WO2011145885A2

WO2011145885A2 - Composition for screening and a screening method for substances active in treating hepatitis b

Info

Publication number: WO2011145885A2
Application number: PCT/KR2011/003685
Authority: WO
Inventors: 류왕식; 왕해봉; 류동균
Original assignee: 연세대학교 산학협력단
Priority date: 2010-05-18
Filing date: 2011-05-18
Publication date: 2011-11-24
Also published as: WO2011145885A3; KR20110127086A

Abstract

The present invention is for screening for substances that selectively inhibit binding between DDX3 proteins and HBV polymerase as distinct from the molecules of the virus itself, and it can be expected that not only will there be only a very remote possibility of mutant viruses occurring that have resistance to avoid the same but also that the cytotoxicity with respect to host cells will be extremely low since the invention entails a target that is formed specifically only in infected cells. In particular, there is the advantage that the invention is outstanding in terms of cytotoxicity and the low occurrence of resistant mutations in chronic hepatitis B requiring long-term treatment of 1 year or more.

Description

【Specification】

[Name of invention]

Compositions and Screening Methods for Screening Hepatitis B Therapeutic Active Materials

Technical Field

The present invention relates to a composition for screening and a method for screening a hepatitis B therapeutically active substance, and more specifically, to identify a mechanism by which the polymerase of hepatitis B virus inhibits the induction of interferon and a new concept through the mechanism. The present invention relates to a screening composition and a screening method capable of screening a hepatitis B therapeutically active substance.

Background Art

Hepatitis B virus (HBV) is a virus that causes chronic hepatitis, as well as acute hepatitis, and is a clinically significant virus that currently infects about 400 million people (Seeger, Zoulim, and Mason, 2007). . HBV is a DNA virus, but has a very specific replication mechanism that replicates DNA genes through reverse transcriptase activity possessed by HBV polymerase. The genome of HBV has a circular DNA gene of about 3.2k bp and is composed of core protein (C, core), polymerase (P, polymerase), surface antigen (S, surface antigen), and X protein (ΗΒχ). Code four viral protein genes. These four _. Among the four viral proteins, core protein (C) and polymerase (Ρ) are essential for gene replication of hepatitis B virus. Hepatitis B virus synthesizes DNA through a reverse transcription process similar to retrovirus (Nassal, 2008).

Nucleoside analogues, such as interferon or lamivudine, have been developed as antiviral agents, but their effects are insufficient due to side effects and the production of resistant viruses. Therefore, the development of new chronic hepatitis B treatment is urgently needed (Zoulim and Locarnini, 2009).

Viral infections, on the other hand, often produce an innate immune response that induces the expression of interferons (IFNs) with antiviral activity. In recent years, the cellular mechanism of interferon generation by recognizing virus invasion has been identified in detail. Specifically, viruses, as well as bacteria and bearheads, are recognized by receptors called PRRs (Akira, Uematsu, and Takeuchi, 2006; Pichlmair and Reis e Sousa, 2007). . Often, nucleic acid constituting the viral genome is pattern-associated molecular (PAMPs) recognized by PR. patterns). PRJ? Is divided into two families. First of all, the first household

Toll-like receptors (TLR) include TLR3, TLR7, TLR 8, TLR9 and the like and are located in endosomes such as dendritic cells. Recognition of PAMPs by TLR activates signaling processes such as IRF signaling to activate transcription factors such as IRF3 / 7 and NF-kB. Specifically, TLR employs an adapter protein called TRIF (TIR-doma in-containing adapter protein inducing IFN 3), which in turn activates ΤΒΚΙ / ΙΚΚε (TAN-binding kinase 1/1 Β kinase-ε). Phosphorylation of the factor IRF3 / 7 induces the expression of interferon (Akira, Uematsu, and Takeuchi, 2006; Honda and Taniguchi, 2006).

The second PRR line is called retinoic-acid inducible gene I (RIG-I) -like receptors (RLRs), which belong to RIG ᅳ I and MDA-5 (melanoma different iat ion-associated gene 5) (Pichlmair and Reis). e Sous a, 2007). Like TLR, viral nucleic acid recognition by RLR activates IRF signaling and the like, and activates transcription factors such as IRF3 / 7 and NF-kB. In particular, RLR binds to the adapter IPSGFNP -promoter stimulator 1, or MAVS, VISA, or Cardif).

Through ΤΒΚΙ / ΙΚΚε kinases. This in turn activates the transcription factors IRF3 / 7, NF-kB (Akira, Uematsu, and Takeuchi, 2006). Eventually, both family receptors activate ΤΒΚΙ / ΙΚΚε kinases, effector kinases for IRF3 signaling, which phosphorylate transcription factors such as IRF3, IRF7, and NF-kB, and promote nuclear migration, while activating all of them. It is an aspect.

To combat viral infections, cells try to suppress the proliferation of viruses by innate immune function. In other words, after recognizing the virus infection, the interferon is expressed by activating IRF signaling to suppress virus growth. However, HBV infection is somewhat exceptional. Infection studies of chimpanzee animal models did not detect the expression of genetically related genes (Wieland et al., 2004; Wieland and Chisari, 2005). The results suggest that HBV infection is not recognized by the innate immune system and has been called "Stealth virus." Unlike these early reports, there are reports of observations of innate immunity to actual HBV infection. For example, the response of natural killer cells, a characteristic of innate immunity, has been observed in patients with acute infection during initial infection (Fisicaro et al., 2009). In addition, HepaRG cells, a cell line sensitive to HBV infection, have observed that HBV infection induces a strong congenital immune response and inhibits HBV DNA replication (Lucifora et al., 2010). Based on the above results, in real HBV infection, the innate immune response is strongly induced but It can be inferred that the protein inhibits it.

The DEAD-box RNA helicase, on the other hand, is a large protein family with about 38 genes found in the human genome (Under, 2006). DEAD-box RNA helicase acts on various RNA metabolism in cells such as transcription, RNA splicing, RNA nuclear release, and translation (Rocak and Linder, 2004). DDX3, a member of the DEAD-box RNA helicase family, has also been reported to be involved in various RNA targets.

Specifically, DDX3 (GeneBank accession No. 丽 001356.3) also works in the life history of various viruses. First, it acts on the proliferation of human immunodeficiency virus (HIV). Specifically, it acts on the Rev / RRE pathway and is essential for nuclear release of HIV RNA (Yedavalli et al., 2004). DDX3 also plays an essential role in RNA genome proliferation of hepatitis C virus (Ariurai et al., 2007; Owsianka and Patel, 1999). On the other hand, in contrast, the present inventors have recently reported that DDX3 inhibits HBV genome proliferation through binding to HBV polymerase (Wang, Kim, and Ryu, 2009). On the other hand, in addition to the relevance of RNA metabolism, DDX3 has been reported to promote congenital immune response (Schroder, Bar an, and Bowie, 2008; Soul at et al., 2008). Specifically, DDX3 promotes innate immunity by amplifying ΤΒΚΙ / ΙΚΚε kinase activators through the combination of ΤΒΚΙ / ΙΚΚε, an effector kinase of IRF signaling.

[Detailed Description of the Invention]

[Technical problem]

The present invention has been made to solve the above-mentioned problems, the first problem to be solved of the present invention is a screening composition that can screen a hepatitis B therapeutically active substance of a new concept that can prevent the generation of resistance mutations and It is to provide a screening method.

Technical Solution

In order to achieve the first object to be solved, the present invention is a) a base sequence encoding the HBV polymerase, the first recombinant expression vector; b) a second recombinant expression vector comprising a reporter gene for measuring the activity of an interferon beta (IFNi3) promoter and an interferon beta (IFNp) promoter operably linked thereto; It provides a composition for.

According to a preferred embodiment of the present invention, the composition may comprise c) an animal cell having the ability to produce interferon during virus infection.

According to another preferred embodiment of the present invention, the composition is d) interferon It may include a third recombinant expression vector comprising a gene encoding a gene for stimulating factor.

According to another preferred embodiment of the present invention, the interferon-producing stimulus is TRIF (GeneBank accession No. NM_182919.2), RIG-I (GeneBank accession No. 丽 _014314.3) / ¾ «-5, IPS-1 And TBK1 ((GeneBank accession No. # _004180.2) / IKK ε (GeneBank accession No. NM_014002.3) molecules.

According to another preferred embodiment of the present invention, the reporter gene is E. coli β-galactosidase, luciferase, alkaline phosphatase, secretory placental alkaline Phosphatase (secreted placental alkaline phosphatase) or chloramphenicol

Chloramphenicol acetyltransferase (CAT). According to another preferred embodiment of the present invention, (1) a first recombinant expression vector and an interferon beta (ΙΡΝβ) promoter encoding a sequence encoding HBV polymerase in an animal cell having an interferon generating ability during virus infection And transfecting a second recombinant expression vector operably linked thereto and comprising a reporter gene for measuring activity of an interferon beta (IFNi3) promoter; (2) adding an interferon production promoter to the animal cell; (3) measuring the activity of the interferon beta (IFNP) promoter by adding candidates to the animal cells; And (4) provides a method for screening the hepatitis B therapeutically active substance comprising the step of selecting a candidate substance with increased activity of the promoter when the candidate substance is added compared to the case where the candidate substance is not added.

According to a preferred embodiment of the present invention, the interferon production promoter may be poly (I: C). The embodiment of the present invention was performed only for the TLR3 receptor and poly (I: C), which is a ligand of TLR3, but the present invention is not limited thereto and includes all other ten kinds of TLR family receptors and various ligands accordingly. For example, the effect of the present invention will be the same even if the TLR9 receptor and CpG DNA which is a ligand of TLR9 are used.

According to another preferred embodiment of the present invention, in the step (1), the animal cell may further comprise a third recombinant expression vector comprising a gene encoding an interferon generating stimulation factor, in this case more preferred Preferably the interferon production stimulus The rule may be any one or more selected from the group consisting of TRIF, RIG-I / MDA-5, IPS-1, ΤΒΙ / Ι κε molecules.

According to another preferred embodiment of the present invention, the reporter gene is Ε. coli β-galactosidase, luci f erase, alkaline phosphatase, secreted placental alkaline phosphatase or chloramphenicol

Chloramphenicol acetyltransferase (CAT).

According to another preferred embodiment of the present invention, (1) in an animal cell having the ability to produce interferon during virus infection, a) the base sequence encoding the HBV polymerase is a first recombinant expression vector, b) interferon beta ( IFNP) promoter and a second recombinant expression vector operably linked thereto comprising a reporter gene for measuring the activity of the interferon beta (IFN 3) promoter and c) a gene encoding the interferon production promoter and interferon production stimulating factor. Transfecting any one or more substances selected from the group consisting of a third expression vector comprising a; (2) measuring the activity of the interferon beta (IFNP) promoter by adding candidates to the animal cells; And (3) selecting a candidate material having increased IFNp promoter activity when the candidate material is added as compared with the case where the candidate material is not added. In another preferred embodiment of the present invention, According to (1) an animal cell having an interferon-producing ability during virus infection, the nucleotide sequence encoding HBV polymerase is included in the recombinant expression vector and the nucleotide sequence encoding the IRF3-GAL4 fusion protein. Transfection of an expression vector and a fourth expression vector comprising a gene encoding an interferon generator stimulating factor and a third recombinant expression vector comprising a nucleotide sequence operably linked to the expression vector and a pFR promoter (transfection); (2) measuring the activity of the pFR promoter by adding a candidate to the animal cells; And (3) selecting candidate substances in which the activity of the IFNp promoter is increased when the candidate substances are added as compared with the case where the candidate substances are not added. According to another preferred embodiment of the present invention, screening of a hepatitis B therapeutically active substance for selecting a candidate substance that inhibits binding of HBV polymerase and DDX3 by adding a candidate substance to an animal cell expressing HBV polymerase Provide a system

According to another preferred embodiment of the present invention, to express HBV polymerase A candidate system is added to animal cells to inhibit the binding of HBV polymerase and DDX3 to provide a screening system for a hepatitis B therapeutically active substance that selects candidates that induce binding of DDX3 with ΤΒΚΙ / ΙΚΚε. Hereinafter, terms used in the present invention will be described.

The term "having the ability of interferon to produce virus infection" refers to a cell that promotes interferon expression by viral infection to produce interferon.

The term "operably linked" means a functional binding between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or transcriptional regulator binding site) and another nucleic acid sequence, whereby the regulatory sequence is The term “IFNp promoter” refers to a DNA sequence that can regulate the expression of interferon, which is an IRF3 / 7 binding site and an NF-kB transcription factor binding site.

It is composed.

The term “reporter gene” refers to a DNA molecule that encodes an expression product (such as an enzyme) that is readily measurable, for example, in biological activity assays.

The term “pFR promoter” is a promoter having a GAL4 binding site, which is a transcription factor of yeast, and is widely used for measuring GAL4 dependent transcription.

The term "candidate" refers to a substance that inhibits the interaction of HBV polymerase with DDX3 and inhibits the inhibition of interferon expression by HBV plymerase, and thus is expected to recover the original expression of interferon expression. Such candidates include, but are not limited to, for example, low molecular weight organic compounds, high molecular weight organic compounds, nucleic acid molecules (eg, DNA, RNA, PNA and aptamers), proteins, sugars, and lipids.

Advantageous Effects

Since the present invention screens for substances that selectively inhibit binding between HBV polymerase-DDX3 molecules, the likelihood of resistance mutant viruses avoiding them will be very slim. In addition, since the binding of HBV polymerase and DDX3 molecules is a target formed specifically for infected cells, the cytotoxicity of a substance that inhibits the binding is expected to be extremely low. In the case of chronic hepatitis B, which requires long-term treatment for more than 1 year, low resistance mutagenesis and cytotoxicity is an excellent antiviral agent.

[Brief Description of Drawings]

La to lc are cleavage maps of the recombinant polamide used in the present invention. Specifically, la is a cleavage map of HBV polymerase expression vector (pcDNA-HBV Pol). The P gene of the ayw genotype (GenBank accession V01460) is inserted at multiple cloning sites of the pcDNA3.0 vector (In Vitrogen Inc.) and expressed from a cytomegalovirus (CMV) pyroelectric promoter. The FLAG tag linked to the N-terminus of the HBV polymerase gene was inserted for detection. Lb is an expression vector of the HBx protein (pCMV7-HBx), in which the X gene of the HBV ayw genotype (GenBank accession V01460) is inserted into a multiple cloning site of the pCMV7 vector (Sigma Inc.) and expressed from a cytomegalovirus (CMV) superelectric promoter. . The FLAG tag linked to the C-terminus of the X gene is inserted for detection. Figure lc shows the expression vector of core protein (Core) (pcDNA-Core) of the gene of the HBV ayw genotype (GenBank accession V01460) is inserted into the multiple cloning site of the pcDNAl / Amp vector (In Vitrogen Inc.) (MV (cytoraegalovirus) Figure 2 shows the inhibition of interferonogenesis by HBV polymerase, Figure 2 shows the transfection of the protein expression vector of ΙΡΝβ Luc if erase reporter plasmid and HBV in HepG2 cells, liver cancer cell lines, respectively. Luc if erase activity was measured in comparison with the control group and expressed in multiples compared with the control group, and poly (I: C) was added to the medium to activate TLR signaling, and the expression of TLR3 was deficient. TLR3 expression plasmids were transfected together to complement the function of HepG2 cells.

3 to 4 show the inhibition of interferon production according to the increase in the amount of HBV polymerase-expressing plasmid. After adding poly (I: C) to the medium of HepG2 cells, which is a hepatic cancer cell line, IFNP Luc if erase After transfecting the reporter plasmid and the HBV polymerase expressing plasmid, respectively, lucif erase activity was measured in comparison with the control group and expressed in multiples compared with the control group. 4 is transfected poly (I: C) to HepG2 cells, hepatocellular carcinoma cell lines, transfected IFNP Lucif erase reporter plasmid and HBV polymerase expression plasmid, respectively, and measured luciferase activity in comparison with the control group. It is expressed in multiples compared to.

Figure 5 shows the inhibition of IRF signaling according to HBV polymerase expression, HepG2 cells were added poly (I: C) to the medium, and after transfection of the HBV polymerase expression plasmid, cells were harvested 3 days later . Samples were electrophoresed by SDS-PAGE and phosphorylated IRF was detected by Western blot using phosphorylated IRF antibodies.

6, 7 shows the increase in the amount of HBV polymerase expression plasmid

As a result showing the decrease in IRF3-lucif erase activity, Figure 6 shows that after transfecting the RIG-I expression plasmid, IRF3 Luciferase reporter fulllasmid and HBV polymerase expressing plasmid in HepG2 cells, the luciferase activity was compared with the control group. Measure it and It is expressed in multiples compared to the control group. Figure 7 After transfection of the TRIF expression plasmid, IRF3 Luciferase reporter plasmid and HBV polymerase expression plasmid in HepG2 cells, respectively, luciferase activity was measured in comparison with the control group and expressed in multiples compared with the control group.

8 to 12 show that the target of HBV polymerase is a ΤΒΚΙ / ΙΚΚε molecule of IRF signaling, and FIG. 8 shows the interrelationship of molecules involved in IRF signaling. In particular, HBV polymerase shows the activity of ΤΒΚΙ / ΙΚΚε. Target DDX3 to promote 9 shows transfection of TBK1 expression plasmid, IRF3 Luciferase reporter pollamide and HBV polymerase expression plasmid, respectively, in HepG2 cells.

Luciferase activity was measured in comparison with the control group and expressed in multiples compared with the control group.

10 is transfected ΙΚΚ ε expression plasmid, IRF3 Luciferase reporter plasmid and HBV polymerase expression plasmid in HepG2 cells, respectively, luciferase activity was measured in comparison with the control group and expressed in multiples compared with the control group.

11 is a transfection of ΙΚΚε expression full psmid, ISRE Luciferase reporter plasmid and HBV holyrylase expression plasmid in HepG2 cells, respectively, measured luciferase activity compared to the control group and expressed in multiples compared with the control group .

12 was transfected with IRF3 expressing fulllasmid, ISRE Luciferase reporter plasmid and HBV polymerase expressing plasmid in HepG2 cells, respectively, and luciferase activity was measured in comparison with the control group and expressed in folds compared with the control group.

13 to 15 show the antagonism of DDX3 on IRF signaling inhibition of HBV polymerase. FIG. 13 shows TBK1 expression plasmid and IRF3 in HepG2 cells.

After transfecting Luciferase reporter full lasmid and HBV polymerase expressing full lasmid, respectively, luciferase activity was measured in comparison with the control group and expressed in multiples compared with the control group. At this time, the effect of increasing the amount of DDX3 expression plasmid was observed. FIG. 14 was transfected with ΙΚΚε expression fulllasmid, IRF3 Luciferase reporter plasmid and HBV polymerase expression plasmid in HepG2 cells, respectively, and luciferase activity was measured in comparison with the control group and expressed in folds compared with the control group. At this time, the effect of increasing the amount of DDX3 expression plasmid was observed. Figure 15 shows the protein precipitated by Western blot (IB, immunoblot) after the immunoprecipitation (IP, immunoprecipitation) to investigate the interaction between DDX3-ΙΚΚε. It is observed. Transfected ΙΚΚε and DDX3 Expression Plasmids in HepG2 Cells After that, immunoprecipitation with ant i -Flag antibody and anti_HA antibody

Western blot (lane 3). Transfection of the HBV polymerase expression plasmid reduced the precipitated DDX3 (lane 4).

Figure 16 is a schematic diagram showing the screening mechanism of the chronic hepatitis B therapeutic agent according to one feature of the present invention.

Figure 17 shows the luciferase activity decreased as the amount of HBV polymerase expression plasmid increased as a result of IFN | 3-Luc reporter assay following treatment with 10 candidate compounds. Since all 300 compounds performed in this example showed similar results, only the results of 10 compounds were illustrated.

[Best form for implementation of the invention]

Hereinafter, the present invention will be described in more detail.

As described above, since the conventional hepatitis B therapeutic agent targets the virus molecule itself, the treatment effect is limited because of the resistance of the mutant strain to generate high frequency mutations. Therefore, in the present invention, HBV polymerase inhibits the interaction of the ΤΒΚΙ / ΙΚΚε molecule and the DDX3 protein that is already bound when interacting with the DDX3 protein. We have developed a composition and a screening method that can screen for hepatitis B therapeutics.

In this case, if the therapeutic agent added is a substance that selectively inhibits the interaction between HBV polymerase and DDX3 protein, the DDX3 protein that inhibited the interaction with HBV polymerase binds to the ΤΒΚΙ / Ι Κε molecule to Induction of activation leads to an increase in the activity of the promoter, and a therapeutic candidate for such an activity can be used as a hepatitis B therapeutic. To this end, according to the first preferred embodiment of the present invention, 1) the sequence encoding the HBV polymerase in an animal cell having the ability to generate interferon upon virus infection with a recombinant expression vector and an interferon beta (IFN3) promoter Adding and transfecting a second recombinant expression vector operably linked thereto and comprising a reporter gene for measuring the activity of interferon beta (IFN | 3); (2) adding an interferon production promoter to the animal cell; (3) attaching candidate substances to the animal cells; .

10

Adding to measure the activity of the interferon beta (IFN 3) promoter; And (4) selecting a candidate substance having increased activity of the interferon beta (IFNI3) promoter when the candidate substance is added as compared with the case where the candidate substance is not added. To provide. Specifically, in step 1), the nucleotide sequence encoding the HBV polymerase to an animal cell having interferon-producing ability during virus infection is operably linked to the first recombinant expression vector and the IFNP promoter, and to measure the activity with the IFNP promoter. Adding and transfecting a second recombinant expression vector comprising a reporter gene for transfection.

First, an animal cell having interferon-producing ability during viral infection expresses DDX3 protein, and animal cells belonging to it may be used without limitation, but preferably

HE 293, HeLa, HepG2 cells and the like can be used. Next, a recombinant expression vector of Crab 1 is added to the nucleotide sequence encoding the HBV polymerase. HBV polymerase, when transfected into animal cells, interacts with DDX3 in animal cells and plays a role of inhibiting promoter activity. On the other hand, if a non-HBV polymerase contains a nucleotide sequence encoding a HBV core portion or other region of HBV, HBV polymerase must be used since it does not interact with DDX3 to inhibit the expression of IFN | 3 and the activity of the promoter. The base sequence encoding should be added with a first recombinant expression vector (see Example 1).

Meanwhile, the nucleotide sequence encoding the HBV polymerase may be the nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Various HBV polymerases may be used, and further, FLAG at the N-terminus of the HBV polymerase for expression measurement. Adding sequences and the like will also be apparent to those skilled in the art.

On the other hand, any recombinant expression vector for use in the invention are commercially available, or inserting a specific DNA sequence in a plasmid that can be easily obtained in the laboratory or as a recombinant plasmid that can be easily ^obtained, in a commercial or laboratory, and its production method is generally a recombinant expression vector It can be produced according to the method for manufacturing, which is obvious to those skilled in the art.

Next, a second recombinant expression vector comprising a reporter gene for measuring the activity of the promoter and the IFNp promoter is operably linked to the animal cell. Add. Promoter (eg, Lin et al., Mol. Cell Biol. 20: 6342 (2000)) sequences capable of modulating the expression of interferon that can be used in the present invention can be used without limitation. Reporter genes that can be used in the second recombinant expression vector of the present invention include E. coli β-galactosidase [An et al. , Mol. Cell. Biol. 2: 1628 (1982)], lucif erase (lucif erase) [De Wet et al. , Mol. Cell. Biol. 7: 725 (1987)], alkaline phosphatase [Henthorn et al., Proc. Natl. Acad. Sci. USA 85: 6342 (1988)], secretory placental alkaline

Phosphatase (secreted lacental alkaline phosphatase) [Berger et al., Gene 66: 1 (1988)] and chloramphenicol acetyltransferase (chloramphenicol

acetyl transferase, CAT) [Gorman et al. , Mol. Cell. Biol. 2: 1044 (1982); Tsang et al., Proc. Natl. Acad. Sci. USA 85: 8598 (1988)], including but not limited to. These reporter genes are commercially available. In the method of the present invention, luciferase may be used as the reporter gene. According to another preferred embodiment of the present invention, in the step (1), the animal cell may further comprise three recombinant expression vectors comprising a gene encoding the interferon stimulating factor. The interferon production stimulating factor may be any one or more selected from the group consisting of TRIF, RIG-I / MDA-5, IPS-1, and ΤΒΚΙ / Ικε protein molecules. Interferon production stimulating factor is a protein molecule that can replace the function of poly (I: C), an interferon production promoter. It is also possible to transfect animal cells with poly (I: C) RNA. On the other hand, by interacting with the HBV polymerase DDX3 protein inhibits the interaction of already bound TBK1 / IKK ε molecule and DDX3 protein to inhibit the expression of interferon, resulting in side reactions that inhibit innate immunity, Interferon-producing stimulating factor that can be used is preferably the interferon generating stimulating factor, including the ΤΒΚΙ / ΙΚΚε molecule, as shown in the center of Figure 9, preferably the interferon generating stimulating factor is TRIF, RIG-I / MDA— 5, IPS—1, ΤΒΚΙ / ΙΚΚε may be any one or more selected from the group consisting of, but not limited to, interferon generation stimulating factors not disclosed in FIG. It can be used. Then, the above-described recombinant expression vector on the animal cell Transfection and transfection can be carried out using a commonly known method. (2) Interferon-producing promoter is added to the animal cells. Interferon production promoter is a substance that is recognized by PRR receptors such as TLR or RIG-I and triggers the IRF signal pathway when it is processed into cells. It is a poly (I: C), which is a replacement nucleic acid of double stranded RNA, and a DNA replacement nucleic acid. CpG and the like are used. Although poly (I: C) is used in the present invention, the present invention is not limited thereto. Next, (3) the activity of the promoter is measured by adding a candidate substance to the animal cells. The candidate substance refers to a substance expected to activate the promoter or the expression of interferon by inhibiting the binding (binding) or interaction of HBV polymerase and DDX3. Such candidates include, but are not limited to, for example, low molecular weight organic compounds, high molecular weight organic compounds, nucleic acid molecules (eg, DNA, RNA, PNA and aptamers), protein sugars and lipids.

The activity of the IFNp promoter can be confirmed by measuring the expression product of the reporter gene and can be measured using conventional reporter gene standard methods. (4) selecting the candidate material whose promoter activity is increased when the candidate material is added as compared with the case where the candidate material is not added. As described above, HBV polymerase transfected and expressed in animal cells blocks the induction of interferon against viral infection through interaction with _DDX3 molecules, so if the added candidate increases the activity of the promoter. It can be interpreted as selectively inhibiting the action of HBV polymerase that inhibits the binding of DDX3 and TBK1 / IKK ε molecule. This may be assumed to be an active substance that inhibits the binding or interaction between DDX3 and HBV polymerase, and may be used as a therapeutic agent for hepatitis Β.

. Screening system according to the present invention in the development of new drugs, high-speed processing

It can be used for high-throughput screening (HTS). The high-speed search method is a search method that can reduce the size of existing search units or automate repetitive tasks to obtain superior results in terms of quality and quantity compared to conventional search methods. Tablets in the form of search Binding affinity for the protein receptor, enzyme activity search, or cell-level functional search can be divided into. According to a second preferred embodiment of the present invention, (1) in an animal cell having the ability to produce interferon upon virus infection, a) a base sequence encoding the HBV polymerase is first recombinant expression vector, b) interferon beta (IFNP) A second recombinant expression vector operably linked to the promoter and comprising a reporter gene for measuring the activity of the interferon beta (IFN 3) promoter and c) a gene encoding the interferon production promoter and interferon production stimulating factor. Transfecting any one or more substances selected from the group consisting of a third expression vector comprising; (2) measuring the activity of the interferon beta (IFNP) promoter by adding candidates to the animal cells; And (3) selecting a candidate substance having an increased activity of an IFNP promoter when the candidate substance is added as compared with the case where the candidate substance is not added. do.

Referring to the second embodiment centering on the difference from the first embodiment, the first embodiment adds an interferon production promoter such as poly (I: C) after transfection, but in the second embodiment Instead, the method further comprises transfecting any one or more substances selected from the group consisting of a third expression vector including a gene encoding an interferon production-promoting agent and an interferon-producing stimulating factor in step (1). Thus, the same effects as in the first embodiment can be achieved. Meanwhile, poly (I: C) is a commonly used nucleic acid material instead of double stranded RNA, and when added to the medium, it acts as a ligand of TLR, while transfecting into cells acts as a ligand of RIG-I to promote interferon production. do. According to a third preferred embodiment of the present invention, (1) a base sequence encoding HBV polymerase is encoded by a first recombinant expression vector, an IRF3-GAL4 fusion protein, into an animal cell having an interferon production capacity during virus infection. A second recombinant expression vector comprising a nucleotide sequence and a pFR promoter and a gene encoding a third recombinant expression vector and an interferon generator stimulating factor operably linked thereto and comprising a base sequence encoding pFR luciferase. Transfecting a fourth expression vector comprising; (2) measuring the activity of the pFR promoter by adding candidates to the animal cells; And (3) selecting a candidate substance whose activity of the IFN13 promoter is increased when the candidate substance is added as compared with the case where the candidate substance is not added. Provide a method for cleaning.

Here, the third recombinant expression vector comprising a nucleotide sequence encoding pFR luciferase means a reporter plasmid having a GAL4 binding site at a promoter site and promoting transcription in a GAL4-dependent manner.

Specifically, the screening method of the first embodiment of the present invention described above is in communication with an interferon beta promoter and a second recombinant expression vector in place of a second recombinant expression vector comprising a reporter gene for measuring the activity of the interferon beta promoter. There is a difference in adding a second recombinant expression vector comprising a nucleotide sequence encoding a GAL4 fusion protein and a system 3 recombinant expression vector comprising a nucleotide sequence encoding an IRF3 GAL4 dependent pFR luciferase. Here, the IRF3-GAL4 fusion protein serves as a transcriptional promoter, and the pFR luciferase third recombinant expression vector having a GAL4 binding site serves as a reporter. On the other hand, according to a fourth embodiment of the present invention a) the base sequence encoding the HBV polymerase is a first recombinant expression vector; b) provides a composition for screening a hepatitis B therapeutically active substance comprising a IFN13 promoter and a second recombinant expression vector operably linked thereto and comprising a reporter gene for measuring the activity of the IFNP promoter. The activity of the promoter can be measured by adding to the animal cells having the ability to produce interferon upon infection with Irol virus and transfecting it, and then adding candidates. Antivirals screened through the screening methods of the present invention will be more effective than the nucleoside derivatives currently in use. Because they act on new protein surface structures formed by binding between viral proteins and host proteins, they inhibit the formation of binding between proteins, making it very unlikely that a mutant virus will be created that avoids them. Furthermore, since the target provided by the present invention is a target specifically formed only for virus-infected cells, antiviral compounds selected by the present screening method are expected to have low toxicity to host cells. This is a significant advantage in the case of chronic hepatitis B, which requires a long-term treatment of the drug for more than one year to obtain a therapeutic effect. Moreover, by inhibiting the proliferation of hepatitis B virus, ultimately, hepatitis B virus can be removed to prevent cirrhosis and liver cancer caused by chronic hepatitis B virus. In addition, the antiviral active substance discovered in the present invention can be used in combination with an existing antiviral agent and may also be co-administered with interferon. [Form for implementation of invention]

The present invention will be described in more detail with reference to specific examples.

The transfection technique, luciferase activity assay, immunoprecipitation method, and immunostaining method used to establish the assay for developing the antiviral agent provided by the present invention used experimental materials and experimental methods frequently used by the literature and experts in this field. . In the present invention, the following abbreviations are used:

M (molar), mM. (mi 11 imolar), μ 1 (microliters), ml (milliliters), u g

(micrograms), mg (milligrams), bp (kilo base pairs), 0RF (open reading frame), PC (polymerase chain react ion), PEG (polyethylene glycol),

Hepatitis B virus (HBV), interferon (IFN), pattern recognition receptor (PR), I RFC interferon regulatory factor (TRF), TIR one domain—containing adapter protein inducing IFN | 3), TBK (TRAF 一 associated NF ᅳ kB activator -binding kinase, IB kinase-ε (IKK ε), pattern recognition molecular pattern (PAMP), toll 1-1 ike receptor (TLR), melanoma differentiat ion—associated gene 5 (MDA-5), human immunodeficiency virus ),

<Example 1> Hepatitis B virus can (HBV) demonstrate the generation of interferon inhibition by polymerase ^'IFNP Luciferase reporter assay

1-1. Inhibition of IFNp Promoter Activity by HBV Polymerase

First, it was examined whether three viral proteins such as core protein (core), polymerase (Pol), and X protein (HBx) encoded by HBV inhibit interferon production (FIG. 1). This measurement utilizes the IFN | 3 luciferase reporter, an interferon beta promoter reporter that is well known in the literature (Schroder, Bar an, and Bowie, 2008).

. ^{"Specifically,} the cells were used as H _e pG2 cells, using DMEM medium supplemented with 10% fetal added 5¾> were cultured in the incubator C¾. Transfection is

PEI (polyethylenimine) was followed and transfected with about 12 ug of plasmid DNA in a 60 mm diameter plate (Ryu, Kim, and Ryu, 2008).

The plasmids used in the experiment were each given to the following researchers: IFN β-Luc reporter (Bowie, Trinity Col lege Dubl in, Ireland). Meanwhile, the plastic below The smid was previously used in the inventor's laboratory.

(1) HBV polymerase expression fulllasmid (Ryu, Kim, and Ryu, 2008) has a cleavage map of the HBV polymerase expression plasmid shown in FIG. La, and encodes a gene encoding HBV polymerase represented by SEQ ID NO: 1. Include. The HBV polymerase expression polamide may be prepared by inserting a gene encoding HBV polymerase represented by SEQ ID NO: 1 into pcDNA3 polamide (InVitrogen Inc.).

(2) HBV core protein expression plasmid (Ryu, Kim, and Ryu, 2008) is a nucleotide sequence encoding the HBV core protein gene in pcDNA3 plasmid (InVitrogen Inc) (SEQ ID NO:

2) and has a cleavage map shown in FIG.

(3) HBx expressing plasmid (Cha et al., 2004) for pcDNA3

Nucleotide sequence encoding HBV X gene moiety to plasmid (InVitrogen Inc) (SEQ ID NO:

3) and has a cleavage map shown in FIG.

(4) The Y Li D mutation expression plasmid of HBV polymerase is the same as that of said HBV polymerase. However, aspartic acid (D) residue of YMDD motif, which is an enzyme residue, is substituted with histidine (H) residue to express HBV polymerase that has lost enzymatic activity.

The reporter assay was carried out according to the method of the manufacturer (Promega Inc.) after harvesting cells 24 hours after transfection of the following plasmid in a 24-well plate. Specifically, after transfecting the protein expression vector of HBV to HepG2 cells, liver cancer cell line, luciferase activity was measured in comparison with the control group and expressed in multiples compared with the control group. At this time, poly (I: C), a substitute for double-stranded RNA, was added to the medium to activate TLR signaling, a major receptor for innate immunity. Meanwhile, TLR3 expression plasmids were transfected together to complement the function of HepG2 cells lacking TLR3 expression (Li et al., 2005).

Figure 2 transfected IFNP Luciferase reporter plasmid and HBV protein expression vector in HepG2 cells, liver cancer cell lines, respectively, luciferase activity was measured in comparison with the control group and expressed in multiples compared with the control group. In addition, poly (I: C) was added to the medium to activate TLR signaling, and TLR3 expression plasmids were transfected together to complement the function of HepG2 cells deficient in TLR3 expression.

According to the experimental results of FIG. 2, it was observed that HBV polymerase reproducibly inhibited at least about 10 times the promoter active ol. Core and X proteins, on the other hand, are IFNp promoters. It did not inhibit the activity. In addition, YMHD, a mutant of the YMDD motif, an active site of HBV polymerase, also inhibited IFNP promoter activity. Therefore, the reverse transcription activity of HBV polymerase is not required for IFNP promoter activity inhibition.

Example 2 IFNP Luc if erase reporter assay showing that HBV polymerase inhibits induction of interferon expression by TLR3 and RIG-I receptors

2-1. HBV polymerase concentration dependence of IFNP promoter activity inhibition.

In order to clarify the inhibition of IFN13 promoter activity by HBV polymerase, it was investigated how the promoter activity changes with HBV polymerase concentration. Specifically, the cells were HepG2 cells and were cultured in a 5% C0 ₂ incubator using DMEM medium with 10% fetal calf added. Transfection is

The method using PEI (polyethylenimine) was followed, and about 12 ug of plasmid DNA was transfected into a 60 mm diameter plate (Ryu, Kim, and Ryu, 2008). The plasmids used in the experiment were each given to the following researchers: IFNb-Luc reporter (Bowie, Trinity College Dublin, Ireland). The following Polismid was purchased from the manufacturer: HA-TLR3 (InvivoGen Inc). On the other hand, the following plasmids were conventionally used in the inventor's laboratory: (1) HBV Pol expression fulllasmid (Fig. La).

The reporter assay was carried out according to the method of the manufacturer (Promega) after harvesting cells 24 hours after transfection of the following plasmid in 24-well full rate. Specifically, unlike the embodiment of 1-1, after transfecting while increasing the amount of HBV polymerase expression vector plasmid in HepG2 cells by doubling, the luc if erase activity was measured in comparison with the control group and the control group It was expressed in comparison to multiples (FIG. 3).

3 is a poly (I: C) added to the HepG2 cells, a hepatocellular carcinoma cell line, transfected IFNp Lucif erase reporter polamide and HBV polymerase expression plasmid, respectively, and luciferase activity was measured in comparison with the control group. And expressed as a multiple compared with the control group. As a result, it can be seen that IFNP promoter activity decreased in proportion to the HBV polymerase concentration. 2-2. Inhibition of promoter activity by HBV polymerase.

To further clarify the inhibition of promoter activity by HBV polymerase, we investigated how the promoter activity varies with HBV polymerase concentration when poly (I: C) transfection is performed. Specifically, the cells were HepG2 cells, and cultured in a 5% C02 incubator using DMEM medium added with 10% fetal calf. Transfection is

PEI (polyethylenimine) was followed, and about 12 ug of plasmid DNA was transfected into 60 μF folate (Ryu, Kim, and Ryu, 2008). The plasmids used in the experiment were each given to the following researchers: IFNb-Luc reporter (Bowie, Trinity College Dublin, Ireland). On the other hand, the following plasmid was conventionally used in the inventor's laboratory: (1) HBV Pol expression plasmid (FIG. La). Reporter assay was carried out according to the manufacturer (Promega) method after harvesting cells 24 hours after transfection of the following plasmid in 24-well full rate. 4 is after transfecting poly (I: C) into HepG2 cells, a liver cancer cell line,

After transfecting the Luc if erase reporter plasmid and the HBV polymerase expression plasmid, the hicif erase activity was measured in comparison with the control group and expressed in multiples compared with the control group. Is located in the cytoplasm: (C I) ^'More specifically, poly (I:: C), unlike the embodiment of Figure 3 haejun added to the medium, poly p is ₀ ly case of the (I C) trans faction. In this case, RIG-I / MDA5 molecules act as receptors instead of TLR3 (Saito and Gale, 2008). Specifically, after transfection while increasing the amount of HBV polymerase expression vector plasmid in HepG2 cells by two-fold, luciferase activity was measured in comparison with the control group and displayed in multiples compared with the control group (FIG. 4). As a result, promoter activity decreased in proportion to HBV polymerase concentration. In other words, HBV polymerase inhibited not only TLR3 but also IRF signaling activated by RIG—I / MDA5.

Example 3 HBV Polymerase Inhibits IRF Signaling

Interferon induction is performed through IRF signaling. However, two major signs of IRF signaling are phosphorylation of IRF and nuclear transfer of IRF. Therefore, IRF may be used to determine whether innate immunity inhibition by HBV polymerase occurs via IRF signaling. Anger was investigated.

3-1. Inhibition of IRF Phosphorylation by HBV Polymerase

In order to investigate the inhibition of IRF phosphorylation by HBV polymerase, transfected poly (I: C) to HepG2 cells, liver cancer cell line, and then IRF phosphorylation according to HBV polymerase expression was performed by Western blot using IRF phosphorylation specific antibody. It was investigated.

3-1.1 Experimental Materials and Methods

Cells were HepG2 cells and were cultured in a 5% C02 incubator using DMEM medium with 10% fetal calf added. Transfection was followed by PEI (polyethylenimine) and transfected about 12 ug of plasmid DNA into a 60 mm diameter plate (Ryu, Kim, and Ryu, 2008). The polelasme used in the experiment was received from the following researchers: DDX3 (KT Jeang, National Institute of Health, USA). The following antibodies were each purchased from the following manufacturers: anti-IRF3 (Zymed Inc.) and ant i-phospho-Ser396-IRF3 (Cell Signaling Inc.). ^' Western blot complies with the standard method. Specifically, after 2 days of transfection, the cells were prepared as a complete solution containing 1% NP-40 (50 niM Tris-Cl [pH 7.4], 50 mM NaCl, 5 mM EDTA, 1 The protein was electrophoresed with 12% SDS-PAGE gel after harvesting with% NP-40), the protein was blotted with PVDF membrane (Immobi lon-P; Millipore), and then detected with antibody. Figure 5 HepG2 cells were added to the poly (I: C) medium, and after transfection of the HBV polymerase expression plasmid, cells were harvested three days later. Samples were electrophoresed by SDS-PAGE and phosphorylated IRF was detected by Western blot using phosphorylated IRF antibodies. As a result, phosphorylated IRF was detected after transfection of poly (I: C), but phosphorylated IRF was not detected when HBV polymerase was expressed (FIG. 5). This result indicates that HBV polymerase inhibits IRF signaling.

Example 4 HBV polymerase target in IRF signaling was identified as a subordinate of RIG-I or TIRF molecule

In order to identify the target of IRF signaling by HBV polymerase, RIG-I and TRIF molecules were expressed instead of poly (I: C) to trigger IRF signaling and investigated IRF signaling inhibition by HBV polymerase. It was. 4-1. Inhibition of IRF Signaling Activated by RIG-I

The expression of RIG-I molecules triggered IRF signaling and investigated the inhibition of IRF signaling by HBV polymerase. Unlike FIG. 4, IRF3-luci f erase reporter _plasmids were used to investigate IRF signaling inhibition (Schroder, Bar an, and Bowie, 2008). Here, the IRF3-lucif erase reporter plasmid means a combination of an IRF3 / 7-GAL4 fusion protein expression plasmid and a pFR-luc if erase reporter plasmid having a GAL4 dependent promoter. Specifically, the cells were HepG2 cells, and cultured in a 5% C02 incubator using DMEM medium added with 10% fetal calf. Transfection is

PEI (polyethylenimine) was followed, and about 12 ug of plasmid DNA was transfected into a 60 mm diameter plate (Ryu, Kim, and Ryu, 2008). The plasmids used in the experiments were each obtained from the following researchers: IRF3-GAL4, IRF7-GAL4, pFR luciferase reporter polelasmid (Dr. Bowie, Trinity College Dublin, Ireland) and Flag-RIG-I (Dr. Fujita, Kyoto University). ). pFR luciferase reporter polamide is a reporter plasmid with GAL4 binding site and is designed to measure the transcriptional activity of IRF3 / 7 by binding IRF3 / 7-GAL4 fusion protein through GA binding site. For example, in order to measure IRF3 / 7 promoter activity, transfection was performed together with an IRF3 / 7-GAL4 fusion protein expression plasmid and a pFR-luciferase reporter plasmid (Stack et al., 2005). Specifically, transfection of RIG-I molecule expression plasmid and IRF3-luci f erase construct in HepG2 cells and transfection with gradual increase of HBV polymerase expression vector plasmid were performed. Measured and expressed in multiples compared to the control group (FIG. 6). As a result, the IRF3 promoter activity decreased in proportion to the HBV polymerase concentration. In other words, this result means that the target of HBV polymerase is located under the RIG-I molecule.

4-2. Inhibition of IRF Signaling Activated by TRIF

The expression of TRIF molecules triggered IRF signaling and investigated the inhibition of IRF signaling by HBV polymerase. Unlike FIG. 3, IRF3_luci f erase construct IRF signaling inhibition was investigated (Schroder, Bar an, and Bowie, 2008). Cells were HepG2 cells and were cultured in 5) C02 incubator using DMEM medium added with 10% fetal calf. Transfection followed the method using PEI (polyethylenimine) and transfected about 12 ug of plasmid DNA on a 60 mm diameter plate (Ryu, Kim, and Ryu, 2008). The polyamides used in the experiment were each obtained from the following researchers: IRF3-GAL4, IRF7-GAL4, pFR luciferase reporter (Dr. Bowie, Trinity College Dublin, Ireland) and Flag-TRIF (Dr. Akira, Osaka).

Universi ty). Specifically, after transfecting TRIF molecule expression plasmid and IRF3_luci f erase plasmid into HepG2 cells, and transfecting gradually increasing the amount of HBV polymerase expression vector plasmid, luciferase activity was measured in comparison with the control group. And expressed as a multiple compared with the control group (FIG. 7). As a result, the IRF3 promoter activity decreased in proportion to the HBV polymerase concentration. In other words, this result means that the target of HBV polymerase is located below the TRIF molecule in IRF signaling.

Example 5 Results clarifying that the target of HBV polymerase is TBKl / IKKe in IRF signaling

In order to identify the target of IRF signaling by HBV polymerase, the expression of TBKl / ΙΚΚε and IRF3 molecules instead of poly (I: C) triggered IRF signaling and investigated the inhibition of IRF signaling by HBV polymerase. Specifically Example 5 shows that HBV polymerase targets ΤΒΚΙ / ΙΚΚε molecules.

5-1. Conceptual Diagram of IRF Signaling

As shown in FIG. 8, the interrelationship between signaling molecules involved in IRF signaling has already been identified (Bowie and Unterholzner, 2008). In particular, it has recently been reported that DDX3, a DEAD-box RNA helicase, promotes the activity of ΤΒΚΙ / ΙΚΚε (Schroder, Bar an, and Bowie, 2008; Soul at et al., 2008).

5-2. Inhibition of IRF Signaling Activated by TBK1

To identify the targets of HBV polymerase, the expression of TBK1 molecule was used to generate IRF signaling. After triggering, we investigated the inhibition of IRF signaling by HBV polymerase. Unlike FIG. 4, IRF signaling inhibition was investigated using an IRF3-luc if erase construct (Schroder, Bar an, and Bowie, 2008). HepG2 cells were used and cultured in a 5% CO ₂ incubator using DMEM medium supplemented with 10% fetal calf. Transfection followed the method using polyethylenimine (PEI) and transfected approximately 12 ug of polamide DNA into a 60 mm diameter plate (Ryu, Kim, and Ryu, 2008). The plasmids used in the experiment were each obtained from the following researchers: IRF3-GAL4, IRF7-GAL4, pFR luc if erase reporter (Dr. Bowie, Trinity College Dublin, Ireland), TBK-Flag, (Dr. Fitzgerald,

University of Massachusetts Medical School, Worcester, MA). Specifically, after transfecting TBK1 molecule expression plasmid and IRF3_luci f erase plasmid into HepG2 cells and gradually increasing the amount of HBV polymerase expression vector plasmid, the kic if erase activity was controlled with the control group. Measured by comparison and expressed in multiples compared with the control group (FIG. 9). As a result, the IRF3 promoter activity decreased in proportion to the HBV polymerase concentration. In other words, this result means that the target of HBV polymerase is at or below the TBK1 molecule.

5-3. Inhibition of IRF Signaling Activated by ΙΚΚε

In order to identify the target of HBV polymerase, IRF signaling was stimulated by expressing ΙΚΚε molecules and examined for inhibition of IRF signaling by HBV polymerase. Unlike FIG. 3, IRF3—lucif erase reporter plasmid using IRF3

Signaling inhibition was investigated (Schroder, Bar an, and Bowie, 2008). Specifically, the cells were HepG2 cells, and cultured in a 5% C02 incubator using DMEM medium added with 10% fetal calf. Transfection is

PEI (polyethylenimine) was followed, and about 12 ug of plasmid DNA was transfected into a 60 micrometer plate (Ryu, Kim, and Ryu, 2008). The plasmids used in the experiments were each given to the following researchers: IRF3-GAL4, IRF7-GAL4, pFR luci ferase reporter (Dr. Bowie, Trinity Col lege Dublin, Ireland) and IKK ε-Flag (Dr. Fitzgerald, University of Massachusetts) Medical School, Worcester, MA). Specifically, transfected ÎΚΚε molecule expression plasmid and IRF3-luci ferase reporter plasmid into HepG2 cells and transfected with increasing amounts of HBV polymerase expression vector full plasmid gradually, and then compared luci ferase activity with control group. Measured and expressed in multiples compared to the control group (FIG. 10). As a result, the IRF3 promoter activity decreased in proportion to the HBV polymerase concentration. In other words, this result means that the target of the HBV polymerase is at or below the ΐΚΚε molecule.

5-4. Inhibition of IRF Signaling Activated by ΙΚΚε

In order to identify the target oligomers of HBV polymerase, IRF expression was triggered by expressing ΙΚΚε molecule and then IRF signaling inhibition by HBV polymerase was investigated. Unlike FIG. 2, IRF3-luci ferase reporter plasmid was used to investigate IRF signaling inhibition (Schroder, Bar an, and Bowie, 2008). Cells were HepG2 cells and were cultured in a 5% CO ₂ incubator using DMEM medium supplemented with 10% fetal calf. Transfection followed the method using polyethylenimine (PEI) and transfected approximately 12 ug of plasmid DNA into a 60 mm diameter plate (Ryu., Kim, and Ryu, 2008). Each of the polelasmids used in the experiment was given by the following researchers: ΙΚΚε -Flag (Dr.

Massachusetts Medical School, Worcester, MA). The following plasmids were purchased from each manufacturer: ISRE-luci ferase construct (Stratagene Inc.). Specifically, transfecting ΙΚΚε molecule expression plasmid and ISRE-luci ferase reporter plasmid into HepG2 cells and transfecting with increasing amounts of HBV polymerase expression vector plasmid gradually, luciferase activity was measured and compared with the control group. It was expressed in multiples compared to the control group (FIG. 11). As a result, the ISRE promoter activity decreased in proportion to the HBV polymerase concentration. In other words, this result means that the target of HBV polymerase is at or below the ΙΚΚε molecule.

5-5. Inhibition of IRF Signaling Activated by IRF3

In order to identify the target of HBV polymerase, the expression of TRIF molecules triggered IRF signaling and the inhibition of IRF signaling by HBV polymerase was investigated. Unlike FIG. 4, IRF using an ISRE-luci ferase reporter plasmid Signaling inhibition was investigated (Schroder, Bar an, and Bowie, 2008). Specifically, HepG2 cells were used and cultured in a 5% C¾ incubator using DMEM medium containing 10% fetal calf. Transfection is

PEI (polyethylenimine) was followed, and about 12 ug of plasmid DNA was transfected into a 60 mm diameter folate (Ryu, Kim, and Ryu, 2008). Each of the polelasmids used in the experiment was given to the following researchers: IRF3 (Dr. Bowie, Trinity College Dublin, Ireland). The following plasmids were purchased from each manufacturer: ISRE-luci f erase construct (Stratagene Inc.). Specifically, after transfecting IRF3 expressing plasmid and ISRE-luci f erase reporter plasmid in HepG2 cells, and transfecting gradually increasing the amount of HBV polymerase expression vector polamide, luc if erase activity was compared with control group. Was measured and expressed in multiples compared with the control group (FIG. 12). As a result, the ISRE promoter activity decreased in proportion to the HBV polymerase concentration. In other words, this result means that the target of HBV polymerase is located on top of the ISRE molecule.

Example 6 Results Showing Antagonistic Action of DDX3 on IRF Signaling Inhibition of HBV Polymerase

The results of Example 5 suggest that the target of HBV polymerase is a TBK1 / IKK ε molecule of IRF signaling. Recently, DDX3 has been reported to promote the activity of TBK1 / IKK ε serine kinase, an effector kinase of IRF signaling (Schroder, Bar an, and Bowie, 2008; Soul at et al., 2008). This suggests that IRF signaling inhibition of HBV polymerase is the result of the interaction of DDX3 with HBV polymerase. Example 6 shows an experimental result that is consistent with the above prediction.

6-1. Results showing antagonism of DDX3 against HBV polymerase I.

It was investigated whether the expression of DDX3 restored IRF signaling inhibition by HBV polymerase.

Specifically, HepG2 cells were used and cultured in a 5% CO ₂ incubator using DMEM medium with 10% fetal calf added. Transfection is

PEI (polyethylenimine) was followed, and about 12 ug in a 60 薩 diameter plate. Plasmid DNA was transfected (Ryu, Kim, and Ryu, 2008). The plasmids used in the experiment were obtained from the following researchers, respectively: IRF3-GAL4, IRF7-GAL4, pFR luciferase reporter, IFNP-Luc reporter (Dr. Bowie, Trinity Col lege Dublin, Ireland), TBKl-Flag (Dr. Fitzgerald, University of Massachusetts Medical School, Worcester, MA), DDX3 (Dr. KT Jeang, National Institute of Health, USA). Specifically, the expression of TBK1 molecules in HepG2 cells triggered IRF signaling, and the inhibition of IRF signaling by HBV polymerase was investigated by gradually increasing the amount of DDX3 expressing plasmid. 13 is transfected TBK1 expressing full plasmid, IRF3 luciferase reporter plasmid and HBV polymerase expressing plasmid in HepG2 cells, and luciferase activity was measured in comparison with the control group and expressed in folds compared with the control group. At this time, the effect of increasing the amount of DDX3 expression plasmid was observed. As a result, the IRF3 promoter activity was restored in proportion to the expression of DDX3. In other words, this result indicates that DDX3 antagonizes IRF signaling inhibition by HBV polymerase. Unlike Example 6-1, instead of TBK1, the expression of ΙΚΚε triggered IRF signaling, and the amount of DDX3 expressing plasmid was gradually increased, thereby inhibiting IRF signaling inhibition by HBV polymerase.

Cells were HepG2 cells and were cultured in a 5% CO ₂ incubator using DMEM medium supplemented with 10% fetal calf. Transfection followed the method using PEI (polyethylenimine) and transfected about 12 ug of polamide DNA into a 60 mm diameter plate (Ryu, Kim, and Ryu, 2008). The plasmids used in the experiment were each given to the following researchers: IRF3-GAL4, IRF7-GAL4, pFR luciferase reporter (Dr. Bowie, Trinity College Dublin, Ireland), IKK ε-Flag (Dr. Fitzgerald,

University of Massachusetts Medical School, Worcester, MA), DDX3 (Dr. KT Jeang, National Institute of Health, USA). FIG. 14 transfected ΙΚΚε expression plasmid, IRF3 Luciferase reporter plasmid and HBV polymerase expression plasmid in HepG2 cells, respectively, and measured luciferase activity in comparison with the control group and expressed it in folds compared with the control group. this When the amount of DDX3 expression plasmid was increased, the effect was observed. As a result, similar to Example 6-1, IRF3 promoter activity was restored in proportion to the expression of DDX3 (FIG. 14). In other words, this result indicates that DDX3 antagonizes the inhibition of IRF signaling by HBV polymerase.

6-3. Results show that HBV polymerase inhibits IKKe_DDX3 binding.

The results of Examples 6-1 and 6-2 predict that HBV polymerase inhibits IRF signaling by blocking the interaction between DDX3- ΙΚΚε. Immunoprecipitation was performed to investigate this hypothesis. Cells were HepG2 cells and were cultured in a C02 incubator using DMEM medium supplemented with 10% fetal calf. Transfection followed the method using PEI (polyethylenimine) and transfected about 12 ug of plasmid DNA on a 60 mm diameter plate (Ryu, Kim, and Ryu, 2008). The pollamides used in the experiments were each obtained from the following researchers: ΙΚΚε -Flag (Dr. Fitzgerald, University of

Massachusetts Medical School, Worcester, MA), DDX3 (Dr. K. T. Jeang, National Institute of Health, USA). Immunoprecipitation was followed by standard methods (Pyronnet et al., 1999), specifically 3 days after transfection, cells were lysed in lye buffer buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, and 1% NP-40], and then prepared by adding, culturing and centrifuging protein G-agarose beads to the cell lysate first. The anti-Flag antibody, a primary antibody, was added thereto, followed by culturing and centrifugation to harvest an immunoconjugate. The harvested immunoprecipitates are washed five times or more with the complete solution, and then dissolved in Lae 隱 Π sample solution and analyzed by electrophoresis with 2 SDS-PAGE.

Specifically, after transfection of DDX3 expressing plasmid and ΙΚΚε expressing plasmid in the cells, the mutual binding between DDX3-ΙΚΚε molecules was confirmed through immunoprecipitation method (FIG. 15, lane 3). On the other hand, when the transfection of the HBV polymerase expression plasmid in the experimental group, the cross-linking between DDX3-IKKe was significantly reduced (Fig. 15, lane 4). This result demonstrates that HBV polymerase inhibits IRF signaling by blocking the interaction between DDX3-IKI molecules (FIG. 16).

<Example 7> Results showing application of hepatitis B treatment active substance screening The experiment of Example 2 may be carried out on a 96 well plate to screen for the hepatitis B therapeutic active substance. Using the compound library containing about 300 organic small molecules obtained from the inventor's laboratory, the active substance screening for hepatitis B treatment was carried out by the following method-The compound library is azaguanine-8, al lantoin, acetazol amide, metformin hydrochloride , atracurium besylate, isof lupredone acetate, amilor ide hydrochloride, amprol ium hydrochloride, hydrochlorothiazide and sul faguanidine. Specifically, HEK293 cells were used, and cultured in a 5¾ C0 ₂ incubator using DMEM medium containing 10% fetal calf. Transfection is

The method using polyethylenimine (PEI) was followed, and the cleavage map of IFN β-Luc reporter plasmid (Dr. Bowie, Trinity College Dublin, Ireland), TLR3 expression plasmid (InvivoGen Inc.), FIG. HBV polymerase expressing plasmids having been transfected intracellularly. After 2 days, poly (I: C) was treated with medium for about 12 hours, and then luciferase activity was measured through a reporter assay. The reporter assay was carried out according to the method of "(Victor 3 Inc. Perk in-Elmer Inc.) when the following plasmid was prepared 24 hours after transfection in a 96 well plate. IFNp luc if erase reporter fulllasmid IFNP promoter activity was designed to be conveniently measured using luciferase and is a well known plasmid. Specifically, the IFNP promoter is composed of an IRF3 / 7 binding site and an NF-kB binding site to measure the activation of the two transcription factors. Specifically, as in Example 3 of Example 2, as the amount of HBV polymerase expression plasmid increased, luciferase activity decreased (FIG. 17), and Compounds # 1 to 10 of the compound library. In the case of treatment, luciferase activity was not restored.In conclusion, the 300 compounds investigated in this example did not recover promoter activity. It is judged that there is no activity as a therapeutic agent for hepatitis B. This example is described for the purpose of illustration of chronic hepatitis B virus therapeutically active substance screening.

Industrial Applicability The screening system of hepatitis B therapeutically active substance according to the present invention can easily detect drugs that induce secretory activity of innate immunity (interferon) by inhibiting the interaction of HBV primerase and DDX3. It can be usefully used for developing hepatitis therapeutics.

Claims

[Range of request]

[Claim 1]

a) a nucleotide sequence encoding HBV polymerase comprises a first recombinant expression vector; b) a hepatitis B therapeutically active substance comprising an interferon beta (IFNP) promoter and a recombinant expression vector comprising a reporter gene operably linked thereto and for measuring the activity of the interferon beta (IFNP) promoter. Composition for screening.

[Claim 2]

The method of claim 1,

The composition is c) a composition for screening hepatitis B therapeutically active material comprising animal cells having the ability to produce interferon during virus infection.

[Claim 3]

The method of claim 1,

The composition is d) a composition for screening a hepatitis B therapeutically active material comprising a third recombinant expression vector comprising a gene encoding an interferon production stimulating factor.

[Claim 4]

The method of claim 3,

The interferon generation stimulating factor is TRIF, RIG- I / MDA-5, IPS-1, ΤΒ Ι / ε ε for the screening composition of the hepatitis B therapeutic active material, characterized in that any one or more selected from the group consisting of molecules. .

[Claim 5]

The method of claim 1 wherein ^'

The reporter gene is E. coli β-galactosidase, luciferase, alkaline ine phosphatase, secreted placental alkaline phosphatase or A chloramphenicol acetyltransferase (CAT) composition for screening a hepatitis B therapeutically active substance.

[Claim 6]

A method for screening a hepatitis B therapeutically active substance comprising the following steps.

(1) The first recombinant expression vector and the interferon beta (IFNP) promoter and the interferon beta (IFNP) promoter are operably linked to an animal cell having interferon-producing ability during viral infection. Transfection of a second recombinant expression vector comprising a reporter gene for measuring activity of the Doing;

(2) adding an interferon production promoter to the animal cell;

(3) measuring the activity of the interferon beta (IFN | 3) promoter by adding candidates to the animal cells; And

(4) selecting a candidate material in which the activity of the IFNp promoter is increased when the candidate material is added as compared with the case where the candidate material is not added.

[Claim 7]

(1) In an animal cell having an interferon-producing ability during virus infection, a) a sequence encoding a HBV polymerase sequence 1 recombinant expression vector, b) an interferon beta (IFNP) promoter and operably linked thereto. (IFN | 3) a second recombinant expression vector comprising a reporter gene for measuring promoter activity and c) a third expression vector comprising an interferon-producing promoter and a gene encoding an interferon-producing stimulating factor. Transfecting any one or more substances selected from the group;

(2) measuring the activity of the interferon beta (IFN3) promoter by adding candidates to the animal cells; And

(3) selecting a candidate substance whose activity of the IFNp promoter is increased when the candidate substance is added as compared with the case where the candidate substance is not added.

[Claim 8]

The method according to claim 6 or 7,

The interferon production promoter is characterized in that the poly (I: C).

[Claim 9]

The method of claim 6,

In the step (1), further comprising a third recombinant expression vector comprising a gene encoding the interferon production stimulating factor.

[Claim 10]

The method according to claim 7 or 9,

The interferon production stimulating factor is any one or more selected from the group consisting of TRIF, RIG- I / MDA-5, IPS-1, ΤΒΙ / ΙΚΚε molecules.

[Claim 11]

The method according to claim 6 or 7, The reporter gene is E. coli β-galactosidase, luciferase, alkaline ine phosphatase, secreted placental alkaline phosphatase Or chloramphenicol acetyltransferase (CAT).

[Claim 12]

(1) a second recombinant expression vector comprising a base sequence encoding an HBV polymerase, a first recombinant expression vector, a base sequence encoding an IRF3-GAL4 fusion protein, in an animal cell having an interferon-producing ability during virus infection; transfecting a third recombinant expression vector comprising a pFR promoter and a nucleotide sequence encoding pFR luciferase and a fourth expression vector comprising a gene encoding an interferon stimulating factor step;

(2) measuring the activity of the pFR promoter by adding a candidate to the animal cells; And

(3) selecting the candidate material having increased activity of the ΙΡΝβ promoter when the candidate material is added as compared with the case where the candidate material is not added.

[Claim 13]

The method of claim 12,

The interferon generation stimulating factor is at least one selected from the group consisting of TRIF, RIG- I / MDA-5, IPS-1, ΤΒΚΙ / Ιε molecule.

[Claim 14]

Screening system for treatment of hepatitis B therapeutic activity by adding candidates to HBV polymerase-expressing animal cells to select candidates that inhibit binding of HBV polymerase and DDX3

[Claim 15]

A screening system for treating hepatitis B therapeutics, which selects candidates that inhibit the binding of HBV polymerase to DDX3 by adding candidates to animal cells expressing HBV polymerase, thereby inducing binding of DDX3 to ΤΒΚΙ / ΙΚε.