Use of asparagine for the preparation of a medicament for the treatment of an immunodeficiency disease and / or for the treatment of a viral infection

Abstract

This invention relates to the application of asparagine in the preparation of antiviral drugs and / or drugs for treating immunodeficiency diseases, belonging to the field of biomedical technology. This invention is the first to discover that asparagine can induce TBK1 activation, increasing the expression of type I interferon in the body under viral infection conditions, enabling the body to rapidly respond to host defense mechanisms and enhance immunity when subjected to viral infection, thereby achieving a broad-spectrum antiviral effect. It also discloses a new use of asparagine as a TBK1 agonist, which can be used in the preparation of antiviral drugs, drugs for treating immunodeficiency diseases, and products for enhancing immunity. Furthermore, asparagine is a non-essential amino acid for the human body, possessing characteristics such as being green, safe, and environmentally friendly; it can be used as a functional additive in pharmaceuticals, health products, or food products.

Description

Technical Field

[0001] This invention relates to the application of asparagine in the preparation of antiviral infection drugs and / or drugs for treating immunodeficiency diseases, and belongs to the field of biomedical technology. Background Technology

[0002] Viral infections pose a serious threat to human health, and sudden large-scale infectious diseases can even disrupt social order. Therefore, studying the mechanisms of viral infection and intervention strategies has become a major concern in the global medical field. The interaction between viral infection and host immunity is key to determining the outcome of viral infections.

[0003] After a virus invades the body, it replicates within host cells and activates the immune system. Innate immunity is the host's first line of defense against viral infection, with TBK1-mediated innate immunity playing a crucial role in clearing the virus. Viral infection-mediated activation of different pattern recognition receptors (PRRs) recruits and activates IRF3 by activating the common key kinase TBK1, thereby inducing the production of type I interferon (IFN-α / β) and initiating an antiviral immune response. The interaction between the virus and the host immune system is key to determining the outcome of viral infection.

[0004] With a deeper understanding of the tertiary structure and biological functional range of TBK1 kinase, TBK1 has become a potential target for the treatment of viral diseases, inflammatory diseases, autoimmune diseases, cancer, metabolic diseases, and neurodegenerative diseases. TBK1 has attracted widespread interest as a novel therapeutic target; however, no TBK1 agonists have been clearly reported to date. The molecular mechanism of action of TBK1 is not fully understood, and further detailed research is needed to elucidate its potential biological functions and mechanisms of action in different diseases and cell types. Given the important role of TBK1 in antiviral immune responses, the search for a novel TBK1 agonist also provides a candidate drug for the treatment of viral diseases. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of asparagine in the preparation of antiviral infection drugs and / or drugs for treating immunodeficiency diseases.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides the use of asparagine in the preparation of antiviral infection drugs.

[0008] According to a preferred embodiment of the present invention, the asparagine increases the expression level of type I interferon by inducing TBK1 activation, thereby inhibiting viral infection.

[0009] According to a preferred embodiment of the present invention, the virus is a DNA virus or an RNA virus.

[0010] More preferably, the DNA virus is herpes simplex virus-1, and the RNA virus is vesicular stomatitis virus.

[0011] Secondly, the present invention provides the use of asparagine in the preparation of drugs for treating immunodeficiency diseases.

[0012] According to a preferred embodiment of the present invention, the asparagine enhances immunity by inducing TBK1 activation, thereby treating immunodeficiency diseases.

[0013] According to a preferred embodiment of the present invention, the immunodeficiency disease is a congenital immunodeficiency disease or a secondary immunodeficiency disease.

[0014] According to a preferred embodiment of the invention, the drug comprises an effective dose of asparagine or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0015] More preferably, the dosage form of the drug is tablet, capsule, granule, pill, liquid preparation, decoction, suspension, dispersant, syrup, suppository, gel, aerosol, or patch.

[0016] Thirdly, the present invention provides the application of asparagine in the preparation of products for the prevention or treatment of viral infections and the enhancement of immunity.

[0017] According to a preferred embodiment of the present invention, the product is a drug, health product, or food.

[0018] Fourthly, this invention provides the application of formulations that increase asparagine levels in the body in the preparation of products for treating or preventing viral infections and enhancing immunity.

[0019] More preferably, the preparation that increases the level of asparagine in the body is an asparagine salt or its derivative that can be hydrolyzed to form free asparagine after entering the body.

[0020] Beneficial effects:

[0021] 1. This invention is the first to discover and disclose a new use of asparagine as a TBK1 agonist, which can be used to prepare antiviral infection drugs, drugs for treating immunodeficiency diseases, and products to improve immunity.

[0022] 2. This invention has discovered that increasing asparagine levels at the cellular and mouse levels can help improve the body's immunity and enhance its antiviral capabilities. The main mechanism is that asparagine can induce TBK1 activation, increasing the expression of type I interferon under viral infection conditions. This allows the body to rapidly respond to host defense mechanisms when exposed to viral infection, improving immunity and thus achieving a broad-spectrum antiviral effect. It has significant potential in the preparation of antiviral drugs, drugs for treating immunodeficiency diseases, and products that enhance immunity. Furthermore, asparagine is a non-essential amino acid for the human body, possessing characteristics such as being green, safe, and environmentally friendly. It can be used as a functional additive in pharmaceuticals, health products, and food products. Attached Figure Description

[0023] Figure 1 The results show the asparagine content in peritoneal macrophages of mice after viral infection;

[0024] In the figure, A is a graph showing the change in asparagine content detected by targeted metabolomics; B is a graph showing the change in asparagine content in cells at different infection times detected by ELISA.

[0025] Figure 2 Asparagine promotes antiviral immune responses;

[0026] In the figure, A~C represent the secretion of IFN-β in peritoneal macrophages of different mice and... Ifnb mRNA expression levels; D and E represent the expression levels of different mouse peritoneal macrophages detected by qPCR. Mx1 , Rantes , Isg15 , Isg54 and Isg56 mRNA expression level; F represents the viral titer in the supernatant of peritoneal macrophages in different mice as detected by viral plaque assay.

[0027] Figure 3 Graph showing the effect of asparagine on enhancing antiviral immune response in vivo;

[0028] In the figure, A shows the detection of IFN-β secretion in serum using ELISA; B shows the detection of IFN-β in tissues using qPCR. Ifnb C represents the expression level of mRNA; D represents the viral titer in the tissue homogenate detected by the viral plaque assay; and D represents the survival curve plotted using the Mantel-Cox method.

[0029] Figure 4 The diagram shows the results of asparagine targeting and promoting TBK1 activation;

[0030] In the figure, A shows the activation of the IFN-β promoter in different HEK293T cells as detected by a dual-luciferase reporter gene assay; B shows the Western blot results of HEK293T cells transfected with TBK1 plasmid after asparagine pretreatment; C shows the kinase activity of purified TBK1 protein after asparagine treatment as detected by an in vitro kinase assay; and D is a micrograph of TBK1 droplets formed by purified TBK1 and 250 mM NaCl in the presence of Asn. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] In this invention, the drug comprises asparagine or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0033] In this invention, the drug is any pharmaceutically acceptable dosage form made by using asparagine or its pharmaceutical salt as the active pharmaceutical ingredient and combining it with pharmaceutically acceptable excipients.

[0034] In this invention, the dosage form is tablet, capsule, granule, pill, liquid preparation, decoction, suspension, dispersant, syrup, suppository, gel, aerosol, or patch.

[0035] The present invention does not impose any special limitations on the preparation method of the drug for the prevention or treatment of viral infections containing asparagine or its pharmaceutical salts; any pharmaceutical method commonly used by those skilled in the art can be followed.

[0036] The asparagine is available from Sigma (catalog number: A8381); the asparaginase used in the in vitro experiments is available from ChemeGen (catalog number: CP60465).

[0037] The DNA virus is herpes simplex virus type I (HSV-1), and the RNA virus is vesicular stomatitis virus (VSV). Both HSV-1 and VSV were donated to Professor Cao Xuetao of Nankai University, and both are routine laboratory viruses available from biotechnology companies.

[0038] The C57BL / 6 mice were available from Tonglihua Animal Technology Co., Ltd. All animals were used at 6–8 weeks of age. Animal experiments were conducted in accordance with the "Guidelines for Laboratory Animal Care and Use" issued by the National Institutes of Health and approved by the Ethics Committee of the School of Basic Medical Sciences, Shandong University.

[0039] Experimental method for targeted metabolomics detection: Cells were collected into 1.5 mL centrifuge tubes. 100 μL of ultrapure water was added to resuspend the cell microspheres. 50 μL of the cell suspension was frozen in liquid nitrogen, thawed three times, and the supernatant was incubated at 12000 × 10⁻⁶. g Centrifuge for 10 min. Determine protein concentration using a BCA protein assay kit (Pierce, Thermo Fisher Scientific). Add the remaining 50 μL of cell suspension to 200 μL of cold methanol, vortex for 2 min, freeze in liquid nitrogen for 5 min, place on ice for 5 min, and repeat 3 times. Incubate the sample at 12,000 × 10⁻⁶ at 4 °C. g Centrifuge for 10 min, transfer 200 μL of the resulting supernatant to a new centrifuge tube, incubate at 20 °C for 30 min, and then centrifuge at 12,000 × 10⁻⁶. g Centrifuge for 10 min, and then pass 180 μL of the supernatant through a protein precipitation plate for LC-MS analysis. The sample extract was analyzed using an LC-ESI-MS / MS system. The T3 method was performed using a Waters ACQUITY UPLCHSS T3 C18 (100 mm × 2.1 mm; inner diameter 1.8 μm) high-performance liquid chromatography (HPLC) column. The mobile phase consisted of solvent A (water containing 0.05% formic acid) and solvent B (acetonitrile containing 0.05% formic acid). The gradient started at 5% B (0 min), increased to 95% B (8–9.5 min), and finally decreased to 5% B (9.6–12 min); the flow rate was 0.35 mL / min; the temperature was 40 °C; and the injection volume was 2 μL. For the amide method, an ACQUITY UPLC BEH amide HPLC column (100 mm × 2.1 mm; diameter 1.7 μm) was used. The mobile phase consisted of solvent A (water containing 10 mM ammonium acetate and 0.3% ammonium hydroxide) and solvent B (90% acetonitrile / water [v / v]). The gradient started at 95% B (0–1.2 min), decreased to 70% B (8 min), then decreased to 50% B (9–11 min), and finally returned to 95% B (11.1–15 min). The flow rate was 0.4 mL / min, the temperature was 40 °C, and the injection volume was 2 μL.

[0040] The experimental method for quantitative analysis of asparagine is as follows: using an asparagine detection kit (GraceBiotechnology), the asparagine content in cells is quantitatively detected according to the instructions.

[0041] Experimental methods for ELISA and qPCR analysis: The IFN-β content in cell supernatant and mouse serum was detected using the IFN-β ELISA kit (BioLgend) according to the instructions. Total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen). RNA was reverse transcribed into cDNA using HiScript III RT SuperMix (Vazyme). PCR analysis was performed using SYBR Green (Vazyme). Data were normalized using β-actin as an internal reference gene.

[0042] Experimental methods for viral plaques: HSV-1 viral titers were detected using Vero cells, and VSV viral titers were detected using HEK293T cells. Supernatants from virus-infected cells or mouse tissue homogenates were serially diluted and added to Vero or HEK293T cells. After 2 hours, the supernatants were discarded, and the cells were cultured in DMEM supplemented with 1% methylcellulose for 48 hours. Cells were then fixed with 4% paraformaldehyde for 15 minutes, stained with 1% crystal violet for 30 minutes, and plaques were counted. Viral titers were determined in pfu / ml.

[0043] Method for detecting TBK1 kinase activity in vitro: TBK1 kinase activity was detected using the ADP-Glo ​​and TBK1 kinase system (Promega) according to the instructions for use. TBK1 protein (100 ng) was incubated at room temperature with solvent (Ctrl) or asparagine (Asn, 100 μM) for 30 min, followed by incubation with 2.5-fold ATP / MBP substrate for 1 h. ADP-Glo™ reagent was added to the reaction system, and incubation was carried out at room temperature for 40 min, followed by the addition of the kinase detection reagent. Chemiluminescence was detected after 30 min of incubation at room temperature.

[0044] In vitro phase separation detection method: Purified His-hTBK1 protein (70 μM) was mixed with sodium chloride (250 mM) and asparagine (500 μM) in a 35 mm confocal culture dish (WHB SCIENTIFIC), which was coated with 50 mg / ml BSA (Sigma). Images were acquired after incubation at 37 °C for 5 min. The phase-separated droplets were imaged under an OLYMPUS IX53 fluorescence microscope.

[0045] Western blotting procedure: Cells were washed with phosphate-buffered saline, lysed with RIPA protein extraction reagent (Pierce, Thermo Fisher Scientific) and added with protease inhibitors (phenylmethylsulfonyl fluoride, Beyotime) and phosphatase inhibitors (CWBIO), and then incubated at 4°C at 12,000 × 10⁻⁶. gCentrifuge for 15 minutes. Determine the protein concentration in the supernatant using the BCA protein assay kit. Adjust the lysate to the same concentration and perform electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel. Then transfer the protein to a PVDF membrane for Western blotting. Incubate the membrane overnight with primary antibody dilution buffer. The next day, wash the membrane three times with TBST and incubate for 1 hour with secondary antibody (Immunoway) dilution buffer (1:4000). Image the protein bands using an enhanced chemiluminescence kit (NCM Biotech) according to the manufacturer's protocol.

[0046] Example 1: Determination of asparagine content in mouse peritoneal macrophages after viral infection

[0047] 1. Mice were injected intraperitoneally with 3% mercaptoacetate. Three days after the injection, primary mouse peritoneal macrophages (PMs) were isolated in a clean bench. The isolated macrophages were cultured in high-glucose DMEM (Pronoseu) medium containing 10% fetal bovine serum (Pronoseu, FBS) at 37°C and 5% CO2 saturated humidity to obtain mouse peritoneal macrophages.

[0048] Then, HSV-1 was added to mouse peritoneal macrophages for 24 hours to obtain HSV-1 infected macrophages (24h).

[0049] 2. HSV-1-infected macrophages and uninfected macrophages were collected into 1.5 mL centrifuge tubes, and then changes in metabolite content were detected using targeted metabolomics. The results are as follows: Figure 1 As shown in Figure A.

[0050] 3. HSV-1-infected macrophages and VSV-infected macrophages were collected at different infection times (0, 4, 8, 12, 24 h). The asparagine content in different cells was detected using an asparagine detection kit. The results are as follows: Figure 1 As shown in B.

[0051] 4. Experimental Results and Analysis

[0052] Depend on Figure 1 As shown in A, the content of metabolites in mouse peritoneal macrophages changed significantly before and after HSV-1 virus infection, with asparagine content decreasing significantly 24 hours after HSV-1 infection.

[0053] Depend on Figure 1 As shown in B, the asparagine content in macrophages gradually decreases with the duration of viral infection.

[0054] Example 2: Asparagine enhances the antiviral immune response of mouse peritoneal macrophages in vitro.

[0055] 1. Asparagine (Asn, final concentration 100 μM) was added to mouse peritoneal macrophages and pre-incubated at 37°C for 48 hours, forming the experimental group (Asn). Mouse peritoneal macrophages without asparagine were used as the control group (Ctrl). HSV-1 and VSV were then added to the experimental and control groups, respectively. After stimulation for 8 hours, cell supernatants were collected, with an equal volume of double-distilled water (DDW) added as a blank control (mock). Finally, the IFN-β content in the supernatant of each group was measured using an ELISA kit (BioLegend) via enzyme-linked immunosorbent assay (ELISA). The results are shown below. Figure 2 As shown in Figure A. Next, qPCR analysis was used to quantitatively detect the presence of macrophages in the peritoneal cavity of mice in each group. Ifnb mRNA expression levels, results as follows Figure 2 As shown in B~C.

[0056] 2. Asparagine (Asn, final concentration 100 μM) was added to mouse peritoneal macrophages and pre-incubated at 37°C for 48 hours, forming the experimental group (Asn). Mouse peritoneal macrophages without asparagine were used as the control group (Ctrl). HSV-1 and VSV were then added to the experimental and control groups, respectively, and stimulated for 12 hours. Subsequently, type I interferon (e.g., 100 μM) was quantitatively detected by qPCR. Ifnb mRNA) and interferon-stimulated genes (e.g.) Mx1, Rantes, Isg15, Isg54 and Isg56 The expression level of mRNA was measured, and the results were as follows: Figure 2 As shown in D and E.

[0057] 3. Asparagine (Asn, final concentration 100 μM) was added to mouse peritoneal macrophages and pre-incubated at 37°C for 48 hours, forming the experimental group (Asn). Mouse peritoneal macrophages without asparagine were used as the control group (Ctrl). HSV-1 and VSV were then added to the experimental and control groups, respectively, and stimulated for 12 hours. Cell supernatants were then collected from each group, and viral titers were detected using the plaque assay. The results are shown below. Figure 2 As shown in F.

[0058] 4. Experimental Results and Analysis

[0059] Depend on Figure 2 As shown in A~C, compared with the control group, the secretion of IFN-β in the macrophage supernatant after asparagine supplementation was... Ifnb The expression level of mRNA increased significantly.

[0060] Depend on Figure 2 As shown in D~E, compared with the control group, the level of type I interferon (e.g., ) in macrophages was significantly reduced after asparagine supplementation. Ifnb mRNA) and interferon-stimulated genes (e.g.) Mx1, Rantes, Isg15, Isg54and Isg56 The transcriptional level of mRNA was significantly increased.

[0061] Depend on Figure 2 As shown in F, the viral plaque assay revealed a significant decrease in viral titer in the cell supernatant.

[0062] Example 3: Asparagine enhances antiviral immune response in mice

[0063] C57BL / 6J mice were divided into two groups. The first group was fed a diet without asparagine and was given drinking water without asparagine. The second group was fed a diet without asparagine and was given drinking water containing 10 mM asparagine. After 20 days of continuous feeding, the mice in each group were divided into two subgroups. Groups 2 and 4 were infected with HSV-1 or VSV by intraperitoneal injection. Groups 1 and 3 were given an equal amount of PBS buffer.

[0064] The expression changes of IFN-β in the serum and spleen tissue of mice in each group were detected by ELISA and qPCR assays, respectively. The results are as follows: Figure 3 As shown in A and B, the viral titer in mouse lung and spleen tissues was detected using the viral plaque assay, and the results are as follows. Figure 3 As shown in C. The survival rate of mice under viral infection was also observed, and the results are as follows. Figure 3 As shown in D.

[0065] Depend on Figure 3 As shown in A~B, compared with the control group, the expression of IFN-β in the serum and tissues of mice was significantly increased after supplementation with asparagine.

[0066] Depend on Figure 3 C indicates that, compared with the control group, the viral titer in the tissue homogenate of the lungs and spleen of mice supplemented with asparagine was significantly reduced.

[0067] Depend on Figure 3 As shown in D, compared with the control group, asparagine supplementation can significantly prolong the survival rate of mice under viral infection.

[0068] All the above results demonstrate that, at the cellular and mouse levels, asparagine supplementation can significantly enhance the host's antiviral immune function.

[0069] Example 4: Asparagine significantly enhances the production of type I interferon induced by DNA and RNA viruses.

[0070] 1. HEK293T cells were cultured to the logarithmic growth phase. Then, innate immune response adaptor molecules such as cyclic GMP-AMP synthase-interferon gene stimulating factor (cGAS-STING), mitochondrial antiviral signaling protein (MAVS), retinoic acid inducible gene I receptor (RIG-I), TANK-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3-5D) were transfected into HEK293T cells. Asparagine was then added to each group of cells to a final concentration of 100 μM. The cells were pre-incubated at 37°C for 24 hours and designated as experimental groups: cGAS-STING+Asn, MAVS+Asn, RIG-I+Asn, TBK1+Asn, and IRF3-5D+Asn.

[0071] HEK293T cells were cultured to the logarithmic growth phase, and then transfected with innate immune response adaptor molecules cGAS-STING, MAVS, RIG-I, TBK1, and IRF3-5D, respectively. Each group of cells was then pre-incubated with double-distilled water equal to the amount of asparagine at 37°C for 24 hours. These groups were designated as control groups: cGAS-STING+Ctrl, MAVS+Ctrl, RIG-I+Ctrl, TBK1+Ctrl, and IRF3-5D+Ctrl.

[0072] HEK293T cells were cultured to the logarithmic growth phase, and then an empty vector plasmid with an equal amount of innate immune response adaptor molecule was transfected into HEK293T cells. The cells were then divided into two groups. One group was given asparagine to a final concentration of 100 μM, and the other group was given an equal amount of double-distilled water. The cells were pre-incubated at 37°C for 24 hours and were designated as the blank group (mock+Asn) and the mock+Ctrl group.

[0073] Cells from the experimental group, control group, and blank group were collected, and the activity of the IFN-β promoter in the cells was detected using dual-luciferase assay. The results are as follows: Figure 4 As shown in Figure A.

[0074] Depend on Figure 4 As shown in A, asparagine does not affect the activity of IRF3-5D in enhancing the IFN-β promoter, indicating that asparagine targets TBK1.

[0075] 2. TBK1 plasmid was transfected into HEK293T cells, and asparagine was added to a final concentration of 100 μM. The cells were pre-incubated at 37°C for 24 hours. The expression level of p-TBK1 protein was detected by Western blotting, with Actin as the internal control. Results are shown below. Figure 4 As shown in B.

[0076] The TBK1 plasmid was constructed by Jinan Boshan Biotechnology Co., Ltd.

[0077] Depend on Figure 4 As shown in B, asparagine can promote the autophosphorylation of TBK1.

[0078] 3. The purified His-hTBK1 protein (70 μM) was co-incubated with asparagine (100 μM) for 48 h under in vitro conditions. The TBK1 kinase activity was then detected using a TBK1 kinase assay kit. The results are as follows: Figure 4 As shown in B.

[0079] Depend on Figure 4 As shown in C, asparagine can significantly promote the kinase activity of TBK1.

[0080] 4. The purified His-hTBK1 protein (70 μM) was mixed with sodium chloride (250 mM) and asparagine (500 μM) in a 35 mm confocal culture dish (WHB SCIENTIFIC), which was coated with 50 mg / ml BSA (Sigma). Images were acquired after incubation at 37°C for 5 min. The results are shown below. Figure 4 As shown in D.

[0081] Depend on Figure 4 As shown in D, asparagine induces liquid-phase separation of TBK1.

[0082] The above results indicate that asparagine targets TBK1 and induces its activation, making it a novel TBK1 agonist.

[0083] The embodiments described above are merely preferred implementations of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of asparagine for the preparation of a medicament for the treatment of a viral infection, characterized in that, The virus in question is either herpes simplex virus-1 or vesicular stomatitis virus.

Images