Application of fructose-1,6-diphosphate in the preparation of drugs for preventing and treating herpes simplex virus type 1 infection

Abstract

The present invention belongs to the field of medicine, and specifically relates to the use of fructose-1,6-diphosphate in the preparation of drugs for preventing and treating herpes simplex virus type 1 infection. Fructose-1,6-diphosphate (FBP) can inhibit the replication of RNA and DNA viruses both in vitro and in vivo. Experiments have shown that this inhibitory effect is largely independent of type I interferon-mediated innate immunity, but occurs through HMGB1. After viral infection, FBP reduces the protein level of HMGB1 by promoting lysosomal degradation of HMGB1. In addition, FBP can also directly inhibit the binding of HMGB1 to the viral genome, thereby inhibiting viral replication. The present invention highlights the key role of FBP in inhibiting viral replication and proposes potential targets for controlling viral infection.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of fructose-1,6-diphosphate in the preparation of a drug for preventing and treating herpes simplex virus type 1 infection. Background Art

[0002] The viral replication process can be divided into five consecutive stages: adsorption, entry, uncoating, replication, and assembly and release. When the virus attaches to the cell surface, the viral nucleic acid enters the cytoplasm through the cell membrane, mainly through fusion and endocytosis. During the process of penetrating the cell, the viral capsid can be removed by lysosomal enzymes in the phagosome. After entering the cell, the virus uses the raw materials in the host cell for viral nucleic acid replication and viral protein synthesis, and then assembles into complete viral particles and releases for further infection. Viral infection can cause damage to the body, resulting in varying degrees of damage or viral diseases depending on the type of virus and the state of the body. The mortality rate of infectious diseases caused by viruses accounts for a large proportion of the global death toll; the study of virus-host interactions is particularly important for the development of new strategies to combat viral infectious diseases.

[0003] When recognizing invading viruses, pattern recognition receptors (PRRs) recruit the adaptor proteins TRIF, MAVS, and STING to activate the TBK1 / IKKε kinase. TBK1 / IKKε phosphorylates IRF3 and nuclear factor κB (NF-κB) and induces their translocation into the nucleus, thereby regulating the expression of type I interferons and inflammatory cytokines. Type I interferons activate the JAK-STAT signaling pathway by binding to the type I interferon receptor complex, promoting the expression of interferon-stimulated genes ISGs to resist viral infection and coordinate adaptive immunity. Viral infection can regulate the metabolism of host cells, which in turn affects the survival or clearance of the virus. During the infection process, the virus always exploits and reprograms cellular components to form an environment that is most suitable for its replication. In addition, host cells can also actively readjust cellular metabolism to inhibit viral entry and replication, or enhance antiviral immune responses. For example, when cells are stimulated by RNA viruses, glycolytic metabolism is inhibited, resulting in reduced lactate production, dissociation of lactate from the MAVS protein, increased binding of the pattern recognition receptor RIG and the recruitment adaptor protein MAVS, activation of the RIG-I-MAVS-mediated signaling pathway, promotion of type I interferon production and enhancement of antiviral innate immunity.

[0004] Fructose-1,6-bisphosphate (FBP) is an important metabolite in the glycolysis pathway. Fructose-6-phosphate kinase 1 (PFK1) catalyzes the conversion of fructose-6-phosphate (F6P) to FBP, which undergoes a series of reactions to generate phosphoenolpyruvate (PEP) and pyruvate, participating in glucose metabolism. Fructose-1,6-bisphosphatase 1 (FBP1), a key enzyme in gluconeogenesis, can break down FBP into F6P and inorganic phosphate. Several studies have shown that FBP has a protective effect against injury and plays a protective role in a variety of cells and tissues, including the brain, kidney, intestine, liver, and heart. Due to its widely recognized cytoprotective properties during stress, FBP has been used in many countries as an adjunct therapy for heart and brain diseases, especially in conditions of oxygen deprivation or reduced blood flow.

[0005] High-mobility group box 1 (HMGB1) is a highly conserved and abundant non-histone chromatin protein involved in a variety of cellular processes, including DNA replication, transcription, recombination, and repair. HMGB1 has two homologous DNA / RNA binding domains (called A-box and B-box, each 75 amino acids long) and a highly negatively charged C-terminal domain consisting of a continuous stretch of glutamic acid and aspartic acid residues. HMGB1 can act as a damage-associated molecular pattern (DAMP) and trigger inflammatory responses by binding to, for example, Toll-like receptors.

[0006] Viral infectious diseases have a significant impact on public health, and the development of effective treatments can help reduce the burden of these diseases on individuals and communities. Currently, no application of FBP in the prevention and treatment of herpes simplex virus type 1 infection has been found, nor has its new role in inhibiting viral replication been revealed. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides the use of fructose-1,6-diphosphate in the preparation of a drug for preventing and treating herpes simplex virus type 1 infection.

[0008] In order to achieve the purpose of the present invention, the technical solution adopted is as follows:

[0009] Application of fructose-1,6-diphosphate as an active ingredient in the preparation of drugs for preventing and treating herpes simplex virus type 1 infection.

[0010] Preferably, the medicament promotes lysosomal degradation of HMGB1 under viral infection.

[0011] Preferably, the drug prevents the binding of HMGB1 to the viral genome.

[0012] Preferably, the medicament reduces the amount of HSV-1 in the brain.

[0013] Preferably, the medicament reduces the infiltration of inflammatory cells.

[0014] Preferably, the medicament alleviates pathological changes in the lungs infected by the virus.

[0015] Preferably, the drug inhibits viral infection independently of the type I interferon signaling pathway.

[0016] Preferably, the medicament inhibits the replication of herpes simplex virus type 1.

[0017] Another object of the present invention is to provide the use of fructose-1,6-bisphosphate in the preparation of HMGB1 blockers or anticancer drugs targeting HMGB1.

[0018] Another object of the present invention is to provide a drug for preventing and treating herpes simplex virus type 1 infection, wherein the raw materials of the drug include fructose-1,6-diphosphate and pharmaceutically acceptable excipients.

[0019] Preferably, the dosage form of the drug includes tablets, pills, granules, powder suspensions, capsules, suppositories, oral solutions, emulsions, injections or aerosols.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention found that fructose-1,6-bisphosphate (FBP) inhibited the infection of human cell lines in vitro with the DNA virus herpes simplex virus type 1 (HSV-1). The effects of FBP on the viability of various cell types were further explored. The results showed that the inhibition of viral infection by FBP was not due to its nonspecific cytotoxicity.

[0022] (2) Fructose-1,6-bisphosphate (FBP) has an inhibitory effect on herpes simplex virus type 1 infection in an in vivo mouse model, reduces the HSV-1 burden in the mouse brain, reduces the infiltration of inflammatory cells, and alleviates pathological changes in the lungs of virus-infected mice.

[0023] (3) The present invention further explores the mechanism of action of fructose-1,6-bisphosphate (FBP) in viral infection. The results show that fructose-1,6-bisphosphate (FBP) itself, rather than other glycolysis products produced in the body, plays a key role in inhibiting viral infection.

[0024] (4) Further studies in the present invention show that fructose-1,6-bisphosphate (FBP) inhibits viral replication through HMGB1 rather than HMGB2.

[0025] (5) Fructose-1,6-bisphosphate promotes the lysosomal degradation of HMGB1 to reduce its protein level, and can directly bind to HMGB1 to block the binding of HMGB1 to the viral genome.

[0026] (6) Studies have found that fructose-1,6-bisphosphate can be used as an inhibitor drug of HMGB1. Fructose-1,6-bisphosphate is a new HMGB1 ligand and can become a cancer drug targeting HMGB1. High mobility group protein 1 (HMGB1) is a highly conserved and abundant non-histone chromatin protein that participates in a variety of cellular processes, including DNA replication, transcription, recombination, and repair. HMGB1 has two homologous DNA / RNA binding domains (called A box and B box, each 75 amino acids long) and a highly negatively charged C-terminal domain, which consists of continuous extended glutamic acid and aspartic acid residues. Fructose-1,6-bisphosphate can reduce the oligomerization of HMGB1 by reducing the binding of HMGB1 A box and C terminus, thereby enhancing the interaction between the P53 gene and HMGB1 and reducing the affinity of HMGB1 for DNA. Therefore, fructose-1,6-bisphosphate (FBP) can aggravate chemotherapy-induced cancer cell death. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a graph showing the results of Example 1 showing the inhibition of HSV-1, VSV and HCV viral infection by fructose-1,6-bisphosphate (FBP) in vitro; **p<0.01, ***p<0.001.

[0028] Figure 2 This is the result of fructose-1,6-bisphosphate (FBP) infection in HSV-1 and VSV mice in Example 2. From left to right, the figure shows the VSV titer result in the liver and the HSV-1 titer result in the brain; *p<0.05, **p<0.01.

[0029] Figure 3 Representative H&E-stained lung tissue sections of mice infected with HSV-1 and VSV in Example 2.

[0030] Figure 4 After BMDMs were treated with 0 mM FBP and 1 mM FBP in Example 3, Ifb1 Results of mRNA expression levels; ***p<0.001.

[0031] Figure 5 This is the result of qPCR analysis of Example 3. After IFNAR1-WT and IFNAR1-KO BMDMs were treated with 5 mM FBP for 12 hours, they were infected with HSV-1 (MOI, 0.1), and the level of HSV-1 gDNA was detected; ***p<0.001.

[0032] Figure 6 Example 3 qPCR analysis of brain Ifb1 Results of mRNA levels; *p<0.05.

[0033] Figure 7 For Example 5, 293T cells were transfected with siControl, siHMGB1 and siHMGB2, and then treated with 5 mMFBP for 12 hours and infected with HSV-1 for 10 hours. The mRNA levels of HMGB1 and HMGB2 and the gDNA level of HSV-1 were evaluated by qPCR. The results are as follows; *p<0.05, **p<0.01, ***p<0.001.

[0034] Figure 8 This is a graph showing the results of qPCR evaluation of Hmgb1 mRNA levels in RAW264.7 cells treated with FBP and infected with HSV-1 in Example 6.

[0035] Fig. 9 This is a graph showing the expression results of HMGB1 by western blot analysis after RAW264.7 cells were treated with FBP and infected with HSV-1 in Example 6.

[0036] Fig.10 This is the result of western blot analysis of HMGB1 expression in A549 cells after pretreatment in Example 6.

[0037] Fig.11 This is a diagram of Example 7, in which the binding of HSV-1 gDNA to HMGB1 was analyzed by combining chromatin immunoprecipitation with qPCR (Chip-qPCR).

[0038] Fig.12 This is a diagram of the binding of HSV-1 gDNA and HMGB1 detected by qPCR; ***p<0.001.

[0039] Fig.13 The binding of HSV-1 gDNA and HMGB1 was detected by qPCR after adjusting the HMGB1 level; *p<0.05. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific implementation modes.

[0041] The raw materials, equipment and reagents used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0042] Example 1 In vitro inhibitory effect of fructose-1,6-diphosphate on herpes simplex virus type 1

[0043] Experimental methods:

[0044] (1) Mouse bone marrow-derived macrophages (BMDMs) were treated with or without 1 mM FBP for 12 h and then incubated with the DNA virus herpes simplex virus type 1 (HSV-1, MOI, 0.1) for 10 h. The cells were then collected and the gDNA level of HSV-1 was assessed using qPCR.

[0045] (2) Mouse BMDMs were treated with or without 1 mM FBP for 12 h and then infected with VSV (MOI, 0.1) for 10 h. Cells were collected and the RNA level of VSV was evaluated by qPCR.

[0046] (3) HLCZ01 cells were infected with HCV (MOI, 0.01) for 60 hours and then treated with or without 1 mM FBP for 12 hours. The cells were harvested and the HCV RNA level was assessed by qPCR, with UI as a reference.

[0047] After different cell types were treated with different concentrations of FBP for 0, 12, and 24 hours, the cell viability was determined using the CCK-8 assay.

[0048] Results Figure 1 The results show that fructose-1,6-bisphosphate (FBP) significantly inhibits the infection of human cell lines with the DNA virus herpes simplex virus type 1 (HSV-1), and its inhibitory effect is significantly better than that of HCV and VSV.

[0049] Further using CCK-8 experiments, we explored the effects of FBP on the viability of multiple cell types including A549, RAW264.7, BMDM, 293T, E0771, HLCZ01, and Huh7.5. The results showed that 1 mM and 5 mM FBP showed no cytotoxicity during the 12-24 hour treatment period, indicating that FBP inhibited viral infection not due to its nonspecific cytotoxicity.

[0050] Example 2 Inhibitory effect of fructose-1,6-diphosphate on herpes simplex virus type 1 in vivo

[0051] Experimental methods:

[0052] C57BL / 6 mice were pretreated with FBP (500 mg / kg) or PBS (every 12 hours for two days) and then injected intraperitoneally with VSV or HSV-1 (2 × 10 per mouse). 8 PFU). The uninfected (UI) group was injected with PBS. During the viral infection period, FBP or PBS was injected at the same frequency.

[0053] Mice were sacrificed 24 hours after VSV infection to analyze VSV mRNA by qPCR and determine VSV titer in liver by TCID50 assay. Representative H&E-stained lung tissue section images.

[0054] Mice were sacrificed 48 hours after infection with HSV-1 to analyze HSV-1 gDNA by qPCR and determine HSV-1 titers in the brain by TCID50 assay. Representative H&E-stained lung tissue section images.

[0055] Experimental results:

[0056] Results Figure 2-Figure 3 In this example, a mouse model infected with VSV and HSV-1 was constructed. FBP reduced the VSV load in the liver and the HSV-1 burden in the brain of mice, reduced the infiltration of inflammatory cells, and alleviated the pathological changes in the lungs of virus-infected mice.

[0057] In summary, the results indicate that FBP has a significant inhibitory effect on DNA virus herpes simplex virus type 1 infection both in vitro and in vivo.

[0058] Example 3 FBP inhibits viral infection independently of type I interferon signaling pathway

[0059] Experimental method: Mouse bone marrow-derived macrophages BMDMs were treated with 0 mM FBP or 1 mM FBP for 12 hours and then infected with HSV-1 (MOI, 0.1) for 10 hours. Cells were collected and evaluated using qPCR. Ifb1 mRNA levels, experimental results see Figure 4 , UI was used as the internal reference. The results showed that FBP inhibited the production of IFN-β induced by HSV-1.

[0060] After IFNAR1-WT and IFNAR1-KO BMDMs were treated with 5 mM FBP for 12 h, they were infected with HSV-1 (MOI, 0.1) for the indicated times, and HSV-1 gDNA levels were assessed using qPCR. Figure 5 , FBP inhibited HSV-1 infection in IFNAR1-knockout mouse bone marrow-derived macrophages (BMDMs).

[0061] Further experiments: C57BL / 6 mice were pretreated with FBP (500 mg / kg) or PBS (once every 12 hours for two days) and then injected intraperitoneally with HSV-1 (2 × 10 per mouse). 8 PFU). During the virus infection, FBP or PBS was injected at the same frequency. Mice were sacrificed 48 hours after infection, and the brain was analyzed by qPCR. Ifb1mRNA levels. Experimental results are shown in Figure 6 In in vivo experiments, FBP reduced IFN-β production in tissues following HSV-1 infection.

[0062] The above results indicate that the inhibitory effect of FBP on viral infection is independent of the IFN-I signaling pathway.

[0063] Example 4 Fructose-1,6-bisphosphate (FBP) itself, rather than its metabolites, plays a key role in inhibiting viral infection

[0064] Experimental methods: 293T cells were transfected with siControl, siFBP1, empty vector (EV), PFKP overexpression plasmid, FBP1 overexpression plasmid, siControl or siPFKP, FBP1 enzyme activity deletion mutant (FBP1-G260R), nuclear localization mutant (FBP1-NLS), and nuclear localization and activity deletion mutant (FBP1-NLS-G260R). Cells were treated with 5 mM FBP for 12 h and then infected with HSV-1 (MOI, 0.1) for 10 h. The expression of FBP1 and PFKP was detected by Western blot. The intracellular FBP content was measured using a microplate reader, and the level of HSV-1 gDNA was evaluated by qPCR.

[0065] The results showed that fructose-1,6-bisphosphatase 1 (FBP1) or phosphofructokinase-1 (PFK1), which regulates FBP production or consumption in cells, was knocked down or overexpressed. Knockdown of FBP1 significantly increased the level of intracellular FBP and inhibited HSV-1 infection in 293T cells. Addition of exogenous FBP further increased the FBP content in cells and enhanced this inhibitory effect. Overexpression of PFKP subunits had a similar effect.

[0066] In addition, overexpression of FBP1 significantly reduced the level of intracellular FBP and enhanced HSV-1 infection. The addition of exogenous FBP further increased the FBP content in the cells and inhibited this enhanced infection effect. PFKP knockdown had a similar effect. To further explore the effect of FBP on viral infection, we constructed the FBP1 enzyme-mediated deletion mutant FBP1-G260R, the nuclear localization mutant FBP1-NLS, and the nuclear localization and enzyme-mediated deletion mutant FBP1-NLS-G260R. Wild-type FBP1 reduced the intracellular FBP level and enhanced HSV-1 infection when treated with exogenous FBP. Similar results were not obtained in FBP1 overexpression mutants.

[0067] Taken together, the results suggest that FBP itself, rather than the production of other glycolysis products, plays a key role in inhibiting viral infection.

[0068] Example 5 HMGB1 plays an important role in FBP-mediated inhibition of viral replication

[0069] 293T cells were transfected with siControl, siHMGB1, and siHMGB2, and subsequently treated with 5 mM FBP for 12 h and infected with HSV-1 for 10 h. Then, the mRNA levels of HMGB1, HMGB2, and HSV-1 were evaluated by qPCR.

[0070] The results are as follows Figure 7 , the results showed that this antiviral effect still existed when only HMGB2 was specifically knocked down. Knocking down HMGB1, but not HMGB2, eliminated the inhibitory effect of FBP on HSV-1 replication, as manifested by a reduction in HSV-1 mRNA in 293T cells. These results suggest that HMGB1, but not HMGB2, plays an important role in FBP's inhibition of viral replication.

[0071] Example 6 FBP promotes lysosomal degradation of HMGB1 to reduce its protein level

[0072] RAW264.7 cells were treated with 0, 1, or 5 mM FBP for 12 h and infected with HSV-1 (MOI, 0.1) for 10 h. After harvesting the cells, the mRNA level of Hmgb1 was assessed by qPCR (see Figure 8 ). The expression of HMGB1 was analyzed by western blot (see Fig. 9 ).

[0073] A549 cells were treated with 0 or 1 mM FBP for 12 h, infected with HSV-1 (MOI, 0.1) for 10 h, and then treated with MG132 (25 μM), NH4Cl (10 mM), or 3-MA (5 μM) for 6 h, after which the expression of HMGB1 was detected by western blot (see Fig.10 ).

[0074] The results showed that under HSV-1 infection, exogenous FBP reduced the protein level of HMGB1 but had no effect on the mRNA level of HMGB1, indicating that HMGB1 is regulated posttranscriptionally.

[0075] HMGB1 can be degraded through proteasome-dependent and lysosome-dependent pathways. The study found that the proteasome inhibitor MG132 could not prevent FBP-mediated HMGB1 degradation, while the lysosome inhibitors NH4Cl and 3MA effectively restored the expression of HMGB1. The above data indicate that FBP reduces the protein level of HMGB1 mainly by promoting lysosomal degradation of HMGB1 during viral infection.

[0076] Example 7 FBP directly binds to HMGB1 and blocks the binding of HMGB1 to the viral genome.

[0077] HMGB1 consists of two homologous DNA / RNA binding domains (A box and B box) and an acidic C-terminal domain. This example focuses on whether FBP inhibits viral replication by affecting the direct binding of HMGB1 to the viral genome.

[0078] 293T cells were treated with FBP for 12 h and then infected with HSV-1 (MOI, 0.5) for 10 h. Immunoprecipitation was performed using anti-HMGB1 antibody and immunoblot analysis was performed using the indicated antibodies. The results are shown in Fig.11 shown.

[0079] The binding of HSV-1 gDNA to HMGB1 was detected by qPCR. Fig.12 As shown. The experiment showed that FBP inhibited the binding of HMGB1 to HSV-1 gDNA.

[0080] In order to exclude the effect of decreased HMGB1 levels after FBP treatment leading to reduced binding of HMGB1 to HSV-1, we took FBP-treated or untreated cell lysates containing the same amount of HMGB1 and treated them with anti-HMGB1 antibodies for ChIP experiments. Fig.13 After adjusting the HMGB1 level, FBP still inhibited the binding of HMGB1 to HSV-1 gDNA, indicating that the inhibition of HSV-1 gDNA binding by FBP was not only due to the reduction of HMGB1 protein level by FBP, but FBP could also directly inhibit the binding of HMGB1 to HSV-1.

[0081] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. Application of fructose-1,6-diphosphate as an active ingredient in the preparation of drugs for preventing and treating herpes simplex virus type 1 infection.

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