Application of compound AJ2-30 in the preparation of antiviral drugs

Abstract

The present invention belongs to the field of biotechnology and specifically relates to the use of a compound AJ2-30 in the preparation of antiviral drugs. The present invention unexpectedly discovered that adding compound AJ2-30 to the culture medium for ASFV can reduce the replication level of ASFV, indicating that compound AJ2-30 has the effect of inhibiting ASFV replication. It can be used to prepare drugs or adjuvants for anti-ASFV infection and inhibit ASFV replication, and has broad application prospects.

Description

Technical Field

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

[0002] African swine fever (ASF) is an acute, 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, organ failure, and bleeding. It is highly contagious and pathogenic, with a mortality rate as high as 100%. Currently, there is no commercially available ASF vaccine, and its pathogenic and immune mechanisms remain incompletely understood.

[0003] Different vaccines have been tested against ASFV, primarily inactivated, live attenuated, subunit, and DNA vaccines. Inactivated vaccines, however, fail to stimulate cellular immunity and therefore fail to induce effective neutralizing antibodies. Compared to inactivated vaccines, traditional attenuated vaccines can stimulate both cellular and humoral immune responses, but offer poor cross-protection. Furthermore, strict dosing is crucial. Attenuated vaccines can cause side effects such as pneumonia, joint swelling, and skin ulcers, and even death in some animals. While subunit vaccines offer a high level of safety, identifying antigens that provide effective immunogenicity and protective efficacy remains challenging. Furthermore, due to the large ASFV genome, vaccines formulated with a single protein may not generate sufficient antibodies, leading to alterations in the antigen gene, resulting in a lack of antigen-antibody binding and protection against virulent strains. DNA vaccines can induce neutralizing antibodies or T-cell-specific antiviral responses. Gene-deletion vaccines generally address the issue of efficacy, but they carry biosafety risks. While inactivated, live vector-based, and subunit vaccines offer safety, the core issue of ineffective protection remains unresolved. In the absence of an effective commercial vaccine, and facing the ongoing outbreak and spread of ASF, the development of anti-ASFV drugs is an alternative approach to ASF prevention and control. However, research on chemical substances that inhibit SVA replication has not yet been reported.

[0004] Compound AJ2-30 is a white to off-white powder and is a SLCl5A4 inhibitor that can inhibit TLR9-mediated B cell activation; it can block endogenous NOD signaling in human and mouse macrophages; and 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 ASFV replication and can be used to prepare drugs or adjuvants against ASFV infection to inhibit the replication of ASFV. Summary of the Invention

[0006] In response to the above technical problems, the present invention aims to provide a method for preparing an antiviral drug using compound AJ2-30. Specifically, the method includes the following:

[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 medicament 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 anti-viral infection drugs or adjuvants for inhibiting the replication of African swine fever virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 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 treating Pam cells with different concentrations of AJ2-30, the mRNA levels of ASFV structural protein p30 showed different detection results;

[0024] Figure 3 Figure 3. Flow cytometry analysis of ASFV-GFP in Pam cells after treatment 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, unless otherwise specified, are all conventional methods; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent companies.

[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 Foot-and-Mouth Disease Epidemiology Team Laboratory 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 using 1640 medium containing 10% FBS. After 16 h, the supernatant was discarded and inoculated with ASFV-GFP (0.1 MOI). The plates were then incubated at 37°C for 1 h.

[0033] 2. Preparation of AJ2-30-containing Culture Medium

[0034] AJ2-30 was serially diluted with 2% FBS-containing culture medium and added to a 24-well plate that had been infected for 1 hour, with final concentrations of 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 ASFV structural protein p30 mRNA levels 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, when different concentrations of AJ2-30 were added during the PAM cell culture process, the protein levels of ASFV P54 and P72 were reduced in a dose-dependent manner.

[0042] The results of the ASFV structural protein p30 mRNA level detection were as follows Figure 2 As shown in the data, during the PAM cell culture process, when different concentrations of AJ2-30 were added, the mRNA level of ASFV P30 decreased 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 in Figure 2, 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 seeded in a 96-well plate with a cell suspension of 100 μL / well. The plate was placed in a 37°C incubator. After 24 hours, the culture medium in the plate was discarded and 1640 culture medium containing different concentration gradients of AJ2-30 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 using a microplate reader.

[0048] The cell viability test results 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 this 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-described embodiments merely illustrate the implementation methods of the present invention. Although the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art may make other modifications without departing from the scope of the present invention, and such modifications are within the scope of protection of the present invention.

Claims

1. Use of compound AJ2-30 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating African swine fever virus 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 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.

3. The use according to claim 2, characterized in that The dosage forms include powder injection, capsule, tablet and suspension.

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