Screening method of RNA helicase inhibitor
The inhibitory activity of RNA helicase was detected through the ADP-GloTM kinase detection kit, which solved the problem of difficulty in screening RNA helicase inhibitors in the prior art, and achieved high-throughput and high-sensitivity RNA helicase 5 and RNA Helicase 17 inhibitor screening has important clinical and market value.
Patent Information
- Application Number
- CN202411292302.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2024-12-17
AI Technical Summary
The existing technology is difficult to effectively screen inhibitors of RNA helicase, especially in the development of inhibitors targeting RNA helicase 5 or RNA helicase 17 in tumor treatment, and there is a lack of high-throughput and sensitivity detection methods.
The ADP-GloTM kinase detection kit was used to screen RNA helicase inhibitors. By mixing RNA helicase with the sample to be tested, adding dsRNA and adenosine triphosphate, terminating the reaction and consuming excess ATP, and then adding kinase detection reagent, the ADP is converted into ATP, and the inhibitory activity of the sample to be tested on RNA helicase is judged based on the fluorescence intensity.
Provides a stable, reliable, high-throughput and universal RNA helicase inhibitor screening method, which can be used for inhibitor screening of RNA helicase 5 and RNA helicase 17, with high sensitivity and suitable for Related enzymatic kinetics research.
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Figure CN119144693A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biochemistry, and in particular to a method for screening RNA helicase inhibitors. Background Art
[0002] RNA helicase 5 is a shuttling protein in the nucleus and cytoplasm. It belongs to the DEAD-box RNA helicase family and has ATP-dependent RNA unwinding activity. The protein is expressed in most tissues and cells and plays a role in regulating normal physiological functions. RNA helicase 5 is involved in almost all RNA metabolic processes, including alternative splicing of mRNA, biogenesis of micro RNAs and ribosomes, degradation of mRNA, interaction with long non-coding RNA, and coordinated regulation of transcriptional activity. In addition, different post-translational modifications of RNA helicase 5, such as phosphorylation, acetylation, ubiquitination, and sumoylation, give it an important regulatory function in the occurrence and progression of tumors. When the expression of RNA helicase 5 or its post-translational modification is dysregulated, the normal cell signaling network collapses, leading to the occurrence and development of a variety of pathological conditions, including the occurrence and development of tumors. Studies have shown that RNA helicase 5 is highly expressed in most tumors, and its abnormal expression is closely related to the occurrence and development of tumors and poor prognosis of patients. Inhibiting the activity of RNA helicase 5 can effectively inhibit tumor growth.
[0003] RNA helicase 17 belongs to the DEAD-box RNA helicase family, has ATP-dependent RNA unwinding activity, participates in RNA processing and modification, affects mRNA splicing and stability, and then regulates gene expression. RNA helicase 17 can regulate the expression of proto-oncogenes and tumor suppressor genes, and is therefore closely related to processes such as cell cycle, apoptosis, and cell proliferation. RNA helicase 17 is upregulated in a variety of cancers, such as breast cancer, non-small cell lung cancer, colorectal cancer, hepatocellular carcinoma, and prostate cancer. Its increased expression level may promote tumor growth, migration, and invasion. In the occurrence and development of tumors, RNA helicase 17 can regulate the expression of tumor-related genes, affecting cell cycle control, apoptosis, and DNA damage repair. Studies have found that high expression of RNA helicase 17 is associated with poor prognosis of tumors. Therefore, RNA helicase 17 is considered a potential target for cancer treatment.
[0004] The development of small molecule inhibitors targeting the activity of RNA helicase 5 or RNA helicase 17 is expected to become a new type of tumor targeted drug. Therefore, the development of an easy-to-implement high-throughput screening method for RNA helicase inhibitors has important clinical and market value.
[0005] In view of this, the present invention is proposed. Summary of the invention
[0006] The purpose of the present invention is to provide a method for screening RNA helicase inhibitors to solve the above technical problems.
[0007] In order to achieve the above objectives, the following technical solutions are adopted:
[0008] In a first aspect, the present invention provides a method for screening RNA helicase inhibitors using ADP-Glo TM The kinase assay kit is used to perform the assay, which includes the following steps:
[0009] a. Mixing the RNA helicase solution and the sample solution to be tested and incubating;
[0010] After the incubation in step ba, a substrate solution containing dsRNA and adenosine triphosphate is added to react;
[0011] After the reaction in step cb is completed, ADP Glo reagent is added to terminate the reaction and consume excess ATP;
[0012] After the dc step, a kinase detection reagent is added to convert ADP into ATP, and the inhibitory activity of the sample to be tested on RNA helicase is determined based on the fluorescence intensity.
[0013] As a further technical solution, the RNA helicase includes RNA helicase 5 or RNA helicase 17.
[0014] As a further technical solution, the concentration of RNA helicase in the RNA helicase solution is 18-22 nM;
[0015] The RNA helicase solution is a buffer containing RNA helicase;
[0016] The buffer is an aqueous solution containing 4-6 mM tris(hydroxymethyl)aminomethane, 1.8-2.2 mM dithiothreitol and 9-11 mM MgCl2;
[0017] The RNA helicase solution and the sample solution to be tested are mixed in equal volumes.
[0018] As a further technical solution, the number of the sample solutions to be tested is at least 1. When the number of the sample solutions to be tested is greater than 1, the concentrations of the sample solutions to be tested are different.
[0019] As a further technical solution, in step a, the incubation temperature is 24-28°C and the time is 18-22 minutes.
[0020] As a further technical solution, in the substrate solution, the concentration of dsRNA is 250-350 nM, and the concentration of adenosine triphosphate is 250-350 μM;
[0021] The substrate solution is an aqueous solution, and further comprises 4-6 mM tris(hydroxymethyl)aminomethane, 1.8-2.2 mM dithiothreitol and 9-11 mM MgCl2;
[0022] The solution after incubation in step a is mixed with an equal volume of substrate solution.
[0023] As a further technical solution, in step b, the reaction pH is 7-8, the temperature is 24-28°C, and the reaction time is 110-130 min.
[0024] As a further technical solution, the volume ratio of the RNA helicase solution to the ADPGlo reagent is 1:2;
[0025] Add ADP Glo reagent 50-70 minutes before proceeding to step d.
[0026] As a further technical solution, the volume ratio of the RNA helicase solution to the kinase detection reagent is 1:4;
[0027] 50-70 min after adding the kinase detection reagent, the fluorescence intensity was measured.
[0028] As a further technical solution, in step d, the half inhibition concentration of the sample to be tested is obtained according to the fluorescence intensity of the sample to be tested at different concentrations, and then the inhibitory activity of the sample to be tested on RNA helicase is determined.
[0029] Compared with the prior art, the present invention has the following beneficial effects:
[0030] The screening method for RNA helicase inhibitors provided by the present invention is stable and reliable, has high sensitivity, and has both high throughput and universality, and can be used for studies on enzymatic kinetics related to the screening of RNA helicase 5 inhibitors and RNA helicase 17 inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0032] Figure 1 The present invention is a dose-effect curve for detecting RNA helicase 5 inhibitors by the method of the present invention;
[0033] Figure 2 This is a dose-effect curve for detecting RNA helicase 17 inhibitors using the method of the present invention. DETAILED DESCRIPTION
[0034] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0035] In a first aspect, the present invention provides a method for screening RNA helicase inhibitors using ADP-Glo TM The kinase assay kit is used to perform the assay, which includes the following steps:
[0036] a. Mixing the RNA helicase solution and the sample solution to be tested and incubating;
[0037] After the incubation in step ba, a substrate solution containing dsRNA and adenosine triphosphate is added to react;
[0038] After the reaction in step cb is completed, ADPGlo reagent is added to terminate the reaction and consume excess ATP;
[0039] After the dc step, a kinase detection reagent is added to convert ADP into ATP, and the inhibitory activity of the sample to be tested on RNA helicase is determined based on the fluorescence intensity.
[0040] The screening method for RNA helicase inhibitors provided by the present invention is stable and reliable, has high sensitivity, and has both high throughput and universality, and can be used for studies on enzymatic kinetics related to the screening of RNA helicase 5 inhibitors and RNA helicase 17 inhibitors.
[0041] In some optional embodiments, the RNA helicase includes but is not limited to RNA helicase 5 or RNA helicase 17.
[0042] In some optional embodiments, the concentration of the RNA helicase in the RNA helicase solution may be, for example, but not limited to, 18 nM, 19 nM, 20 nM, 21 nM or 22 nM;
[0043] The RNA helicase solution is a buffer containing RNA helicase;
[0044] The buffer solution is an aqueous solution containing tris(hydroxymethyl)aminomethane, dithiothreitol and MgCl2; wherein the concentration of tris(hydroxymethyl)aminomethane may be, for example, but not limited to 4mM, 5mM or 6mM; the concentration of dithiothreitol may be, for example, but not limited to 1.8mM, 2mM or 2.2mM; the concentration of MgCl2 may be, for example, but not limited to 9mM, 10mM or 11mM.
[0045] The RNA helicase solution and the sample solution to be tested are mixed in equal volumes.
[0046] In some optional embodiments, the number of the sample solutions to be tested is at least 1. When the number of the sample solutions to be tested is greater than 1, the concentrations of the sample solutions to be tested are different.
[0047] When the number of the test sample solution is 1, a positive control group (known RNA helicase inhibitor) can be set up, and the inhibitory activity of the test sample on RNA helicase can be determined by comparing the fluorescence signal intensity of the test sample with that of the positive control group.
[0048] When there are multiple samples to be tested, the half-inhibitory concentration of the sample to be tested can be obtained according to the fluorescence intensity of the samples to be tested with different concentrations, and then the inhibitory activity of the sample to be tested on RNA helicase can be determined.
[0049] In some optional embodiments, in step a, the incubation temperature may be, but is not limited to, 24° C., 26° C. or 28° C., and the incubation time may be, but is not limited to, 18 min, 20 min or 22 min.
[0050] In some optional embodiments, in the substrate solution, the concentration of dsRNA may be, for example, but not limited to 250 nM, 300 nM or 350 nM, and the concentration of adenosine triphosphate may be, for example, but not limited to 250 μM, 300 μM or 350 μM;
[0051] The substrate solution is an aqueous solution, and further comprises an aqueous solution of tris(hydroxymethyl)aminomethane, dithiothreitol and MgCl2; wherein the concentration of tris(hydroxymethyl)aminomethane may be, for example, but not limited to 4mM, 5mM or 6mM; the concentration of dithiothreitol may be, for example, but not limited to 1.8mM, 2mM or 2.2mM; the concentration of MgCl2 may be, for example, but not limited to 9mM, 10mM or 11mM.
[0052] The solution after incubation in step a is mixed with an equal volume of substrate solution.
[0053] In some optional embodiments, in step b, the pH of the reaction may be, for example, but not limited to 7, 7.5 or 8, the temperature may be, for example, but not limited to 24°C, 26°C or 28°C, and the time may be, for example, but not limited to 11 min, 120 min or 130 min.
[0054] In some optional embodiments, the volume ratio of the RNA helicase solution to the ADPGlo reagent is 1:2;
[0055] Add ADPGlo reagent 50-70 minutes before proceeding to step d.
[0056] In some optional embodiments, the volume ratio of the RNA helicase solution to the kinase detection reagent is 1:4;
[0057] 50-70 min after adding the kinase detection reagent, the fluorescence intensity was measured.
[0058] In some optional embodiments, in step d, the half inhibition concentration of the sample to be tested is obtained according to the fluorescence intensity of the sample to be tested at different concentrations, and then the inhibitory activity of the sample to be tested on RNA helicase is determined.
[0059] In some optional embodiments, in step d, GraphPad Prism 6.0 is used for data processing.
[0060] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.
[0061] It should be noted that in the following examples, room temperature refers to 25°C.
[0062] Example 1 Suramin sodium inhibits RNA helicase 5
[0063] Experimental consumables:
[0064] 384-well was purchased from Greiner, and the pipette chip was purchased from Thermo.
[0065] Experimental instruments:
[0066] The microplate reader was purchased from Thermo (model: VarioskanLUX); the microplate centrifuge was purchased from Xiangyi (model: TDZ5-WS); and the compound pipette was purchased from Thermo (model: Multidrop Pico8).
[0067] Experimental Materials:
[0068] RNA helicase 5 (FL, 1-614) protein was purified and extracted from insect cell lines, and the protein electrophoresis purity reached more than 90%; dsRNA was purchased from Sangon Biotech (Shanghai) Co., Ltd. (sequence: forward 5'-CAUUAUCGGAUAGUGGAACCUAGCUUCGACUAUCGGAUAAUC-3'
[0069] (SEQ ID NO.1); reverse 5'-GCUAGGUUCCACUAUCCGAUAAUG-3'(SEQ ID NO.2)), ADP-GLO (catalog number V9102) reagent was purchased from Promega; inhibitor suramin sodium reagent was purchased from MCE (catalog number HY-B0879A).
[0070] 1. Use an automatic sampler (Thermo, specification: MultidropPico8) to add the test compound (suramin sodium) solution to the Greiner 384-well: dilute the test compound suramin sodium stock solution to 10mM, the solvent is DMSO. Set the program to 100μM as the highest compound concentration, and dilute it from left to right at a dilution ratio of 1:3. Use DMSO for normalization (solvent concentration normalization, software program operation), a total of 9 compound dilution gradients, add 200nL of compound to each well, and do not add compounds to the 1st and 11th columns of the 384-well plate as the control group. Centrifuge at 1000rmp for 1min at room temperature after the compound is added. Set up two replicates for each well.
[0071] 2. Dilute RNA helicase 5 with buffer (5mM Tris pH 7.5, 10mM MgCl2, 2mM DTT) to a final concentration of 20nM, take 5μL and add it to Greiner 384-well. Do not add enzyme solution to the first column, but use buffer instead, as a low control; add enzyme solution to columns 2-11, and do not add compound to column 11, as a high control. Mix well, centrifuge at 1000rmp at room temperature for 1min, and incubate in a constant temperature box at 26℃ for 20min.
[0072] 3. Dilute dsRNA and ATP with buffer (5mM Tris pH 7.5, 10mM MgCl2, 2mM DTT) to a final concentration of 300nM and 300μM, respectively. Take 5μL of the diluted dsRNA and ATP premix and add it to the above Greiner 384-well. Mix well, centrifuge at room temperature for 1min, and incubate in a 26℃ incubator for 120min;
[0073] 4. Return the ADP-Glo reagent to room temperature, add 10 μL to each well, centrifuge at 1000 rpm for 1 min, let stand at room temperature for 60 min to terminate the reaction;
[0074] 5. Return the kinase detection reagent to room temperature, add 20 μL to each well, centrifuge at 1000 rpm for 1 min, and stand at room temperature in the dark for 60 min;
[0075] 6. Use a Thermo microplate reader (model: VarioskanLUX) to detect the luminescence signals of the corresponding samples in 384 wells, and the time interval for reading each well is set to 1000ms.
[0076] 7. Calculate using GraphPad Prism software, inhibition rate of each well (%) = 100 × (average High control - average sample well) / (average High control - average Low control), (High control represents the reading of the well without compound in the 11th column, Low control represents the reading of the well without enzyme solution in the 1st column, and sample well represents the reading of the well with test compound added in columns 2-10). Use GraphPad Prism to perform nonlinear four-parameter fitting to obtain the dose-effect curve of RNA helicase 5 inhibitor ( Figure 1 ). The horizontal axis is the logarithm of the molar concentration, and the vertical axis is the inhibition rate. As can be seen from the figure, the inhibitory activity IC of suramin sodium targeting RNA helicase 5 50 It was 36.3nM, indicating that the screening method was effective and reliable.
[0077] Example 2 Suramin sodium inhibits RNA helicase 17
[0078] Experimental consumables:
[0079] 384-well was purchased from Greiner, and the pipette chip was purchased from Thermo.
[0080] Experimental instruments:
[0081] The microplate reader was purchased from Thermo (model: VarioskanLUX); the microplate centrifuge was purchased from Xiangyi (model: TDZ5-WS); and the compound pipette was purchased from Thermo (model: Multidrop Pico8).
[0082] Experimental Materials:
[0083] RNA helicase 17 (80-712) protein was purified and extracted from insect cell lines, and the protein electrophoresis purity reached more than 90%; dsRNA was purchased from Sangon Biotech (Shanghai) Co., Ltd. (sequence: forward 5'-CAUUAUCGGAUAGUGGAACCUAGCUUCGACUAUCGGAUAAUC-3'
[0084] (SEQ ID NO.1); reverse 5'-GCUAGGUUCCACUAUCCGAUAAUG-3'(SEQ ID NO.2)), ADP-GLO (catalog number V9102) reagent was purchased from Promega; inhibitor suramin sodium reagent was purchased from MCE (catalog number HY-B0879A).
[0085] Experimental steps:
[0086] 1. Use an automatic sampler (Thermo, specification: MultidropPico8) to add the test compound solution to the Greiner 384-well: dilute the test compound suramin sodium stock solution to 10mM, set the program to 100μM as the highest compound concentration, and dilute it from left to right at a dilution ratio of 1:3. Use DMSO for normalization (solvent concentration normalization, software program operation), a total of 9 compound dilution gradients, add 200nL of compound to each well, and do not add compounds to the 1st and 11th columns of the 384-well plate as the control group. Centrifuge at 1000rmp for 1min at room temperature after the compound is added. Set up two replicates for each well.
[0087] 2. Dilute RNA helicase 17 with buffer (5mM Tris pH 7.5, 10mM MgCl2, 2mM DTT) to a final concentration of 20nM, take 5μL and add it to Greiner 384-well. Do not add enzyme solution to the first column, but use buffer instead, as a low control; add enzyme solution to columns 2-11, and do not add compound to column 11, as a high control. Mix well, centrifuge at 1000rmp at room temperature for 1min, and incubate in a constant temperature box at 26℃ for 20min.
[0088] 3. Dilute dsRNA and ATP with buffer (5mM Tris pH 7.5, 10mM MgCl2, 2mM DTT) to a final concentration of 300nM and 300μM, respectively. Take 5μL of the diluted dsRNA and ATP premix and add it to the above Greiner 384-well. Mix well, centrifuge at room temperature for 1min, and incubate in a 26℃ incubator for 120min;
[0089] 4. Return the ADP-Glo reagent to room temperature, add 10 μL to each well, centrifuge at 1000 rpm for 1 min at room temperature, and place in a 26°C incubator for 60 min;
[0090] 5. Return the kinase detection reagent to room temperature, add 20 μL to each well, centrifuge at 1000 rpm for 1 min at room temperature, and place in a 26°C thermostat away from light for 60 min;
[0091] 6. Use a Thermo microplate reader (model: VarioskanLUX) to detect the luminescence signals of the corresponding samples in 384 wells, and the time interval for reading each well is set to 1000ms.
[0092] 7. Calculate using GraphPad Prism software, inhibition rate of each well (%) = 100 × (average High control - average sample well) / (average High control - average Low control), (High control represents the reading of the wells without adding compound in the 11th column, Low control represents the reading of the wells without adding enzyme solution in the 1st column, and sample well represents the reading of the wells with the test compound added in columns 2-10). Use GraphPad Prism to perform nonlinear four-parameter fitting to obtain the dose-effect curve of RNA helicase 17 inhibitor ( Figure 2 ). The horizontal axis is the logarithm of the molar concentration, and the vertical axis is the inhibition rate. As can be seen from the figure, the inhibitory activity IC of suramin sodium targeting RNA helicase 17 50 It was 56.2nM, indicating that the screening method was effective and reliable.
[0093] Example 3 Suramin sodium inhibits RNA helicase 5
[0094] The difference from Example 1 is that the experimental steps are as follows:
[0095] 1. Use an automatic sampler (Thermo, specification: MultidropPico8) to add the test compound (suramin sodium) solution to the Greiner 384-well: dilute the test compound suramin sodium stock solution to 10mM, the solvent is DMSO. Set the program to 100μM as the highest compound concentration, and dilute it from left to right at a dilution ratio of 1:3. Use DMSO for normalization (solvent concentration normalization, software program operation), a total of 9 compound dilution gradients, add 200nL of compound to each well, and do not add compounds to the 1st and 11th columns of the 384-well plate as the control group. Centrifuge at 1000rmp for 1min at room temperature after the compound is added. Set up two replicates for each well.
[0096] 2. Dilute RNA helicase 5 with buffer (4mM Tris pH 7.5, 9mM MgCl2, 1.8mM DTT) to a final concentration of 18nM. Take 5μL and add it to Greiner 384-well. Do not add enzyme solution to the first column, but use buffer instead, as a low control. Add enzyme solution to columns 2-11, and do not add compound to column 11, as a high control. Mix well, centrifuge at 1000rmp at room temperature for 1min, and incubate in a constant temperature box at 24℃ for 22min.
[0097] 3. Dilute dsRNA and ATP with buffer (5mM Tris pH 7.5, 10mM MgCl2, 2mM DTT) to a final concentration of 250nM and 250μM, respectively. Take 5μL of the diluted dsRNA and ATP premix and add it to the above Greiner 384-well. Mix well, centrifuge at room temperature for 1min, and incubate in a 24℃ incubator for 130min;
[0098] 4. Return the ADP-Glo reagent to room temperature, add 10 μL to each well, centrifuge at 1000 rpm for 1 min, let stand at room temperature for 50 min to terminate the reaction;
[0099] 5. Return the kinase detection reagent to room temperature, add 20 μL to each well, centrifuge at 1000 rpm for 1 min, and stand at room temperature in the dark for 50 min;
[0100] 6. Use a Thermo microplate reader (model: VarioskanLUX) to detect the luminescence signals of the corresponding samples in 384 wells, and the time interval for reading each well is set to 1000ms.
[0101] 7. Calculate using software GraphPad Prism, inhibition rate of each well (%) = 100 × (average High control - average sample well) / (average High control - average Low control), (High control represents the reading of the well without compound in the 11th column, Low control represents the reading of the well without enzyme solution in the 1st column, and sample well represents the reading of the well with test compound added in columns 2-10), use GraphPad Prism to perform nonlinear four-parameter fitting, and obtain the dose-effect curve of RNA helicase 5 inhibitor. The results show that suramin sodium has inhibitory activity against RNA helicase 5.
[0102] Example 4 Suramin Sodium Inhibits RNA Helicase 5
[0103] The difference from Example 1 is that the experimental steps are as follows:
[0104] 1. Use an automatic sampler (Thermo, specification: MultidropPico8) to add the test compound (suramin sodium) solution to the Greiner 384-well: dilute the test compound suramin sodium stock solution to 10mM, the solvent is DMSO. Set the program to 100μM as the highest compound concentration, and dilute it from left to right at a dilution ratio of 1:3. Use DMSO for normalization (solvent concentration normalization, software program operation), a total of 9 compound dilution gradients, add 200nL of compound to each well, and do not add compounds to the 1st and 11th columns of the 384-well plate as the control group. Centrifuge at 1000rmp for 1min at room temperature after the compound is added. Set up two replicates for each well.
[0105] 2. Dilute RNA helicase 5 with buffer (6mM Tris pH 7.5, 11mM MgCl2, 2.2mM DTT) to a final concentration of 22nM. Take 5μL and add it to Greiner 384-well. Do not add enzyme solution to the first column, but use buffer instead, as a low control. Add enzyme solution to columns 2-11, and do not add compound to column 11, as a high control. Mix well, centrifuge at 1000rmp at room temperature for 1min, and incubate in a constant temperature box at 28℃ for 18min.
[0106] 3. Dilute dsRNA and ATP with buffer (6mM Tris pH 7.5, 11mM MgCl2, 2.2mM DTT) to a final concentration of 350nM and 350μM, respectively. Take 5μL of the diluted dsRNA and ATP premix and add it to the above Greiner 384-well. Mix well, centrifuge at room temperature for 1min, and incubate in a 28℃ incubator for 110min;
[0107] 4. Return the ADP-Glo reagent to room temperature, add 10 μL to each well, centrifuge at 1000 rpm for 1 min, let stand at room temperature for 70 min, and terminate the reaction;
[0108] 5. Return the kinase detection reagent to room temperature, add 20 μL to each well, centrifuge at 1000 rpm for 1 min, and stand at room temperature in the dark for 70 min;
[0109] 6. Use a Thermo microplate reader (model: VarioskanLUX) to detect the luminescence signals of the corresponding samples in 384 wells, and the time interval for reading each well is set to 1000ms.
[0110] 7. Calculate using software GraphPad Prism, inhibition rate of each well (%) = 100 × (average High control - average sample well) / (average High control - average Low control), (High control represents the reading of the well without compound in the 11th column, Low control represents the reading of the well without enzyme solution in the 1st column, and sample well represents the reading of the well with test compound added in columns 2-10), use GraphPad Prism to perform nonlinear four-parameter fitting, and obtain the dose-effect curve of RNA helicase 5 inhibitor. The results show that suramin sodium has inhibitory activity against RNA helicase 5.
[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for screening RNA helicase inhibitors, characterized in that: Using ADP-Glo TM The kinase assay kit is used to perform the assay, which includes the following steps: a. Mixing the RNA helicase solution and the sample solution to be tested and incubating; After the incubation in step ba, a substrate solution containing dsRNA and adenosine triphosphate is added to react; After the reaction in step cb is completed, ADPGlo reagent is added to terminate the reaction and consume excess ATP; After the dc step, a kinase detection reagent is added to convert ADP into ATP, and the inhibitory activity of the sample to be tested on RNA helicase is determined based on the fluorescence intensity.
2. The screening method according to claim 1, characterized in that The RNA helicase includes RNA helicase 5 or RNA helicase 17.
3. The screening method according to claim 1, characterized in that The concentration of RNA helicase in the RNA helicase solution is 18-22 nM; The RNA helicase solution is a buffer containing RNA helicase; The buffer is an aqueous solution containing 4-6 mM tris(hydroxymethyl)aminomethane, 1.8-2.2 mM dithiothreitol and 9-11 mM MgCl2; The RNA helicase solution and the sample solution to be tested are mixed in equal volumes.
4. The screening method according to claim 1, characterized in that The number of the sample solutions to be tested is at least 1. When the number of the sample solutions to be tested is greater than 1, the concentrations of the sample solutions to be tested are different.
5. The screening method according to claim 1, characterized in that In step a, the incubation temperature is 24-28° C. and the incubation time is 18-22 min.
6. The screening method according to claim 1, characterized in that In the substrate solution, the concentration of dsRNA is 250-350 nM, and the concentration of adenosine triphosphate is 250-350 μM; The substrate solution is an aqueous solution, and further comprises 4-6 mM tris(hydroxymethyl)aminomethane, 1.8-2.2 mM dithiothreitol and 9-11 mM MgCl2; The solution after incubation in step a is mixed with an equal volume of substrate solution.
7. The screening method according to claim 1, characterized in that In step b, the reaction pH is 7-8, the temperature is 24-28°C, and the reaction time is 110-130 min.
8. The screening method according to claim 1, characterized in that The volume ratio of the RNA helicase solution to the ADPGlo reagent is 1:2; Add ADP Glo reagent 50-70 minutes before proceeding to step d.
9. The screening method according to claim 1, characterized in that The volume ratio of the RNA helicase solution to the kinase detection reagent is 1:4; 50-70 min after adding the kinase detection reagent, the fluorescence intensity was measured.
10. The screening method according to claim 1, characterized in that In step d, the half inhibition concentration of the sample to be tested is obtained according to the fluorescence intensity of the sample to be tested at different concentrations, and then the inhibitory activity of the sample to be tested on RNA helicase is determined.