Nav channel protein binding agent screening and identifying method based on ligand-induced protein stability change
By combining a ligand-induced protein stability change-based method with protein restriction enzyme digestion and immunoblotting analysis, the high cost and low throughput of existing Nav channel protein binding agent screening and identification methods have been solved, enabling rapid and low-cost screening and affinity assessment of Nav channel protein binding agents.
Patent Information
- Application Number
- CN202511157157.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-19
- Publication Date
- 2025-11-21
AI Technical Summary
Existing methods for screening and identifying Nav channel protein binders rely on large instruments, resulting in low throughput, high cost, and significant technical challenges. These methods are not efficient, rapid, or low-cost for screening and identifying the affinity of compounds for Nav channel proteins.
Using a ligand-induced protein stability-based approach, Nav channel protein binding agents, including agonists and inhibitors, were screened and identified through Nav channel protein activity extraction, protein restriction enzyme digestion in the presence of surfactants, and Western blot analysis.
This enables rapid, efficient, and low-cost screening, identification, and affinity assessment of Nav channel protein binders, avoiding reliance on large instruments, improving screening efficiency, and reducing costs.
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Figure CN120992921A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of target screening and identification, and particularly relates to a Na v channel protein binding agent screening and identification method based on ligand-induced protein stability change. BACKGROUND
[0002] Voltage-gated sodium ion channel protein, hereinafter referred to as Na v channel protein, is a kind of multi-subunit transmembrane channel protein complex responsible for the rapid influx of sodium ions in the process of cell membrane depolarization, involved in the generation and propagation of action potential, and plays a key role in important physiological functions such as nerve conduction, muscle contraction, pain perception, etc. The development of high-efficiency binding agents for Na v channel protein is of great significance for the development of drugs for the treatment of diseases such as epilepsy, arrhythmia, pain, etc.
[0003] Electrophysiological methods are currently the gold standard for screening Na v channel protein binding agents, but the method relies on large instruments, has low detection throughput, high cost, and great technical difficulty, which limits the screening efficiency of new Na v channel protein binding agents. Therefore, the research on the screening and identification method of Na v channel protein binding agents has important significance for accelerating the screening of high-activity Na v channel protein binding agents, improving the discovery efficiency of Na v channel targeting drug lead compounds, and accelerating the development of related drugs.
[0004] At present, there is a lack of Na v channel protein binding agent screening and identification methods based on affinity. Conventional patch clamp technology, high-throughput real-time fluorescence detection analysis technology, and other Na v channel protein binding agent screening methods belong to screening and identification methods based on activity, which cannot directly detect the affinity of compounds and Na v channel protein; surface plasmon resonance, isothermal titration calorimetry, and other screening and identification methods based on affinity require pre-modification or labeling of active molecules or Na v channel protein, which is complicated and difficult in technology. At the same time, both methods rely on expensive special large instruments, which are high in cost and difficult to meet the requirements of efficient, rapid, and low-cost development of new Na v channel protein binding agents.
[0005] Therefore, it is necessary to develop a Na v channel protein binding agent screening method to effectively improve the existing Na v channel protein binding agent screening efficiency and cost, and to provide technical support for the development of new Na v channel protein targeting drugs. SUMMARY
[0006] The purpose of the present application is to solve the problems of the existing Na v channel protein binding agent screening method relies on large instruments, low detection flux, high cost and high technical difficulty, and provides a Na v channel protein binding agent screening method based on ligand-induced protein stability changes. v channel protein activity extraction, protein restriction enzyme digestion under the condition of surfactant and immunoblotting analysis technology, realizing the rapid, efficient and low-cost screening and identification of Na v channel protein binding agent (including agonists and inhibitors) and affinity evaluation.
[0007] The technical solutions adopted by the present application are as follows:
[0008] A Na v channel protein binding agent screening method based on ligand-induced protein stability changes, comprising the following steps:
[0009] S1 protein lysis: using a buffer containing 0.5-2% dodecyl-beta-D-maltoside, 0.05-0.5% cholesteryl hemisuccinate and trimethylammonium methane as a protein lysis solution, mixed with Na v channel protein expression cells to obtain a mixed solution by complete lysis;
[0010] Centrifuge the mixed solution to obtain the protein lysis solution; detect the concentration of the protein lysis solution;
[0011] S2 incubation: incubate the protein lysis solution with gradient concentrations of test compounds and DMSO at room temperature, as experimental and control groups;
[0012] S3 restriction enzyme digestion: according to m (蛋白裂解液) : m (蛋白酶)=1000:1~10000:1, the protease is added to the protein lysate of the experimental group and the control group, after incubation at room temperature, SDS is added to make the final mass concentration 0.5~5%, and the enzyme hydrolysis is continued at 30~40℃; then the protein denaturation loading buffer is added, the protein denaturation loading buffer includes SDS with a mass concentration of 5~15%, glycerol with a mass concentration of 30~60%, 2~8% β-mercaptoethanol, and incubation at 90~98℃, so as to completely inactivate the protease and complete the sample preparation;
[0013] S4 enzyme hydrolysis detection: the sample is detected to analyze the enzyme hydrolysis ratio of Na v The concentration difference of the channel protein;
[0014] If there is a significant difference in the concentration between the control group and the experimental group, the tested compound has specific binding with Na v 1.5 The channel protein has specific binding.
[0015] If there is no significant difference, it indicates that the tested compound has no specific binding with Na v 1.5 The channel protein has no specific binding.
[0016] Preferably, the step S4 adopts the immunoblotting analysis method, and the specific steps are as follows:
[0017] In step S3, the protein denaturation loading buffer also includes 0.1~1% electrophoretic tracking dye;
[0018] Step S4: prepare 5~15% separation gel and 2~6% concentration gel, load the protein lysate in each well, and perform constant voltage electrophoresis until the tracking dye migrates to the bottom of the gel; after soaking and activating the PVDF membrane, use wet transfer constant flow membrane transfer, 2~8% skimmed milk powder is blocked at room temperature; dilute the Na v channel protein polyclonal antibody with 2~8% skimmed milk powder, 0.2~0.8% Tween 20 in TBST buffer, and incubate overnight at 2~8℃; dilute the HRP-labeled secondary antibody with 0.2~0.8% Tween 20 in TBST buffer, and incubate at room temperature; cover the PVDF membrane with ECL, develop using a chemiluminescence imager, and analyze the enzyme hydrolysis ratio of Na v channel protein, and β-actin is used as an internal reference protein.
[0019] Further, in step S4, whether there is a statistically significant difference in the enzyme hydrolysis ratio of Na v channel protein between the experimental group and the control group is determined.
[0020] Preferably, the electrophoretic tracking dye is bromophenol blue.
[0021] Preferably, the Na vThe channel protein expression cell is a human colorectal cancer cell line SW620 cell.
[0022] Preferably, the protein lysis solution comprises 100 mM Tris-HCl, 1% dodecyl-beta-D-maltoside, 0.1% cholesteryl hemisuccinate tris-hydroxymethyl aminomethane.
[0023] Preferably, the protein denaturation loading buffer is a 250 mM Tris-HCl buffer containing 10% SDS, 0.5% electrophoretic tracking dye, 50% glycerol and 5% beta-mercaptoethanol.
[0024] Preferably, in step S1, after the protein lysis solution is added, the syringe is repeatedly aspirated several times to ensure complete lysis.
[0025] Preferably, in step S1, the protein lysis solution concentration is detected by BCA.
[0026] Preferably, the protease is proteinase K.
[0027] The Na v The screening principle of the channel protein binding agent is that the ligand induces changes in protein stability. During the protein limited hydrolysis process, surfactant-type allosteric agents are contained in the lysis solution, and the technical effect is achieved through a specially designed protein denaturation loading buffer.
[0028] The beneficial effects of the present application are:
[0029] The Na v The channel protein binding agent screening and identification method has the advantages of being based on affinity to carry out Na v The channel protein binding agent screening and identification method has the advantages of being based on affinity to carry out Na v The channel protein binding agent screening and identification method cannot directly detect the affinity of the compound to the Na v Channel protein based on affinity screening and identification methods such as surface plasmon resonance and isothermal titration calorimetry require pre-modification or labeling of active molecules or Na v Channel protein, the steps are complicated and the technical difficulty is great. At the same time, both methods rely on expensive special large instruments, the cost is high, the detection throughput is low, and it is difficult to meet the efficient, rapid and low-cost development of new Na v Channel protein binding agent discovery problems. BRIEF DESCRIPTION OF DRAWINGS
[0030] Figure 1Western blotting image of the effect of flecainide on the anti-enzymatic stability of SCN5A protein
[0031] Figure: SCN5A, voltage-gated channel alpha subunit 5 protein.
[0032] Figure 2 Relative retention amount of SCN5A protein after treatment with different concentrations of flecainide
[0033] Figure: **, there was a statistical difference between the drug administration group and the DMSO control group, p<0.01; ***, there was a statistical difference between the drug administration group and the DMSO control group, p<0.001. Statistical analysis was performed using two-tailed t-test.
[0034] Figure 3 Western blotting image of the effect of bullet ant toxin on the anti-enzymatic stability of SCN5A protein
[0035] Figure 4 Relative retention amount of SCN5A protein after treatment with different concentrations of bullet ant toxin
[0036] Figure: **, there was a statistical difference between the drug administration group and the DMSO control group, p<0.01; ***, there was a statistical difference between the drug administration group and the DMSO control group, p<0.001. Statistical analysis was performed using two-tailed t-test.
[0037] Figure 5 Western blotting image of the effect of staurosporine on the anti-enzymatic stability of SCN5A protein
[0038] Figure: SCN5A, voltage-gated channel alpha subunit 5 protein.
[0039] Figure 6 Relative retention amount of SCN5A protein after treatment with different concentrations of staurosporine
[0040] Figure: n.s, there was no statistical difference between the drug administration group and the DMSO control group. Statistical analysis was performed using two-tailed t-test. DETAILED DESCRIPTION
[0041] The application will be further described below in conjunction with the accompanying drawings and examples.
[0042] In the following examples, the protein lysis solution uses 100 mM Tris-HCl buffer (pH 7.5, containing 1% dodecyl-β-D-maltoside, 0.1% cholesteryl hemisuccinate trimethylammonium methane); the protein denaturation loading buffer uses 250 mM Tris-HCl buffer (pH 6.8, containing 10% SDS, 0.5% bromophenol blue, 50% glycerol and 5% β-mercaptoethanol).
[0043] Example 1
[0044] Example 1 provides a method for identifying the affinity of a fluorocarbazamine to Na v 1.5 Affinity identification test of channel protein.
[0045] SW620 cells were cultured in Leibovitz's L-15 medium (containing 10% FBS, 1% P / S), and when the cells in the T25 cell culture flask reached 80% confluence, the cells were washed with PBS three times, 200 μL of protein lysate was added to lyse the cells, and the syringe was repeatedly sucked for 10 times to ensure sufficient lysis. The lysate was collected and centrifuged at 13,000g for 10 min in a low-temperature high-speed centrifuge. The supernatant was taken and the concentration of the above protein lysate was detected by BCA. 40 μL of the protein lysate was incubated with DMSO (control group) and fluorocarbazamine (FLE) at a final concentration of 5, 10, 20, 40 and 80 μM at room temperature for 15 min. The above solution was then divided into two groups (group A and group B) in equal volume. 5 μL of PBS was added to each tube in group A, and 5 μL of proteinase K was added to each tube in group B. After incubation at room temperature for 5 min, 2% SDS was added, and the incubation was continued at 37°C for 30 min. Protein denaturation loading buffer was added, and the incubation was continued at 95°C for 10 min.
[0046] 10% separation gel and 4% concentration gel were prepared, and 20 μg of protein lysate was loaded on each well. Electrophoresis was carried out at a constant voltage of 100 V until the bromophenol blue migrated to the bottom of the gel. After the PVDF membrane was soaked in 100% methanol for 1 min, wet transfer was performed at a constant current of 300 mA for 2 h, and 5% skimmed milk powder was blocked at room temperature for 1 h. The primary antibody diluent was used to dilute the Na v 1.5 Polyclonal antibody of channel protein (SCN5A), incubated at 4°C overnight. The HRP-labeled secondary antibody was diluted in TBST buffer containing 0.5% Tween 20, and incubated at room temperature for 2 h.
[0047] ECL covers the PVDF membrane, and the chemiluminescence imager is used for development. The results of immunoblotting analysis are shown in Figure 1 The change in gray value of the band indicates that the hydrolysis of Na v channel protein can be significantly inhibited by fluorocarbazamine treatment. v 1.5 Hydrolysis of channel protein, Figure 2 , indicating that fluorocarbazamine can specifically bind to Na v 1.5 channel protein.
[0048] Example 2
[0049] Example 2 provides a method for identifying the affinity of a bullet ant toxin to Na v channel protein:
[0050] SW620 cells were cultured in Leibovitz's L-15 medium (containing 10% FBS, 1% P / S), and when the cells in the T25 cell culture bottle reached 80% confluence, the cells were washed with PBS three times, 200 μL of protein lysate was added to lyse the cells, and the syringe was repeatedly sucked for 10 times to ensure complete lysis. The lysate was collected and centrifuged at 13,000 g for 10 min in a low-temperature high-speed centrifuge. The supernatant was taken, and the concentration of the above protein lysate was detected by BCA. 40 μL of protein lysate was incubated with DMSO (control group) or bullet antitoxin (PoTX) at a final concentration of 3.125, 6.25, 12.5, 25, and 50 μM at room temperature for 15 min. The above solution was then divided into two groups (group A and group B) in equal volume. 5 μL of PBS was added to each tube in group A, and 5 μL of proteinase K was added to each tube in group B. After incubation at room temperature for 5 min, 2% SDS was added, and the incubation was continued at 37°C for 30 min. Protein denaturation loading buffer was added, and the incubation was continued at 95°C for 10 min.
[0051] 10% separation gel and 4% concentration gel were prepared, and 20 μg of protein lysate was loaded on each well. Electrophoresis was performed at 100 V constant voltage until the bromophenol blue migrated to the bottom of the gel. After the PVDF membrane was activated by soaking in 100% methanol for 1 min, wet transfer was performed at 300 mA constant current for 2 h, and 5% skim milk was blocked at room temperature for 1 h. The primary antibody diluent was used to dilute the Nav1.5 channel protein (SCN5A) polyclonal antibody, and the incubation was performed at 4°C overnight. The HRP-labeled secondary antibody was diluted in TBST buffer containing 0.5% Tween 20, and the incubation was performed at room temperature for 2 h.
[0052] ECL was used to cover the PVDF membrane, and the chemiluminescence imager was used for development. The results of Western blot analysis are shown in Figure 3 The proportion of Na v channel protein enzymolysis was analyzed by band gray value change, and β-actin was used as an internal reference protein. The treatment of bullet antitoxin can significantly inhibit the hydrolysis of Na v 1.5 channel protein, Figure 4 , indicating that the bullet antitoxin can specifically bind to Na v 1.5 channel protein.
[0053] Example 3
[0054] Example 3 provides a method for identifying the affinity of starfishin to Na v 1.5 channel protein.
[0055] SW620 cells were cultured in Leibovitz's L-15 medium (containing 10% FBS, 1% P / S), and when the cells in a T25 cell culture flask reached 80% confluence, the cells were washed with PBS three times, 200 μL of protein lysate was added to lyse the cells, and the syringe was repeatedly sucked for 10 times to ensure sufficient lysis. The lysate was collected, centrifuged at 13,000g for 10 min in a low-temperature high-speed centrifuge, and the supernatant was taken, and the concentration of the above protein lysate was detected by BCA. 40 μL of the protein lysate was incubated with DMSO (control group) or staphylinosin at a final concentration of 6.25, 12.5, 25, 50 and 100 μM at room temperature for 5 min, and then the above solution was equally divided into two groups (group A and group B). 5 μL of PBS was added to each tube in group A, and 5 μL of proteinase K was added to each tube in group B, and then incubated at room temperature for 5 min, and then 2% SDS was added, and then incubated at 37°C for 30 min. Protein denaturation loading buffer was added, and then incubated at 95°C for 10 min.
[0056] 10% separation gel and 4% concentration gel were prepared, 20 μg of protein lysate was loaded on each well, and electrophoresis was performed at 100V constant voltage until the bromophenol blue migrated to the bottom of the gel. After the PVDF membrane was soaked in 100% methanol for 1 min, wet transfer was performed at 300mA constant current for 2h, and 5% skim milk was blocked at room temperature for 1h. The Na v 1.5 polyclonal antibody was diluted with the first antibody diluent, and then incubated at 4°C overnight. The HRP-labeled secondary antibody was diluted with TBST buffer containing 0.5% Tween 20, and then incubated at room temperature for 2h.
[0057] ECL was used to cover the PVDF membrane, and then developed by using a chemiluminescence imager, and the results of the Western blot analysis are shown in Figure 5 The Na v actin as an internal reference protein, and staphylinosin treatment had no obvious effect on the hydrolysis of Na v 1.5 channel protein, as shown in Figure 6 , indicating that the staphylinosin molecule does not specifically bind to Na v 1.5 channel protein.
[0058] Compared with the traditional electrophysiological method, the method of the present application can effectively improve the detection and screening rate, and does not require large experimental equipment, and the screening cost is low.
Claims
1. A Na v channel protein binding agent screening method characterized by, The steps include the following: S1 protein cleavage: using 0.5-2% dodecyl-β-D-maltoside, 0.05-0.5% cholesteryl hemisuccinate tris-hydroxymethyl aminomethane buffer as protein cleavage solution, with Na v Channel protein expression cell mixture, fully lysed to get mixed solution; The mixture is centrifuged to obtain a protein lysate; and the concentration of the protein lysate is detected; S2 incubation: the protein lysate is incubated with gradient concentrations of the test compound and DMSO at room temperature, as the experimental group and the control group; S3 restriction enzyme digestion: according to m (蛋白裂解液) : m (蛋白酶) =1000:1~10000:1, the protease is added to the protein lysate of the experimental group and the control group, and after incubation at room temperature, SDS is added to make the final mass concentration 0.5~5%, and the enzyme digestion is continued at 30~40℃; then protein denaturation loading buffer is added, which includes SDS with a mass concentration of 5~15%, glycerol with a mass concentration of 30~60%, 2~8% β-mercaptoethanol, and incubation at 90~98℃ to completely inactivate the protease and complete sample preparation; S4 enzymatic detection: The samples were detected to analyze the concentration of Na v The concentration difference of channel proteins; If there is a significant difference in the concentration between the control and experimental groups, then the compound to be tested binds to Na v 1.5 The presence of a channel protein is specific for the binding; If there is no significant difference, it indicates that the compound to be tested does not bind to Na v 1.5 Non-specific binding of the channel protein.
2. The Na based on ligand-induced protein stability changes as described in claim 1 v A method for screening and identifying channel protein binders, characterized in that, The step S4 adopts the immunoblotting analysis method, and the specific steps are as follows: In step S3, the protein denaturation loading buffer further includes 0.1-1% electrophoretic tracking dye. Step S4: prepare 5-15% separation gel, 2-6% concentration gel, load protein lysate per well, constant voltage electrophoresis until the tracking dye migrates to the bottom of the gel; after soaking and activating the PVDF membrane, use wet transfer constant current transfer membrane, 2-8% skim milk powder at room temperature for blocking; dilute Na v Channel protein polyclonal antibody, incubate overnight at 2-8°C; dilute HRP labeled secondary antibody with TBST buffer containing 0.2-0.8% Tween 20, incubate at room temperature; cover the PVDF membrane with ECL, develop using a chemiluminescence imager, and analyze the change in gray scale of the band to determine the Na v Enzymatic ratio of channel protein, β-actin as internal reference protein.
3. The Na based on ligand-induced protein stability changes as described in claim 1 v A method for screening and identifying channel protein binders, characterized in that, In the step S4, the Na v Whether the statistical difference exists in the enzymolysis ratio of the channel protein is determined.
4. The Na based on ligand-induced protein stability changes as described in claim 1 v A method for screening and identifying channel protein binders, characterized in that, The electrophoretic tracking dye is bromophenol blue.
5. The Na based on ligand-induced protein stability changes according to claim 1 v A method for screening and identifying channel protein binders, characterized in that, Na v The channel protein-expressing cells were human colorectal cancer cell line SW620 cells.
6. The Na based on ligand-induced protein stability changes according to claim 1 v A method for screening and identifying channel protein binders, characterized in that, The protein lysate contains 100 mM Tris-HCl, 1% dodecyl-beta-D-maltoside, and 0.1% cholesteryl hemisuccinate tris-hydroxymethyl aminomethane.
7. The ligand-based protein stability change inducing Na v The screening method for identifying a channel protein binding agent characterized by, The protein denaturation loading buffer is 250 mM Tris-HCl buffer containing 10% SDS, 0.5% electrophoretic tracking dye, 50% glycerol, and 5% beta-mercaptoethanol.
8. The ligand-based protein stability change inducing Na v The screening method for identifying a channel protein binding agent characterized by, In step S1, after the protein lysate is added, a syringe is repeatedly aspirated several times.
9. The ligand-based protein stability change inducing Na v The screening method for identifying a channel protein binding agent is characterized by, In step S1, the concentration of the protein lysate is detected by BCA.
10. The Na based on ligand-induced protein stability changes according to claim 1 v A method for screening and identifying channel protein binders, characterized in that, The protease is proteinase K.