Method for identifying catechol drug targets based on oxidative cross-linking and boron affinity enrichment

Abstract

The present invention relates to a new method for screening catechol drug targets. This method utilizes the covalent reaction between the highly reactive electrophilic orthoquinone generated after the oxidation activation of catechol and the nucleophilic amino acid side chains to fix the interaction between the drug and the target protein, and then uses the principle of specific binding of phenylboronic acid to 1,2-diol to separate and enrich the drug-target complex. The enriched sample is quantitatively analyzed by a liquid chromatography-mass spectrometry system to achieve in-situ screening and identification of catechol drug targets in cells. This method does not require modification of the drug, can achieve in-situ target identification in cells, has high accuracy in target identification and strong practical applicability, and provides an effective tool for the identification of interacting proteins of catechol small molecules widely present in natural products.

Description

Technical Field

[0001] The present invention relates to a novel drug target screening method suitable for identifying catechol small molecule drug target proteins. By utilizing the redox reversible conversion between catechol and electrophilic o-benzoquinone, it covalently binds to the target proteins of the drug, and boron affinity enrichment is performed on the immobilized drug target complex to reveal the target proteins, action mechanisms, and binding kinetics of catechol drugs. It has the advantages of not requiring drug modification, high reaction efficiency, and a wide coverage of target proteins, and has significant advantages for in-situ drug target identification in cells. Background Art

[0002] Complex protein networks are crucial for maintaining cellular systems. Dysregulation of protein signaling cascades will cause healthy cells to transform into a diseased state, thereby driving the cellular system to threaten the health of the entire organism (Cell, 2011, 144(5), 646–674). Small molecule drugs have been widely used to control or compensate for abnormal protein networks by interacting with target molecules in the body, enabling the organism to restore and maintain a healthy homeostasis. With in-depth research, it has gradually been found that most drugs do not act on a single target, but rather a single drug often has multiple targets. Therefore, identifying the target proteins of small molecule drugs is a prerequisite and key step in drug research and development. Once the target proteins of a drug can be determined, the structure of the drug can be optimized through structure-activity relationships to improve drug efficacy and predict possible side effects, reducing the risk of new drug development (Nature Chemical Biology, 2013, 9(4), 232–240). In addition, screening for drug targets also helps to explore new uses of drugs and realize the reuse of old drugs, which will greatly shorten the research and development cycle and cost of traditional new drugs.

[0003] Currently, the developed proteome-based drug target screening technologies can be mainly divided into two categories: one is the affinity- or activity-based target identification method (ABPP) that requires drug modification (Nature Communications, 2015, 6:1-11), and the other is the energy-based drug target screening technology that does not require drug modification. The ABPP technology can effectively screen for low-affinity targets. However, this strategy is not applicable to small molecules with a tight structure-activity relationship or complex natural products because it requires drug modification to introduce cross-linking and enrichment groups, and even a slight modification may destroy the biological activity of the original compound due to the change in its drug efficacy (TrAC-Trends in Analytical Chemistry, 2020, 124:115574). Therefore, in recent years, a series of energy-based drug target analysis methods have been developed, which identify drug target proteins by directly measuring the changes in specific protein properties after drug binding, such as thermal stability (Nature Biotechnology, 2020, 38(3):303–308), proteolytic sensitivity (Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(51):21984–21989), etc. However, the common problem of these technologies is the low coverage of the identification of membrane protein targets because they usually extract proteins by mild lysis methods in PBS in order not to destroy the protein structure and activity, and a large number of membrane proteins will be lost during this process.

[0004] Due to the specific reactive catechol group of catechol drugs, this group can be oxidized into a highly electrophilic o-quinone structure, and the o-quinone reacts covalently with amino acids with nucleophilic side chains, thus fixing the interaction between the drug and the target. And after the reaction, the small molecule returns from o-quinone to the catechol form again. Therefore, based on the high binding affinity between phenylboronic acid and vicinal diol, the target protein modified with catechol drugs can be enriched and identified. Based on this characteristic, this patent has developed a drug target screening method for catechol drugs that does not require drug modification and can comprehensively explore targets in situ in cells. Summary of the Invention

[0005] The present invention develops a drug target identification method based on oxidative cross-linking and boron affinity enrichment for the catechol group with specific reactive activity of catechol drug small molecules. Isoproterenol and its known targets superoxide dismutase 1 (SOD1) and β-adrenergic receptor are used as method validation, and the method is applied to screening the medicinal target protein of danshensu isopropyl ester (IDHP) in the treatment of vascular dementia and myocardial fibrosis, which can not only obtain target information with lower affinity, expand the identification range of the target, but also improve the accuracy of target identification without modifying the drug.

[0006] The method for identifying catechol drug targets based on oxidative cross-linking and boron affinity enrichment comprises the following steps:

[0007] (1) Incubate the SILAC-labeled cells with catechol drugs at 37°C and 5% CO2 for 0.5-24 hours, and the drugs reversibly bind to the target protein through conformational and non-covalent interactions; add oxidants to the cells and incubate again. Under the conditions of physiological pH 7-8 (preferably 7.4) and 37°C, the oxidants quickly and fully convert catechol into highly active o-benzoquinone, thereby covalently binding to the nucleophilic amino acid side chains near the site, fixing the interaction between the small molecule drug and its target protein, forming a drug-target complex, and obtaining an experimental group. SILAC-labeled cells that were incubated without the addition of catechol drugs or incubated again without the addition of oxidants were used as the control group;

[0008] The amino acids with nucleophilic side chains include one or more of cysteine, histidine, lysine, tyrosine, tryptophan and the like.

[0009] (2) The cells of the experimental group and the control group were collected and an ionic liquid containing 0.5-5% (v / v) protease inhibitor cocktail was added at 3×10 6 Add 200-600 μL of ionic liquid to each cell, disrupt the cells by ultrasound, centrifuge at 10000-16000 g for 5-30 minutes, and take out the supernatant protein complex sample;

[0010] The protein in the supernatant is subjected to high-temperature denaturation reduction using a denaturant, dithiothreitol or tris(2-carboxyethyl)phosphine, wherein the temperature of the high-temperature denaturation is 37-95°C for 5 min-2 h; iodoacetamide at a concentration twice that of the denaturant is added, and alkylation treatment is performed at room temperature in the dark for 20-40 min.

[0011] (3) Based on the binding affinity between phenylboronic acid and vicinal diol, the drug-target complex is enriched using a material immobilized with cyclic phenylboronic acid, and the enriched drug-target complex is eluted under the condition of pH 3 to 6.5;

[0012] Synthesize the material Fe3O4 / PDA / GO / PEI / CBX with cyclic phenylboronic acid immobilized. Prepare the Fe3O4 magnetic nanomaterial by solvothermal method; under weakly alkaline conditions, bond a polydopamine layer on the surface of the magnetic spheres to obtain the Fe3O4 / PDA nanomaterial; utilize the π-π interaction between polydopamine and graphene oxide to self-assemble into the Fe3O4 / PDA / GO nanomaterial; electrostatically self-assemble the negatively charged graphene oxide and the positively charged polymer PEI to form the Fe3O4 / PDA / GO / PEI nanomaterial; finally, introduce the phenylboronic acid monomer by amidation of the carboxyl group of the cyclic phenylboronic acid monomer CBX and the amino group of PEI to prepare the boronate affinity enrichment magnetic nanomaterial Fe3O4 / PDA / GO / PEI / CBX;

[0013] Disperse the Fe3O4 / PDA / GO / PEI / CBX material in 10 - 100 mM phosphate buffer (pH 7.4), then mix it with the protein sample after denaturing reduction alkylation treatment, adjust the pH to 7 - 8 (preferably 7.4), and incubate at 4 - 37 °C for 6 - 12 hours. Recover the material enriched with the protein modified with catechol drugs by magnetic separation. Disperse the recovered magnetic material with 10 - 100 mM phosphate buffer (pH 7.4), incubate at 4 - 37 °C for 2 - 5 minutes, discard the supernatant by magnetic separation, and repeat 2 - 4 times. Add it to the eluent with pH 3 - 6.5 (ACN:H2O:TFA = 50:49:1 - 80:19:1, v / v / v), incubate at 4 - 37 °C for 1 hour, separate with a magnet, retain the supernatant, repeat once, combine the two supernatants and perform lyophilization. After lyophilization, redissolve the protein with 8 M guanidine hydrochloride.

[0014] (4) Dilute the enriched and redissolved protein sample to 6 - 10 times its volume with 100 mM PBS, pH 8.0; add trypsin and perform enzymatic digestion for 12 - 16 hours at an enzyme:protein complex ratio of 1:20 - 1:50 (w / w); desalt the enzymatically digested sample using a C18 desalting column; lyophilize the desalted sample and redissolve the peptide segments with an aqueous solution of formic acid with a volume fraction of 0.1%.

[0015] (5) Collect mass spectrometry data for the obtained peptide segment sample using a liquid chromatography - mass spectrometry system, separate the sample using a C18 chromatographic column, and collect data using a high - resolution mass spectrometer; search the spectra using Proteome Discoverer (Thermo Fisher), Maxquant (https: / / www.maxquant.org / ), perform SILAC intensity quantification to remove non - specific adsorption, and preliminarily screen and identify potential target proteins of catechol drugs;

[0016] The screening rules include: 1) The labeling intensity ratio of the experimental group to the control group is greater than or equal to 2; 2) The protein has at least 3 Unique peptides; 3) It is quantified at least twice in 3 biological replicates.

[0017] (6) Use search engines such as Uniprot (https: / / www.uniprot.org / ), DAVID Bioinformatics Resources (https: / / david.ncifcrf.gov / ), GORILLA (http: / / cbl-gorilla.cs.technion.ac.il / ), PANTHER Classification System (http: / / pantherdb.org / ), KEGG (https: / / www.genome.jp / kegg / ) to perform gene ontology annotation and enrichment analysis (GO analysis) and KEGG pathway analysis on the preliminarily identified potential target proteins, collect annotation information on molecular functions, biological processes, cellular components, and KEGG pathways with a p-value less than or equal to 0.05 or an FDR less than or equal to 1%, and obtain the targeted protein pathway information and its distribution information within the cell;

[0018] Furthermore, based on the obtained information, determine the corresponding diseases that catechol drugs can treat and / or prevent, or determine the application of catechol drugs in drugs for treating and / or preventing the corresponding diseases.

[0019] The present invention utilizes the covalent reaction between the highly reactive electrophilic ortho-quinone generated after the oxidation activation of catechol and the nucleophilic amino acid side chains to fix the interaction between the drug and the target protein, and then utilizes the principle of the specific binding of phenylboronic acid to 1,2-diols to separate and enrich the drug-target complex. The enriched sample is analyzed by liquid chromatography-mass spectrometry combined with quantitative analysis to achieve the in-situ target screening and identification of catechol drugs in cells. This method does not require the modification of the drug, can achieve in-situ target identification in cells, has high target identification accuracy and strong practical applicability, and provides an effective tool for the identification of interacting proteins of catechol small molecules widely present in natural products.

[0020] This patent has the following advantages:

[0021] (1) Strong targeting and good biocompatibility;

[0022] (2) The drug dosage is relatively low, and the drug can only bind to the protein when the conformation matches, and then oxidative cross-linking occurs after binding, reducing the occurrence of non-specific cross-linking;

[0023] (3) Due to the double-electron oxidation activity of sodium periodate, the oxidative cross-linking can fully react within 10 min, reducing the perturbation to the proteome;

[0024] (4) It can label various amino acids such as cysteine, histidine, lysine, tyrosine, and tryptophan, with a wide protein coverage;

[0025] (5) Utilizing the strong binding force of cyclic phenylboronic acid to catechol and its binding characteristics at physiological pH, the drug-target complex can be enriched under conditions of good biocompatibility;

[0026] (6) There is no need to modify the drug, ensuring an accurate reflection of the original activity of the drug;

[0027] (7) Using the oxidative cross-linking method to fix the interaction between the drug and the target protein is convenient for identifying target proteins with relatively low affinity. Description of the Drawings

[0028] Figure 1 . Schematic diagram of the principle and process of the drug-target identification strategy based on oxidative cross-linking and boronate affinity enrichment.

[0029] Figure 2 . a is a schematic diagram of the Fe3O4 / PDA / GO / PEI / CBX material; b is the Zeta potential characterization result, (1) is Fe3O4; (2) is Fe3O4 / PDA; (3) is Fe3O4 / PDA / GO; (4) is Fe3O4 / PDA / GO / PEI; (5) is Fe3O4 / PDA / GO / PEI / CBX; c is the XPS characterization result.

[0030] Figure 3 . Verify the effect of IDHP oxidative cross-linked peptides (taking the peptide Ac-DHMASSIER as an example, the gray is the molecular ion peak, and the red is the modified peak). Detailed Embodiments

[0031] The following describes the embodiments of the present invention in detail with reference to the attached drawings and tables: This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0032] Example 1

[0033] 1. In vitro experiment - verify the effectiveness based on catechol oxidative cross-linking

[0034] 1.1 Verify the reaction activity of catechol oxidative cross-linking at the peptide level

[0035] To a peptide solution with a final concentration of 0.5 mM (dissolved in different ratios of DMSO and water according to the hydrophilicity and hydrophobicity of the peptide), an aqueous solution of IDHP with a final concentration of 2.5 mM and an aqueous solution of sodium periodate with a final concentration of 2.5 mM were added. The mixture was incubated at 37 °C for 30 min, and then diluted 20-fold with acetonitrile containing 0.1% (v / v) TFA. 2 μL of the diluted solution was spotted on the target, and the matrix was 2,5-dihydroxybenzoic acid (DHB). Analysis was performed using an Ultraflex III MALDI-TOF / TOF MS (Bruker Daltonics Inc.). The peptide sequences and properties used are shown in Table 1. In the mass spectrometry results, peptides containing histidine showed obvious modified peaks (attached Figure 3 ).

[0036] 1.2 Taking isoprenaline (ISO) and its target protein superoxide dismutase 1 (SOD1) as examples, the modification effect of catechol oxidative cross-linking on proteins was verified

[0037] 1) Three proteins, SOD1, Myoglobin, and HRP, were mixed at a molar ratio of 1:100:100 (dissolved in 1×PBS at working concentration), with the final concentration of SOD1 being 3 μM. ISO with a final concentration of 30 μM was added, and the mixture was incubated at 37 °C for 1 h. The sample was transferred to a FASP membrane (2k, pre-washed once with water), and centrifuged at 16000 g at 4 °C for 30 - 40 min to remove the excess ISO that did not bind to the protein. 100 μL of PBS and sodium periodate with a final concentration of 10 μM were added to the membrane. After incubation at 37 °C for 10 min, it was centrifuged at 16000 g at 4 °C for 30 - 40 min. It was washed once with PBS again. Tris(2-carboxyethyl)phosphine (TCEP) with a final concentration of 10 mM (dissolved in 50 mM ABC) was added for reduction, and the reaction was carried out at 37 °C for 1 h. TCEP was removed, and then iodoacetamide (IAA) with a final concentration of 20 mM (dissolved in 50 mM ABC) was added for alkylation, and the reaction was carried out in the dark at room temperature for 30 min. It was washed 3 times with 50 mM ABC. It was digested in 10 mM ABC, and trypsin was added at a ratio of 1:30 (w / w). The reaction was carried out at 37 °C for 12 - 16 h. It was centrifuged at 16000 g at 4 °C for 30 - 40 min to obtain a peptide solution, and the membrane was washed 2 times with 50 μL of 10 mM ABC solution. The washes were combined, freeze-dried, and stored at -80 °C.

[0038] 2) NanoLC-MS / MS analysis. An EasyNano HPLC and Q-ExactiveTM combined quadrupole Orbitrap mass spectrometer was used to construct a 1D-nano-RPLC-ESI-MS / MS system for sample analysis. The peptide segments were redissolved in an aqueous solution of 0.1% formic acid by volume, and the peptide segment concentration was measured using a NanoDrop one ultra-micro spectrophotometer (Thermo Fisher), and 1-2 μg was loaded. Liquid chromatography conditions: Mobile phase A: aqueous solution containing 2% ACN and 0.1% FA (by volume), Mobile phase B: aqueous solution containing 98% ACN and 0.1% FA (by volume). The gradient conditions are shown in Table 2.

[0039] 3) Data analysis. All the data collected by mass spectrometry were searched using pFind. The spectral ratios of the peptide segments containing ISO Michael addition modification in the three proteins were calculated. The ratio in the SOD1 protein reached about 8.5%, and the number of ISO-modified spectra of the other two proteins was small.

[0040] 2. Synthesis and characterization of boronate affinity enrichment magnetic nanomaterials

[0041] 2.1 Synthesis of Fe3O4 / PDA / GO / PEI / CBX boronate affinity enrichment magnetic nanomaterials

[0042] Refer to Example 1 in the patent "Phenylboronic Acid Functionalized Graphene Oxide Composite Nanomaterials and Their Preparation and Application" (CN201510552575, CN106475068A).

[0043] 1) Synthesis of Fe3O4 magnetic spheres. Weigh 1.08 g of FeCl3·6H2O + 0.20 g of sodium citrate and disperse them in 20 mL of ethylene glycol, and ultrasonically dissolve them completely. Add 1.20 g of sodium acetate and magnetically stir for 30 minutes to form a brown suspension. Transfer the above solution to a stainless steel reaction kettle with a 100 mL polytetrafluoroethylene inner liner and react at 200 °C for 10 h. After the reaction, collect the black particles in the reaction kettle with the assistance of a magnet, and wash them 3 times with distilled water and 3 times with ethanol under ultrasonic conditions. Vacuum dry at room temperature to obtain the product Fe3O4 nanomaterials.

[0044] 2) Synthesis of Fe3O4 / PDA nanomaterials. In a 100 mL three-necked flask, disperse 100 mg of Fe3O4 nanomaterials in 50 mL of 10 mM Tris-HCl buffer (pH 8.5). Under mechanical stirring, add 100 mg of dopamine hydrochloride (DA). React at 25 °C in a water bath for 2 h. Collect the particles in the flask with the assistance of a magnet, and wash them 3 times with distilled water and 3 times with ethanol under ultrasonic conditions. Vacuum dry at room temperature to obtain the product Fe3O4 / PDA nanomaterials.

[0045] 3) Synthesis of Fe3O4 / PDA / GO nanomaterials. Weigh 100 mg of graphene oxide (GO), add 100 mL of water, and ultrasonicate for 1 h to completely exfoliate GO, obtaining a GO dispersion (1 mg / mL). Disperse 100 mg of Fe3O4 / PDA nanomaterials in 20 mL of aqueous solution to form a 5 mg / mL dispersion. Mix the above two dispersions and react under mechanical stirring at room temperature for 8 h for self-assembly. Collect the reaction product with the assistance of a magnet and wash it 6 times with distilled water under mild sonication conditions. Vacuum dry at room temperature to obtain the product Fe3O4 / PDA / GO nanomaterials.

[0046] 4) Synthesis of Fe3O4 / PDA / GO / PEI nanomaterials. Mix 10 mL of 100 mg / mL polyethyleneimine (PEI) aqueous solution with 50 mL of 2 mg / mL Fe3O4 / PDA / GO dispersion and react under mechanical stirring at room temperature for 10 h for electrostatic self-assembly. Collect the reaction product with the assistance of a magnet and wash it 6 times with distilled water under mild sonication conditions. Vacuum dry at room temperature to obtain the product Fe3O4 / PDA / GO / PEI nanomaterials.

[0047] 5) Synthesis of Fe3O4 / PDA / GO / PEI / CBX nanomaterials. Weigh 150 mg of 6-aminobenzo[c][1,2]oxaborole-1(3H)-ol hydrochloride (CBX) and disperse it in 30 mL of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.6) in a 100 mL three-necked flask, and ultrasonicate to dissolve it completely. Then transfer it to a 45 °C water bath, stir mechanically, add 640 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 198 mg of N-hydroxysuccinimide (NHS) for CBX carboxyl activation, and react for 45 min. Add 60 mL of 0.1 M Na2HPO4 to adjust the pH to neutral. Under mechanical stirring, add 15 mg of Fe3O4 / PDA / GO / PEI dispersion and react at room temperature for 4 h. Collect the reaction product with the assistance of a magnet and wash it 6 times with distilled water under mild sonication conditions. Vacuum dry at room temperature to obtain the product Fe3O4 / PDA / GO / PEI / CBX nanomaterials.

[0048] 2.2 Characterization of Fe3O4 / PDA / GO / PEI / CBX Boron Affinity Enrichment Magnetic Nanomaterials

[0049] 1) Transmission Electron Microscopy (TEM) Characterization

[0050] Use a JEM-2000EX transmission electron microscope (JEOL, Tokyo, Japan) to perform transmission electron microscopy (TEM) characterization on the above 5-step materials (0.3 mg / mL, dissolved in ethanol).

[0051] 2) Zeta potential characterization

[0052] The surface Zeta potential of the above five-step materials (0.3 mg / mL, dissolved in water) was measured using a Malvern Nano zs90 Zeta sizer (Malven, Worcester, U.K.) (Appendix Figure 2 B).

[0053] 3) X-ray energy spectrum characterization

[0054] The surface carbon, nitrogen, oxygen, iron, and boron element compositions of the Fe3O4 / PDA / GO / PEI / CBX nanomaterials were analyzed using an ESCALAB250Xi X-ray photoelectron spectrometer (Thermo, Waltham, USA) (Appendix Figure 2 C).

[0055] 3. In vitro experiments - verifying the effectiveness of the boron affinity enrichment material

[0056] Three proteins, SOD1, Myoglobin, and HRP, were mixed at a molar ratio of 1:100:100 (dissolved in PBS), with the final concentration of SOD1 being 3 μM. IDHP with a final concentration of 30 μM was added, and the mixture was incubated at 37 °C for 1 h. Subsequently, sodium periodate with a final concentration of 20 μM was added, and the mixture was incubated at 37 °C for 10 min. Desalting was performed using a 0.5 mL Zeba desalting column. 1.5 mg of the Fe3O4 / PDA / GO / PEI / CBX nanomaterials were taken and activated twice with 2 mL of loading buffer (1×PBS, pH 7.4). The protein sample was added to the materials, and the volume was diluted to 2 mL with the loading buffer, followed by incubation at 25 °C for 6 h. Then, it was washed three times with 2 mL of the loading buffer to remove non-specifically adsorbed proteins. Subsequently, 200 μL of eluent (ACN:H2O:TFA = 50:49:1, v / v / v) was added, and the mixture was incubated at 25 °C for 1 h. This was repeated once, and the supernatants were combined. After freeze-drying the supernatant, it was re-dissolved with a small volume of an aqueous solution with a volume concentration of 0.1% FA. 2 μL was taken for spotting on the target, with sinapic acid (SA) as the matrix, and analyzed using an Ultraflex III MALDI-TOF / TOF MS (Bruker Daltonics Inc.).

[0057] Example 2

[0058] In vivo experiments - verifying the effectiveness of catechol oxidation cross-linking and boron affinity enrichment

[0059] (1) Culture of SILAC-labeled HepG2 cells. Under the conditions of 37 °C and air containing 5% CO2 by volume, HepG2 cells were cultured with SILAC DMEM medium containing natural isotope amino acids (L-lysine-HCl and L-arginine-HCl) and heavy isotope-labeled amino acids (D8-L-lysine-HCl and 13 C6 15 N4-Argine), 10% fetal bovine serum (FBS) by volume, and 1% penicillin-streptomycin by volume. After more than 6 passages, the cells were completely labeled to obtain SILAC light-labeled (natural isotope amino acid-labeled) and heavy-labeled HepG2 cells respectively. When the density of the stably labeled cells reached about 80%, the experiment was carried out.

[0060] (2) Discard the culture medium of the light-labeled HepG2 cells and wash them once with 1×PBS buffer at working concentration. Dilute ISO to 20 μM with PBS buffer and add it to the culture dish. Incubate with the cells in an incubator at 37 °C and containing 5% CO2 by volume for 1 h. After incubation, wash 3 times with PBS buffer. Then add PBS solution containing 10 μM sodium periodate and incubate with the cells in an incubator at 37 °C and containing 5% CO2 by volume for 15 min. After incubation, wash 3 times with PBS buffer. The blank control group uses heavy-labeled HepG2 cells and replaces 20 μM ISO with PBS, and the rest is the same as above. Or, the blank control group uses heavy-labeled HepG2 cells and replaces 10 μM sodium periodate with PBS, and the rest is the same as above.

[0061] (3) Harvest cells by scraping. Discard the medium of the above cells, add 1 mL of PBS pre-cooled at 4 °C to scrape the cells, collect them into an ice-bath centrifuge tube, and wash the culture dish once with PBS pre-cooled at 4 °C. Combine the two liquids, centrifuge at 500 g and 4 °C for 5 min, wash 2 times with PBS, and count the cells. Harvest the cells of the experimental group and the control group respectively according to the above steps.

[0062] (4) Extract proteins. Add ionic liquid (1-dodecyl-3-methylimidazolium chloride) containing 1% (v / v) protease inhibitor cocktail to the cells of the experimental group and the control group respectively. Add 300 μL of ionic liquid to every 3×10 6 cells, and extract the protein complex by ultrasonic wave at a power of 120 W for 2 min. Determine the protein concentration by BCA method. Mix the samples of the light and heavy labeling groups according to the protein mass ratio of 1:1 (w:w).

[0063] (5) Denaturation, reduction, and alkylation. Add tris(2-carboxyethyl)phosphine (TCEP) with a final concentration of 50 mM (dissolved in PBS) to the protein sample for reduction, and react at 56 °C for 30 min. Then add iodoacetamide (IAA) with a final concentration of 100 mM (dissolved in PBS) for alkylation, and react in the dark at room temperature for 30 min.

[0064] (6) Enrichment. Activate the Fe3O4 / PDA / GO / PEI / CBX nanomaterial twice with the loading buffer (1×PBS, pH 7.4), and finally disperse the material in the loading buffer, with 2 mL of loading buffer corresponding to every 1 mg of material. Mix the protein sample with the material dispersion and incubate at 25 °C for 12 h. Every 100 μg of protein corresponds to 2 mg of material. After enrichment, combine with magnetic separation and wash the material 3 times with the loading buffer to remove non-specifically adsorbed proteins. 2 mL of loading buffer corresponds to every 1 mg of material.

[0065] (7) Elution. Add the elution solution (ACN:H2O:TFA = 50:49:1, v / v / v) to the washed material, incubate at 25 °C for 1 h, repeat once, and combine the supernatants. 0.2 mL of elution solution corresponds to every 1 mg of material. After freeze-drying the supernatant, redissolve it with 20 μL of 8 M guanidine hydrochloride.

[0066] (8) Enzymatic digestion. Dilute the above-redissolved sample with 180 μL of 10 mM PBS (pH 8.0), add trypsin (mass spectrometry grade, promega) at a mass ratio of 1:25 (enzyme / protein, w / w), and digest at 37 °C for 16 h. Add 20 μL of 10% formic acid aqueous solution by volume to terminate the enzymatic digestion.

[0067] (9) Desalting. Desalt with a C18 trapping column and freeze-dry the sample.

[0068] (10) NanoLC-MS / MS analysis. Construct a 1D-nano-RPLC-ESI-MS / MS system using an Easynano HPLC equipped with a Q-ExactiveTM combined quadrupole Orbitrap mass spectrometer to analyze the sample. Redissolve the peptide segments in a 0.1% formic acid solution by volume, load 1 - 2 μg, and the gradient is shown in Table 3.

[0069] (11) Data analysis. All mass spectrometry data were searched using MaxQuant_1.6.5.0 with the built-in Andromeda search engine. SILAC quantitative database searching was performed for the experimental group and the blank control group. The Perseus_1.5.8.5 software was used to process the database search results to find differential proteins with a quantitative intensity ratio greater than 2 and a number of unique peptides greater than or equal to 3. Information such as the name and cellular distribution of the proteins was annotated on the Uniprot website and the DAVID website. Among the differential proteins, the target proteins β-adrenergic receptor protein and SOD1 reported by ISO were identified, and their quantitative intensity ratios showed significant differences. The identified differential proteins mainly belonged to membrane proteins.

[0070] Example 3

[0071] Mouse hippocampal neuron cells HT22 were cultured to screen and identify the target proteins and action mechanisms of isopropyl tanshinonate (IDHP) drug in the treatment of vascular dementia.

[0072] 1) Hypoxia and glucose deprivation treatment was performed on SILAC-stably labeled HT22 cells to simulate the cell growth environment of cerebral ischemia. HT22 hypoxia and glucose deprivation cell culture: Discard the normal cell culture medium of HT22 cells, wash twice with pre-cooled working concentration 1×PBS at 4°C, add DMEM sugar-free medium, and then place it in a 37°C triple gas incubator containing 94% N2 + 5% CO2 + 1% O2 for 6 h. Replace the DMEM sugar-free medium with the normal cell DMEM medium, place it in a 37°C, 5% CO2 incubator for continued culture. At the same time, the experimental group (light-labeled cells) was treated with IDHP at a final concentration of 20 μM, and incubated in a 37°C, 5% CO2 incubator for 6 h. After completion, wash 3 times with PBS buffer. Then add a PBS solution with a periodate concentration of 10 μM, and co-incubate with the cells in a 37°C, 5% CO2 incubator for 15 min. After completion, wash 3 times with PBS buffer. The control group (heavy-labeled HT22 cells) used PBS instead of 20 μM IDHP or PBS instead of 10 μM periodate, and the rest was the same as above. After extracting the protein from the cells and measuring the concentration, equal amounts of protein were mixed from the experimental group and the control group. Subsequently, steps such as denaturation, reduction, alkylation, enrichment, elution, enzymatic digestion, and desalting were carried out. Finally, mass spectrometry analysis was performed. The operation was the same as steps (3)-(11) of Example 1. 27 credible differential proteins were identified, which were mainly involved in the phosphatidylinositol-3-kinase (PI3K) / protein kinase B (Akt) pathway to treat cognitive dysfunction caused by vascular dementia.

[0073] Example 4

[0074] Primary rat cardiac fibroblasts were cultured to screen and identify the target proteins and action mechanisms of isopropyl tanshinonate (IDHP) drug in the treatment of cardiac fibrosis.

[0075] (1) Culture of SILAC-labeled primary rat cardiac fibroblasts. Under the conditions of 37 °C and air containing 5% CO2 by volume concentration, primary rat cardiac fibroblasts were cultured with SILAC DMEM / F12 medium containing natural isotope amino acids (L-lysine-HCl and L-arginine-HCl) and heavy isotope-labeled amino acids (D8-L-lysine-HCl and 13 C6 15 N4-Argine), 10% fetal bovine serum (FBS) by volume concentration, and 1% penicillin-streptomycin by volume concentration. After more than 6 passages to completely label the cells, primary rat cardiac fibroblasts with SILAC light label (labeled with natural isotope amino acids) and heavy label were obtained respectively. When the density of the stably labeled cells reached about 80%, the experiment was carried out.

[0076] (2) The experimental group (light-labeled cells) was treated with IDHP at a final concentration of 20 μM, incubated in an incubator at 37 °C and 5% CO2 for 12 h. After that, it was rinsed 3 times with PBS buffer. Then, a PBS solution with sodium periodate at 10 μM was added, and the cells were co-incubated in an incubator at 37 °C and 5% CO2 for 15 min. After that, it was rinsed 3 times with PBS buffer. The control group (heavy-labeled cells) used PBS to replace 20 μM IDHP or used PBS to replace 10 μM sodium periodate, and the rest was the same as above. After extracting the protein from the cells and measuring the concentration, equal amounts of protein were mixed from the experimental group and the control group. Subsequently, steps such as denaturation reduction alkylation, enrichment elution, enzymatic digestion, and desalting were carried out. Finally, mass spectrometry analysis was performed. The operation was the same as steps (3)-(11) of Example 1. 36 highly credible differential proteins were identified. IDHP mainly inhibits the occurrence of cardiac fibrosis by regulating the NOX2 / ROS / p38 signaling pathway.

[0077] Table 1: Peptide sequences and related properties for verifying the effect of catechol oxidative cross-linking

[0078]

[0079] Table 2: NanoLC-MS / MS gradient conditions for proteomics perturbation experiments

[0080]

[0081] Table 3: NanoLC-MS / MS gradient conditions for verifying the effectiveness of catechol oxidative cross-linking and boronate affinity enrichment in in vivo experiments

[0082]

Claims

1. A method for identifying catechol drug targets based on oxidative cross-linking and boron affinity enrichment, characterized in that, It includes the following steps: 1) Add catechol drugs to the SILAC-labeled cells for incubation; add an oxidant to the cells for re-incubation to obtain an experimental group; use the SILAC-labeled cells without adding catechol drugs for incubation or without adding an oxidant for re-incubation as a control group; 2) Disperse the experimental group and control group cells in an ionic liquid for disruption, extract protein complexes using the ionic liquid, and perform disulfide bond reduction and alkylation treatment on the proteins; 3) Based on the binding affinity between phenylboronic acid and vicinal diol, use a material immobilized with cyclic phenylboronic acid to enrich the drug-target complex, and elute the enriched drug-target complex under the condition of pH < 5; 4) Digest the obtained protein complex into peptide segments, and collect mass spectrometry data using a liquid chromatography-mass spectrometry system; Combined with SILAC labeling quantification, preliminarily screen and identify potential target proteins of catechol drugs through the difference in labeling intensity; 5) Perform gene ontology clustering analysis, i.e., GO analysis and KEGG pathway analysis, on the preliminarily identified potential target proteins to obtain the biological processes involved in their target proteins; The catechol drugs mentioned are: one or more of isoprenaline, noradrenaline, dopamine, dobutamine, danshensu, isopropyl danshensu, borneol danshensu, drugs containing catechol groups; The oxidants include: one or more of sodium periodate, hydrogen peroxide, tyrosinase; Add dithiothreitol or tris(2-carboxyethyl)phosphine to the extracted protein complex sample, denature and reduce it at a high temperature, the temperature of the high-temperature denaturation is 37 - 95 °C, and the time is 5 minutes - 2 hours; Add iodoacetamide to the denatured protein sample for light-shielded alkylation for 15 - 30 minutes; The material immobilized with cyclic phenylboronic acid is Fe3O4 / PDA / GO / PEI / CBX, which is obtained by sequentially coating polydopamine, monolayer graphene oxide, polyethyleneimine, and 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid on the magnetic sphere of iron oxide; SILAC labeling quantification is a quantitative method based on precursor ions. The sample is labeled with stable isotopes of light, medium, and heavy, or the sample is labeled with stable isotopes of light and heavy. After mass spectrometry acquisition, relative quantitative analysis is achieved by extracting the peak areas and peak intensities of paired precursor ion peaks with light, medium, and heavy labels, or paired precursor ion peaks with light and heavy labels, in the first-order spectrum; Preliminarily screen and identify potential target proteins of catechol drugs through the difference in labeling intensity: Among them, compared with the peak intensities of the experimental group and the control group, proteins with a peak intensity fold difference greater than 2 are considered proteins identified by labeling, thereby removing non-specifically adsorbed proteins; At the same time, use the second-order spectrum to perform sequence determination on the peptide segments of the identified proteins for protein identification; The cells include: one or more of tumor cells, hippocampal neuron cells, cardiac fibroblasts; 2. The method according to claim 1, wherein: Understand the mechanism of action of the drug through the biological processes and action pathways involved in its targets; 3. The method according to claim 1, characterized in that: The SILAC labeling reagent described above includes one or more of the following: SILAC DMEM, SILAC IMDML, SILAC DMEM / F12 media lacking L-lysine and L-arginine; or 13 C, 15 [N2]-L-lysine-HCl, 13 C6, 15 [N4]-L-arginine-HCl, L-lysine-HCl, L-arginine-HCl, D4-L-lysine-HCl, 13 C6-L-lysine-HCl, 13 C6-L-arginine-HCl, D8-L-lysine-HCl; or one or more of these The drug incubation conditions are: in an incubator at 37 °C with 5% CO2 air by volume concentration, incubate for 0.5 - 24 hours.

4. The method according to claim 1, wherein: The ionic liquid includes: one or more of 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroacetate, 1-butyl-3-methylimidazolium trifluoromethanesulfonate, 1-butyl-3-methylimidazolium bromide tetrafluoroborate, 1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium chloride, 1-octyl-3-methylimidazolium chloride, 1-dodecyl-3-methylimidazolium chloride, 1-allyl-3-methylimidazolium chloride, 1-aminopropyl-3-methylimidazolium tetrafluoroborate, 1-hydroxyethyl-3-methylimidazolium tetrafluoroborate, N-butylpyridinium chloride, N-butylpyridinium tetrafluoroborate; The ratio of cells to ionic liquid is: 3×10 6 cells are added to 200 - 600 μL of ionic liquid; The proportion of protease inhibitor cocktail in the ionic liquid is 0.5-5% (v / v); Add the ionic liquid containing protease inhibitor cocktail to the cells, ultrasonically disrupt the cells, the ultrasonic power is 80-160 W, and the ultrasonic time is 1-4 minutes; centrifuge at 10000-16000 g for 5-30 minutes, and take out the supernatant sample.

5. The method according to claim 1, wherein: The enrichment and elution method includes: incubating the magnetic material with the protein sample at 4-37 °C for 6-12 hours under the condition of pH 7-8.5; washing the material with 10-100 mM PBS at pH 7-8.5, separating with a magnet at 4-37 °C, discarding the supernatant, and repeating 2-4 times; Disperse the washed material into the eluent ACN:H2O:TFA = 50:49:1~80:19:1 (v / v / v) at pH 3-6.5, incubate at 4-37 °C for 1 hour, separate with a magnet, retain the supernatant, repeat 1 time, combine the two supernatants and freeze-dry, and redissolve the protein with 8M guanidine hydrochloride after freeze-drying.

6. The method according to claim 1, wherein: The enzymatic digestion method includes: adding protease, and performing enzymatic digestion for 12-16 hours at a protease:protein complex ratio of 1:20-1:50 (w / w); Using a C18 desalting column to desalt the enzymatically digested sample; freeze-drying the desalted sample; The protease includes one or more of trypsin, lysyl endopeptidase, chymotrypsin, lysine arginine N-terminal protease.

7. The method according to claim 1, wherein: SILAC labeling, namely stable isotope labeling technology under cell culture conditions, is to add light, medium and heavy stable isotope-labeled essential amino acids lysine and arginine to different cell culture systems respectively, or add light and heavy stable isotope-labeled essential amino acids to different cell culture systems respectively. Through normal metabolism during cell incubation, the newly synthesized proteins are labeled with stable isotope tags; extract the proteins contained in the cells after cell incubation, mix equal masses of the proteins extracted from different labeled cell culture systems, perform enzymatic digestion and then perform mass spectrometry quantitative analysis, and the essential amino acids are lysine and arginine.

8. The method according to claim 1, characterized in that: Using one or more of Uniprot, DAVID Bioinformatics Resources, GORILLA, PANTHER Classification System, and KEGG search engines to perform gene ontology annotation, enrichment analysis, and KEGG pathway analysis on the preliminarily identified potential target proteins, collecting annotation information of molecular functions, biological processes, cell components, and KEGG pathways with p-value less than or equal to 0.05 or FDR less than or equal to 1%, and obtaining the targeted protein pathway information and its distribution information within the cell.

9. The method according to claim 1 or 8, characterized in that: Further, by obtaining the information, determining the corresponding diseases that catechol drugs can treat and / or prevent, or determining the application of catechol drugs in the drugs for treating and / or preventing the corresponding diseases.

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