阅读说明：本技术 马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用 (Application of indacaterol maleate serving as cGAS-STING pathway targeted agonist ) 是由 何庆瑜 李杨葭 于 2021-09-29 设计创作，主要内容包括：本发明公开了马来酸茚达特罗在作为cGAS-STING通路靶向激动剂中的应用。本发明通过体外水平验证、生物素光亲和标记,结合蛋白质组学、生物信息学、分子生物学结果表明马来酸茚达特罗可以靶向激活cGAS-STING通路,抑制癌细胞增殖,对于辅助治疗结直肠癌相关疾病具有良好的治疗前景。(The invention discloses an application of indacaterol maleate serving as a cGAS-STING pathway targeted agonist. The results of in vitro level verification, biotin photoaffinity labeling, proteomics, bioinformatics and molecular biology show that the indacaterol maleate can activate a cGAS-STING pathway in a targeted manner, inhibit cancer cell proliferation and has good treatment prospect for adjuvant treatment of colorectal cancer-related diseases.)

1. The indacaterol maleate is applied to being used as a cGAS-STING pathway targeted agonist.

2. The indacaterol maleate is applied to the application as a cGAS-STING pathway targeted drug.

3. Use of indacaterol maleate in the treatment of diseases associated with the cGAS-STING pathway.

4. The use according to claim 3, wherein the disease is rectal cancer.

5. The use according to claim 3, wherein the medicament is a medicament for the treatment of rectal cancer.

6. Use according to claims 1-2, characterized in that the indacaterol maleate is present in a concentration of 0-30 um.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of indacaterol maleate serving as a cGAS-STING pathway targeted agonist.

Background

The indacaterol maleate has the following structural formula:

chronic Obstructive Pulmonary Disease (COPD) is a persistent, progressive airflow-limiting disease [1 ]. COPD patients often suffer from symptoms of dyspnea, muscle weakness, etc. due to factors such as impaired peripheral muscle function, impaired ventilation, impaired cardiopulmonary function, etc., further causing impaired exercise tolerance, resulting in a decreased quality of life [2 ]. Indacaterol maleate is used as a bronchodilator, is a long-acting beta 2 receptor agonist, can continuously relax bronchial smooth muscle and relieve excessive lung expansion, thereby improving airflow limitation and relieving symptoms of COPD patients [3 ].

The cyclic GMP-AMP synthase (cGAS) and interferon gene-stimulating factor (STING) pathways are pattern recognition pathways, and activation of STING leads to activation of inflammatory pathways by sensing cytoplasmic double-stranded DNA through cGAS [4 ]. cGAS is capable of catalyzing the production of cyclic guanine nucleotide-adenine nucleotide (cyclic GMP-AMP, cGAMP) which further activates STING and its downstream pathways [5 ]. The targeting activator of the cGAS-STING pathway has strong application value [6 ].

At present, no article exists for showing the relationship between indacaterol maleate and cGAS-STING pathway, and no patent application is made.

Disclosure of Invention

The invention aims to show the application of indacaterol maleate serving as a cGAS-STING pathway targeted agonist, and provides an idea for treating diseases such as colorectal cancer-related tumors.

The indacaterol maleate can target and activate a cGAS-STING passage through in vitro experiments and by combining proteomics, bioinformatics and molecular biology results.

The indacaterol maleate targeting cGAS-STING pathway is identified by using biotin photoaffinity labeling and proteomics: the indacaterol maleate is combined with a photo-crosslinking probe, the combination of the indacaterol maleate-probe and a potential target protein is identified by in-situ labeling of living cells, and the indacaterol maleate potential target protein is identified by combining a pull-down mass spectrometry.

In vitro levels were used to verify that indacaterol maleate targets cGAS: and verifying the indacaterol maleate targeting cGAS by using a pull-down-western blotting method, and verifying the indacaterol maleate targeting cGAS by knocking out cGAS at a cellular level.

In vitro levels were used to verify that indacaterol maleate activates the cGAS-STING pathway: and (3) identifying the cGAMP level in the colorectal cancer cells after the indacaterol maleate is treated by a cGAMP enzyme-linked immunosorbent assay, identifying the cGAS-STING downstream protein level after the indacaterol maleate is treated by a Western blotting method, and identifying the IFN beta level in the supernatant of the colorectal cancer cells after the indacaterol maleate is treated by an IFN beta enzyme-linked immunosorbent assay.

The invention provides a new application of indacaterol maleate, and provides a new medicine source for adjuvant therapy of cancer.

Compared with the prior art, the invention has the following beneficial effects: indacaterol maleate is a drug approved by the FDA for marketing for the treatment of chronic obstructive pulmonary disease. The research finds that the indacaterol maleate can target a cGAS-STING pathway besides the existing action, and as an activator of the indacaterol maleate, compared with the development of a new medicament, the indacaterol maleate has low cost, high safety and good development prospect.

Drawings

FIG. 1 shows the identification of indacaterol maleate targeting cGAS-STING pathway by biotin photoaffinity labeling in combination with proteomics.

Fig. 2 is a graph demonstrating that indacaterol maleate targets cGAS at the in vitro level.

FIG. 3 demonstrates that indacaterol maleate activates the cGAS-STING pathway at the in vitro level.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

Example 1

In order to achieve the purpose, the invention adopts the following technical scheme:

1. the biotin photoaffinity labeling is combined with proteomics to identify the indacaterol maleate targeting cGAS-STING pathway. The method comprises the following specific steps:

(1) indacaterol maleate was conjugated to a photocrosslinking probe. 50mg of indacaterol maleate (from Shanghai Toolff Biochemical Co., Ltd.) was dissolved in 5mL of Dimethylformamide (N, N-dimethyl formamide, DMF), and 25.8 mg of probe dipyridyl (3- (but-3-yn-1-yl) -3- (2-iodoethyl) -3H-diazine) (from Shanghai Biao medical science Co., Ltd.) and 26mg of potassium carbonate were added to mix the solution and stirred at 60 ℃ for 12 hours. After the reaction was completed, 10mL of ultrapure water was added to cool down, and extracted by ethyl acetate (2 times, 10mL each), and the organic layer was combined with 10mL of brine and washed twice, and dried over anhydrous sodium sulfate. The residue remaining after evaporation of the solvent was purified by flash column chromatography (dichloromethane: methanol ═ 30:1) to give indacaterol maleate with photocrosslinking probe (7 mg).

(2) The binding of the indacaterol maleate-probe and potential target protein is identified by in situ labeling of living cells. Colorectal cancer cells HT29 (purchased from ATCC) were plated in 6-well plates, and after the cell density reached 80% -90%, the medium was removed, serum-free 1640 medium (purchased from GIBCO) containing indacaterol maleate-probe concentrations of 0,7.5,15, and 30. mu.M was added, respectively, and incubated for 4.5 hours at 37 ℃/5% CO2 in an incubator. After incubation, the medium was removed, washed twice with pre-cooled PBS, and the 6-well plate was placed on ice and illuminated with 365nm UV light for 10 minutes. After the end of the light exposure, the cells were lysed with RIPA lysate (purchased from shanghai Binyan biotechnology limited), centrifuged at 14000 × g for 20 minutes at 4 ℃ per well, and the protein supernatant was collected and assayed for protein concentration with BCA reagent (purchased from Thermo Fisher Scientific). Equal amounts of protein were taken from each group and freshly prepared Click Chemistry reagents (50. mu.M TAMEA-N3, 0.1mM TBTA, 1mM TCEP, 1mM CuSO4, from Sigma-Aldrich and Click Chemistry Tools) were added and reacted for two hours at room temperature on a mixer. After the reaction, the reaction was terminated with 500. mu.M pre-chilled acetone, and the protein was precipitated by incubation at-20 ℃ for 1 hour. The precipitated proteins were separated by centrifugation at 4 ℃/14000 Xg and re-solubilized with 30. mu.L of SDS lysate containing 1 Xloading buffer and water bath at 95 ℃ for 10 minutes. An equal amount of sample from each group was run on a 10% SDS-PAGE gel, and the gel after completion of electrophoresis was imaged by fluorescence scanning using a Typhoon 9500 fluorescence gel scanner (Amersham Biosciences), stained with Coomassie Brilliant blue, and photographed.

(3) And identifying the potential target protein of the indacaterol maleate by using a combination of a pull-down mass spectrum and a mass spectrum.

Colorectal cancer cells HT29 (purchased from ATCC) were plated on 10cm cell dishes until the cell density reached 80% -90%, the medium was removed, serum-free 1640 medium (purchased from GIBCO) containing indacaterol maleate-probe concentration of 0, 30. mu.M was added, respectively, and incubated for 4.5 hours at 37 ℃/5% CO2 in an incubator. After incubation, the medium was removed, washed twice with pre-cooled PBS, and the cell dish was placed on ice and illuminated with 365nm UV light for 10 minutes. After the light exposure, the cells were lysed with RIPA lysate (purchased from shanghai Binyan biotechnology limited), centrifuged at 14000 × g for 20 minutes at 4 ℃ per dish, and the protein supernatant was collected and assayed with BCA reagent (purchased from Thermo Fisher Scientific). Equal amounts of protein were taken from each group and freshly prepared Click Chemistry reagents (50. mu.M Biotin-N3, 0.1mM TBTA, 1mM TCEP, 1mM CuSO4, from Sigma-Aldrich and Click Chemistry Tools) were added and reacted for two hours at room temperature on a mixer. After the reaction, the reaction was terminated with 500. mu.M pre-chilled acetone, and the protein was precipitated by incubation at-20 ℃ for 1 hour. The protein was re-solubilized with 1% SDS-containing PBS and incubated with 80. mu.L streptavidin agarose beads overnight at 4 ℃. Agarose beads were collected by centrifugation at 4 ℃/2500rpm for 10 minutes and proteins were lysed with 50 μ L SDS lysate.

Equal volume of 8M urea solution was added to each group, and DTT solution with a final concentration of 50mM was added thereto, followed by reaction in water bath at 37 ℃ for 1 hour. A final concentration of 150mM IAA solution was added and the reaction was carried out for 30 minutes at room temperature with the exclusion of light. The reacted solution was added to a 30kD ultrafilter tube previously rinsed with 50mM TEAB (purchased from Sigma-Aldrich) and rinsed twice with 200. mu.L urea and five times with 200. mu.L TEAB, respectively. Mu.g of mass-spectrometric grade pancreatin (purchased from Beijing Huali science, Ltd.) was added to the ultrafiltration tube and incubated overnight at 37 ℃. The digested peptide fragment solution was desalted by MonoTIPTM C18 desalting column (from GL Sciences) and mass spectrometry was performed using Orbitrap Fusion Lumos mass spectrometer (from Thermo Fisher Scientific), and the raw data were pooled using Spectronaut software (Omicsolution Co., Ltd.).

2. The indacaterol maleate was validated to target cGAS at the in vitro level. The method comprises the following specific steps:

(1) the combination of the pull-down western blotting method and the indacaterol maleate target cGAS is verified.

Colorectal cancer cells HT29 (purchased from ATCC) were plated on 10cm cell dishes until the cell density reached 80% -90%, the medium was removed, serum-free 1640 medium (purchased from GIBCO) containing indacaterol maleate-probe concentration of 0, 30. mu.M was added, respectively, and incubated for 4.5 hours at 37 ℃/5% CO2 in an incubator. After incubation, the medium was removed, washed twice with pre-cooled PBS, and the cell dish was placed on ice and illuminated with 365nm UV light for 10 minutes. After the light exposure, the cells were lysed with RIPA lysate (purchased from shanghai Binyan biotechnology limited), centrifuged at 14000 × g for 20 minutes at 4 ℃ per dish, and the protein supernatant was collected and assayed with BCA reagent (purchased from Thermo Fisher Scientific). Equal amounts of protein were taken from each group and freshly prepared Click Chemistry reagents (50. mu.M Biotin-N3, 0.1mM TBTA, 1mM TCEP, 1mM CuSO4, from Sigma-Aldrich and Click Chemistry Tools) were added and reacted for two hours at room temperature on a mixer. After the reaction, the reaction was terminated with 500. mu.M pre-chilled acetone, and the protein was precipitated by incubation at-20 ℃ for 1 hour. The protein was re-solubilized with 1% SDS-containing PBS and incubated with 80. mu.L streptavidin agarose beads overnight at 4 ℃. The agarose beads were collected by centrifugation at 4 ℃/2500rpm for 10 minutes and the proteins were lysed with 50 μ L of SDS lysate containing 1 × loading buffer. Equal amounts of samples from each group were run on a 10% SDS-PAGE gel for Western blot analysis, and bands were incubated with cGAS antibody, the protein of interest, and visualized.

(2) And knocking out cGAS at a cellular level to verify that the indacaterol maleate targets the cGAS.

The sgcGAS plasmid was used to construct HT29 cell line which knocks out endogenous cGAS protein. The sgcGAS plasmid was purchased from ayaki biotechnology, guangzhou, CGCATCCCTCCGTACGAGAA for the sgRNA fragment and lentiCRISPRv2 for the plasmid vector. 293T cells (purchased from ATCC) in logarithmic phase were seeded at 50% density in 6-well plates and cultured for 12 hours; mixing sgcGAS plasmid with packaging plasmids PSPAX2 (from Addge) and PMD2G (from Addge) in opti-MEM (from Thermo Fisher Scientific), adding p3000 (from Life Science), adding another tube of opti-MEM into Lipo3000(Life Science), mixing and standing for 5 min, mixing and standing for 20 min; the 293T cells were replaced with the above mixed transfection reagent, cultured in an incubator for 6 hours, replaced with fresh complete medium for 48 hours, and the cell supernatant was collected, centrifuged to remove cell debris, and filtered through a 0.45 μm filter to completely remove debris. The filtered supernatant was added to a 6-well plate inoculated with HT29 cells at 30% density 1 day before infection for 24 hours, and the virus-containing medium was removed and replaced with fresh medium. And after 48 hours, adding puromycin (1 mu g/mL) for screening, changing the solution every two days, changing the solution into a normal culture medium for culture after screening for one week, detecting the cGAS protein knockout condition, and preserving the seeds for later use.

And (3) respectively cracking the cGAS-knocked-out HT29 cells and control group cells to obtain proteins, measuring the concentration by using a BCA method, adding 5 × loading buffer in equal amount to prepare samples, performing a western blot experiment by using 10% SDS-PAGE gel, and incubating a band after membrane transfer with a cGAS antibody (purchased from ABClonal) to detect the cGAS knocking-out condition. HT29 cells from the control and cGAS-knockout groups were plated into 96-well plates, 3000 cells per well. After 12 hours of culture, the medium was removed, the two groups were treated for 24 hours at two indacaterol maleate treatment concentrations (0,15 μ M), and cell proliferation was detected using CCK8 reagent (purchased from Shanghai pottery Biochemical technologies, Ltd.).

3. The in vitro level verifies that indacaterol maleate activates the cGAS-STING pathway. The method comprises the following specific steps:

(1) and (3) identifying the intracellular cGAMP level of the colorectal cancer after the indacaterol maleate is treated by a cGAMP enzyme-linked immunosorbent assay. HT29 cells were plated in 6-well plates at 1.2X 106 cells per well and cultured for 12 hours. The medium containing indacaterol maleate with concentration of 0,15,30 μ M was replaced with 1640 complete medium and cultured for 48 hours. After the treatment was completed, the medium was removed, washed twice with PBS, the cells were lysed with M-PER MAMMALIAN PROTEIN EXTRACTION REAGENT lysate (from Thermo Fisher Scientific), and intracellular cGAMP levels were measured using cGAMP ELISA kit (from Cayman Chemical).

(2) And (3) identifying the protein level of cGAS-STING downstream after indacaterol maleate treatment by a western blot method. HT29 cells are paved into a 6-well plate, and after the cell density reaches 60%, 1640 complete culture media containing indacaterol maleate with the concentrations of 0,15 and 30 mu M are respectively added for culturing for 48 hours. After the treatment is finished, each group of cells are respectively lysed to obtain protein, after the concentration is measured by using a BCA method, an equal amount of 5 × loading buffer is added to prepare a sample, a 10% SDS-PAGE gel is used for carrying out a western blot experiment, and a strip after membrane transfer is used for incubating a pSTING antibody, a pTBK1 antibody, a pIRF3 antibody (all purchased from ABCloncal) and a beta-Actin antibody (purchased from Bioworld) to detect the downstream activation condition of the cGAS-STING pathway.

(3) And (3) identifying the IFN beta level in the supernatant of the colorectal cancer cells after the indacaterol maleate treatment by an IFN beta enzyme-linked immunosorbent assay. HT29 cells were plated in 6-well plates at 1.2X 106 cells per well and cultured for 12 hours. The medium containing indacaterol maleate with the concentration of 0,15 and 30 μ M was replaced and cultured in serum-free 1640 medium for 48 hours. After the treatment, the cell supernatants were collected, centrifuged at 4 ℃/1000 Xg to remove cell debris, and the IFN β levels in the cell culture supernatants were measured using a Human IFN- β ELISA kit (available from Hangzhou Union Biotechnology, Inc.).

The biotin photoaffinity labeling is combined with proteomics to identify the indacaterol maleate targeting cGAS-STING pathway.

The hydrogen nuclear magnetic resonance spectrum of the indacaterol maleate combined with the photocrosslinking probe is shown in FIG. 1A, and the result shows that the indacaterol maleate and the photocrosslinking probe are successfully combined. The in situ labeling experiment result of the living cells is shown in fig. 1B, in the fluorescence imaging (left), the amount of the protein pulled down shows an increasing trend along with the increase of the concentration of the indacaterol maleate-probe, and the coomassie brilliant blue stained holoprotein (right) is used as an internal reference, which indicates that the indacaterol maleate-probe can pull down the potential target protein bound with the indacaterol maleate-probe. As shown in fig. 1C, cGAS is an indacaterol maleate potential target protein that could be detected by pulldown-mass spectrometry in the indacaterol maleate probe, but could not be detected in the control group.

The indacaterol maleate was validated to target cGAS at the in vitro level.

As shown in fig. 2A, in all protein lysates of the control and indacaterol maleate-probe sets, equal amounts of cGAS protein could be detected, whereas in both the control and the protein lysate pulled down with indacaterol maleate-probe, only the latter could detect cGAS protein, indicating that indacaterol maleate-probe could specifically bind to cGAS protein and pull down it. As shown in fig. 2B, the cGAS endogenous protein knockout was successful by detecting cGAS protein expression of HT29 cell line and control cell line from which endogenous cGAS protein was knocked out by western blotting; as shown in fig. 2C, cGAS-knocked-out HT29 cells were less sensitive to indacaterol maleate than control cells, and cGAS-knocked-out HT29 cells were more survivable under the same concentration and time of indacaterol maleate treatment, indicating that indacaterol maleate inhibits the growth of HT29 cells through cGAS.

The in vitro level verifies that indacaterol maleate activates the cGAS-STING pathway.

As shown in fig. 3A, as the concentration of indacaterol maleate treated increases, the intracellular cGAMP level also shows an increasing trend, indicating that indacaterol maleate can target the activation of cGAS to generate cGAMP, which further activates STING and downstream pathways. As shown in FIG. 3B, with the increase of the treated concentration of indacaterol maleate, the amounts of activated forms pSTING, pTBK1 and pIRF3 proteins of the cGAS-STING pathway STING, TBK1 and IRF3 proteins all showed an increasing trend, which indicates that indacaterol maleate can activate the cGAS-STING pathway. As shown in fig. 3C, with the increase of indacaterol maleate treatment concentration, IFN β levels in cell supernatants also showed an increasing trend, indicating that indacaterol maleate treatment can activate cGAS-STING pathway and thus downstream production of type I interferon.

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the foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.