Bee individual mortality inhibitor

Abstract

The present invention provides a composition or method for suppressing individual bee mortality or improving the survival rate of honeybees, or a technique for inhibiting factors related to the mechanism of individual bee mortality caused by mite-borne viral infections. [Solution] A composition for suppressing individual bee mortality and a composition for improving the survival rate of honeybees are provided, comprising a substance that inhibits the activity of proteases derived from Israeli acute paralysis virus (IAPV). Alternatively, a method for suppressing individual bee mortality and a method for improving the survival rate of honeybees are provided, comprising inhibiting the activity of proteases derived from IAPV. Furthermore, a composition for inhibiting the activity of proteases derived from IAPV is provided.

Description

Technical Field

[0001] The present invention relates to a technique useful for suppressing the individual death of honeybees and its utilization.

Background Art

[0002] Honeybees are agricultural insects necessary for pollinating crops. Honeybees are responsible for pollinating most fruits, many vegetables, seed production, livestock forage, etc., and pollination is essential for their production. Therefore, the presence of honeybees is indispensable in the field of agricultural production.

[0003] In recent years, a large-scale death of honeybees has occurred worldwide due to the emergence of Varroa destructor, a mite parasitic on honeybees. The most major cause of the death of honeybee colonies reported by beekeepers in the United States in recent years is Varroa destructor (Non-Patent Document 1). Varroa destructor has spread its distribution from Asia to all over the world along with the artificial movement of honeybees. If such a situation is left unattended, many agricultural products such as vegetables and fruits will not be able to be produced. The large-scale death of honeybees that play a role in pollination is a major problem in agriculture and beekeeping, and is also an important problem from the perspective of biodiversity.

[0004] Recently, it has been reported that by administering bacteria that have been genetically recombined to have an RNAi function to honeybees, it is possible to reduce the viruses infecting honeybees and the number of parasitic mites (Non-Patent Document 2). However, although this report shows that it is technically possible to reduce the number of viruses and mites, it is a technology that cannot be easily used in actual beekeeping sites because it involves genetically modified organisms.

[0005] Solving the problem of the large-scale death of honeybees leads to a stable supply of agricultural products and has become an important issue in agriculture. However, so far, the pathogens causing the large-scale death of honeybees, the pathogenesis mechanism of those pathogens, and the therapeutic agents for them have not been clarified at all. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Steinhauer, N., et. al., “United States honey bee colony losses 2022-23: Preliminary results from the Bee Informed Partnership”, [online], June 22, 2020, Bee Informed Partnership, [searched March 10, 2020], Internet <https: / / beeinformed.org / 2023 / 06 / 22 / united-states-honey-bee-colony-losses-2022-23-preliminary-results-from-the-bee-informed-partnership / > [Non-Patent Document 2] Leonard, SP, et. al. Engineered symbionts activate honey bee immunity and limit pathogens, Science. 2020 Jan 31;367(6477):573-576. doi: 10.1126 / science.aax9039. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention aims to identify the mechanism of individual bee death caused by mite infestation and to provide a composition or method for suppressing individual bee death or improving the survival rate of honeybees.

[0008] Alternatively, the present invention aims to provide a technique for inhibiting factors related to the mechanism of individual bee death due to mite-borne viral infection. [Means for solving the problem]

[0009] The inventors diligently conducted research to solve the above problems and revealed that the pathogen causing individual death of honeybees due to mite infestation is Israeli acute paralysis virus (IAPV). Furthermore, they discovered that intestinal damage caused by IAPV-derived proteases is associated with individual death of honeybees due to IAPV infection. In addition, the inventors screened compounds having inhibitory activity against IAPV-derived proteases in silico and found that administering the compounds identified through screening to honeybees suppressed individual death associated with mite infestation, thus completing the present invention.

[0010] In other words, the following invention is provided. [1] A composition for suppressing individual bee mortality, comprising a substance that inhibits the activity of a protease derived from Israeli acute paralysis virus (IAPV). [2] The composition according to [1], wherein the death of individual bees is caused by mite infestation of the bees. [3] A composition for improving the survival rate of honeybees, comprising a substance that inhibits the activity of IAPV-derived proteases. [4] The composition according to any one of [1] to [3], wherein the substance that inhibits the activity of IAPV-derived proteases is one or more compounds selected from compounds 1 to 416 listed in Tables I-1 to I-36 of the specification, or a salt thereof. [5] A method for suppressing individual bee mortality, comprising inhibiting the activity of IAPV-derived proteases. [6] The method described in [5] relating to the death of individual bees caused by mite infestation of bees. [7] A method for improving the survival rate of honeybees, comprising inhibiting the activity of IAPV-derived proteases. [8] The method according to any one of [5] to [7], wherein inhibiting the activity of IAPV-derived proteases is the use of a substance that inhibits the activity of IAPV-derived proteases. [9] The method according to [8], wherein the substance that inhibits the activity of IAPV-derived proteases is one or more compounds selected from compounds 1 to 416 listed in Tables I-1 to I-36 of the specification, or a salt thereof.

[10] A composition for inhibiting the activity of IAPV-derived proteases, comprising one or more compounds selected from compounds 1 to 416 listed in Tables I-1 to I-36 of the specification, or a salt thereof. [Effects of the Invention]

[0011] The present invention provides a composition or method for suppressing individual bee mortality. Alternatively, the present invention provides a composition or method for improving the survival rate of honeybees. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows a schematic diagram of the sample preparation procedure for MinION analysis. (Example 1) [Figure 2] Figure 2 shows a heatmap illustrating the quantitative values ​​of each viral load calculated using MinION analysis. (Example 1) [Figure 3] Figure 3 shows the results of quantification of honeybee viruses by RT-PCR. The horizontal axis represents the sample number (honeybee population No.), and the vertical axis represents the number of viral copies per 1 ng RNA. (Example 2) [Figure 4] Figure 4 shows the quantitative results of viral response factors and antimicrobial peptides in honeybees. The horizontal axis represents the gene name, and the vertical axis represents the expression level ratio (Fold Change; FC). (Example 3) [Figure 5] Figure 5 shows the quantitative results of intestinal repair factors in honeybees. The horizontal axis represents the gene name, and the vertical axis represents the expression level ratio (Fold Change; FC). (Example 3) [Figure 6] Figure 6 shows the results of RNAi testing of the virus in honeybees. The horizontal axis represents the number of days since the start of administration, and the vertical axis represents the survival rate, with the number of individuals on day 0 set to 100 and the number of individuals on each day shown as a percentage. (Example 4) [Figure 7] Figure 7 shows the detection results of pH3 antibody-positive cells in honeybees. It shows the immunostaining images and Merge images of DAPI and pH3 of the microscopic images of the midguts of the virus-infected group and the non-infected group. (Example 5) [Figure 8] Figure 8 shows the analysis results of pH3 antibody-positive cells after dsRNA administration. The horizontal axis represents the administration group, where "None" is the control group, "GFPi" is the GFP dsRNA administration group, "IAPVi" is the IAPV dsRNA administration group, and "DWVi" is the DWV dsRNA administration group, and the vertical axis represents the number of pH3 antibody-positive cells in the midgut per individual. (Example 5) [Figure 9] Figure 9 shows the results of suppressing the individual death of honeybees in virus-infected group No. 5 by administration of E-64. The horizontal axis is the number of days elapsed since the start of administration, the vertical axis is the survival rate, and the number of individuals on day 0 is taken as 100, and the number of individuals on each day is shown as a percentage. (Example 9) [Figure 10] Figure 10 shows the results of suppressing the individual death of honeybees in virus-infected group No. 2 by administration of E-64. The horizontal axis is the number of days elapsed since the start of administration, the vertical axis is the survival rate, and the number of individuals on day 0 is taken as 100, and the number of individuals on each day is shown as a percentage. (Example 9)

Mode for Carrying Out the Invention

[0013] The present invention will be described in detail below. Each feature and each element of the present invention described below can be arbitrarily selected and combined.

[0014] <Composition> One embodiment of the present invention relates to a composition (hereinafter sometimes referred to as the composition of the present invention).

[0015] [Active Ingredient] The composition of the present invention contains as an active ingredient a substance that inhibits the activity of proteases derived from Israeli acute paralysis virus (IAPV). Examples of IAPV-derived proteases include those having the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, as well as those resulting from mutations. For example, the amino acid sequence shown in SEQ ID NO: 1, 2, or 3 may have 1 to 40, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, or more preferably 1 to 3 amino acid residues deleted, added, inserted, or substituted, and may be a polypeptide having degrading activity against peptides that are substrates for IAPV proteases derived from the sequence of an IAPV-derived polyprotein (SEQ ID NO: 4) (for example, one or more selected from PNDIVDVTMQ (SEQ ID NO: 5), TMQMWKDQVA (SEQ ID NO: 6), and IAPV-derived polyprotein (SEQ ID NO: 4), etc.). Furthermore, the amino acid sequences of IAPV-derived proteases have 50% or more identity with the amino acid sequences of SEQ ID NOs: 1, 2, or 3, for example, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, or 99.9% or more, and include variants consisting of amino acid sequences that have degrading activity against peptides that are substrates for IAPV proteases derived from the sequence of IAPV-derived polyprotein (SEQ ID NO: 4) (for example, one or more selected from PNDIVDVTMQ (SEQ ID NO: 5), TMQMWKDQVA (SEQ ID NO: 6), and IAPV-derived polyprotein (SEQ ID NO: 4), etc.). Furthermore, examples of cDNA sequences encoding IAPV-derived proteases include the sequence shown in SEQ ID NO: 7, the sequence shown in SEQ ID NO: 8, and the sequence shown in SEQ ID NO: 9. However, other sequences resulting from polymorphism or mutations are also included, such as those in which 1 to 50, preferably 1 to 20, more preferably 1 to 5, and more preferably 1 to 3 bases are deleted, substituted, and / or added in the sequence of SEQ ID NO: 7, 8, or 9.Furthermore, the nucleotide sequences of the cDNA encoding the IAPV-derived protease include variants consisting of nucleotide sequences that have 50% or more identity with the nucleotide sequence of SEQ ID NO: 7, 8, or 9, for example, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99% or more, 99.5% or more, 99.7% or more, or 99.9% or more. The identity of the nucleotide sequences can be determined using known algorithms such as BLAST or FASTA. Furthermore, the nucleotide sequences of the cDNA encoding the IAPV-derived protease include variants consisting of nucleotide sequences that hybridize under stringent conditions with the nucleotide sequence of SEQ ID NO: 7, 8, or 9. Examples of stringent hybridization conditions include, for example, the following conditions for post-hybridization washing: typically "5×SSC, 1%SDS, 37°C", "1×SSC, 0.1%SDS, 37°C", "0.5×SSC, 0.1%SDS, 42°C", and "5×SSC, 1%SDS, 65°C". As shown in this embodiment, IAPV-derived proteases are involved in viral intestinal damage in honeybees.

[0016] Sequence ID 1 VAQRLITNRVLTNLYKVCLVHNDDRITPLLNGLFVRSNLMLIPGHLLGFIAEYDTIEIRNLFDVVFRVPWKDVKKIPIVNALGESKEAALLAFPKFVCQHSDLVKHFQN AESMSKFKRCEVTLPVLRFSEKVGKFLSTLIECDRVEAYDRPYTLNDSQKGQYILRQGLEYTMPTTNGDCGSPLIINETQVLRKIAGIHVAGATTGKAYAESITQKDLE

[0017] Sequence ID 2 PNDIVDVTMQMWKDQVAQRLITNRVLTNLYKVCLVHNDDRITPLLNGLFVRSNLMLVPGHLLGFIAEYDTIEIRNLFDVVFRVPWKDVKKILIVNALGESKEAALLAFPKFVCQHSDLVKHFQNAESMSKFKRCE VTLPVLRFSEKVGKFLSTLIECDRVEAYDRPYTLNDSQKGQYILRQGLEYTMPTTNGDCGSPLIINETQVLRKIAGIHVAGATTGKAYAESITQKDLERAFTKIDVSMQIQLDLDSTLDFSKPEPKLPSGTEFGP

[0018] Sequence ID 3 SSGDCVTRKQQVQRVIEAFASSDAVTLKKSTAKFVESDPNDIVDVTMQMWKDQVAQRLITNRVLTNLYKVCLVHNDDRITPLLNGLFVRSNLMLVPGHLLGFIAEYDTIEIRN LFDVVFRVPWKDVKKIPIVNALGESKEAALLAFPKFVCQHSDLVKHFQNAESMSKFKRCEVTLPVLRFSEKVGKFLSTLIECDRVEAYDRPYTLNDSQKGQYILRQGLEYTMP TTNGDCGSPLIINETQVLRKIAGIHVAGATTGKAYAESITQKDLERAFTKIDVSMQIQLDLDPTLDFSKPEPKLPSGTEFGPEDLSFCDLPALKMLPVGKLSEPLFEPGKTDI RPSLVHGQISDIKTKPAYLRNVIKDGIFVNMKHKNLMKCAMDTPYIDKDMIDEAYQLTKSVWLKGMRDELKKVLTYEEAICGSEVSEYISSINRSSSPGYPWIKDRTKGTKGKQ

[0019] Sequence ID 4

[0020] sequence no. 7 GTTGCCCAACGATTGATAACCAACCGTGTGTTAACAAACTTGTATAAGGTTTGTTTAGTGCATAATGATCGCGCATAACACCTCTTTTGAATGGACTTTTTGTTCGTCAAATTTGATGTTAATTCCTGGACATCTTCTTGGATTTATTGCTGAGTATGATACAATAGAAATTCGAAATCTCTTTGACGTGGTATTTAGAGTACCATGGAAGATGTAAAGAAGATTCCAATTGTTAATGCTCTCGGTGAATCTAAGGAGGCAGCGTTTGGCCTTTCCAAATTCGTTTGTCAACATTCTGACTTGGTCAAGCATTTCCAAAACG CTGAATCAATGTCTAAATTCAAACGTTGTGAAGTAACCCTCCCAGTATTACGTTTCTCTGAGAAAGTTGGTAAATTTTTATCAACACTCATAGAATGCGATCGGGTGGAGGCTTATGACAGACCATATACGCTGAATGACTCTCAAAAGGACAATATATTCTACGTCAAGGCTTAGAATATACGATGCCAACGACCAATGGTGATTGTGGTTCGCCACTAATCATCAATGAAACCCAAGTTTTGAGGAAAATTGCTGGTATTCATGTGGCAGGACTACAAGGTAAGGCCTATGCTGAATCTAAACCCAAAAGATCTTGAGTAAC

[0021] sequence number 8 CCAAATGACATAGTTGATGTTACAATGCAAATGTGGAAGGATCAAGTTGCTCAACGACTGATAACTAACCGTGTGTTAACAAACTTGTATAAGGTTTGTTTAGTGCATAATGACGATCGCATTACACCTCTTTGAATGGACTTTTTGTTCGTTCGAATTTGATGTTAGTTCCTGGACATCTTCTTGGATTTATTGCTGAGTA TGATACAATAGAAATTCGAAATCTCTTTGACGTGGTATTTAGAGTACCATGGAAGATGTAAAGAAGATTCTAATTGTTAATGCTCTCGGTATCTAAGGAGGCAGCGTTATTGGCCTTTCCAAATTCGTTTGTCAACATTCTGACTTGGTCAAGCATTTCCAAAATGCTGAATCAATGTCTAAATTCAAACGATGTGAAG TAACCCTCCCAGTATTACGTTTCTCTGAGAAAGTTGGTAAATTTTTATCAACACTCATAGAATGCGATCGGGTGGAGGCTTATGACAGACCATATACGCTGAATGACTCTCAAAAAGGACAATATATTCTACGTCAAGGCTTAGAATATACGATGCCAACGACCAATGGTGATTGTGGTTCGCCACTAATCATCAACGAAACC CAAGTTTTGAGGAAATTGCTGGTATTCATGTGGCAGGAGCTACAACCGGTAAGGCCTATGCTGAATCTATAACCCAAAAGATCTTGAGCGAGCATTTACTAAAATAGATGTGAGCATGCAGATCCAATTGGATTTGGATTCGACATTGGACTTCTCAAAACCTGAACCTAAATTGCCTTCTGGTACTGAATTTGGTCCTTAA

[0022] sequence number 9

[0023] Substances that inhibit the activity of IAPV-derived proteases can be any substance that has the effect of inhibiting the activity of IAPV-derived proteases, such as compounds that inhibit the enzymatic activity of IAPV-derived proteases, such as antibodies, carbohydrates, lipids, proteins, and peptides, with organic small molecule compounds being preferred. Other examples include compounds that suppress the expression of genes encoding IAPV-derived proteases, such as nucleic acids (siRNA, dsRNA, antisense RNA, etc., against IAPV-derived protease genes).

[0024] The protease activity derived from IAPV can be measured using known or novel protease activity measurement methods. For example, it can be measured using a method that utilizes the degradation of a substrate (e.g., casein, synthetic substrate, etc.). A method utilizing substrate degradation can be used, for example, by adding IAPV-derived protease to a substrate in the presence of the test substance, and then quantifying the undegraded substrate and / or degraded substrate after a certain period of time. The quantification of the undegraded substrate and / or degraded substrate can be performed, for example, by using a fluorescently labeled substrate, electrophoresis, or absorbance measurement. When using a method utilizing substrate degradation, contact of the test substance with the IAPV-derived protease may be performed in a protease substrate solution, or the IAPV-derived protease may be further contacted with the substrate in another solvent after contact with the test substance. A higher amount of undegraded substrate indicates lower protease activity, while a higher amount of substrate degradation products indicates higher protease activity. "Inhibitory activity against IAPV-derived proteases" refers to the activity that inhibits (suppresses) the activity of IAPV-derived proteases, and can be evaluated by determining that the activity of IAPV-derived proteases that have come into contact with the test substance is lower than the activity of IAPV-derived proteases that have not come into contact with the test substance. The comparison between the activity of IAPV-derived proteases that have come into contact with the test substance and the activity of control IAPV-derived proteases that have not come into contact with the test substance is preferably performed based on whether or not there is a statistically significant difference. Examples of such methods include the following: A peptide that is a substrate of IAPV proteases derived from the sequence of IAPV-derived polyprotein (SEQ ID NO: 4) (e.g., PNDIVDVTMQ (SEQ ID NO: 5), TMQMWKDQVA (SEQ ID NO: 6), etc.) is labeled with a fluorescent substance, and the peptide is reacted with the IAPV-derived protease under conditions in which the IAPV-derived protease can act and in the presence of the substance to be evaluated, and the evaluation can be performed by detecting a signal based on the degradation of the peptide by the activity of the protease. Furthermore, IAPV-derived protease activity can also be evaluated using the following evaluation methods.The protease activity of the IAPV-derived protease can be evaluated by reacting a polypeptide chain expressing the cDNA base sequence (SEQ ID NO: 10) of an IAPV-derived polyprotein (SEQ ID NO: 4) in E. coli or other bacteria with an IAPV-derived protease for a certain period of time, and then measuring the degradation of the IAPV-derived polyprotein by analyzing the reaction product using methods such as gel staining after SDS-PAGE.

[0025] Sequence ID 10

[0026] (compound) In one embodiment, the substance that inhibits the activity of IAPV-derived proteases is one or more compounds selected from compounds 1 to 416 listed in Tables I-1 to I-36, or a pharmaceutically acceptable salt thereof.

[0027] [Table 1]

[0028] [Table 2]

[0029] [Table 3]

[0030] [Table 4]

[0031] [Table 5]

[0032] [Table 6]

[0033] [Table 7]

[0034] [Table 8]

[0035] [Table 9]

[0036] Table 10

[0037] Table 11

[0038] Table 12

[0039] Table 13

[0040] Table 14

[0041] Table 15

[0042] Table 16

[0043] Table 17

[0044] Table 18

[0045] Table 19

[0046] Table 20

[0047] Table 21

[0048] Table 22

[0049] Table 23

[0050] Table 24

[0051] Table 25

[0052] Table 26

[0053] Table 27

[0054] Table 28

[0055] Table 29

[0056] Table 30

[0057] [Table 31]

[0058] [Table 32]

[0059] [Table 33]

[0060] [Table 34]

[0061] [Table 35]

[0062] [Table 36]

[0063] The above compounds can be commercially available. Some examples include compound 240 (product number: HY-102087), compound 2 (product number: HY-100350), compound 257 (product number: HY-132850), compound 280 (product number: HY-109001), compound 285 (product number: HY-18236), compound 153 (product number: HY-145155), and compound 327 (product number: HY-100522), which are available from Medchemexpress, and compound 211 (product number: RG00110), which is available from Thermo Scientific (Maybridge). Alternatively, the above compounds may be synthesized using conventional methods.

[0064] In one embodiment, a substance that inhibits the activity of IAPV-derived proteases is one or more compounds selected from compounds 1, 2, 153, 240, 211, 257, 280, 285, 327, 26, 37, 71, 83, 191, 220, 284, 313, 325, 328, 352, 353, 375, 381, 387, 405, and 413, or a pharmaceutically acceptable salt thereof. In another embodiment, the substance that inhibits the activity of IAPV-derived proteases is one or more compounds selected from compounds 1, 2, 153, 240, 211, 257, 280, 285, 327, 26, 37, 71, 83, 191, 220, 284, 313, 325, 328, 352, 353, 375, 381, 387, 405, and 413, or a pharmaceutically acceptable salt thereof.

[0065] (Pharmacologically acceptable salts) pharmaceutically acceptable salts of compounds are, for example, acid addition salts or base addition salts. Examples of acid addition salts include, but are not limited to, mineral salts such as hydrochloride, hydrobromide, nitric acid, and sulfuric acid; organic carboxylate salts such as formic acid, acetic acid, citric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, malic acid, tartaric acid, aspartic acid, trichloroacetic acid, and trifluoroacetic acid; and sulfonates such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid. Examples of base addition salts include, but are not limited to, alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium and magnesium; ammonium salts; and nitrogen-containing organic base salts such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-efenamine, and N,N'-dibenzylethylenediamine.

[0066] [Application] (1. Suppression of individual bee mortality) The composition of the present invention can be used to suppress individual bee mortality. Suppressing individual bee mortality means, for example, that when the composition of the present invention is administered or ingested by honeybees, the mortality rate of the honeybee population decreases or the survival rate improves compared to an appropriate control (for example, in the case of no treatment; when a substance without pharmacological activity such as water or a sugar-containing aqueous solution is administered or ingested, etc.). As an example of a specific evaluation method for the mortality rate and survival rate of a honeybee population, as shown in this embodiment, the mortality rate can be evaluated by measuring the change in the number of individuals in a honeybee population raised in an environment suitable for growth, and measuring the percentage of individuals that have died at any point during the rearing period, with the number of individuals in the honeybee population at the start of rearing set to 100, and the survival rate can be evaluated by measuring the percentage of individuals that are still alive at any point during the rearing period, with the number of individuals in the honeybee population at the start of rearing set to 100. Furthermore, the effect of the composition of the present invention on suppressing individual bee mortality can be confirmed by measuring, as shown in this example, that in a population of honeybees that have ingested the composition of the present invention under conditions suitable for growth, the mortality rate of the honeybee population at any point after ingestion is lower, or the survival rate of the honeybee population is higher, compared to a population that has not ingested the composition of the present invention.

[0067] In the present invention, individual bee death may be caused by mite infestation. As shown in this example, it is suggested that mites transmit IAPV infection to honeybees. The type of mite is not particularly limited as long as it transmits IAPV infection, but examples include Varroa mites (e.g., Varroa ventricosa, Varroa japonica), Varroa woodi mites, and Spiny mites. The fact that individual bee death is caused by mite infestation can be confirmed by the presence of mites on the surface of the honeybee's body, or by a higher amount of IAPV virus in the midgut of the honeybee compared to the uninfected case. The amount of IAPV virus in the midgut of the honeybee can be appropriately measured by methods known to those skilled in the art, and such methods may include, for example, a method of quantifying the amount of IAPV mRNA in RNA extracted from the midgut by quantitative real-time PCR or sequencing analysis, as shown in this example.

[0068] (2. Improving the survival rate of honeybees) The compositions of the present invention can also be used to improve the survival rate of honeybees. Improving the survival rate of honeybees means, similar to the suppression of individual honeybee mortality described above, that when honeybees are administered or ingested the compositions of the present invention, the survival rate of the honeybee population is improved compared to an appropriate control (for example, in the case of no treatment; when a substance without pharmacological activity such as water or a sugar-containing aqueous solution is administered or ingested). As an example of a specific method for evaluating the survival rate of a honeybee population, as shown in this embodiment, the survival rate can be evaluated by measuring the change in the number of individuals in a honeybee population raised in an environment suitable for growth, and measuring the percentage of surviving individuals at any point in time during the rearing period, with the number of individuals in the honeybee population at the start of rearing set to 100. Furthermore, the effect of the composition of the present invention on improving the survival rate of honeybees can be confirmed by measuring, as shown in this example, that the survival rate of a honeybee population that has been given the composition of the present invention under conditions suitable for growth is higher at any point after ingestion compared to a population that has not been given the composition of the present invention.

[0069] (subject) When using the composition of the present invention to suppress individual bee mortality or improve the survival rate of honeybees, the target to which the composition is administered or ingested is honeybees. In the present invention, honeybees refer to insects belonging to the genus Apis. Examples of species belonging to the genus Apis include, but are not limited to, the Eastern honeybee (Apis cerana), the European honeybee (Apis mellifera), the mackerel honeybee (Apis koschevnikovi), the Kinabalu mountain honeybee (Apis nuluensisi), the black-banded honeybee (Apis nigrocincta), the giant honeybee (Apis dorsata), the Himalayan giant honeybee (Apis laboriosa), the dwarf honeybee (Apis florea), and the black dwarf honeybee (Apis andreniformis).

[0070] (3. Inhibition of IAPV-derived protease activity) In one embodiment, the composition of the present invention can be used to inhibit the activity of IAPV-derived proteases. The activity of IAPV-derived proteases can be measured using known or novel protease activity measurement methods, for example, by a method that utilizes the degradation of a substrate (e.g., casein, synthetic substrate, etc.). A method that utilizes substrate degradation can be used, for example, by adding IAPV-derived protease to a substrate in the presence of a test substance and quantifying the undegraded substrate and / or degraded substrate after a certain period of time. The quantification of undegraded substrate and / or degraded substrate can be performed, for example, by a method using a fluorescently labeled substrate, electrophoresis, or absorbance measurement. When using a method that utilizes substrate degradation, contact of the test substance with the IAPV-derived protease may be performed in a protease substrate solution, or the IAPV-derived protease may be further contacted with the substrate in another solvent after contact with the test substance. A higher amount of undegraded substrate results in lower protease activity, while a higher amount of substrate degradation products results in higher protease activity. "Inhibitory activity against IAPV-derived proteases" refers to the activity that inhibits (suppresses) the activity of IAPV-derived proteases, and can be evaluated by determining that the activity of IAPV-derived proteases that have come into contact with the test substance is lower than the activity of IAPV-derived proteases that have not come into contact with the test substance. The comparison between the activity of IAPV-derived proteases that have come into contact with the test substance and the activity of control IAPV-derived proteases that have not come into contact with the test substance is preferably performed based on whether or not there is a statistically significant difference. Examples of such methods include the following: A peptide that is a substrate of IAPV protease derived from the sequence of IAPV-derived polyprotein (SEQ ID NO: 4) is labeled with a fluorescent substance, and the peptide is reacted with the IAPV-derived protease under conditions in which the IAPV-derived protease can act and in the presence of the substance to be evaluated, and the activity can be evaluated by detecting a signal based on the degradation of the peptide by the activity of the protease. In addition, IAPV-derived protease activity can also be evaluated by the following evaluation methods.The protease activity of the IAPV-derived protease can be evaluated by reacting a polypeptide chain expressing the cDNA base sequence (SEQ ID NO: 10) of an IAPV-derived polyprotein (SEQ ID NO: 4) in E. coli or other bacteria with an IAPV-derived protease for a certain period of time, and then measuring the degradation of the IAPV-derived polyprotein by analyzing the reaction product using methods such as gel staining after SDS-PAGE.

[0071] (subject) When the composition of the present invention is used to inhibit the activity of IAPV-derived proteases, the target to which the composition is administered or ingested may be honeybees. Alternatively, the composition of the present invention can be used not only in vivo but also, for example, in vitro to inhibit the activity of IAPV-derived proteases.

[0072] [Content] The content of the active ingredient in the composition of the present invention is not particularly limited and can be, for example, 0.1 w / w% or more, 1 w / w% or more, or 10 w / w% or more, while it can be, for example, 99.9 w / w% or less, 90 w / w% or less, or 80 w / w% or less. The above values ​​may be combined as appropriate to express a range of content.

[0073] [Dosage / Intake] The dosage or intake of the composition of the present invention can be appropriately set depending on the application. When the composition of the present invention is mixed with a sugar-containing aqueous solution and administered orally to honeybees, the concentration of the active ingredient in the aqueous solution can be, for example, 1 μM or more, 10 μM or more, or 50 μM, or on the other hand, it can be, for example, 1 mM or less, 500 μM or less, or 200 μM or less. The above values ​​may be appropriately combined and expressed as a range of dosage or intake (concentration).

[0074] [Method of administration / method of intake] The composition of the present invention can be administered orally or ingested. When administering or ingesting the composition of the present invention to honeybees orally, for example, the composition of the present invention can be mixed with feed (e.g., a sugar-containing solution such as sucrose, pollen, honey, artificial feed, etc.) and given to the honeybees through water absorption, feeding, etc.

[0075] The composition of the present invention can also be administered to or ingested by honeybees by spraying, dipping, coating, fumigating, or scattering it onto honeybees, beehives, plants that serve as nectar sources for honeybees, or suitable materials. The material is attached inside or around the beehive, and the composition of the present invention is administered to the honeybees when they come into contact with it.

[0076] [Additives, other ingredients] The composition of the present invention may further contain pharmaceutically acceptable carriers or additives in addition to the active ingredient. Examples of pharmaceutically acceptable carriers or additives include, but are not limited to, sugars such as sucrose, dextrin, and glucose; surfactants such as sodium lauryl sulfate, laurylamine acetate, and polyoxyethylene lauryl alcohol; lubricants such as talc and calcium stearate; stabilizers such as tocopherol, ascorbic acid, and vitamins; and thickeners such as starch, xanthan gum, and polyvinylpyrrolidone.

[0077] [Dosage form / form] The composition of the present invention can be prepared in the form of a liquid, suspension, emulsion, wettable powder, aerosol, fumigant, powder, granules, tablet, etc., depending on the application and administration method. Preferably, the composition of the present invention is in the form of a liquid. The manufacturing method for each of the above-mentioned dosage forms is not particularly limited, and techniques that can be used for general agricultural chemical formulations can be used.

[0078] [Preparation method] The compositions of the present invention can be prepared, for example, by mixing an active ingredient with pharmaceutically acceptable carriers and additives. Examples of pharmaceutically acceptable carriers and additives include, but are not limited to, sugars such as sucrose, dextrin, and glucose; surfactants such as sodium lauryl sulfate, laurylamine acetate, and polyoxyethylene lauryl alcohol; lubricants such as talc and calcium stearate; stabilizers such as tocopherol, ascorbic acid, and vitamins; and thickeners such as starch, xanthan gum, and polyvinylpyrrolidone.

[0079] <Methods to suppress individual bee mortality> One embodiment of the present invention relates to a method for suppressing individual bee mortality (hereinafter sometimes referred to as the "method for suppressing individual bee mortality of the present invention"). Suppression of individual bee mortality can be evaluated or confirmed as described in the [Applications] section above.

[0080] In this invention, the death of a honeybee may be caused by mite infestation. The type of mite is not particularly limited as long as it transmits IAPV infection, but examples include Varroa mites (e.g., Varroa ventricosa, Varroa japonica), Varroa woodi mites, and Spiny mites. The fact that the death of a honeybee is caused by mite infestation can be confirmed by the presence of mites on the surface of the honeybee's body and by the fact that the amount of IAPV virus in the honeybee's midgut is higher than in the case of non-infected honeybees.

[0081] <Methods to improve the survival rate of honeybees> One embodiment of the present invention relates to a method for improving the survival rate of honeybees (hereinafter, it may be referred to as the method for improving the survival rate of honeybees of the present invention). Improving the survival rate of honeybees can be evaluated or confirmed as described in the [Applications] section above.

[0082] The present invention provides a method for suppressing individual bee mortality and improving the survival rate of honeybees, which includes inhibiting the activity of IAPV-derived protease. Inhibiting the activity of IAPV-derived protease in the present invention's method for suppressing individual bee mortality and improving the survival rate of honeybees may involve using a substance that inhibits the activity of IAPV-derived protease. The substance that inhibits the activity of IAPV-derived protease can be selected as described in the [Active Ingredient] section above.

[0083] The present invention's method for suppressing individual bee mortality and improving the survival rate of honeybees may include administering or allowing honeybees to ingest a substance that inhibits the activity of IAPV-derived protease. In this case, the honeybees are as described in the (Target) section above, and the method of administration or ingestion, dosage, etc., can be selected as described in the [Dosage / Ingestion], [Administration Method, Ingestion Method], etc. sections above. [Examples]

[0084] The present invention will be described in more detail below with reference to examples. The following examples are merely specific illustrations of the present invention and do not limit the scope of the present invention in any way.

[0085] <Example 1. Quantitative determination of honeybee viruses using MinION> (The honeybees that were targeted) The study targeted 14 honeybee colonies (populations No. 1-14) from 13 apiaries across Japan, including colonies that were dying due to mites and healthy colonies.

[0086] (Sample preparation) The midgut of honeybees was collected, and total RNA was extracted using ISOGEN II (Nippon Gene Co., Ltd.). 30 μg of RNA was treated with E. coli Poly(A) Polymerase (New England Biolab Japan (NEB)) to add polyA to the RNA. Subsequently, Oligotex was used.TM -dT30 <super>PolyA-additive RNA was purified using the mRNA Purification Kit (Takara Bio Inc.). Furthermore, excess rRNA was removed using the NEB Next rRNA Depletion Kit v2 (Human / Mouse / Rat) with RNA Sample Purification Beads (NEB Inc.) to obtain sample RNA containing viral RNA. A schematic diagram of the sample preparation procedure for MinION analysis is shown in Figure 1.

[0087] (MinION analysis method) The prepared RNA was converted to cDNA using the Direct cDNA Sequencing Kit (Oxford Nanopore Technologies) and Native Barcoding Expansion 1-12 (PCR-free) (Oxford Nanopore Technologies), and the RNA converted to cDNA was quantified using a MinION flow cell (Oxford Nanopore Technologies: FLO-MIN106D). The sequence information of the detected viruses was mapped to the viral library information, and quantitative values ​​for each viral load were calculated. The quantitative results, shown as a heatmap, are shown in Figure 2.

[0088] As shown in Figure 2, it became clear that the main viruses were Israeli acute paralysis virus (IAPV) and deformed wing virus (DWV).

[0089] <Example 2. Quantification of honeybee viruses by RT-PCR> (Sample preparation and RT-PCR analysis method) The midguts of honeybees from colonies No. 1-14 were collected, and total RNA was extracted using ISOGEN II (manufactured by Nippon Gene Co., Ltd.). One Step PrimeScript was extracted from the total RNA. TM III. The virus contained in the midgut RNA of honeybees was quantified using RT-qPCR Mix (Takara Bio Inc.) and the probes and primers shown in Table II. For quantification, the number of virus copies present in the midgut RNA of honeybees was calculated from the relationship between the viral copy number and the CT value of RT-PCR, using DNA fragments based on each RNA virus sequence. The results are shown in Figure 3. The base sequences (DNA) of the positive control viruses are shown in Tables III-1 and III-2.

[0090] As shown in Figure 3, virus levels were high in honeybee populations No. 1, 2, 3, and 5, which originated from colonies dying due to mites. Among these, IAPV and DWV were the main viruses found in virus-infected populations No. 2, No. 3, and No. 5, with IAPV levels being extremely high, more than 10 times higher than DWV.

[0091] [Table 37]

[0092] [Table 38]

[0093] [Table 39]

[0094] <Example 3. RNA-seq analysis of biofactors in honeybees and quantification of each factor> (Sample preparation and RT-PCR analysis method) Midgut samples were collected from honeybee individuals in virus-infected groups No. 1, 2, 3, and 5, and from virus-uninfected groups No. 6, 7, 9, and 14. Total RNA was extracted using ISOGENII (Nippon Gene Co., Ltd.). mRNA was purified with oligo-dT, cDNA was synthesized using random primers, and a sequencing library was prepared. Sequencing analysis (DNBSEQ-G400 100bp paired-end sequencing) was performed on the obtained library. Data analysis was performed from the sequence information of the analysis results to quantify the amount of mRNA in the midgut of honeybee individuals from groups No. 1, 2, 3, 5, 6, 7, 9, and 14. The number of factors whose gene expression increased and decreased in virus-infected groups No. 1, 2, 3, and 5 compared to virus-uninfected groups No. 6, 7, 9, and 14 was listed. A list of differentially expressed genes is shown in Table IV. Figure 4 shows the quantitative results for viral response factors and antimicrobial peptides, and Figure 5 shows the quantitative results for intestinal repair factors. The quantitative values ​​for each factor shown in Figure 5 were calculated from the quantitative values ​​obtained by RNA-seq analysis.

[0095] As shown in Table 3, in the vivo state of virus-infected honeybees, a decrease in the expression of immune factors and metabolic factors, and an increase in the expression of apoptosis-related factors were observed. This revealed that IAPV or DWV induces individual death of honeybees by causing a decrease in immune and metabolic function, cell death and tissue degradation through changes in the expression of these factors. Furthermore, analysis including the histochemical analysis of Figures 4 and 5 and Example 5 described later revealed that these viruses also cause a decrease in immune and metabolic function, cell death and tissue degradation by allowing bacteria to enter the body by creating holes in the intestinal tract and inducing sepsis.

[0096] [Table 40]

[0097] <Example 4. RNAi test of honeybee virus> (dsRNA preparation method and RNAi test method) Israeli acute paralysis virus (IAPV) was cloned using two sets of primers (IAPV Cloning primer1-F,R (SEQ ID NOs. 47, 48) and IAPV Cloning primer2-F,R (SEQ ID NOs. 49, 50)), and DWV (DWV) was cloned using one set of primers (DWV Cloning primer-F,R (SEQ ID NOs. 51, 52)). Based on the DNA fragments, IAPV dsRNA (a mixture of IAPV dsRNA1 and IAPV dsRNA2) and DWV dsRNA were synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System (Promega) with the primers for each virus (IAPV T7 primer1-F,R (SEQ ID NOs. 53, 54) and IAPV T7 primer2-F,R (SEQ ID NOs. 55, 56), and DWV T7 primer-F,R (SEQ ID NOs. 57, 58). For GFP, a GFP-linked vector (pEGFP-C3) and primer (GFP T7) were used. GFP dsRNA was synthesized using primer-F,R (SEQ ID NOs. 59, 60). Each dsRNA was dissolved in 50% sucrose solution at a concentration of 100 μg / ml and administered to honeybees from honeybee colony No. 5 that were infected with the virus. The 50% sucrose solution containing the dsRNA was changed daily, and the daily change in the number of honeybees was measured. The control group was administered a 50% sucrose solution without dsRNA. Aseptic in vivo drug evaluation rearing was used.

[0098] [Table 41]

[0099] (Aseptic in vivo drug evaluation rearing method) A novel aseptic in vivo drug evaluation rearing method for honeybees was established. Incubators and rearing hives were cleaned and then sterilized with 70% ethanol. 30-35 honeybees were placed in each hive and fed 50% sucrose solution (aseptically dissolved in sterile water) and pollen dissolved in 50% sucrose solution. The rearing temperature was maintained at 30°C. The 50% sucrose solution was changed daily, and the number of individuals was counted at that time. Drugs were administered after being dissolved in 50% sucrose solution. When the survival rate fell below the initial 50%, total RNA was collected and histochemical analysis of the midgut and other tissues was performed on the surviving individuals. Pharmacological analysis was performed on the collected RNA, tissue, and blood.

[0100] As shown in Figure 6, RNAi testing revealed that IAPV RNAi suppressed individual bee mortality and sepsis after tick infection, while DWV RNAi was ineffective. These results clearly indicate that IAPV is the pathogen responsible for mass bee deaths.

[0101] <Example 5. Analysis of intestinal tract regeneration in the midgut of honeybees> (Sample preparation and detection methods) After dissecting the midgut of honeybees, the samples were fixed with 4% paraformaldehyde for 30 minutes, washed three times in PBS containing 0.2% TritonX-100 (hereinafter sometimes referred to as PBX) at room temperature for 20 minutes each, and then reacted with 2% BSA / PBX for 1 hour to block. Next, they were reacted with 0.5% BSA / PBX containing Phospho-Histone H3 (pH3) antibody (Cell Signaling Technology) at 4°C for 8 hours. After washing three times in PBX at room temperature for 20 minutes each, they were reacted with 0.5% BSA / PBX containing a secondary antibody at 4°C for 8 hours. After washing three times in PBX at room temperature for 20 minutes each, the samples were mounted on glass slides and the number of pH3 antibody-positive cells was counted using a fluorescence microscope. Microscopic images were observed using a confocal laser microscope. In addition, dsRNA of IAPV, DWV, or GFP from Example 4 was administered, and the number of pH3 antibody-positive cells in the midgut of each individual on day 7 of rearing was counted.

[0102] Microscopic images of the midgut of the virus-infected group (honeybee population No. 5) and the uninfected group are shown in Figure 7. Figure 8 shows the number of pH3 antibody-positive cells in the midgut of each group on day 7 of rearing, after administration of IAPV, DWV, or GFP dsRNA. Intestinal regeneration cells were detected by the pH3 antibody. This indicates that the intestinal tract is damaged, and therefore intestinal regeneration is observed; this result signifies intestinal damage. As shown in Figure 7, the detection of intestinal regeneration cells in the virus-infected group confirmed midgut damage, and the involvement of the protease of the infecting virus was presumed. As shown in Figure 8, it was confirmed that midgut damage was suppressed by IAPV dsRNA.

[0103] <Example 6. Evaluation of IAPV protease inhibitory activity by in silico screening> [method] 1. Protein structure prediction and thermodynamic spatial sampling of binding spaces To perform in silico screening and predict the three-dimensional structure of IAPV proteases, we used Alphafold2. Furthermore, since it was unknown where the protein ends are cleaved when the protein performs its enzymatic function, we predicted the structure using multiple sequences and selected those that could maintain binding space / binding interaction against E-64, which has been experimentally shown to have inhibitory effects. As the IAPV protease, we used the polypeptide consisting of the amino acid sequence of SEQ ID NO: 65.

[0104] Sequence ID 65 PGHLLGFIAEYDTIEIRNLFDVVFRVPWKDVKKIPIVNALGESKEAALLAFPKFVCQHSDLVKHFQNAESMSKFKRCEVTLPVLRFSEKVGKFLSTLIECDRVEAYDRPYTLNDSQKGQYILRQGLEYTMPTTNGDCGSPLIINETQVLRKIAGIHVAGATTGKAYAESITQKDLERAFIKI

[0105] For selected IAPV proteases, hydrogen addition and optimization of the hydrogen bond network were performed using prepwizward (Schroedinger, LLC, New York, NY, 2024; Impact, Schroedinger, LLC, New York, NY; Prime, Schroedinger, LLC, New York, NY, 2024). For molecular dynamics (MD) calculations, counterions were placed to cancel charges within the MD cell space, and water molecules were positioned to create a 12 Å relaxation region from the protein. This structure was used as the initial structure. After relaxing the structure by energy minimization, the temperature was gradually increased to 310 K (in vivo temperature) over a sufficient length to equilibrate the structure. Afterward, the protein structure was sampled over a time interval of 1 ns. All these calculations were performed using the Amber 18 Program Package (DA Case, RM Betz, DS Cerutti, TE Cheatham, III, TA Darden, RE Duke, TJ Giese, H. Gohlke, AW Goetz, N. Homeyer, S. Izadi, P. Janowski, J. Kaus, A. Kovalenko, TS Lee, S. LeGrand, P. Li, C. Lin, T. Luchko, R. Luo, B. Madej, D. Mermelstein, KM Merz, G. Monard, H. Nguyen, HT Nguyen, I. Omelyan, A. Onufriev, DR Roe, A. Roitberg, C. Sagui, CL Simmerling, WM Botello-Smith, J. Swails, RC Walker, J. Wang, RM Wolf, X. Wu, L. Xiao and PA Kollman (2016), AMBER (2016, University of California, San Francisco)Furthermore, the force fields used were Amber 99 (Wang J, Cieplak P, Kollman PA. How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J Comp Chem 2000;21(12):1049-74.) and TIP3P (Jorgensen WL, Chandrasekhar J, Madura JD. Comparison of simple potential functions for simulating liquid water. J Chem Phys 1983;79(2):926-35. https: / / doi.org / 10.1063 / 1.445869).

[0106] 500 conformations were extracted from the sampled protein trajectories at equal intervals, superimposed on the amino acids of the enzyme active site of the IAPV protease, and then conformational similarity analysis was performed. The 32 representative conformations used for compound screening were determined using the penalty function by LA Kelley et al. (LA Kelley, et al., Protein Engineering, 11, 1063, 1996).

[0107] 2. Compound Screening To perform in silico screening, grid potentials were created for the enzyme active site of each representative structure of IAPV proteases. For the compound groups of E-64 derivatives (613 compounds: obtained by structural search using SciFinder with E-64 substructures as queries) and Calpain inhibitor derivatives (343 compounds: obtained by searching the database CDDI (Cortellis Drug Discovery Intelligence) with the keyword Calpain Inhibitors), docking calculations were first performed on the initial structures with restricted ligand flexibility. Subsequently, docking calculations considering ligand flexibility were performed on the narrowed-down compounds (416 compounds) using all representative conformations. The structures of the 416 compounds are shown in Tables VI-1 to VI-36. The docking calculations in this study were performed using Schroedinger's glide software (Schroedinger, LLC, New York, NY, 2024; Impact, Schroedinger, LLC, New York, NY; Prime, Schroedinger, LLC, New York, NY, 2024).

[0108] Screening was conducted based on three scores, as shown below: G-score, D-score, and Binding Efficiency. The G-score represents an energy index score (which can be called the interaction energy score between the ligand and protein) obtained by weighting physical interactions such as van der Waals (hydrophobic) interactions, hydrogen bonds, and electrostatic interactions according to the situation. The D-score represents a score corrected based on the G-score, taking into account the state of the ligand. Binding Efficiency represents a value equivalent to the size of the molecule, for example, a value obtained by dividing by the number of atoms other than hydrogen (this allows for equivalent comparison, considering that interaction energy is additive and cannot be equivalently compared to the size of the molecule). (Position swapped) Note that for G-score, D-score, and Binding Efficiency, a lower value indicates a stronger binding between the enzyme protein and the compound.

[0109] [Table 42]

[0110] [Table 43]

[0111] [Table 44]

[0112] [Table 45]

[0113] [Table 46]

[0114] [Table 47]

[0115] Table 48

[0116] Table 49

[0117] Table 50

[0118] Table 51

[0119] Table 52

[0120] Table 53

[0121] Table 54

[0122] Table 55

[0123] Table 56

[0124] Table 57

[0125] Table 58

[0126] Table 59

[0127] Table 60

[0128] Table 61

[0129] Table 62

[0130] Table 63

[0131] Table 64

[0132] Table 65

[0133] Table 66

[0134] Table 67

[0135] Table 68

[0136] [Table 69]

[0137] [Table 70]

[0138] [Table 71]

[0139] [Table 72]

[0140] [Table 73]

[0141] [Table 74]

[0142] [Table 75]

[0143] [Table 76]

[0144] [Table 77]

[0145] [result] The G-score, D-score, and Binding Efficiency of each compound are shown in Tables VII-1 to VII-12. Among G-score, D-score, and Binding Efficiency, since G-score most reflects the binding of enzyme protein and compound, compounds were evaluated centered around G-score. E-64 (Compound No. 1) showed inhibitory activity against IAPV protease (Example 8) and suppressed the individual death of honeybees infected with virus (IAPV) (Example 9). In the analysis of the present invention, the pharmacological effects of drugs were evaluated centered around E-64. In order to find compounds that bind more strongly to IAPV protease than E-64 and show high enzyme inhibitory activity, screening was carried out with G-score ≤ -7.0, which is a value lower than the G-score of E-64, as the threshold.

[0146]

Table 78

[0147]

Table 79

[0148]

Table 80

[0149]

Table 81

[0150]

Table 82

[0151]

Table 83

[0152]

Table 84

[0153] [Table 85]

[0154] [Table 86]

[0155] [Table 87]

[0156] [Table 88]

[0157] [Table 89]

[0158] <Example 7. Protein expression of IAPV protease> (Experimental method) RNA was extracted from the midgut of virus-infected honeybees using ISOGEN II (Nippon Gene), and this RNA was reverse transcribed using PrimeScript RT reagent Kit (Takara) to obtain midgut-derived cDNA. Next, the midgut-derived cDNA was subjected to PCR using IAPV protease Primer-F1 (SEQ ID NO: 61) and IAPV protease Primer-R (SEQ ID NO: 62) on KOD FX Neo (TOYOBO) (30 cycles of 30 seconds at 98°C, 30 seconds at 60°C, and 2 minutes at 68°C) to obtain a cDNA fragment of IAPV protease (consisting of the nucleotide sequence of SEQ ID NO: 64, which codes for the amino acid sequence of SEQ ID NO: 63). The cDNA fragment of IAPV protease was restricted enzyme-treated with BamHI and HindIII, and the expression vector pET21a (Merck) was also restricted enzyme-treated with BamHI and HindIII. Restriction enzyme-treated IAPV protease cDNA fragments and pET21a DNA fragments were ligated using Ligation High Ver.2 (TOYOBO) at 16°C for 1 hour to ligate the vectors and cDNA fragments, and E. coli was transformed. Colony PCR was performed to obtain an expression vector containing the IAPV protease cDNA fragment. The IAPV protease expression vector was transformed into E. coli Roseta-gami B (Merck), pre-cultured in 50 ml of LB medium containing ampicillin, then subcultured in 500 ml of LB medium containing ampicillin, and cultured until the absorbance at 600 nm was 0.5. Isopropyl β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM was added, and the cells were cultured at 15°C for 24 hours. The culture medium was centrifuged (8000 rpm, 10 min, 4°C) to collect E. coli cells. These cells were then dissolved in 10 ml of sonication buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT), and the E. coli cells were disrupted by sonication. The resulting lysate was centrifuged (12000 rpm, 10 min, 4°C) to obtain the supernatant. IAPV protease was purified from this supernatant to obtain a purified protein solution.Purification was performed by cation exchange chromatography using a buffer of 20 mM Tris-HCl (pH 7.5) containing 20 mM Tris-HCl (pH 7.5) and 1 M NaCl and HiPrep SP XL 16 / 10 (Cytiva), and gel filtration chromatography using a 50 mM phosphate buffer (pH 7.0) containing 0.15 M NaCl and HiLoad 16 / 600 Superdex 200 (Cytiva).

[0159] SEQ ID NO: 61 CGCGGATCCCCAAATGACATAGTTGATGTTACAATGCAA

[0160] SEQ ID NO: 62 CCCAAGCTTTTATATTTTAGTAAATGCTCGCTC

[0161] SEQ ID NO: 63 VAQRLITNRVLTNLYKVCLVHNDDRITPLLNGLFVRSNLMLVPGHLLGFIAEYDTIEIRNLFDVVFRVPWKDVKKILIVNALGESKEAALLAFPKFVCQHSDLVKHFQNAESMSKFKRCEVTLPVLRFSEKVGKFLSTLIECDRVEAYDRPYTLNDSQKGQYILRQGLEYTMPTTNGDCGSPLIINETQVLRKIAGIHVAGATTGKAYAESITQKDLERAFTKI

[0162] SEQ ID NO: 64 GTTGCTCAACGACTGATAACTAACCGTGTGTTAACAAACTTGTATAAGGTTTGTTTAGTGCATAATGACGATCGCATTACACCTCTTTTGAATGGACTTTTTGTTCGTTCGAATTTGATGTTAGTTCCTGGACATCTTCTTGGATTTATTGCTGAGTATGATACAATAGAAATTCGAAATCTCTTTGACGTGGTATTTAGAGTACCATGGAAAGATGTAAAGAAGATTCTAATTGTTAATGCTCTCGGTGAATCTAAGGAGGCAGCGTTATTGGCCTTTCCAAAATTCGTTTGTCAACATTCTGACTTGGTCAAGCATTTCCAAAATGCTGAATCAATGTCTAAATTCAAACGATGTGAAGTAACCCTCCCAGTATTACGTTTCTCTGAGAAAGTTGGTAAATTTTTATCAACACTCATAGAATGCGATCGGGTGGAGGCTTATGACAGACCATATACGCTGAATGACTCTCAAAAAGGACAATATATTCTACGTCAAGGCTTAGAATATACGATGCCAACGACCAATGGTGATTGTGGTTCGCCACTAATCATCAACGAAACCCAAGTTTTGAGGAAAATTGCTGGTATTCATGTGGCAGGAGCTACAACCGGTAAGGCCTATGCTGAATCTATAACCCAAAAAGATCTTGAGCGAGCATTTACTAAAATATAA

[0163] <Example 8. Measurement of IAPV protease activity by purified enzyme> [Method] The inhibitory activity against IAPV protease was evaluated using the compounds screened in Example 6, as shown in Table 6. For the IAPV protease activity measurement, synthesized substrates, Dnp-PNDIV DVTMQK(Nma)-NH2 or Nma-TMQMWKDQVAK(Dnp)-NH2, consisting of a peptide and a fluorescent substrate, were used. The activity measurement substrate fluoresces when cleaved by the protease. The activity measurement substrate was prepared to 20 μM in assay buffer (50 mM citrate buffer pH 6.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT). The purified protein solution of the IAPV protease obtained in Example 10 was used as the enzyme solution. 50 μl of assay buffer containing the activity measurement substrate and 50 μl of enzyme solution were mixed and reacted in a 96-well black plate at 37°C for 30 minutes in the presence or absence of each compound shown in Table 6. The fluorescence intensity was then measured using a TECAN fluorescence plate reader (TECAN Japan) at an excitation wavelength of 340 nm and an emission wavelength of 440 nm. Each inhibitor was prepared to a final concentration of 1 mM, pre-incubated at 37°C for 1 hour, and then the enzyme reaction was carried out.

[0164] [result] Table VIII shows the relative activity of IAPV proteases, calculated from the fluorescence intensities measured in the presence of various inhibitors, with the fluorescence intensity measured in the absence of the compound ("None" in Table 6) set as 100% relative activity. Each E-64 analog (E-64, E-64_comp_5, and CA-074Me) and each calpain inhibitor (dazcapistat (Calpain_comp_2) and alicapistat (Calpain_comp_3)) used in the experiment were shown to have IAPV protease inhibitory activity. From these results, it is indicated that the compounds screened in silico in Example 6 have a high probability of having IAPV protease inhibitory activity.

[0165] [Table 90]

[0166] E-64 was manufactured by Peptide Laboratories, while E-64_comp_5, CA-074Me, alpain_comp_2, and Calpain_comp_3 were manufactured by MedChemExpress.

[0167] Furthermore, Calpain_comp_8 (compound No. 153), Calpain_comp_103 (compound No. 327), Calpain_comp_324 (compound No. 285), Calpain_comp_126 (compound No. 211), Calpain_comp_266 (compound No. 26), Calpain_ comp_199 (Compound No. 37), Calpain_comp_154 (Compound No. 71), Calpain_comp_311 (Compound No. 83), Calpain_comp_309 (Compound No. 191), Calpain_comp_222 (Compound No. 220), Calpain_comp_69 (Compound N By performing the same tests as in Example 8 using each of the following compounds, it can be confirmed that they possess IAPV protease inhibitory activity: o.284), Calpain_comp_128 (compound No. 313), Calpain_comp_102 (compound No. 325), Calpain_comp_129 (compound No. 328), E-64_comp_347 (compound No. 352), E-64_comp_124 (compound No. 353), E-64_comp_148 (compound No. 375), Calpain_comp_101 (compound No. 381), E-64_comp_36 (compound No. 387), E-64_comp_417 (compound No. 405), or E-64_comp_113 (compound No. 413).

[0168] <Example 9. Suppression of individual bee mortality by administration of screened compounds> (Experimental method and administration method) From the compounds screened in Example 6, E-64 (Compound No. 1) ([(2S,3S)-3-carboxyoxiran-2-carbonyl]-L-leucine (4-guanidinobutyl)amide hemihydrate, manufactured by Peptide Research Institute Co., Ltd.) was selected. This compound was dissolved in 50% sucrose solution at concentrations of 200 μM, 100 μM, and 50 μM, and administered to honeybee populations No. 2 and No. 5 that showed viral infection. The control group (None) was administered only 50% sucrose. The solution containing E-64 was changed daily, and the change in the number of honeybees was measured. The results are shown in Figures 9 and 10. The aseptic in vivo drug evaluation rearing method described above was used for rearing.

[0169] As shown in Figures 9 and 10, E-64 was divided into two lines derived from colonies that were dying due to mites. When administered, E-64 suppressed individual bee deaths caused by mite infestations, and it became clear that the proliferation of the pathogenic virus and the damage to the intestinal tract were caused by the viral protease. Based on these results, E-64 can be used as an antiviral agent to suppress mass bee deaths by inhibiting IAPV protease.

[0170] Furthermore, Calpain_comp_8 (Compound No. 153), Calpain_comp_103 (Compound No. 327), Calpain_comp_324 (Compound No. 285), Calpain_comp_126 (Compound No. 211), Calpain_comp_266 (Compound No. 26), Calpain_comp_19 9 (Compound No. 37), Calpain_comp_154 (Compound No. 71), Calpain_comp_311 (Compound No. 83), Calpain_comp_309 (Compound No. 191), Calpain_comp_222 (Compound No. 220), Calpain_comp_69 (Compound No. 284), Calpain_ By performing the same tests as in Example 9 using each of the following compounds, it is possible to confirm the effect of suppressing individual bee deaths due to mite infestation.

[0171] The results above indicate that the compounds screened in silico in Example 6 are highly likely to possess IAPV protease inhibitory activity and are highly likely to have an effect in suppressing individual bee mortality caused by mite infection.

[0172] Furthermore, this research according to the present invention was supported by a Grant-in-Aid for Scientific Research (Research Project / Area Number: 22K05681).< / super>

Claims

1. A composition for suppressing individual bee mortality, comprising a substance that inhibits the activity of a protease derived from Israeli acute paralysis virus (IAPV).

2. The composition according to claim 1, wherein the death of individual honeybees is caused by mite infestation on the honeybees.

3. A composition for improving the survival rate of honeybees, containing a substance that inhibits the activity of IAPV-derived protease.

4. The composition according to any one of claims 1 to 3, wherein the substance that inhibits the activity of IAPV-derived protease is one or more compounds selected from compounds 1 to 416 listed in Tables I-1 to I-36 of the specification or a pharmaceutically acceptable salt thereof.

5. A method for suppressing individual bee mortality, including inhibiting the activity of IAPV-derived protease.

6. The method according to claim 5, wherein the death of the honeybee is caused by mite infestation on the honeybee.

7. A method for improving the survival rate of honeybees, including inhibiting the activity of IAPV-derived proteases.

8. The method according to any one of claims 5 to 7, wherein inhibiting the activity of IAPV-derived protease is achieved by using a substance that inhibits the activity of IAPV-derived protease.

9. The method according to claim 8, wherein the substance that inhibits the activity of IAPV-derived protease is one or more compounds selected from compounds 1 to 416 listed in Tables I-1 to I-36 of the specification or a pharmaceutically acceptable salt thereof.

10. A composition for inhibiting the activity of IAPV-derived proteases, comprising one or more compounds selected from compounds 1 to 416 listed in Tables I-1 to I-36 of the specification, or a pharmaceutically acceptable salt thereof.

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