Use of zeste degrader of ezrin (zak) inhibitors in the preparation of a medicament for treating ev71 viral infection

Abstract

This invention discloses the application of ZAK inhibitors in the preparation of drugs for treating EV71 virus infection, relating to the biomedical field. The ZAK inhibitors exert their effects by inhibiting the ZAK-p38 MAPK signaling pathway and by inhibiting the ZAKα-mediated ribosomal toxicity stress response (RSR). The ZAK inhibitor is nilotinib or a pharmaceutically acceptable salt thereof. Cellular and animal experiments of this invention demonstrate that EV71 infection activates the ZAKα-mediated p38 MAPK pathway (RSR), promoting viral replication and leading to multi-organ damage. ZAK inhibitors (such as nilotinib) can inhibit this pathway, reduce the expression of the viral protein VP1, and improve infection-induced renal function damage, metabolic disorders, and muscle damage. Therefore, ZAK inhibitors can be used to prepare anti-EV71 drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of ZAK inhibitors in the preparation of drugs for treating EV71 virus infection. Background Technology

[0002] Enterovirus 71 (EV71) is one of the main pathogens causing hand-foot-and-mouth disease in infants and young children. Some infections can lead to serious neurological complications and even death. Currently, there are no specific antiviral drugs for EV71 infection.

[0003] Therefore, developing new treatment strategies and drugs is of great significance.

[0004] ZAK (also known as MLK7) is a MAP3K kinase and a key upstream kinase for the ribotoxic stress response (RSR).

[0005] RSR can be triggered by certain toxins that damage ribosomes, ultimately mediating cellular stress responses through the p38 MAPK pathway.

[0006] Currently, there is a lack of existing technologies regarding the role of the ZAK-p38 MAPK pathway in EV71 virus infection and its potential as a therapeutic target.

[0007] Therefore, this invention proposes the application of ZAK inhibitors in the preparation of drugs for treating EV71 virus infection. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of ZAK inhibitors in the preparation of drugs for treating EV71 virus infection.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: Application of ZAK inhibitors in the preparation of drugs for treating EV71 virus infection.

[0010] Preferably, the ZAK inhibitor works by inhibiting the ZAK-p38 MAPK signaling pathway.

[0011] Preferably, the ZAK inhibitor works by inhibiting the ZAKα-mediated ribosomal toxicity stress response (RSR).

[0012] Preferably, the ZAK inhibitor is Nilotinib or a pharmaceutically acceptable salt thereof.

[0013] Preferably, the treatment includes reducing the level of EV71 viral protein VP1 in the subject.

[0014] Preferably, the treatment includes improving renal function impairment caused by EV71 infection, which is indicated by elevated blood urea nitrogen (BUN) and / or serum creatinine (SCr) levels.

[0015] Preferably, the treatment includes improving metabolic disorders caused by EV71 infection, which are indicated by hypoglycemia and / or high lactate levels.

[0016] Preferably, the treatment includes improving muscle damage caused by EV71 infection, which is indicated by elevated creatine kinase (CK) levels.

[0017] A pharmaceutical composition for treating EV71 infection comprising a therapeutically effective amount of a ZAK inhibitor and a pharmaceutically acceptable carrier.

[0018] Preferably, the ZAK inhibitor is Nilotinib.

[0019] The beneficial effects of this invention are as follows: Clear antiviral effects: Cell experiments have confirmed that inhibiting the kinase activity of ZAKα by chemical inhibitors (Nilotinib) or gene knockdown (siRNA) can effectively inhibit the ZAK-p38 MAPK signaling pathway activated by EV71 infection, significantly reduce the phosphorylation level of downstream p38, and consequently reduce the expression of viral protein VP1, thereby directly inhibiting viral replication.

[0020] Significant systemic protective effects in vivo: Animal experiments have shown that EV71 infection leads to multi-organ dysfunction in suckling mice, including renal impairment (elevated BUN and SCr), metabolic disorders (hypoglycemia and hyperlactatemia), and muscle damage (elevated CK). Treatment with the ZAK inhibitor nilotinib comprehensively and significantly improved these abnormal biochemical indicators, demonstrating its systemic organ protective function.

[0021] The mechanism of action is clear: Experiments have shown that during EV71 infection, the activation of p38 phosphorylation is mainly mediated by ZAKα, and inhibiting this target can effectively block signal transduction. Simultaneously, viral replication levels (VP1 protein levels) and the activation state of the ZAK-p38 MAPK pathway show synergistic changes, further clarifying the direct correlation between targeting this pathway and antiviral activity.

[0022] This invention provides a new treatment strategy for EV71 infection: For the first time, this invention clarifies the key role of the ZAK-p38 MAPK pathway (i.e., ribosomal toxic stress response, RSR) in the pathogenesis of EV71 infection, and expands the scope of ZAK inhibitors (such as the marketed Nilotinib) into potential anti-EV71 drugs, providing new candidate drugs and targets for clinical treatment. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the activation of the ZAK-p38 MAPK pathway and viral replication after EV71 infection of RD cells according to the present invention. Figure 2 This is a schematic diagram illustrating the effect of the ZAK kinase inhibitor Nilotinib of the present invention on p38 phosphorylation and viral replication in EV71-infected RD cells; Figure 3 This is a schematic diagram illustrating the effect of ZAKα gene knockdown on the ZAK-p38 MAPK pathway and viral replication in EV71-infected RD cells. Figure 4 This is a schematic diagram illustrating the effect of Nilotinib of the present invention on serum biochemical parameters in EV71-infected ICR neonatal mice; Figure 5 This is a schematic diagram of the mouse blood biochemical results of the present invention; Figure 6 This is a schematic diagram of the immunohistochemical (IHC) staining results of viral protein VP1 in skeletal muscle tissue of EV71-infected ICR suckling mice according to the present invention. Figure 7 This is a schematic diagram of the brain tissue IHC (positive granule is VP1) results of the present invention; Figure 8 This is a schematic diagram of the results of muscle tissue IHC (positive particles are VP1) according to the present invention. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0025] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0026] Example 1: 1. Cell Experiment The cells used were RD cells (human malignant embryonic rhabdomyosarcoma cells), and the virus was EV71 (enterovirus 71).

[0027] Experiments revealed that EV71 virus infection promotes ZAK phosphorylation, and ZAK transmits signals down the MAPK pathway and activates p38. Activation of the ZAK-p38 MAPK pathway is a key signaling pathway for ribosomal toxic stress response (RSR).

[0028] Treatment of infected cells with the ZAK kinase inhibitor nilotinib at different concentrations and infection times effectively suppressed p38 phosphorylation levels, with the inhibitory effect increasing with increasing inhibitor concentration. Simultaneously, the expression of the viral protein VP1 gradually decreased. This demonstrates that EV71 infection activates the ZAK-p38 MAPK pathway, and inhibiting ZAK kinase activity reduces viral replication.

[0029] To avoid potential off-target effects of inhibitors, three different ZAKα-siRNAs (all at 20 pmol) were designed for gene knockdown experiments. Comparing the ZAKα-knockdown infection group with the non-knockdown infection group, it was found that after ZAKα knockdown, viral replication was difficult to maintain, producing only a small amount of VP1 protein, and p38 phosphorylation level only slightly increased. Comparing the blank control group with the knockdown infection group, it was found that while the upstream ZAKα reduction did not affect the downstream p38 itself, p38 phosphorylation level still decreased significantly. This indicates that the increase in p38 phosphorylation level during viral infection is mainly mediated by ZAKα, rather than other upstream kinases in the MAPK pathway. Furthermore, changes in ZAKα and p38 phosphorylation levels showed a synergistic effect with changes in the viral protein VP1. These results demonstrate that ZAKα-mediated RSR is triggered during EV71 infection, and inhibiting ZAKα activity can reduce viral replication (ZAK has two subtypes, α and β, but only ZAKα is associated with RSR).

[0030] 2. Animal experiments The experimental animals were one-week-old ICR suckling mice, divided into four groups: control group, EV71 infection group, low-dose Nilotinib (40 mg / kg) treatment group, and high-dose Nilotinib (80 mg / kg) treatment group. Viral solution was administered intramuscularly during model establishment, and the drug was administered intraperitoneally during treatment.

[0031] Analysis of serum biochemical parameters in mice showed that: (1) Renal function: Compared with the control group, the blood urea nitrogen (BUN) and serum creatinine (SCr) of mice in the virus infection group were significantly increased, indicating impaired glomerular filtration function. The levels of BUN and SCr in both treatment groups decreased, indicating that Nilotinib treatment can effectively alleviate renal function damage caused by EV71.

[0032] (2) Metabolic function: The virus infection group showed hypoglycemia and hyperlactatemia, indicating systemic metabolic disorder. Drug treatment could effectively relieve hypoglycemia and hyperlactatemia.

[0033] (3) Muscle damage: Creatine kinase (CK) levels were significantly elevated after viral infection, while creatine kinase isoenzyme (CK-MB) levels showed no significant change, indicating that the damage mainly occurred in skeletal muscle. After treatment, CK levels decreased significantly, indicating that the medication alleviated muscle damage.

[0034] (4) Liver-related indicators: Alanine aminotransferase (ALT) and alkaline phosphatase (ALP) both increased after viral infection. The drug only had a significant therapeutic effect on ALP, but not on ALT, suggesting that the hepatocellular damage caused by EV71 infection was atypical, and the elevated ALP may have originated from the bones or bile duct.

[0035] In summary, EV71 infection leads to multi-organ dysfunction, with the kidneys, metabolic system, and muscular system being most significantly affected. Nilotinib treatment comprehensively improves these abnormal indicators, demonstrating that targeting the ZAK-p38 MAPK pathway can exert a systemic protective effect in vivo.

[0036] 3. Organizational Level Validation The results of Western blotting were consistent with those of the cell experiments: viral infection increased p38 phosphorylation levels, which decreased after nilotinib treatment, and the expression of the viral protein VP1 also decreased. This indicates that nilotinib can still effectively inhibit viral replication and reduce viral replication in vivo.

[0037] Immunohistochemical (IHC) analysis of brain and muscle tissues (antibody: VP1) revealed a large number of dark brown VP1-positive granules in the cytoplasm of infected cells. The number of positive granules decreased after both low- and high-dose nilotinib treatment. This confirms, from a histopathological perspective, that the drug effectively inhibits viral replication in vivo.

[0038] The above experiments fully demonstrate that ZAK inhibitors (taking Nilotinib as an example) can effectively inhibit EV71 virus replication and improve the damage to the body caused by viral infection by inhibiting the ZAK-p38 MAPK pathway. Therefore, they can be used to prepare drugs for the treatment of EV71 infection.

[0039] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Application of ZAK inhibitors in the preparation of drugs for treating EV71 virus infection.

2. The application according to claim 1, characterized in that, The ZAK inhibitor works by inhibiting the ZAK-p38 MAPK signaling pathway.

3. The application according to claim 1, characterized in that, The ZAK inhibitors exert their effects by inhibiting the ZAKα-mediated ribosomal toxicity stress response (RSR).

4. The application according to any one of claims 1-3, characterized in that, The ZAK inhibitor is Nilotinib or a pharmaceutically acceptable salt thereof.

5. The application according to claim 1, characterized in that, The treatment included reducing the level of the EV71 viral protein VP1 in the subject's body.

6. The application according to claim 1, characterized in that, The treatment includes improving renal function impairment caused by EV71 infection, which is indicated by elevated blood urea nitrogen (BUN) and / or serum creatinine (SCr) levels.

7. The application according to claim 1, characterized in that, The treatment includes improving metabolic disorders caused by EV71 infection, which are indicated by hypoglycemia and / or high lactate levels.

8. The application according to claim 1, characterized in that, The treatment includes improving muscle damage caused by EV71 infection, which is indicated by elevated creatine kinase (CK) levels.

9. A pharmaceutical composition for treating EV71 infection, characterized in that, It contains a therapeutically effective amount of a ZAK inhibitor and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that, The ZAK inhibitor is Nilotinib.

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