Determination method of ABHD6 enzyme activity

Abstract

The invention relates to the technical field of biological detection, in particular to a determination method of ABHD6 enzyme activity. The method comprises the following steps: a) catalyzing 2-arachidonyl glycerol (2-AG) to hydrolyze by ABHD6 to generate glycerol; b) adding glycerol kinase and adenosine triphosphate (ATP), and enabling the glycerol to generate glycerol-1-phosphoric acid (G-1-P) and adenosine diphosphate (ADP) under the catalysis of the glycerol kinase; and c) carrying out quantitative detection on ADP by adopting an ADP-Glo detection kit, and deducing the ABHD6 enzyme activity through the chemiluminescence signal intensity. Compared with an existing ABHD6 activity detection method based on radioactive labeling, mass spectrometry or fluorescence coupling, the ABHD6 activity detection method has the advantages that the ABHD6-glycerol kinase-ADP-Glo luminous system is established, so that the detection sensitivity, stability and flux are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a method for determining the activity of the ABHD6 enzyme. Background Technology

[0002] ABHD6 (α / β-hydrolase domain 6) is an endocannabinoid hydrolase that catalyzes the hydrolysis of 2-arachidonicoylglycerol (2-AG) to glycerol and arachidonic acid. ABHD6 is an important regulatory enzyme in the endocannabinoid system, participating in the metabolism of 2-AG along with ABHD12 and monoacylglycerol lipase (MAGL). Changes in its activity are closely related to the pathogenesis of various central nervous system and metabolic diseases. For example, ABHD6 inhibitors have shown potential therapeutic effects in disease models of neuropathic pain, epilepsy, anxiety, depression, Parkinson's disease, Alzheimer's disease, non-alcoholic fatty liver disease, and diabetes. Therefore, establishing a sensitive, stable, and high-throughput ABHD6 enzyme activity detection system is of great significance for screening ABHD6 inhibitors and related drugs.

[0003] Currently, the main technical solutions for detecting enzyme activity targeting ABHD6 are as follows: (1) Radioactive isotope method: using ³H or ¹ 4 Enzyme activity is calculated by measuring changes in radioactivity intensity using C-labeled 2-AG substrates. This method is highly sensitive, but requires stringent experimental conditions and can only be performed in laboratories with radioactivity accreditation. Furthermore, it has low throughput and high cost.

[0004] (2) Mass spectrometry quantification: The concentration change of arachidonic acid, the reaction product, is directly detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to reflect enzyme activity. Although this method can accurately quantify, the equipment is expensive, the operation is complicated, and the sample pretreatment is cumbersome, making it unsuitable for high-throughput screening.

[0005] (3) Fluorescently coupled enzyme method: ABHD6 hydrolyzes 2-AG to generate glycerol, which is then converted to glycerol-1-phosphate (G-1-P) by glycerol kinase, and then oxidized to hydrogen peroxide by glycerol-3-phosphate oxidase (GPO). Finally, under the catalysis of horseradish peroxidase (HRP), the substrate Amplex Red is converted to the red fluorescent product Resorufin. This method can achieve high throughput, but the system involves multiple coupling enzymes and substrates, the reaction chain is long, and it is easily affected by external conditions and the autofluorescence of the compound, resulting in unstable signals and false positive results.

[0006] In summary, existing methods for ABHD6 enzyme activity detection still have significant limitations in terms of sensitivity, stability, and applicability to high-throughput screening. In particular, fluorescence-coupled systems, due to the large number of coupled enzymes and complex interfering factors, struggle to obtain highly reliable results in drug screening. Therefore, there is an urgent need to develop a new method for ABHD6 enzyme activity detection that is simple in system design, highly sensitive, robust against interference, and suitable for high-throughput drug screening, in order to address the problems of existing technologies, such as operational complexity, signal instability, and limited detection throughput. Summary of the Invention

[0007] This invention covers the following technical solutions: One aspect of the present invention relates to a method for determining ABHD6 enzyme activity, comprising: a) ABHD6 catalyzes the hydrolysis of 2-arachidonicylglycerol (2-AG) to glycerol; b) Add glycerol kinase and adenosine triphosphate (ATP) to catalyze the glycerol to generate glycerol-1-phosphate (G-1-P) and adenosine diphosphate (ADP). c) ADP was quantitatively detected using the ADP-Glo ​​assay kit, and ABHD6 enzyme activity was inferred from the intensity of the chemiluminescent signal.

[0008] Another aspect of the present invention relates to an ABHD6 enzyme activity assay kit, comprising: 2-AG, glycerol kinase, ATP, and components of the ADP-Glo ​​assay kit.

[0009] Another aspect of the present invention relates to the use of the method described above, or the kit described above, in screening drugs related to ABHD6 enzyme activity.

[0010] Compared with existing methods for detecting ABHD6 activity based on radiolabeling, mass spectrometry, or fluorescence coupling, this invention significantly improves the sensitivity, stability, and throughput of detection by establishing an ABHD6–glycerol kinase–ADP-Glo ​​luminescent system. Attached Figure Description

[0011] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0012] Figure 1 : Schematic diagram of the ABHD6 ADP-Glo ​​experimental principle; Figure 2 ABHD6 protein titration results; Figure 3 : Km test plot of substrate 2-AG; Figure 4 : Graph of glycerol kinase titration results; Figure 5 : Results of ATP concentration titration; Figure 6 ABHD6 protein titration results; Figure 7 Results of experimental stability tests. Detailed Implementation

[0013] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0014] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0015] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the protein and biochemical, molecular biology-related terms and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0016] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0017] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0018] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0019] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0020] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0021] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0022] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0023] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0024] The first aspect of the present invention relates to a method for determining ABHD6 enzyme activity, comprising: a) ABHD6 catalyzes the hydrolysis of 2-arachidonicylglycerol (2-AG) to glycerol; b) Add glycerol kinase and adenosine triphosphate (ATP) to catalyze the glycerol to generate glycerol-1-phosphate (G-1-P) and adenosine diphosphate (ADP). c) ADP was quantitatively detected using the ADP-Glo ​​assay kit, and ABHD6 enzyme activity was inferred from the intensity of the chemiluminescent signal.

[0025] This method has at least one of the following technical effects: -High detection sensitivity and low protein dosage: The ADP-Glo ​​luminescent system indirectly reflects the enzyme activity of ABHD6 by detecting the amount of ADP generated by glycerol kinase, with the signal intensity showing a linear correlation with ADP concentration. In an exemplary embodiment, the detection limit of this system for ADP is approximately 8 nM, which is 1-2 orders of magnitude higher than that of traditional fluorescence methods. This significantly reduces the required concentrations of both ABHD6 and the substrate 2-AG, thereby saving on protein preparation and reagent costs.

[0026] - The system is simple, stable, and highly repeatable. This invention employs only a two-step enzymatic reaction (ABHD6 and glycerol kinase), reducing cascade errors and reaction uncertainties caused by multi-enzyme coupling in traditional fluorescence methods. Verification has shown that, in the exemplary embodiment, the detection window value Z′=0.83, with high signal stability, is suitable for long-term batch screening experiments.

[0027] -Strong anti-interference capability and high reliability of results: The fluorescence of the compound itself does not affect the chemiluminescence signal, which can effectively avoid the false positive or signal drift problems caused by the autofluorescence of candidate compounds in traditional fluorescence detection, thereby improving the accuracy of inhibitor screening and the reliability of structure-activity relationship (SAR) analysis.

[0028] -Easy to operate, with an excellent balance between cost and throughput: This system requires no radiolabeling or mass spectrometry instruments, relying solely on a conventional microplate reader for readings. Experiments are quick and scalable to 384-well or even 1536-well plates, enabling high-throughput screening. Furthermore, the cost of reagents and consumables is significantly lower than that of radioactive and LC-MS methods, making it highly economical.

[0029] - Wide range of applications: The detection system of this invention can be widely used in the discovery and screening of ABHD6 inhibitors or agonists, and is particularly suitable for the early development of drugs related to central nervous system diseases and metabolic diseases (such as neuropathic pain, Alzheimer's disease, non-alcoholic fatty liver disease, diabetes, etc.).

[0030] In this invention, ADP-Glo ​​refers to the ADP test kit provided by Promega, with the trade name ADP-Glo™ Kinase Assay or other variants such as ADP-Glo™ Max Assay, and its catalog number is V9102. There are also other specifications such as V9101 and V6930.

[0031] In some embodiments, the reaction system of the method is a buffer system comprising the following components: 0.5-1.5mM EDTA, 50-150mM Na + 3-7 mM Mg 2+ And 0.3%-0.7% BSA, pH=6.5-8.5.

[0032] The method for determining ABHD6 enzyme activity described in this invention employs an optimized buffer system to balance the enzymatic activities of ABHD6 and glycerol kinase. The reaction system includes EDTA and Na... + Components such as Mg²⁺ and BSA can provide the ionic environment and cofactors required for the reaction while maintaining the stability of the protein structure.

[0033] Furthermore, the concentration of EDTA in the buffer system is preferably 0.5-1.5 mM, such as 0.7 mM, 1.0 mM, or 1.3 mM, to chelate trace metal ions and reduce non-specific enzyme activity.

[0034] Furthermore, Na + The preferred concentration is 50-150 mM, such as 70 mM, 90 mM, 100 mM, 110 mM, or 130 mM, to maintain the ionic strength of the reaction and the spatial conformation of the protein.

[0035] Furthermore, the Mg²⁺ concentration is preferably 3-7 mM, such as 4 mM, 5 mM, or 6 mM, which can serve as an essential cofactor for ATP-dependent reactions.

[0036] Furthermore, the BSA concentration is preferably 0.3%-0.7%, for example 0.4%, 0.5%, or 0.6%; this can be used to prevent proteins from adsorbing onto the reaction plate wall under low concentration conditions and to maintain system stability.

[0037] As used herein, the term "buffer system" refers to an aqueous solution or composition that resists changes in pH when an acid or base is added. This resistance to pH changes is due to the buffering properties of such solutions. Therefore, solutions or compositions exhibiting buffering activity are called buffer solutions or buffered solutions. Buffer solutions generally do not have the ability to maintain the pH of a solution or composition indefinitely. Instead, they are generally able to maintain a pH within a specific range, typically 6.5–8.5, for example, pH = 7.0, which maintains optimal conditions for ABHD6 catalysis of 2-AG while avoiding the inhibition of ADP-Glo ​​luminescence by excessive Mg²⁺ concentrations. Generally, buffer solutions are able to maintain pH within their pKa and the next logarithm (see, for example, Mohan, Buffers, A guide for the preparation and use of buffers in biological systems, CALBIOCHEM, 1999). Buffer solutions and buffered solutions are generally prepared from buffer salts or preferably nonionic buffer components such as TRIS and HEPES. The buffer solution that can be used in the method of the present invention is preferably selected from phosphate buffer, phosphate-buffered saline (PBS), 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer, TRS buffered saline (TBS) and TRS / EDTA (TE).

[0038] By achieving a comprehensive balance between the aforementioned ions and protein stabilizers, both ABHD6 and glycerol kinase are kept in their high-activity range, resulting in a detection system with high signal-to-noise ratio and good repeatability.

[0039] In some embodiments, the final concentration of ABHD6 is 600-1000 nM, the final concentration of 2-AG is 150-250 μM, the final concentration of glycerol kinase is 0.1-0.3 U / mL, and the final concentration of ATP is 20-40 μM.

[0040] Specifically, the final concentration of ABHD6 is preferably 600-1000 nM, such as 700 nM, 800 nM, 900 nM, or 1000 nM. This concentration range ensures a linear relationship between the reaction rate and signal intensity without enzyme saturation. The final concentration of 2-AG is preferably 150-250 μM, such as 180 μM, 200 μM, or 220 μM. This concentration corresponds to a substrate concentration close to the enzyme kinetic constant (Km value), which can effectively amplify the inhibitor screening signal window. The final concentration of glycerol kinase is preferably 0.1-0.3 U / mL, such as 0.15 U / mL, 0.2 U / mL, or 0.25 U / mL, to ensure complete glycerol phosphorylation while avoiding excessively high background signals. The final concentration of ATP is preferably 20-40 μM, such as 25 μM, 30 μM or 35 μM. This range can meet the substrate requirements of the glycerol kinase reaction and avoid the interference of excessive ATP in the system on the ADP-Glo ​​luminescence reaction.

[0041] Under the optimized concentration conditions described above, the intensity of the resulting luminescent signal showed a good linear relationship with the amount of ADP generated.

[0042] In some embodiments, the reaction is carried out at 20-30 °C for 1.5-2.5 hours. Specifically, the reaction temperature is preferably 20-30 °C, such as 22 °C, 25 °C, or 28 °C, with 25 °C being the optimal reaction condition, which can obtain the highest luminescence intensity while ensuring enzyme stability. The reaction time is preferably 1.5-2.5 hours, such as 1.5 hours, 2.0 hours, or 2.5 hours. Within this range, the ADP generation rate is linearly correlated with the reaction time, and the system is stable without significant background drift.

[0043] Under the above temperature and time conditions, the hydrolysis reaction of 2-AG catalyzed by ABHD6 can proceed completely, and the signal intensity of the reaction system reaches its peak at 2 hours without significant decrease. A second aspect of the present invention relates to an ABHD6 enzyme activity assay kit, comprising: 2-AG, glycerol kinase, ATP, and components of the ADP-Glo ​​assay kit.

[0044] In this disclosure, the term "kit" may refer to any article (e.g., packaging or container) that includes at least one device and comprises the detection reagents as described in this disclosure. In some embodiments, the kit may further include instructions for use in the methods or steps described in this disclosure, and preferably may also include supplementary reagents and / or components or assemblies.

[0045] The components of the kit can be packaged individually or in combination as needed. The components can be packaged in solution, solid, or test strip form. In some embodiments, at least one component of the kit is a solid, including at least one of lyophilized microspheres, lyophilized cakes, lyophilized powder, and spots dependent on a solid medium. Therefore, components required for ABHD6 enzyme activity detection, particularly various enzyme, peptide components, and reaction buffer components, can be provided in lyophilized form.

[0046] A third aspect of the invention relates to the use of the method described above, or the kit described above, in screening drugs related to ABHD6 enzyme activity.

[0047] In some embodiments, the drug is an agonist or inhibitor of the ABHD6 enzyme.

[0048] In some embodiments, the drug is a candidate drug for central nervous system diseases or metabolic diseases targeting ABHD6.

[0049] In some embodiments, the central nervous system disease candidate drug is used for diseases associated with imbalances in the endocannabinoid system. In some embodiments, these include one or more of the following: neuropathic pain, epilepsy, depression, anxiety disorders, Parkinson's disease, Alzheimer's disease, multiple sclerosis, post-traumatic stress disorder, ischemic brain injury, spinal cord injury, and neuroinflammatory-related diseases. In some embodiments, the metabolic disease candidate drug is used for diseases associated with lipid metabolism disorders or insulin resistance; preferably including one or more of obesity, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD / NASH), atherosclerosis, metabolic syndrome, and insulin-resistant hyperglycemia.

[0050] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0051] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0052] Example 1: ABHD6 enzyme activity assay Experimental preparation: Obtaining ABHD6 protein, substrates and other related reagents. For details of the reagents, consumables and equipment involved, please refer to Table 1. 1) The experimental buffer solution was: 50 mM Tris (pH 7.5), 1 mM EDTA, 100 mM NaCl, 5 mM MgCl2, and 0.5% BSA. 2) Prepare 3× ABHD6 protein working solution and dilute it with experimental buffer; 3) Prepare 3× 2-AG and dilute it with experimental buffer; 4) Prepare a mixed working solution of 3× glycerol kinase and ATP, and dilute it with experimental buffer.

[0053] Experimental steps: 1) Transfer 2 μL of 3× ABHD6 protein working solution (final concentration 800 nM) to an OptiPlate 384-well plate and centrifuge at 1000 rpm for 1 min; 2) Transfer 2 μL of 3× 2-AG working solution (final concentration 200 μM) to an OptiPlate 384-well plate and centrifuge at 1000 rpm for 1 min; 3) Pre-incubate in a 25 ℃ constant temperature incubator for 30 min; 4) Transfer 2 μL of a mixture of 3× glycerol kinase and ATP (final concentration of glycerol kinase: 0.2 U / ml, final concentration of ATP: 30 μM) to an OptiPlate 384-well plate and centrifuge at 1000 rpm for 1 min. 5) Incubate at 25 ℃ for 2 hours; 6) Read the Luminescence signal value using a BMG microplate reader.

[0054] The experimental reagents, consumables, and instruments used in this embodiment are shown in the table below.

[0055]

[0056]

[0057] Example 2: Optimization process of ABHD6 enzyme activity assay 1) ABHD6 protein titration test To determine the appropriate ABHD6 concentration, protein titration was performed, starting at 2000 nM and serially diluted twofold, resulting in nine concentration points. A control well containing only experimental buffer and no protein was included. The 2-AG concentration was set at 50 μM, the final glycerol kinase concentration at 1 U / ml, and the final ATP concentration at 25 μM. The reaction time was 2 h. Experimental data showed that ABHD6 protein remained within the linear range up to 2000 nM, and the experimental window was approximately 5-fold at 1000 nM. This concentration was chosen for the substrate 2-AG titration.

[0058] 2) 2-AG concentration titration test To determine the Km value of substrate 2-AG, a 2-AG assay was performed. The 2-AG concentration was set at nine concentration points, starting from 2000 μM and serially diluted 2-fold. A control without ABHD6 protein was included. ABHD6 protein was used at a concentration of 1000 nM. The final concentration of glycerol kinase was 1 U / ml, and the final concentration of ATP was 25 μM. The reaction time was 2 h. The results showed that the Km of 2-AG was 206 μM. 200 μM of 2-AG will be used for further testing.

[0059] 3) Glyceryl kinase titration test To determine the optimal concentration of glycerol kinase, different concentrations of glycerol kinase were tested, starting at 2 U / ml and diluting twice for seven concentration points. A control well without ABHD6 protein was used as a low control. ABHD6 protein was added at 1 μM, and 2-AG at 200 μM. ATP was added at 25 μM, and the reaction time was 2 h. Experimental data showed that the glycerol kinase concentration remained relatively constant up to 0.13 U / ml, and a concentration of 0.2 U / ml was selected for further testing.

[0060] 4) ATP concentration titration test To confirm the appropriate ATP concentration, different ATP concentrations were tested, starting at 250 μM and diluting 2-fold at seven concentration points. A control well without ABHD6 protein was used as a low control. In the experiment, ABHD6 protein was used at 1 μM, and 2-AG at 200 μM. Glyceryl kinase concentration was 0.2 U / ml, and the reaction time was 2 h. The results showed that the Km of ATP was 104 μM. A concentration of 30 μM, with a lower Km, was selected for subsequent tests, resulting in a larger experimental window.

[0061] 5) ABHD6 protein titration test Because the concentrations of 2-AG, glycerol kinase, and ATP differed from those in the initial protein titration, a second protein titration was performed to better confirm the protein concentration. Different protein concentrations were set, starting with ABHD6 at 2000 nM, with seven 2-fold dilutions, and a low control group without ABHD6 protein was included. The 2-AG concentration was 200 μM, glycerol kinase was 0.2 U / ml, and ATP was 30 μM. After 2 hours of reaction, data showed that the ABHD6 protein concentration remained linear within 2000 nM. 800 nM ABHD6 protein was selected as the final test concentration.

[0062] 6) ABHD6 ADP-Glo ​​Experimental Stability Test To verify the stability of the experiment, optimized conditions were used for testing. The final concentration of ABHD6 was 800 nM, and a low control group without ABHD6 protein was set up. The concentration of 2-AG was 200 μM, glycerol kinase was 0.2 U / ml, and ATP was 30 μM. The reaction time was 2 h. The results showed that the experiment was very stable, with a Z' value of 0.83.

[0063] The results of the above experiment are as follows Figures 2-7 As shown.

[0064] Example 3: Comparison with traditional detection methods 1. Comparison of experimental throughput and cost: As shown in the table below, in terms of experimental reagents, only the coupled fluorescence method uses a relatively large number of reagents, resulting in a higher unit price; the reagent costs for the other three methods are comparable. In terms of experimental consumables, the LC-MS method is the most expensive, while the fluorescence method and the ADP-Glo ​​method are comparable. Except for the LC-MS method, the other three methods can all perform high-throughput testing. Due to the special nature of radioactive experiments, the ADP-Glo ​​method offers the most suitable balance between cost and high throughput.

[0065]

[0066] 2. Comparison of anti-interference performance The data are as follows: Two molecules were added simultaneously to the experimental buffer, and the fluorescence values ​​were read at Ex / Em=530 / 590 and on the chemiluminescence assay program, respectively. The 0 concentration point of compound 1 served as a blank control. The results are as follows: The high concentration points of compounds 1 and 2 in the fluorescence assay showed significantly higher readings than the blank control, indicating that the compounds exhibit autofluorescence, which interferes with the fluorescence assay readings. The chemiluminescence assay readings of the compounds were consistent with the blank control, indicating that the compounds themselves do not interfere with the assay.

[0067]

[0068] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Methods for determining ABHD6 enzyme activity, including: a) ABHD6 catalyzes the hydrolysis of 2-arachidonicylglycerol (2-AG) to glycerol; b) Add glycerol kinase and adenosine triphosphate (ATP) to catalyze the glycerol to generate glycerol-1-phosphate (G-1-P) and adenosine diphosphate (ADP). c) ADP was quantitatively detected using the ADP-Glo ​​assay kit, and ABHD6 enzyme activity was inferred from the intensity of the chemiluminescent signal.

2. The method according to claim 1, wherein the reaction system of the method is a buffer system comprising the following components: 0.5 - 1.5 mM EDTA, 50 - 150 mM Na 2+ , 3 - 7 mM Mg 2+ and 0.3% - 0.7% BSA, pH = 6.5 - 8.

5.

3. The method according to claim 1 or 2, wherein the final concentration of ABHD6 is 600-1000 nM, the final concentration of 2-AG is 150-250 μM, the final concentration of glycerol kinase is 0.1-0.3 U / mL, and the final concentration of ATP is 20-40 μM.

4. The method according to claim 3, wherein the reaction is carried out at 20-30 °C for 1.5-2.5 hours.

5. An ABHD6 enzyme activity assay kit, comprising: 2-AG, glycerol kinase, ATP, and components of the ADP-Glo ​​assay kit.

6. The method according to any one of claims 1-4, or the kit according to claim 5, in the screening of drugs related to ABHD6 enzyme activity.

7. The application according to claim 6, wherein the drug is an agonist or inhibitor of the ABHD6 enzyme.

8. The application according to claim 6, wherein the drug is a candidate drug for central nervous system diseases or metabolic diseases targeting ABHD6.

9. The application according to claim 8, wherein the central nervous system disease candidate drug is used for diseases associated with imbalances in the endocannabinoid system; preferably comprising: Neuropathic pain, epilepsy, depression, anxiety, Parkinson's disease, Alzheimer's disease, multiple sclerosis, post-traumatic stress disorder, ischemic brain injury, spinal cord injury, and neuroinflammatory-related diseases.

10. The application according to claim 8, wherein the metabolic disease candidate drug is used for diseases associated with lipid metabolism disorders or insulin resistance; preferably including one or more of obesity, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD / NASH), atherosclerosis, metabolic syndrome, and insulin-resistant hyperglycemia.

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