A method for screening protein targets of traditional Chinese medicine

Abstract

The present invention provides a method for screening protein targets of Chinese medicine, belonging to the field of biomedicine technology. The present invention utilizes a probe to compete with a Chinese medicine chemical system, and the Chinese medicine molecules bound to the target will weaken the binding of the probe. After the target binds to the Chinese medicine molecule, the signal intensity will decrease, thereby judging whether the Chinese medicine chemical system binds to the target, and being able to clearly define the binding site, solving the problem in the prior art that the Chinese medicine chemical system is complex and it is difficult to achieve the labeling of all compound molecules, and thus the target screening cannot be completed. The present invention does not require labeling of Chinese medicine molecules, and can detect the target screening of a complex chemical system of Chinese medicine in a relatively short time. The method is economical and fast, and the detection result is based on the competitive molecule, with strong specificity and stable signal, and is suitable for promotion and application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for screening protein targets of traditional Chinese medicine. Background Art

[0002] There are a wide range of small molecules that bind to targets in traditional Chinese medicine. The chemical components of traditional Chinese medicine bind to target proteins to play an important pharmacological role. Small molecules can affect the structure and function of proteins by directly binding to the three-dimensional structure of proteins, thereby regulating the cascade reaction of the body to play an important role. Traditional Chinese medicine and natural products come from nature and have drug activity, but their targets are unknown. Discovering and confirming their drug targets is of great value for studying their mechanism of action and drugability modification.

[0003] Biochip technology is a conventional means of identifying small molecule drug targets. In the process of using drug target chips to determine the targets of small molecules, small molecules must be labeled and tracked. However, this method requires that the structure-activity relationship (SAR) of the small molecule is clear and can be labeled. Given that traditional Chinese medicine is a complex chemical system that contains a large number of different types of compound molecules, it is extremely challenging to label all compound molecules. Therefore, the application of biochip technology in the field of complex system targets of traditional Chinese medicine has encountered a technical bottleneck.

[0004] Chinese patent CN101034060A discloses a method for combining quantum dot nanotechnology with biochip technology to find the target of the effective ingredients of traditional Chinese medicine. The quantum dots are connected with the effective ingredients of traditional Chinese medicine to form a "quantum dot-effective ingredients of traditional Chinese medicine" fluorescent probe, and then the fluorescent probe is directly incubated with the biochip. After washing, the specific binding sites (i.e., targets) between the drug and the known proteins fixed on the chip are determined based on the fluorescence emitted by the quantum dots on the biochip when excited by light, and then the proteins at these binding sites are analyzed by computer software. The present invention can easily and quickly find the target proteins that the effective ingredients of traditional Chinese medicine produce on cells, and can provide efficient technical means for studying the pharmacology of traditional Chinese medicine and high-throughput screening of new traditional Chinese medicines.

[0005] However, existing technologies have not yet been able to circumvent the complexity and difficulty of labeling the active ingredients of traditional Chinese medicine, and thus cannot achieve effective target screening and prediction. Summary of the invention

[0006] The purpose of the present invention is to provide a method for screening target protein of traditional Chinese medicine, which can realize simpler and more efficient detection of target protein of complex chemical system of traditional Chinese medicine.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for screening target sites of Chinese medicine proteins, comprising the following steps:

[0009] Adding the protein to the microarray chip to obtain a loaded microarray chip;

[0010] The microarray chips after loading are divided into a drug group and an inhibitor group;

[0011] In the drug group, the microarray chip was incubated with the Chinese medicine analyte and the protein inhibitor after loading.

[0012] In the inhibitor group, the microarray chip was only incubated with protein inhibitors after loading;

[0013] The protein inhibitor is capable of specifically binding to the protein;

[0014] After the protein inhibitor is incubated, the binding signal intensity is presented through a visual reaction;

[0015] Compare the binding signal intensities of the drug group and the inhibitor group:

[0016] Compared with the inhibitor group, the binding signal intensity of the drug group was lower, indicating that the protein was the target of the Chinese medicine analyte;

[0017] The protein inhibitor is a chemical probe modified with a fluorescent group.

[0018] Preferably, the fluorescent group is selected from one or more of Alexa Fluor dye series, FAM, Cy2, Cy3, Cy5, TRITC, FITC, Hoechst 33258, DAPI, YOYO-1, SYTOX Green, TET, HEX, VIC, ROX, Texas Red, Allophycocyanin, Propidium Iodide, Ethidium Bromide or GFP.

[0019] Preferably, the protein is a kinase protein, a transcription factor protein, a G protein coupled receptor protein, an ion channel protein, a nuclear receptor protein, a protease or an oxidoreductase.

[0020] Preferably, the protein is a kinase protein, and the protein inhibitor is a broad-spectrum kinase probe, more preferably a XO44 alkynyl probe.

[0021] Preferably, the kinase protein is MAPK1.

[0022] Preferably, the protein is a transcription factor protein, and the protein inhibitor is an iodoacetamide alkynyl probe, more preferably an IAA alkynyl probe.

[0023] Preferably, the transcription factor protein is NfkB1-p50 protein.

[0024] Preferably, the protein is an oxidoreductase, and the protein inhibitor is an iodoacetamide alkynyl probe, more preferably an IAA alkynyl probe.

[0025] Preferably, the oxidoreductase is IDH1 protein.

[0026] Beneficial effects of the present invention:

[0027] The present invention utilizes probes to compete with complex systems of traditional Chinese medicine. The traditional Chinese medicine molecules bound to the target will weaken the binding of the probe. After the target binds to the traditional Chinese medicine molecules, the signal intensity will decrease, thereby judging whether the complex system of traditional Chinese medicine is bound to the target, and being able to clearly identify the binding site. Compared with traditional detection methods, there is no need to label the traditional Chinese medicine molecules, and the target screening of the complex chemical system of traditional Chinese medicine can be detected in a shorter time. The method is economical and fast, and data can be obtained without the use of large instruments. The detection results are based on competitive molecules, have strong specificity, and stable signals, and are suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a technical principle diagram of the present invention;

[0029] Figure 2 This is the result of using XO44 probe combined with biochip to screen small molecule compounds for kinase protein targets;

[0030] Figure 3 This is the result of using XO44 probe combined with biochip to screen kinase protein targets in complex chemical systems of traditional Chinese medicine;

[0031] Figure 4 This is the result of using IAA probe combined with biochip to screen protein targets of small molecule compounds;

[0032] Figure 5 This is the result of using IAA probe combined with biochip to screen protein targets in complex chemical systems of traditional Chinese medicine. DETAILED DESCRIPTION

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] The sources of some reagents used in the present invention are as follows:

[0035] XO44 probe, purchased from Sigma-Aldrich, catalog number PZ0306;

[0036] IAA probe was purchased from Targetmol Company with the catalog number T15543.

[0037] Example 1 Screening of small molecule compounds and MAPK1 kinase protein targets in traditional Chinese medicine complex systems by using kinase-binding XO44 alkynyl probe combined with biochip

[0038] The steps for preparing the MAPK1 kinase protein chip are as follows:

[0039] (1) Preparation of 3D substrate

[0040] The three-dimensional substrate is an optical-grade polymer three-dimensional substrate covered with a bottom film and modified with aldehyde groups, and is purchased from Bio-Jingxin's polymer three-dimensional optical-grade three-dimensional substrate H.

[0041] (2) Detection of kinase indicators and spot sample preparation

[0042] A total volume of 10 μL of kinase protein (1 μg / μL) was mixed with the spotting solution and transferred to a 384-well plate for sample loading.

[0043] (3) Chip spotting

[0044] The sample spotter is a Jingxin microarray chip sample spotter, and the index is sampled in a spotting manner. First, the sample is cleaned before spotting. The cleaning procedure is as follows:

[0045] Wash for 5 seconds; ultrasonicate for 5 seconds; dry for 5 seconds; repeat 3 times.

[0046] Then, load the 384-well plate, place the sample sheet and the pre-spotted sample sheet, and spot multiple arrays on the three-dimensional substrate, with a spot diameter of 0.01 mm and a spot spacing of 0.01 mm. After spotting, fix, package, and store at 4°C.

[0047] The experimental steps for MAPK1 inhibitor validation are as follows:

[0048] 1. Blocking: Use a chip incubation box, add 10 mL of blocking solution, take the chip out from -80°C, and place it in the incubation box with the front side facing up; incubate on a side-swinging shaker at 50 rpm, room temperature, for 1 hour;

[0049] 2. Inhibitor (XO44 with blocked alkyne groups) incubation: discard the blocking solution, quickly add 3 mL of the pre-prepared probe incubation solution (final sample concentration <10 μM), shake on a side-to-side shaker at 20 rpm, at room temperature, for 1 hour, and avoid light.

[0050] 3. Cleaning: Take out the chip, place it in a chip cleaning box with cleaning solution, place it on a horizontal shaker, 60rpm, clean it 3 times, 5 minutes each time, and avoid light;

[0051] 4. XO44 probe incubation: Take out the chip and incubate with XO44 probe (final concentration <10 μM) on a side-swing shaker at 20 rpm, room temperature, for 1 hour. Keep the chip away from light.

[0052] 5. Cleaning: Take out the chip and place it in a chip cleaning box with cleaning solution. Shake horizontally at 60 rpm and clean it 3 times, 5 minutes each time. Keep it away from light.

[0053] 6. After completion, use the XO44 probe to connect to Cy3 azide using click chemistry reaction;

[0054] 7. Drying: Place the chip in a chip dryer and centrifuge to dry;

[0055] 8. Scanning: Use 532nm and 635nm to scan.

[0056] The results are as follows Figure 2 As shown, the ATP competition probe (XO44) can compete with the MAPK1 inhibitor (XO44 with blocked alkyne group), and the competition effect becomes more obvious as the inhibitor concentration increases, and the XO44 signal intensity becomes lower, indicating that the system can work.

[0057] Example 2 Screening protein targets of complex Chinese medicine systems using covalently bound XO44 alkynyl probes combined with biochips

[0058] (1) Blocking: Use a chip incubation box, add 10 mL of blocking solution, take the chip out from -80°C, and place it in the incubation box with the front side facing up;

[0059] (2) Sample incubation: discard the blocking solution, add the Chinese herbal medicine Inula odorata extract (concentration unit: μg / mL) and incubate on a side-swinging shaker at 20 rpm, at room temperature, for 1 h;

[0060] (3) Cleaning: Take out the chip and place it on a horizontal shaker at 60 rpm. Clean it three times, 5 min each time.

[0061] (4) Incubation with Chinese herbal medicine extract: Take out the chip and incubate it on a side-swing shaker at 20 rpm, room temperature, for 1 h;

[0062] (5) Cleaning: Take out the chip and clean it three times on a horizontal shaker at 60 rpm, each time for 5 min;

[0063] (6) XO44 probe incubation: Take out the chip and incubate with XO44 probe (final concentration <10 μM) on a side-to-side shaker at 20 rpm, at room temperature, for 1 h. Keep the chip away from light.

[0064] (7) Wash twice with ddH2O, 5 min each time, and avoid light;

[0065] (8) Drying: Place the chip in a chip dryer and centrifuge to dry it;

[0066] (9) Scanning: Use 532nm scanning.

[0067] The results are as follows Figure 3 As shown, different Chinese herbal extracts showed different binding strengths to MAPK1, reflecting different signal intensities. The stronger the binding ability, the weaker the signal intensity, reflecting that MAPK1 is a potential target of Chinese herbal extracts.

[0068] Example 3 Screening of covalently bound drug molecules using covalently bound IAA probes

[0069] The chip experiment operation steps are as shown in Example 1, except that the inhibitor is andrographolide (NfkB1-p50 protein is covalently bound), the target is NfkB1-p50 protein (containing a covalent binding site), and the probe is an IAA alkynyl probe. The results are shown in FIG. Figure 4 As shown, after the IAA probe binds to the NfkB1-p50 protein, the protein shows a signal intensity. When the inhibitor andrographolide is added, the protein signal is weakened, which proves that the IAA probe is suitable for the method provided by the present invention.

[0070] Example 4 Screening of covalently bound drug molecules for IDH1 targets using covalently bound IAA alkynyl probes

[0071] The chip experiment operation steps are as shown in Example 2, except that the inhibitor used is an IDH1 inhibitor (the protein is covalently bound), the target is an IDH1 protein (containing a covalent binding site), and the probe is an IAA alkynyl probe. The results are as follows Figure 5 As shown, after the IAA probe (10 μM) binds to the IDH1 protein, the protein shows a signal intensity. When different Chinese herbal extracts (10 μg / mL) or inhibitors are added, the protein signal is weakened, which proves that the IAA probe is suitable for the method of the present invention. The extracts of Chinese herbal medicines 4, 6, and 9 can significantly compete with the IAA probe, and the target may be IDH1.

[0072] It can be seen from the above embodiments that the screening method provided by the present invention can directly detect the target protein in the complex chemical system of traditional Chinese medicine without labeling the effective ingredients of traditional Chinese medicine; through the competitive binding between the probe and the target protein, the target protein bound to the complex system of traditional Chinese medicine can be quickly identified. This method simplifies the experimental steps, avoids the complicated labeling process, and improves the experimental efficiency; since it does not require the use of large instruments, the method is cost-effective and easy to operate, and the accuracy and repeatability of the test results are ensured through the specific binding of competitive molecules. The screening method of the present invention is applicable to a variety of complex systems of traditional Chinese medicine and has a wide range of applicability. Through experimental verification, the method of the present invention can effectively screen out traditional Chinese medicine molecules that bind to specific protein targets, providing a new technical means for the pharmacological research of traditional Chinese medicine and the development of new drugs.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for screening protein targets of traditional Chinese medicine, characterized in that: The following steps are involved: Adding the protein to the microarray chip to obtain a loaded microarray chip; The microarray chips after loading are divided into a drug group and an inhibitor group; In the drug group, the microarray chip was incubated with the Chinese medicine analyte and the protein inhibitor after loading. In the inhibitor group, the microarray chip was only incubated with protein inhibitors after loading; The protein inhibitor is capable of specifically binding to the protein; After the protein inhibitor is incubated, the binding signal intensity is presented through a visual reaction; Compare the binding signal intensities of the drug group and the inhibitor group: Compared with the inhibitor group, the binding signal intensity of the drug group was lower, indicating that the protein was the target of the Chinese medicine analyte; The protein inhibitor is a chemical probe modified with a fluorescent group; The protein is a kinase protein, a transcription factor protein, a G protein coupled receptor protein, an ion channel protein, a nuclear receptor protein, a protease or an oxidoreductase.

2. The screening method according to claim 1, characterized in that The fluorescent group is selected from one or more of Alexa Fluor dye series, FAM, Cy2, Cy3, Cy5, TRITC, FITC, Hoechst 33258, DAPI, YOYO-1, SYTOX Green, TET, HEX, VIC, ROX, Texas Red, Allophycocyanin, Propidium Iodide, Ethidium Bromide or GFP.

3. The screening method according to claim 1, characterized in that The protein is a kinase protein, and the protein inhibitor is a broad-spectrum kinase probe.

4. The screening method according to claim 3, characterized in that The kinase protein is MAPK1.

5. The screening method according to claim 4, characterized in that The protein is a transcription factor protein, and the protein inhibitor is an iodoacetamide alkynyl probe.

6. The screening method according to claim 5, characterized in that The transcription factor protein is NfkB1-p50 protein.

7. The screening method according to claim 1, characterized in that The protein is an oxidoreductase, and the protein inhibitor is an iodoacetamide alkyne probe.

8. The screening method according to claim 7, characterized in that The oxidoreductase is IDH1 protein.

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