New use of compound HTH-01-015 for preventing or treating african swine fever

Abstract

The present application belongs to the technical field of African swine fever treatment, and particularly relates to a new use of a compound HTH-01-015 for preventing or treating African swine fever. The present application surprisingly finds that the compound HTH-01-015 can inhibit the transcription level of a structural protein P72 of an African swine fever virus and inhibit the replication of the African swine fever virus, and has an effect of inhibiting the infection of the African swine fever virus; and the compound HTH-01-015 has no influence on the replication of FMDV, can specifically inhibit the African swine fever virus, and can be used for preparing a medicine or an adjuvant against the infection of the African swine fever virus and for inhibiting the replication of the African swine fever virus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel use of compound HTH-01-015 for the prevention or treatment of African swine fever. Background Technology

[0002] African swine fever (ASF) is an acute and highly contagious disease caused by African swine fever virus (ASFV), characterized by fever and hemorrhage in various organs of the pig. The mortality rate in domestic pigs can reach 100%. ASFV is the only member of the Asfarviridae family, belonging to the nucleoplasmic large DNA virus (NCLDV) family, and is the only known DNA arbovirus transmitted by soft ticks (Ornithodoros). The ASFV particle is an enveloped icosahedral structure with a genome ranging from 170 to 190 kb, encoding more than 150 open reading frames (ORFs) depending on the strain.

[0003] Since there are still no effective vaccines or specific treatments for African swine fever (ASF), outbreaks can only be controlled through culling. However, this method not only results in economic losses but also fails to meet the needs of large-scale pig farming in my country. Therefore, effectively controlling ASF is one of the biggest challenges facing the global pig industry and a critical strategic issue that urgently needs to be addressed in my country's ASF prevention and control efforts. Thus, while developing vaccines, finding drugs that can prevent or treat ASF is equally important.

[0004] p30 and p72 are key structural proteins in ASFV, the main structural proteins that make up viral particles, and important surface antigens. They are closely related to host cell tropism, pathogenicity, and immunogenicity, participate in viral internalization, and are involved in viral invasion of host cells.

[0005] Compound HTH-01-015 is a selective NUAK1 / ARK5 inhibitor. This invention unexpectedly discovered that while compound HTH-01-015 does not inhibit foot-and-mouth disease virus replication, it can reduce the replication level of African swine fever virus and inhibit the expression of African swine fever virus structural proteins, thus exhibiting an inhibitory effect on African swine fever virus infection. Therefore, it can be used as an inhibitor of African swine fever virus for the prevention or treatment of African swine fever. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a novel use of compound HTH-01-015 for the prevention or treatment of African swine fever, specifically including the following:

[0007] In a first aspect, the present invention provides the use of compound HTH-01-015 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing African swine fever virus infection, wherein the structural formula of compound HTH-01-015 is shown in formula (Ⅰ) below:

[0008]

[0009] Preferably, the compound HTH-01-015 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

[0010] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, suspensions, and injectable solutions.

[0011] Secondly, the present invention provides the use of compound HTH-01-015 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating African swine fever virus infection, wherein the structural formula of compound HTH-01-015 is shown in formula (Ⅰ) below:

[0012]

[0013] Preferably, the compound HTH-01-015 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

[0014] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, suspensions, and injectable solutions.

[0015] Thirdly, the present invention provides the application of compound HTH-01-015 in the preparation of reagents for inhibiting the expression of African swine fever virus P72 gene / protein; the structural formula of compound HTH-01-015 is shown in formula (Ⅰ) below:

[0016]

[0017] The beneficial effects of this invention are as follows: This invention unexpectedly discovered that compound HTH-01-015 can reduce the replication level of African swine fever virus (ASFV), inhibit the expression of ASFV structural proteins, and has the effect of inhibiting ASFV infection; moreover, compound HTH-01-015 has no effect on FMDV replication, can specifically inhibit ASFV, and can be used to prepare drugs or adjuvants against ASFV infection for inhibiting ASFV replication. Attached Figure Description

[0018] Figure 1 The figure shows the experimental results of the inhibitory effect of compound HTH-01-015 on ASFV-eGFP.

[0019] Figure 2 The figure shows the experimental results of HTH-01-015 inhibiting ASFV-eGFP replication.

[0020] Figure 3 Figure 1 shows the experimental results of the effect of compound HTH-01-015 on the cell viability of PAM cells.

[0021] Figure 4 The figure shows the experimental results of the effect of compound HTH-01-015 on FMDV replication. Detailed Implementation

[0022] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention is further described below in conjunction with specific embodiments. However, the scope of protection of this invention is not limited to the embodiments described below.

[0023] The experiments described in the following examples obtained biosafety clearance and African swine fever laboratory activity clearance:

[0024] In accordance with the requirements for a Biosafety Level 3 (BSL-3) laboratory and related biosafety for African swine fever, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, through a hierarchical reporting process involving the Lanzhou Veterinary Research Institute's Biosafety Committee, Laboratory Animal Ethics Committee, the Chinese Academy of Agricultural Sciences' Biosafety Committee, the Lanzhou Veterinary Research Institute's Laboratory Animal Ethics Committee, and the Lanzhou Veterinary Research Institute's Biosafety Committee, obtained permission from the Ministry of Agriculture and Rural Affairs to conduct research on highly pathogenic ASFV pathogens and related animals. This permit has been registered with the Ministry of Agriculture and Rural Affairs and meets national biosafety level requirements.

[0025] The structural formula of compound HTH-01-015 described in the following examples is shown in formula (Ⅰ). The compound was purchased from Taoshu Biotechnology (TargetMol, L2510).

[0026]

[0027] Unless otherwise specified, all cells and reagents described in the following examples are commercially available.

[0028] Example 1: Preliminary screening of the effect of compound HTH-01-015 on African swine fever virus.

[0029] Dilute compound HTH-01-015 stock solution (10 mM) to 10 μM with RPMI 1640 (10% FBS, 1% triple antibody) serially, and set aside; dilute ASFV-eGFP fluorescent toxin 1:50 with RPMI 1640 (10% FBS, 1% triple antibody), and set aside; resuscitate porcine primary alveolar macrophages (PAMs) at 1×10 5Each compound was seeded per well in a 96-well plate. After incubating at 37°C and 5% CO2 for 6 hours, the culture medium was discarded. 100 μL of diluted compound HTH-01-015 and 100 μL of diluted ASFV-eG FP fluorescent toxin were added, and the plate was incubated at 37°C and 5% CO2 for another 48 hours. Three replicates of each compound were used, with a DMSO group as a control. Significant fluorescence changes in all three replicates were considered indicative of efficacy.

[0030] The results are as follows Figure 1 As shown, compared with the DMSO control group, the ASFV fluorescence in the HTH-01-015 treatment group was significantly reduced, indicating that HTH-01-015 can inhibit ASFV replication.

[0031] Example 2: qPCR verification of the effect of compound HTH-01-015 on African swine fever virus.

[0032] The experimental method in Example 1 was repeated. After 48 hours, the cells and supernatant were repeatedly frozen and thawed three times at -80°C and 4°C to release the virus from the cells. The transcription level of P72 was detected by qPCR. The qPCR detection system is as follows:

[0033] Upstream primer: 5'-CGTGATGT G / A GC G / A AGAATGAAGAA-3', downstream primer: 5'-C G / T GGAAACGCA C / T GAGCAGTATC-3', Probe: FAM-AGCTCCACGA A / G AA A / G The GTGTCGAG-BHQ1 assay system (25 μL) consisted of 1 μL each of forward and reverse primers, 1 μL of probe, 3 μL of virus solution, and 19 μL of ddH2O. qPCR conditions were: 95℃ for 2 min; 95℃ for 7 s, 60℃ for 15 s, 3 cycles; 95℃ for 6 s, 57℃ for 11 s, 40 cycles. The standard curve was calculated as Ct = -3.3652lgcopies + 36.272. Copy numbers were calculated and compared based on the standard curve after the assay.

[0034] The results are as follows Figure 2 As shown, compound HTH-01-015 significantly inhibited the P72 transcription level of ASFV.

[0035] Example 3: CCK-8 assay to detect the effect of compound HTH-01-015 on the cell viability of PAM cells.

[0036] Resuscitate PAM cells at a rate of 1×10 5Cells were seeded into 96-well cell culture plates and cultured at 37°C for 6 hours in a 5% CO2 incubator. Cells were then divided into control and experimental groups. Control group cells were supplemented with 0.1% (v / v) dimethyl sulfoxide (DMSO), while experimental group cells were supplemented with RPMI 1640 medium containing 0.5, 1, 2, 5, 10, and 20 μM of compound HTH-01-015, respectively. A blank control group containing the same dose of DMSO as the control group was also included. Cells were incubated for 48 hours. After incubation, 10 μL of CCK-8 reagent was added to each well, the cell plate was gently tapped to mix the reagent, and the cells were incubated at 37°C for another 1 hour. After incubation, the absorbance at 450 nm was measured using a microplate reader. The cell viability at the corresponding compound concentration was calculated using the formula: [(Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance)] × 100% = Cell viability percentage.

[0037] The results are as follows Figure 3 As shown, compound HTH-01-015 had no significant effect on cell viability at 0.5-10 μM, indicating good safety.

[0038] Example 4: Specificity experiment of compound HTH-01-015 on the inhibition of African swine fever virus proliferation in PAM cells. 1. Infection and the effect of the compound:

[0039] PK-15 was seeded into 12-well cell plates and cultured at 37°C for 12 hours. HTH-01-015 (10mM) was diluted to 10μM with DMEM containing 2% serum and added to the cells along with FMDV, 500μL each, for a total volume of 1mL / well. The FMDV infection dose was 1MOI. A 0.1% DMSO group was set up as a control group. The cells were cultured at 37°C and 5% CO2 for 16-20 hours. The cells were harvested when significant cytopathic effects were observed.

[0040] 2. Extract total RNA:

[0041] Discard the culture medium and extract total RNA according to the Nucleozol (MACHEREY-NAGEL) procedure. Add 400 μL of RNase-free water, vortex for 15 seconds, incubate at room temperature for 15 minutes, and centrifuge at 12,000 g for 15 minutes at room temperature. Transfer the supernatant to a new centrifuge tube, add isopropanol at a 1:1 ratio, invert to mix, incubate at room temperature for 10 minutes, and centrifuge at 12,000 g for 10 minutes at room temperature. Discard the supernatant, add 500 μL of 75% ethanol to resuspend the precipitate, centrifuge at 8,000 g for 3 minutes at room temperature, discard the ethanol, and dry the precipitate. Dissolve the precipitate in 100-200 μL of RNase-free water.

[0042] 3. Reverse transcription:

[0043] An equal amount of extracted RNA was used for reverse transcription, following the steps of Rever Tra Ace qPCR RTMaster Mix with gDNA Remover (Takara).

[0044] 4. qPCR:

[0045] Take equal amounts of reverse transcription product and perform qPCR. qPCR enzyme is... Next qPCR Mix (Takara) was used with primers 3D-F: 5'-TGGGACCATACAGGAGAAGT-3' and 3D-R: 5'-TTCACCCATCGCAGGTAAAG-3'. The reagent mixture consisted of 10 μL Mix, 0.8 μL 3D-F / R, 2 μL cDNA, and 6.4 μL ddH2O. The qPCR conditions were 95℃ for 30 s (pre-denaturation), 95℃ for 5 s, and 95℃ for 10 s (40 cycles). The melting curves were 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. Each sample was run in triplicate. Results were converted to the logarithm of genome copy number according to the standard curve.

[0046] 5. Plot the standard curve:

[0047] The standard plasmid pCDNA3.1-3D containing the FMDV 3D gene was diluted to concentrations of 10... 5 10 6 10 7 10 8 10 9 Using the plasmid as a template for the standard curve, perform the same qPCR as in "4", with three replicates for each concentration. Plot the standard curve with the logarithm of the plasmid concentration on the x-axis and the Ct value on the y-axis.

[0048] The results are as follows Figure 4 As shown, compound HTH-01-015 has no significant effect on the replication of FMDV in PK-15, which also indicates that compound HTH-01-015 described in this application can specifically inhibit ASFV and is used to combat African swine fever virus infection.

[0049] In summary, the HTH-01-015 of the present invention can inhibit the replication of ASFV in a dose-dependent manner. It can specifically inhibit the replication of ASFV and can be used as an inhibitor of African swine fever virus for the prevention or treatment of African swine fever.

Claims

1. The use of compound HTH-01-015 or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention of African swine fever virus infection, wherein the structural formula of compound HTH-01-015 is shown in formula (Ⅰ): ， Equation (Ⅰ).

2. The application as described in claim 1, characterized in that, The compound HTH-01-015 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

3. The application as described in claim 2, characterized in that, The dosage forms include tablets, sprays, granules, capsules, oral liquids, suspensions, and injections.

4. The use of compound HTH-01-015 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating African swine fever virus infection, wherein the structural formula of compound HTH-01-015 is shown in formula (Ⅰ) below: ， Equation (Ⅰ).

5. The application as described in claim 4, characterized in that, The compound HTH-01-015 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipients to form any pharmaceutically acceptable dosage form.

6. The application as described in claim 5, characterized in that, The dosage forms include tablets, sprays, granules, capsules, oral liquids, suspensions, and injections.

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