CN115778946B - Application of Compound ZPP in the Preparation of Antiviral Drugs - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a compound ZPP in preparation of antiviral drugs. The invention surprisingly discovers that the compound ZPP can reduce the replication level of foot-and-mouth disease viruses and the Sein card viruses, inhibit the expression of structural proteins of the foot-and-mouth disease viruses and the Sein card viruses, has the effect of inhibiting the infection of the foot-and-mouth disease viruses and the Sein card viruses, can be used for preparing medicines or adjuvants for resisting the infection of the foot-and-mouth disease viruses and the Sein card viruses, and is used for inhibiting the replication of the foot-and-mouth disease viruses and the Sein card viruses. However, the invention is not limited to foot-and-mouth disease virus and sai-kava virus, and on the basis that the compound ZPP can inhibit the replication of foot-and-mouth disease virus and sai-kava virus, the compound ZPP can also inhibit the replication of other viruses of the picornaviridae family, and can also be used for inhibiting the replication of other viruses to prepare medicaments or adjuvants for resisting viral infection.

Description

Application of Compound ZPP in the Preparation of Antiviral Drugs

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of a compound ZPP in the preparation of antiviral drugs.

Background technique

Foot and mouth disease virus (FMDV) belongs to the Picornaviridae family and the genus Aphthovirus. Its genome is a single-stranded positive-strand RNA with a length of about 8500 nt. FMDV mainly infects artiodactyls such as pigs, cattle and sheep. Causes acute, hot, high-contact and severe animal infectious diseases that can spread quickly and over long distances. There are more than 70 species of susceptible animals; cause blisters and ulcers on the skin or hairless parts of the oral mucosa, hooves and breasts , leading to reduced or lost productivity. The disease can cause huge economic losses and social and political impacts in animal husbandry. It is listed as a legally notifiable animal disease by the World Organization for Animal Health (WOAH), and it is also a Class I animal disease that my country focuses on prevention. FMDV contains 7 serotypes (A, O, C, SAT1, SAT2, SAT3, and Asia1) and more than 80 subtypes of strains, and there is no effective cross-protection between different serotypes, which makes the prevention and control of FMD more difficult . Currently, immunization is the most effective measure against FMD. However, research on the host immunity and pathogenic mechanism caused by its pathogenic variation still needs a breakthrough. Therefore, it is very important for the screening and research of drugs to inhibit foot-and-mouth disease virus infection.

Senecavirus A (Senecavirus A, SVA) belongs to Picornaviridae (Picornaviridae), a member of the genus Senecavirus (Senecavirus), and is one of the pathogens that cause vesicular disease in pigs. The first SVA strain, SVV-001, was isolated from conventional cell culture in 2002. SVA contains a single-stranded positive-sense RNA genome that encodes a polypeptide that is cleaved into the leader protein L and three precursor proteins, P1, P2, and P3. Subsequently, P1 is cleaved into VP4, VP2, VP3 and VP1 structural proteins, while P2 and P3 are cleaved into 2A, 2B, 2C, 3A, 3B, 3C and 3D nonstructural proteins. SVA can cause vesicular damage to the oral and nasal mucosa of pigs, lethargy, anorexia, lameness, and even acute death of piglets. Because SVA induces vesicular disease similar to FMDV, porcine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV), it is difficult to differentiate clinically.

N-Benzyloxycarbonyl-prolyl-prolinal (Z-pro-prolinal, ZPP) is a small molecular compound, which is an effective and selective inhibitor of prolyl oligopeptidase.

The present invention unexpectedly finds that the compound ZPP can reduce the replication level of FMDV and SVA, inhibit the expression of viral structural proteins, and has the effect of inhibiting virus infection, and can be used to prepare drugs or adjuvants against picornavirus infection, and to inhibit virus infection. copy.

Contents of the invention

The invention provides an application of compound ZPP in the preparation of antiviral infection medicine. Specifically include the following:

In the first aspect, the present invention provides the application of a compound ZPP or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing viral infection. The structural formula of the compound ZPP is shown in the following formula (I):

Preferably, the virus is a Picornaviridae virus.

Preferably, the virus is foot-and-mouth disease virus, Seneca virus.

Preferably, the compound ZPP or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and/or adjuvant to prepare any pharmaceutically acceptable dosage form.

Preferably, the dosage forms include powder injections, capsules, tablets, and suspensions.

In a second aspect, the present invention provides a compound ZPP or a pharmaceutically acceptable salt thereof in the preparation of a drug for the treatment of viral infection, the structural formula of the compound ZPP is shown in the following formula (I):

Preferably, the virus is a Picornaviridae virus.

Preferably, the virus is foot-and-mouth disease virus, Seneca virus.

Preferably, the compound ZPP or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and/or adjuvant to prepare any pharmaceutically acceptable dosage form.

Preferably, the dosage forms include powder injections, capsules, tablets, and suspensions.

The beneficial effects of the present invention are: the present invention unexpectedly finds that the compound ZPP can reduce the replication level of FMDV and SVA, inhibit the expression of viral structural proteins, have the effect of inhibiting virus infection, and can be used to prepare drugs or adjuvants against picornavirus infection. agent to inhibit virus replication.

Description of drawings

The impact result of Fig. 1 compound ZPP on FMDV titer;

The result figure that Fig. 2 compound ZPP inhibits FMDV replication in IBRS-2 cells;

The result figure that Fig. 3 compound ZPP inhibits the expression of FMDV structural protein VP1;

The impact result of Fig. 4 compound ZPP on SVA titer;

Figure 5 Compound ZPP inhibits the results of SVA replication in IBRS-2 cells;

Fig. 6 Compound ZPP inhibits the result figure of SVA structural protein VP2 expression;

Figure 7 Safety of compound ZPP.

Detailed ways

In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments. But the scope of protection of the present invention is not limited to the following examples.

The experiments described in the following examples were licensed for Biosafety and FMD Reference Laboratory Activities:

Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences, in accordance with the requirements of biosafety level 3 laboratory (BSL-3) and biosafety, has been approved by the Biosafety Committee of Lanzhou Veterinary Research Institute, the Experimental Animal Ethics Committee, the Biosafety Committee of Chinese Academy of Agricultural Sciences, Lanzhou Veterinary Research The Experimental Animal Ethics Committee and the Biosafety Committee of Lanzhou Veterinary Research Institute reported to each level, and obtained the permission from the Ministry of Agriculture to carry out research on FMDV and other pathogens and animals, and have been filed with the Ministry of Agriculture and Rural Affairs, meeting the requirements of the national biosafety level.

Sources of experimental cells and viruses described in the following examples:

Cell culture: IBRS-2 cells were derived from porcine (Sus scrofa) animals and BHK-21 cells were preserved in our laboratory; DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibody was placed in a 5 The cultures were carried out in a % CO ₂ incubator (37°C).

Virus source: FMDV strain (O/BY/CHA/2010) and SVA strain (CH/FJ/2017) are preserved in the Foot-and-Mouth Disease and Emerging Epidemiology Team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences and the National Reference Laboratory of Foot-and-Mouth Disease.

Compound ZPP, purchased from Enzo Company, item number PI112;

The experimental methods in the following examples, unless otherwise specified, are conventional methods; the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies.

Example 1 Compound ZPP inhibits the replication of FMDV

1. Preparation of compound-containing ZPP medium

ZPP dry powder was dissolved in DMSO to prepare a stock solution with a concentration of 10 mM. Before the experiment, different doses of the compound ZPP were added to the DMEM medium, so that the concentrations were 10 μM, 50 μM, 100 μM and 150 μM, respectively.

2. Sample preparation of compound ZPP-treated cells

IBRS-2 cells were counted and spread in six-well plates. When the cells grew to 70%-80% confluence, the experimental group treated the cells with different concentrations of ZPP (10 μM, 50 μM, 100 μM and 150 μM) for 4 hours, and then infected the control group. After the cells were treated with DMSO (1%) for 4 h; then inoculated with FMDV (MOI=0.1), 1 h after inoculation, the inoculation liquid was discarded, the cells were washed once with PBS, and the maintenance liquid was supplemented, and the supernatant and cells were collected 12 to 14 h after inoculation. Cells not inoculated with FMDV were used as Mock control; DMSO (without compound ZPP) was used as blank control.

3. FMDV titer determination

Measure the TCID ₅₀ of the supernatant obtained in step 2 above, and perform virus titer analysis.

The measurement steps of TCID ₅₀ are as follows: BHK-21 cells were inoculated into 96-well plates 16 hours in advance, after the cells formed a single layer of cells, the cells were washed 3 times with PBS, and the supernatant collected in step 2 (10 ^-1 to 10 ^-10 times Ratio dilution) was used as the infection well, and two negative control wells were set up. Add 100 μL of virus filtrate or double-diluted virus dilution to the infection well, adsorb at 37°C for 1 hour, and shake gently every 10 minutes to ensure that the virus is evenly adsorbed. After 1 h of adsorption, the supernatant was sucked off, and the plate was gently washed once with PBS. After adding virus maintenance solution, after 48 hours, the cell lesion was observed every 12 hours. After 72 hours, the number of lesion wells was recorded, and the TCID ₅₀ was calculated, measured in parallel three times, and the average value was taken as the final virus titer.

The experimental results are shown in Figure 1. During the culture process, after the cells were treated with the compound ZPP, the TCID ₅₀ of FMDV was significantly reduced in a dose-dependent manner. It was shown that the compound ZPP significantly reduced the titer of FMDV in cells and inhibited the replication of FMDV.

4. Determination of FMDV Replication

Determine the viral RNA copy number in the supernatant obtained in step 2 above, and perform viral RNA replication analysis.

Viral RNA replication assay steps are as follows: Extract the viral RNA in the harvested sample according to the instructions of the viral RNA extraction kit (OMEGA company), use the one-step qPCR kit (TAKARA company) to perform fluorescent quantitative PCR amplification, and detect the RNA-dependent RNA of FMDV Copy number of replicase 3D gene.

qPCR reaction system: a total volume of 20 μL, including 0.4 μL of 10 μM upstream and downstream primers and fluorescently labeled probes, 10 μL of 2×One Step RT-PCR Buffer Ⅲ, 0.4 μL of TaKaRa Ex Taq HS (5U/μL), 0.4 μL of PrimeScript RTEnzyme Mix Ⅱ, 90ng RNA 2μL, RNase Free dH2O 5.6μL. The reaction conditions are: 42°C, 5min, 95°C, 10s, 1Cycle; 95°C, 5s, 60°C, 20s, 40cycles.

The primer and probe sequences of qPCR are: upstream primer 5'-ACTGGGTTTTACAAACCTGTGA-3', downstream primer 5'-GCGAGTCCTGCCACGGA-3', fluorescent labeling probe 5'-FAM-TCCTTTGCACGCCGTGGGAC-TAMRA-3'.

The results are shown in Figure 2. During the culture of IBRS-2 cells, after the compound ZPP was added to treat the cells, the RNA replication of FMDV was significantly reduced in a dose-dependent manner. It was shown that the compound ZPP significantly inhibited the RNA replication of FMDV. 5. Expression of FMDV structural protein VP1

The expression of FMDV structural protein VP1 in the cell sample was measured, and the expression level of viral structural protein was analyzed.

The analysis steps of virus structural protein expression level are as follows: after counting IBRS-2 cells, spread them in a 35mm culture dish, and when the cells grow to 80% confluence, the experimental group treats the cells with different concentrations of ZPP (50 μM and 100 μM) for 4 hours, and then infects the cells. The control group treated the cells with DMSO (1%) for 4 hours; then inoculated with FMDV (MOI=0.1), discarded the inoculation liquid 1 hour after inoculation, washed the cells once with PBS, supplemented with maintenance solution, and harvested the cells 12 to 14 hours after inoculation. Cells not inoculated with FMDV were used as Mock control; DMSO (without compound ZPP) was used as blank control. The total protein was extracted, and the Western-blotting experiment was performed to detect the expression difference of FMDV VP1 protein.

The results are shown in Figure 3. During the culture of IBRS-2 cells, the expression of FMDV structural protein VP1 was significantly reduced in a dose-dependent manner after the compound ZPP was added to treat the cells. It shows that the compound ZPP significantly inhibits the expression of FMDV structural protein VP1, thereby inhibiting the replication and infection of FMDV.

Example 2 Compound ZPP inhibits the replication of SVA

1. Preparation of compound-containing ZPP medium

ZPP dry powder was dissolved in DMSO to prepare a stock solution with a concentration of 10 mM. Before the experiment, different doses of the compound ZPP were added to the DMEM medium, so that the concentrations were 10 μM, 50 μM, 100 μM and 150 μM, respectively.

2. Sample preparation of compound ZPP-treated cells

IBRS-2 cells were counted and spread in six-well plates. When the cells grew to 70%-80% confluence, the experimental group treated the cells with different concentrations of ZPP (10 μM, 50 μM, 100 μM and 150 μM) for 4 hours, and then infected the control group. Treat the cells with DMSO (1%) for 4 hours; then inoculate SVA virus (MOI=0.1), discard the inoculation liquid 1 hour after inoculation, wash the cells with PBS once, supplement the maintenance solution, and collect the supernatant and cells respectively 12-14 hours after inoculation . Cell wells not inoculated with SVA were used as Mock control; DMSO (without compound ZPP) was used as blank control.

3. SVA titer determination

Measure the TCID ₅₀ of the supernatant obtained in step 2 above, and perform virus titer analysis.

The measurement steps of TCID ₅₀ are as follows: Inoculate IBRS-2 cells in 96-well plates 16 hours in advance, wash the cells with PBS three times after the cells form a monolayer, and inoculate the supernatant collected in step 2 (10 ^-1 to 10 ^-10 times Ratio dilution) was used as the infection well, and two negative control wells were set up. Add 100 μL of virus filtrate or double-diluted virus dilution to the infection well, adsorb at 37°C for 1 hour, and shake gently every 10 minutes to ensure that the virus is evenly adsorbed. After 1 h of adsorption, the supernatant was sucked off, and the plate was gently washed once with PBS. After adding virus maintenance solution, after 48 hours, the cell lesion was observed every 12 hours. After 72 hours, the number of lesion wells was recorded, and the TCID ₅₀ was calculated, measured in parallel three times, and the average value was taken as the final virus titer.

The experimental results are shown in Figure 4. During the culture process, after the IBRS-2 cells were treated with the compound ZPP, the TCID ₅₀ of SVA decreased significantly in a dose-dependent manner. It was shown that the compound ZPP significantly reduced the titer of SVA in cells and inhibited the replication of SVA.

4. Determination of SVA Replication

Determine the viral RNA copy number in the supernatant obtained in step 2 above, and perform viral RNA replication analysis.

The virus RNA replication assay steps are as follows: extract the viral RNA in the harvested sample according to the instructions of the viral RNA extraction kit (OMEGA company), use the one-step qPCR kit (TAKARA company) to perform fluorescent quantitative PCR amplification, and detect the RNA-dependent RNA of SVA Copy number of replicase 3D gene.

qPCR reaction system: a total volume of 20 μL, including 0.4 μL of 10 μM upstream and downstream primers and fluorescently labeled probes, 10 μL of 2×One Step RT-PCR Buffer Ⅲ, 0.4 μL of TaKaRa Ex Taq HS (5U/μL), 0.4 μL of PrimeScript RTEnzyme Mix Ⅱ, 90ng RNA 2μL, RNase Free dH2O 5.6μL. The reaction conditions are: 42°C, 5min, 95°C, 10s, 1Cycle; 95°C, 5s, 60°C, 20s, 40cycles.

The primers and probe sequences of qPCR are: upstream primer 5'-GCCAACGTCCCTATCAACC-3', downstream primer 5'-CTAATGGCGTAGGGCAAACC-3', fluorescent labeling probe 5'-FAM-AGCAATCCTGGGCATCCCTGGA-TAMRA-3'.

The results are shown in Figure 5. During the culture of IBRS-2 cells, after the compound ZPP was added to treat the cells, the RNA replication of SVA was significantly reduced in a dose-dependent manner. It was shown that the compound ZPP significantly inhibited the RNA replication of SVA.

5. Expression of SVA structural protein VP2

The expression of the SVA structural protein VP2 in the cell sample was measured, and the expression level of the viral structural protein was analyzed.

The analysis steps of virus structural protein expression level are as follows: after counting IBRS-2 cells, spread them in a 35mm culture dish, and when the cells grow to 80% confluence, the experimental group treats the cells with different concentrations of ZPP (50 μM and 100 μM) for 4 hours, and then infects the cells. The control group treated the cells with DMSO (1%) for 4 hours; then inoculated with SVA (MOI=0.1), discarded the inoculation solution 1 hour after inoculation, washed the cells once with PBS, supplemented with maintenance solution, and harvested the cells 12 to 14 hours after inoculation. Cells not inoculated with SVA were used as Mock control; DMSO (without compound ZPP) was used as blank control. The total protein was extracted, and the Western-blotting experiment was performed to detect the expression difference of SVAVP2 protein.

The results are shown in Figure 6. During the culture of IBRS-2 cells, the expression of SVA structural protein VP2 was significantly reduced in a dose-dependent manner after the compound ZPP was added to treat the cells. It was shown that the compound ZPP significantly inhibited the expression of SVA structural protein VP2, thereby inhibiting the replication and infection of SVA.

The cytotoxicity of embodiment 3 compound ZPP

The toxicity of small molecular compound ZPP to cells was detected by CCK-8 assay. Prepare IBRS-2 cells (2×10 ⁵ /well) in a 96-well plate with DMEM+10% FBS medium, culture overnight, discard the cell culture medium, and add different concentrations of ZPP (1 μM, 2 μM, 5 μM) to the wells , 8 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM), and set blank wells (only containing cell culture medium), control wells (containing cells and medium), and incubated the culture plate in the incubator for 24 hours, and then poured Add 10 μL of CCK-8 solution to each well, incubate the culture plate in the incubator for 1-4 hours, and mix gently on the shaker before reading the plate. Then the absorbance at 450nm was measured with a microplate reader, and the cell viability was calculated.

The results are shown in Figure 7, the compound ZPP has little toxicity to cells, and even when the dose reaches 100 μM, the cell activity can still reach more than 90%, with low cytotoxicity and good safety.

In summary, the embodiment of the present invention takes the host cell IBRS-2 as an example, and the research proves that the compound ZPP can inhibit the replication of FMDV and SVA in the host cell IBRS-2, indicating that the compound ZPP of the present invention can inhibit the replication of FMDV and SVA. The replication of SVA can be used to prepare medicines or adjuvants against Picornaviridae virus infection.

It should be noted that Picornaviridae mainly includes the following five genera: Enterovirus, Rhinovirus, Cardiovirus, Oral Virus, and Seneca Virus, while FMDV belongs to Oral Virus, and SVA belongs to Serene Virus. Cavirus. Due to the high degree of conservation of viral structural proteins among the various genera of Picornaviridae. Although the present invention takes FMDV and SVA of picornaviridae virus as an example, it has been proved that compound ZPP can inhibit the replication of FMDV and SVA, but the present invention is not limited to FMDV and SVA, and compound ZPP described in the present invention can inhibit FMDV On the basis of the replication of SVA virus, it can also inhibit the replication of other viruses of Picornaviridae, and can also be used to inhibit the replication of other viruses, prepare antiviral infection drugs, or adjuvants.

Claims (4)

1. The application of a compound ZPP in preparing a medicament for preventing virus infection is provided, wherein the structural formula of the compound ZPP is shown as the following formula (I):

the virus is foot-and-mouth disease virus or saikaki virus.

2. The application of a compound ZPP in preparing a medicament for treating virus infection is provided, wherein the structural formula of the compound ZPP is shown as the following formula (I):

the virus is foot-and-mouth disease virus or saikaki virus.

3. The use according to claim 1 or 2, wherein the compound ZPP is formulated into any pharmaceutically acceptable dosage form by adding pharmaceutically acceptable carriers and/or excipients.

4. The use according to claim 3, wherein the dosage form comprises a powder for injection, a capsule, a tablet, a suspension.