WO2023138481A1 - 一种筛查乙酰胆碱酯酶抑制剂的方法 - Google Patents

一种筛查乙酰胆碱酯酶抑制剂的方法 Download PDF

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WO2023138481A1
WO2023138481A1 PCT/CN2023/071987 CN2023071987W WO2023138481A1 WO 2023138481 A1 WO2023138481 A1 WO 2023138481A1 CN 2023071987 W CN2023071987 W CN 2023071987W WO 2023138481 A1 WO2023138481 A1 WO 2023138481A1
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acetylcholinesterase
nfatc1
egfp
mixed
acetylcholine
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French (fr)
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马波
王莉莉
李治
郭磊
徐华
谢剑炜
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中国人民解放军军事科学院军事医学研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C12Q1/46—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase involving cholinesterase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the disclosure belongs to the fields of food, environment, pharmacy and medicine, and specifically relates to a method for screening acetylcholinesterase inhibitors, and also relates to a kit and system for screening acetylcholinesterase inhibitors.
  • Acetylcholinesterase also known as true cholinesterase, or AChE for short, is a key enzyme in the process of biological nerve transmission.
  • AChE can degrade acetylcholine (ACh) between cholinergic nerve synapses, terminate the excitatory effect of neurotransmitters on postsynaptic membranes, and ensure the normal transmission of nerve signals in organisms.
  • organophosphate (OPs) neurotoxins, carbamate (CM) pesticides, and Alzheimer's disease (AD) therapeutic drugs all target AChE, which induce neurotoxic effects or improve cognitive function in AD by inhibiting normal and pathologically elevated AChE activity, respectively.
  • OPs are the largest class of AChE inhibitors (AChEIs), and related compounds are currently found in hundreds of products used worldwide, including insecticides, defoliants, flame retardants, industrial solvents, lubricants, plasticizers, fuel additives, neurotoxic chemical warfare agents, and the AD prescription drug metrifolipin. Thanks to the great advantages of pesticides and other pesticides in improving agricultural productivity and controlling deadly vector-borne diseases, and with the rapid development of industrialization, OPs substances are widely used. According to statistics, about 1 billion pounds of OPs are released into the environment, food and water supply globally every year, making OPs one of the most common synthetic chemicals that can be detected in the environment as well as in animal and human tissues.
  • AChE inhibitors ACChE inhibitors
  • AChEI screening methods there are mainly three types of commonly used AChEI screening methods, namely, the determination based on the known OP structure, the analysis based on the inhibition of AChE activity, and the immunoassay based on the immunogenicity of OP. Due to the limitation of preparing antibodies with strong affinity and high specificity, immunoassay methods not only face challenges in successfully detecting low-level OPs, but also fail to distinguish OPs with different structures.
  • the analysis based on chemical structure mainly adopts 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), with high sensitivity (detection limit in the nanomolar concentration range), are the preferred methods for trace quantitative analysis, but this method is only suitable for known molecules, low throughput, not suitable for large-scale screening, and the technology is time-consuming and expensive, and must be performed by trained technicians.
  • the enzyme activity inhibition method is based on the inhibition of AChE, and AChEI can be detected by measuring the enzyme activity before and after exposure to the test sample.
  • Enzyme activity is typically measured by Ellman colorimetric assays, radiometric assays, fluorometric/electrochemical assays, or chemiluminescent assays. These methods are low-cost, high-throughput, and fast in analysis, and are the most promising alternatives to classical methods (GC/MS, LC/MS), however, the method is not sensitive to changes in enzyme activity after exposure to some OPs compounds, which also severely limits the detection sensitivity of these methods. In addition, from the perspective of efficient screening of AChEI, the above-mentioned methods can only provide a single characterization of the chemical to be tested at the known structure or enzyme molecular level, and cannot provide biological characterization of potentially toxic molecules, multi-dimensional information for accurate early warning and efficient treatment guidance.
  • the present disclosure relates to a method of screening for acetylcholinesterase inhibitors, comprising:
  • step 2) mixing the mixed system obtained in step 1) with acetylcholine;
  • the activation degree of the reporter cell M3:NFATc1-EGFP U2OS is expressed as the degree of translocation of NFATc1-EGFP in the reporter cell from the cytoplasm to the nucleus. If it is reported that intracellular NFATc1-EGFP translocates from the cytoplasm to the nucleus, it is judged that the sample to be screened contains an acetylcholinesterase inhibitor. The inhibitory activity of the acetylcholinesterase inhibitor on acetylcholinesterase can also be quantified according to the number of intracellular NFATc1-EGFP translocated from the cytoplasm to the nucleus.
  • the samples to be screened in the present disclosure may be chemicals, food (such as various meats, vegetables, tea, etc.), organophosphorus pesticides in the environment, AD treatment drugs, nerve agents, etc.
  • the acetylcholine is iodoacetylcholine
  • the acetylcholinesterase is recombinant human acetylcholinesterase.
  • step 1) of the method for screening for acetylcholinesterase inhibitors described in the present disclosure the sample to be screened is mixed with acetylcholinesterase in a first solvent.
  • step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure the mixed system obtained in step 1) is mixed with acetylcholine in a second solvent.
  • the first solvent and the second solvent are the same or different, each independently being a medium suitable for the normal growth of the reporter cell M3:NFATc1-EGFP U2OS, such as a DMEM high-glucose medium.
  • 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 (concentration when not reacting with acetylcholine) is 0.005-0.018 U/mL, preferably 0.007-0.015 U/mL, more preferably 0.01 U/mL.
  • 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 10-1000 nM, preferably 30-800 nM, more preferably 30-500 nM, further preferably 30-400 nM, further preferably 30-30 0 nM, more preferably 30 to 200 nM, still more preferably 30 to 100 nM.
  • step 1) of the method for screening acetylcholinesterase inhibitors described in the present disclosure the sample to be screened is mixed with acetylcholinesterase at a temperature of 25-40°C (such as 30°C, 32°C, 35°C, 37°C) for 10-60 minutes, such as 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 50 minutes.
  • step 2) of the method for screening acetylcholinesterase inhibitors described in the present disclosure the mixed system obtained in step 1) is mixed with acetylcholine at a temperature of 25-40°C (such as 30°C, 32°C, 35°C, 37°C) for 10-60min, such as 20min, 25min, 30min, 35min, 40min, 50min.
  • 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 (concentration when not reacting with acetylcholine) is 0.005-0.018 U/mL, and the initial concentration of acetylcholine in the obtained mixed system is 10-1000 nM (for example, 30-800 nM, 30-50 0nM, 30-400nM, 30-300nM, 30-200nM, or 30-100nM).
  • 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 (concentration when not reacting with acetylcholine) is 0.007-0.015 U/mL, and the initial concentration of acetylcholine in the obtained mixed system is 10-1000 nM (for example, 30-800 nM, 30-50 0nM, 30-400nM, 30-300nM, 30-200nM, or 30-100nM).
  • 10-1000 nM for example, 30-800 nM, 30-50 0nM, 30-400nM, 30-300nM, 30-200nM, or 30-100nM.
  • 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 (concentration when not reacting with acetylcholine) is 0.01 U/mL, and the initial concentration of acetylcholine in the obtained mixed system is 10-1000 nM (eg, 30-800 nM, 30-500 nM, 30-400 nM, 30-300 nM, 30-200 nM, or 30-100 nM).
  • 10-1000 nM eg, 30-800 nM, 30-500 nM, 30-400 nM, 30-300 nM, 30-200 nM, or 30-100 nM.
  • 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, and the initial concentration of acetylcholine in the obtained mixed system is 30 nM.
  • the mixed system obtained in step 1) is mixed with acetylcholine for 10 min.
  • 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, and the initial concentration of acetylcholine in the obtained mixed system is 100 nM.
  • the mixed system obtained in step 1) is mixed with acetylcholine for 20 min.
  • step 3) of the method for screening acetylcholinesterase inhibitors described in the present disclosure the reporter cell M3:NFATc1-EGFP U2OS is in contact with the mixed system obtained in step 2) for 10-60 min, such as 20 min, 25 min, 30 min, 35 min, 40 min, 50 min.
  • step 4 of the method for screening acetylcholinesterase inhibitors described in the present disclosure, further comprising:
  • Stain eg, fluorescent stain
  • the nucleus and/or cytoskeleton actin filaments, microtubules
  • the method for screening acetylcholinesterase inhibitors described in the present disclosure uses a high-content cell imaging analysis system to detect the degree of activation of the reporter cell M3:NFATc1-EGFP U2OS.
  • the present disclosure also relates to a kit comprising reporter cells M3:NFATc1-EGFP U2OS, acetylcholine (such as iodoacetylcholine), acetylcholinesterase (such as recombinant human acetylcholinesterase) and fluorescent dyes (such as Hoechst 33342, phalloidin).
  • acetylcholine such as iodoacetylcholine
  • acetylcholinesterase such as recombinant human acetylcholinesterase
  • fluorescent dyes such as Hoechst 33342, phalloidin
  • the kit also includes a medium suitable for the growth of the reporter cell M3:NFATc1-EGFP U2OS, such as DMEM high glucose medium.
  • a medium suitable for the growth of the reporter cell M3:NFATc1-EGFP U2OS such as DMEM high glucose medium.
  • the kit further includes a phosphate saline (PBS) buffer substance (in aqueous or anhydrous form), formaldehyde.
  • PBS phosphate saline
  • the kit further includes instructions describing the method of screening for acetylcholinesterase inhibitors described in the present disclosure.
  • the present disclosure also relates to the use of the kit for screening acetylcholinesterase inhibitors.
  • the present disclosure also relates to a system for screening acetylcholinesterase inhibitors, including the kit and a high-content cell imaging analysis system.
  • the reporter cell M3:NFATc1-EGFP U2OS described in this disclosure is a U2OS cell stably expressing human muscarinic M3 receptor (muscarinic receptor3, M3) and human NFATc1 (Nuclear Factor Of Activated T Cells 1) fused to the C - terminus of enhanced green fluorescent protein (EGFP). Translocates rapidly from the cytoplasm to the nucleus.
  • the cells are commercially available or can be constructed according to existing techniques.
  • a method for high-throughput screening of AChE inhibitors based on ACh activity is constructed by using M3 receptor-activated reporter cells as detectors and optimizing the rhAChE reaction system.
  • the method was successfully used in the screening of OPs pesticides, nerve agents and AD drug cholinesterase inhibitors.
  • the detection limit of paraoxon was 1nM
  • the detection limit of donepezil was 1nM
  • the detection limit of GD (soman) was 0.1nM
  • the detection limit of VX was 30pM.
  • this method can achieve high throughput, high sensitivity, and low cost of time and reagent consumption.
  • this method can obtain the phenotypic profile information associated with the analyte, cytotoxicity and cellular effects in a single experiment, provide more abundant characteristic information for the identification of potential drugs or toxic substances, and provide a new and efficient tool for the screening of a large number of neurotoxic compounds.
  • Fig. 1 shows the detection system of the method for screening AChEI constructed by the embodiment of the present disclosure
  • Figure 2 shows that iodo-ACh concentration-dependently activates the reporter cell line and induces nuclear translocation of EGFP-NFATc1 in M3:NFATc1-EGFP U2OS human osteosarcoma cells;
  • Figure 3A shows the effect of different concentrations of rhAChE and iodo-ACh incubated for 10 min on the nuclear translocation of EGFP-NFATc1;
  • Figure 3B shows the effect of 0.01U/mL rhACh enzyme and different concentrations of ACh incubated for different times on EGFP-NFATc1 nuclear translocation;
  • Figure 3C shows the effect of VX on EGFP-NFATc1 nuclear translocation in two reaction systems
  • Fig. 4 shows that the method for screening AChEI constructed by the embodiment of the present disclosure is used for the detection of organophosphorus pesticide class AChEI.
  • Test results where A is the test result of paraoxon; B is the test result of dichlorvos; C is the test result of chlorpyrifos;
  • Fig. 5 shows the test result of AD treatment drug class AChEI using the method for screening AChEI constructed by the embodiment of the present disclosure, wherein A is the test result of donepezil; B is the test result of rivastigmine;
  • Figure 6 shows the test results of the nerve agent AChEI using the method for screening AChEI constructed by the embodiments of the present disclosure, wherein A is the test result of tabun; B is the test result of sarin; C is the test result of soman;
  • Fig. 7 shows the results of characterization and analysis of the AChEI cell phenotype profile by applying the method for screening AChEI constructed in the embodiments of the present disclosure.
  • the main instrument used in the embodiments of the present disclosure Perkin Elmer Opera Phenix TM high-content cell imaging analysis system.
  • M3:NFATc1-EGFP U2OS human osteosarcoma cell line (Thermo Company) was cultured in DMEM high-glucose medium containing 10% FBS at 37° C. in a 5% CO 2 incubator.
  • DMEM high-glucose medium containing 10% FBS at 37° C. in a 5% CO 2 incubator.
  • 0.5 mg/mL G418, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin were added to the culture solution.
  • Acetylcholine iodide (ACh) was prepared as a 5 mM stock solution with sterile deionized water, and diluted with a serum-free medium during the experiment to be the target working solution.
  • Paraoxon, dichlorvos, and chlorpyrifos were prepared as 500 ⁇ M stock solution with methanol, and diluted with serum-free medium to be the target working solution during the experiment.
  • Donepezil hydrochloride Donepezil
  • rivastigmine tartrate rivastigmine tartrate
  • the detection system of the method for screening AChEI constructed in the embodiment of the present disclosure is shown in Figure 1, including 1 a reporter cell line based on cholinergic receptors; 2 AChE reaction system, and a high content screening (High Content Screening, HCS) detection system 3 composed of 1 and 2.
  • 1 is the reporter cell line of cholinergic receptor M3:NFATc1-EGFP U2OS, which is a U2OS cell that stably expresses human muscarinic choline receptor 3 (M3) and human NFATc1 fused to the C-terminus of enhanced green fluorescent protein (EGFP).
  • M3 When M3 is activated by the agonist ACh, intracellular Ca 2+ is released, and the increase in calcium level leads to dephosphorylation of NFATc1 and rapid translocation from the cytoplasm to the nucleus, whereby the activity of the agonist can be quantitatively analyzed;
  • It is a detection system containing recombinant human AChE (rhAChE) and ACh and reporter cell lines with optimized and determined content;
  • 3 is to detect AChEI using the optimized HCS detection system.
  • the present disclosure uses acetylcholine iodide (ACh) at an optimized concentration as an agonist; pre-treats AChE with AChEI, then adds ACh to incubate for a certain period of time to treat the reporter cell line, and uses the HCS system to quantitatively measure the activity of the reporter cells after AChEI is added to the AChE reaction system, and thus determines the inhibitory activity of AChEI on AChE and its unique cell response characteristics.
  • ACh acetylcholine iodide
  • This embodiment investigates whether ACh can stably activate M3:EGFP-NFATc1 cells, select iodo ACh, and adopt A high-content cell imaging analysis system was used to collect the cell images of the reporter cell line treated with ACh, and the degree and concentration range of ACh to activate the M3 receptor were determined by quantitatively analyzing the degree of EGFP-NFATc1 nuclear translocation. Specific steps are as follows:
  • an AChE reaction system suitable for the reporter cell line was constructed. Based on the results of the quantitative analysis of the activation of the reporter cell line, the effects of different substrate (iodo-ACh) concentrations, different enzyme (rhAChE) concentrations, and different enzyme-substrate reaction times on the reaction system were investigated. Specific steps are as follows:
  • ⁇ is the standard deviation
  • ⁇ is the mean signal
  • c+ is the positive control
  • c- is the negative control.
  • Z' factor should be between 0 and 1.
  • Z'factor When the Z'factor is 0, it indicates that the experimental system is not established; when 0 ⁇ Z'factor ⁇ 0.5, it indicates that the stability of the system is poor and unreliable; when 0.5 ⁇ Z'factor ⁇ 1, it indicates that the experimental system is stable and reliable; if the Z'factor is 1, it indicates that the standard deviation between the positive control and the negative control is 0, which 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).
  • concentration range of iodo-ACh activation reporter cell line five iodo-ACh concentration points were selected in the range of 10-1 ⁇ 10 5 nM, and the optimal AChE content and reaction time in the AChE reaction system were investigated under these conditions.
  • the concentration is the initial concentration of rhAChE in the AChE reaction system, that is, the enzyme concentration in the system when it does not react with iodo-Ach
  • rhAChE were incubated with different concentrations of iodo-ACh for 10 minutes, the results of the activation of the reporter cell lines by each reaction system are shown in Figure 2A.
  • Too low enzyme content ( ⁇ 0.003U/mL) cannot effectively hydrolyze iodo-ACh, while Excessively high enzyme content (0.02U/mL) resulted in excessive hydrolysis of iodo-ACh, and the difference between the two conditions was limited in reporter cell analysis; under the condition of 0.01U/mL rhAChE, iodo-ACh showed a concentration-dependent induction of EGFP-NFATc1 nuclear translocation, and the concentration range was 10-1000nM.
  • the iodo-ACh in the above concentration range has nearly 100% cell activation effect in the enzyme-free system, suggesting that within 10 minutes, the amount of iodo-ACh hydrolyzed by 0.01 U/mL rhAChE can be quantitatively expressed by the reporter cell line. Therefore, the suitable initial concentration of rhAChE in the reaction system may be 0.005-0.018 U/mL, preferably 0.007-0.015 U/mL, and more preferably 0.01 U/mL.
  • the initial concentrations of iodo-ACh substrates in the AChE reaction system were set to 10, 30, 100, and 300 nM within a concentration-dependent range, and the effects of different incubation times of 0.01 U/mL enzyme and substrate on the results were investigated.
  • Results As shown in Figure 2b, under the minimum concentration of the substrate (10nm), 5min hydrolysis is completed, and the feasibility of subsequent experiments is low; at the highest concentration (300nm), the substrate residual amount of the substrates is too high and the differences are significantly different. The subsequent experiments lack sufficient windows and stability. When the substrate concentration is 30nm, the enzyme incubation is 10min, and the concentration of the substrate is.
  • iodine ACH hydrolysis is greater than 90 %, and the system has basically reached stability. This situation not only satisfies the requirement of the maximum window value for inhibitor screening, but is also beneficial to the stability of the system and has strong operability.
  • VX can increase the activity of the cholinergic receptors of the reporter cells in a concentration-dependent manner, that is, induce the nuclear translocation of EGFP-NFATc1 in the reporter cells, and the minimum detection concentration is 0.1 nM;
  • the Z' factors of the two reaction system composition methods are 0.716 and 0.614, respectively, that is, the combination of the two reaction systems with the reporter cell lines can sensitively and reliably reflect the inhibitory activity of VX on AChE.
  • the Z'factor was higher when incubated with 0nM ACh for 10min. Therefore, this system was selected as the best reaction system.
  • the method for screening cholinesterase inhibitors (AChEI) consisting of this system and reporter cell line has high-throughput testing capabilities, and has good stability and sensitivity.
  • Example 3 The method for screening AChEI constructed in the present disclosure evaluates the inhibitory activity of different types of AChEI on AChE
  • This example uses the method established in this disclosure to determine the inhibitory activity of organophosphorus pesticides paraoxon (Paraoxon), dichlorvos (DDVP), chlorpyrifos (Chlopyrifos), AD treatment drugs Donepezil, rivastigmine, nerve agents sarin (GB), tabun (GA), soman (GD) and VX on AChE commonly used in agricultural production. Specific steps are as follows:
  • the evaluation results of the above three types of AChEI show that the method established in the present disclosure can screen different types of AChEI through-through, and has the characteristics of high sensitivity, strong stability, convenient operation, and low consumption.
  • Example 4 Application of the method for screening AChEI constructed in the present disclosure in characterizing the phenotypic characteristics of AChEI cells
  • the method for screening AChEI constructed in the present disclosure is not only used for screening AChEI and detecting the enzyme inhibitory effect of AChEI, but also can combine the fluorescent staining of the nucleus and cytoskeleton (microfilaments, microtubules) to characterize the cell phenotype characteristics of the measured AChEI, and prompt the specific cell activity and toxicity information of the analyte.
  • the specific method is as follows:
  • AChEI has various types and structures, and its biological effects are not the same. Studies have 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 chronic OP exposure and long-term CNS effects of developmental neurotoxicity, and the interaction of AChE with these targets is also becoming part of the cumulative risk assessment of OPs.
  • the method for screening AChEI constructed in the present disclosure can not only detect and identify AChEI through the degree of EGFP-NFATc1 nuclear translocation, but also can quantitatively detect the changes in cells, subcellular morphology, signaling pathways and target proteins induced by the molecule to be tested in the same experiment through compatible fluorescent labels, and obtain the unique cell phenotype spectrum characteristics of the molecule to be tested. This provides richer information for screening and identifying new AChEIs.
  • 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 are different.
  • the slopes of VX and paraoxon were basically the same, and the slope of donepezil was significantly smaller, suggesting that the way donepezil binds to AChE is different from that of VX and paraoxon (basically consistent with literature reports).
  • the three showed their own phenotypic changes; VX and paraoxon, which are both OPs, had more similar cell phenotype characteristics, but they were quite different from the AD drug donepezil. Accordingly, according to the screening purpose, the phenotypic characteristics can be enriched by expanding time points and different cell phenotypes, and finally obtain the unique phenotype spectrum of each analyte, providing more basis for screening and identification.

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Abstract

本公开涉及一种筛查乙酰胆碱酯酶抑制剂的方法，还涉及一种用于筛查乙酰胆碱酯酶抑制剂的试剂盒及系统。

Description

一种筛查乙酰胆碱酯酶抑制剂的方法
本申请是以CN申请号为202210059880.4，申请日为2022年1月19日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本公开属于食品、环境、药学与医学领域，具体涉及一种筛查乙酰胆碱酯酶抑制剂的方法，还涉及一种用于筛查乙酰胆碱酯酶抑制剂的试剂盒及系统。
背景技术
乙酰胆碱酯酶，又称真性胆碱酯酶，简称AChE，是生物神经传导过程中的一种关键性酶。AChE在胆碱能神经突触间能降解乙酰胆碱(ACh)，终止神经递质对突触后膜的兴奋作用，保证神经信号在生物体内的正常传递。目前已知的有机磷酸盐类(OPs)神经毒物质、氨基甲酸酯(CM)农药和阿尔茨海默病(AD)治疗药物都是以AChE为重要靶点的，它们分别通过抑制正常和病理性升高的AChE活性诱发神经毒效应或改善AD的认知功能。OPs是最大的一类AChE抑制剂(AChEI)，目前在全世界使用的数百种产品中都发现了相关化合物，包括杀虫剂、落叶剂、阻燃剂、工业溶剂、润滑剂、增塑剂、燃料添加剂、神经毒类化学战剂以及AD处方药美曲磷脂。得益于杀虫剂等农药在提高农业生产力和控制致命病媒传播疾病的巨大优势，并且伴随快速的工业化的发展，OPs类物质被广泛使用。据统计，每年全球约有10亿磅的OPs被释放到环境、食品和水供应中，使OPs已成为能在环境以及动物和人体组织中检测到的最常见的合成化学品之一。由此带来日益严重的环境、生态问题，对人类健康和公共安全构成巨大威胁。另外，面对日益严峻的全球人口老龄化问题和远未满足的AD治疗需求，作为确证的AD治疗药物，新型AChEI(非有机磷类)仍然是AD药物研发的重点。因此，筛查和精准表征已知和潜在的AChEI(包括有机磷类神经毒剂)对于环境、食品安全的预警、潜在毒性物质的认识、中毒的精准救治以及更高效AD新药的研究都具有重要的意义。
迄今为止，常用的AChEI的筛查方法主要有三类，即基于已知OP结构的测定、基于AChE活性抑制的分析以及基于OP免疫原性的免疫分析。由于制备强亲和性和高特异性抗体受限，免疫分析方法不仅在成功检测低水平的OPs面临挑战，也无法区别不同结构的OPs。基于化学结构的分析主要采用色谱法，如液相色谱(LC)、液相色谱-质谱(LC-MS)、 液相色谱-串联质谱(LC-MS/MS)、气相色谱(GC)和气相色谱-质谱(GC-MS)，灵敏度高(纳摩尔浓度范围内的检测限)，是痕量定量分析的首选方法，但该方法仅适合已知分子，通量低、不适合大规模筛查，且技术耗时、设备昂贵，须由训练有素的技术人员执行。酶活性抑制法是基于AChE被抑制，通过测定暴露于待测样品之前和之后的酶活性可以检测AChEI。酶活性通常通过埃尔曼比色分析法、放射性分析法、荧光分析法\电化学分析法、或化学发光法进行测量。这些方法成本低、通量高、分析速度快，是经典方法(GC/MS、LC/MS)的最有希望的替代方法，然而，该方法对一些OPs化合物暴露后酶活性的变化不敏感，也严重限制了这些方法的检测灵敏度。此外，从高效筛查AChEI的角度来看，上述各种方法仅能在已知结构或酶分子水平上对待测化学品提供单一表征，无法提供潜在有毒分子的生物学表征、为准确预警和高效救治的指导提供多维信息。
公开内容
本公开涉及一种筛查乙酰胆碱酯酶抑制剂的方法，包括：
1)使待筛查样品与乙酰胆碱酯酶混合；
2)使步骤1)中所得混合体系与乙酰胆碱混合；
3)使报告细胞M3:NFATc1-EGFP U2OS与步骤2)所得混合体系接触；
4)检测报告细胞M3:NFATc1-EGFP U2OS被激活的程度。
在某些实施方案中，所述报告细胞M3:NFATc1-EGFP U2OS的激活程度表示为报告细胞内NFATc1-EGFP从胞质转位至细胞核的程度。若报告细胞内NFATc1-EGFP从胞质转位至细胞核，判断待筛查样品中含有乙酰胆碱酯酶抑制剂。还可以根据细胞内NFATc1-EGFP从胞质转位至细胞核的数目对乙酰胆碱酯酶抑制剂对于乙酰胆碱酯酶的抑制活性进行定量。
在某些实施方案中，本公开所述待筛查样品可以为化学品、食品(例如各种肉类、蔬菜、茶叶等)、环境中的有机磷农药、AD治疗药物、神经毒剂等。
在某些实施方案中，所述乙酰胆碱为碘代乙酰胆碱，所述乙酰胆碱酯酶为重组人乙酰胆碱酯酶。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤1)中，使待筛查样品与乙酰胆碱酯酶在第一溶剂中混合。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤2)中，使步骤1)中所得混合体系与乙酰胆碱在第二溶剂中混合。
在某些实施方案中，所述第一溶剂、第二溶剂相同或不同，各自独立地为适合报告细胞M3:NFATc1-EGFP U2OS正常生长的培养基，例如DMEM高糖培养基。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤2)中，步骤1)中所得混合体系与乙酰胆碱混合后，乙酰胆碱酯酶在所得混合体系中的初始浓度(未与乙酰胆碱反应时的浓度)为0.005～0.018U/mL，优选为0.007～0.015U/mL，进一步优选为0.01U/mL。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤2)中，步骤1)中所得混合体系与乙酰胆碱混合后，乙酰胆碱在所得混合体系中的初始浓度(未与乙酰胆碱酯酶反应时的浓度)为10～1000nM，优选为30～800nM，进一步优选为30～500nM，进一步优选为30～400nM，进一步优选为30～300nM，进一步优选为30～200nM，进一步优选为30～100nM。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤1)中，待筛查样品与乙酰胆碱酯酶在25～40℃(例如30℃，32℃，35℃，37℃)的温度条件下混合10～60min，例如20min，25min，30min，35min，40min，50min。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤2)中，步骤1)中所得混合体系与乙酰胆碱在25～40℃(例如30℃，32℃，35℃，37℃)的温度条件下混合10～60min，例如20min，25min，30min，35min，40min，50min。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤2)中，步骤1)中所得混合体系与乙酰胆碱混合后，乙酰胆碱酯酶在所得混合体系中的初始浓度(未与乙酰胆碱反应时的浓度)为0.005～0.018U/mL，乙酰胆碱在所得混合体系中的初始浓度为10～1000nM(例如30～800nM，30～500nM，30～400nM，30～300nM，30～200nM，或30～100nM)。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤2)中，步骤1)中所得混合体系与乙酰胆碱混合后，乙酰胆碱酯酶在所得混合体系中的初始浓度(未与乙酰胆碱反应时的浓度)为0.007～0.015U/mL，乙酰胆碱在所得混合体系中的初始浓度为10～1000nM(例如30～800nM，30～500nM，30～400nM，30～300nM，30～200nM，或30～100nM)。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤2)中，步骤1)中所得混合体系与乙酰胆碱混合后，乙酰胆碱酯酶在所得混合体系中的初始浓度(未与乙酰胆碱反应时的浓度)为0.01U/mL，乙酰胆碱在所得混合体系中的初始浓度为 10～1000nM(例如30～800nM，30～500nM，30～400nM，30～300nM，30～200nM，或30～100nM)。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤2)中，步骤1)中所得混合体系与乙酰胆碱混合后，乙酰胆碱酯酶在所得混合体系中的初始浓度为0.01U/mL，乙酰胆碱在所得混合体系中的初始浓度为30nM，优选步骤1)中所得混合体系与乙酰胆碱混合10min。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤2)中，步骤1)中所得混合体系与乙酰胆碱混合后，乙酰胆碱酯酶在所得混合体系中的初始浓度为0.01U/mL，乙酰胆碱在所得混合体系中的初始浓度为100nM，优选步骤1)中所得混合体系与乙酰胆碱混合20min。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤3)中，报告细胞M3:NFATc1-EGFP U2OS与步骤2)所得混合体系接触10～60min，例如20min，25min，30min，35min，40min，50min。
在某些实施方案中，在本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的步骤4)之前，还包括：
用磷酸盐(PBS)缓冲液稀释的甲醛溶液固定报告细胞；
对报告细胞的细胞核和/或细胞骨架(微丝、微管)进行染色(例如荧光染色)。
在某些实施方案中，本公开所述的筛查乙酰胆碱酯酶抑制剂的方法采用高内涵细胞成像分析系统检测报告细胞M3:NFATc1-EGFP U2OS被激活的程度。
本公开还涉及一种试剂盒，包括报告细胞M3:NFATc1-EGFP U2OS、乙酰胆碱(例如碘代乙酰胆碱)、乙酰胆碱酯酶(例如重组人乙酰胆碱酯酶)和荧光染料(例如Hoechst 33342、鬼笔环肽)。
在某些实施方案中，所述试剂盒还包括适合报告细胞M3:NFATc1-EGFP U2OS生长的培养基，例如DMEM高糖培养基。
在某些实施方案中，所述试剂盒还包括磷酸盐(PBS)缓冲物质(以水溶液形式存在或无水形式存在)、甲醛。
在某些实施方案中，所述试剂盒还包括记载本公开所述的筛查乙酰胆碱酯酶抑制剂的方法的说明书。
本公开还涉及所述的试剂盒用于筛查乙酰胆碱酯酶抑制剂的用途。
本公开还涉及一种用于筛查乙酰胆碱酯酶抑制剂的系统，包括所述的试剂盒和高内涵细胞成像分析系统。
本公开所述报告细胞M3:NFATc1-EGFP U2OS是稳定表达人毒蕈碱M3受体(muscarinic receptor3,M3)和融合到增强型绿色荧光蛋白(EGFP)C端的人NFATc1(Nuclear Factor Of Activated T Cells 1)的U2OS细胞，当M3被激动剂ACh激活，细胞内Ca2+释放，钙水平升高导致NFATc1去磷酸化并从细胞质快速易位至细胞核。该细胞可商购获得，也可根据现有技术构建。
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。
本公开的有益效果
本公开以M3受体激活的报告细胞为检测器，通过优化rhAChE反应体系，构建了一种基于ACh活性的高通量筛查AChE抑制剂的方法。该方法成功用于OPs农药、神经毒剂以及AD药物胆碱酯酶抑制剂的筛选，对对氧磷的检测限为1nM，对多奈哌齐的检测限为1nM，对GD(梭曼)的检测限0.1nM,对VX的检测限30pM。与传统方法相比，该方法可高通量，灵敏度高，时间和试剂消耗成本低。尤其是，该方法可在单一实验获得待测物与细胞毒性和细胞效应关联表型谱信息，为潜在药物或毒性物质的识别提供更为丰富的特征信息，为大量神经毒化合物的筛查提供新的高效的工具。
附图说明
图1示出了本公开实施例构建的筛查AChEI的方法的检测体系；
图2示出了碘代ACh浓度依赖地激活报告细胞系，诱导M3:NFATc1-EGFP U2OS人骨肉瘤细胞内EGFP-NFATc1发生核转位；
图3A示出了不同浓度rhAChE和碘代ACh孵育10min对EGFP-NFATc1核转位的影响；
图3B示出了0.01U/mL rhACh酶与不同浓度ACh孵育不同时间对EGFP-NFATc1核转位的影响；
图3C示出了VX在两种反应体系中对EGFP-NFATc1核转位的影响；
图4示出了应用本公开实施例构建的筛查AChEI的方法对有机磷农药类AChEI的测 试结果，其中A为对氧磷的测试结果；B为敌敌畏的测试结果；C为毒死蜱的测试结果；
图5示出了应用本公开实施例构建的筛查AChEI的方法对AD治疗药物类AChEI的测试结果，其中A为多奈哌齐的测试结果；B为卡巴拉汀的测试结果；
图6示出了应用本公开实施例构建的筛查AChEI的方法对神经性毒剂AChEI的测试结果，其中A为塔崩的测试结果；B为沙林的测试结果；C为梭曼的测试结果；
图7示出了应用本公开实施例构建的筛查AChEI的方法对AChEI的细胞表型谱的表征分析结果。
具体实施方式
下面将结合本公开实施例中的附图，对本公开实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本公开一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，不作为对本公开及其应用或使用的任何限制。基于本公开中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
在这里示出和讨论的所有示例中，任何具体值应被解释为仅仅是示例性的，而不是作为限制。因此，示例性实施例的其它示例可以具有不同的值。
下面通过一个具体实施例对本公开进行详细说明。
除非特别指明，本公开中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley & Sons,Inc.，1995中所述的方法进行。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本公开，且不意欲限制本公开所要求保护的范围。
本公开实施例所用主要药品及试剂：DMEM高糖培养基、胎牛血清(FBS)，G418(geneticin，50mg/mL)、青霉素/链霉素混合溶液(100×)、0.05％胰蛋白酶-EDTA溶液、1×PBS磷酸盐缓冲溶液(pH＝7.4)、10×PBS磷酸盐缓冲溶液(pH＝7.4)购于Gibco公司；牛血清白蛋白(BSA)购于Amresco公司；碘代乙酰胆碱(AChI)购于Selleck公司；重组人乙酰胆碱酯酶(rhAChE)购于Sigma-Aldrich公司；Hoechst 33342荧光染料购于Invitrogen公司；633标记鬼笔环肽购于Solarbio公司；小鼠抗人α-tubulin一抗购于Thermo公司；Alexa Flour 546标记驴抗小鼠二抗购于Invitrogen公司；对氧磷、敌敌畏、 毒死蜱购于Merck公司；多奈哌齐、卡巴拉汀购于Selleck公司；沙林、塔崩、梭曼和VX购于防化研究院；其他试剂均为分析纯试剂。
本公开实施例所用主要仪器：Perkin Elmer Opera PhenixTM高内涵细胞成像分析系统。
本公开实施例所用细胞系及其培养条件：M3:NFATc1-EGFP U2OS人骨肉瘤细胞系(Thermo公司)用含10％FBS的DMEM高糖培养基在37℃，5％CO2培养箱中培养。此外，培养液中还添加0.5mg/mL G418、100U/mL青霉素和100μg/mL链霉素。
本公开实施例中溶液的配制：
1.重组人乙酰胆碱酯酶(rhAChE)使用含0.1％BSA的10×PBS缓冲液(pH＝7.4)配制成1U/mL的母液，实验时用无血清的培养基稀释为目标工作液。
2.碘代乙酰胆碱(ACh)使用无菌去离子水配制为5mM母液，实验时用无血清的培养基稀释为目标工作液。
3.对氧磷、敌敌畏、毒死蜱使用甲醇配置为500μM母液，实验时用无血清的培养基稀释为目标工作液。
4.盐酸多奈哌齐(Donepezil)和酒石酸卡巴拉汀(Rivastigmine)使用无菌去离子水配制为5mM母液，实验时用无血清的培养基稀释为目标工作液。
5.塔崩(GA)、沙林(GB)、梭曼(GD)、VX使用乙腈配制为0.1M母液，实验时用无血清的培养基稀释为目标工作液。
本公开实施例构建的筛查AChEI的方法的检测体系如图1所示，包括①基于胆碱能受体的报告细胞系；②AChE反应体系，以及①和②共同组成的高内涵筛选(High Content Screening，HCS)检测体系③。其中①是胆碱能受体的报告细胞系M3:NFATc1-EGFP U2OS，该细胞是稳定表达人毒蕈碱胆碱受体3(M3)和融合到增强型绿色荧光蛋白(EGFP)C端的人NFATc1的U2OS细胞，当M3被激动剂ACh激活，细胞内Ca2+释放，钙水平升高导致NFATc1去磷酸化并从细胞质快速易位至细胞核，据此可对激动剂的活性进行定量分析；②是含有经优化确定含量的重组人AChE(rhAChE)和ACh与报告细胞系的检测系统；③为利用优化的HCS检测体系检测AChEI。本公开采用优化浓度的碘代乙酰胆碱(ACh)为激动剂；用AChEI预处理AChE，随后加入ACh孵育一定时间后处理报告细胞系，并使用HCS系统定量测定AChEI加入AChE反应体系后报告细胞的活性，并由此确定AChEI对于AChE的抑制活性及其特有的细胞反应特征。
实施例1 ACh激活M3:NFATc1-EGFP U2OS报告细胞系的高内涵分析
本实施例考察ACh是否可以稳定激活M3:EGFP-NFATc1细胞，选择碘代ACh，采 用高内涵细胞成像分析系统采集ACh处理报告细胞系后的细胞图像，并通过定量分析EGFP-NFATc1核转位程度，确定ACh激活M3受体的程度及浓度范围。具体步骤如下：
制备1×105个/mL的细胞悬液，按100μL/孔接种于黑色透底的96孔培养板中，培养24h后，吸弃培养上清，使用无血清培养基洗涤细胞2遍，并按100μL/孔分别加入碘代ACh梯度溶液(无血清培养液稀释，终浓度分别为0、0.01、0.03、0.1、0.3、1、3、10、30、100、300、1000、3000、10000、30000、100000nmol/L，每个浓度3孔)，37℃，5％CO2培养箱中放置20min；随后按50μL/孔加入含12％甲醛的1×PBS溶液(甲醛终浓度为4％)室温避光固定20min；使用1×PBS溶液漂洗细胞2遍后，加入200μL/孔含有1μM Hoechst 33342的1×PBS溶液，37℃避光孵育30min；用1×PBS溶液漂洗细胞2遍，再加入100μL 1×PBS/孔。上机检测，选用Alexa 488(吸收波长:495nm,发射波长:519nm)及DAPI(吸收波长:340nm,发射波长:488nm)通道，20×物镜采集细胞图像。每孔9个视野。采用Harmony 4.9软件定量分析细胞核数目和细胞内NFATc1-EGFP从胞质转位至细胞核情况，以3复孔，每孔不少于1000个细胞的平均每个细胞中NFATc1-EGFP核转位程度表示ACh的激活效应，并用Prism 8计算EC50值。
不同浓度的碘代ACh处理M3:NFATc1-EGFP U2OS后，细胞内EGFP-NFATc1转位的高内涵分析结果如图2所示，碘代ACh在0.1～100nM范围内浓度依赖地激活报告细胞系,当碘代ACh的浓度为0.3nM时，细胞出现了激活反应，至1nM时，EGFP-NFATc1核转位强度即约为最大强度的40％，当ACh浓度达10nM时，EGFP-NFATc1核转位程度接近最大强度，30nM达最大效应。利用Prism 8.0软件非线性拟合log(agonist)vs.response获得S型曲线，进而分析得到碘代ACh的EC50值为1.76±0.34nM，Z’因子为0.685，表明碘代Ach作为激动剂的胆碱能受体报告细胞分析模型是一个灵敏度高、稳定性强的HTS方法。
实施例2 AChE反应体系的构建
本实施例构建适于报告细胞系的AChE反应体系，以报告细胞系激活定量分析结果为依据，考察不同底物(碘代ACh)浓度、不同酶(rhAChE)浓度以及不同酶-底物反应时间对反应体系的影响。具体步骤如下：
首先，将梯度设置的rhAChE工作液与碘代ACh工作液交叉组合，按1：1(v/v)在96孔板中混匀，分别在37℃反应5、10、15、20min后，将混合液100μL/孔加至预接种报告细胞系的黑色透底96孔板中，孵育20min后，按实施例1所述方法定量分析报告细胞系的激活程度，并计算Z’因子，以获得最佳的实验体系。
Z’因子的计算公式为：
其中，“σ”为标准差(standard deviation)；“μ”为平均值(meansignal)；“c+”为阳性对照(positive control)；“c-”为阴性对照(negative control)。
Z’因子的值应在0～1之间。当Z’因子为0时，表明实验体系不成立；当0＜Z’因子＜0.5时，表明体系稳定性较差，不可靠；当0.5≤Z’因子＜1时，表明实验体系稳定性好，可靠性好；若Z’因子为1，表明阳性对照与阴性对照的标准差为0，是一种理想的实验体系(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)。
根据碘代ACh激活报告细胞系的浓度范围，在10～1×105nM区间内选取了5个碘代ACh浓度点，并在此条件下考察AChE反应体系中最佳的AChE含量与反应时间。
当0.001、0.003、0.01和0.02U/mL(该浓度为AChE反应体系中rhAChE的初始浓度，即未与碘代Ach反应时体系中的酶浓度)的rhAChE分别与不同浓度的碘代ACh孵育时间10min时，各反应体系对报告细胞系激活的结果如图2A所示，过低酶含量(≤0.003U/mL)无法有效水解碘代ACh，而过高酶含量(0.02U/mL)则过度水解碘代ACh，两种情况在报告细胞分析中差异有限；在0.01U/mL rhAChE条件下，碘代ACh呈现浓度依赖地诱导EGFP-NFATc1核转位的作用，浓度范围为10～1000nM。而上述浓度范围的碘代ACh在无酶体系中均具有近100％的细胞激活效应，提示在10min中内，0.01U/mL rhAChE水解底物碘代ACh的量可通过报告细胞系定量表示。因此，反应体系中rhAChE的适合的初始浓度可以为0.005～0.018U/mL，优选为0.007～0.015U/mL，进一步优选为0.01U/mL。
考虑筛选拮抗剂实验的可行性，在浓度依赖的范围内设置AChE反应体系中碘代ACh底物的初始浓度为10、30、100、300nM，考察了0.01U/mL酶与底物孵育不同时间对结果的影响。结果如图2B所示，底物最低浓度(10nM)下，5min水解完成，后续实验可行性低；最高浓度(300nM)下，各个时间点底物残余量过高、差异显著，后续实验缺乏足够的窗口和稳定性；在底物浓度为30nM时，酶孵育10min后，以及底物浓度为100nM时，酶孵育20min后两种情况下，碘代ACh水解大于90％，体系基本达到稳定。此种情况既满足了抑制剂筛选的最大窗口值的需求、有利于系统的稳定性，可操作性强。由此， 初步确定0.01U/mL rhAChE和30nM ACh孵育10min，0.01U/mL rhAChE和100nM ACh孵育20min两个反应体系为优选。
在此基础上，进一步用已知的乙酰酯酶抑制剂VX评估了上述两个筛选体系。结果如图2C所示，在两种反应体系下，VX均能浓度依赖地增加报告细胞的胆碱能受体的活性，即诱导报告细胞EGFP-NFATc1核转位，最低检测浓度为0.1nM；两种反应体系组成方法的Z’因子分别为0.716、0.614，即两种反应体系分别与报告细胞系配合均能灵敏可靠地反映VX对于AChE的抑制活性，其中在0.01U/mL rhAChE和30nM ACh孵育10min条件下Z’因子更高，因此，选择这一体系为最佳反应体系，由该体系与报告细胞系构成的筛查胆碱酯酶抑制剂(AChEI)的方法具有高通量测试能力，并具有较好的稳定性和灵敏度。
实施例3 本公开构建的筛查AChEI的方法对于不同类别AChEI对于AChE抑制活性的评价
本实施例使用本公开实施例建立的方法测定农业生产中常用的有机磷农药对氧磷(Paraoxon)、敌敌畏(DDVP)、毒死蜱(Chlopyrifos)，AD治疗药物多奈哌齐(Donepezil)、卡巴拉汀(Rivastigmine)，神经毒剂沙林(sarin，GB)、塔崩(tabun，GA)、梭曼(soman，GD)和VX对于AChE的抑制活性。具体步骤如下：
于每孔30μL rhAChE工作液中对应加入30μL梯度浓度的AChEI，37℃孵育30min，使有AChEI与rhAChE充分作用，随后加入60μL碘代ACh工作液，37℃反应10min；之后，吸取100μL反应液加至预接种报告细胞系的黑色透底96孔板中，孵育20min后，按实施例1所述方法定量分析报告细胞系的激活程度，以此确定AChEI对于AChE的抑制活性。
有机磷农药对rhAChE活性的抑制作用结果如图4所示，对氧磷、敌敌畏、毒死蜱均能通过抑制细胞检测系统中的rhAChE，降低体系内碘代ACh的水解，进而激活报告细胞的M3受体，促进EGFP-NFATc1核转位。三者最低的检测浓度分别为1nM、0.3μM、3μM，IC50值分别为：4.75±0.05nM、0.93±0.04μM、13.28±0.87μM。提示，三者的抑制活性的排序为，对氧磷〉敌敌畏〉毒死蜱。这一顺序与已报导的结果一致，表明本公开实施例建立的方法能够用于不同OP农药的乙酰胆碱酯酶抑制活性的筛查。
多奈哌齐和卡巴拉汀对rhAChE活性的抑制作用结果如图5所示，多奈哌齐和卡巴拉汀均能通过抑制细胞检测系统中的rhAChE，促进EGFP-NFATc1核转位，其IC50值分别为：26.21±1.45nM、4.05±1.26μM，该结果与文献报道的结果基本一致。
OP神经毒剂对rhAChE抑制作用结果如图6和图3C所示，塔崩、沙林、梭曼和VX 均显著抑制细胞检测系统中的rhAChE，促进EGFP-NFATc1核转位；IC50值分别为：3.75±0.03nM(GA)；0.62±0.05nM(GB)；0.19±0.05nM(GD)、0.19±0.03nM(VX)。其中，VX最小检测浓度达到30pM。这一结果与本实验室采用Ellman法的结果一致(其中塔崩有部分降解)。
综合以上三大类AChEI的评价结果表明，本公开所建立的方法能够通量化筛查不同类型的AChEI，具有灵敏度高、稳定性强、操作便捷、消耗小的特点。
实施例4 本公开构建的筛查AChEI的方法在表征AChEI细胞表型特征中的应用
本公开构建的筛查AChEI的方法除用于筛查AChEI、检测AChEI的酶抑制作用外，还可以结合细胞核、细胞骨架(微丝、微管)的荧光染色，表征所测定AChEI的细胞表型特征，提示待测物特有的细胞活性和毒性的信息。具体方法如下：
按实施例1所述的方法对处理后细胞进行Hoechst 33342染色后，用200μL含0.3％Triton X-100的1×PBS溶液于37℃避光透膜30min，之后加入50μL/孔633-标记的鬼笔环肽37℃避光染色30min；弃去上清，使用1×PBS溶液漂洗细胞3遍，每孔加入200μL封闭液(含5％BSA的PBS溶液)，室温封闭细胞1h；弃去封闭液，每孔加入50μL小鼠抗人α-Tubulin单克隆抗体作为一抗，37℃避光孵育1h；回收一抗，使用封闭液漂洗细胞3遍后，每孔加入50μL Alexa Flour 546标记的驴抗小鼠IgG二抗，室温避光孵育30min；使用1×PBS溶液漂洗细胞2遍后，于孔内加入100μL 1×PBS溶液。用高内涵细胞成像分析系统获取细胞图像，并对细胞核形态、微丝和微管的状态进行分析。
不同外源物质靶点不同、诱发细胞变化不同，其细胞表型特征有所不同。AChEI种类繁多、结构多样，生物效应不尽相同。已有研究表明，除特异性的靶点AChE外，OPs还影响其他的靶点，如，数百种酶、受体和其他蛋白质，其中一些靶点可能与慢性OP暴露和发育性神经毒性的长期CNS效应特别相关，AChE与这些靶点的相互作用也正成为OPs的累积风险评估的一部分。本公开构建的筛查AChEI的方法，不仅可以通过EGFP-NFATc1核转位程度检测鉴别AChEI，还可以在相同实验中，通过兼容的荧光标记，定量检测待测分子诱发的细胞、亚细胞形态结构、信号通路与靶蛋白的变化，获得待测分子特有的细胞表型谱特征。这为筛查和鉴别新的AChEI提供了更为丰富的信息。
在本实施例中，以不同类型AChEI为代表(神经毒剂VX、OP农药对氧磷和AD药物多奈哌齐)，示例性地在检测EGFP-NFATc1核转位确定靶点特异性活性的同一分析中，通过不同通道荧光标记，定量分析了细胞核、微丝和微管的变化。它们既可以反映外源物扰动引发的细胞、亚细胞形态和细胞骨架的快速反应特征，也是毒性及潜在机制的重要指 征。不同类型AChEI处理后细胞四参数荧光成像及定量结果见图7，如EGFP-NFATc1核转位图片所示，VX、对氧磷和多奈哌齐均能浓度依赖地抑制AChE的活性，但浓度-活性曲线的范围和斜率有所不同。VX和对氧磷的斜率基本一致，多奈哌齐的斜率明显更小，提示多奈哌齐与AChE结合的方式与VX和对氧磷不同(与文献报道基本一致)。虽然三者均浓度依赖地提高细胞微丝(Actin)的表达水平(图7中的A，B)，但曲线斜率相差较大；三者均增加细胞核面积(图7中的A，C)，但浓度效应曲线则不同；另外，经三者处理后，微管(Tubulin)在细胞中的分布状态明显不同(图7中的A)，多奈哌齐从1nM开始，就浓度依赖地增大微管的细胞分布，而VX、对氧磷同样随着浓度的增大使微管的细胞分布略有减小。这些结果显示，除抑制AChE活性外，三者呈现各自的表型变化特征；同为OPs的VX、对氧磷类似的细胞表型特征更多，而与AD药物多奈哌齐则差别较大。据此，可根据筛查目的，通过扩大时间点、不同细胞表型，使表型特征更加丰富，最终获得每个待测物特有的表型谱，为筛查与鉴别提供更多的依据。
尽管本公开的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本公开的保护范围之内。本公开的全部分为由所附权利要求及其任何等同物给出。

Claims (11)

  1. 一种筛查乙酰胆碱酯酶抑制剂的方法，包括：
    1)使待筛查样品与乙酰胆碱酯酶混合；
    2)使步骤1)中所得混合体系与乙酰胆碱混合；
    3)使报告细胞M3:NFATc1-EGFP U2OS与步骤2)所得混合体系接触；
    4)检测报告细胞M3:NFATc1-EGFP U2OS被激活的程度。
  2. 权利要求1所述的方法，其中报告细胞M3:NFATc1-EGFP U2OS的激活程度表示为报告细胞内NFATc1-EGFP从胞质转位至细胞核的程度，
    若报告细胞内NFATc1-EGFP从胞质转位至细胞核，判断待筛查样品中含有乙酰胆碱酯酶抑制剂，
    优选地，根据细胞内NFATc1-EGFP从胞质转位至细胞核的数目对乙酰胆碱酯酶抑制剂对于乙酰胆碱酯酶的抑制活性进行定量。
  3. 权利要求1或2所述的方法，其中所述乙酰胆碱为碘代乙酰胆碱，所述乙酰胆碱酯酶为重组人乙酰胆碱酯酶。
  4. 权利要求1或2所述的方法，其中：
    步骤1)中，使待筛查样品与乙酰胆碱酯酶在第一溶剂中混合；
    步骤2)中，使步骤1)中所得混合体系与乙酰胆碱在第二溶剂中混合；
    所述第一溶剂、第二溶剂相同或不同，各自独立地为适合报告细胞M3:NFATc1-EGFP U2OS正常生长的培养基，例如DMEM高糖培养基，
    优选地，步骤2)中，步骤1)中所得混合体系与乙酰胆碱混合后，乙酰胆碱酯酶在所得混合体系中的初始浓度为0.005～0.018U/mL，优选为0.007～0.015U/mL，进一步优选为0.01U/mL，
    优选地，步骤2)中，步骤1)中所得混合体系与乙酰胆碱混合后，乙酰胆碱在所得混合体系中的初始浓度为10～1000nM，优选为30～800nM，进一步优选为30～500nM，进一步优选为30～400nM，进一步优选为30～300nM，进一步优选为30～200nM，进一步优选为30～100nM。
  5. 权利要求1-3任一项所述的方法，其中
    优选地，步骤1)中，待筛查样品与乙酰胆碱酯酶在25～40℃(例如30℃，32℃， 35℃，37℃)的温度条件下混合10～60min，例如20min，25min，30min，35min，40min，50min，
    优选地，步骤2)中，步骤1)中所得混合体系与乙酰胆碱在25～40℃(例如30℃，32℃，35℃，37℃)的温度条件下混合10～60min，例如20min，25min，30min，35min，40min，50min。
  6. 权利要求1-4任一项所述的方法，其中步骤3)中，报告细胞M3:NFATc1-EGFP U2OS与步骤2)所得混合体系接触10～60min，例如20min，25min，30min，35min，40min，50min。
  7. 权利要求1-5任一项所述的方法，其中在步骤4)之前，还包括：
    用磷酸盐缓冲液稀释的甲醛溶液固定报告细胞；
    对报告细胞的细胞核和/或细胞骨架(微丝、微管)进行染色(例如荧光染色)。
  8. 权利要求1-6任一项所述的方法，其中采用高内涵细胞成像分析系统检测报告细胞M3:NFATc1-EGFP U2OS被激活的程度。
  9. 一种试剂盒，包括报告细胞M3:NFATc1-EGFP U2OS、乙酰胆碱(例如碘代乙酰胆碱)、乙酰胆碱酯酶(例如重组人乙酰胆碱酯酶)和荧光染料(例如Hoechst 33342、鬼笔环肽)，
    优选地，所述试剂盒还包括适合报告细胞M3:NFATc1-EGFP U2OS生长的培养基，例如DMEM高糖培养基，
    优选地，所述试剂盒还包括磷酸盐缓冲物质(以水溶液形式存在或无水形式存在)、甲醛，
    可选地，所述试剂盒还包括记载权利要求1-7任一项所述的方法的说明书。
  10. 权利要求8所述的试剂盒用于筛查乙酰胆碱酯酶抑制剂的用途。
  11. 一种用于筛查乙酰胆碱酯酶抑制剂的系统，包括权利要求8所述的试剂盒和高内涵细胞成像分析系统。
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