Application of compound AJ2-30 in preparation of antiviral drugs

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a compound AJ2-30 in preparation of antiviral drugs. The invention accidentally finds that the compound AJ2-30 is added into a culture medium for culturing the ASFV, so that the replication level of the ASFV can be reduced, which indicates that the compound AJ2-30 has the effect of inhibiting the ASFV replication, can be used for preparing a medicine or an adjuvant for resisting ASFV infection, is used for inhibiting the replication of the ASFV, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of a compound AJ2-30 in the preparation of antiviral drugs. Background Art

[0002] African swine fever (ASF) is an acute and highly contagious disease of wild boars and domestic pigs caused by the African swine fever virus (ASFV). Symptoms of ASF infection include high fever, loss of appetite, cyanosis of the skin, systemic organ failure, and bleeding. It is highly contagious and highly pathogenic, with a mortality rate of up to 100%. There is currently no commercial ASF vaccine, and its pathogenic and immune mechanisms are still not fully understood.

[0003] Different vaccines have been tested for ASFV, mainly inactivated vaccines, live attenuated vaccines, subunit vaccines and DNA vaccines. Among them, inactivated vaccines cannot stimulate the body's cellular immunity and cannot induce effective neutralizing antibodies to produce protection. Compared with inactivated vaccines, traditional attenuated vaccines can stimulate the body to produce cellular immune responses and humoral immune responses, but the cross-immunity protection effect is poor. At the same time, the vaccination dose must be strictly controlled during vaccination. Attenuated vaccines may cause side effects such as pneumonia, joint swelling and skin ulcers, and even cause death in a few animals. Although subunit vaccines have high safety, they face difficulties in identifying antigens that provide good immunogenicity and protection efficiency for the host. At the same time, there are also problems such as the large ASFV genome, the vaccine prepared with one protein cannot produce enough antibodies, the antigen gene changes, and then the antigen and antibody cannot combine, and cannot resist the attack of strong strains. DNA vaccines can induce neutralizing antibodies or T cell-specific antiviral immune responses. In general, gene-deficient vaccines solve the problem of immune efficacy, but there are biosafety risks. Although inactivated vaccines, live vector vaccines and subunit vaccines are safe, the core problem of "immunity without protection" of these vaccines has not been solved. In the absence of an effective commercial vaccine, the development of anti-ASFV drugs is an alternative method for ASF prevention and control in the face of the continuous outbreak and spread of ASF. However, there are no reports on the use of chemical substances to inhibit SVA replication.

[0004] Compound AJ2-30 is a white to off-white powder. It is a SLCl5A4 inhibitor that can inhibit TLR9-mediated B cell activation; it can block endogenous NOD signaling in human and mouse macrophages; and it can also be used for inflammation research.

[0005] The present invention unexpectedly discovered that adding AJ2-30 to the culture medium for culturing ASFV can reduce the replication level of ASFV, indicating that AJ2-30 has the effect of inhibiting the replication of ASFV and can be used to prepare drugs or adjuvants against ASFV infection to inhibit the replication of ASFV. Summary of the invention

[0006] In view of the above technical problems, the purpose of the present invention is to provide a use of compound AJ2-30 in the preparation of antiviral infection drugs. Specifically including the following contents:

[0007] In a first aspect, the present invention provides a use of a compound AJ2-30 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating viral infection; the structural formula of the compound AJ2-30 is shown in the following formula (I);

[0008]

[0009] Preferably, the virus is a DNA virus.

[0010] Preferably, the virus is a double-stranded DNA virus.

[0011] Preferably, the virus is African swine fever virus.

[0012] Preferably, the AJ2-30 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.

[0013] Preferably, the dosage forms include powder injection, capsule, tablet, and suspension.

[0014] In a second aspect, the present invention provides a use of a compound AJ2-30 or a pharmaceutically acceptable salt thereof in the preparation of a viral vaccine adjuvant, wherein the structural formula of the compound AJ2-30 is shown in the following formula (I);

[0015]

[0016] Preferably, the virus is a DNA virus.

[0017] Preferably, the virus is a double-stranded DNA virus.

[0018] Preferably, the virus is African swine fever virus.

[0019] Preferably, the AJ2-30 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.

[0020] Preferably, the dosage forms include powder injection, capsule, tablet, and suspension.

[0021] The beneficial effect of the present invention is: the present invention unexpectedly found that adding AJ2-30 to the culture medium for culturing ASFV can inhibit the replication of ASFV, indicating that AJ2-30 has the effect of inhibiting the replication of ASFV and can be used to prepare drugs or adjuvants against viral infections to inhibit the replication of African swine fever virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The protein level detection results of ASFV structural proteins P54 and P72 after Pam cells were treated with different concentrations of AJ2-30;

[0023] Figure 2 After Pam cells were treated with different concentrations of AJ2-30, the mRNA levels of ASFV structural protein p30 showed different detection results;

[0024] Figure 3 The results of flow cytometry detection of ASFV-GFP after Pam cells were treated with different concentrations of AJ2-30;

[0025] Figure 4 TCID 50 Measurement results;

[0026] Figure 5 Cytotoxicity assay results. DETAILED DESCRIPTION

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0028] AJ2-30 was purchased from MCE.

[0029] The African swine fever virus strain is the genotype II African swine fever virus strain ASFV CN / GS / 2018 isolate, which was isolated by our team and preserved in the laboratory of the Foot-and-Mouth Disease Epidemiology Team of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0030] Example 1 Compound AJ2-30 inhibits ASFV replication

[0031] 1. ASFV vaccination

[0032] PAM cells were plated in 24-well plates with 1640 containing 10% FBS. After 16 h, the supernatant was discarded, ASFV-GFP (0.1 MOI) was inoculated, and the plates were placed in a 37°C incubator for 1 h.

[0033] 2. Preparation of AJ2-30-containing culture medium

[0034] AJ2-30 was serially diluted with a medium containing 2% FBS and added to a 24-well plate that had been infected for 1 hour, so that the final concentrations were 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM, respectively, with 6 replicate wells for each gradient.

[0035] 3. ASFV inhibition level determination

[0036] Collect samples after 24 hours of culture and perform the following tests:

[0037] (1) Detect the protein levels of ASFV structural proteins P54 and P72 by Western Blot;

[0038] (2) Detection of the mRNA level of ASFV structural protein p30 by qPCR;

[0039] (3) detection of the proportion of ASFV-GFP by flow cytometry;

[0040] (4) Collect cell supernatant and analyze by TCID 50 Determine and analyze the virus titer.

[0041] The protein level detection results of ASFV structural proteins P54 and P72 are as follows Figure 1 As shown in the data, during the PAM cell culture process, when different concentrations of AJ2-30 were added, the protein levels of ASFV P54 and P72 were reduced in a dose-dependent manner.

[0042] The results of the mRNA level detection of ASFV structural protein p30 are as follows Figure 2 As shown, during the PAM cell culture process, when different concentrations of AJ2-30 were added, the mRNA level of ASFV P30 was reduced in a dose-dependent manner.

[0043] The results of flow cytometry detection of ASFV-GFP are as follows Figure 3 As shown, the horizontal axis FITC represents the ratio of ASFV-GFP, indicating that during the PAM cell culture process, when different concentrations of AJ2-30 were added, the ASFV in the PAM cells decreased with the increase of concentration, indicating that AJ2-30 inhibited the replication of ASFV in a dose-dependent manner.

[0044] TCID 50 The results of the test are as follows Figure 4 As shown, when different concentrations of AJ2-30 were added, the TCID 50 decreased in a dose-dependent manner.

[0045] The above results show that AJ2-30 inhibits the expression of ASFV structural proteins P30, P54 and P72, reduces the titer of ASFV, and inhibits the replication of ASFV; and as the AJ2-30 content increases, the inhibitory effect on ASFV replication becomes stronger, showing a dose-dependent relationship.

[0046] Example 2 Effect of Compound AJ2-30 on PAM Cell Activity

[0047] PAM cells were inoculated in a 96-well plate with a cell suspension of 100 μL / well and placed in a 37°C incubator. After 24 hours, the culture medium in the well plate was discarded and 1640 culture medium containing AJ2-30 of different concentration gradients was added. Five replicate wells were set for each gradient. After 24 hours of culture, culture medium containing 10% CCK8 was added. After incubation for 30 minutes, the OD value was detected with a microplate reader.

[0048] The results of cell viability assay were as follows Figure 5 As shown, when the concentration of AJ2-30 is 50 μM, the viability of PAM cells is greater than 50%, and when the concentration of AJ2-30 is less than or equal to 25 μM, the viability of PAM cells is greater than 90%, indicating that the compound AJ2-30 described in the present application has good safety.

[0049] In summary, the embodiments of the present invention take the host cell PAM cells as an example, and the study proves that AJ2-30 can inhibit the replication of ASFV in the host cell PAM cells, indicating that the AJ2-30 described in the present invention can inhibit the replication of ASF virus and can be used to prepare drugs or adjuvants against ASF virus infection; it can also be used to prepare anti-antiviral drugs, and has broad application prospects.

[0050] The above-mentioned embodiments are only embodiments of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, other improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. Use of compound AJ2-30 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating viral infection; the structural formula of the compound AJ2-30 is shown in the following formula (I):

2. The use according to claim 1, characterized in that The virus is a DNA virus.

3. The use according to claim 2, characterized in that The virus is a double-stranded DNA virus.

4. The use according to claim 3, characterized in that The virus is African swine fever virus.

5. Use of compound AJ2-30 or a pharmaceutically acceptable salt thereof in the preparation of a viral vaccine adjuvant, wherein the structural formula of the compound AJ2-30 is shown in the following formula (I):

6. The use according to claim 5, characterized in that The virus is a DNA virus.

7. The use according to claim 6, characterized in that The virus is a double-stranded DNA virus.

8. The use according to claim 7, characterized in that The virus is African swine fever virus.

9. The use according to any one of claims 1 to 8, characterized in that: The AJ2-30 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.

10. The use according to claim 9, characterized in that The dosage forms include powder injection, capsule, tablet and suspension.

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