CN115778946B - Application of compound ZPP in preparation of antiviral drugs - Google Patents
Application of compound ZPP in preparation of antiviral drugs Download PDFInfo
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- CN115778946B CN115778946B CN202211375256.1A CN202211375256A CN115778946B CN 115778946 B CN115778946 B CN 115778946B CN 202211375256 A CN202211375256 A CN 202211375256A CN 115778946 B CN115778946 B CN 115778946B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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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
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a compound ZPP in preparation of antiviral drugs.
Background
Foot and mouth disease virus (Foot and mouth disease virus, FMDV) belongs to Picornaviridae (Picornaviridae) and Aphthovirus (Aphthovirus), the genome is single-strand positive strand RNA, about 8500nt, FMDV mainly infects artiodactyls such as pigs, cows and sheep to cause acute, febrile, highly contagious and rapidly and remotely transmissible animal virulent infectious diseases, and more than 70 susceptible animals; causes the skin or hairless parts of the mouth mucosa, hooves, breasts and the like to have the symptoms of blisters and ulcers, and leads to the reduction or loss of productivity. The disease can cause huge economic loss and social political influence of animal husbandry, is an animal epidemic disease which is listed as legal report by the world animal health organization (WOAH), and is also a class I animal epidemic disease which is important to prevent in China. FMDV comprises strains of more than 7 serotypes (A, O, C, SAT, SAT2, SAT3 and Asia 1), 80 subtypes, there is no effective cross protection between the different serotypes, which makes control of FMD more difficult. Currently, vaccination is the most effective measure against FMD. However, research on host immunity, pathogenesis and the like caused by pathogenic variation is still to be broken through. Therefore, the method is of great importance to screening and research of drugs for inhibiting foot-and-mouth disease virus infection.
Senecavirus A (SVA) belongs to Picornaviridae, members of Senecavirus, and is one of the causative agents of swine vesicular disease. The first SVA strain, SVV-001, was isolated from conventional cell culture in 2002. SVA comprises a single-stranded positive sense RNA genome that encodes a polypeptide that is cleaved into 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, somnolence, anorexia, lameness, and even acute death of piglets to the oral and nasal mucosa of the pig. Since SVA induces vesicular disease similar to FMDV, porcine vesicular disease virus (SVDV) and Vesicular Stomatitis Virus (VSV), it is clinically difficult to distinguish.
N-Benzyloxycarbon-prolyl-prolinal (ZPP) is a small molecule compound that is a potent and selective inhibitor of prolyl oligopeptidase.
The invention surprisingly discovers that the compound ZPP can reduce the replication level of FMDV and SVA, inhibit the expression of virus structural proteins, has the effect of inhibiting virus infection, and can be used for preparing medicaments or adjuvants for resisting picornavirus infection and inhibiting the replication of viruses.
Disclosure of Invention
The invention provides an application of a compound ZPP in preparing an antiviral infection medicine. The method specifically comprises the following steps:
in a first aspect, the invention provides an application of a compound ZPP or pharmaceutically acceptable salt thereof in preparing a medicament for preventing virus infection, wherein the structural formula of the compound ZPP is shown as the following formula (I):
preferably, the virus is a picornaviridae virus.
Preferably, the virus is a foot-and-mouth disease virus, a sai card virus.
Preferably, the compound ZPP or pharmaceutically acceptable salt thereof is added with pharmaceutically acceptable carriers and/or auxiliary materials to prepare any pharmaceutically acceptable dosage form.
Preferably, the dosage forms comprise powder injection, capsules, tablets and suspensions.
In a second aspect, the invention provides an application of a compound ZPP or pharmaceutically acceptable salt thereof in preparing a medicament for treating viral infection, wherein 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 a foot-and-mouth disease virus, a sai card virus.
Preferably, the compound ZPP or pharmaceutically acceptable salt thereof is added with pharmaceutically acceptable carriers and/or auxiliary materials to prepare any pharmaceutically acceptable dosage form.
Preferably, the dosage forms comprise powder injection, capsules, tablets and suspensions.
The beneficial effects of the invention are as follows: the invention surprisingly discovers that the compound ZPP can reduce the replication level of FMDV and SVA, inhibit the expression of virus structural proteins, has the effect of inhibiting virus infection, and can be used for preparing medicaments or adjuvants for resisting picornavirus infection and inhibiting the replication of viruses.
Drawings
The effect of ZPP compound on FMDV titer results of fig. 1;
FIG. 2 is a graph of the results of inhibition of FMDV replication by ZPP in IBRS-2 cells;
FIG. 3 is a graph showing the results of inhibiting the expression of FMDV structural protein VP1 by ZPP;
FIG. 4 results of the effect of compound ZPP on SVA titer;
FIG. 5 is a graph of the results of inhibition of SVA replication by ZPP in IBRS-2 cells;
FIG. 6 is a graph showing the results of the inhibition of VP2 expression by the structural SVA protein by ZPP;
figure 7 safety of the ZPP compound.
Detailed Description
The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand. The scope of the invention is not limited to the examples described below.
The experiments described in the examples below obtained biosafety permissions and foot-and-mouth disease reference laboratory activity permissions:
the national institute of agricultural science, the biological safety committee of the national institute of veterinary science, the laboratory animal ethics committee of the national institutes of agricultural science, the biological safety committee of the national institutes of agricultural science, the laboratory animal ethics committee of the national institute of veterinary science, the biological safety committee of the national institute of veterinary science, the method obtains the research permission of the agricultural department on developing pathogens such as FMDV and animals, and has been recorded in the agricultural rural department, thereby meeting the requirements of the national biosafety level.
Experimental cells, viral sources as described in the examples below:
cell culture: IBRS-2 cells were derived from animals of the porcine family (Sus scrofa) and BHK-21 cells were stored in the laboratory; the culture medium containing 10% Fetal Bovine Serum (FBS) and 1% diabody was placed in DMEM medium containing 5% CO 2 Culturing was performed in an incubator (37 ℃).
Viral origin: FMDV strains (O/BY/CHA/2010) and SVA strains (CH/FJ/2017) were saved in the national institute of veterinary science, department of agriculture, academy of china, foot-and-mouth disease and new disease epidemiology team and national foot-and-mouth disease reference laboratory.
Compound ZPP, available from enco company under the product number PI112;
the experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent company.
EXAMPLE 1 Compound ZPP inhibits replication of FMDV
1. Preparation of Compound ZPP-containing Medium
The ZPP dry powder was dissolved in DMSO to prepare a stock solution at a concentration of 10 mM. Before the experiment, the ZPP compounds were added in different doses to the DMEM medium at concentrations of 10. Mu.M, 50. Mu.M, 100. Mu.M, 150. Mu.M, etc.
2. Sample preparation of Compound ZPP-treated cells
IBRS-2 cells were counted and plated in six well plates, after treatment of the experimental group with ZPP (10 μm, 50 μm, 100 μm and 150 μm) at different concentrations for 4h and after treatment of the control group with DMSO (1%) for 4h when the cells were grown to 70% -80% confluency; FMDV (moi=0.1) was then inoculated, the inoculation liquid was discarded 1h after inoculation, the cells were washed once with PBS, the maintenance liquid was supplemented, and the supernatant and cells were harvested 12-14 h after inoculation, respectively. Taking cells which are not inoculated with FMDV as a Mock control; DMSO (without compound ZPP) was used as a blank.
Fmdv titer assay
Determining TCID of the supernatant obtained in the above step 2 50 Virus titer analysis was performed.
TCID 50 The measurement steps of (a) are as follows: BHK-21 cells were seeded into 96-well plates 16h in advance, after the cells formed a monolayer of cells, the cells were washed 3 times with PBS, and the supernatant collected in step 2 was seeded (10 -1 ～10 -10 Multiple dilution) was used as an infection well, and two additional rows of negative control wells were provided. 100 mu L of virus filtrate or dilution of the virus dilution with a multiple ratio is added into the infection hole, and the virus is adsorbed for 1h at 37 ℃ and is gently shaken every 10min, so that the virus adsorption is ensured to be uniform. After 1h of adsorption, the supernatant was aspirated and the plate was gently washed 1 time with PBS. Adding virus maintenance solution, observing cytopathic effect every 12h after 48h, recording pathological hole number after 72h, and calculating TCID 50 Three replicates were assayed and the average was taken as the final viral titer.
The experimental results are shown in FIG. 1, in which FMDV TCID was obtained after the cells were treated with ZPP compound during the culture 50 Significantly reduced and dose dependent. The compound ZPP was shown to significantly reduce intracellular FMDV titers, inhibiting FMDV replication.
Determination of FMDV replication
The viral RNA copy number in the supernatant obtained in step 2 above was measured, and viral RNA replication analysis was performed.
The viral RNA replication assay steps are as follows: viral RNA in the harvested samples was extracted according to the instructions of the viral RNA extraction kit (OMEGA Co.) and fluorescent quantitative PCR amplification was performed using the one-step qPCR kit (TAKARA Co.) to detect the copy number of the RNA-dependent RNA replicase 3D gene of FMDV.
qPCR reaction system: the total volume was 20. Mu.L, including 10. Mu.M of each of the upstream and downstream primers and the fluorescent-labeled probe, 0.4. Mu.L, 10. Mu.L of 2 XOne Step RT-PCR Buffer III, 0.4. Mu.L of TaKaRa Ex Taq HS (5U/. Mu.L), 0.4. Mu.L of PrimeScript RT Enzyme Mix II, 90ng of RNA 2. Mu.L, and 5.6. Mu.L of RNase Free dH 2O. The reaction conditions are as follows: 42 ℃, 5min,95 ℃, 10s,1cycle;95 ℃, 5s,60 ℃, 20s,40cycles.
Wherein the primer and probe sequences of qPCR are: upstream primer 5'-ACTGGGTTTTACAAACCTGTGA-3', downstream primer 5'-GCGAGTCCTGCCACGGA-3', fluorescent labeled probe 5'-FAM-TCCTTTGCACGCCGTGGGAC-TAMRA-3'.
The results are shown in figure 2, where the RNA replication of FMDV was significantly reduced and dose dependent after addition of compound ZPP to the treated cells during IBRS-2 cell culture. The compound ZPP was shown to significantly inhibit RNA replication of FMDV. Expression of FMDV structural protein VP1
And (3) measuring the expression of the FMDV structural protein VP1 in the cell sample, and carrying out virus structural protein expression level analysis.
The viral structural protein expression level analysis steps are as follows: IBRS-2 cells were counted and spread in 35mm dishes, and after treatment of the cells with ZPP (50 μm and 100 μm) at different concentrations for 4h in the experimental group and DMSO (1%) in the infected control group for 4h when the cells were grown to 80% confluency; then, FMDV (MOI=0.1) was inoculated, the inoculated liquid was discarded 1h after inoculation, the cells were washed once with PBS, the maintenance liquid was supplemented, and the cells were harvested 12-14 h after inoculation, respectively. Taking cells which are not inoculated with FMDV as a Mock control; DMSO (without compound ZPP) was used as a blank. Extracting total protein, and carrying out Western-blotting experiment to detect the expression difference of FMDV VP1 protein.
The results are shown in FIG. 3, in which the expression of FMDV structural protein VP1 was significantly reduced and dose-dependent after the addition of compound ZPP to the cells during the cultivation of IBRS-2 cells. The compound ZPP is shown to significantly inhibit the expression of FMDV structural protein VP1, thereby inhibiting replication and infection of FMDV.
EXAMPLE 2 Compound ZPP inhibits replication of SVA
1. Preparation of Compound ZPP-containing Medium
The ZPP dry powder was dissolved in DMSO to prepare a stock solution at a concentration of 10 mM. Before the experiment, the ZPP compounds were added in different doses to the DMEM medium at concentrations of 10. Mu.M, 50. Mu.M, 100. Mu.M, 150. Mu.M, etc.
2. Sample preparation of Compound ZPP-treated cells
IBRS-2 cells were counted and plated in six well plates, after treatment of the experimental group with ZPP (10 μm, 50 μm, 100 μm and 150 μm) at different concentrations for 4h and after treatment of the control group with DMSO (1%) for 4h when the cells were grown to 70% -80% confluency; then SVA virus (MOI=0.1) was inoculated, the inoculation liquid was discarded 1h after inoculation, the cells were washed once with PBS, the maintenance liquid was supplemented, and the supernatant and the cells were collected 12-14 h after inoculation, respectively. Taking a cell hole which is not inoculated with SVA as a Mock control; DMSO (without compound ZPP) was used as a blank.
SVA titre assay
Determining TCID of the supernatant obtained in the above step 2 50 Virus titer analysis was performed.
TCID 50 The measurement steps of (a) are as follows: IBRS-2 cells were seeded 16h in advance in 96-well plates, after cell formation into monolayer cells, the cells were washed 3 times with PBS, and the supernatant collected in step 2 was seeded (10 -1 ～10 -10 Multiple dilution) was used as an infection well, and two additional rows of negative control wells were provided. 100 mu L of virus filtrate or dilution of the virus dilution with a multiple ratio is added into the infection hole, and the virus is adsorbed for 1h at 37 ℃ and is gently shaken every 10min, so that the virus adsorption is ensured to be uniform. After 1h of adsorption, the supernatant was aspirated and the plate was gently washed 1 time with PBS. Adding virus maintenance solution, observing cytopathic effect every 12h after 48h, recording pathological hole number after 72h, and calculating TCID 50 Three replicates were assayed and the average was taken as the final viral titer.
The experimental results are shown in FIG. 4, in which the IBRS-2 cells were treated with ZPP compound during the culture process, and the TCID of SVA 50 Significantly reduced and dose dependent. The compound ZPP was shown to significantly reduce the titer of intracellular SVA, inhibiting the replication of SVA.
Assay for SVA replication
The viral RNA copy number in the supernatant obtained in step 2 above was measured, and viral RNA replication analysis was performed.
The viral RNA replication assay steps are as follows: viral RNA in the harvested samples was extracted according to the protocol of the viral RNA extraction kit (OMEGA Co.) and fluorescent quantitative PCR amplification was performed using the one-step qPCR kit (TAKARA Co.) to detect the copy number of the RNA-dependent RNA replicase 3D gene of SVA.
qPCR reaction system: the total volume was 20. Mu.L, including 10. Mu.M of each of the upstream and downstream primers and the fluorescent-labeled probe, 0.4. Mu.L, 10. Mu.L of 2 XOne Step RT-PCR Buffer III, 0.4. Mu.L of TaKaRa Ex Taq HS (5U/. Mu.L), 0.4. Mu.L of PrimeScript RT Enzyme Mix II, 90ng of RNA 2. Mu.L, and 5.6. Mu.L of RNase Free dH 2O. The reaction conditions are as follows: 42 ℃, 5min,95 ℃, 10s,1cycle;95 ℃, 5s,60 ℃, 20s,40cycles.
Wherein the primer and probe sequences of qPCR are: upstream primer 5'-GCCAACGTCCCTTATCAACC-3', downstream primer 5'-CTAATGGCGTAGGGCAAACC-3', fluorescent labeled probe 5'-FAM-AGCAATCCTGGGCATCCCTGGA-TAMRA-3'.
The results are shown in FIG. 5, where RNA replication of SVA was significantly reduced and dose dependent after addition of compound ZPP to the cells during IBRS-2 cell culture. The compound ZPP was shown to significantly inhibit RNA replication of SVA.
Expression of SVA structural protein VP2
And measuring the expression of the SVA structural protein VP2 in the cell sample, and carrying out virus structural protein expression level analysis.
The viral structural protein expression level analysis steps are as follows: IBRS-2 cells were counted and spread in 35mm dishes, and after treatment of the cells with ZPP (50 μm and 100 μm) at different concentrations for 4h in the experimental group and DMSO (1%) in the infected control group for 4h when the cells were grown to 80% confluency; then SVA (MOI=0.1) was inoculated, the inoculation liquid was discarded 1h after inoculation, the cells were washed once with PBS, the maintenance liquid was supplemented, and the cells were harvested 12-14 h after inoculation, respectively. Taking cells which are not inoculated with SVA as a Mock control; DMSO (without compound ZPP) was used as a blank. Extracting total protein, and carrying out Western-blotting experiment to detect the expression difference of SVA VP2 protein.
The results are shown in FIG. 6, where SVA structural protein VP2 expression was significantly reduced and dose dependent after the addition of compound ZPP to the cells during IBRS-2 cell culture. The compound ZPP was shown to significantly inhibit the expression of the structural protein VP2 of SVA, thereby inhibiting replication and infection of SVA.
EXAMPLE 3 cytotoxicity of Compound ZPP
The toxicity of the small molecule compound ZPP to cells was tested using the CCK-8 assay. IBRS-2 cells (2X 10) were prepared in 96-well plates with DMEM+10% FBS medium 5 Well), cell culture medium was discarded, ZPP (1 μm, 2 μm, 5 μm, 8 μm, 10 μm, 20 μm, 50 μm, 80 μm, 100 μm) was added to the wells while blank wells (containing cell culture medium alone), control wells (containing cells and medium) were set, after incubation of the plates in the incubator for 24h, 10 μl of CCK-8 solution was added to each well of the cell plates, the plates were incubated in the incubator for 1-4 h, and gentle mixing on a shaker was possible before reading the plates. The absorbance at 450nm was then measured with an enzyme-labeled instrument to calculate the cell viability.
As shown in FIG. 7, the compound ZPP has small toxicity to cells, and even when the dosage reaches 100 mu M, the cell activity can still reach more than 90%, so that the compound ZPP has small cytotoxicity and good safety.
In summary, the embodiment of the invention takes host cell IBRS-2 as an example, and researches prove that the compound ZPP can inhibit the replication of FMDV and SVA in the host cell IBRS-2, and the compound ZPP disclosed by the invention can inhibit the replication of FMDV and SVA and can be used for preparing medicaments or adjuvants for resisting virus infection of picornaviridae.
The picornaviridae mainly includes the following five genera: enteroviruses, rhinoviruses, cardioviruses, aphthoviruses, and saikovirus, whereas FMDV belongs to aphthoviruses, SVA belongs to saikovirus. Due to the high degree of conservation of viral structural proteins between the various genera of the picornaviridae. Although the invention takes FMDV and SVA of picornaviridae virus as examples, the compound ZPP can inhibit the replication of FMDV and SVA, but the invention is not limited to FMDV and SVA, and on the basis that the compound ZPP can inhibit the replication of FMDV and SVA virus, the compound ZPP can also inhibit the replication of other viruses of picornaviridae virus, and can also be used for inhibiting the replication of other viruses, and preparing antiviral infected medicines 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.
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| CA1335079C (en) * | 1988-07-25 | 1995-04-04 | Kiyoshi Takatsuki | Preventive and curative agents for acquired immune deficiency syndrome (aids) |
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| CN109758447B (en) * | 2019-03-11 | 2021-06-08 | 中国农业科学院兰州兽医研究所 | Application of teriflunomide in preparation of drugs for preventing foot-and-mouth disease virus infection |
| CN109820852B (en) * | 2019-03-11 | 2021-02-19 | 中国农业科学院兰州兽医研究所 | Application of nimodipine in preparation of drugs for preventing foot-and-mouth disease virus infection |
| CN114177176B (en) * | 2021-12-04 | 2022-07-12 | 中国农业科学院兰州兽医研究所 | Application of compound cyproheptadine hydrochloride in preparation of drug for preventing or treating African swine fever |
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