics of cell and subcellular morphology and cytoskeleton caused by the interference of exogenous substances, but can also be important indicators of toxicity and potential mechanisms. The fluorescence imaging and quantitative results of four parameters of cells treated with different types of AChEI are shown in Figure 7. As shown in the nuclear translocation images of EGFP-NFATc1, VX, paraoxon, and donepezil can all inhibit the activity of AChE in a concentration-dependent manner, but the range and slope of the concentration-activity curves were different. The slopes of VX and paraoxon were essentially the same, while the slope of donepezil was significantly smaller, suggesting that donepezil binds to AChE in a different way from VX and paraoxon (which was essentially consistent with literature reports). All three showed a concentration-dependent increase in cellular actin (actin) expression levels (Figures 7A and 7B), but the slopes of the curves were quite different; all three showed an increase in nuclear area (Figures 7A and 7C), but the concentration-effect curves were different; furthermore, the three showed obvious differences in intracellular tubulin distribution after treatment (Figure 7A), i.e., donepezil showed a concentration-dependent increase in tubulin distribution starting from 1 nM, while VX and paraoxon showed a slight decrease in cellular tubulin distribution as the concentration increased. These results showed that in addition to inhibiting AChE activity, the three also exhibited their own phenotypic change characteristics; VX and paraoxon are both OPs, and they had more similar cell phenotypic characteristics, which were quite different from the AD drug donepezil. Therefore, according to the purpose of screening, the phenotypic characteristics can be highlighted by extending the time points and changing the cell phenotype, and finally, the unique phenotypic spectrum of each test substance can be obtained, which can provide more basis for screening and identification.

[0063] Although the specific embodiments of the present disclosure have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the details based on all the teachings disclosed, and these modifications are within the scope of protection of the present disclosure. The entirety of the present disclosure can be obtained from the appended claims and their equivalents.

[0064] This application claims the benefit of priority from Chinese Application No. 202210059880.4, filed on January 19, 2022, the disclosure of which is incorporated herein by reference in its entirety.

Claims

1. 1. A method for screening for an acetylcholinesterase inhibitor, comprising: 1) mixing a sample to be screened with acetylcholinesterase; 2) mixing the mixture obtained in step 1) with acetylcholine; 3) contacting the reporter cell M3:NFATc1-EGFP U2OS with the mixture obtained in step 2); 4) Detecting the activation degree of the reporter cell M3:NFATc1-EGFP U2OS A method comprising:

2. The activation degree of the reporter cell M3:NFATc1-EGFP U2OS is expressed as the translocation degree of NFATc1-EGFP from the cytoplasm to the nucleus of the reporter cell; When the NFATc1-EGFP in the reporter cell is translocated from the cytoplasm to the nucleus, the sample to be screened is determined to contain an acetylcholinesterase inhibitor; The method according to claim 1, wherein the inhibitory activity of an acetylcholinesterase inhibitor against acetylcholinesterase is preferably quantified according to the number of intracellular NFATc1-EGFP translocated from the cytoplasm to the nucleus.

3. 3. The method of claim 1 or 2, wherein the acetylcholine is acetylcholine iodide and the acetylcholinesterase is recombinant human acetylcholinesterase.

4. In step 1), the sample to be screened is mixed with the acetylcholinesterase in a first solvent; In step 2), the mixture obtained in step 1) is mixed with the acetylcholine in a second solvent; The first solvent and the second solvent are the same or different, and each independently is a medium suitable for normal growth of the reporter cell M3:NFATc1-EGFP U2OS, for example, DMEM high glucose medium; Preferably, in step 2), after mixing the mixture obtained in step 1) with acetylcholine, the initial concentration of acetylcholinesterase in the obtained mixture is 0.005 to 0.018 U / mL, preferably 0.007 to 0.015 U / mL, more preferably 0.01 U / mL; The method according to claim 1 or 2, wherein, in step 2), after the mixed system obtained in step 1) is mixed with acetylcholine, the initial concentration of acetylcholine in the obtained mixed system is 10 to 1000 nM, preferably 30 to 800 nM, more preferably 30 to 500 nM, more preferably 30 to 400 nM, more preferably 30 to 300 nM, more preferably 30 to 200 nM, and more preferably 30 to 100 nM.

4. Preferably, in step 1), the sample to be screened is mixed with the acetylcholinesterase at a temperature of 25 to 40° C. (e.g., 30° C., 32° C., 35° C., 37° C.) for 10 to 60 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or 50 minutes; The method according to any one of claims 1 to 3, wherein in step 2), the mixture obtained in step 1) is mixed with the acetylcholine at a temperature of 25 to 40°C (e.g., 30°C, 32°C, 35°C, 37°C) for 10 to 60 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 50 minutes.

5. The method according to any one of claims 1 to 4, wherein in step 3), the reporter cells M3:NFATc1-EGFP U2OS are contacted with the mixture obtained in step 2) for 10 to 60 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or 50 minutes.

6. Before step 4), Fixing the reporter cells with a formaldehyde solution diluted in a phosphate buffer; Staining (e.g., fluorescent staining) the nucleus and / or cytoskeleton (actin, tubulin) of the reporter cells. The method of any one of claims 1 to 5, further comprising:

7. The method according to any one of claims 1 to 6, wherein a high content cell imaging and analysis system is used to detect the activation degree of the reporter cell M3:NFATc1-EGFP U2OS.

8. Reporter cell M3: containing NFATc1-EGFP U2OS, acetylcholine (e.g., acetylcholine iodide), acetylcholinesterase (e.g., recombinant human acetylcholinesterase) and a fluorescent dye (e.g., Hoechst 33342, phalloidin), Preferably, the reporter cell M3:NFATc1-EGFP U2OS further comprises a medium suitable for growth, for example, a DMEM high glucose medium; Preferably, it further comprises a phosphate buffer substance (in aqueous or anhydrous form), and formaldehyde, Optionally, the kit further comprises instructions describing the method according to any one of claims 1 to 7.

9. Use of the kit according to claim 8 for screening acetylcholinesterase inhibitors.

10. A system for screening acetylcholinesterase inhibitors, comprising the kit of claim 8 and a high-content cellular imaging and analysis system.

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