Application of CUL4B activator in preparation of anti-influenza virus drugs

Abstract

The invention provides an application of a CUL4B activator in preparation of anti-influenza virus drugs, and belongs to the technical field of medicines. The invention provides an application of a CUL4B activator in preparation of an anti-influenza virus drug, and the action target of the CUL4B activator comprises an E3 ligase compound formed by a CUL4B-DDB1-RBX1 compound and a DCAF (DDB1 and CUL4 related factor) family protein. The invention provides a new medicine and a new way for effective treatment of influenza.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of a CUL4B activator in the preparation of an anti-influenza virus drug. Background Art

[0002] Influenza virus belongs to the family Orthomyxoviridae and is an enveloped negative-sense single-stranded RNA virus. Influenza virus can cause infection in hosts including humans, poultry, pigs, and horses. In the virological classification, influenza viruses are divided into four categories: A, B, C, and D (A, B, C, and D). Influenza A virus can be further divided into several subtypes, such as H1N1, H3N2, and H5N1, based on the antigenicity of hemagglutinin (HA) and neuraminidase (NA) on the surface of the virus. Influenza A virus, mainly H1N1 and H3N2, and influenza B virus cause seasonal influenza epidemics. Influenza virus repeatedly causes human respiratory diseases every year, placing a heavy burden on social economy and human health. According to data released by the WHO, the incidence of influenza in adults is 5%-10%, and the incidence in children is 20%-30%. The number of deaths from related respiratory diseases caused by seasonal influenza reaches 650,000 each year. The main symptoms of influenza include cough, fever, chills, headache, etc.

[0003] The most effective way to prevent influenza is to get vaccinated. Although vaccines have been used to prevent serious illness and death caused by influenza viruses, there are still some obstacles in practice. Currently, the influenza vaccines approved for marketing are mainly inactivated vaccines and live attenuated vaccines. Due to the rapid antigenic variation of influenza viruses, the multiple subtypes and the differences in human individual immune responses, the protective effect of vaccines in specific populations is still unsatisfactory. Vaccines only have a preventive effect on existing virus subtypes, and are ineffective against new strains produced by antigenic drift or antigenic conversion. It is difficult for the produced vaccines to be consistent with the strains that are about to become popular. Therefore, effective antiviral small molecule drugs have become a good choice for intervening in influenza virus infection. Currently, four antiviral drugs have been approved for the treatment of influenza, including neuraminidase (NA) inhibitors (oseltamivir, zanamivir and peramivir) and PA inhibitors (baloxavir). However, due to the emergence of drug-resistant strains, antiviral drugs have gradually lost their efficacy. For example, oseltamivir-resistant H1N1 strains began to appear in the 2007-2008 influenza season. Therefore, it is urgent to find new drugs to treat influenza viruses. Summary of the invention

[0004] In view of the above shortcomings of the prior art, the present invention provides the use of a CUL4B activator in the preparation of an anti-influenza virus drug. The CUL4B activator has good in vitro and in vivo anti-influenza virus activity and has broad application prospects in the prevention and treatment of influenza virus infection.

[0005] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0006] In a first aspect, the present invention provides use of a CUL4B activator in the preparation of an anti-influenza virus drug.

[0007] The amino acid sequence of CUL4B is shown in SEQ ID NO. 1.

[0008] Furthermore, the targets of the CUL4B activator include the E3 ligase complex formed by the CUL4B-DDB1-RBX1 complex and the DCAF (DDB1 and CUL4 associated factor) family protein.

[0009] Furthermore, the CUL4B activator includes Etoposide or a pharmaceutically acceptable salt thereof.

[0010] The structural formula of the Etoposide is as follows:

[0011]

[0012] In a second aspect, the present invention provides an anti-influenza virus drug, wherein the active ingredient of the drug is a CUL4B activator.

[0013] Furthermore, the drug's targets include the E3 ligase complex formed by the CUL4B-DDB1-RBX1 complex and the DCAF family protein.

[0014] Furthermore, the CUL4B activator includes Etoposide or a pharmaceutically acceptable salt thereof.

[0015] Furthermore, in the mouse experiment level, the effective dosage of Etoposide is 10-50 mg / kg per day.

[0016] Furthermore, the drug also includes a pharmaceutically acceptable carrier and / or excipient.

[0017] Furthermore, the dosage form of the drug includes but is not limited to one of tablets, granules, capsules, pills, sustained-release preparations, oral liquid preparations, and injections.

[0018] Compared with the prior art, the present invention is beneficial in that:

[0019] 1. The present invention provides an application of a CUL4B activator in the preparation of an anti-influenza virus drug. The target of the CUL4B activator includes an E3 ligase complex formed by a CUL4B-DDB1-RBX1 complex and a DCAF (DDB1 and CUL4 associated factor) family protein. Silencing of any one of CUL4B, DDB1, and RBX1 will promote the replication of influenza virus PA protein, while the CUL4B activator will promote the degradation of PA protein. The present invention provides a new drug and approach for the effective treatment of influenza.

[0020] 2. The present invention confirms through experimental studies at the cell level that the CUL4B activator Etoposide can enhance the polyubiquitination of the K48 type of influenza virus polymerase PA subunit, promote the ubiquitin-proteasome degradation of PA, reduce the activity of the viral polymerase, and thus inhibit the infection of the influenza virus; in vivo experiments confirm that the CUL4B activator Etoposide has low toxicity, shows very good antiviral activity against influenza virus, can significantly inhibit the replication of influenza virus in mouse lung tissue, and reduce virus-induced lung damage, and has broad application prospects in the prevention and treatment of influenza virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The CUL4B-DDB1-RBX1 complex participates in the degradation of influenza virus polymerase PA. DCAF family proteins act as substrate recognition receptors and form CUL4B with the CUL4B-DDB1-RBX1 complex. DCAF Results of the E3 ubiquitin ligase targeted PA degradation experiment; Figure 1 A shows the effects of knocking down CUL4A, CUL4B, DDB1 and RBX1 on PA protein levels; Figure 1 B shows the effects of knocking down CUL4A, CUL4B, DDB1 and RBX1 on PB2 protein levels; Figure 1 C shows the effects of knocking down CUL4A, CUL4B, DDB1 and RBX1 on PB1 protein levels; Figure 1 D indicates that DCAF family proteins act as substrate recognition receptors and form CUL4B-DDB1-RBX1 complex DCAF Model for E3 ubiquitin ligase targeting PA for degradation;

[0022] Figure 2 The results of the in vitro anti-influenza virus activity test of Etoposide;

[0023] Figure 3 This is the research result of the mechanism of action of Etoposide against influenza virus; Figure 3A is the results of Western blot analysis after HEK293T cells were treated with proteasome inhibitor MG132 or Etoposide; Figure 3 B is the result of Western blot analysis after HEK293T cells were treated with Etoposide; Figure 3 C is the dual luciferase activity result of HEK293T cells after treatment with Etoposide;

[0024] Figure 4 The results of the in vivo anti-influenza virus activity test of Etoposide; Figure 4 A is an influenza virus infection mouse model; Figure 4 B shows the weight changes of mice after oral administration of Etoposide for 6 days; Figure 4 C is the weight change of mice infected with influenza virus within 6 days; Figure 4 D is the RT-qPCR experimental results of mouse lung tissue; Figure 4 E is the HE staining result of mouse lung tissue on the 6th day of infection. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The present invention provides application of a CUL4B activator in preparing an anti-influenza virus drug.

[0027] The amino acid sequence of CUL4B is shown in SEQ ID NO. 1.

[0028] The targets of the CUL4B activator include the E3 ligase complex formed by the CUL4B-DDB1-RBX1 complex and the DCAF family protein.

[0029] The CUL4B activator includes Etoposide or a pharmaceutically acceptable salt thereof.

[0030] In the following specific examples, the structural formula of the Etoposide is as follows:

[0031]

[0032] Example 1 Effect of CUL4B on influenza virus

[0033] 1. Effect of CUL4B-DDB1-RBX1 complex on influenza virus PA protein

[0034] Cullin4-RING (CRL4) E3 ligase is a multiprotein complex that can specifically recognize target proteins and perform ubiquitination-dependent degradation. CRL4 E3 ubiquitin ligase is composed of Cullin4 (CUL4A / CUL4B), DNA damage-binding protein-1 (DDB1) and RING-box protein 1 (RBX1). CUL4A or CUL4B acts as a scaffold protein to recruit DDB1 and RBX1 to assemble the DDB1-CUL4-RBX1 complex. Among them, RBX1, as a RING finger protein, binds to the E2 ubiquitin conjugating enzyme, while DDB1 acts as an adaptor to recruit DDB1 and CUL4-associated factors (DCAFs) to form a complete Cullin4-RING E3 ubiquitin ligase complex. DCAFs are responsible for recognizing substrates as substrate recognition receptors in Cullin4-RING E3 ubiquitin ligase and determining substrate specificity. This example uses siRNAs to identify whether CUL4A or CUL4B plays a role in the degradation of influenza virus PA protein.

[0035] HEK293T cells were transfected with siRNAs targeting host CUL4A, CUL4B, DDB1, and RBX1 or negative control (NC) and Flag-tagged PA expression plasmids, and Western blotting was performed 48 h after expression. The experimental results are shown in Figure 1 A. Figure 1 A shows that the knockdown efficiency of siRNAs is about 70%-90%. Compared with the control group, the expression of PA protein increased significantly after knocking down CUL4B, while knocking down CUL4A had no significant effect on the expression level of PA protein. This result shows that CUL4B is responsible for regulating the degradation of PA protein in influenza virus. In addition, the expression of PA protein also increased significantly after knocking down DDB1 or RBX1. The above results show that the CUL4B-DDB1-RBX1 complex is involved in the regulation of the stability of influenza virus PA protein expression.

[0036] 2. Effect of CUL4B-DDB1-RBX1 complex on influenza virus PB1 or PB2 protein

[0037] HEK293T cells were transfected with siRNAs targeting host CUL4A, CUL4B, DDB1 and RBX1 or negative control (NC) and Flag-tagged PB1 or PB2 protein expression plasmids, and protein immunoblotting was performed after 48 h of expression. The experimental results are shown in Figure 1 B and Figure 1 C. Figure 1 B and Figure 1 C shows that compared with the control group, knockdown of the DDB1-CUL4-RBX1 complex had no significant effect on the expression levels of PB1 / PB2 proteins, indicating that the CUL4B-DDB1-RBX1 complex had no obvious effect on the expression stability of PB1 or PB2.

[0038] 3. In addition to the CUL4B-DDB1-RBX1 complex, the formation of the complete CUL4B-RING E3 ubiquitin ligase also requires substrate recognition receptors DCAFs. DDB1 can act as an adaptor to recruit DDB1 and CUL4-associated factors (DCAFs). Taken together, these results indicate that host DCAF family proteins, CUL4B, DDB1, and RBX1 form a complete CRL4B DCAF The E3 ubiquitin ligase complex is responsible for the degradation of polymerase PA protein (e.g. Figure 1 D).

[0039] From the above, it can be seen that the CUL4B-DDB1-RBX1 complex is involved in the degradation of influenza virus polymerase PA, and DCAF family proteins act as substrate recognition receptors and form CUL4B-DDB1-RBX1 complexes DCAF E3 ubiquitin ligase targets PA for degradation.

[0040] Example 2 In vitro anti-influenza virus activity and mechanism of action of Etoposide

[0041] 1. Etoposide's anti-influenza virus activity in vitro

[0042] The antiviral activity of Etoposide was evaluated by the reduction of intracellular influenza virus nucleoprotein (NP) after infection of A549 cells. A549 cells were seeded in 12-well plates and infected with influenza H1N1 PR8 The virus (MOI = 0.1) was infected at 37°C for 1 h, and then the virus-containing culture medium was removed and culture medium containing Etoposide (50 μM) was added; after 24 h of culture, the cells were lysed to obtain protein samples, and the viral NP and PA protein levels were detected by western blotting experiments using NP and PA specific antibodies to reflect viral replication and confirm the effect of Etoposide on viral infection.

[0043] The results of the in vitro anti-influenza virus activity test of Etoposide are shown in Figure 2 .Depend on Figure 2 It can be seen that Etoposide has good anti-influenza virus activity at the cellular level.

[0044] 2. Mechanism of action of Etoposide against influenza virus

[0045] The specific mechanism of Etoposide inhibiting influenza virus infection was explored through cell and biochemical experiments. Flag-PA expression plasmid (1 μg) was transfected into HEK293T cells, and after 36 h of expression, it was treated with proteasome inhibitor MG132 (20 μM) or Etoposide (50 μM) for 12 h, followed by protein immunoblotting analysis. The experimental results are shown in Figure 3 A. The autophagy-lysosome pathway and the ubiquitin-proteasome pathway are the main protein degradation pathways in eukaryotic cells, and MG132 is an inhibitor of the proteasome. Figure 3 A shows that only MG132 treatment will upregulate the expression of influenza virus polymerase PA protein; only Etoposide treatment will promote the degradation of PA protein; when MG132 and Etoposide are used for treatment at the same time, MG132 blocks the degradation-promoting effect of Etoposide. This result shows that Etoposide can degrade PA through the ubiquitin-proteasome pathway and reduce its protein level.

[0046] Subsequently, we tested whether Etoposide promoted the degradation of viral PA subunits by increasing ubiquitin binding. Flag-PA was co-transfected with HA-Ub (K48) expression plasmid or empty vector into HEK293T cells for expression for 36 h, and then treated with Etoposide (50 μM) for 12 h. After cells were lysed with RIPA lysis buffer, immunoprecipitation (IP) experiments were performed with Flag antibody, and the immunoprecipitates were used for protein immunoblot analysis. The experimental results are shown in Figure 3 B, The figure shows that Etoposide increases the K48 ubiquitination of PA protein.

[0047] The influenza virus minireplicon system was used to detect the effect of Etoposide on the overall viral replication and transcription. The minireplicon system plasmid was co-transfected into HEK293T cells. 36 h after transfection, the cells were treated with Etoposide (50 μM) for 12 h (DMSO was used as a control), and the dual luciferase activity of the cells was detected. The experimental results are shown in Figure 3 C, The figure shows that Etoposide significantly reduces the activity of influenza virus polymerase.

[0048] The above results indicate that Etoposide can enhance the polyubiquitination of K48 type of influenza virus polymerase PA subunit, promote the ubiquitin-proteasome degradation of PA, reduce the activity of viral polymerase, and thus inhibit influenza virus infection.

[0049] Example 3 Anti-influenza virus activity of Etoposide in vivo

[0050] Etoposide down-regulated the expression of influenza virus PA protein in vitro, limiting influenza virus infection, but its effect in mice has not yet been identified. Next, we will verify the anti-influenza virus activity of Etoposide in vivo.

[0051] Influenza virus infection mouse model Figure 4 As shown in A, 6-8 week old C57BL / 6J mice were orally treated with Etoposide (20 mg / kg) once a day for 6 days. After being anesthetized with isoflurane anesthetic, they were infected with the mouse-adapted PR8 virus through the nasal route. The body weight of the mice was monitored for several consecutive days. The mice were euthanized on the 6th day of infection, and the lung tissues were taken for viral load determination and pathological observation. The mice were only treated with the drug orally, and the in vivo toxic effects of Etoposide were determined by monitoring the changes in the weight of the mice for several consecutive days. The experimental results are shown in Figure 4 B, 4C; among them, Figure 4 B shows that compared with the control group, no significant weight loss was observed in mice that received continuous oral treatment with Etoposide (20 mg / kg) for 6 days, indicating that Etoposide has no obvious drug toxicity in mice; Figure 4 C shows that mice not treated with Etoposide orally exhibited more significant weight loss after virus infection than mice treated with Etoposide orally.

[0052] In order to further understand why Etoposide can reduce the weight loss of infected mice, lung tissues of mice on the 6th day of infection were obtained, broken by tissue crusher, and RNA was extracted using TRIzol reagent, and then the lung viral load was detected by RT-qPCR. Figure 4 D, the figure shows that the abundance of viral NP RNA in the lung tissue of mice treated with Etoposide was significantly lower than that in the control group, indicating that Etoposide treatment can reduce the viral load in the lung tissue of mice. At the same time, HE staining was performed on the lung tissue of mice on the 6th day of infection. The experimental results are shown in Figure 4 E, The figure shows that compared with the control group, the pathological changes in the lung tissue of mice that did not receive oral treatment with Etoposide were more severe, with more cells and protein debris infiltration in the alveoli and obvious alveolar necrosis.

[0053] The above results confirmed that Etoposide can inhibit the replication of influenza virus in mice and reduce lung damage caused by influenza virus.

[0054] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Application of CUL4B activators in the preparation of anti-influenza virus drugs.

2. Use of the CUL4B activator according to claim 1 in the preparation of anti-influenza virus drugs, characterized in that: The targets of the CUL4B activator include the E3 ligase complex formed by the CUL4B-DDB1-RBX1 complex and the DCAF family protein.

3. Use of the CUL4B activator according to claim 1 or 2 in the preparation of anti-influenza virus drugs, characterized in that: The CUL4B activator includes Etoposide or a pharmaceutically acceptable salt thereof.

4. A drug for resisting influenza virus, characterized in that: The active ingredient of the drug is a CUL4B activator.

5. The anti-influenza virus drug according to claim 4, characterized in that: The targets of the CUL4B activator include the E3 ligase complex formed by the CUL4B-DDB1-RBX1 complex and the DCAF family protein.

6. An anti-influenza virus drug according to claim 4 or 5, characterized in that: The CUL4B activator includes Etoposide or a pharmaceutically acceptable salt thereof.

7. The anti-influenza virus drug according to claim 6, characterized in that: The effective dosage of Etoposide is 10-50 mg / kg per day.

8. The anti-influenza virus drug according to claim 4, characterized in that: The medicament further comprises a pharmaceutically acceptable carrier and / or excipient.

9. The anti-influenza virus drug according to claim 4, characterized in that: The dosage form of the drug includes, but is not limited to, tablets, granules, capsules, pills, sustained-release preparations, oral liquid preparations or injections.

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