KR101221777B1 - Composition for treating liver diseases comprising a Hsp90 inhibitor and Method for screening an agent for treating liver diseases through inhibition of Hsp90 and Core protein interaction - Google Patents

Abstract

The present application provides a method for screening a liver disease therapeutic agent comprising a Hsp90 inhibitor and inhibiting the interaction between Hsp90 and a core protein. The therapeutic agents herein may be used effectively in the treatment of liver diseases, which may be caused in particular by hepatitis B virus infection.

Description

Composition for treating liver diseases comprising a Hsp90 inhibitor and method for screening an agent for treating liver diseases through inhibition of Hsp90 and Core protein interaction}

The present invention relates to the treatment of liver disease and the field of screening thereof.

An estimated 350 million people worldwide are infected with Hepatitis B Virus (HBV), of which 5 to 10% suffer from chronic hepatitis B for life, with chronic hepatitis B (Hepatocellular Carcinoma, HCC) (Ahn et al., 2005; Montalto, G. et al., 2002). Hepatocellular carcinoma is the fourth most incidence cancer in Korea (National Cancer Information Center, 2003-2005). The main treatments for liver cancer include liver resection, hepatic artery embolization, chemotherapy, radiotherapy, and liver transplantation, but there are no anticancer drugs that can act specifically on liver cells (National Cancer Information Center). In order to develop anticancer drugs that can act specifically on liver cancer cells, the development of therapeutic methods based on the understanding of liver cancer mechanisms at the molecular biological level should be prioritized.

HBV, which causes chronic liver cancer, is a 3.2 kb circular DNA virus whose genome is partially double-stranded, with four overlapping translation frames, from which surface proteins (HBs), core proteins (HBc), and polymerases (HBV) pol), and X protein (HBx) is expressed (Seeger, C. et al., 2000). Core proteins are involved in the packaging of HBV pol, pregenomic RNA (pgRNA) and other proteins and are known to play an important role in the HBV lifecycle ( ibid .). As the production of viruses requires the assembly of core proteins, viral genomes and other proteins, the assembly of core proteins is an important step in HBV replication.

The core protein consists of 183-185 amino acids with two regions consisting of the N-terminal site required for the core assembly and the C-terminal that regulates viral replication ( ibid . ). The core protein forms the capsid of the virus, which plays an essential role in the protection of the packaged virus and host factor. Therefore, in order to develop an effective liver disease treatment agent, it is urgent to develop a target that interacts with the core protein and affects its capsid formation and a drug that targets the protein, but a protein that interacts with the core protein and affects HBV capsid formation. There is no known mechanism in detail and the development of therapeutic methods based thereon.

The present invention provides a therapeutic agent for liver disease and a screening method thereof capable of inhibiting the production of hepatitis virus by targeting the host protein Hsp90 and a protein interacting with it.

The present application provides a composition for treating liver disease due to hepatitis virus infection comprising an Hsp90 inhibitor as an active ingredient.

In addition, the present application, the Hsp90 inhibitor is a substance that inhibits the expression and / or activity of Hsp90, the substance is a low molecular compound, antibodies, antisense oligonucleotides, siRNA, shRNA, miRNA, and polypeptides, hepatitis virus infection It provides a composition for treating liver disease.

The application also provides the steps of providing Hsp90 and HBV core proteins; Contacting the protein with a test substance expected to be able to selectively inhibit the interaction of the Hsp90 with the HBV core protein; And detecting the interaction between the Hsp90 and the HBV core protein, wherein the interaction between the Hsp90 and the HBV core protein is reduced when contacted with the test substance in comparison with the control group not in contact with the test substance. Provided is a method for screening a therapeutic agent for liver disease that selectively inhibits the interaction of Hsp90 and HBV core protein gene, comprising selecting a substance as a candidate.

The present application also provides that the Hsp90 protein comprises at least residues 520-615 based on the full length or the sequence of Table 1, wherein the HBV core protein comprises at least residues 1-149 or residues 111-130 based on the full length or sequence of Table 1. To provide a method for screening a liver disease therapeutic substance.

The present application also provides a method for screening a liver disease therapeutic substance, wherein the Hsp90 and HBV core proteins are provided in the form of cells expressing the same.

The present application also provides a method for screening a liver disease therapeutic substance, wherein the cells are HepG2, Huh7, Hep3B or PLC / PRF / 5.

The present application also provides a method for screening a therapeutic agent for liver disease, wherein the interaction of the Hsp90 and HBV core proteins is measured by extracellular HBV nucleic acid concentration.

Inhibition of HBV production by inhibiting the activity and / or expression of Hsp90 can be effectively used for the treatment of diseases caused by hepatitis virus infection.

Figure 1 shows the results showing the binding between Hsp90 and HBV core protein. (A) Coimmunoprecipitation result of anti-HBV core Ab and anti-Hsp90 Ab in human liver cancer cell line Huh7 expressing HBV core protein; (B) In vitro binding of HBV core protein Cp140 dimer / capsid with Hsp90 and co-immunoprecipitation using anti-HBV core Ab and anti-Hsp90 Ab to confirm binding (core protein dimer; top panel, capsid; bottom) panel); (C) Immunization with anti-HBV core Ab and anti-Hsp90 Ab after binding assay with core protein Cp140 dimer (top panel) / capsid (bottom panel) using His-tagged Hsp90 and Ni-NTA agarose columns As a result of the blot, each lane is as follows: lane 1, purified Cp149; Lane 2, purified Hsp90; Lane 3. Ni-NTA agarose alone; Lane 4, Hsp90 immobilized on agarose; Lane 5, flow through; Lanes 6-8, 50 mM imidazole wash (to show protein that does not bind agarose); Lane 9, 500 mM imidazole eluate.
FIG. 2 shows the results of Hsp90 incorporation into HBV capsid through interaction with HBV core protein. (A) After mixing HSP90 and Cp149 dimer, the capsid assembly was subjected to sucrose density gradient analysis and immunoblot using anti-HBV core Ab and anti-Hsp90 Ab; (B) Dot blot (top panel), unmodified 0.9% agarose gel electrophoresis (middle panel) and 15% SDS PAGE analysis for sucrose concentration gradient fractions 3 and 8 (lanes 3 and 4, respectively) of A) Post-immunoblotting with anti-HBV core Ab and anti-Hsp90 Ab; (C) Dot blot results using anti-HBV core Ab and anti-Hsp90 Ab showing that Hsp90 was incorporated into the capsid.
3 shows that Hsp90 promotes HBV core protein assembly. 3A shows 0.9% agarose gel electrolysis after reacting Cp149 dimer (A) and its point mutation (C61A) (B) under assembly conditions (Hsp90, p23, 0.5 mM ATP-γ-S, and 2 μM GA) Immunoblot results using anti-HBV core antibody after electrophoresis were analyzed for core, total Cp149 dimer and C61A by 15% SDS PAGE. Figure 3B shows (C) sucrose density gradient fraction analysis and anti-capsid formation of Cp149 dimers with negative controls (BSA top panel), ATP-activated Hsp90 (middle panel) and GA-inactivated Hsp90 (bottom panel). As a result of immunoblot with Hsp90 Ab, the line graph shows the band pattern of each panel fraction, and the bar graph shows the amount of capsid formed in fractions 7 to 10. Figure 3b (D) is a result of observing the capsid formation in vitro by immunoblot over time, Cp149 and negative control (BSA, top panel), ATP-activated Hsp90 (middle panel) and Hsp90 (N190 △) Capsid formation with (bottom panel) is shown and line graph band strength is shown. Intensity was used as the value of 1 as a standard based on the reaction of BSA and Cp149 dimer for 15 minutes. The experiment was repeated three times and the error bar was the standard deviation of the three experiment results.
4 is a result showing that Hsp90 promotes HBV core assembly over a wide range of temperatures. (A) Cp149 dimer was incubated with BSA (control protein; left panel) at 30 ° C. for 30 minutes, and then cultured when the temperature was changed for 30 minutes. ATP-γ-S (0.5 mM) was added to Hsp90 at 30 ° C. and reacted for 30 minutes (middle panel), GA (2 μM) was added to Hsp90 and reacted at 30 ° C. for 30 minutes (right panel). Each mixture was reacted for 30 minutes at Cp149 dimer and at various temperatures indicated in the figure. The control group (C) was reacted with only Cp149 dimer at 30 ° C. for 30 minutes. The amount of capsid produced was detected by immunoblot using anti-HBV core antibody after electrophoresis of the mixture on 15% SDS-PAGE. The graph below is a graph showing the quantitative band intensities of the immunoblot gel photographs, and the control value was set to 1. (B) Huh7 cells were transfected with pCMV / Flag-core followed by addition of 4 μM GA (right panel) or without GA treatment (left panel) for 2 hours at 30, 35, 37, 40. and 43 ° C. And then cultured. Capsid was subjected to immunoblot using anti-FLAG M2 antibody after electrophoresis on 0.9% agarose gel. Hsp90, betaactin and HBV core protein expression was immunoassayed after electrophoresis on 15% SDS-PAGE. The graph below shows the intensity of each band in the gel photograph, and the band intensity of the sample incubated at 37 ° C. described in the left panel was 1, and other values were expressed based on this. The experiment was repeated three times and the error bar was the standard deviation of the three experiment results.
5 shows that Hsp90 inhibits HBV capsid dissociation under surfactant treatment conditions. FIG. 5A shows Cp149 dimers at 37 ° C. for 30 minutes to form capsids followed by addition of (0-3M) urea, followed by HSA 90 treated with BSA, ATP-activated Hsp90, or GA and 30 at 37 ° C. FIG. After incubation for min, electrophoresis on 0.9% agarose gel, followed by immunoblot with anti-HBV core antibody (top panel), stained with Coomassie blue (bottom panel). FIG. 5B shows (B) 0.9% agarose gel immediately after treatment with a capsid at 37 ° C. for 30 minutes with different concentrations of SDS (0-0.5%) in the presence of BSA, ATP-activated Hsp90, or GA treated Hsp90. This is the result of electrophoresis analysis. Capsid and denatured capsid were quantified by staining with Coomassieblue. In Figures 5a and 5b the graph below shows the band strength on the gel, with the intensity of C (the amount of capsid not treated with surfactant) being 1, and the other values are indicated against this. The experiment was repeated three times and the error bar was the standard deviation of the three experiment results. 5C is a capsid electron micrograph, C is a detergent untreated sample, D is a capsid photograph treated with Hsp90 or ATP-activated Hsp90 inactivated with GA in 3M urea, E is 0.05% SDS under the same conditions as D A picture of the capsid when treated. Arrows indicate capsids degraded by surfactants (SDS or urea).
6 shows that down expression and inhibition of Hsp90 reduces the amount of extracellular HBV DNA in HepG2.2.15. (A) HepG2.2.15 cells were treated with GA or 3TC, then transfected with shRNA-Hsp90 for 24 hours, and the cells were fused and analyzed by 0.9% native agarose gel electrophoresis to determine HBV capsid (top Panel) and 15% SDS-PAGE to analyze the HBV core protein (bottom panel). (B) The intensity of the HBV capsid band was analyzed by software (ImageMaster 2D Elite 4.01) (black bars) and HBV DNA of cell culture medium was analyzed by quantitative PCR (gray bars) (ABI 7300, Applied Biosystems). . (C) Cell lysates are the result of immunoblot analysis using anti-Hsp90 antibody and anti-HBV core antibody after electrophoresis on 15% SDS-PAGE. Beta-actin was used as a control, the experiment was repeated three times, the error bar is the standard deviation of the results of the three experiments. NCshRNA: negative control of shRNA (pLK0.1-scramble vector).
Figure 7 is an immunoblot analysis showing the binding site between Hsp90 and HBV core protein. 7A shows a series of Hsp90 deletion variants (left panel) and Cp149 dimers at 4 ° C. for 1 hour, followed by immunoblot analysis with the same antibody after coimmunoprecipitation with an anti-HBV core antibody and an anti-His antibody. (Right panel). FIG. 7B shows a series of Cp149 deletion variants (left panel) and point mutations (C61A) and Hsp90 at 4 ° C. for 1 hour, and then the mixture was co-precipitated with anti-HBV core antibody and anti-His antibody to the same antibody. Immunoblot analysis (right panel). Input was 8% total protein mixture and was used as a control. IgG is a negative control. White arrow: protein of interest. Black arrows: heavy and light chains. IP: immunoprecipitation, IB: immmuno lot.

HBV capsid assembly is essential for HBV replication and HBV formation. During capsid assembly, host factors such as heat shock proteins and protein kinases are also packaged into the HBV capsid along with the HBV pol / pgRNA complex to enable HBV replication (Bartenschlager and Schaller, 1992; Hirsch et al., 1990; Hu et. al., 2004; Hu et al., 1997; Kann and Gerlich, 1994; Nassal, 1999). One of the host factors, Hsp90, is known to affect the priming of polymerase and the formation of HBV pol / pgRNA complexes through interaction with HBV pol (polymerase). However, the relationship between Hsp90 and HBV core proteins and their effects on capsid assembly are unknown.

It was found herein that the host factor Hsp90 catalyzes and inhibits the degradation of the capsid assembly of hepatitis virus through interaction with the HBV core protein, ultimately promoting HBV formation and production and stabilizing the capsid. Furthermore, it was found that inhibition of Hsp90 and downregulation of expression can lead to inhibition of HBV production. Therefore, the substance targeting Hsp90 according to the present invention can be effectively used for the treatment and prevention of various diseases caused by hepatitis virus by fundamentally preventing the replication of HBV. In view of this, the present invention can be usefully used for developing a liver disease treatment system due to hepatitis virus infection targeting Hsp90 and / or proteins interacting with it.

In one aspect, the present application provides a composition for treating liver disease due to hepatitis virus, including an Hsp90 inhibitor. Heat Shock Protein 90 (Hsp90) is a 90 kDa chaparon protein consisting of an N-terminal ATP-binding region, an intermediate region, and a C-terminal dimerization region (Terasawa et al. 2005). Hsp90 function is ATP dependent and involved in protein folding through interaction with other proteins in the cell, such as kinases and transcription factors (Cho et al., 2000a, b; Hu et al., 2004). .

As used herein, the term “liver disease” is a concept that encompasses both acute and chronic liver disease caused by viral infection, such as hepatitis, fatty liver, cirrhosis, liver cancer. In one embodiment, the liver disease is caused by a hepatitis B virus infection.

As used herein, the term “treatment” is a concept that includes inhibiting, eliminating, alleviating, alleviating, ameliorating, and / or preventing a disease or condition or condition resulting from the disease.

The present application relates to the inhibition of HBV production targeting Hsp90, including various known Hsp90 inhibitors having this function. For example, the inhibitor of the present invention is a substance capable of inhibiting the expression (transcription and translation) and / or activity of Hsp90, a protein including small molecule compounds, antibodies and polypeptides, and nucleic acid molecules including RNA and DNA or It is to include any combination of. As used herein, "expression" refers to the transcription of a gene and the expression of a transcript into a protein, and "activity" refers to a biological action that allows the expressed protein to function in the cell. In one embodiment of the present application, the Hsp90 inhibitor is a low molecular weight compound, for example geldanamycin (benzoquinone ansamycin) and its various derivatives or analogs, such as 17-Allylamino-17-demethoxygeldanamycin, BDGA (4,4- difluoro-4-bora-3α, 4α-diaza-s-indacene-geldanamycin) and derivatives or analogs thereof, but are not limited thereto. Geldanamycin binds to the ATP binding region of Hsp90, interferes with ATP binding and inhibits interaction with other proteins. Known materials that act as such a mechanism, as well as Hsp90 inhibitors by other mechanisms, are included herein.

In other embodiments herein, the Hsp90 inhibitor is an antisense oligokyuleide, siRNA (small interfering RNA), shRNA (small hairpin RNA) that can specifically inhibit transcription of the gene encoding it and / or protein synthesis of the transcript ) Or miRNA (microRNA). siRNA, shRNA and miRNA can be transcribed through RNA interference, for example, short-length interfering RNA (siRNA) binds specifically to a transcript to form an RNA Induced Silencing Complex (RISC). The RNA cannot be expressed as a protein in the cell (silencing). siRNA, shRNA or miRNA have a sequence that is significantly complementary to its target sequence. By highly complementary sequence is meant at least about 70% complementarity, at least about 80% complementarity, at least about 90% complementarity, or about 100% complementarity with at least consecutive 15 base length sequences of the target gene. In one embodiment, an antisense oligo that targets Hsp90 genes from a variety of genes may be included as long as the siRNA used for silencing Hsp90 is not limited thereto. Nucleotides, siRNAs, shRNAs and / or miRNAs may be used, including biological equivalents, derivatives and analogs thereof. Antisense oligonucleotides are as known in the art and are, for example, short synthetic nucleic acids that bind to any coding sequence of a target protein, thereby inhibiting / reducing the expression of the target protein. The antisense RNA may have an appropriate length depending on the target gene and the method of delivery, for example about 6, 8 or 10 to 40, 60 or 100 nucleotides. In one embodiment the siRNA, shRNA, antisense oligonucleotide and / or miRNA are derived from the human Hsp90 gene, for example human derived Hsp90 gene is NM_007355 (GI: 20149593), NM_003299 (GI: 4507676), NM_001017963 (GI: 153792589 ), Or NM_005348 (GI: 154146190) (NCBI Accesion number), but is not limited thereto. Hsp90 protein sequences include, but are not limited to, GI: 56204416.0 (Embl: AL139392.10) NP_03181 (GI: 20149594.0) or NP_005339.3 (GI: 154146191.0). In one embodiment the Hsp90 gene sequence as used herein is the NM_007355 protein sequence is NP_03181.

In other embodiments herein, the Hsp90 inhibitor is an antibody that can specifically recognize it. As used herein, the term antibody is intended to include intact antibodies, antigen-binding fragments of antibody molecules and antibodies or fragments functionally equivalent to these. In addition, the antibody of the present invention may be of the IgG, IgM, IgD, IgE, IgA or IgY type, and may be of the IgGl, IgG2, IgG3, IgG4, IgA1 or IgA2 class or subclass thereof. Antibodies of the invention include monoclonal, polyclonal, chimeric, single chain, bispecific, amatogenic and humanized antibodies and active fragments thereof.

In other embodiments herein, the Hsp90 inhibitor is a polypeptide that can interfere with, interfere with and / or inhibit its activity and / or action / mechanism. The term polypeptide, as used herein, refers to a polymer of amino acids, either natural or synthetic, and does not refer to a particular length as long as it has this action, and includes peptides, oligopeptides. In one embodiment the polypeptide is a polypeptide comprising all or a portion of residues 520 to 615 of Hsp90 based on the sequences set forth in Table 1, which polypeptide binds to the HBV core protein competitively with normal Hsp90 to It can function as a competitive inhibitor that acts to hinder the action of. Polypeptides include those that are naturally or artificially modified, such as glycosylation, acetylation, phosphorylation, and the like.

The therapeutic agent herein may further contain at least one active ingredient exhibiting the same or similar function in addition to the aforementioned Hsp90 inhibitor, or a compound which maintains / increases the solubility and / or absorption of the active ingredient. In addition, the therapeutic agents herein may be used alone or in combination with methods using surgery, drug treatment and biological response modifiers for the treatment or prevention of liver disease. The therapeutic agent herein may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-mentioned active ingredients. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The method of administration of the therapeutic agent of the present application is not particularly limited, and known administration methods of Hsp90 inhibitors may be applied, and parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, or topical) may be applied according to a desired method. Oral administration, or parenteral administration can be administered via a patch-type, nasal / respiratory tract to the skin, intravenous injection is preferred to obtain a rapid therapeutic effect. Dosage varies widely depending on the patient's weight, age, sex, health status, diet, time of administration, method of administration, rate of excretion, and severity of the disease, the dosage of a known Hsp90 inhibitor can be applied. Parenteral administration may be preferred for protein preparations including siRNAs, miRNAs, antisense oligonucleotides, shRNA preparations and polypeptides targeting Hsp90, but do not exclude other routes and means. For typical drugs, dosage units include, for example, about 0.01 mg to 100 mg but do not exclude the below and above ranges. The daily dose may be about 1 μg to 10 g, and may be administered once to several times a day.

In another aspect, the present invention provides a method for screening a substance capable of treating liver disease by inhibiting the interaction between Hsp90 and a core protein, wherein the substance selected by the method is particularly useful as a liver disease inhibitor / therapeutic agent due to hepatitis virus infection. Do. The method comprises providing Hsp90 and a core protein; Contacting the protein with a test substance that is expected to inhibit the interaction between Hsp90 and the core protein; And it provides a method for screening a substance that inhibits the interaction between the Hsp90 and the core protein comprising the step of comparing the degree of interaction between the Hsp90 and the core protein in the control group not in contact with the test substance.

The core protein of hepatitis B virus consists of 183-185 amino acids in two regions consisting of the N-terminal region (amino acids 1-149; Cp149) required for assembly and the C terminus (amino acids 150-183 or 185) that regulate viral replication. (Seeger et al., 2000). Cp149, from which the 34 C-terminal residues have been cut, forms capsids in vitro and in vivo under appropriate conditions (Kim et al., 2001).

Hsp90s that can be used in the screening methods of the present application are as described above, Hsp90 and core proteins may be used in the present method from a variety of host, depending on the specific method of screening, including both full length or truncated form . In one embodiment Hsp90 comprises at least residues 520-615 based on the sequence set forth in Table 1. In other embodiments Hsp90 comprises at least all or a portion of residues 520-615 based on the sequences set forth in Table 1. In addition, there may be sequence variations according to a specific individual, region, environment, etc., even if they are derived from the same host, for example, humans, and of course, some sequences have been modified (deleted, substituted, added), but all functionally equivalent variants are present. It can be used in the present invention. In one embodiment, hepatitis B virus is derived from a human host. For example, the genes of the hepatitis B virus core protein include the C protein coding gene (1814-2452) included in the GenBank Accession No HQ684848 sequence, and the protein sequence encoded therein (ADV 29803.1), GenBank Accession No AB537951.1 and Protein sequences encoded thereby (BAJ53082.1), or GenBank: BAE79771.1 or BAD91266.1, but are not limited thereto. In one embodiment, the core protein comprises at least 111-130 residues based on the sequence of Table 1. In other embodiments, the core protein may comprise all or part of residues 111-130 based on the sequences in Table 1. In other embodiments, the core protein may be used comprising at least residues 1 to 149 based on the sequence of Table 1 instead of the full length.

In other embodiments, the screening methods herein include full length Hsp90 and full length or HBV core proteins of 1-149 residues (full length sequences illustratively shown in Table 1), Hsp90 proteins comprising all or part of the residues 520-615 above The HBV core protein comprises a full length or 1-149 residues, or the Hsp90 protein is full length, the HBV core protein comprises all or part of the aforementioned residues 111-130, or the Hsp90 protein comprises all or part of the residues 520-615 The HBV core protein may be used in a form comprising residues 1-149 or all or part of the aforementioned residues 111-130.

As used herein, Hsp90 and core proteins can be prepared using methods known in the art. In particular, the method for producing a protein used for screening is to use a genetic recombination technique. For example, the plasmid containing the corresponding gene encoding the protein can be delivered to prokaryotic or eukaryotic cells such as insect cells and mammalian cells, overexpressed and purified. For example, the plasmid may be cloned into a vector such as pET28b (Novagen) as used in an exemplary embodiment of the present application and transferred to a cell line, followed by purification of the expressed protein, but not limited thereto. no.

Or by expressing a DNA or RNA sequence encoding a protein used for screening in a suitable host cell to make the cell lysate or translating the mRNA of the screening protein in vitro and then screening the protein by a protein isolation method known in the art. Can be purified. Usually, in order to remove cell debris and the like, the cell lysate or the result of in vitro translation is centrifuged, followed by precipitation, dialysis, and various column chromatography. Ion exchange chromatography, gel-permeation chromatography, HPLC, reverse phase-HPLC, preparative SDS-PAGE, affinity columns and the like are examples of column chromatography. Affinity columns can be made, for example, using anti-screening protein antibodies.

The contact of the purified proteins can be used directly in vitro to confirm binding in the absence or presence of the test substance. In this case, the protein may be labeled using a variety of markers such as protein tags, biotin, fluorescent materials, acetylation, radioisotopes or commercially available protein labeling kits for ease of detection, It can be detected using a detector suitable for the labeled material. Protein-protein interactions can be measured using various methods known in the art. For example, the East to Hybrid method and the Confocal Microscopy method for confirming the binding / interaction of intracellular proteins. Co-immunoprecipitation, surface plasma resonance (SPR) and spectroscopy methods include, but are not limited to, further references on comparisons and detailed test methods for these methods. Berggard et al., (2007) "Methods for the detection and analysis of protein-protein interactions ", PROTEOMICS Vol7: pp 2833-2842.

In one embodiment, Hsp90 and the core protein may be provided in the form of cells expressing it. Mammalian cells expressing, for example, but not limited to, Hsp90 and core proteins, such as but not limited to HepG2, Huh7, Hep3B, PLC / PRF / 5, 293T cells, cell culture plates After culturing in, the test substance is added thereto, and after a certain time, the total protein is extracted from the cells, and for example, the interaction between Hsp90 and the core protein can be confirmed through co-immunoprecipitation and immunofluorescence. . Inhibition of the interaction between Hsp90 and the core protein can be selected as a candidate for the treatment of liver disease as compared to the control (untreated test material).

The amount of protein used in the present method, the type of cells and the amount and type of test substance vary depending on the specific test method used and the type of test substance, and those skilled in the art will be able to select an appropriate amount. As a result of the experiment, a substance which inhibits the interaction between Hsp90 and the core protein in the presence of the test substance is selected as compared with the control group which is not in contact with the test substance. Less than about 99%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60% A reduction, about 55% reduction, about 50% or less reduction, about 45% or less reduction, about 40% or less reduction, about 30% or less reduction, or about 20% or less reduction, but the range is not excluded.

In the present invention, interaction refers to contact / binding between proteins, and includes both interactions by covalent and non-covalent bonds. As used herein, the term "test substance" refers to a substance that is expected to inhibit the interaction (binding) of the Hsp90 protein and the core protein, and thus, low molecular weight compounds, high molecular weight compounds, nucleic acid molecules (eg, DNA, RNA, PNA and Aptamers), proteins, sugars and lipids, and the like. The test substance may be a polypeptide having two or more amino acid residues, such as 6, 10, 12, 20 or less or more than 20 such as 50 amino acid residues. The protein may comprise an antibody against Hsp90 or a core protein.

For the purpose of screening drugs for inhibiting, treating and / or preventing the occurrence of liver disease, the compound may have a low molecular weight therapeutic effect. For example, compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and small molecule compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely illustrative. It is not limited to this.

Protein-to-protein interactions of the method can be determined by the amount of HBV nucleic acid formed extracellularly. When the interaction is tested intracellularly, the method may further comprise selecting a substance that reduces the amount of HBV nucleic acid compared to the control which is not treated with the test substance. When Hsp90 and the core protein interact with each other, the assembly action of the next step is interrupted, which eventually reduces the amount of HBV produced as described in the Examples herein, and a compound having such an effect is a candidate for treating liver disease. Can be screened. The amount of the HBV nucleic acid may be, but is not limited to, various known methods, for example, real-time quantification, DNA chips, and the like. Such methods may be referred to as appropriate in the literature list described herein.

In addition, the protein-to-protein interaction of the method can be determined by measuring the concentration of the specific protein constituting the generated HBV particles or HBV capsid. For example, reference may be made to appropriate ones in the literature list described herein. In addition, for example, methods using “antigen-antibody complex” formation between polymerases extracted from HBV virus and specific antibodies thereof, Western blot, Enzyme Linked ImmunoSorbant Assay (ELISA), radioimmunoassay, radioimmunodiffuse method, tissue immunostaining, Immunoprecipitation assays, complement fixation assays, FACS (Fluorescent Activated Cell Sorter), protein chips, and the like, but are not limited thereto. Through the above detection methods, the amount of HBV protein expression in the control group and the amount of expression in the test group treated with the test substance are compared and the substance whose amount is reduced is selected as a candidate to inhibit the interaction between Hsp90 and the core protein. do. Such materials may be useful as treatment / inhibitors of liver disease due to liver cancer, in particular hepatitis B virus infection.

In another aspect, the present invention provides a method of preparing a cell comprising: providing a cell expressing Hsp90 and a core protein; Contacting said cell with a test substance; And measuring and comparing the degree of interaction and / or the amount of HBV produced (nucleic acid, and / or protein) in a control group not in contact with the cell in contact with the test substance. do. As mentioned above, substances that inhibit the interaction of Hsp90 and core proteins may be useful candidates for the treatment of liver disease. Cell lines that can be used in the method are as described above, in particular, but not limited to animal cells, more particularly hepatocytes, even more particularly HepG2, Huh7, Hep3B, PLC / PRF / 5. The cell line was transfected with a plasmid expressing Hsp90 and a plasmid expressing the core protein, or a plasmid expressing both the proteins as mentioned above, and then after a certain time, for example 48 hours, HBV was removed from the cell medium. Extraction, the amount of HBV nucleic acid is detected and compared to a control not treated with the candidate. Compounds that reduce the amount of HBV nucleic acid as compared to the control is a substance capable of treating the liver disease targeted in the present invention. The method for measuring the concentration of HBV nucleic acid in the cell extract is as mentioned above, or may be performed according to the method described in this example. The amount of test substance depends on the specific test method used and the type of test substance, and a person skilled in the art can select an appropriate amount. As a result of the test, compared with the control group not in contact with the test substance, in the presence of the test substance, less than about 99% reduction, less than about 95% reduction, about 90% reduction, about 85% reduction, about 80% reduction, about 75% Decrease, about 70% less, about 65% less, about 60% less, about 55% less, about 50% less, about 45% less, about 40% less, about 30% less, about 20 It means a reduction of less than%, but does not exclude the range beyond.

The invention may be practiced using conventional techniques that are within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombination techniques, and immunology, unless otherwise noted. See also the following books and literature for a more detailed description of general techniques. For general methods of molecular biology and biochemistry, see Molecular Cloning: A Laboratory Manual, 3rd Ed. Sambrook et al., Cold Spring Harbor Laboratory Press 2001; Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. Eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., Eds.); And Recombinant DNA Methodology (Wu, ed., Academic Press). See also Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., Eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986); Protein Methods (Bollag et al., John Wiley & Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. Eds., Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., Academic Press 1995); Immunology Methods Manual (Lefkovits ed., Academic Press 1997); And Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). For general techniques regarding cell culture and medium, see Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); And Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251).

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

Materials and Methods

Cell culture and transduction transfection )

Human liver cancer cell lines, Huh7 and HepG2.2.15 were cultured in DMEM (Dulbecco's modified Eagle's medium, Welgene, Korea) containing 10% fetal bovine serum (FBS, Invitrogen, USA) at 37 ° C., 5% CO ₂ . Cells were transfused using Fugene 6 (Roche, Germany) using the manufacturer's instructions.

Cp149 , Cp149  Point mutation construction p23  Protein expression and purification

Cp149 was cloned into pET30a vector (Novagen, USA) and then cloned into BL21 (DE3) + pLysS E. coli After transformation into Novagen, proteins were isolated as previously described (Choi et al., 2005). The core protein point mutation C61A (61 th cysteine residue replaced with alanine residue) was constructed using the site directed mutation protocol (Qiagen, Germany). Using pCMV / Flag-core (Kang et al., 2008) as a template, a forward primer (F) 5'-CAGGCAAGCTATTCTGGCTTGGGGTGAGTTGATG-3 'and a reverse primer (R) 5'CATCAACTCACCCCAAGCCAGAATAGCTTGCCTG-3' were used.

Purified core protein dimer was stored in 100 mM glycine (pH 9.5) which can stably store the protein. For Cp149 assembly, reaction buffer (50 mM Hepes, pH 7.5), 5 mM MgCl ₂ , 15 mM NaCl, and 10 mM CaCl ₂ was mixed with Cp149 and reacted for 30 minutes at 37 ° C. (Choi et al., 2005 Reference). p23 protein was cloned into pET28b vector (Novagen) and then BL21 (DE3) + pLysS E. coli After transformation into Novagen, the protein was isolated ( ibid ) as previously described.

Cp149  defect Mutant  Construction, Expression and Purification

Cp149 deletion variants as described in FIG. 7B were cloned into pET30a vector (Novagen), followed by BL21 (DE3) + pLysS E. coli (Novagen) and then purified by expression (Choi et al., 2005). Deletion variants were amplified using the following primer set (forward-F, reverse-R) with pCMV / Flag-core (Kang et al., 2008) as a template: Cp130 (1-130), F-5 '-CGGAATTCATGGACATTGACCCGTATAAAG-3', R-5'-GCCGTCGACTAGGGAGGAGTGCGAATCC-3 '; Cp 111 (1-111), F-5′-CGGAATTCATGGACATTGACCGTATAAAG-3 ′, R-5′-GCCGTCGACTATCCAAAAGTAAGGCAGG-3 ′.

Hsp90  And Hsp90  defect Mutant  Construction, Expression and Purification

Human Hsp90β was cloned into the pET28b vector (Novagen) as previously described (Cho et al., 2000a). Human Hsp90 deletion variants were directly cloned into pET28b (Cho et al., 2000a, b) and were cloned after PCR amplification using pET28b-Hsp90β as a template and the following primer sets: N190Δ ( 191-724), F-5'-GCGGTCGACCAGAGTACCTAGAAGAGAGG-3 ', R-5'-CCGCGGCCGCCTAATCGACTTCTTCC-3'; N327Δ (328-724), F-5'-ACGAATTCAGGGCATTGCTA-3 ', R-5'-CCGCGGCCGCCTAATCGACTTCTTCC-3'; N413Δ (414-724), F-5'-ACGAGCTCTTCTCTGAGCTG-3 ', R-5'-CCGCGGCCGCCTAATCGACTTCTTCC-3'; C520Δ (191-519), F-5'-GCGGTCGACCAGAGTACCTAGAAGAGAGG-3 ', R-5'-ACGCGGCCGC TTACTCGTCAATGGGCTCGGTC-3'; C616Δ (191-615), F-5 'GCGGTCGACCAGAGTACCTAGAAGAGAGG-3', R-5'-ACGCGGCCGCTTAGGAGTTGTCCCGAAGTGC-3 '. This construct had six histidine tags added to the N terminus of Hsp90 (Cho et al., 2000b), and the Hsp90 deficient mutant was found in E. coli in 2xYT (Yeast Extract Tryptone) media. Overexpressed using BL21 (DE3) cells. In summary 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the cell culture and further incubated at 22 ° C. for 20 hours to induce protein expression. Cells were then lysed in lysis buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% (v / v) glycerol) and centrifuged at 18,000 g for 60 minutes. The supernatant was suspended on a nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) affinity column and the protein was eluted with a lysis buffer containing 500 mM imidazole.

HBV  Core protein Hsp90 Co-immunoprecipitation of

For coimmunoprecipitation (Co-IP) of HBV core protein and Hsp90, Huh7 cells were transfected into pCMV / Flag-core. After 48 hours, cells were lysed in lysis buffer and mouse monoclonal anti-HBV core antibody (Ab) (Santa Cruz, sc-23945) and goat polyclonal anti-Hsp90 Ab (Santa Cruz, sc-1057) Co-immunoprecipitation was added by addition at 4 ° C. for 2 hours. Immunoprecipitated lysates were electrophoresed in 15% Sodium Dodecyl Sulfate (SDS) -Polyacrylamide gel electrophoresis (PAGE), followed by mouse monoclonal anti-FLAG M2 Ab (1: 5000; Sigma, F3165) and chlorine polyclonal Western blot was performed with anti-Hsp90 Ab (1: 1000; Santa Cruz, sc-1057). In the case of coimmunoprecipitation of Cp149 dimer or capsid with Hsp90, 20 μM of Cp149 dimer or capsid was added to 20 μM of Hsp90, and then the sample was reacted at 30 ° C. for 1 hour. Cp149 dimer (or capsid) and Hsp90 were coimmunoprecipitated as mentioned above. Western blot was performed using the rabbit polyclonal anti-HBV core Ab (1: 4000; Dako, B0586) and the goat polyclonal anti-Hsp90 Ab (1: 1000; Santa Cruz, sc-1057).

Hsp90 and HBV  Binding Analysis of Core Proteins

To analyze the binding of Hsp90 to HBV Cp149 dimer and capsid, 5 μg of His-tagged Hsp90 was bound with 100 μl of Ni-NTA agarose at 4 ° C. for 1 hour. The Hsp90-Ni-NTA complex was added dropwise to the column with the bottom of the column locked, and the bottom was opened to allow liquid to drain. 5 μg of each Cp149 dimer and capsid were then added to the column. Ni-NTA agarose coupled protein was washed four times with TN buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl) and eluted with TN buffer containing 500 mM imidazole. Eluate was subjected to electrophoresis on 15% SDS-PAGE followed by goat polyclonal anti-Hsp90 Ab (1: 1000; Santa Cruz, sc-1057) and rabbit polyclonal anti-HBV core antibody (1: 4000; Dako, Western blot was performed using B0586).

Sucrose Density gradient  Analysis and Dotblat  analysis

Assembly reaction was carried out for 30 minutes in 37 ℃ reaction buffer. After assembly, ultracentrifugation (P55ST2 rotor CP-100α) (Hitachi Koki Co., Ltd, Tokyo, Japan) was performed at 160,000 X g for 4 hours 30 minutes at 20 ° C., and a sucrose density gradient analysis was performed. 10.50% (w / v) sucrose density gradient in mM Hepes, pH 7.5. Fractions were then collected and subjected to electrophoresis on 15% SDS-PAGE, followed by rabbit polyclonal anti-HBV core antibody (1: 4000; Dako, B0586) and goat polyclonal anti-Hsp90 Ab (1: 1000; Western blotting was performed using Santa Cruz, sc-1057). Dot blots were performed according to the Bio-Dot Microfiltration Apparatus manual (Bio-Rad Laboratories Inc., USA).

Undenatured Agarose  Gel electrophoresis HBV Capsid  detection

To determine the effect of Hsp90 on HBV capsid formation, 20 μM Cp149 dimer and 20 μM Hsp90 were added to 20 mM Na.₂MoO₄, 1 mM DTT, 0.01% NP-40, and 0.5 mM adenosine 5'-OIn a reaction buffer containing-(3-thiotriphosphate) (ATP- [gamma] -S), the reaction was performed at 37 [deg.] C. for 30 minutes (see Sullivan et al., 1997). After the reaction, the proteins were separated by electrophoresis on a 0.9% unmodified agarose gel, and Western blot was performed with the rabbit polyclonal anti-HBV core antibody (1: 4000; Dako, B0586) (see Kang et al., 2006). ). Capsid bands were analyzed with ImageMaster 2D Elite software 4.01 (Amersham Pharmacia Biotechnology).

Huh7  In a cell Hsp90  Treatment with Inhibitors and Temperature Changes

Hsp90 inhibitor, geldanamycin (GA; 4 μΜ, A.G. Scientific, Inc., USA) was added to Huh7 cells, and then cells were transfected with pCMV / Flag-core expressing core protein. The cells were then subjected to CO at 30, 35, 37, 40, and 43 ° C.₂ Thermal shock was given by placing in the incubator for 2 hours. The cells were then lysed in lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, and 0.5% NP-40) and 16,000 ×gCell debris was removed by centrifugation for 15 minutes at. Supernatants were subjected to electrophoresis on 0.9% unmodified agarose gel followed by Western blot with monoclonal anti-FLAG M2 Ab (1: 5000; Sigma, F3165).

Urea  And SDS  After processing HBV Capsid  Stability Detection

Assembled Cp149 and Hsp90 were incubated for 30 minutes at 37 ° C. in urea (0.3M) or SDS (0.1%). Samples were then subjected to electrophoresis on 0.9% unmodified agarose gel followed by Western blot with rabbit polyclonal anti-HBV core antibody (1: 4000; Dako, B0586).

Urea  And SDS  After processing HBV Capsid  Transmission electron microscope for detection of breakdown

Cp149 in vivo assembly was performed followed by urea and SDS treatment. For negative staining, 10 μl of solution containing assembled Cp149 was applied to the carbon coated grid and left for 1 minute. The grid was then stained with 2% uranyl acetate for 1 minute and washed with water. Transmission electron micrographs were taken at 80 kV using JEM 1010 (JEOL, Tokyo, Japan) (NICEM (National Instrumentation Center for Environmental Management, Korea).

Real time quantification PCR Using HBV DNA Quantification of

HepG2.2.15 cells were treated with 4 μM GA or 85 μM lamivudine (3TC) (HBV pol inhibitor) (Allen et al., 1998; Doong et al., 1991; Severini et al., 1995). Transfer was with shRNA-Hsp90 (Sigma, MISSION shRNA NM_007355). PLK0.1 plasmid was used for the negative control. After 24 hours, the medium was collected to collect the released HBV virus. After phenol extraction, DNA was precipitated with ethanol. The extracted DNA was mixed with real-time PCR SYBR green reaction mixture (Qiagen, USA). The reaction mixture contained HotStar Taq polymerase to prevent false positives, and the primers used were forward 5'-GTGTCTGCGGCGTTTTATCA-3 'and reverse 5'-GACAAACGGGCAACATACCTT-3'. The primers amplify 379 to 476 sites of the HBV genome to produce a 98bp product. PCR conditions were repeated 15 times at 95 ℃, 40 times in a cycle of 15 seconds at 94 ℃, 30 seconds at 55 ℃ and 30 seconds at 72 ℃. The HBV concentration was determined by the pHBV 1.2 x described in Guidotti et al., 1995 with 5.28 x 10 ⁶ copies / ml (cpm), 5.28 x 10 ⁵ cpm, 5.28 x 10 ⁴ cpm, 5.28 x 10 ³ cpm, 5.28 x10 ² cpm and Serial dilutions were made and standard curves were prepared and compared.

Example 1 Hsp90 and HBV  Core protein Dimer  Binding and site binding

Coimmunoprecipitation (co-IP) analysis was performed to determine the association between Hsp90 and HBV core protein. The results are in A of FIG. 1 and show the binding between the two proteins. Using a purified protein to determine the binding site between the two proteins, co-IP analysis showed that Hsp90 binds to Cp149 dimers (B and C top panels in FIG. 1), but not to the capsid surface. (Panels B and C in Figure 1).

To identify the binding sites on Hsp90 and HBV Cp149, a series of deletion variants were made from each protein as described in the Experimental Materials and Methods section to identify the binding sites. In order to confirm the binding site on Hsp90, it was cultured by mixing its defective variant and Cp149, and to confirm the binding site on the Cp149 dimer, it was cultured by mixing its defective variant and Hsp90. The results are in FIGS. 7A and 7B. The residues are based on the sequences set forth in Table 1 below. As shown in FIG. 7, it was confirmed that 520 to 615 residues of Hsp90 and 111-130 residues of Cp149 dimer were binding sites.

order HSP90
Beta
(human) 1 mpeevhhgee evetfafqae iaqlmsliin tfysnkeifl relisnasda ldkiryeslt
61 dpskldsgke lkidiipnpq ertltlvdtg igmtkadlin nlgtiaksgt kafmealqag
121 adismigqfg vgfysaylva ekvvvitkhn ddeqyawess aggsftvrad hgepigrgtk
181 vilhlkedqt eyleerrvke vvkkhsqfig ypitlyleke rekeisddea eeekgekeee
241 dkddeekpki edvgsdeedd sgkdkkkktk kikekyidqe elnktkpiwt rnpdditqee
301 ygefyksltn dwedhlavkh fsvegqlefr allfiprrap fdlfenkkkk nniklyvrrv
361 fimdscdeli peylnfirgv vdsedlplni sremlqqski lkvirknivk kclelfsela
421 edkenykkfy eafsknlklg ihedstnrrr lsellryhts qsgdemtsls eyvsrmketq
481 ksiyyitges keqvansafv ervrkrgfev vymtepidey cvqqlkefdg kslvsvtkeg
541 lelpedeeek kkmeeskakf enlcklmkei ldkkvekvti snrlvsspcc ivtstygwta
601 nmerimkaqa lrdnstmgym makkhleinp dhpivetlrq kaeadkndka vkdlvvllfe
661 tallssgfsl edpqthsnri yrmiklglgi dedevaaeep naavpdeipp legdedasrm
721 eevd
HBV
Core protein
(human) 1 mdidpykefg asvellsflp sdffpsirdl ldtasalyre alespehcsa hhtalrqail
61 cwgelmnlat wvgsnledpa srelvvsyvn vnmglklrqi lwfhiscltf gretvleylv
121 sfgvwirtpq ayrppnapil stlpettvvr rrgrsprrrt psprrrrsqs prrrrsqsre
181 sqc

The amino acid sequence is represented by a single letter code, and amino acids corresponding to the single letter are known in the art.

Example 2 Cp149 Dimer  Through binding Hsp90 of HBV Incorporation into

Hsp90 was observed in fractions 8 to 10 as a result of sucrose density gradient analysis by mixing Hsp90 with Cp149 dimer. After the assembly reaction, capsid was also observed in the fraction (A in FIG. 2). The mixing of Hsp90 with BSA (control) was observed in fractions 2-4 (results not shown). Two samples of fractions 3 and 8, respectively, were analyzed under unmodified and denaturing conditions to confirm that Hsp90 was packaged into the capsid. SDS-PAGE shows that Hsp90 is present in fractions 3 and 8 (B, lanes 3 and 4 in FIG. 2, bottom panel). However, it was found that the dot blot and unmodified agarose gel analysis did not interfere with the formation of capsids and that Hsp90 was not detected in fractions 3 and 8 (B, top and middle panels in FIG. 2). Since Hsp90 is located in the capsid, it may not be detected in fraction 8. Since unmodified agarose gel electrophoresis can only detect capsid, Cp149 can only be detected in fraction 8 (FIG. 2B, lane 4 of the middle panel). In addition, a dot blot analysis detected Cp149 dimers (C in FIG. 2) to show that Hsp90 was mixed into the capsid during assembly. From the above results, it can be seen that Hsp90 is packaged into the capsid through binding to the Cp149 dimer.

Example 3 Activated Hsp90 On by HBV  Promotes Core Protein Assembly

HBV core assembly begins with the formation of Cp149 homodimer via cysteine disulfide bonds between the 61st amino acids of each subunit (Nassal et al., 1992; Zheng et al., 1992). Hsp90 activity requires protein p23 binding and ATP, which promotes maturation of client proteins (Sullivan et al., 1997; Woo et al., 2009). To determine whether activated Hsp90 affects HBV core assembly, assembly reactions with Cp149 dimer or point mutation (C61A) core protein were performed in the presence of Hsp90. As shown in FIG. 3A, activated Hsp90 promoted the formation of C61A as well as Cp149 dimers; However, C61A capsid was not formed when Hsp90 was absent or when Hsp90 was inactivated due to GA treatment (see B of FIG. 3A). Further capsid formation was increased in the presence of ATP activated Hsp90 by sucrose density gradient analysis, but was not affected by Hsp90 inactivated by BSA or GA (FIG. 3B C). To reconfirm HBV core assembly activity by activated Hsp90, activated Hsp90, Hsp90 mutations (N190Δ- ATPase site deletion) and BSA (control) were independently added to Cp149, and capsid formation over time was investigated. It can be seen that in the presence of activated Hsp90, capsid formation proceeded rapidly compared to Hsp90 mutants or BSA. Hsp90 deletion mutants (N190Δ) and BSA progressed slowly until saturation at 120 minutes (D in FIG. 3B). 15 minutes after the assembly reaction, the strength of the Cp149 dimer was set to 1, and the relative intensities of the other bands which were compared with each other were calculated. Since the same amount of Cp149 was used, the capsid formation in all samples was saturated at 120 minutes (D. right graph in FIG. 3B).

Example 4 Activated by Temperature Change Hsp90 On by HBV Capsid  Regulation of formation

The results in FIG. 3 demonstrate that activated Hsp90 promotes the formation of capsids. Hsp90 activity is known to inhibit stress-protein aggregation by temperature, to prevent further folding delays of unfolded proteins and to help rapid recovery (Freeman and Morimoto, 1996; Yonehara et al., 1996). HBV capsid formation from HBV core proteins is temperature dependent (Hilmer et al., 2008). Therefore, in order to investigate the effect of activated Hsp90 on the formation of HBV capsid with temperature changes, (1) Cp149 dimers and BSA (control), (2) Cp149 dimers and activated Hsp90, and (3) Cp149 dimers And capsid formation over a range of temperatures in the presence of GA treated Hsp90 was investigated in vitro. Capsid formation was highest at 37 ° C. for the control (FIG. 4A, left panel). With Cp149 dimer and activated Hsp90, capsid formation peaked over a wide temperature range from 30 ° C. to 43 ° C. and the amount was double compared to the control (FIG. 4A, middle panel). To determine whether the capsid assembly is entirely dependent on activated Hsp90, GA was added to the Hsp90 and Cp149 dimers. When treated with GA, the effect of Hsp90 disappeared and the same results as with Cp149 and BSA were obtained (FIG. 4A, right panel). In order to investigate the effect of Hsp90 on HBV capsid formation at various temperatures in the cells, the cells were treated with GA after pCMV / Flag-core transduction into Huh7. After thermal shock was applied, the amount of HBV capsid was measured by Western blot. In contrast to the results with purified proteins (FIG. 4A), the extent of capsid formation in cells not treated with GA was constant regardless of temperature (FIG. 4B, left panel). This is because the capsid formation was saturated 48 hours after delivery. However, the thermal shock after GA treatment affected capsid formation, which was relatively reduced compared to capsid formation in untreated GA cells at all temperatures tested except at 37 ° C. Experiments with Hsp90 alone were similar (results not shown). Hsp90 inactivated with GA was unable to promote temperature dependent capsid formation, which is an experimental and aborted pattern with purified proteins. These results demonstrate that they stabilize capsid formation in the range of 30 ° C. to 43 ° C.

Example 5 Activated Hsp90 By surfactant HBV Capsid  Decomposition suppression

Capsids were treated with various concentrations of urea and SDS at 37 ° C. for 30 minutes. When treated with 3M durea, activated Hsp90 exhibited a 2.7-fold higher capsid concentration compared to the absence of Hsp90 (A left panel in FIG. 5A) and the presence of Hsp90 treated with GA (right panel in FIG. 5A). (A middle panel of FIG. 5A). Capsid concentration after 0.05% SDS treatment was 2.4 times higher in the presence of activated Hsp90 compared to negative control (BSA) (FIG. 5B, left panel) and GA treated Hsp90 (FIG. 5B, right panel) (FIG. 5B). Middle panel). This protective effect on capsid degradation by Hsp90 disappeared when the concentration of SDS exceeded 0.1%, and after this point all capsids were decomposed (FIG. 5B). Cp149 dimers assembled in vitro and detergent treated to monitor HBV capsid degradation in response to detergent treatment were observed by transmission electron microscopy as previously described (Astheimer et al.). Previous reports have shown that Cp149 dimers are about 30 nm in size (Newman et al., 2003). The TEM results of this experiment showed a spherical capsid with a compact average diameter of 30 nm for the presence of activated Hsp90 and untreated controls (C, D and E in FIG. 5C, respectively, average diameter, 30.5 nm, 30.75 nm, And 30.87 nm). However, in the presence of GA inactivated Hsp90, 95% of the formed capsids formed an irregular complex or crumpled capsid (D in FIG. 5C), and 42% was an expanding shaped capsid (E in FIG. 5C). ). These results demonstrate that activated Hsp90 reduces HBV capsid degradation due to detergent.

Example 6 Hsp90  By suppression and down regulation Extracellular HBV DNA  A decrease in quantity

HepG2.2.15 cells from HepG2 cells produce HBV (Liu et al., 2009). HBV DNA amount was measured after Hsp90 inhibition and downregulation. To this end, HepG2.2.15 cells were treated with GA and then transfected with shRNA-Hsp90. After 24 hours, HBV DNA was isolated from cell culture medium and quantified by real-time quantitative PCR (qRT-PCR). The amount of intracellular capsids formed in GA treated cells and shRNA-Hsp90 treated cells compared to untreated HepG2.2.15 cells decreased 53% and 49%, respectively (A, lanes 3 and 4 in FIG. 6), and extracellular HBV The amount of DNA also decreased (B, lanes 3 and 4 in FIG. 6, 50% and 48% reduction, respectively). HepG2.2.15 cells were then treated with lamivudine (3TC) for 24 hours to inhibit HBV polymerase (pol) activity. Extracellular HBV DNA was reduced by 83% (B of Figure 6, lane 5). However, there was no change in capsid assembly (FIG. 6A, lane 5). This result indicates that inhibition of polymerase activity by 3TC inhibits HBV DNA synthesis in the cytoplasm, thus reducing extracellular HBV DNA concentration but does not affect capsid assembly. Furthermore, when HepG2.2.15 cells were simultaneously treated with 3TC and GA, the amounts of intracellular capsid and extracellular HBV DNA were reduced by 62% and 63%, respectively, as compared to the treatment with 3TC alone (B, lane of FIG. 6). 6). These results indicate that reduced intracellular capsid concentration by Hsp90 inhibition affects the extracellular HBV DNA amount, provided that polymerase activity is inhibited by 3TC. Except for shRNA-Hsp90 transfected samples, Hsp90 concentrations were the same in all samples. Beta-actin was used as a control (FIG. 6C). These results demonstrate that Hsp90 is a host factor that affects the amount of extracellular HBV DNA through capsid assembly as well as HBV polymerase activity.

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Claims (7)

delete delete A method for screening a therapeutic agent for liver disease that selectively inhibits the interaction between Hsp90 and HBV core protein, comprising the following steps:
Providing Hsp90 and HBV core proteins;
Contacting the protein with a test substance expected to be able to selectively inhibit the interaction of the Hsp90 with the HBV core protein; And
Detecting the interaction between the Hsp90 and the HBV core protein, and when the interaction between the Hsp90 and the HBV core protein is reduced when contacted with the test substance compared to the control group not in contact with the test substance, the test substance Screening method for treating liver disease comprising the step of selecting a candidate. The method of claim 3, wherein the Hsp90 protein comprises at least residues 520-615 based on the full length or the sequence of Table 1, wherein the HBV core protein comprises residues 1-149 or at least residue 111 based on the full length or the sequence of Table 1. To 130, wherein the method for screening a liver disease therapeutic substance. The method according to claim 3 or 4, wherein the Hsp90 and HBV core proteins are provided in the form of cells expressing them. The method of claim 5, wherein the cells are HepG2, Huh7, Hep3B or PLC / PRF / 5. The method of claim 3 or 4, wherein the interaction of the Hsp90 and HBV core protein is measured by extracellular HBV nucleic acid concentration.

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