KR101805394B1 - Pharmaceutical composition for preventing or treating Calicivirus infection - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating calcivirus infection and, more specifically, to a pharmaceutical composition for preventing or treating calcivirus infection, capable of preventing or treating infection by various calcivirus family viruses by comprising a PGE2 inhibitor, specifically a COX-1 selective inhibitor, a COX-2 selective inhibitor, COX-1 and COX-2 nonselective inhibitors and a CREB activation inhibitor.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating calicivirus infection,

The present invention relates to a pharmaceutical composition for preventing or treating calicivirus infection.

Caliciviruses are all viruses belonging to the Caliciviridae family. These viruses have a linear positive sense single strand in the capsid layer of 27 to 40 nm in size. -stranded RNA genome. Caliciviridae has five genera of viruses (Lagovirus, Nebovirus, Norovirus, Sapvorius and Vesivirus), Atlantic salmon calicivirus , Bavovirus, Nacovirus, Recovirus and Valovirus remain as undifferentiated species.

Sapovirus is a virus belonging to genus Sapovirus in calicivirus, which causes diarrhea in humans and pigs.

Norovirus is a virus belonging to the genus Norovirus in calicivirus and is the causative agent of diarrhea in humans, cows, pigs and dogs. In particular, norovirus accounts for 95% of nonbacterial food poisoning Cause. Mouse murine noroviruses cause systemic infections in immunodeficient mice.

Bovine neovirus is a virus belonging to the genus Nebovirus in calicivirus, which causes diarrhea in calves.

Feline calicivirus, a virus belonging to the genus Lagovirus, is a causative agent of upper and lower respiratory infections in cats.

As such, viruses in caliciviruses cause a variety of diseases in humans and animals, and these pathogens are causing enormous damage both socially and economically.

However, there is no treatment, vaccine development or industrialization for viruses in calicivirus.

This is because the virus in the calicivirus is very difficult to culture and much of the life cycle of the calicivirus is unclear. For this reason, only the pig swab virus Cowden strain has been reported as a virus in the established cultured calicivirus, and only a small number of researchers are using pig swab virus Cowden strain.

In addition, some viruses, the production of cAMP / PKA, PI3K / Akt and MEK / activate the ERK signaling pathway cycloalkyl oxygenates Kinases 1 and 2 (cyclooxygenases 1 and 2, COX-1 and COX-2) by expressing the protein PGE ₂ Is increased. In particular, COX-1, COX-2 or PGE ₂ is known to inhibit or increase virus proliferation. In particular, Vesicular Stomatitis Virus increases COX-2 protein expression and increases PGE ₂ production. It is known that PGE ₂ promotes the proliferation of Vesicular Stomatitis Virus by reducing the production of nitric oxide (NO), which has antiviral efficacy.

However, it is not known whether the virus belonging to the calicivirus depends on the above mechanism.

The inventors of the present invention have for the first time discovered that when calicivirus infects cells, COX-1 and COX-2 of cells are activated to produce PGE ₂ and inhibit NO production, thus completing the present invention.

Published Japanese Patent Application No. 10-2015-0109763 (Feb.

The present invention is to provide a pharmaceutical composition capable of preventing or treating a calicivirus infection through a mechanism of calicivirus infection.

The present invention provides a pharmaceutical composition capable of preventing or treating infectious diseases caused by human sandwich virus, porcine sandovirus, human norovirus, porcine norovirus, sonorovirus, gonorovirus, sonnevirus and cats calicivirus will be.

The present invention is to provide a pharmaceutical composition capable of reducing the number of genomes of viruses in a subject infected with calicivirus.

The present invention is to provide a pharmaceutical composition capable of reducing the amount of protein of virus in a subject infected with calicivirus.

The present invention is to provide a pharmaceutical composition capable of reducing the activity of calicivirus.

1. A pharmaceutical composition for preventing or treating a calicivirus infection comprising a PGE ₂ inhibitor.

2. The composition of claim 1 wherein said PGE ₂ inhibitor is at least one selected from the group consisting of a COX-1 selective inhibitor, a COX-2 selective inhibitor, a COX-1 and a COX- A pharmaceutical composition for preventing or treating infectious diseases.

3. The pharmaceutical composition according to item 1 above, wherein the PGE ₂ inhibitor is a non-steroidal anti-inflammatory analgesic, for the prevention or treatment of calicivirus infection.

4. The method of claim 2, wherein said COX-1 selective inhibitor is at least one selected from the group consisting of SC-560, mofezolac, P6, TFAP, resveratrol and COX- ≪ / RTI >

5. The method of claim 2, wherein the COX-2 selective inhibitor is at least one selected from the group consisting of nimesulide, SC-58125, SC-236, NS- 398, COX-2 siRNA and celecoxib. A pharmaceutical composition for preventing or treating infectious diseases.

6. The method of claim 2 wherein said COX-1 and COX-2 nonselective inhibitors are selected from the group consisting of indomethacin, ibuprofen, diclofenac sodium, prubipropene, aspirin, prorostilbene, rofecoxib and naproxen Lt; RTI ID = 0.0 > (I) < / RTI >

7. The pharmaceutical composition according to 2 above, wherein said CREB activation inhibitor is at least one selected from the group consisting of MLD-12330A, PKI, U0126 and bortmannin.

8. The method of 1 above, wherein said calicivirus is selected from the group consisting of human serovirus, porcine sandovirus, human norovirus, porcine norovirus, sonorovirus, canovirus, A pharmaceutical composition for the prevention or treatment of calicivirus infection, which is any one.

The pharmaceutical composition according to the present invention can prevent or treat calicivirus infection.

The pharmaceutical composition according to the present invention can prevent or treat infectious diseases caused by human sapovirus, porcine sapovirus, human norovirus, porcine norovirus, sonorovirus, canovirus, sonevovirus, and feline calicivirus.

The pharmaceutical composition according to the present invention can reduce the number of viruses in a subject infected with calicivirus.

The pharmaceutical composition according to the present invention can reduce the genome number of viruses in a subject infected with calicivirus.

The pharmaceutical composition according to the present invention can reduce the amount of protein of virus in a subject infected with calicivirus.

The pharmaceutical composition according to the present invention can reduce the activity of calicivirus.

1 schematically shows a control scheme for controlling the propagation of sandworms by a COX inhibitor.
Figure 2 shows the production of COX-1, COX-2 and PGE ₂ upon infection with sandovirus.
Figure 3 shows that COX-1 and COX-2 inhibition controls virus proliferation.
Figure 4 shows that reduction of sandovirus proliferative potency by COX-1 and COX-2 inhibitors is restored by the addition of PGE ₂ .
Figure 5 shows that bile acid, a cell culture additive, does not affect COX-2 expression upon sapovirus infection.
FIG. 6 shows that the protein-RNA dependent RNA polymerase (PROSOL) and the viral protein genome-linked (VPg) protein increase COX-2 expression .
FIG. 7 is a graph showing the results of immunohistochemical analysis of cyclic adenosine-3 ', 5'-monophosphate (cAMP) / A-protein kinase A (PKA), phosphoinositide 3-kinase , PI3K) / protein kinase B (Akt) and mitogen-activated protein kinase kinase (MAPKK) / extracellular signal-regulated kinase (ERK) this type shows that sikindaneun to phosphorylate BMP response element binding protein (cAMP response element-binding protein, CREB) transcription factor activation of COX-2 and PGE _2.
Figure 8 shows that PGE ₂ inhibits the antiviral efficacy of nitric oxide (NO).

The present invention relates to a pharmaceutical composition for the prevention or treatment of calicivirus infection, which comprises a PGE ₂ inhibitor, more particularly a COX-1 selective inhibitor, a COX-2 selective inhibitor, a COX-1 and a COX- The present invention also relates to a pharmaceutical composition for preventing or treating calicivirus infection, which can prevent or treat infectious diseases caused by various viruses of calicivirus.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating a calicivirus infection comprising a PGE ₂ inhibitor.

In the present invention, calicivirus means all virus belonging to calicivirus.

Caliciviridae has five genera of viruses (Lagovirus, Nebovirus, Norovirus, Sapvorius and Vesivirus), Atlantic salmon calicivirus , Bavovirus, Nacovirus, Recovirus and Valovirus remain as undifferentiated species.

The calicivirus infection according to the present invention means an infection caused by at least one of the virus belonging to the calicivirus. For example, an infection caused by at least one of human sapovirus infection, porcine sapovirus infection, human norovirus infection, porcine norovirus infection, sonorovirus infection, gonorrho virus infection, sonnevovirus infection and cat calicivirus infection it means.

In the present invention, treatment is a concept including inhibition, elimination, alleviation, alleviation, improvement and / or prevention of a symptom or a condition due to a disease or a disease.

We have found that calicivirus activates cyclooxygenases 1 and 2 (cyclooxygenases 1 and 2, COX-1 and COX-2) in host cells to produce PGE ₂ . In particular, it expresses COX-2 by phosphorylating the CRE Binding Protein (CREB), a transcription factor that binds to cAMP Responseve Element (CRE) through PKA, MEK1 / 2, ERK, PI3K and AKt signaling system. PGE ₂ expressed by caliciviruses reduces the production of nitric oxide (NO), which has antiviral efficacy, and promotes the proliferation of calicivirus.

COX-1 and COX-2 are also called PGE ₂ synthetases, and arachidonic acid is made into PGE ₂ through several steps. The resulting PGE ₂ inhibits the generation of nitric oxide, an antiviral substance.

PGE ₂ inhibitors are substances that inhibit any of the stages in which PGE ₂ is produced. For example, the PGE ₂ inhibitor may be at least one selected from the group consisting of a COX-1 selective inhibitor, a COX-2 selective inhibitor, a COX-1 and a COX-2 nonselective inhibitor, and a CREB activation inhibitor.

COX-1 and COX-2 selective inhibitors are all compounds that are capable of directly or indirectly inhibiting the activity or expression of COX-1 and COX-2, respectively, independently.

COX-1 and COX-2 nonselective inhibitors refer to any compound capable of directly or indirectly inhibiting the activity or expression of COX-1 and COX-2.

The pharmaceutical composition for preventing or treating calicivirus infection according to the present invention may include a COX-1 selective inhibitor.

The COX-1 selective inhibitor selectively inhibits the activity or expression of COX-1, thereby inhibiting the generation of PGE ₂ , preventing the inhibition of nitric oxide production, and treating or preventing a calicivirus infection.

According to one embodiment of the present invention, the COX-1 selective inhibitor may be at least one selected from the group consisting of SC-560, mofezolac, P6, TFAP, resveratrol and COX-1 siRNA.

SC-560 is represented by the formula of 5- (4-chlorophenyl) -1- (4-methoxyphenyl) -3- (trifluoromethyl) -1H-pyrazole.

Mofezolac is represented by the formula [3,4-bis (4-methoxyphenyl) isoxazol-5-yl] acetic acid.

P6 is represented by the formula 3- (5-chlorofuran-2-yl) -5-methyl-4-phenylisoxazole.

TFAP is represented by the formula of N- (5-Amino-2-pyridinyl) -4-trifluoromethylbenzamide.

Resveratrol has the formula 3,5,4'-trihydroxy-trans-stilbene and is a polyphenolic material.

Small interference RNA (siRNA) refers to nucleic acid molecules capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 And WO 00/44914). Because siRNAs can inhibit the expression of target genes, they are provided either as efficient gene knockdown methods or as gene therapy methods. siRNA has been found for the first time in plants, insects, fruit flies and parasites, but recently siRNA has been developed and used in mammalian cell research (Degot S, et al., 2002; et al., 2005).

The siRNA may have a structure in which the sense strand and the antisense strand are located on opposite sides to form a double strand. In addition, the siRNA molecules of the invention may have a single stranded structure with self-complementary sense and antisense strands. The siRNA does not need to have a sequence completely complementary to a part of the mRNA, and it suffices that the siRNA has enough complementarity to the extent that it can hybridize with mRNA and perform its inherent function. The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included.

The COX-1 siRNA of the present invention is an siRNA that inhibits the expression of COX-1. The COX-1 siRNA is complementary to the COX-1 mRNA sequence and does not need to have a completely complementary sequence with a portion of the mRNA, Sufficient complementarity within the range is sufficient.

The pharmaceutical composition for preventing or treating calicivirus infection according to the present invention may include a COX-2 selective inhibitor.

The COX-2 selective inhibitor selectively inhibits the activity or expression of COX-2, thereby inhibiting the generation of PGE ₂ , preventing the inhibition of nitric oxide production, and treating or preventing a calicivirus infection.

According to one embodiment of the present invention, the COX-2 selective inhibitor is selected from the group consisting of Nimesulide, SC-58125, SC-236, NS-398, COX-2 siRNA and celecoxib At least one.

Nimesulide is represented by the formula of N- (4-Nitro-2-phenoxyphenyl) methanesulfonamide.

SC-58125 is represented by the formula of 5- (4-fluorophenyl) -1- [4- (methylsulfonyl) phenyl] -3- (trifluoromethyl) -1H-pyrazole.

SC-236 is represented by the formula 4- [5- (4-chlorophenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl] -benzenesulfonamide.

NS-398 is represented by the formula of N- [2- (Cyclohexyloxy) -4-nitrophenyl] methanesulfonamide.

Celecoxib is represented by the formula 4- [5- (4-Methylphenyl) -3- (trifluoromethyl) pyrazol-1-yl] benzenesulfonamide.

The COX-2 siRNA of the present invention is an siRNA that inhibits the expression of COX-2. The COX-2 siRNA is complementary to the COX-2 mRNA sequence and does not need to have a sequence completely complementary to a partial sequence of the mRNA, and is capable of hybridizing with the mRNA to perform a unique action of the COX-2 siRNA Sufficient complementarity within the range is sufficient.

The pharmaceutical composition for preventing or treating calicivirus infection according to the present invention may include COX-1 and COX-2 non-selective inhibitors.

COX-1 and COX-2 nonselective inhibitors nonselectively inhibit the activity or expression of COX-1 and COX-2, thereby inhibiting the generation of PGE ₂ and preventing the production of nitric oxide, Infection can be treated or prevented.

For example, indomethacin, Ibuprofen, Diclofenac Sodium, Flurbiprofen, aspirin, pterostilbene, rofecoxib, and naproxen (Naproxen).

Indymethacin is represented by the formula of 2-1 - [(4-Chlorophenyl) carbonyl] -5-methoxy-2-methyl-1H-indol-3-ylacetic acid.

Ibuprofen is represented by the formula (RS) -2- (4- (2-Methylpropyl) phenyl) propanoic acid.

Diclofenac sodium 2 - [(2,6-dichlorophenyl) amino] benzeneacetic acid.

Furubipropene is represented by the formula (RS) -2- (2-fluorobiphenyl-4-yl) propanoic acid.

Aspirin is represented by the formula of 2- (acetyloxy) benzoic acid.

Prarostilbene is represented by the formula of 4 - [(E) -2- (3,5-Dimethoxyphenyl) ethenyl] phenol.

Rofecoxib is represented by the formula 4- (4-methylsulfonylphenyl) -3-phenyl-5H-furan-2- one.

Naphroxene is represented by the formula of (+) - (S) -2- (6-methoxynaphthalen-2-yl) propanoic acid.

The pharmaceutical composition for preventing or treating calicivirus infection according to the present invention may contain a CREB activation inhibitor.

The CREB activation inhibitor of the present invention refers to a substance that inhibits phosphorylation of CREB and inhibits the expression of a downstream gene.

According to one embodiment of the present invention, the CREB activation inhibitor may be at least one selected from the group consisting of MLD-12330A, PKI, U0126 and bortmannin.

MDL-12330A is a cAMP inhibitor, and cis-N- (2-Phenylcyclopentyl) azacyclotridec-1-en-2-amine. HCl. ≪ / RTI >

PKI is a PKA inhibitor and may be any substance capable of inhibiting PKA.

U0126 is an inhibitor of ERK and has the formula 1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene.

Bortmannin is an inhibitor of PI3K,

Lt; / RTI >

The present invention can inhibit CREB activation, inhibit COX-1 and COX-2 expression, inhibit the generation of PGE ₂ , inhibit nitric oxide production and prevent or prevent calicivirus infection.

According to one embodiment of the present invention, the PGE ₂ inhibitor may include nonsteroidal anti-inflammatory drugs (NSIDs).

Nonsteroidal anti-inflammatory drugs (NSIDs) are not only analgesic, pain-killing, and antipyretic, fever-reducing, but also have anti-inflammatory effects at high doses. Nonsteroidal anti-inflammatory analgesics inhibit the production of various prostaglandins and thromboxanes by inhibiting cyclooxygenase (COX) 1, 2, or both, depending on the type thereof, .

Thus, when a non-steroidal anti-inflammatory analgesic is used, the inhibition of COX-1 and COX-2 can inhibit the production of PGE ₂ , thereby treating or preventing a calicivirus infection.

Hereinafter, the present invention will be described in more detail by way of examples. It is to be understood that these embodiments are only for explaining the present invention in more detail, and the scope of the present invention is not limited to those exemplified in these embodiments.

Example

The use of porcine sowovirus Cowden strain as a model virus among the caliciviruses capable of cell cultivation and the non-steroidal anti-inflammatory analgesic agent, The use of analgesics was performed as follows.

Cell culture

LLC-PK pig kidney cells were monolayer cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 100 u / ml penicillin and 100 ug / ml streptomycin.

Cowden  strain Titration

In order to measure the potency of porcine sandworm Cowden strain, LLC-PK cells cultured in monolayer were inoculated with porcine sandovirus Cowden strain and then treated with 200 uM / ml glycochenodeoxycholic acid (GCDCA), 100 u / ml penicillin and 100 After adding EMEM supplemented with ug / ml streptomycin, the cells were cultured in a 36 C CO ₂ incubator for 36 hours. Cell culture immunofluorescence assay (CCIF assay) was performed using an antibody against porcine sapovirus VPg protein Respectively. Reverse transcription of porcine sandworm virus Cowden strain was expressed as fluorescence focus units per milliliter (ffu / mL).

A single layer after the culture LLC-PK pig sandpaper virus Cowden strain in the cell inoculation, GCDCA 100 uM, a kind of a bile acid / ml, was added to 100 u / ml penicillin and 100 ug / ml EMEM the streptomycin is added 36C CO ₂ And incubated for 1, 6, 12, 24 and 36 hours in an incubator.

The cytotoxicity of each chemical was measured by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) assay. That is, LLC-PK cells were cultured in a 96-well plate as described above, followed by incubation in a 36C CO ₂ incubator for 24 hours after each diluted chemical was added. After removing the culture medium from each well, 200 μl of MTT solution was added and cultured in a 36 C CO ₂ incubator for 4 hours. 150 μl of DMSO was added to each well, incubated at room temperature for 10 minutes, and absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Cell viability was calculated by the formula [OD (sample) OD (blank) / (OD (control) OD (blank)] X 100 and the experiment was conducted using the non-toxic concentration of each chemical.

chemical  And inhibitor efficacy

Each chemical and inhibitor was dissolved in DMSO to a concentration of 10 mM and each chemical concentration used in the experiment was used by stair-dilution in EMEM. LLC-PK cells were monolayered in 6-well plates or 12-well plates and the efficacy of each chemical or inhibitor was verified. That is, stair-diluted chemicals or inhibitors were treated with each layer of LLC-PK cells cultured in monolayers prior to inoculation, after inoculation, before inoculation, and after inoculation.

In the case of administration of a chemical or control agent prior to inoculation of the virus, LLC-PK cells were cultured in a monolayer culture well, followed by culturing in a 36 C CO ₂ incubator for 24 hours after adding the respective stepwise diluted chemical or control agent . After washing the cells with phosphate buffered saline (PBS, pH 7.4), the cells were treated with a 1 (1 ffu / cell) multiplicity of infection (MOI) determined by immunofluorescent antibody method Sandovirus Cowden strain was inoculated and cultured in 36C CO ₂ incubator for 1, 6, 12, 24 and 36 hours.

In the case of administration of a chemical or an inhibitor after the inoculation of virus, LLC-PK cells were cultured in monolayers of each well and after inoculation of pig swab virus Cowden strain with MOI of 1, Each stair-diluted chemical or control agent was added and incubated for 1, 6, 12, 24, and 36 hours in a 36C CO ₂ incubator.

When the chemicals or control agents were administered before and after the inoculation, the LLC-PK cells were cultured in each well of the monolayer culture, and the cells were incubated in a 36C CO ₂ incubator for 24 hours after each step of diluted chemical or control agent was added . After washing with phosphate buffered saline (PBS, pH 7.4), the cells were inoculated with porcine sowovirus Cowden strain with MOI of 1, and the virus was adsorbed onto the cells for 1 hour, The cells were incubated for 1, 6, 12, 24, and 36 hours in a 36C CO ₂ incubator.

The cultured supernatant was collected before and after the inoculation of pig swab virus Cowden strain, before and after administration of the chemical or inhibitor, and before and after the administration of the siRNA to the LLC-PK cell monolayer culture described above, and then the culture supernatant was collected by PGE ₂ EIA kit (Cayman Chemical, Ann Arbor, MI, USA) The concentration of PGE ₂ was measured. The concentration of PGE ₂ was measured at 405 nm with an ELISA reader.

Cowden  COX-1 and < RTI ID = 0.0 > COX-2 < PGE ₂ of  How to confirm expression

1 and COX-2 and PGE ₂ by the respective genes [p11, p28, p35 (NTPase), p32, p14 (VPg), p70 (ProPol), p60 PCR was performed using pCV4A plasmid containing the entire gene sequence of porcine sandworm Cowden strain and a primer specific for each gene (Table 1). That is, the 5 'end of the forward primer for amplifying each gene of porcine sandwich virus Cowden strain contains the nucleotide sequence of influenza virus hemagglutinin gene polypeptide.

To confirm the expression of COX-1 and COX-2 and PGE ₂ by [p11, p28, p35 (NTPase), p32, p14 (VPg), p70 (ProPol), p60 (VP1), VP2] After PCR was performed using the pCV4A plasmid containing the entire genome sequence of the virus Cowden strain and primers specific for each gene (Table 1), each gene amplified by PCR reaction was purified using PuriGel (Invitrogen, Waltham, MA, USA), and inserted into pUNO cloning vector (InvivoGen, San Diego, CA, USA). The empty plasmids were transfected into LLC-PK cells monolayered in 6-well plates according to the instructions of the Lipofectamine 2000 reagent (Invitrogen). Each cell was harvested at different times and the expression of these genes and proteins was confirmed by real-time PCR and Western blot analysis.

The underlined letter is the forward primer (SalI; reverse primer: NheI), and the italic letter is the nucleotide sequence of the influenza virus hemagglutinin gene polynucleotide. * Indicates the partial nucleotide sequence of the pig COX-1 gene (GenBank accession no. AF207823.1), ** indicates the partial nucleotide sequence of the pig COX-2 gene (GenBank accession no AF207824.1).

Answer on juice  RTI ID = 0.0 > COX-1 < / RTI & PGE ₂ of  How to determine induction of expression

To confirm that the expression of COX-1 and COX-2 and PGE ₂ induced by porcine sandworm Cowden strain is not induced by bile acid added to the cell culture, the whole gene sequence of porcine sandovirus Cowden strain is included PCV4A plasmid was transferred in vitro according to the instructions of the mMESSAGE mMACHINE Kit (Ambion, austin, TX, USA), and LLC-PK cells cultured monolayer in a 6-well plate according to the instructions of Lipofectamine 2000 reagent (Invitrogen) For 4 hours, and the cell culture medium was replaced with EMEM with or without bile acid GCDCA. After 6 days of transfection, the cells were lysed and the expression of COX-1, COX-2, PGE _2, and porcine sapovirus VPg gene and protein were examined by real-time PCR and Western blot analysis.

Cowden  lt; RTI ID = 0.0 > COX-1 < / RTI & PGE ₂ of  Expression induction assays

In order to verify the pig sandpaper virus expression of COX-1 and COX-2 and PGE ₂ induced by Cowden strain, a COX-1 siRNA, COX-2 siRNA or scrambled control siRNA using the guidance of the Lipofectamine 2000 reagent (Invitrogen) The cells were transfected into LLC-PK cells cultured in 70-80% monolayer in 6 well plates, and then swine swine virus Cowden strain with MOI of 1 was inoculated and adsorbed for 1 hour. Subsequently, the culture was replaced with EMEM containing 2.5% fetal bovine serum and 100 μM GCDCA, and incubated in a 36C CO ₂ incubator for 36 hours. The viral genome number, viral titer, viral VPg protein expression, COX- COX-2 protein expression was assessed by real-time PCR, 50% tissue culture infective dose (TCID50) and Western blot analysis.

Methods for quantifying the expression of target proteins

To quantify the expression of the target protein under the above conditions, LLC-PK cells were harvested at different time points, washed twice with PBS, and the proteolytic enzyme and phosphatase inhibitor (Roche, Basel, Switzerland) Cells were thawed using extraction buffer (Invitrogen). After dissolving the dissolved cell extracts with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), the dissolved proteins were transferred to a nitrocellulose blotting membrane (Amersham Protran, GE Healthcare Life Science, Germany). The transfected membranes were incubated with COX-1, COX-2, ERK, phospho-ERK, PI3K, phospho-PI3K, CREB, phospho-CREB, glyceraldehyde 3-phosphate dehydrogenase , GAPDH), hemagglutinin (HA), porcine sapovirus VPg, protease or viral protein 1 (Viral Protein 1, VP1) and then a second antibody against rabbit or mouse IgG .

Immunoreactivity was enhanced by using the chemoluminescence reaction kit (DoGen, Seoul, South Korea), and the reaction phase for each target protein was measured using Davinch-Western Imaging System (Young Ltd., Seoul, South Korea). To confirm whether the same amount of protein was tested, the protein was applied to the transfected membrane as an antibody against GAPDH and then compared with the intensity of the target protein. Immunoreactivity of the COX-1 and COX-2 proteins was also examined by comparing the intensity of GAPDH with that of Image Studio Lite (LI-COR Biotechnology, Lincoln, NE, USA).

Target RNA extraction method

In order to extract the target RNA under the above conditions, LLC-PK cells were washed twice with PBS, the cells were scraped off the plate, and the whole RNA was extracted with PureLink RNA mini kit (Ambion Life technologies, Carlsbad, Calif., USA) Respectively. The amount of extracted RNA was examined at 260 nm wavelength using BioPhotometer plus (Eppendorf, Hamburg, Germany).

How to measure target RNA expression level

In order to measure the expression level of the target RNA under the above conditions, 1 ug of RNA was converted into cDNA using random hexamers (Promega, Madison, Wis., USA), and the primers shown in Table 1 were used to express COX-1 , COX-2 and porcine sapovirus VPg genes were amplified by real-time PCR. The expression levels of COX-1 and COX-2 genes were measured by the program of Rotagene (Corbett, Perth, Australia) compared to the beta gene of the cells. VPg gene expression of porcine sandworm virus The amplified pCV4A plasmid was diluted 10-fold and compared with that amplified by real-time PCR.

virus Potency  How to measure

In order to measure the titer of the virus under the above conditions, 200 μl of the virus culture obtained under the above conditions was inoculated into each well of a 96-well plate in which LLC-PK cells were monolayer cultured, and cultured in a 36 C CO ₂ incubator for 6 days Respectively. The median tissue culture infective dose (TCID50) was calculated by the method of Reed and Muench.

How to measure virus infectivity

In order to measure the infectivity of the virus under the above conditions, a porcine sow virus Cowden strain having an MOI of 1 determined by the immunofluorescent antibody method was inoculated. After the virus was adsorbed on the cells for 1 hour, cells were washed three times with PBS After washing with water, EMEM medium was added and incubated in a 36C CO ₂ incubator for 36 hours. After incubation, the cells were fixed with 4% formaldehyde diluted with PBS and immunofluorescent staining was performed as follows. That is, the cells were treated with 0.2% Triton X-100 for 10 minutes to increase the permeability of the cells. Then, the cells were washed with PBS containing 0.1% fresh calf serum (PBS-neonate calf serum, PBS-NCS). The cells were then treated with a monoclonal antibody against porcine sapovirus capsid protein diluted 40-fold in PBS overnight at 4C, and then the cells were washed 3 times with PBS-NCS. The cells were then stained with 4 ', 6-Diamidino-2-phenylindole (DAPI) and stained with a confocal microscope. FITC-conjugated anti-mouse IgG antibodies were diluted 100-fold with PBS Respectively.

Method for measuring the expression level of nitric oxide in cell culture fluid

The expression level of nitric oxide (NO) in cell culture medium was measured according to the guidelines of Griess reagent system (Promega, Madison, WI, USA) under the above conditions. In other words, the same amount of cell culture solution and sulfanilamide solution were mixed and allowed to react at room temperature for 10 minutes. Then, the same amount of N-1-naphthylethylenediamine dihydrochloride solution was further added and the mixture was allowed to react at room temperature for 10 minutes. The absorbance for each sample was then measured at 540 nm with an ELISA reader. The amount of nitrogen oxides in each sample was measured using linear regression analysis as compared to the amount of standard samples.

In order to increase the reliability of the results under the above conditions, three independent experiments were performed and the results obtained were expressed as standard deviations. Statistical significance was assessed using GraphPad Prism software version 5.03 (GraphPad Software Inc., La Jolla, CA, USA) and one-way ANOVA with a p-value greater than 0.05 was considered statistically significant.

Experimental material

The chemicals used in the present invention, celecoxib, NS-398, SC-58125, SC-236 and nimesulide were purchased from Cayman Chemical (Ann Arbor, 1-ene-2-amine hydrochloride (MDL-12330A) and indomethacin (L-NAME), dimethylsulfoxide (DMSO), LN-Nitroarginine methyl ester (indomethacin) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

COX-1 siRNA, COX-2 siRNA and scrambled siRNA were purchased from Santa Cruz Biotechnology (CA, USA). COX-1 mouse monoclonal antibody and COX-2 rabbit polyclonal antibody were purchased from Abcam (Cambridge, MA, UK). Murine monoclonal antibodies against influenza virus hemagglutinin-tag (HA-tag) gene fragments were purchased from OriGene (Rockville, MD, USA). Mouse monoclonal antibody against pig sapovirus capsid protein and rabbit polyclonal antibody against porcine sapovirus VPg and protease protein were prepared and used. The anti-rabbit immunoglobulin G (IgG) -based goat antibody conjugated with horse-radish hydrogen peroxide (HRP) was detected in cell signaling (Beverly, MA, USA) Were purchased from Santa Cruz, respectively. Goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) was purchased from Jackson Immuno Research Lab (PA, USA). Synthetic PGE ₂ and PKI-14-22 were purchased from Tocris Bioscience (Ellisville, MO, USA).

Experiment result

As shown in Fig. 1, sandwich virus 1) activates the cAMP / PKA, PI3K / Akt and MEK / ERK signaling systems. These signal transduction systems activate COX-2 protein by phosphorylating CREB and converting it into an active form. 3) After phosphorylated CREB is transferred to the nucleus, it binds to the COX-2 promoter. 6) The COX-2 protein increased the expression of PGE ₂ , 7) decreased the production of antiviral nitric oxide, and 8) promoted the propagation of sandwich virus. However, 1) non-steroidal anti-inflammatory analgesics such as indomethacin, COX-1 and COX-2 nonselective inhibitors, and NS-398 and celecoxib, COX-2 specific inhibitors, 2) decreased expression of PGE ₂ , 3) increased production of antiviral nitric oxide, and 4) decreased propagation of sandwich virus.

Thus, COX-1 and COX-2 inhibitors have demonstrated antiviral efficacy in reducing the proliferation of sandworms.

As shown in FIG. 2, (A) COX-1 gene was transiently increased 24 hours after infection in LLC-PK cells infected with sandwich virus, while COX-2 gene was increased in time dependency at 24 hours and 36 hours . (B) Virus genomes are increasing in a time-dependent manner in LLC-PK cells infected with saprophirus. (C) In COX-1 cells infected LLC-PK cells infected with serotypes were transiently increased 24 hours after infection, whereas COX-2 protein was increased time-dependent at 24 hours and 36 hours. In addition, saponovirus VPg protein increased in time-dependent manner after viral infection. (D) PGE ₂ increased in time dependent manner in cell culture infected with sandworm. NS-398 and COX-1 and COX-2 nonselective inhibitor indomethacin were administered before, during, and after infecting the cells with EF (saprophytic virus) PGE ₂ was reduced in a concentration-dependent manner before and after infection. (GH) sampoviruses were transfected with COX-1 siRNA and COX-2 siRNA before infection, and COX-2 siRNA significantly reduced PGE ₂ production than COX-1 siRNA.

Thus, the above results demonstrate that the sandwich virus activates the COX-1 and COX-2 delivery systems in the later stages of infection and ultimately promotes the production of PGE ₂ .

As shown in FIG. 3, when the (AC) sapovirus was administered to the cells before, during and after the infection, and after the infection with the COX-2 inhibitor NS-398, Virus genome number (A), viral titer (B), and viral antigen amount (C) decreased in a concentration dependent manner. (DF) sapomycin was administered to the cells before, during and after infection and after infection with indomethacin, a non-selective inhibitor of COX-1 and COX-2, The number (D), the viral titer (E) and the amount of virus antigen (F) decreased in a concentration dependent manner. (GI) Celecoxib, a COX-2 inhibitor, was administered before, during, and after infection of cells with virus (GI) D), viral titer (E) and virus antigen (F) decreased in a concentration dependent manner. Transmission of COX-1 siRNA and COX-2 siRNA prior to infecting cells with J-O sapovirus resulted in a significant decrease in the number of viral genome, viral titer, and viral antigen compared to COX-1 siRNA.

Thus, indomethacin, NS-398 and celecoxib, the PGE ₂ inhibitors, have been shown to significantly reduce siderovirus proliferation.

As shown in Figure 4, (AB) by administration of the NS-398 COX-2 inhibitor after the infection of sandpaper virus to cell when the next addition of PGE _2, the virus genome (A) and potency (B) the concentration of PGE ₂ It was restored to dependency. (C) and titer (D) were determined by the concentration-dependence of PGE _{2 when} the cells were infected with CDX-1 (CD) sampvirus and then PGE ₂ was administered after the induction of COX-1 and COX- Recovered.

Therefore, PGE ₂ inhibitor over that the sandpaper virus proliferation inhibition by PGE ₂ inhibitor, indomethacin, NS-398 is activated by the addition of PGE ₂ was identified that to suppress the sandpaper virus proliferation activated by PGE _2.

As shown in FIG. 5, when NS-398, a COX-2 inhibitor, was administered after transposing (AB) saponin-infecting particle infection or sandovirus genome, COX-2 (A), NS-398 significantly reduced viral VPg protein (A) and dielectric (B). NS-398, a COX-2 inhibitor, was transfected with a serotonin transporter of the COX-2 gene (C) and the PGE2 gene (C) ₂ protein expression (D), NS-398 significantly reduced transcription of COX-2 gene (C) and expression of PGE ₂ protein (D).

Thus, bile acid, a cell culture additive essential for the propagation of sapvirus, is not a point of action of PGE ₂ inhibitors.

As shown in Fig. 6, (A) 9 amino acids of the influenza virus hemagglutinin gene were tagged at the amino terminal of eight genes (p11, p28, p35, p32, VPg, Prop, VP1 and VP2) pUNO plasmids were prepared. After each plasmid was transfected with cells, the expression of each protein was confirmed by Western blot using antibodies against hemagglutinin. (B) and its gene expression (C) were examined after transposing each plasmid of each plasmid (BC) into the cells, and then the viruses Propol and VPg proteins were analyzed for COX-2 protein (B) C) significantly. (D) Propol and VPg proteins also significantly increased the expression of PGE ₂ , the final product of COX-1 and COX-2.

Therefore, it was found that the propol and VPg proteins increase the expression of COX-1, COX-2 and PGE ₂ among the sapovirus proteins expressed upon intracellular proliferation of the sapovirus.

As shown in FIG. 7, (A) PI3K, ERK and CREB were phosphorylated and converted into the activated form in the latter half of the propagation of the envelope virus (24 or 36 hours of culture). (B) MDL-12330A, a PKA inhibitor, U0126, an ERK inhibitor, and wortmannin, a PI3K inhibitor, significantly reduced the expression of PGE ₂ in cells infected with saprophytes in a concentration-dependent manner. (C), viral titer (D), and viral load (VP) of MDR-12330A, a PKA inhibitor, U0126, an ERK inhibitor, and bortmannin, a PI3K inhibitor, The expression level (EH) of the antigen was decreased.

Thus, we have shown that the COX-1 and COX-2 and PGE ₂ delivery systems activated by the sapovirus infection are activated by the cAMP / PKA, PI3K / Akt and MEK / ERK signaling systems.

As shown in FIG. 8, (A) in the cells infected with the sapoxide, NO was not significantly changed until 24 hours after infection but rapidly increased at 36 hours after infection. (B) The PKA inhibitor PKI, the ERK inhibitor U0126, and the PI3K inhibitor, wortmannin, increased the production of nitric oxide in cells infected with sandworms in a concentration dependent manner. (C) The COX-2 inhibitor, NS-398, increased the amount of nitric oxide in the cells infected with the sapovirus. On the contrary, when the nitric oxide inhibitor L-NAME was administered after pretreatment of NS-398, Production was reduced by concentration-dependence of L-NAME. (D-F) L-NAME, a nitric oxide inhibitor, significantly increased the number of genomes (D), viral titers (E) and viral VPg antigen (F) of viruses reduced by the COX-2 inhibitor NS-398.

Therefore, the signal transduction system of COX-1 and COX-2 and PGE ₂ activated by the sapovirus infection reduced the production of antiviral nitric oxide, and consequently promoted the propagation of sandovirus.

conclusion

The inhibition of sapovirus proliferation was significantly reduced when COX-1 selective inhibitors, COX-2 selective inhibitors, COX-1 and COX-2 nonselective inhibitors and CREB activation inhibitors were administered to cells infected with porcine sapovirus Cowden strain , Stomach inhibitors and inhibitors can be used as anti-sapovirus preventive and therapeutic agents.

Claims (8)

Comprising a COX-2 inhibitor,
Wherein the COX-2 inhibitor is selected from the group consisting of nimesulide, 5- (4-fluorophenyl) -1- [4- (methylsulfonyl) phenyl] -3- (trifluoromethyl) At least one selected from the group consisting of 3- (trifluoromethyl) -1H-pyrazol-1-yl] -benzenesulfonamide, N- [2- (Cyclohexyloxy) -4-nitrophenyl] methanesulfonamide, COX-2 siRNA, celecoxib and indomethacin A pharmaceutical composition for the prevention or treatment of calicivirus infection.
delete delete delete delete delete delete [3] The method according to claim 1, wherein the calicivirus is at least one selected from the group consisting of human serovirus, porcine sambovirus, human norovirus, porcine norovirus, sonorovirus, canovirus, Or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of calicivirus infection.

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