Use of one-carbon metabolism inhibitors in inhibiting viral infection

Abstract

The application relates to the field of preventing veterinary technology, in particular to application of a carbon metabolism inhibitor in inhibiting viral infection, and discloses that SHIN1 has the functions of inhibiting water blistering stomatitis virus and Seneca virus for the first time; through evaluation on viral titer, viral mRNA and protein level, the result shows that the carbon metabolism inhibitor inhibits viral replication; the application provides a scientific experimental basis for prevention and control of viruses and provides a feasible and effective treatment scheme for clinically preventing and treating viral infectious diseases.

Description

Technical Field

[0001] This invention relates to the field of preventive veterinary medicine, specifically the application of one-carbon metabolism inhibitors in inhibiting viral infections. Background Technology

[0002] Serine hydroxymethyltransferase (SHMT) is a key protein regulating one-carbon metabolism, with two isoforms: cytoplasmic (SHMT1) and mitochondrial (SHMT2). ​​Mammalian organisms possess both SHMT1 and SHMT2. SHMT catalyzes the conversion of serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. This reversible conversion provides a carbon unit for nucleotide synthesis and influences DNA methylation and NADH / NADPH production. In SHMT2-deficient cell lines, they support the synthesis of thymidine nucleotides and pyrimidines by catabolizing serine through SHMT1. High-specificity SHMT1 / 2 dual inhibitors... HIN1 inhibits SHMT1 and SHMT2. Regarding the development of one-carbon metabolism inhibitors, the first SHMT inhibitor was NSC127755, but its development was limited due to its adverse reactions. Leukopsin was also shown to inhibit both SHMT subtypes, but it has not been used clinically because it is easily converted into other folic acid analogs in vivo. In 2015, Marani et al. discovered a plant-derived SHMT inhibitor compound 2.12, which induces lung cancer cell death through apoptosis. In 2017, Ducker et al. optimized SHIN1 based on plant-derived inhibitors, and SHIN1 enhanced the activity of SHMT.

[0003] Vesicular stomatitis (VS) is an acute, highly contagious infectious disease of various mammals, including pigs, horses, and cattle, caused by vesicular stomatitis virus (VSV). VSV belongs to the family Rhabdoviridae and the genus Varicavivirus. Its genome is approximately 11 kb in length and is a single-stranded, negative-sense RNA without segmentation. VSV is transmitted through arthropods, infecting livestock and rodents, and can also infect humans. The VSV genome encodes five proteins from the 3′ to 5′ ends: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and viral RNA polymerase (L). In nature, this virus is characterized by its wide transmission range, numerous susceptible animals, and complex morphology. Although the mortality rate of this disease is not high, the harmful consequences on livestock are irreversible, and the economic losses to the livestock industry are enormous. Therefore, the prevention and control of this disease has important socio-economic and public health significance.

[0004] Recent reports indicate that SARS-CoV-2, a model virus, hijacks folic acid and one-carbon metabolism for self-replication. The results show that SHIN1 inhibits SARS-CoV-2 replication. SHIN1 exhibits effective and specific target activity against SHMT in human colorectal adenocarcinoma cells and inhibits the proliferation of a wide range of cancer cell lines. SHIN2 was further optimized based on SHIN1. SHIN2 has an anti-leukemic effect by inhibiting T cell growth through the inhibition of SHMT.

[0005] In light of the current application of one-carbon metabolism inhibitors, this invention addresses the use of one-carbon metabolism inhibitors to inhibit viral replication, and can be used for the prevention or control of viruses. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] To address the shortcomings of existing technologies, this invention provides the application of one-carbon metabolism inhibitors in inhibiting viral infections, which has the advantage of preventing or controlling viruses, thereby solving the aforementioned problems.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the present invention provides the following technical solution: the application of a one-carbon metabolism inhibitor in inhibiting viral infection, wherein the one-carbon metabolism inhibitor is SHIN1, comprising the following steps: S1, the one-carbon metabolism inhibitor inhibits the working concentration of the virus; S2, the one-carbon metabolism inhibitor affects the invasion of the virus; S3, the one-carbon metabolism inhibitor inhibits the replication of the virus.

[0010] Preferably, S1: First, the effect of one-carbon metabolism inhibitors (0 μM–50 μM) on cell viability is screened. Then, working concentrations that do not affect cell viability are used to screen for effective concentrations that inhibit viral replication. In this invention, 0.5 μM–20 μM inhibitors are applied to cells and infect them with the virus. The viral replication, viral mRNA, and protein levels are evaluated using fluorescence microscopy. Finally, the working concentration with the best inhibitory effect on viral replication is applied in this invention.

[0011] Preferably, S2: The present invention further designs a one-carbon metabolism inhibitor to affect the viral invasion stage. The present invention treats cells with the inhibitor before and after viral infection, evaluates viral replication, viral mRNA, and protein levels by observing them under a fluorescence microscope, and finally applies the most effective mechanism of action to the present invention.

[0012] Preferably, S3: The present invention applies the optimal inhibitor concentration and the optimal method of using the inhibitor to virus-infected cells, and evaluates the viral replication, viral titer, viral mRNA and protein levels by observing the virus replication, viral titer, viral mRNA and protein levels by fluorescence microscopy. Ultimately, the one-carbon metabolism inhibitor inhibits viral replication.

[0013] Preferably, the inner surface of the front end of the unloading pipe is provided with a dust dispersion hole, the length of which is greater than half the width of the unloading pipe, to prevent excessive accumulation of dust and extend service life.

[0014] Optionally, the inhibitor can be used to salvage the SHIN1 effect via formate. This invention utilizes formate to assess viral replication, viral titer, viral mRNA and protein levels by observing viral replication under a fluorescence microscope, ultimately allowing the one-carbon metabolism inhibitor to restore the viral replication level.

[0015] (III) Beneficial Effects

[0016] Compared with the prior art, the present invention provides the application of one-carbon metabolism inhibitors in inhibiting viral infection, which has the following beneficial effects:

[0017] (1) Inhibitors do not affect cell activity: The concentration of the carbon metabolism inhibitors used in this invention does not affect cell concentration in the range of 0 μM to 50 μM.

[0018] (2) Significant effect in inhibiting viral replication: The carbon metabolism inhibitor of this invention can achieve a considerable effect in inhibiting viral replication with a concentration of 3 μM.

[0019] (3) Wide range of stages of action: The carbon metabolism inhibitor of the present invention can inhibit viral replication both before and after viral replication, and the effect is best when the inhibitor is used before and after.

[0020] (4) Action against multiple viruses: The carbon metabolism inhibitor of the present invention can inhibit vesicular stomatitis virus and Seneca virus, indicating that it can produce an inhibitory effect on multiple viruses.

[0021] (5) The inhibitory effect can be salvaged: The carbon metabolism inhibitor of the present invention can be salvaged by formate, and the viral replication level is restored. Attached Figure Description

[0022] Figure 1 Screening inhibitor working concentrations (A) The OD value of SHIN1-treated cells at 450 nm was detected by CCK8 assay. The figure shows that 0 μM–50 μM had no effect on cell viability. (BD) Fluorescence microscopy observations, viral mRNA and protein levels showed that the concentrations of the inhibitor SHIN1 (0.5 μM, 1 μM, 3 μM, 5 μM, 8 μM, 10 μM, and 20 μM) all affected viral replication. The concentration of 3 μM selected in this invention showed the best effect.

[0023] Figure 2 Inhibitors affect viral invasion. By treating cells with SHIN1 inhibitors at different time points, fluorescence results show that the inhibitors at different stages have an inhibitory effect on the virus. The combined use of pretreatment and posttreatment in this invention has the best antiviral effect.

[0024] Figure 3 The inhibitor inhibited VSV replication. Cells were infected with the virus before and after treatment with the inhibitor, as shown in the figure. The results of viral titer and protein level are shown. The one-carbon metabolism inhibitor effectively inhibited the replication of vesicular stomatitis virus.

[0025] Figure 4 Formate rescued the SHIN1 effect by applying formate to cells treated with the inhibitor. The results, shown in the figure, were obtained by microscopic observation of viral replication, viral titer, viral mRNA and protein levels. Viral replication levels recovered after formate application.

[0026] Figure 5 The inhibitor suppressed SVA replication. Cells were treated with the inhibitor before and after viral infection. The results, shown in the figure, indicate a decrease in viral titer, viral mRNA, and protein levels. The one-carbon metabolism inhibitor effectively inhibited Seneca virus replication. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Application of one-carbon metabolism inhibitors in vesicular stomatitis virus infection

[0029] like Figure 1-4 As shown:

[0030] 1. Cell Culture

[0031] Porcine kidney (PK-15) cells were preserved in the laboratory. Cells were grown in DMEM medium containing 10% fetal bovine serum and 1% triple antibodies. Cells were also grown in monolayers in tissue culture flasks or dishes under humid conditions of 37°C and 5% CO2.

[0032] 2. Antibodies and reagents

[0033] β-tubulin and GFP antibodies were purchased from Proteintech. SHIN1 was purchased from Tocris. Sodium formate was purchased from Sigma. Horseradish peroxidase-labeled goat anti-rabbit secondary antibody and horseradish peroxidase-labeled goat anti-mouse secondary antibody were purchased from Invitrogen.

[0034] 3. Virus propagation

[0035] The VSV strain was preserved in the laboratory. After PK-15 adhered and grew to 80%–90%, the cells were washed with PBS. VSV was allowed to adsorb for 1 hour at 37°C, followed by another PBS wash. DMEM nutrient solution containing 2% FBS was added, and the cells were cultured in a humidified environment of 5% CO2 at 37°C until cytopathic effects reached 80%–90%. The virus solution was repeatedly frozen and thawed three times, and the supernatant was collected and aliquoted into centrifuge tubes. Centrifuge at 1000 rpm for 10 minutes. Store at -80°C.

[0036] 4. Working concentration for screening inhibitors

[0037] First, the effects of one-carbon metabolism inhibitors (0 μM–50 μM) on cell viability were screened using a CCK-8 assay kit. Then, working concentrations that did not affect cell viability were used to screen for effective concentrations inhibiting viral replication. After concentration screening, cells were treated with inhibitors ranging from 0.5 μM to 20 μM and infected with the virus. Viral replication, viral mRNA, and protein levels were evaluated using fluorescence microscopy. Ultimately, a working concentration of 3 μM showed the best effect. Therefore, we selected 3 μM as the working concentration of the inhibitor in this invention.

[0038] 5. Inhibitors affect viral invasion.

[0039] Cells were treated with inhibitors before infection (-24h), after infection (+1h), and before and after infection (-24h & +1h). Viral replication, viral mRNA, and protein levels were assessed using fluorescence microscopy. The study found that treatment with inhibitors both before and after infection resulted in the most significant inhibition of viral replication. This invention utilizes inhibitors both before and after infection for experimental purposes.

[0040] 6. Inhibitors suppress viral replication.

[0041] Cells were treated with the inhibitor and infected with the virus according to the above working concentration and mode of action. The results were evaluated by observing viral replication, viral titer, viral mRNA and protein levels under a fluorescence microscope. The one-carbon metabolism inhibitor effectively inhibited viral replication.

[0042] 7. Formate rescues the SHIN1 effect

[0043] Cells treated with the inhibitor were then reinfected with formate to rescue the SHIN1 inhibitor effect. Evaluation of viral replication, viral titer, viral mRNA, and protein levels using fluorescence microscopy showed that viral replication levels were restored after formate treatment. Ultimately, the inhibitor selected in this invention can be used with formate to restore viral replication levels.

[0044] Example 2: Application of one-carbon metabolism inhibitors in Seneca virus infection

[0045] like Figure 5 As shown:

[0046] 1. Cells and strains

[0047] Porcine kidney (PK-15) cells were preserved in the laboratory, and the Seneca virus (SVA) strain was derived from this laboratory.

[0048] 2. Inhibitors suppress viral replication.

[0049] Cells were treated with inhibitors before and after infection. The viral titer, viral mRNA and protein levels were evaluated. The results showed that the one-carbon metabolism inhibitor inhibited viral replication. This invention uses a one-carbon metabolism inhibitor to inhibit the replication of Seneca virus.

[0050] In summary, the carbon metabolism inhibitor SHIN1 of this invention plays a role in the prevention or treatment of viral infections; it has the effect of inhibiting vesicular stomatitis virus and Seneca virus.

[0051] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Use of a one-carbon metabolism inhibitor SHIN1 for the manufacture of a medicament for inhibiting viral infection, characterized in that, The virus in question is the Seneca virus.

2. The application according to claim 1, characterized in that, The effective working concentration of SHIN1 in the drug for inhibiting the virus is 0.5 μM to 50 μM.

3. The application according to claim 1, characterized in that, The drug is administered before and / or after viral infection, and exerts its antiviral effect by influencing viral invasion and inhibiting viral replication.

4. The application according to claim 1, characterized in that, The antiviral activity of the drug was evaluated and verified by observing the viral replication status, detecting viral titer, viral mRNA level, and viral protein level using fluorescence microscopy.

5. The application according to claim 1, characterized in that, The antiviral effect of SHIN1 can be reversed by formate, which can restore the level of SHIN1-mediated viral replication inhibition.

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