High-flux toxicity pre-evaluation substitution method based on acetylcholin esterase target

Abstract

The invention discloses a high-flux toxicity pre-evaluation substitution method based on an acetylcholin esterase target. By integrating an SH-SY5Y nerve cell endogenous AChE and recombinant exogenous AChE dual detection system and relying on an automatic screening system constructed by integrating core equipment such as an automatic pipetting workstation, a constant-temperature incubator and a multifunctional microplate reader, a high-flux toxicity pre-evaluation substitution method based on an AChE target is successfully established and is used for evaluating the neurotoxicity of a compound, and the method has the advantages of high sensitivity, high sensitivity, high sensitivity and the like. The method solves the limitation that a single screening method in the prior art cannot evaluate factors such as target inhibition, cell permeability and metabolic transformation at the same time, and the method is simple, convenient, rapid, high in accuracy and suitable for large-scale primary screening of the AChE inhibitor and early warning of neurotoxicity risks.

Description

An alternative method for high-throughput toxicity prediction based on acetylcholinesterase targets Technical Field

[0001] This invention belongs to the field of biodetection technology and relates to a high-throughput alternative method for toxicity pre-evaluation based on acetylcholinesterase targets. Specifically, it involves using a high-throughput detection platform to construct an alternative method for neurotoxicity pre-evaluation based on endogenous and exogenous recombinant expression of acetylcholinesterase, which is used to assess the neurotoxicity risk of compounds. Background Technology

[0002] Toxicology studies the harmful effects and mechanisms of action of exogenous chemicals, physical and biological factors on living organisms, thereby predicting the severity of their harm to humans and the ecological environment and providing a scientific basis for determining safety limits and implementing prevention and control measures. With the advancement of toxicological research, the limitations of traditional toxicity evaluation methods based on animal experiments—such as long cycles and high costs—have become increasingly apparent. Simultaneously, driven by the 3R principle (replace, reduce, optimize), ethical and regulatory requirements for animal experiments are becoming increasingly stringent globally, making the development of alternative methods that can accurately reflect neurotoxic effects in vitro a significant trend in the field of toxicology. In toxicological research, enzymes, as key regulatory factors in organisms, are often used as detection targets. Acetylcholinesterase, as a key enzyme in the nervous system, participates in the hydrolysis of acetylcholine and regulates nerve signal transmission; changes in its activity are important indicators in toxicology for assessing nervous system health and neurotoxicity, and are widely used in toxicity evaluation studies. Evaluating the inhibitory activity of exogenous substances on acetylcholinesterase can serve as an alternative method for neurotoxicity pre-evaluation.

[0003] Existing high-throughput assays for acetylcholinesterase inhibitory activity, while theoretically possessing high-throughput potential, are limited in practical implementation and application by factors such as complex experimental procedures and insufficient automation. Their high-throughput capabilities often remain theoretical and have not yet been truly realized, failing to meet the practical needs of rapid analysis of large-scale compound libraries. Furthermore, while existing in vitro evaluation methods for exogenous acetylcholinesterase inhibitory activity can characterize the direct interaction between compounds and targets, they struggle to reflect the comprehensive effects at the cellular level, involving processes such as osmosis and metabolism. These limitations result in insufficiently comprehensive information provided by existing in vitro assessment systems, thereby restricting their accuracy and reliability.

[0004] Therefore, current methods for testing acetylcholinesterase inhibitory activity are still insufficient to meet the needs of rapid and reliable toxicity early warning tools in chemical substance safety evaluation and drug development. Thus, there is an urgent need to develop a new toxicity assessment method based on acetylcholinesterase that is both efficient and alternative. Summary of the Invention

[0005] The purpose of this invention is to provide an alternative high-throughput toxicity prediction method based on acetylcholinesterase targets. This method aims to overcome the limitations of existing single-screening methods that cannot simultaneously assess target inhibition along with factors such as cell permeability and metabolic transformation by constructing a detection method for inhibitors of intracellular AChE and recombinant AChE. This will enable high-throughput and accurate early warning of the neurotoxicity risk of compounds.

[0006] The inventive concept of this invention is to construct a combined detection method based on the combination of endogenous and exogenous acetylcholinesterase, using human acetylcholinesterase as the target. This is the first time that an endogenous and exogenous acetylcholinesterase detection system has been integrated into the same automated detection platform, achieving joint determination of acetylcholinesterase inhibitory activity. The endogenous system uses human neuroblastoma cells SH-SY5Y as a model to assess changes in intracellular acetylcholinesterase activity; the exogenous system obtains secretory acetylcholinesterase through transfection of HEK293T cells to assess changes in the activity of extracellular secreted enzymes. Both systems operate on the same high-throughput automated platform, which integrates core equipment such as a pipetting workstation, a CO2 incubator, and an ELISA reader, enabling automated completion of operations such as sample addition, reaction, transfer, and reading. The Ellman assay is used for detection, achieving standardization of the reaction system and high-throughput detection through automated operation. Based on this combined detection method, typical positive compounds are measured, and the inhibitory activity data from the endogenous and exogenous systems are cross-validated. The results show that this method has the advantages of good repeatability and simple operation, and can quickly and accurately detect acetylcholinesterase inhibitory activity, making it particularly suitable for early warning of neurotoxicity risks.

[0007] The technical solution adopted in this invention is as follows: A high-throughput alternative method for toxicity pre-evaluation based on human acetylcholinesterase targets, characterized by relying on an automated platform to determine compounds through a combined detection method of endogenous and exogenous acetylcholinesterases, and cross-validating the inhibitory activity data of the endogenous and exogenous systems. The specific steps are as follows: S1. Construction of the endogenous AChE detection system: Human neuroblastoma cells SH-SY5Y are used as the endogenous model and seeded in the culture module of the automated platform. Utilizing their complete cell membrane structure and intracellular metabolic system, the actual response of the compound in a complex intracellular environment is simulated; S2. Construction of the exogenous AChE detection system: HEK293T cells are used as the expression host. After optimized transfection, soluble AChE with its native conformation is efficiently secreted, providing a highly sensitive and accurate target response, facilitating the measurement of the direct inhibitory effect of the compound on recombinant AChE; S3. Automated high-throughput detection: The analyte compound is diluted to a preset concentration gradient, and an automated pipetting workstation is used to incubate the compound with the aforementioned endogenous cells and exogenous recombinant enzyme, respectively. The Ellman assay is employed, and the compound is efficiently measured using a multi-functional microplate reader, with automatic data acquisition. S4. Data processing: The system fits the IC50 value based on the absorbance data of the endogenous and exogenous systems. 50 The curves, by comparing the IC50 values ​​of the same compound in both intrinsic and extrinsic systems, show the results. 50 The difference in values ​​compensates for the limitations of a single detection system, thereby reducing the risk of missed detection, achieving more accurate toxicity warnings, and ensuring the accuracy of screening results.

[0008] Step S1 includes: The system automatically dispenses cell suspension using the 96-channel pipette on the main unit of the pipetting workstation, and sets the SH-SY5Y cell density to 4×10⁻⁶. 4 - 6×10 4 Cells are seeded into each well, and then a robotic gripper automatically transfers the seeded 96-well plate to a guide rail, which transports it to a CO2 incubator. After incubation, the plate is transferred to the pipetting station, where a 96-channel pipette removes the cell culture medium. The system then automatically adds phosphate-buffered saline (PBFS) and DTNB to each well and transfers the plate to the CO2 incubator for incubation. Finally, the plate is transferred back to the pipetting station, and 50 μL of 2-6 mmol / L solution is automatically added. -1 The plate is then ATCI-treated and returned to the CO2 incubator for reaction at 37-42°C for 15-25 minutes. After the reaction, the mechanical gripper automatically transfers the plate to the microplate reader to read the absorbance value and uploads the detection data for processing.

[0009] In step S1, the cell seeding number is 5 × 10⁶. 4Cells / well. The system's automatic dispensing module adds 100 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol·L⁻¹ to each well. -1 DTNB was transferred to a CO2 incubator and incubated at 37°C for 5 minutes.

[0010] In step S1, the substrate ATCI was used at a concentration of 2 mmol·L⁻¹. -1 The reaction time was 20 min and the reaction temperature was 37°C.

[0011] Step S2 includes: seeding cells to a suitable density using a high-throughput automated platform, incubating overnight in a 37°C, 5% CO2 saturated humidity incubator integrated into the workstation; automatically dispensing transfection reagents and plasmid solutions using the 96-well pipette on the pipetting workstation main unit, adding samples according to different transfection ratios; then automatically transferring the seeded plate to the CO2 incubator via a guide rail; after incubation, removing the plate from the incubator and transferring it to the pipetting workstation, collecting the culture supernatant as the source of recombinant AChE solution; replacing the 96-well plate, automatically adding transfection supernatant to each well of the 96-well plate using the high-throughput automated platform, along with phosphate buffer and DTNB; then returning the plate to the CO2 incubator for incubation; after incubation, transferring the plate back to the pipetting workstation, where the system automatically adds 50 μL of a solution with a concentration of 2-6 mmol·L⁻¹. -1 The plate was then incubated at 37-42°C in a 5% CO2 incubator for 15-25 minutes. After the reaction, the plate was transferred to a microplate reader using a mechanical gripper to read the absorbance values, and the detection data was uploaded for processing.

[0012] In step S2, different transfection ratios of 2:1, 3:1, 4:1, and 5:1 were used for sample addition. The system automatically added 15 μL of transfection supernatant to each well of the 96-well plate via a high-throughput automated platform, along with 85 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol / L... -1 DTNB was then applied, and the plate was returned to the CO2 incubator for incubation at 37°C for 5 minutes.

[0013] The substrate ATCI used in step S2 is at a concentration of 2 mmol·L⁻¹. -1 The reaction time was 20 min and the reaction temperature was 37°C.

[0014] In steps S1 and S2, the cell suspension was cultured in a CO2 incubator at 37°C and 5% CO2 for 24 hours.

[0015] Step S3 specifically involves seeding human neuroblastoma cells SH-SY5Y into 96-well plates, with 5 × 10⁶ cells per well.4 Cells were cultured at 37°C and 5% CO2 for 24 hours. After incubation, the culture medium was aspirated from the wells, and control and blank wells were set up. The system prepared stock solutions of the test compounds using DMSO as solvent, and obtained 12 concentration gradients by three-fold dilution. After incubation in a CO2 incubator, the well plates were transferred back to the pipetting station, and 50 μL of 8 mmol·L⁻¹ solution was added. -1 ATCI was performed, and the well plate was then returned to a 37°C, 5% CO2 incubator for 20 minutes. After the reaction, the well plate was transferred to a microplate reader using a mechanical gripper, and the absorbance value was read at 412 nm. The supernatant of HEK293T cell culture containing recombinant human acetylcholinesterase was collected as the enzyme source, and the test compound was prepared into a 1 mmol·L⁻¹ solution using DMSO as the solvent. -1 The stock solution was diluted three-fold to obtain 12 concentration gradients, with control and blank wells included. Using the 96-channel pipette on the main unit of the pipetting station, 15 μL of enzyme supernatant, 75 μL of 0.1 M phosphate buffer, and 10 μL of different concentrations of positive compound solutions were added to a 96-well plate. After incubation in a CO2 incubator, the plate was transferred back to the pipetting station, and 50 μL of 8 mmol·L⁻¹ solution was added using the 96-channel pipette on the main unit. -1 The plate was then returned to the CO2 incubator and reacted at 37°C for 20 minutes. After the reaction, the plate was transferred to a microplate reader by a mechanical gripper to read the absorbance at 412 nm and the data was uploaded for processing.

[0016] Incubation steps for human neuroblastoma cells SH-SY5Y in a CO2 incubator: Add 90 μL of 0.1 M phosphate buffer and 10 μL of different concentrations of compound solutions to each well, transfer to a CO2 incubator, and incubate at 37°C for 30 minutes; after incubation, transfer the plate back to the pipetting station, and add 50 μL of 10 mmol·L⁻¹ solution using the 96-channel pipette of the pipetting station. -1 DTNB was added, and the well plate was then returned to the CO2 incubator and incubated at 37°C for 5 minutes. The incubation steps for recombinant human acetylcholinesterase HEK293T in the CO2 incubator were as follows: 15 μL of enzyme supernatant, 75 μL of 0.1 M phosphate buffer, and 10 μL of positive compound solutions of different concentrations were added to the 96-well plate using the 96-channel pipette of the pipetting workstation. The plate was then incubated at 37°C for 30 minutes. After incubation, the well plate was transferred back to the pipetting workstation, 50 μL of DTNB was added, and then the plate was transferred to the CO2 incubator via the rail system and incubated at 37°C for 5 minutes.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The dual-system AChE of this method are both human-derived, effectively avoiding species differences or expression system defects: Compared with the currently commonly used AChE inhibitor detection methods (which usually use electric eel or rat-derived AChE), there are amino acid sequence differences between animal-derived AChE and human-derived AChE, which may affect the binding of compounds to enzymes, making the inhibitory activity data obtained based on animal-derived enzymes unable to reliably reflect their effect on human-derived AChE; when using prokaryotically expressed AChE (such as E. coli expression), prokaryotic enzymes lack the human-specific glycosylation modification, and the resulting recombinant enzymes may differ from natural enzymes in the human body in terms of stability and binding kinetics, which may affect the accuracy of screening results.

[0018] 2. HEK293T Secretory Enzyme System: HEK293T cells are a human embryonic kidney cell-derived line with extremely strong exogenous gene expression capabilities, and their post-translational modification systems (such as glycosylation and phosphorylation) are highly consistent with those of human cells. By constructing an expression vector containing the AChE secretion signal peptide and transfecting these cells, human AChE with its native conformation can be efficiently secreted. This avoids the species differences associated with extracting AChE from animal tissues and overcomes the limitations of prokaryotic expression systems in post-translational modification, ensuring the reliability of exogenous enzyme activity detection results. Transfected HEK293T cells can directly secrete AChE into the cell culture supernatant, allowing for direct enzyme activity detection without cell lysis, greatly improving experimental efficiency and convenience. The secreted AChE in the supernatant exists in a free state. The system has a simple composition, maintains the structure and function of the natural enzyme, and not only ensures the sensitivity and reproducibility of enzyme activity detection results but also directly reflects the targeted binding ability of compounds to AChE molecules, thus specifically reflecting the direct inhibitory ability of compounds on human AChE.

[0019] SH-SY5Y Cell Endogenous System: SH-SY5Y cells are human neuroblastoma cells that stably express endogenous AChE. The biochemical characteristics of this enzyme are consistent with those of natural AChE in human neurons. By detecting AChE in intact human neuronal cells, the comprehensive effects of compounds after penetration, metabolism, and target binding can be evaluated. Combining the exogenous recombinant AChE detection system based on HEK293T cells with the endogenous AChE detection system based on SH-SY5Y cells allows for a two-way cross-validation of "direct molecular target action + overall effect verification of compounds in the cellular environment," assessing the inhibitory effects of compounds from different levels, thereby improving the comprehensiveness and reliability of the evaluation method.

[0020] 3. The experimental system is highly compatible, ensuring the comparability and standardization of data: The advantages of this combination are: (1) Highly consistent experimental adaptability: SH-SY5Y and HEK293T are both human cell lines that adhere to the wall and have basically the same culture conditions (culture medium formula, CO2 concentration, incubation temperature) without the need for additional adjustments; (2) Platform compatibility: Both can be integrated into the same high-throughput automated platform, and the culture of endogenous cells and the reaction of exogenous enzymes can be completed simultaneously. The experimental parameters of sample addition, incubation and reading are all controlled by standardized automated programs, eliminating operational errors and ensuring the standardization and repeatability of data; (3) Adaptable detection methods: Both use the Ellman method for detection, without the need to change detection reagents and calculation methods, simplifying the experimental operation process.

[0021] 4. This invention is fully adapted to the needs of the entire toxicity screening process. The automated platform supports rapid screening of large-scale compound libraries, meets the high-throughput requirements of toxicity screening, and can effectively support high-throughput screening in the early stages of drug development, improving screening efficiency. At the same time, the use of dual-system data cross-validation can eliminate "non-specific cytotoxicity" that causes enzyme activity to decrease by affecting cells. It can also use an exogenous AChE detection system to directly and efficiently evaluate the specific inhibitory activity of compounds on AChE.

[0022] In summary, this invention successfully establishes a high-throughput alternative method for toxicity pre-evaluation based on AChE targets, which is simple, rapid, and highly accurate, and is suitable for early warning of neurotoxicity risks of compounds and large-scale initial screening of AChE inhibitors. Attached Figure Description

[0023] Figure 1: Schematic diagram of the high-throughput automated platform; Figure labels: 1- CO2 incubator, 2-ELISA reader, 3- Consumables stack, 4- Pipetting workstation host, 5- Linear shuttle rail, 6- 96-channel gantry, 7- 8-channel gantry, 8- 96-channel pipette, 9- 8-channel pipette, 10- Infrared sensor, 11- Mechanical gripper.

[0024] Figure 2: Effect of automated platform optimization of cell seeding density on endogenous AChE activity assay; Figure 3: Effect of automated platform optimization of different substrate concentrations on endogenous AChE activity assay; Figure 4: Effect of automated platform optimization of different incubation times on endogenous AChE activity assay; Figure 5: Effect of automated platform optimization of different incubation temperatures on endogenous AChE activity assay; Figure 6: Validation of the automated platform for the endogenous AChE activity assay system; Figure 7: Automated platform optimization of transfection conditions for exogenous AChE expression system in HEK293T cells; Figure 8: Automated platform optimization of exogenous AChE activity assay at different substrate concentrations. The effects of different incubation times on the determination of exogenous AChE activity by the automated platform; Figure 9: Effect of different incubation times on the determination of exogenous AChE activity by the automated platform; Figure 10: Effect of different incubation times on the determination of exogenous AChE activity by the automated platform; Figure 11: Validation of the cellular endogenous AChE activity detection system by the automated platform; Figure 12: Determination of the effects of Mipafox and Paraoxon on endogenous AChE activity by the automated platform; Figure 13: Determination of the effects of Mipafox and Paraoxon on exogenous AChE activity by the automated platform; Figure 14: Collection of candidate compounds screened by the automated platform for acetylcholinesterase inhibition; Reference numerals: ● indicates IC50 of the compound against endogenous AChE. 50 Value, ■ indicates the IC50 value of the compound for exogenous AChE. 50 Value, ○ indicates that the compound is at 10 -4 At M concentration, there was no significant inhibition of endogenous and exogenous AChE. Detailed Implementation

[0025] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but do not limit the present invention. The following non-limiting embodiments can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way.

[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0027] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. The use of the terms "a" or "an" in this invention does not exclude a plurality. Words such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects.

[0028] Example 1: Construction of the Acetylcholinesterase Inhibitor Screening System (Automated Platform) As shown in Figure 1, the acetylcholinesterase inhibitor screening system includes a pipetting workstation and peripheral equipment, which are connected via connectors. The peripheral equipment includes, but is not limited to: a CO2 incubator (1), an ELISA reader (2), and a consumables stack (3). The connectors include a linear shuttle rail (5) and a mechanical gripper (11). The mechanical gripper and the 96-channel / 8-channel pipette can move automatically along the rail, enabling precise transfer and sample addition of 96-well plates between modules, ensuring the continuity and stability of the experimental process.

[0029] The main unit of the pipetting workstation (4) is the core operating unit of the system. It is equipped with a 96-channel gantry (6) and an 8-channel gantry (7), and is equipped with corresponding 96-channel pipettes (8) and 8-channel pipettes (9). Each pipette is equipped with a mechanical gripper (11), which can automate operations such as tip loading, reagent addition and plate handling between different plate positions.

[0030] The main unit (4) of the pipetting workstation is equipped with a pipette tip cleaning station (such as an 8-channel pipette tip cleaning station or a 96-channel pipette tip cleaning station). The pipette tip cleaning station is used to clean pipette tips and reduce cross-contamination. The ELISA reader is located near the main unit of the pipetting workstation and is used to pick up and put down ELISA plates by mechanical grippers installed on the 96-channel / 8-channel pipette.

[0031] The consumable stack (3) is connected to the pipetting workstation host (4) via a linear shuttle rail (5), and the CO2 incubator (1) is connected to the pipetting workstation host (4) via a second rail.

[0032] The system can be freely configured with plate positions according to experimental needs, including but not limited to: 96-channel pipette tip loading position, oscillation incubation position, microplate loading position for microplate reader, incubator loading and unloading position, pipette tip recycling basket, main unit and track exchange position, consumable stack and main unit exchange position, etc.

[0033] The system is also equipped with an infrared sensor (10), which is embedded in the operation entrance of the liquid handling workstation through a light curtain sensor strip to ensure accurate detection of the movement of objects in the operation area.

[0034] This acetylcholinesterase inhibitor screening system is a one-stop acetylcholinesterase inhibitor screening system with an automated pipetting workstation as its core. It integrates equipment such as a CO2 incubator, consumables stack, and microplate reader, as well as multiple plate positions, providing ample operating space.

[0035] Example 2: Optimization and Establishment of Screening Method for Endogenous Acetylcholinesterase Inhibitors (1) Optimization of Cell Seeding Number To optimize the optimal number of cells seeded in the endogenous acetylcholinesterase reaction system, this invention utilizes a high-throughput automated platform for cell density gradient optimization. The system automatically dispenses cell suspension using a 96-channel pipette on the main unit of the pipetting workstation, setting 1×10 4 2×10 4 3×10 4 4×10 4 5×10 4 6×10 4 Six gradient groups of cells / wells were seeded. A robotic gripper automatically transferred the seeded 96-well plates to a guide rail, which then transported them to a CO2 incubator for 24 hours at 37°C and 5% CO2. After incubation, the plates were transferred to the pipetting workstation according to the program. A 96-well pipette was used to remove the supernatant from the wells, and liquids were added. For blank wells, only 150 μL of 0.1 M phosphate buffer and 50 μL of dithiodinitrobenzic acid (DTNB) were added to each well. The system's automatic dispensing module added 100 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol·L⁻¹ sodium bicarbonate to each well. -1 DTNB was added and transferred to a CO2 incubator, where it was incubated at 37°C for 5 minutes. The plate was then transferred back to the pipetting station, where 50 μL of 8 mmol·L⁻¹ solution was automatically added. -1 Iodothioacetylcholine (ATCI) was added, and then the plate was returned to a CO2 incubator and reacted at 37°C for 20 minutes. After the reaction, a mechanical gripper automatically transferred the plate to a microplate reader to read the absorbance value at 412 nm, and the detection data was uploaded to the system analysis software for processing.

[0036] The results are shown in Figure 2, indicating that the cell seeding number was 5 × 10⁶. 4 The cell / well reaches a stable plateau phase, and the signal stabilizes. Therefore, the optimal cell seeding number is 5 × 10⁶. 4 Cells / well (suitable range: 4 × 10⁻⁶) 4 - 6×10 4 (cells / pores).

[0037] (2) Optimization of substrate concentration: To optimize the optimal concentration of ATCI in the endogenous acetylcholinesterase reaction system, this invention employs a high-throughput automated platform for gradient optimization. The SH-SY5Y cell density was set to 5 × 10⁻⁶ cells / year. 4 Cells were seeded into 96-well plates, and a robotic gripper automatically transferred the seeded plates to a guide rail, which then transported them to a CO2 incubator. The plates were incubated at 37°C and 5% CO2 for 24 hours. The initial concentration was 72 mmol / L.-1 ATCI was then used, followed by a 3-fold dilution and serial dilutions to 5 gradients. After incubation, the plate was transferred to a pipetting station according to the procedure. The supernatant was aspirated using a 96-channel pipette, and 100 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol / L solution were added. -1 DTNB is applied, and then the well plate is transferred to a 37°C, 5% CO2 incubator for 5 minutes. After incubation, the well plate is transferred back to the pipetting station. The system automatically adds 50 μL of ATCI solution of different concentrations through the 96-channel pipette of the pipetting station host according to the set program. Then the well plate is sent back to the 37°C, 5% CO2 incubator for the set time (10-90 minutes). After the reaction is completed, the mechanical gripper automatically transfers the well plate to the microplate reader to read the absorbance value at 412 nm and uploads the detection data to the system analysis software for processing.

[0038] The results are shown in Figure 3, with final substrate concentrations ranging from 2 to 6 mmol·L⁻¹. -1 Within the specified range, the signal is stable and reproducible. Therefore, the preferred final substrate concentration is 2 mmol·L⁻¹. -1 (Suitable range is 2 - 6 mmol·L) -1 ).

[0039] (3) Optimization of reaction time: To optimize the reaction time in the endogenous acetylcholinesterase reaction system, this invention utilizes a high-throughput automated platform for reaction time optimization. The SH-SY5Y cell density is 5 × 10⁻⁶ cells / year. 4 Cells were seeded into 96-well plates, and the mechanical gripper automatically transferred the seeded plates to a guide rail, which then transported them to a CO2 incubator for 24 hours at 37°C and 5% CO2. Different reaction time points were set (10, 20, 30, 40, 50, 60, 70, 80, 90 minutes). In blank wells, only 150 μL of 0.1 M phosphate-buffered saline (PBFS) and 50 μL of DTTN were added. The system automatically aspirated the supernatant from the wells using the 96-channel pipette on the main unit of the pipetting station, and added 100 μL of 0.1 M PBFS and 50 μL of DTTN to each well. The plates were then transferred to the CO2 incubator and incubated at 37°C for 5 minutes. Subsequently, the plates were transferred back to the pipetting station, and the system automatically added 50 μL of 8 mmol·L⁻¹ PBFS. -1 ATCI (with a final concentration of 2 mmol·L⁻¹) -1 The plate was then returned to the CO2 incubator and reacted at 37°C for the set time. After the reaction, the mechanical gripper automatically transferred the plate to the microplate reader and read the absorbance value at 412 nm, then uploaded the detection data to the system analysis software for processing.

[0040] The results are shown in Figure 4. The results indicate that the reaction is in a linear phase when the reaction time is in the range of 15-25 min. Therefore, the optimal reaction time is determined to be 20 min (within a suitable range of 15-25 min).

[0041] (4) Optimization of reaction temperature: To optimize the optimal reaction temperature in the endogenous acetylcholinesterase reaction system, this invention utilizes a high-throughput automated platform for gradient optimization of the reaction temperature. The SH-SY5Y cell density was 5 × 10⁻⁶ cells / year. 4 Cells were seeded into 96-well plates, and the mechanical gripper automatically transferred the seeded plates to a guide rail, which then transported them to a CO2 incubator for 24 hours at 37°C and 5% CO2. Different reaction temperatures were set for the system (22, 30, 37, 42, 46, 52°C). 150 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol·L⁻¹ sodium chloride were added to each blank well. -1 The DTNB system automatically removes the supernatant from each well using the 96-channel pipette on the main unit of the pipetting workstation, and adds 100 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol·L⁻¹ to each well. - 1 DTNB was added, and then the well plate was transferred to a CO2 incubator and incubated for 5 minutes at the set temperature. Subsequently, the well plate was transferred back to the pipetting station, and the system automatically added 50 μL of 8 mmol·L⁻¹ solution. -1 The plates were then subjected to ATCI, and subsequently returned to a CO2 incubator for 20 minutes at the set temperature. After the reaction was complete, a mechanical gripper transferred the plates to a microplate reader to read the absorbance at 412 nm, and the data was uploaded to the system analysis software for processing.

[0042] The results are shown in Figure 5, indicating that the signal is stable when the reaction temperature is in the range of 37-42°C. Therefore, the preferred reaction temperature is determined to be 37°C (suitable range is 37-42°C).

[0043] (5) Establishment of a method for detecting endogenous acetylcholinesterase activity: To detect endogenous acetylcholinesterase activity, human neuroblastoma cells SH-SY5Y were first seeded on a high-throughput automated platform. The system automatically dispensed the cell suspension using a 96-channel pipette on the main unit of the pipetting workstation, at a concentration of 5 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates. A robotic gripper automatically transferred the seeded 96-well plates to a guide rail, which then transported them to a CO2 incubator for 24 hours at 37°C and 5% CO2. After incubation, the plates were transferred back to the pipetting station, and the cell culture medium was automatically aspirated from the wells using a 96-well pipette. Then, the system automatically added 100 μL of 0.1 M phosphate-buffered saline and 50 μL of 10 mmol / L [amount missing] to each well. -1 DTNB was used to transfer the well plate to a CO2 incubator via a guide rail, where it was incubated at 37°C for 5 minutes. After incubation, the well plate was transferred back to the pipetting station, where the system automatically added 50 μL of 8 mmol·L⁻¹ solution to each well. -1 After ATCI, the well plates were returned to a CO2 incubator and reacted at 37°C for 20 minutes. Following the reaction, the plates were transferred to a microplate reader using a mechanical gripper, where absorbance values ​​were read at 412 nm and the data was uploaded to the system analysis software for processing.

[0044] As shown in Figure 6, the control group produced only a very low background signal, while the cell group showed a significantly increased OD value. The difference between the two can be used to characterize the relative activity of endogenous AChE in cells.

[0045] Example 3: Optimization and establishment of screening method for exogenous acetylcholinesterase inhibitors (1) Optimization of transfection reagent and plasmid ratio The cells were seeded into 12-well plates with appropriate cell density using a high-throughput automated platform as input plates and incubated overnight in a saturated humidity incubator with 37°C and 5% CO2 concentration integrated in the workstation. The cell culture plates were placed in the set position in the automated workstation via guide rails and mechanical grippers. The culture medium in the culture plates was replaced, and the system automatically dispensed transfection reagents and plasmid solutions through the 96-well pipette of the pipetting workstation host. Samples were added according to different transfection ratios (2:1, 3:1, 4:1, 5:1). The well plates were then transferred to the guide rails via mechanical grippers and transported to the CO2 incubator. They were cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the well plates were transferred to the pipetting workstation, and the culture supernatant was collected as the source of recombinant AChE solution. After replacing the 96-well plate, the system automatically added 15 μL of transfection supernatant and 85 μL of 0.1 M phosphate-buffered saline (PBFS) to each well using a high-throughput automated platform. In the control wells, the system automatically added 15 μL of untransfected cell supernatant and 85 μL of 0.1 M PBFS. Subsequently, the system used the 96-channel pipette on the pipetting workstation mainframe to add 50 μL of 10 mmol / L [polymerase chain reaction] to each well. -1DTNB was added and the plate was returned to the CO2 incubator and incubated at 37°C for 5 minutes. After incubation, the plate was transferred back to the pipetting station, where the system automatically added 50 μL of 8 mmol·L⁻¹ DTNB. -1 The plates were then incubated at 37°C with 5% CO2 for 20 minutes. After the reaction, the plates were transferred to a microplate reader using a mechanical gripper for absorbance measurement. The reader read the absorbance at 412 nm and uploaded the data to the system analysis software for processing.

[0046] The results are shown in Figure 7. The results show that the Control group only showed a low background signal, while the transfected culture medium showed a significantly increased OD value, proving that the exogenous substance could be successfully secreted into the culture medium and maintain its activity.

[0047] (2) Optimization of substrate concentration: To optimize the optimal concentration of ATCI in the exogenous acetylcholinesterase reaction system, this invention utilizes a high-throughput automated platform for gradient optimization of substrate concentration. The initial concentration was set at 72 mmol·L⁻¹. -1 ATCI was then performed, followed by a 3-fold dilution and serial dilutions to five levels. 150 μL of 0.1 M phosphate buffer was added to each blank well. Using the 96-channel pipette on the workstation, 15 μL of supernatant, 85 μL of 0.1 M phosphate buffer, and 50 μL of 10 mmol / L sodium chloride were automatically added to each well. -1 DTNB was added, and the wells were then transferred to a 37°C, 5% CO2 incubator for 5 minutes. After incubation, the wells were transferred back to the pipetting station, where the system automatically added 50 μL of ATCI at different concentrations using the station's 96-channel pipette according to the pre-programmed sequence, and then returned to the 37°C, 5% CO2 incubator for 20 minutes. After the reaction, the wells were transferred to a microplate reader for absorbance measurement using a mechanical gripper. The reader read the absorbance at 412 nm and uploaded the data to the system's analysis software for processing.

[0048] The results are shown in Figure 8, indicating that the substrate concentration used was 2–6 mmol·L⁻¹. -1 Within the specified range, the signal is stable and reproducible. Therefore, the preferred substrate concentration is determined to be 2 mmol·L⁻¹. -1 (Suitable range is 2 - 6 mmol·L) -1 ).

[0049] (3) Optimization of reaction time: To optimize the reaction time in the exogenous acetylcholinesterase reaction system, this invention utilizes a high-throughput automated platform for gradient optimization of the reaction time. The system is set to different reaction time points (10, 20, 30, 40, 50, 60, 70, 80, 90 minutes). Only 150 μL of 0.1 M phosphate buffer and 50 μL of DTNB are added to each blank well. The system automatically adds 15 μL of supernatant, 85 μL of 0.1 M phosphate buffer, and 50 μL of 10 mmol·L⁻¹ using the 96-channel pipette on the pipetting workstation. -1 DTNB was then used, followed by a mechanical gripper that automatically transferred the well plate to a guide rail, which transported it to a CO2 incubator for incubation at 37°C for 5 minutes. After incubation, the well plate was transferred back to the pipetting station, where the system automatically added 50 μL of 8 mmol·L⁻¹ solution using the station's 96-channel pipette according to a pre-programmed sequence. -1 The plate was ATCI-treated and returned to a CO2 incubator for the set reaction time (10-90 minutes). After the reaction, the plate was transferred to a microplate reader using a mechanical gripper for absorbance detection. The microplate reader reads the absorbance value at 412 nm and uploads the data to the system analysis software for processing.

[0050] The results are shown in Figure 9. The results indicate that the reaction is in a linear phase when the reaction time is in the range of 15-25 min. Therefore, the optimal reaction time is determined to be 20 min (within a suitable range of 15-25 min).

[0051] (4) Optimization of reaction temperature: To optimize the reaction temperature of the exogenous acetylcholinesterase reaction system, this invention utilizes a high-throughput automated platform for gradient optimization of the reaction temperature. Different reaction temperatures (22, 30, 37, 42, 46, 52°C) were set in the system. In each blank well, only 150 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol·L⁻¹ were added. -1 DTNB was used to determine background absorbance. The system automatically added 15 μL of supernatant, 85 μL of 0.1 M phosphate buffer, and 50 μL of 10 mmol / L sodium chloride using the 96-well pipette on the main unit of the pipetting workstation. -1 DTNB is then automatically transferred to a guide rail by a mechanical gripper, and transported to a CO2 incubator for incubation at the set temperature for 5 minutes. After incubation, the plate is transferred back to the pipetting station, where the system automatically adds 50 μL of 8 mmol·L⁻¹ solution using the station's 96-channel pipette according to the set program. -1The plates were ATCI-treated and returned to a CO2 incubator for 20 minutes at the set temperature. After the reaction, the plates were automatically transferred to a microplate reader using a mechanical gripper for absorbance detection. The microplate reader read the absorbance value at 412 nm and uploaded the data to the system analysis software for processing.

[0052] The results are shown in Figure 10, indicating that the signal is stable when the reaction temperature is in the range of 37-42°C. Therefore, the preferred reaction temperature is determined to be 37°C (suitable range is 37-42°C).

[0053] (5) Establishment of a method for detecting exogenous acetylcholinesterase activity: Cells were seeded into 12-well plates of appropriate density using a high-throughput automated platform and incubated overnight in a 37°C, 5% CO2 saturated humidity incubator integrated into the workstation. The cell culture plates were placed in the designated positions on the automated workstation via guide rails and mechanical grippers. The culture medium in the plates was replaced, and the system automatically dispensed transfection reagents and plasmid solutions using the 96-well pipette of the pipetting workstation host, adding samples according to different transfection ratios (2:1, 3:1, 4:1, 5:1). The plates were then transferred to guide rails via mechanical grippers and transported to a CO2 incubator for 24 hours at 37°C and 5% CO2. After incubation, the plates were transferred to the pipetting workstation, and the culture supernatant was collected as the source of recombinant AChE enzyme solution. Blank wells were also prepared (each well containing only 150 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol·L⁻¹). -1 DTNB. The system automatically adds 15 μL of supernatant to each well using the 96-channel pipette on the main unit of the pipetting workstation, followed by the sequential addition of 85 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol·L⁻¹. -1 DTNB was applied, and the wells were then returned to a CO2 incubator for incubation at 37°C for 5 minutes. After incubation, the wells were transferred back to the pipetting station, where the system automatically added 50 μL of 8 mmol·L⁻¹ solution using the station's 96-channel pipette according to the pre-programmed sequence. -1 The plate was then incubated at ATCI and returned to a CO2 incubator at 37°C for 20 minutes. After the reaction, a mechanical gripper transferred the plate to a microplate reader, which read the absorbance at 412 nm and uploaded the data to the system analysis software for processing.

[0054] The results are shown in Figure 11. The results show that the control group had OD 412 The absorbance at nm was low, representing only a basic background signal; however, the absorbance in the cell culture supernatant of transfected AChE was significantly increased, and the OD value was much higher than that of the control group, indicating that recombinant human AChE could be successfully secreted and maintain high enzyme activity.

[0055] Example 4: Verification of the Reliability of the Endogenous Acetylcholinesterase Inhibitor Screening Method Using Positive Compounds To verify the reliability of the endogenous acetylcholinesterase inhibitor screening method established based on a high-throughput automated platform, the positive control compounds Mipafox and Paraoxon were selected for verification. The system used a 96-channel pipette on the main unit of the pipetting workstation to seed human neuroblastoma cells SH-SY5Y into 96-well plates, with 5 × 10⁶ cells per well. 4 Cells were collected. A robotic gripper then automatically transferred the well plate to a guide rail, which transported it to a CO2 incubator where it was incubated at 37°C and 5% CO2 for 24 hours. After incubation, the culture medium was automatically aspirated from the wells. Control and blank wells were set up. 100 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol / L sodium chloride were added to the control wells. -1 DTNB, 50 μL 8mmol·L -1 ATCI; Add 140 μL of 0.1 M phosphate buffer, 10 μL of the test compound solution, and 50 μL of 10 mmol·L⁻¹ to the blank well. -1 DTNB. The system prepared positive compounds Mipafox and Paraoxon into a 1 mmol·L⁻¹ solution using dimethyl sulfoxide (DMSO) as the solvent. -1 The stock solution was three-fold diluted to obtain 12 concentration gradients. The 96-channel pipette of the pipetting workstation main unit added 90 μL of 0.1 M phosphate buffer and 10 μL of different concentrations of positive compound solutions to each well. The plate was transferred to a CO2 incubator via a guide rail and incubated at 37°C for 30 minutes. After incubation, the plate was transferred back to the pipetting workstation, and the system automatically added 50 μL of 10 mmol·L⁻¹ solution according to the preset program using the 96-channel pipette of the pipetting workstation main unit. -1 DTNB was applied, and the wells were then returned to a CO2 incubator for incubation at 37°C for 5 minutes. After incubation, the wells were transferred back to the pipetting station, where the system automatically added 50 μL of 8 mmol·L⁻¹ solution using the station's 96-channel pipette. -1 The plates were incubated at ATCI and then returned to a CO2 incubator for 20 minutes. After the reaction, a mechanical gripper transferred the plates to a microplate reader, which read the absorbance at 412 nm and uploaded the data to the system analysis software for processing, thus determining the IC50 values ​​for Mipafox and Paraoxon. 50 value.

[0056] The results are shown in Figure 12. The results show that Mipafox and Paoraoxon have an effect on the IC50 of endogenous AChE. 50 The values ​​are 2.11 × 10 -6 M and 6.70×10 -9M, and Mipafox-AChE IC in published literature 50 : 13 - 46 μM, Paraoxon-AChE IC 50 When compared with 0.002 - 0.05 μM, the measured IC50 values ​​were... 50 The values ​​are consistent with the range reported in the literature, indicating that this method can effectively detect the inhibitory activity of compounds on acetylcholinesterase, and verifying that this method can be used to accurately identify compounds with neurotoxic targets (AChE).

[0057] Example 5: Verification of the Reliability of the Exogenous Acetylcholinesterase Inhibitor Screening Method Using Positive Compounds To verify the reliability of the exogenous acetylcholinesterase inhibitor screening method established based on a high-throughput automated platform, the positive control compounds Mipafox and Paraoxon were selected for verification. The system collected HEK293T cell culture supernatant containing recombinant human acetylcholinesterase as the enzyme source. The system prepared the positive compounds Mipafox and Paraoxon into a 1 mmol·L⁻¹ solution using dimethyl sulfoxide as the solvent. -1 The stock solution was diluted three-fold to obtain 12 concentration gradients, and control and blank wells were also included. The control wells contained only 15 μL of enzyme solution, 85 μL of 0.1 M phosphate buffer, and 50 μL of 10 mmol / L... - 1 DTNB, 50 μL, 8 mmol·L -1 ATCI; blank wells contain only 140 μL of 0.1 M phosphate buffer, 10 μL of positive compound solution, and 50 μL of 10 mmol·L⁻¹. -1 DTNB. The system uses the 96-channel pipette on the main unit of the pipetting workstation to add 15 μL of enzyme supernatant, 75 μL of 0.1 M phosphate buffer, and 10 μL of positive compound solutions of different concentrations to a 96-well plate. The mechanical gripper then automatically transfers the plate to a guide rail, which transports it to a CO2 incubator for incubation at 37°C for 30 minutes. After incubation, the plate is transferred back to the pipetting workstation, and the system uses the 96-channel pipette on the main unit of the pipetting workstation to automatically add 50 μL of DTNB according to the set program. The plate is then returned to the CO2 incubator for incubation at 37°C for 5 minutes. After incubation, the plate is transferred back to the pipetting workstation, and 50 μL of 8 mmol·L⁻¹ DTNB is added using the 96-channel pipette on the main unit of the pipetting workstation. -1 ATCI was performed, and the well plates were returned to a CO2 incubator at 37°C for 20 minutes. After the reaction, the plates were transferred to a microplate reader using a mechanical gripper. The reader read the absorbance at 412 nm and uploaded the data to the system analysis software for processing, to determine the IC50 values ​​for Mipafox and Paraoxon. 50 value.

[0058] The results are shown in Figure 13. The results show that Mipafox and Paoraoxon have an IC50 of exogenous AChE. 50 The values ​​are 6.11 × 10 -6 M and 7.94×10 -9 M, and Mipafox-AChE IC in published literature 50 : 13 - 46 μM, Paraoxon-AChE IC 50 When compared with 0.002 - 0.05 μM, the measured IC50 values ​​were... 50 The values ​​are consistent with the range reported in the literature, indicating that this method can effectively detect the inhibitory activity of compounds on acetylcholinesterase, and verifying that this method can be used to accurately identify compounds with neurotoxic targets (AChE).

[0059] Example 6: Detection system for the inhibitory activity of the test compound against endogenous and exogenous acetylcholinesterase. Human neuroblastoma cells SH-SY5Y were seeded into 96-well plates using a 96-channel pipette on a pipetting workstation, with 5 × 10⁶ cells per well. 4 Cells were collected, and then the mechanical gripper automatically transferred the well plate to the guide rail, which transported it to a CO2 incubator for 24 hours at 37°C and 5% CO2. After incubation, the culture medium was automatically aspirated from the wells, and control and blank wells were set up. The control wells contained only 100 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol / L sodium chloride solution. -1 DTNB, 50 μL 8mmol·L -1 ATCI; Add 140 μL of 0.1 M phosphate buffer, 10 μL of the test compound solution, and 50 μL of 10 mmol·L⁻¹ to the blank well. -1 DTNB. The system prepares the analyte compound into a 1 mmol·L⁻¹ solution using DMSO as the solvent. -1 The stock solution was diluted threefold to obtain 12 concentration gradients. The 96-channel pipette of the pipetting workstation main unit added 90 μL of 0.1 M phosphate buffer and 10 μL of different concentration compound solutions to each well. The mechanical gripper then automatically transferred the plate to a guide rail, which transported it to a CO2 incubator for incubation at 37°C for 30 minutes. After incubation, the plate was transferred back to the pipetting workstation, and the system automatically added 50 μL of 10 mmol·L⁻¹ solution according to the preset program using the 96-channel pipette of the main unit. -1 DTNB was applied, and the well plate was then returned to the CO2 incubator for incubation at 37°C for 5 minutes. After incubation, the well plate was transferred back to the pipetting station, and the system automatically added 50 μL of 8 mmol·L⁻¹ solution using the 96-channel pipette on the main unit according to the set program.-1 The plates were then incubated at ATCI and returned to a CO2 incubator for 20 minutes at 37°C. After the reaction, the plates were transferred to a microplate reader using a mechanical gripper, and the absorbance was read at 412 nm.

[0060] HEK293T cell culture supernatant containing recombinant human acetylcholinesterase was collected as the enzyme source. The test compound was prepared into a 1 mmol·L⁻¹ solution using DMSO as the solvent. -1 The stock solution was diluted three-fold to obtain 12 concentration gradients, and control and blank wells were also included. The control wells contained only 15 μL of enzyme solution, 85 μL of 0.1 M phosphate buffer, and 50 μL of 10 mmol / L... -1 DTNB, 50 μL, 8 mmol·L -1 ATCI; blank wells contain only 140 μL of 0.1 M phosphate buffer, 10 μL of positive compound solution, and 50 μL of 8 mmol·L⁻¹. -1 DTNB. The system uses the 96-channel pipette on the main unit of the pipetting workstation to add 15 μL of enzyme supernatant, 75 μL of 0.1 M phosphate buffer, and 10 μL of positive compound solutions of different concentrations to a 96-well plate. The mechanical gripper then automatically transfers the plate to a guide rail, which transports it to a CO2 incubator for incubation at 37°C for 30 minutes. After incubation, the plate is transferred back to the pipetting workstation, and the system automatically adds 50 μL of DTNB using the 96-channel pipette on the main unit according to the set program. The plate is then returned to the CO2 incubator for incubation at 37°C for 5 minutes. After incubation, the plate is transferred back to the workstation, and 50 μL of ATCI is added using the 96-channel pipette on the main unit. The plate is then returned to the CO2 incubator for reaction at 37°C for 20 minutes. After the reaction is complete, the mechanical gripper transfers the well plate to the microplate reader, which reads the absorbance value at 412 nm and uploads the detection data to the system analysis software for processing.

[0061] (1) Calculate the relative inhibition rate of acetylcholinesterase activity (%) according to the formula = (A1-A2) / (A3-A4) (2) A1 represents the absorbance with the test sample and recombinant enzyme added; A2 represents the absorbance without the recombinant enzyme added; A3 represents the absorbance without the test sample added; A4 represents the absorbance without the recombinant enzyme and test sample added.

[0062] Example 7: Co-screening of Endogenous and Exogenous Acetylcholinesterase Inhibitors Based on an Automated High-Throughput Platform In this example, the constructed automated platform integrates an automated pipetting workstation, a multi-functional microplate reader, and a central control module. A control program is written to uniformly schedule and coordinate the operation of each functional unit. The control program is used to automate the experimental process, including sample information retrieval, sample addition order setting, volume parameter control, reaction time management, and automatic acquisition and storage of detection data. During the detection process, the control program automatically schedules the endogenous and exogenous AChE detection systems according to a preset procedure. For each analyte compound, measurements are performed in both the endogenous and exogenous AChE detection systems. Twelve concentration gradients are set for each compound, with four replicate wells for each concentration gradient. Under the above experimental conditions, the running time of the automated platform is statistically analyzed. From the start of sample addition to the completion of detection by the microplate reader, the automated system can complete the complete detection process of 12 compounds per hour on average during continuous operation. The automated detection platform built based on this invention tested 49 compounds, and the data were reliable and reproducible, truly achieving high throughput and significantly improving evaluation efficiency. It provides low-cost and highly reliable technical support for subsequent large-scale compound library screening.

[0063] The method of this invention measures the IC50 of Paraoxon, Mipafox, CDP, and TPHP on endogenous AChE. 50 They are 6.70×10 -9 M, 2.11×10 -6 M, 2.58×10 -6 M and 6.53×10 -6 M; correspondingly, its IC50 of exogenous recombinant AChE 50 They are 7.94×10 -9 M, 6.11×10 -6 M, 7.48×10 -6 M and 1.98×10 -6 M. According to published literature, the oral LD50 of Paraoxon in rats... 50 The intraperitoneal LD50 of Mipafox in rats is 1.8 mg / kg. 50 The oral LD50 of CDP and TPHP in rats was 90 mg / kg. 50 The values ​​were 1420 mg / kg and 3500 mg / kg, respectively. Comprehensive analysis showed that compounds with higher acute toxicity levels (such as Paraoxon and Mipafox) exhibited strong inhibitory activity in both endogenous and exogenous AChE systems, while compounds with relatively lower acute toxicity (such as CDP and TPHP) had corresponding IC50 values ​​of 1420 mg / kg and 3500 mg / kg, respectively. 50The values ​​are generally high, and this trend is consistent with the IC values ​​measured in this experiment. 50 The same value indicates stronger inhibitory activity (IC50). 50 The lower the value of a compound, the lower its acute toxicity (LD50) in vivo. 50 The stronger the value, the better, which suggests that the IC... 50 With LD 50 There is a potential association between them, indicating that IC based on AChE inhibition... 50 Assays hold promise as an effective in vitro alternative for assessing the neurotoxicity of compounds, thus providing a viable alternative method for neurotoxicity risk assessment. Therefore, this invention establishes a reliable high-throughput toxicity pre-evaluation alternative method suitable for early warning of neurotoxicity risks in large-scale compound libraries.

Claims

1. A high-throughput alternative method for toxicity pre-evaluation based on human acetylcholinesterase targets, characterized in that, Based on the automated platform, the combined detection of endogenous and exogenous acetylcholinesterase was performed. The specific steps are as follows: S1. Construction of the endogenous AChE detection system: Human neuroblastoma cells SH-SY5Y were used as the endogenous model and seeded into the culture module of the automated platform to simulate the real response of compounds in a complex intracellular environment. S2. Construction of the exogenous AChE detection system: HEK293T cells were used as the expression host. After optimized transfection, soluble AChE with native conformation was efficiently secreted, facilitating the measurement of the direct inhibitory effect of the compound on recombinant AChE; S3. Automated high-throughput detection: The test compound was diluted to a preset concentration gradient. The compound was incubated with the above-mentioned endogenous cells and exogenous recombinant enzyme using an automated pipetting workstation. The Ellman assay was used to efficiently measure the compound using a multi-functional microplate reader, and the reaction data were automatically collected. S4. Data Processing: The system fits IC based on the absorbance data of the internal and external source systems. 50 The curves, by comparing the IC50 values ​​of the same compound in both intrinsic and extrinsic systems, show the results. 50 Differences in values ​​enable toxicity early warning.

2. The method according to claim 1, characterized in that, Step S1 includes: The system automatically dispenses cell suspension using the 96-channel pipette on the main unit of the pipetting workstation, and sets the SH-SY5Y cell density to 4×10⁻⁶. 4 - 6×10 4 Cells were seeded into wells, and the seeded 96-well plates were automatically transferred to a CO2 incubator for culture. After culture, phosphate buffer and DTNB were added to each well via the system's automatic dispensing module, and the plates were then transferred to a CO2 incubator for further incubation. Subsequently, the plates were transferred back to the pipetting station, and 50 μL of 2-6 mmol·L⁻¹ phosphate buffer was automatically added. -1 The plate was then incubated at 37-42°C for 15-25 minutes using an ATCI assay. After the reaction, the plate was automatically transferred to a microplate reader by a mechanical gripper to read the absorbance values ​​and upload the data for processing.

3. The method according to claim 2, characterized in that, In step S1, the cell seeding number is 5 × 10⁶. 4 Cells / well: The system's automatic dispensing module adds 100 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol·L⁻¹ to each well. -1 DTNB was transferred to a CO2 incubator and incubated at 37°C for 5 minutes.

4. The method according to claim 2, characterized in that, In step S1, the substrate ATCI was used at a concentration of 2 mmol·L⁻¹. -1 The reaction time was 20 min and the reaction temperature was 37°C.

5. The method according to claim 1, characterized in that, Step S2 includes: seeding cells to a suitable density using a high-throughput automated platform, incubating overnight in a 37°C, 5% CO2 saturated humidity incubator integrated into the workstation; automatically dispensing transfection reagents and plasmid solutions using the 96-well pipette on the pipetting workstation main unit, adding samples according to different transfection ratios; then automatically transferring the seeded plate to the CO2 incubator via a guide rail; after incubation, removing the plate from the incubator and transferring it to the pipetting workstation, collecting the culture supernatant as the source of recombinant AChE solution; replacing the 96-well plate, automatically adding transfection supernatant to each well of the 96-well plate using the high-throughput automated platform, along with phosphate buffer and DTNB; then returning the plate to the CO2 incubator for incubation; after incubation, transferring the plate back to the pipetting workstation, where the system automatically adds 50 μL of a solution with a concentration of 2-6 mmol·L⁻¹. -1 ATCI was then transferred to a 37-42°C, 5% CO2 incubator for 15-25 minutes. After the reaction, the absorbance value was automatically transferred to a microplate reader by a mechanical gripper and the detection data was uploaded for processing.

6. The method according to claim 5, characterized in that, In step S2, different transfection ratios of 2:1, 3:1, 4:1, and 5:1 were used for sample addition. The system automatically added 15 μL of transfection supernatant to each well of the 96-well plate via a high-throughput automated platform, along with 85 μL of 0.1 M phosphate buffer and 50 μL of 10 mmol·L⁻¹. -1 DTNB was then applied, and the plate was returned to the CO2 incubator and incubated at 37°C for 5 minutes.

7. The method according to claim 5, characterized in that, The substrate ATCI used in step S2 is at a concentration of 2 mmol·L⁻¹. -1 The reaction time was 20 min and the reaction temperature was 37°C.

8. The method according to claim 2 or 5, characterized in that, In steps S1 and S2, the cell suspension was cultured in a CO2 incubator at 37°C and 5% CO2 for 24 hours.

9. The method according to claim 1, characterized in that, Step S3 specifically involves seeding human neuroblastoma cells SH-SY5Y into 96-well plates, with 5 × 10⁶ cells per well. 4 One cell was cultured at 37°C and 5% CO2 for 24 hours. After incubation, the culture medium in the wells was removed, and control and blank wells were set up. The system prepared stock solutions of the test compounds using DMSO as solvent, and obtained 12 concentration gradients by three-fold dilution. After incubation in a CO2 incubator, the well plate was transferred back to the pipetting station, and 50 μL of 8 mmol·L⁻¹ solution was added. -1 ATCI was performed, and the well plate was then returned to a 37°C 5% CO2 incubator for 20 minutes. After the reaction, the well plate was transferred to a microplate reader by a mechanical gripper to read the absorbance at 412 nm. The supernatant of HEK293T cell culture containing recombinant human acetylcholinesterase was collected as the enzyme source, and the test compound was prepared into a 1 mmol·L⁻¹ solution using DMSO as the solvent. -1 The stock solution was diluted three-fold to obtain 12 concentration gradients, with control and blank wells included. Using the 96-channel pipette on the main unit of the pipetting station, 15 μL of enzyme supernatant, 75 μL of 0.1 M phosphate buffer, and 10 μL of different concentrations of positive compound solutions were added to a 96-well plate. After incubation in a CO2 incubator, the plate was transferred back to the pipetting station, and 50 μL of 8 mmol·L⁻¹ solution was added using the 96-channel pipette on the main unit. -1 The plate was then returned to the CO2 incubator and reacted at 37°C for 20 minutes. After the reaction, the plate was transferred to a microplate reader by a mechanical gripper to read the absorbance at 412 nm and the data was uploaded for processing.

10. The method according to claim 9, characterized in that, Incubation steps for human neuroblastoma cells SH-SY5Y in a CO2 incubator: Add 90 μL of 0.1 M phosphate buffer and 10 μL of different concentrations of compound solutions to each well, transfer to a CO2 incubator, and incubate at 37°C for 30 minutes; after incubation, transfer the plate back to the pipetting station, and add 50 μL of 10 mmol·L⁻¹ solution using the 96-channel pipette of the pipetting station main unit. -1 DTNB was added, and then the well plate was returned to the CO2 incubator and incubated at 37°C for 5 minutes. The incubation steps for recombinant human acetylcholinesterase HEK293T in a CO2 incubator were as follows: 15 μL of enzyme supernatant, 75 μL of 0.1 M phosphate buffer, and 10 μL of different concentrations of positive compound solutions were added to a 96-well plate using the 96-channel pipette of the pipetting workstation. The plate was then transferred to the CO2 incubator and incubated at 37°C for 30 minutes. After incubation, the well plate was transferred back to the pipetting workstation, and 50 μL of DTNB was added using the 96-channel pipette of the pipetting workstation. The plate was then returned to the CO2 incubator and incubated at 37°C for 5 minutes.

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