Pharmaceutical composition for preventing or treating disease caused by herpesvirus infection

Abstract

To provide a novel pharmaceutical composition.SOLUTION: The present invention provides a pharmaceutical composition for preventing or treating diseases caused by herpesvirus infection, containing a calmodulin inhibitor as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention broadly relates to novel uses of specific calmodulin inhibitors, etc. [Background technology]

[0002] It is known that the progression of viral gene expression in cultured cells infected with herpes simplex virus (HSV) at a high multiplicity of infection is heterogeneous in each cell. Understanding the factors that cause this heterogeneity may lead to the identification of novel mechanisms of viral gene expression regulation in HSV. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2011 / 118779 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a novel means for regulating gene expression of herpes viruses and thereby preventing or treating diseases caused by herpes virus infection. [Means for solving the problem]

[0005] The present inventors have found that inhibition of calmodulin negatively regulates the gene expression cascade of herpes viruses, and have completed the present invention.

[0006] That is, the present invention includes the following inventions. [1] A pharmaceutical composition for preventing or treating a disease caused by infection with a herpes virus, comprising a calmodulin inhibitor as an active ingredient. [2] The pharmaceutical composition according to [1], wherein the calmodulin inhibitor is a naphthalenesulfonamide compound or a pharma- ceutical acceptable salt thereof. [3] The pharmaceutical composition according to [2], wherein the naphthalenesulfonamide compound is selected from the group consisting of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(6-aminoethyl)-5-chloro-1-naphthalenesulfonamide; and N-(10-aminodecyl)-5-chloro-1-naphthalenesulfonamide. [4] The pharmaceutical composition according to [2], wherein the pharma- ceutical acceptable salt of the naphthalenesulfonamide compound is a hydrochloride salt. [5] The pharmaceutical composition according to any one of [1] to [4], wherein the disease is caused by expression of the β gene group or later in the gene expression cascade of a herpes virus. [6] The pharmaceutical composition described in [5], wherein the expression of the β gene group and / or the γ gene group is inhibited by inhibiting the binding of the herpes virus ICP4 protein to calmodulin. [7] The pharmaceutical composition described in [6], wherein binding to the IQ-like motif in the ICP4 protein of a herpes virus is inhibited. [8] The pharmaceutical composition according to any one of [1] to [7], wherein the disease is selected from the group consisting of herpes labialis, genital herpes, encephalitis, neonatal herpes, keratitis, and whitlow. [9] The pharmaceutical composition according to any one of [1] to [8], which is administered into the brain of a subject.

[10] A method for controlling the infection heterogeneity of a herpes virus, the method comprising the step of contacting the herpes virus with a calmodulin inhibitor. Effect of the Invention

[0007] Calmodulin (CaM) binds to ICP4 (Infected Cell Protein 4), positively controls the binding to β and γ gene promoters, and promotes the gene expression cascade of herpes viruses, which is thought to contribute to efficient viral proliferation of herpes viruses. The present inventors administered a naphthalenesulfonamide compound, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), to mice infected with herpes simplex virus, and evaluated the pathogenicity and intracerebral viral titer. As a result, it was revealed that W-7 significantly inhibited the survival rate of mice and viral proliferation in the brain compared to the control.

[0008] Effective antiviral drugs such as acyclovir have been developed for herpes virus infections, but many herpes virus infections remain a problem because the herpes virus is latent. Anti-herpes virus drugs developed to date target virus-infected cells in the virus proliferation phase (lytic infection phase). Therefore, existing anti-herpes virus drugs are completely ineffective against latently infected cells. In other words, once a person is infected with the herpes virus, it is impossible for existing anti-herpes virus drugs to remove the herpes virus from the site of latent infection.

[0009] Unlike existing antiviral drugs such as acyclovir that inhibit the gene replication of viruses, calmodulin inhibitors, particularly naphthalenesulfonamide compounds, inhibit the gene expression cascade of herpes viruses, thereby realizing the treatment or prevention of diseases caused by herpes virus infection. Through an action mechanism different from that of such existing antiviral drugs, it is possible to provide a novel pharmaceutical composition that exerts unprecedented effects.

[0010] WO2011 / 118779 discloses that myosin light chain kinase (MLCK) inhibitors prevent herpes virus from invading cells. Although the publication describes W-7 as an MLCK inhibitor, it does not describe or suggest that it controls the expression of the β gene group and subsequent genes in the herpes virus gene expression cascade.

[0011] The heterogeneity of herpes virus infection is thought to result from the heterogeneity of the progression of the gene expression cascade. The present inventors have identified the CALM1 gene, which encodes calmodulin, as the gene that controls the heterogeneity of infection. Identification of host factors that control such heterogeneity of infection and elucidation of the cause of heterogeneity are expected to lead to an understanding of the virus proliferation mechanism and a method for controlling virus proliferation. [Brief description of the drawings]

[0012] [Figure 1A] The target sequence of sgRNA and the sequence of each allele in CALM1 / 2 KO cells created by CRISPR / Cas9. The parent sequence is SEQ ID NO:1, and the allele sequences are SEQ ID NO:2 and 3. [Figure 1B] Western blotting analysis of calmodulin expression levels in wild-type, CALM1 KO, and CALM1 / 2 KO cells. [Figure 1C] Quantitative results in Figure 1B. [Figure 1D] HeLa cells were infected with the wild-type HSV-1 strain at an MOI of 5, and the mRNA levels of the indicated HSV-1 genes were analyzed by qPCR 6 hours later. [Figure 1E] In Fig. 1D, the fold reduction in expression levels in CALM1 KO and CALM1 / 2 KO cells compared to wild-type HeLa cells. [Figure 1F] Viral titers 12 hours after infection of wild-type, CALM1 KO, and CALM1 / 2 KO cells with the HSV-1 wild-type strain at an MOI of 5. [Figure 1G]Viral titers 48 hours after infection of wild-type, CALM1 KO, and CALM1 / 2 KO cells with the HSV-1 wild-type strain at an MOI of 0.05. [Figure 2A] 1. Wild-type or CALM1 KO HeLa cells were infected with rICP47 / vUs11 at an MOI of 5, and the infected cells were analyzed by flow cytometry 12 hours later. Panel A shows the mean fluorescence intensity (MFI) of TagRFP. [Figure 2B] Venus MFI. [Figure 2C] 2D plot of TagRFP and Venus. [Figure 2D] Percentage of virus-producing cells (VPCs). [Figure 2E] 2. Wild-type, CALM1 KO, and CALM1 / 2 KO HeLa cells were infected with rICP47 / vUs11 at an MOI of 5, and the infected cells were analyzed by flow cytometry 12 hours later. Figure 2E shows the MFI of TagRFP. [Figure 2F] Venus MFI. [Figure 2G] 2D plot of TagRFP and Venus. [Figure 2H] Percentage of VPCs. [Figure 3A] An amino acid region that is highly homologous to the IQ motif in ICP4. [Figure 3B] HeLa cells were infected with the wild-type HSV-1 strain at an MOI of 5, fixed 9 hours later, permeabilized, stained with ICP4 and calmodulin antibodies, and observed under a confocal microscope. [Figure 3C] HeLa / puroR and HeLa / Flag-CaM were infected with HSV-1 at an MOI of 5 and immunoprecipitated with Flag antibody. [Figure 3D] Design of ICP4 / ΔIQ expressed in 293FT cells. The underlined sequences in the figure indicate amino acids that are defined by the consensus sequence of the IQ motif and that are identical to the amino acid sequence of ICP4. [Figure 3E]The results of pull-down of ICP4 / WT, ICP4 / ΔIQ, and EGFP expressed in 293FT cells using CaM-beads or Protein A beads. [Figure 3F] Band quantification in Figure 3E. [Figure 4A] HeLa cells were infected with HSV-1 wild-type strain (HSV-1(F)), ICP4 / ΔIQ, or ICP4 / ΔIQ-repair at an MOI of 5, and the mRNA levels of each gene listed in the figure were analyzed by qPCR. [Figure 4B] HeLa cells were infected with ICP4 / ΔIQ or its revertant strain, ICP4 / ΔIQ-repair, at an MOI of 5, and a ChIP assay was performed using an ICP4 antibody 3.5 hours later. [Figure 4C] Wild-type or CALM1 KO HeLa cells were infected with the wild-type HSV-1 strain at an MOI of 5, and ChIP assay was performed 3.5 hours later using an ICP4 antibody. [Figure 5A] (125 μg of W-7 was inoculated into a 3-week-old female ICR mouse, and 2 hours later, 500 PFU of the wild-type HSV-1 and 50 μg of W-7 were simultaneously administered into the brain. Figure 5A shows the survival curve of the mice. [Figure 5B] Viral titers in the brain. [Figure 5C] Survival curve when 500 PFU of HSV-1 wild-type strain and the indicated amounts of W-7 and acyclovir (ACV) were simultaneously inoculated into the brain of 3-week-old female ICR mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, aspects or embodiments of the present invention will be described, but the scope of the present invention is not interpreted as being limited to the following aspects or embodiments.

[0014] Pharmaceutical Composition In a first aspect, there is provided a pharmaceutical composition for preventing or treating a disease caused by infection with a herpes virus, the pharmaceutical composition comprising a calmodulin inhibitor as an active ingredient.

[0015] There are three known types of human calmodulin: calmodulin 1 (e.g., NCBI Reference Sequence: NP_001350598.1), calmodulin 2 (e.g., NP_001292553.1), and calmodulin 3 (e.g., NP_001316850.1). These are encoded by the CALM1 gene, CALM2 gene, and CALM3 gene, respectively, but are encoded as the same protein, and therefore, as used herein, the term "calmodulin inhibitor" refers to an inhibitor of calmodulins 1 to 3.

[0016] There are various compounds as calmodulin inhibitors, but from the viewpoint of preventing or treating diseases caused by herpes virus infection, particularly from the viewpoint of controlling gene expression of herpes virus, naphthalenesulfonamide calmodulin inhibitors are preferred. Various compounds are known as naphthalenesulfonamide calmodulin inhibitors, but N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (CAS registration number 61714-27-0) (W-7 hydrochloride, hereinafter also simply referred to as "W-7") or its free form, or compounds having a structure similar thereto, are particularly preferred. Examples of compounds similar to W-7 include compounds having the same number of carbon atoms in the side chain bonded to the naphthalene skeleton as W-7, such as N-(6-aminoethyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (CAS Registry No. 61714-25-8) (W-5 hydrochloride; hereinafter, also simply referred to as "W-5") or its free form; side chain analogs of W-7, such as N-(6-aminoethyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (CAS Registry No. 78957-85-4) (A-3 hydrochloride; hereinafter, also simply referred to as "A-3") or its free form; and N-(10-aminodecyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (CAS Registry No. 79127-24-5) (A-7 hydrochloride; hereinafter, also simply referred to as "A-7") or its free form.

[0017] As long as the calmodulin inhibitor has the desired activity, it may be in the form of a free form (sometimes called an original form), a medicamentously acceptable salt, a solvate, a hydrate, etc. Examples of such salts include, but are not limited to, inorganic hydrochlorides such as hydrochloride and sulfate, and organic acid salts such as fumarate, succinate, oxalate, acetate, etc.

[0018] In one embodiment, the calmodulin inhibitor is a naphthalenesulfonamide compound or its hydrochloride salt.

[0019] In one embodiment, a particularly preferred calmodulin inhibitor is N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or a pharma- ceutically acceptable salt thereof, among others, W-7.

[0020] Calmodulin inhibitors are used as active ingredients for the prevention or treatment of diseases caused by herpesvirus infection. Herpesviruses are phylogenetically divided into three subfamilies: Alphaherpesvirinae, Betaalphaherpesvirinae, and Gammaherpesvirinae. Nine types of herpesviruses that infect humans have been identified: herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, human cytomegalovirus, Epstein-Barr (EB) virus, human herpesvirus 6A, human herpesvirus 6B, human herpesvirus 7, and Kaposi's sarcoma-associated herpesvirus.

[0021] In one embodiment, the disease caused by infection with a herpes virus is a disease caused by infection with a virus belonging to the subfamily Alphaherpesvirinae, such as herpes simplex virus, in particular infection with herpes simplex virus type 1 (HSV-1).

[0022] Herpes virus genes are broadly classified into immediate early (IE, α) genes, early (E, β) genes, and late (L, γ) genes according to the time of expression. Viral gene expression is controlled in a cascade manner starting from the α gene to the γ gene. The α gene product contains five viral factors (ICP0, ICP4, ICP22, ICP27, and ICP47), many of which play extremely important roles in herpes virus replication and infection.

[0023] ICP4 is an essential protein for viral proliferation and plays a major role in the expression of β and γ genes by recruiting basic transcription factors to the β and γ gene promoters. Calmodulin binds to ICP4, positively regulates its binding to the β and γ gene promoters, and promotes the HSV gene expression cascade, thereby contributing to efficient viral proliferation. ICP4 is widely conserved among herpes viruses in the Alphaherpesvirinae subfamily. Viruses belonging to the Alphaherpesvirinae subfamily that cause human infectious diseases include herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV).

[0024] The present inventors found that only ICP4 in the α protein group has a calmodulin-binding motif, the IQ motif (X1QX2X3X4X5GX6X7X8X9X 10 X 11 X 12 X1 is any one of F, I, L, or V, and X2 to X4, X6 to X8, and X 10 and X 11 each represents any amino acid, which may be the same or different from each other; X5 and X9 are R or K; 12 The IQ motif is a sequence that is highly homologous to the nucleotide sequence of ... 1060 VQCAVRWPAARDL 1072" (SEQ ID NO: 4), it may be "VQCAVRWPAAR" (SEQ ID NO: 5). Specific calmodulin inhibitors, such as W-7, are thought to inhibit the binding between ICP4 and calmodulin, and inhibit the expression of the β gene group and subsequent genes in the herpes virus gene expression cascade. Therefore, such specific calmodulin inhibitors can be suitably used for the treatment or prevention of diseases caused by ICP4 expression in the herpes virus gene expression cascade, and thus by the expression of the β gene group and subsequent genes.

[0025] Calmodulin inhibitors can also be used as inhibitors of ICP4 expression, or inhibitors of the expression of the β gene group and beyond.

[0026] After initial infection, herpes viruses proliferate in mucosal epithelial cells at the site of infection. It is rare for herpes viruses to cause pathology during the proliferative phase (lytic infection phase), and it is thought that most lytic infections are asymptomatic. Herpes viruses that proliferate locally infect the sensory nerve terminals that control the infected site, regardless of whether pathology develops or not. Then, viral particles are transported retrogradely within the axon, reach the trigeminal ganglion or sacral ganglion, and after transient proliferation, transition to latent infection. Latent herpes viruses are reactivated by certain host changes (ultraviolet light exposure, cold, menstruation, immunosuppression, stress), and the production of viral particles begins. The reactivated viruses are transported anterogradely within the axon and cause pathology locally again. In this way, herpes viruses repeat latency and reactivation, and persist in the host for life.

[0027] There are many diseases caused by herpes virus infection, and examples of diseases caused by herpes simplex virus include herpes labialis, genital herpes, encephalitis, neonatal herpes, keratitis, whitlow, encephalitis, skin diseases, eye diseases, etc. Calmodulin inhibitors significantly inhibit the neuropathogenicity of herpes viruses and the proliferation of viruses in the brain, and are therefore considered to be effective in treating or preventing herpes encephalitis, among the above diseases.

[0028] As used herein, "disease caused by herpes virus infection" refers to a disease associated with efficient herpes virus proliferation due to promotion of herpes virus gene expression cascade. In other words, the active ingredient calmodulin inhibitor is intended to suppress the onset of symptoms after infection, but is not intended to block the virus from infecting cells.

[0029] In one embodiment, the disease caused by herpes virus infection is a disease caused by the heterogeneity or neuropathogenicity of herpes virus infection, including herpes virus encephalitis.

[0030] Calmodulin inhibitors can be used for other purposes besides the treatment or prevention of the above diseases. For example, wild-type herpes simplex virus (HSV) expresses genes from the β gene onwards, and therefore cannot be used as an active ingredient in vaccines against HSV from the viewpoint of safety, but can be rendered harmless by using it in combination with a calmodulin inhibitor.

[0031] The calmodulin inhibitor is preferably administered simultaneously with or immediately before infection with a herpes virus. "Immediately before infection" means several tens of minutes to 5 hours, preferably 1 to 3 hours, more preferably 2 hours before infection.

[0032] A pharmaceutical composition containing a calmodulin inhibitor can be administered to humans or non-human mammals (e.g., mice, rats, rabbits, dogs, pigs, cows, horses, monkeys, etc.) for the purpose of preventing or treating herpes virus infections.

[0033] The amount of the active ingredient contained in the pharmaceutical composition of the present invention can be appropriately determined depending on the type of calmodulin inhibitor, the type of subject, and the type of disease to be treated or prevented. For example, in the case of a naphthalenesulfonamide compound such as W-7, the dosage for humans can be, but is not limited to, 0.01 to 50 mg / kg, preferably 0.1 to 25 mg / kg, more preferably 0.5 to 10 mg / kg, and further preferably 1 to 5 mg / kg.

[0034] Calmodulin inhibitors can also be used in combination with known components used in the prevention or treatment of herpes virus infections, such as acyclovir, valacyclovir, famciclovir, etc. It is also envisioned that calmodulin inhibitors can be used in combination with substances that inhibit alpha protein. Such substances are not limited to low molecular weight compounds, but may be polymeric compounds such as nucleic acid molecules, proteins, peptides, etc.

[0035] Calmodulin inhibitors can also be used for diseases other than herpes infection. For example, by simultaneously administering an optimized amount of calmodulin inhibitor to an oncolytic herpes simplex virus and a tumor, it is possible to reduce the toxicity of the oncolytic herpes simplex virus, which is usually highly toxic to humans, and as a result, it is possible to obtain the ideal oncolytic effect of the herpes simplex virus.

[0036] Examples of nucleic acid molecules include siRNAs and antisense nucleic acids that can functionally suppress the expression of α, β, or γ genes. For example, LAT (latency associated transcript) is a transcript of about 8.3 kb, which is spliced ​​to express stable introns of about 2 kb and about 1.5 kb, and small non-coding RNAs encoded in the LAT gene region can suppress the gene expression of α genes ICP4 and ICP0, and therefore, it is considered that the expression of α protein is further inhibited by using it in combination with a calmodulin inhibitor.

[0037] The pharmaceutical compositions can be administered orally or parenterally, systemically or locally. Parenteral administration includes, but is not limited to, intraperitoneal, intracerebral, intravenous, subcutaneous, intradermal, intramuscular, transdermal, intranasal, transmucosal, etc.

[0038] In one embodiment, the administration route is intracerebral and / or intraperitoneal.When the calmodulin inhibitor is administered multiple times, the administration route may be the same or different.For example, when the calmodulin inhibitor is administered twice, it is preferable to administer the calmodulin inhibitor intraperitoneally before infection and administer the calmodulin inhibitor intracerebrally at the same time as infection.

[0039] The administration method may be selected from injection, suppository, enema, oral enteric-coated drug, etc. The administration route and method may be appropriately selected depending on the disease to be treated or prevented.

[0040] Pharmaceutical compositions may contain pharmacy-acceptable carriers such as preservatives and stabilizers. The pharmacy-acceptable carriers may be any that can be administered together with the active ingredient, and are not particularly limited. For example, water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants, excipients, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc. are included, but are not limited thereto.

[0041] The pharmaceutical composition can be formulated into any form using the above-mentioned carrier. Such a formulation is appropriately prepared using the above-mentioned carrier. The form of the formulation is not particularly limited, and is appropriately selected and used according to the purpose of treatment. Representative examples include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, syrups, suppositories, injections (liquids, suspensions, emulsions), etc. These formulations may be manufactured by commonly used methods.

[0042] (Methods for controlling infection heterogeneity) In a second aspect, a method of controlling infection heterogeneity of a herpes virus is provided, the method comprising the step of contacting a herpes virus with a calmodulin inhibitor.

[0043] As used herein, "herpes virus infection heterogeneity" means that the progression of infection is heterogeneous.

[0044] Such heterogeneity is due to differences in the efficiency of expression transition from the viral immediate-early genes to the early and late genes. Furthermore, since the three calmodulin-encoding genes CALM1, CALM2, and CALM3 are all thought to be genes that control infection heterogeneity, calmodulin inhibitors are expected to control infection heterogeneity to a uniform state.

[0045] In the method for controlling the infection heterogeneity of herpes viruses, a substance other than a calmodulin inhibitor may be added. For example, the homogeneity can be negatively controlled by adding a calmodulin agonist. Such an agonist includes CALP1 (Calcium-like Peptide 1) and the like.

[0046] The timing of contact with the calmodulin inhibitor may be any of before, during, or after infection, but when the purpose is to achieve a uniform state, it is preferably simultaneous with or immediately before infection. The calmodulin inhibitor may be contacted with the herpes virus itself or with cells infected with the herpes virus.

[0047] The degree of infection heterogeneity can also be determined by subjecting infected cells to a means capable of evaluating cell characteristics such as flow cytometry, measuring the expression level of herpesvirus genes in each cell, and evaluating the degree of variation in the expression level between cells (e.g., Gini coefficient or coefficient of variation). Methods for determining the expression level include single-cell transcriptome analysis and FACS analysis. If the degree of variation in the expression level of herpesvirus genes decreases upon contact with a calmodulin inhibitor, the heterogeneous state can be considered to have become homogeneous.

[0048] (Treatment method) In a third aspect, there is provided a method of preventing or treating a disease resulting from infection with a herpes virus in a subject, the method comprising administering to the subject a calmodulin inhibitor.

[0049] Prevention or treatment of a disease caused by infection with a herpes virus can be achieved by administering a calmodulin inhibitor to a subject in need thereof. The dosage form and administration route of the calmodulin inhibitor are not limited to those described above and can be appropriately determined by those skilled in the art.

[0050] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0051] CaM is encoded by three genes, CALM1, CALM2, and CALM3, all of which have the same amino acid sequence. To analyze CaM expression levels and HSV-1 gene expression, we used CRISPR / Cas9 to knock out CALM2 from CALM1 KO HeLa cells to generate CALM1 / CALM2 double knockout cells (Fig. 1A). At this time, triple knockout cells of CALM1 / CALM2 / CALM3 could not be generated because the cells were lethal. Western blotting analysis of CaM expression levels in WT, CALM1KO, and CALM1 / 2 KO cells showed a decrease in CaM expression levels depending on the number of CALM genes knocked out (Fig. 1B, C).

[0052] These cells were infected with HSV-1, and 6 hours later, the expression of the α, β, and γ genes was analyzed by qPCR. The results showed that the mRNA levels of the α genes ICP27 and ICP0 were not significantly decreased in CALM1 KO HeLa cells, but were significantly decreased in CALM1 / 2 KO HeLa cells (Fig. 1D). On the other hand, the mRNA levels of the β and γ genes UL50, ICP8, VP22, and Us2 were significantly decreased in CALM1 KO HeLa cells, and were further decreased in CALM1 / 2 KO HeLa cells (Fig. 1D). The reduction in the expression of each gene in CALM1 / 2 KO HeLa cells compared to WT was about 1.6-fold for the α gene, and about 2- to 12-fold for the β and γ genes (Fig. 1E). It was also found that the amount of progeny virus production was reduced depending on the CaM expression level in the cell population (Fig. 1F and G). These results suggest that CaM is required for efficient gene expression of HSV-1, especially for β and γ gene expression, and that CaM contributes to efficient virus replication.

[0053] The inventors have demonstrated through analysis using a reporter virus (rICP47 / vUs11) in which tagRFP was added to the α gene ICP47 and Venus was added to the γ gene Us11 that gene expression in HSV-1-infected HeLa cells was heterogeneous even at the protein level, even under high MOI conditions. In other words, although it would be expected that a high multiplicity of infection would eliminate heterogeneity at least at the protein level, this was not actually the case (Moeka Nobe et al., "Direct Relationship between Protein Expression and Progeny Yield of Herpes Simplex Virus 1 Unveils a Rate-limiting Step for Virus Production", 2023, bioRxiv, https: / / doi.org / 10.1101 / 2023.06.07.544155). This paper also shows that progeny virus production in a cell population depends on the proportion of cells in the cell population whose gamma gene expression level exceeds a threshold (virus producing cells: VPCs); in other words, there is what can be called a positive correlation between the proportion of VPCs and progeny virus production; that is, when the proportion of VPCs in a cell population is low, the cell population produces less progeny virus, and when the proportion of VPCs in a cell population is high, the cell population produces more progeny virus.

[0054] The results in Figure 1 also suggested that the intracellular CaM expression level correlated with the viral gene expression level, suggesting that CaM preferentially upregulates HSV-1 β and γ genes. Therefore, it is possible that the heterogeneity of CaM expression level is involved in the heterogeneity of β and γ gene expression in a cell population, and as a result, it may control the proportion of VPCs in the cell population. To analyze the relationship between CaM expression level and the proportion of VPCs in a cell population, WT or CALM1 KO HeLa cells were infected with rICP47 / vUs11 at an MOI of 5 and the proportion of VPCs was analyzed by flow cytometry 12 hours later.

[0055] Since rICP47 / vUs11 is a virus in which tagRFP is added to the α gene ICP47 and Venus is added to the γ gene Us11, the fluorescence intensity of TagRFP in infected cells indicates the expression of the α gene, and the fluorescence intensity of Venus indicates the expression of the γ gene. As with the results of qPCR, the MFI of tagRFP, which is an indicator of the expression of the α gene in the entire cell population, was not significantly decreased in CALM1KO cells, whereas the fluorescence intensity of Venus, which is an indicator of the expression of the γ gene, was significantly decreased (Fig. 2A, B). At this time, the proportion of VPCs was significantly decreased in CALM1KO cells (Fig. 2C, D). In addition, analysis using WT, CALM1 KO, and CALM1 / 2 KO showed that the MFI of Venus and the proportion of VPCs in rICP47 / vUs11-infected cells decreased according to the number of CALM gene knockouts (Fig. 2E-H). These results suggest that the reduced progeny virus production in CALM1 KO cells is due to a change in the heterogeneity of viral gene expression in the cell population, resulting in a decrease in the proportion of VPCs (cells in which the expression level of the γ gene exceeded the amount required for progeny virus production).

[0056] The results so far indicate that CaM is highly required for the expression of α, β, and γ genes, especially β and γ genes. It is known that the expression of α protein is necessary for the expression of β and γ genes. Therefore, it is considered that CaM may bind to a specific α protein required for the expression of β and γ proteins and positively regulate their function. Therefore, we searched the CaM-binding motifs present in ICP0, ICP27, ICP4, and ICP22, which have been reported to be necessary for the expression of β and γ genes, using the CaM database, and found that ICP4 contains an amino acid sequence that is highly homologous to the IQ motif, which is a CaM-binding motif (Figure 3A).

[0057] HeLa cells were infected with HSV-1 wild-type at an MOI of 5, fixed 9 hours later, permeabilized, stained with ICP4 and calmodulin antibodies, and observed by confocal microscopy. In HSV-1-infected cells, CaM was colocalized with ICP4 in the replication compartment (the site of HSV gene expression in the nucleus) (Fig. 3B). In addition, HeLa / puroR and HeLa / Flag-CaM were infected with HSV-1 at an MOI of 5, and immunoprecipitated with Flag antibody. It was found that ICP4 was co-precipitated with the infected cell lysate of HeLa cells that constitutively express Flag-CaM (Fig. 3C). These results suggest that ICP4 is indeed associated with CaM in infected cells.

[0058] Next, to examine whether CaM binds to ICP4 via the IQ-like motif in ICP4, we analyzed the effect of the deletion of the IQ-like motif on the binding of ICP4 to CaM by immunoprecipitation of ICP4 / WT, ICP4ΔIQ, and EGFP expressed in 293FT cells with CaM beads. The design of ICP4 / ΔIQ expressed in 293FT cells is shown in Figure 3D. The control EGFP was not pulled down by CaM beads or Protein A beads, but ICP4 / WT was pulled down only by CaM beads, indicating that ICP4 has the ability to directly bind to CaM (Figure 3E, F). In this case, the binding of ICP4 / ΔIQ to CaM was significantly weakened, suggesting that ICP4 binds to CaM via the IQ-like motif (Figure 3D-F).

[0059] To analyze the significance of ICP4 binding to CaM in HSV-1 gene expression, we created ICP4 / ΔIQ, in which the IQ motif of ICP4 was deleted, and its revertant, ICP4 / ΔIQ-repair, and analyzed the expression of the α, β, and γ genes by quantitative PCR. HeLa cells were infected with HSV-1 wild-type strain (HSV-1(F)), ICP4 / ΔIQ, and ICP4 / ΔIQ-repair at an MOI of 5, and the mRNA levels of the α, β, and γ genes were analyzed by qPCR. The results are shown in Fig. 4A. The mRNA levels of the α gene, ICP27, and ICP0 in cells infected with the ICP4 / ΔIQ mutant virus were significantly reduced compared to the wild-type strain and the revertant, but the extent of the reduction was slight. On the other hand, the mRNA levels of the β genes, ICP8, UL50, and the γ genes, VP22 and Us2, in ICP4 / ΔIQ mutant virus-infected cells were significantly reduced compared to the wild type and revertant strain, and the reduction fold was greater than that of the α genes, ICP27 and ICP0. These results were very similar to the phenotype in CALM1 / 2 KO cells shown in Figure 1.

[0060] ICP4 activates the expression of β and γ genes by binding to the viral genome. To examine whether the binding of ICP4 to CaM controls the binding ability of ICP4 to the viral genome, chromatin immunoprecipitation (ChIP) assays were performed with ICP4 antibody on ICP4 / ΔIQ or ICP4 / ΔIQ-repair infected cells. HeLa cells were infected with ICP4 / ΔIQ or its revertant ICP4 / ΔIQ-repair at an MOI of 5, and ChIP assays were performed with ICP4 antibody 3.5 hours later. The introduction of the ICP4ΔIQ mutation attenuated the binding ability of ICP4 to the HSV genome (ICP27, ICP8, and VP22 promoter regions) (Fig. 4B). Furthermore, when WT or CALM1 KO HeLa cells were infected with HSV-1 wild-type strain at MOI 5 and a ChIP assay of ICP4 was performed 3.5 hours later, the CALM1 KO HeLa cells showed a phenotype similar to that of ICP4 / ΔIQ (Fig. 4C). These results suggest that CaM binds to ICP4 and promotes the binding of ICP4 to the viral genome, thereby upregulating HSV-1 gene expression.

[0061] These results suggest that CaM regulates the binding ability of ICP4 to the viral genome, and thus increases the proportion of VPCs in the cell population by positively regulating the expression of HSV β and γ genes, thereby contributing to viral proliferation. In other words, CaM could be a therapeutic target for HSV-1. W-7 is known as a CaM inhibitor that inhibits the binding of CaM to its binding proteins. These results suggest that W-7 may inhibit the binding of ICP4 to CaM, suppress the expression of β and γ genes, and thereby reduce the proportion of VPCs (change the heterogeneity), thereby inhibiting the pathology of HSV.

[0062] In the HSV-1 intracerebral inoculation model, we investigated the pathological manifestation of the virus when W-7 was inoculated and the effect on the viral titer in the brain. 125 μg of W-7 (Merck) was intraperitoneally administered to 3-week-old female ICR mice, and 2 hours later, 500 PFU of the wild-type HSV-1 strain and 50 μg of W-7 were simultaneously administered into the brain (Figure 5A). As a result, W-7 almost completely inhibited the lethality of mice caused by HSV-1 and the viral proliferation in the mouse brain (Figure 5A, B).

[0063] Next, we investigated the inhibitory effect of W-7 on pathological expression in comparison with acyclovir, which is widely used as an anti-HSV drug. 500 PFU of HSV-1 wild-type strain and W-7 (50 μg) or acyclovir (50 μg, 150 μg) (Merck) were administered simultaneously into the brains of 3-week-old female ICR mice. As a result, the inhibitory effect of 50 μg of W-7 on pathological expression was higher than that of 150 μg of acyclovir (Figure 5C). Therefore, it was suggested that CaM could be a therapeutic target for HSV-1 infection, and furthermore, that W-7 and its derivatives could be used as antiviral agents against HSV-1 infection. In particular, while acyclovir, an existing anti-HSV drug, inhibits HSV DNA replication, W-7 is thought to inhibit the expression of the β gene, which is a precursor to DNA replication.

Claims

1. A pharmaceutical composition for preventing or treating a disease caused by infection with a herpes virus, comprising a calmodulin inhibitor as an active ingredient.

2. 2. The pharmaceutical composition according to claim 1, wherein the calmodulin inhibitor is a naphthalenesulfonamide compound or a pharma- ceutical acceptable salt thereof.

3. 3. The pharmaceutical composition of claim 2, wherein the naphthalenesulfonamide compound is selected from the group consisting of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(6-aminoethyl)-5-chloro-1-naphthalenesulfonamide; and N-(10-aminodecyl)-5-chloro-1-naphthalenesulfonamide.

4. 3. The pharmaceutical composition of claim 2, wherein the pharma- ceutical acceptable salt of the naphthalenesulfonamide compound is a hydrochloride salt.

5. The pharmaceutical composition according to claim 1 or 2, wherein the disease is caused by expression of the β gene group or later in the gene expression cascade of herpesvirus.

6. The pharmaceutical composition according to claim 5, wherein the expression of the β gene group and / or the γ gene group is inhibited by inhibiting the binding of the ICP4 protein of the herpes virus to calmodulin.

7. The pharmaceutical composition according to claim 6, wherein binding to an IQ-like motif in an ICP4 protein of a herpes virus is inhibited.

8. 3. The pharmaceutical composition according to claim 1 or 2, wherein the disease is selected from the group consisting of herpes labialis, genital herpes, encephalitis, neonatal herpes, keratitis and whitlow.

9. The pharmaceutical composition according to claim 1 or 2, which is administered into the brain of a subject.

10. A method for controlling the infection heterogeneity of a herpes virus, the method comprising the step of contacting the herpes virus with a calmodulin inhibitor.

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