Application of sodium oxamate in preparation of anti-influenza virus product

Abstract

The invention discloses an application of sodium oxamate in preparation of anti-influenza virus products, and provides an application of sodium oxamate as a lactic dehydrogenase A inhibitor in preparation of anti-influenza virus products, the sodium oxamate has broad-spectrum antiviral activity, and can play a unique action mechanism by regulating host cell metabolism (such as protein lactylation). And a new direction is provided for overcoming virus resistance.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and in particular to the application of sodium oxalate in the preparation of anti-influenza virus products. Background Technology

[0002] Influenza viruses, belonging to the Orthomyxoviridae family, are important pathogens causing seasonal epidemics and periodic global pandemics. Among them, influenza A virus (IAV) poses the most significant public health threat due to its broad host spectrum (covering humans, birds, pigs, etc.) and high mutation rate. The biological characteristics of this virus are mainly reflected in its structure and mutation mechanisms. Two key glycoproteins are distributed on the viral envelope surface: hemagglutinin (HA) mediates the binding of the virus to receptors on the host cell surface and the membrane fusion process, while neuraminidase (NA) is responsible for hydrolyzing the sialic acid linkage between progeny virus particles and host cells, promoting viral release and spread. Furthermore, the influenza virus genome consists of multiple segmented single-stranded RNAs, a structural feature that provides the molecular basis for its genetic variation.

[0003] The mutation mechanisms of influenza viruses mainly fall into two categories. The first is antigenic drift, where point mutations in the HA and NA proteins gradually accumulate during viral replication, leading to progressive changes in antigenic characterization. This mechanism weakens the immune memory established by previous infection or vaccination in the host, thus becoming a major cause of annual seasonal influenza epidemics. The second is antigenic shift, where different subtypes of influenza viruses (e.g., human and avian viruses) co-infect the same host cell, and their segmented genomes may reassort, forming recombinant viruses containing entirely new HA or NA combinations. Because the population generally lacks pre-existing immunity against such novel viruses, antigenic shifts can lead to transregional or even global influenza pandemics. Therefore, influenza viruses continue to evolve through these two mechanisms, posing a long-term and dynamic challenge to disease control.

[0004] Although antiviral drugs, such as neuraminidase inhibitors (e.g., oseltamivir), are widely used in clinical practice, the development of novel antiviral drugs remains urgent, primarily due to three challenges: drug resistance, limitations in the efficacy of existing drugs, and unmet needs in response to pandemics. First, viruses can evolve drug-resistant mutations under drug selection pressure. For example, M2 ion channel inhibitors have been withdrawn from clinical use due to widespread resistance, and oseltamivir-resistant strains have also been reported. Therefore, it is necessary to stockpile antiviral drugs with different mechanisms of action to expand treatment options. Second, existing drugs typically require early intervention to achieve optimal efficacy, limiting their effectiveness in treating severe cases. Finally, for novel pandemic strains potentially caused by antigenic shifts in viruses such as avian influenza, the development and deployment of existing drugs and vaccines may lag behind. There is an urgent need to develop treatments such as polymerase inhibitors (e.g., baloxavir) with broad-spectrum antiviral activity that can be rapidly deployed and effectively inhibit viral replication.

[0005] In conclusion, the continuous development of new antiviral drugs is a strategic necessity to address the ongoing mutation of the virus, enhance clinical treatment capabilities, and strengthen pandemic preparedness. Summary of the Invention

[0006] In view of this, the purpose of this invention is to propose the application of sodium oxalate in the preparation of anti-influenza virus products. This method has found that sodium oxalate can significantly inhibit the replication of multiple influenza virus subtypes in A549 cells, demonstrating promising application prospects.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows: This protocol proposes sodium oxamate as an inhibitor of lactate dehydrogenase A (LDH-A) (LDHi), which can effectively reduce intracellular lactate levels and its mediated protein lactation modification by inhibiting the activity of LDH-A.

[0008] The chemical structural formula of sodium oxamate is shown below: .

[0009] This protocol used sodium oxalate as a lactate dehydrogenase A inhibitor (LDHi) to pretreat A549 cells for 1 h, followed by infection with different subtypes of influenza A virus A / PR / 8 / 34 (PR8), A / Chicken / Fujian / MQ01 / 2015 (H9N2), and A / HuNan / 01 / 2014 (H3N2) strains at a multiplicity of infection (MOI) of 0.01. Cell protein samples and supernatants were collected 24 h post-infection (hpi). The expression of viral NP and PB2 proteins was detected by Western blotting, and the viral titer in the supernatant was detected by TCID50. Cell protein samples were collected at 12 h, 18 h, and 24 h post-PR8 infection, and the level of intracellular lactation modification was detected by Western blotting. The results showed that LDHi could reduce the level of intracellular lactation modification and significantly inhibit influenza virus replication.

[0010] This study reveals a novel inhibitor, sodium oxalate, as a lactate dehydrogenase A inhibitor that effectively and broadly inhibits the replication of multiple influenza A virus subtypes. Given its favorable biosafety profile, this compound demonstrates potential as an antiviral drug candidate.

[0011] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art: The present solution proposes the application of sodium oxalate as a lactate dehydrogenase A inhibitor in the preparation of anti-influenza virus products. It has broad-spectrum antiviral activity and can exert a unique mechanism of action by regulating host cell metabolism (such as protein lactation), providing a new direction for overcoming viral drug resistance. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 The results of the LDHi cytotoxicity assay show a comparison of the cytotoxicity of A549 cells incubated with different concentrations of LDHi for 24 h, as detected by the CCK-8 cell assay kit. Figure 2This is the result of LDHi pretreatment inhibiting PR8 virus replication. A549 cells were pretreated with LDHi for 1 hour, then inoculated with PR8 virus. Cell samples and culture supernatants were collected 24 hours post-infection. The expression levels of viral NP and PB2 proteins were detected by Western blotting, and TCID45 was used. 50 Method for determining viral titer in culture supernatant (B); Figure 3 This is the result of LDHi pretreatment inhibiting H9N2 virus replication. A549 cells were pretreated with LDHi for 1 hour, then inoculated with H9N2 virus. Cell samples and culture supernatants were collected 24 hours post-infection. The expression levels of viral NP and PB2 proteins were detected by Western blotting, and TCID45 was used. 50 Method for determining viral titer in culture supernatant (B); Figure 4 This result reflects the inhibition of H3N2 virus replication by LDHi pretreatment. A549 cells were pretreated with LDHi for 1 hour before being inoculated with H3N2 virus. Cell samples and culture supernatants were collected 24 hours post-infection. The expression levels of viral NP and PB2 proteins were detected by Western blotting, and TCID45 was used to analyze the results. 50 Method for determining viral titer in culture supernatant (B); Figure 5 This is the result of LDHi pretreatment inhibiting intracellular lactation levels. A549 cells were pretreated with LDHi for 1 h and then inoculated with PR8 virus. Cell samples were collected at 12, 18 and 24 hours post-infection, and the expression changes of viral proteins NP and PB2 and intracellular total protein lactation modification levels were detected by Western blotting. Detailed Implementation

[0014] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] I. Materials and Methods 1.1 Cell Culture and Viral Infection The human lung epithelial cells (A549) and canine kidney cells (MDCK) used in this embodiment were routinely cultured in the applicant's laboratory. The influenza virus strains A / PR / 8 / 34 (PR8), A / WSN / 33 (WSN), A / Chicken / Fujian / MQ01 / 2015 (H9N2), and H3N2 used were all stored in the applicant's laboratory. All cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2. For virus infection, the cells were washed twice with phosphate-buffered saline (PBS), then inoculated with influenza virus at a multiplicity of infection (MOI) of 0.01 and incubated at 37°C for 1 hour. After removing the virus solution, the cells were washed twice more with PBS, and the medium was replaced with DMEM medium containing 0.2 μg / mL TPCK-trypsin, and cultured for the specified time.

[0016] 1.2 Cytotoxicity test A549 cells were seeded in 96-well plates and cultured overnight at 37°C with 5% CO2. The following day, different concentrations of the drug were added to treat the cells for 24 hours. After treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for another 30 minutes. Finally, the absorbance of each well was measured at 450 nm using a microplate reader.

[0017] 1.3 Western blotting Cells were lysed with NETN lysis buffer, and protein samples were collected, separated by SDS-PAGE electrophoresis, and then transferred to nitrocellulose membranes (NC membranes). The transferred membranes were blocked with 5% skim milk and then incubated with the corresponding specific primary antibodies for target protein detection. The antibodies used were: NP (prepared in our laboratory), Pan-Lac (Jingjie, PTM-1401RM, China), GAPDH (Zhongshan Jinqiao, TA-08, China), and PB2 (GeneTex, GTX125926, USA).

[0018] 1.4 Half-maximal dose of infection in tissue culture (TCID) 50 ) MDCK cells were seeded in 96-well plates and cultured overnight at 37°C with 5% CO2. The supernatant from influenza virus-infected A549 cells was collected and serially diluted. 100 µL of each dilution was seeded into a monolayer of MDCK cells and incubated at 37°C for 1 hour. After removing the virus solution, the medium was replaced with DMEM containing 1 µg / mL TPCK-trypsin and cultured for another 72 hours. Viral titers were calculated using the Reed-Muench method, and results were expressed as logarithmic values. 10 TCID 50 / mL represents the volume of water.

[0019] II. Experimental Results To determine the safe concentration of compound LDHi (sodium oxalate as a lactate dehydrogenase A inhibitor (LDHi)), this example first performed a cytotoxicity test in A549 cells. A549 cells were seeded in 96-well plates and cultured overnight at 37°C with 5% CO2. Cells were then treated with 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM LDHi for 24 hours, respectively. After treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 30 minutes. The absorbance at 450 nm was then measured using a microplate reader. The results are as follows: Figure 1 As shown, LDHi concentrations up to 75 mM did not exhibit significant cytotoxicity against A549 cells.

[0020] Based on the above CCK-8 experimental results (reference) Figure 1 In this embodiment, A549 cells were pretreated with 50 mM LDHi for 1 hour, followed by inoculation with H1N1 subtype strain A / PR / 8 / 34 at a multiplicity of infection (MOI) of 0.01. Cell protein samples were collected 24 hours after viral infection, and the expression levels of viral proteins NP and PB2 were detected by Western blotting. The results showed that LDHi treatment significantly reduced the expression levels of NP and PB2 proteins in H1N1 virus. Figure 2 A). Collect cell culture supernatant after viral infection and use TCID50. 50 The method involves detecting the viral titer. For example... Figure 2 As shown in Figure B, the viral titer in the culture supernatant of A549 cells treated with LDHi was significantly lower than that in the untreated control group.

[0021] In this embodiment, A549 cells were pretreated with LDHi for 1 hour, followed by infection with the H9N2 strain. Twenty-four hours after infection, cell protein samples and culture supernatant were collected. Viral protein expression was detected by Western blotting and analyzed using TCID45. 50Viral titers were determined using the LDHi method. Results showed that LDHi treatment significantly inhibited the expression of NP and PB2 proteins in the H9N2 strain. Figure 3 A). Meanwhile, the viral titer in the cell supernatant of the LDHi-treated group was significantly lower than that of the untreated control group ( Figure 3 B).

[0022] In this embodiment, A549 cells were pretreated with LDHi for 1 hour, followed by infection with the H3N2 strain. Twenty-four hours after infection, cell protein samples and culture supernatant were collected. Viral protein expression was detected by Western blotting and analyzed using TCID45. 50 Viral titers were determined using the LDHi method. Results showed that LDHi treatment significantly inhibited the expression of NP and PB2 proteins in the H3N2 strain. Figure 4 A). Meanwhile, the viral titer in the cell supernatant of the LDHi-treated group was significantly lower than that of the untreated control group ( Figure 4 B). The above results indicate that LDHi has a significant inhibitory effect on the replication of multiple influenza virus subtypes.

[0023] To further explore the mechanism of action of LDHi, this study examined its effect on the overall protein lactation modification level in cells. Cell samples were collected 1 hour after LDHi pretreatment, at 12, 18, and 24 hours post-virus infection, and changes in protein lactation levels were detected. The results showed that LDHi treatment significantly reduced the total protein lactation level in A549 cells, accompanied by a significant inhibition of influenza virus replication. Figure 5 ).

[0024] These results indicate that LDHi affects the replication process of influenza virus by regulating the metabolic modification state of host cells.

[0025] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. The application of sodium oxalate in the preparation of anti-influenza virus products, characterized in that, The chemical structural formula of the sodium oxalate is shown below: 。

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