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WO2019230654A1 - Traitement antiviral d'une infection à flavivirus - Google Patents

Traitement antiviral d'une infection à flavivirus Download PDF

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Publication number
WO2019230654A1
WO2019230654A1 PCT/JP2019/020916 JP2019020916W WO2019230654A1 WO 2019230654 A1 WO2019230654 A1 WO 2019230654A1 JP 2019020916 W JP2019020916 W JP 2019020916W WO 2019230654 A1 WO2019230654 A1 WO 2019230654A1
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substance
flavivirus
nucleolus
cells
activity
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Japanese (ja)
Inventor
洋一 宮本
正啓 岡
悦啓 米田
岡本　徹
善治 松浦
詢 徳永
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National Institutes of Biomedical Innovation Health and Nutrition
University of Osaka NUC
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Osaka University NUC
National Institutes of Biomedical Innovation Health and Nutrition
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Priority to JP2020522183A priority Critical patent/JP6996720B2/ja
Priority to SG11202011775VA priority patent/SG11202011775VA/en
Publication of WO2019230654A1 publication Critical patent/WO2019230654A1/fr
Anticipated expiration legal-status Critical
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
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    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A—HUMAN NECESSITIES
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
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    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to the treatment of flavivirus infections and flavivirus related diseases in mammals, particularly humans.
  • the present invention is particularly used as a target for new therapeutic protocols for the treatment and / or prevention of infections and diseases caused by flaviviruses and for the identification and development of substances with new anti-flavivirus activity
  • a screening method is provided.
  • Flaviviridae Flavivirus is a Japanese encephalitis virus (JEV) that causes infectious diseases that are a public health problem worldwide, such as Japanese encephalitis, dengue fever, dengue hemorrhagic fever, and Zika fever. It contains many pathogens such as dengue virus (DENV) and Zika virus (ZIKV). Flaviviridae viruses are mainly arthropod viruses and often cause significant morbidity and mortality in mammals and birds (Non-Patent Document 1). JEV is distributed in the south and southeastern regions of Asia, and is a zoonotic infection between pigs or birds and mosquitoes (Non-Patent Documents 1 and 2).
  • the Flaviviridae contains more than 70 viruses, most of which are mediated by blood-sucking arthropods.
  • Mosquito-borne flaviviruses include Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), etc. , Tick-borne encephalitis virus (TBEV) and the like (Non-patent Document 3).
  • JEV Japanese encephalitis virus
  • DEV dengue virus
  • ZIKV Zika virus
  • WNV West Nile virus
  • YFV yellow fever virus
  • TBEV Tick-borne encephalitis virus
  • 14 types of flaviviruses with unknown vectors have been reported.
  • Flavivirus is composed of an enveloped virus whose genome is a + -strand RNA having a total length of about 11 kb, and has a cap structure at the 5 'end and no poly A structure at the 3' end (Non-patent Document 4).
  • Non-patent Document 4 There is a single open reading frame on this genome, and polyproteins translated from the genome are cleaved simultaneously and post-translationally by the host and viral proteases, resulting in 3 core, precursor membrane (prM) and envelope (E) proteins. This results in two structural proteins, and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Non-patent Document 5).
  • Non-Patent Documents 6 and 7 protease activity inhibitors
  • Non-Patent Documents 8 and 9 helicase activity inhibitors
  • an effect is also observed with respect to NS5 methyltransferase activity (Non-Patent Documents 10 and 11) and NS5 RNA-dependent RNA polymerase inhibitor (Non-Patent Documents 12 and 13).
  • Non-patent Document 14 E protein, which is one of structural proteins
  • Non-Patent Document 15 and 16 a drug that inhibits structural changes in E protein during infection is searched for.
  • Non-patent Document 17 St. Louis encephalitis virus infection
  • DEV dengue virus
  • JEV Japanese encephalitis virus
  • Non-patent Document 21 the effect of suppressing viral replication has been observed due to GTP depletion by inosine monophosphate dehydrogenase inhibition.
  • Non-patent Document 23 no significant effect was observed in clinical trials for JEV infection in humans.
  • Non-patent Document 24 it has been reported that a nucleoside analog ETAR similar to ribavirin suppresses replication of flaviviruses such as DENV (Non-patent Document 24), and further studies are expected.
  • Non-Patent Documents 25 and 26 RNAi for degrading target RNA
  • Patent Documents 27 and 28 morpholino-oligo
  • Certain antiviral effects have been observed in animal models.
  • siRNA against JEV with a rabies virus-derived peptide Non-patent Document 29
  • siRNA against DENV with a peptide that specifically binds to a ligand on a dendritic cell Non-Patent Document 30
  • These technologies can be applied only to specific organs / cells where the target virus is infected and proliferated, and it is considered that side reactions can be reduced.
  • Patent Document 1 Non-Patent Document 31
  • a pan-caspase inhibitors for example, pan-caspase inhibitors, cyclin-dependent kinase (CDK) inhibitors, category B anthelmintics or proteasome inhibitors
  • CDK cyclin-dependent kinase
  • Patent Document 31 Non-Patent Document 31
  • Knipe DM Howley PM (Ed), Lippincott-Raven, Philadelphia, PA, 1102-53, 2007 Stoermer MJ, et al. J. Med. Chem. 51: 5714-21, 2008 Yang CC, et al. Antimicrob. Agents. Chemother. 55: 229-38, 2011 Zhang N, et al. J. Med. Chem. 46: 4149-64, 2003 Borowski P, et al. Eur. J. Biochem. 270: 1645-53, 2003 Luzhkov VB, et al. Med. Chem. 15: 7795-802, 2007 Lim SP, et al. J. Biol. Chem. 286: 6233-40, 2011 Yin Z, et al.
  • Flaviviridae Flaviviridae, Flaviviridae, are non-human mammals and birds that are possessed hosts and amplified animals and are mediated by mosquitoes or ticks. In human life, it is difficult to completely eliminate contact with infected mosquitoes or infected ticks, so it is impossible to completely eliminate flavivirus infection. Accordingly, the present inventors have sought antiviral treatment for flavivirus infection, have intensively studied, and have obtained surprising findings that inhibit viral replication.
  • Antiviral pharmaceutical composition comprising a substance that affects the morphology of nucleolus in a biological cell.
  • An anti-flavivirus pharmaceutical composition comprising a substance that affects the morphology of nucleolus in a biological cell.
  • the anti-flavivirus pharmaceutical composition according to [1] comprising a substance that affects the nucleolus morphology in a biological cell as an active ingredient having anti-flavivirus activity.
  • the pharmaceutical composition according to [1] or [2], wherein the substance that affects nucleolus morphology in biological cells is a substance that causes nucleolus morphological abnormalities.
  • composition according to any one of [1] to [3], wherein the substance that affects the morphology of the nucleolus in the biological cell also involves a change in localization of the viral core protein in the biological cell.
  • composition according to any one of [1] to [4], wherein the substance that affects the morphology of nucleoli in biological cells causes viral replication inhibition.
  • the medicament according to any one of [1] to [5], wherein the flavivirus is an anti-flavivirus activity selected from Japanese encephalitis virus, dengue virus, Zika virus, West Nile virus, yellow fever virus Composition.
  • the substance that affects the nucleolus morphology in biological cells is a substance having CDK inhibitory activity, a substance having rRNA transcription inhibitory activity, a substance having DNA topoisomerase inhibitory activity, or a substance having GSK-3 inhibitory activity
  • the pharmaceutical composition according to any one of [1] to [6].
  • a substance having CDK inhibitory activity is 2-cyanoethylartelopaulone, 5-amino-3-((4- (aminosulfonyl) phenyl) amino) -N- (2,6-difluorophenyl) -1H-1, 2,4-triazole-1-carbothioamide, (4- (2-amino-4-methylthiazol-5-yl) pyrimidin-2-yl)-(3-nitrophenyl) amine, P276-00, A-674563 , SU9516, SNS-032 (BMS-387032), dinacrib, flavopiridol, AT7519, flavopiridol hydrochloride, AT7519 HCl, AZD5438, R547, and kaempaurone, [7] [9] A substance having CDK inhibitory activity is 2-cyanoethylartelopaulone, 5-amino-3-((4- (aminosulfon)
  • composition according to [10] The pharmaceutical composition according to [7], wherein the substance having rRNA transcription inhibitory activity is selected from actinomycin D, doxorubicin, and aclarubicin.
  • substance having DNA topoisomerase inhibitory activity is selected from doxorubicin, aclarubicin, ellipticine, idarubicin, idarubicin hydrochloride, epirubicin hydrochloride, mitoxantrone hydrochloride, and daunorubicin hydrochloride.
  • a method for screening a substance having anti-flavivirus activity comprising the following steps: (A) contacting a biological cell with a candidate compound of a substance having anti-flavivirus activity in vitro; (B) investigating whether the candidate compound affects the nucleolus morphology in the biological cell; (C) A method in which the candidate compound is identified as a substance having anti-flavivirus activity if it has an effect. [15] [14] The method according to [14], wherein in step (c), the influence of the candidate compound is a nucleolus morphological abnormality in a biological cell.
  • a change in the formation and / or localization of a protein localized in the nucleolus is a change compared to the effect of a control compound in the protein localized in the nucleolus, wherein the candidate compound has a The method according to [16], wherein it is determined that an influence is exerted.
  • the protein localized in the nucleolus in biological cells is one or more proteins selected from necrofosmin, nucleolin, fibrillarin, RNA polymerase I and flavivirus core protein [14 ] To [17].
  • Biological cells are established from cultured cell lines derived from eukaryotes including humans such as Huh7 cells derived from liver, primary cultured cells, primate derived cell lines such as monkey kidney derived Vero cells, and mosquitoes such as C6 / 36 cells.
  • the present invention discloses a compound effective for prevention and treatment of flavivirus infection, and further provides a screening method that provides a novel action mechanism, infection prevention, inhibitory efficacy, and usefulness as a drug for a target.
  • a screening method that provides a novel action mechanism, infection prevention, inhibitory efficacy, and usefulness as a drug for a target.
  • there are approved vaccines for humans against JEV, YFV, and TBEV infections but thousands to tens of thousands of patients each year are still reported worldwide. Since flaviviruses are possessed and amplified by non-human animals and it is difficult to completely eliminate contact with infected mosquitoes or infected ticks, the development of specific therapies and vaccines is an important issue.
  • ADVANTAGE OF THE INVENTION According to this invention, a new anti- flavivirus therapeutic agent can be created and a new therapeutic substance can be provided.
  • FIG. 1 (A) shows the results of transient expression of Japanese encephalitis virus core protein: JEV core, dengue virus core protein: DENV core, Zika virus core protein: ZIKV core, and West Nile core protein: WNV core. Indicates localization.
  • FIG. 1 (B) shows intracellular localization when various flavivirus core proteins are microinjected into the cytoplasm of cells.
  • FIG. 1C shows localization in the cytoplasm, cell nucleus (genomic DNA) and nucleolus when the Japanese encephalitis virus core protein is microinjected into the cytoplasm of the cell.
  • FIG. 2 (A) shows a graph of the amount of core protein present in the cell nucleus.
  • FIG. 2 (B) shows the nuclear localization of Japanese encephalitis virus core protein upon treatment with DMSO (control) and three CDK inhibitors.
  • Cdk1 / 2 inhibitor III (5-amino-3-((4- (aminosulfonyl) phenyl) amino) -N- (2,6-difluorophenyl) -1H-1,2,4-triazole-1-carbohydrate It is a photograph replacing a drawing, showing that thioamide) changes the morphology of nucleolus in a cell.
  • FIG. 3 (A) shows that Cdk1 / 2 inhibitory substance III treatment causes abnormal nucleolus morphology as compared with DMSO treatment.
  • FIG. 3 shows the localization in the cytoplasm, cell nucleus (genomic DNA), and nucleolus when Japanese encephalitis virus core protein is microinjected into the cytoplasm of cells treated with 2-cyanoethylalsteropaulone. It is a graph which shows that the specified CDK inhibitor suppresses replication of Japanese encephalitis virus at a specific concentration, but does not kill biological cells.
  • FIG. 5 is a photograph replacing a drawing, showing a morphological change of a nucleolus caused by a substance having rRNA transcription inhibitory activity. The fluorescence image at the time of processing DMSO (control) and three rRNA transcription inhibitors is shown.
  • FIG. 6 (A) is a photograph replacing a drawing, showing the morphological change of the nucleolus by the substance 1-azaken palon having GSK-3 inhibitory activity.
  • FIG. 6 (B) is a graph showing growth inhibition of Japanese encephalitis virus, dengueurus and zika virus by 1-azaken pauron. It is a photograph instead of a drawing showing morphological changes of nucleolus by substances having DNA topoisomerase inhibitory activity: ellipticine, idarubicin, idarubicin hydrochloride, epirubicin hydrochloride, mitoxantrone hydrochloride and daunorubicin hydrochloride.
  • the present invention provides a method for screening a substance having anti-flavivirus activity, comprising the following steps: (A) contacting a biological cell with a candidate compound of a substance having anti-flavivirus activity in vitro; (B) investigating whether the candidate compound affects the nucleolus morphology in the biological cell; (C) If the candidate compound has an effect, a method is provided in which the candidate compound is identified as a substance having anti-flavivirus activity. This will be specifically described below.
  • flavivirus means a virus belonging to the genus Flaviviridae that is composed of an envelope virus having a plus-strand RNA as a genome, and includes 70 or more viruses currently known.
  • mosquito-borne flaviviruses include Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), St.
  • tick-borne flaviviruses include tick-borne encephalitis virus (TBEV) (Gubler DJ, Kuno G, Markoff L: Flaviviruses. In: Fields virology (Fifth edition). Knipe DM, Howley PM (Ed), Lippincott-Raven, Philadelphia, PA, 1153-252, 20071). In addition, 14 kinds of flaviviruses whose vectors are unknown have been reported.
  • “Flaviviruses” are typically Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), more typically Japanese encephalitis virus (JEV) dengue virus (DENV). Zika virus (ZIKV). JEV spreads to dead-end hosts including humans when bitten by mosquitoes infected with JEV, causing central nervous system infection with high mortality (Burke, DS, and TP Monath. 2001. Flaviviruses, p. 1043- 1125. In DM Knipe, PM Howley, DE Griffin, RA Lamb, MA Martin, B. Roizman, and SE Straus (ed.), Fields virology, 4th ed., Vol. 1.
  • JEV has a single stranded plus-strand RNA genome of approximately 11 kb in length, which is capped at the 5 ′ end, but lacks modification at the 3 ′ end due to polyadenylation (Lindenbach, BD, and CM). 2001. Flaviviridae: The viruses and their replication, p. 991-1041.In DM Knipe, PM Howley, DE Griffin, RA Lamb, MA Martin, B. Roizman, and SE Straus (ed.). Fields virology, 4th ed., vol. 1. Lippincott Williams & Wilkins, Philadelphia, Pa.).
  • Genomic RNA encodes a single large open reading frame, and polyproteins translated from the genome are cleaved simultaneously and post-translationally by the host and viral proteases to produce core, precursor membrane (prM) and envelope (E) protein. It yields three structural proteins and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Sumiyoshi, H., et al. Virology 161: 497-510).
  • the JEV core protein (aka C protein or capsid protein) has little amino acid homology with other flaviviruses, for example only 25% homology with tick-borne encephalitis virus (TBEV), Hydrophobic profile, number of basic amino acid residues and secondary structure are very similar (Dokland, T., et al. Structure 12: 1157-1163 .; Jones, CT, et al. J. Virol 77: 7143-7149 .; Ma, L., et al. Proc. Natl. Acad. Sci. USA 101: 3414-3419).
  • the flavivirus core protein commonly contains two hydrophobic sequences in the middle and at the carboxyl terminus, with the carboxyl terminal hydrophobic region serving as the signal sequence for prM.
  • the signal anchor sequence is cleaved by the viral protease NS2B-3, and this cleavage is necessary for subsequent release of the prM amino terminus by the host signal peptidase (Lobigs, M., and E. Lee. 2004. J. Virol. 78: 178-186; Stocks, CE, and M. Lobigs. 1998. J. Virol. 72: 2141-2149; Yamshchikov, VF, and RW Compans. 1994. J. Virol. 68: 5765-5771).
  • the mature core protein released from the endoplasmic reticulum (ER) membrane is thought to bind to genomic RNA via amino-terminal and carboxyl-terminal basic amino acid clusters to form nucleocapsids (Khromykh, AA, and EG Westaway. 1996. Kunjin. Arch. Virol. 141: 685-699).
  • the central hydrophobic region of the core protein may be associated with the ER membrane, and this interaction facilitates the assembly of the nucleocapsid with the two membrane proteins prM and E and appears to budding into the ER lumen as a virion (Markoff, L., B. Falgout, and A. Chang. 1997. Virology 233: 105-117).
  • anti-flavivirus or “anti-flavivirus activity” in the present invention means that flavivirus replication, cell entry and / or cell flavivirus infection is reduced, inhibited, blocked or prevented, and consequently flavivirus. It means any effect that suppresses or inhibits the viral life cycle and extinguishes or reduces viral activity.
  • the “substance having anti-flavivirus activity” or “active ingredient having anti-flavivirus activity” in the present invention refers to a substance that can treat or prevent a subject affected or infected with flavivirus by eliminating or reducing flavivirus. means.
  • the terms “affected by a flavivirus” or “infected with a flavivirus” are used in connection with a subject to mean infected with a flavivirus and having a flavivirus-related disease.
  • flavivirus-related disease is meant any disease or disorder known or suspected to be associated with and / or caused directly or indirectly by a flavivirus.
  • flavivirus-related diseases fever, headache, muscle pain, and joint pain are the main symptoms, especially hemorrhagic fever due to dengue virus, encephalitis due to Zika virus, Japanese encephalitis virus, West Nile virus, tick-borne encephalitis virus, Including, but not limited to, microcephaly due to Zika virus.
  • the term “candidate compound” refers to any natural or non-natural molecule, for example, a biological macromolecule such as a nucleic acid, polypeptide or protein, an organic or inorganic molecule, or an organism to test for the activity of interest. It means an extract prepared from a biological material, such as cells or tissues of bacteria, fungi, plants or animals (particularly including mammals and humans).
  • candidate compounds are evaluated for their ability to have a predetermined anti-flavivirus activity.
  • it refers to natural or non-natural compound libraries, medicinal plant extracts, peptides, antibodies, nucleic acid random dance rally, etc. possessed by universities and companies.
  • a “biological cell” as used in the screening methods of the present invention refers to a substantially homogeneous cell population, preferably at least about 80%, more preferably at least about 100% of the cells in the population are of the same cell type. is there.
  • Examples of cell types include, but are not limited to, platelets, lymphocytes, T cells, B cells, natural killer cells, endothelial cells, tumor cells, epithelial cells, granulocytes, monocytes, mast cells, nerve cells, and the like.
  • Preferred examples include cultured cell lines and primary cultured cells derived from eukaryotes including mammals such as human liver-derived Huh7 cells and monkey kidney-derived Vero cells, and cells established from mosquitoes such as C6 / 36 cells.
  • the screening method of the present invention includes, as one embodiment, a first stage for confirming abnormal nucleolus morphology and a second stage for confirming virus replication.
  • the “biological cells” used in the first stage are all cells that can observe nucleolus, and the “biological cells” used in the second stage are preferably virus-sensitive cells, more preferably. Are flavivirus sensitive cells.
  • a “virus sensitive cell” refers to any cell that can be infected with a virus, including but not limited to brain cells, primary cells, dendritic cells, placental cells, endometrial cells, lymph node cells, lymphoid cells (B and T cells), peripheral blood mononuclear cells, skin cells, Langerhans cells, and monocytes / macrophages.
  • the cells used in the screening methods of the present invention may be prepared by techniques well known in the art, eg, the cells may be obtained by drawing blood from a patient or healthy person or by biopsy, or by immunization and microbial suppliers, such as You can purchase it from American Type Culture Collection, Manassas, VA.
  • the cells used in the present invention can be cultured according to standard cell culture techniques.
  • the cells are grown in a sterile environment at 37 ° C. in a suitable container in an incubator containing humidified 95% air-5% CO 2 .
  • the container may contain a stirred or stationary medium.
  • Various cell culture media may be used, including medium containing undefined biological fluids (such as fetal calf serum) as well as fully established media such as 293 SFM serum-free media (Invitrogen Corp., Carlsbad, CA) )including.
  • the screening method of the present invention is performed using cells contained in a plurality of wells of a multi-well assay plate.
  • assay plates are commercially available from, for example, Strategene® Corp. (LaJolla, CA) and Corning® Inc. (Acton, MA), including, for example, 48-well, 96-well, 384-well, and 1536-well plates. .
  • Contact in vitro means, for example, adding a candidate compound of a substance having anti-flavivirus activity adjusted to a predetermined concentration to cells contained in a plurality of wells of a multi-well assay plate. Next, the candidate compound is examined for a predetermined “anti-flavivirus activity”.
  • Predetermined anti-flavivirus activity (B) investigating whether the candidate compound affects the nucleolus morphology in the biological cell; (C) If the candidate compound has an influence, it is identified as a substance having anti-flavivirus activity. Specifically, in the step (c), the influence of the candidate compound is a morphological abnormality of a nucleolus in a biological cell.
  • the method for determining the predetermined anti-flavivirus activity in the candidate compound is based on the following first findings by the present inventors.
  • the present inventors sought and sought antiviral treatment for flavivirus infection, and initially sought a different target.
  • the event that inhibits viral replication is not the essence of the target, and the nucleus in biological cells I accidentally discovered that the small bodies are related.
  • the nucleolus present in the nucleus of biological cells is a structure unique to eukaryotes that can be clearly observed with an optical microscope, and is mainly responsible for the biosynthesis of ribosomes.
  • the main components are ribosomal DNA (rDNA), ribosomal RNA (rRNA), ribosomal protein, etc., and proteomic analysis of the constituent proteins using the proteomics technique is progressing (Anderson et al., Nature volume 433: 77-83 ). From observation with an electron microscope, the structure of the nucleolus can be divided into three regions depending on the difference in electron density.
  • FC fiber center
  • DFC dense fibrillar component
  • GC granular component
  • the flavivirus is considered to be essential for viral replication, as one of the viral replication functions, the core protein, which is one of the constituent proteins, utilizes the nucleolus function of the host cell.
  • the core protein which is one of the constituent proteins, utilizes the nucleolus function of the host cell.
  • morphological abnormalities of the nucleolus adversely affect the function of the core protein.
  • the nucleolus is a site for transcription of ribosomal RNA (rRNA) and synthesis of ribosome, and in a normal state, the core protein is involved in host protein synthesis by localizing in the nucleolus.
  • nucleolus relatively enhances viral protein translation by promoting the synthesis of virus-derived proteins or suppressing the synthesis of host cell proteins.
  • the effect on the nucleolus morphology of certain substances found in the present invention is thought to result from the action of reducing or destroying this normal nucleolus function.
  • the predetermined anti-flavivirus activity that is, the morphological abnormality of the nucleolus in a biological cell is caused by the presence and / or locality of a protein or nucleic acid localized in the nucleolus. It is discriminated by the change of the present.
  • a change in the presence and / or localization of a protein or nucleic acid localized in the nucleolus is an alternative to a change in the formation state of the nucleolus itself, found compared to the effect of the control compound.
  • a protein localized in a nucleolus in a biological cell means a protein endogenous to the nucleolus or a protein that specifically binds to the nucleolus.
  • Nucleophosmin also known as NPM, NPM1, B23, NO38, Numatrin
  • nucleolin also known as C23, NCL
  • fibrillarin also known as FBL
  • proteins that specifically bind to the nucleolus include polymerase type I (RNA polymerase 1, also known as Pol1), flaviviruses such as Japanese encephalitis virus and hepatitis C virus, core protein of coronavirus, Other names include, but are not limited to, capsid proteins (Rawlinson and Moseley, 2015.
  • Necrofosmin is a protein that binds to ribonucleic acid (RNA) present in the granular element (GC) region located on the outermost side of the nucleolus structure.
  • Nucleolin is a protein that is mainly localized in the dense fibrous element (DFC) of the nucleolus and is involved in transcription of ribosomal deoxyribonucleic acid (rDNA).
  • Fibrillarin is a protein that is localized in the fibrous element (FC) and high-density fibrous element (DFC) of the nucleolus, binds to RNA, and functions to process and modify the rRNA precursor.
  • RNA polymerase I is an enzyme (RNA polymerase) that catalyzes a reaction (transcription) that reads the base sequence of a DNA template strand (single strand) to synthesize complementary RNA.
  • RNA polymerase I is present in the nucleolus and synthesizes the rRNA precursor.
  • anti-necrofosmin antibody (anti-NPM1 antibody) (ab10530, Abcam, UK), anti-nucleolin antibody (anti-NCL antibody) (A300-710A, BethylatorLaboratories, TX), anti-fibrillar antibody (GTX24566, TGeneTex, CA), anti-RNA polymerase I antibody (anti-POLI antibody) (13635-1-AP, Proteintech, IL)
  • these proteins bind nucleic acids that encode these proteins and nucleic acids that encode fluorescent substances such as green fluorescent protein (GFP), and introduce the combined nucleic acid into cells, or fuse endogenous proteins and fluorescent substances.
  • the observed recombinant protein can be observed by a fluorescence imaging method by microinjecting into the cell.
  • Nucleic acid localized in a nucleolus in a biological cell means a nucleic acid endogenous to the nucleolus or a nucleic acid that specifically binds to the nucleolus.
  • Examples of the nucleic acid inherent in the nucleolus include ribosomal deoxyribonucleic acid (also known as ribosomal DNA or rDNA).
  • rDNA is a repetitive sequence consisting of 43 kb (coding region 13.7 kb, non-coding region 29.3 kb) as a unit. In humans, it is a short sequence of chromosomes 13, 14, 15, 21, 21 and 22. It is the DNA present in the nuclear short arm of the arm-type chromosome.
  • FISH Fluonecsence In situ Hybridization
  • the FISH method is a technique in which, for example, an oligonucleotide probe obtained by labeling a specific gene sequence of rDNA with a fluorescent substance is used to hybridize with a target gene and detected with a fluorescence microscope.
  • the core protein of flavivirus can express nucleolus morphology by visualizing the localization of nucleolus in biological cells (Tsuda et al., 2006 Microbiol. Immunol., 50, 225-234).
  • the use of the flavivirus core protein in the present invention to determine the effect on the morphology of the nucleolus is based on this finding.
  • the core protein of flavivirus is expressed in cells using a gene transfer reagent such as lipofectamine (ThermoFisher), or recombinant proteins purified from E. coli and insect cells are microinjected using a glass needle. Inject into the cytoplasm and observe its localization. Flavivirus core protein is introduced into E.
  • coli or baculovirus by introducing a plasmid containing DNA encoding the core protein.
  • E. coli sf9 insect cells are infected in the cells, and in the case of baculovirus, the insect cells are infected.
  • the protein expressed by crushing the cells and insect cells and the tag protein such as a specific antibody or glutathione-S-transferase are adsorbed and purified with Sepharose beads cross-linked with a specific binding protein.
  • Changes in the localization of flavivirus core proteins include, for example, immunofluorescence antibody methods using anti-Flag antibodies to core proteins fused with anti-flavivirus antibodies or Flag tags, and fusion proteins fused with fluorescent proteins such as GFP. Can be observed with a fluorescence microscope, a confocal microscope, a super-resolution microscope, an electron microscope, or the like.
  • the protein localized in the nucleolus in the biological cell is selected from necrophosmin, nucleolin, fibrillarin, RNA polymerase I, and flavivirus core protein. Included are methods of the invention that are one or more proteins. The use of two or more proteins localized in the nucleolus is preferable from the viewpoint of the certainty of the candidate compound.
  • the method of the present invention in a particular embodiment, (1) contacting a biological cell with a candidate compound of a substance having anti-flavivirus activity in vitro, under conditions sufficient for the candidate compound to affect the morphology of the nucleolus in the biological cell And incubating over time (2) investigating whether the candidate compound has an effect on the morphology of nucleolus in the biological cell; (3) The candidate compound having an influence is identified as a substance having anti-flavivirus activity.
  • Candidate compounds that affect nucleolar morphology in the biological cell are identified as potential anti-flavivirus drugs.
  • the invention also provides in a preferred embodiment (1) contacting a biological cell with a candidate compound of a substance having anti-flavivirus activity in vitro, under conditions sufficient for the candidate compound to affect the morphology of the nucleolus in the biological cell And incubating over time (2) investigating whether the candidate compound has an effect on the morphology of nucleolus in the biological cell; (3) selecting the candidate compounds that are influencing, and (4) examining the changes in localization of the core protein of the virus in biological cells for the selected candidate compounds, (5) If the localization of the core protein is changed along with the influence on the morphology of the nucleolus, it is identified as a substance having anti-flavivirus activity.
  • the screening method of the present invention leads to the discovery and development of anti-flavivirus drugs that exert their effects by affecting the morphology of nucleoli in biological cells. These drugs may be potentially useful in the treatment and / or prevention of flavivirus infections and / or flavivirus related diseases and conditions.
  • the present invention includes any compound identified as a substance having anti-flavivirus activity by the screening method of the present invention.
  • nucleolus morphology in biological cells found by the present inventors can be used as targets for the identification, design and development of new anti-flavivirus drugs.
  • Substances that affect nucleolus morphology in biological cells useful as anti-flavivirus drugs belong to a wide variety of molecular families, including but not limited to organic compounds (eg, small molecules, saccharides, steroids, etc.) , Monoclonal antibodies or polyclonal antibodies (eg, antibodies that bind to the nuclear translocation of flavivirus core protein), peptides, polypeptides, nucleic acid molecules (eg, antisense compounds, ribozymes, triple helix molecules, SELEX RNA, etc.).
  • Library screening provides compounds with the desired but not optimized biological activity, usually referred to as “hit” or “lead” compounds.
  • the next step in developing useful drug candidates usually involves analyzing the relationship between the chemical structure of the hit compound and its biological or pharmacological activity. Molecular structure and biological activity can be related by observing the results of global structural modifications for a given biological index. The structure-activity relationship information available from the initial screen can then be used to generate a small compound library, which is then screened for compounds with higher affinity.
  • the process of performing synthetic modification of biologically active compounds to meet the stereoelectronic, physicochemical, pharmacokinetic and toxicological factors required for clinical utility is lead optimization
  • Candidate compounds identified by the screening methods of the present invention can be applied early in lead optimization as well.
  • Drug candidates that have been chemically modified and improved by lead optimization can be included in the present invention to the extent appropriate.
  • the appropriate range is a range that can be understood by those skilled in the art that drug candidate compounds that affect the nucleolus morphology of biological cells in the present invention have a certain range of chemical structures and show similar activities.
  • the screening method described in this specification can be made into a kit.
  • the biological cells disclosed herein can be packaged in a variety of containers, such as vials, tubes, microtiter well plates, bottles, and other reagents can be included in separate containers for kits.
  • a positive control sample or compound, a negative control sample or compound, a buffer solution, a cell culture solution, a specific detection probe and the like can be used as a whole.
  • the present invention provides an anti-flavivirus pharmaceutical composition comprising a substance that affects nucleolus morphology in biological cells, preferably a substance that affects nucleolus morphology in biological cells.
  • an anti-flavivirus pharmaceutical composition preferably a nucleolus-form comprising a substance that causes a morphological abnormality of the nucleolus
  • an anti-flavivirus pharmaceutical composition comprising a substance causing an abnormality as an active ingredient having anti-flavivirus activity. This will be specifically described below.
  • flavivirus has the aforementioned meanings, and the terms “anti-flavivirus” or “anti-flavivirus activity” are used interchangeably to refer to flavivirus replication, cell entry and / or cell flavivirus. It means any effect that reduces, inhibits, blocks or prevents viral infection, consequently suppresses or inhibits the flavivirus life cycle and extinguishes or reduces viral activity.
  • the “anti-flavivirus pharmaceutical composition” means a pharmaceutical composition capable of treating or preventing a subject suffering from or infected with flavivirus by eliminating or reducing flavivirus.
  • the terms “affected by a flavivirus” or “infected with a flavivirus” are used in connection with a subject to mean infected with a flavivirus and having a flavivirus-related disease.
  • the term “flavivirus infection” can refer to the introduction of flavivirus genetic information into target cells (eg, by fusion of the target cell membrane with flavivirus or flavivirus envelope glycoprotein positive cells).
  • “Flavivirus-related disease” refers to any disease or disorder known or suspected to be associated with and / or caused directly or indirectly by a flavivirus.
  • the main flavivirus-related diseases are fever, headache, muscle pain, and joint pain. Particularly severe hemorrhagic fever due to dengue virus, encephalitis due to Zika virus, Japanese encephalitis virus, West Nile virus, tick-borne encephalitis virus, Zika Including but not limited to viral microcephaly.
  • the flavivirus is an anti-flavivirus activity selected from Japanese encephalitis virus, dengue virus, Zika virus, West Nile virus, and yellow fever virus.
  • Treatment refers to (1) delaying or preventing the onset of a disease or condition (eg, flavivirus infection or flavivirus related disease); (2) progression, exacerbation of symptoms of the disease or condition, or Refers to a method or process intended to slow or stop exacerbation; (3) provide amelioration of symptoms of the disease or condition; or (4) cure the disease or condition. Treatment may be given as a preventive measure prior to the onset of the disease or condition, or alternatively, treatment may be given after the onset of the disease.
  • a disease or condition eg, flavivirus infection or flavivirus related disease
  • preventing, inhibiting or blocking flavivirus infection refers to the amount of flavivirus genetic information introduced into a sensitive cell or population of sensitive cells when a substance having anti-flavivirus activity is used. It means to reduce compared to the amount that would be introduced in the absence.
  • “Pharmaceutical composition” as used herein comprises an effective amount of at least one substance having anti-flavivirus activity, and at least one pharmaceutically acceptable carrier or excipient.
  • the term “effective amount” refers to a compound or composition that is sufficient to meet its intended purpose, eg, a desired biological or pharmacological response in a cell, tissue, system, or subject. Means any amount.
  • the objective is to have anti-flavivirus activity, prevent flavivirus infection, prevent the onset of flavivirus-related diseases, progression of symptoms of flavivirus-related diseases, Means slowing, reducing or stopping exacerbations or exacerbations, providing amelioration of disease symptoms, and / or curing flavivirus-related diseases.
  • “Pharmaceutically acceptable carrier or excipient” refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient and is not excessively toxic to the host at the concentration at which it is administered. .
  • the term includes solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, adsorption retarders and the like. The use of such media and drugs for pharmaceutically active substances is well known in the art.
  • the “substance affecting the morphology of the nucleolus in a biological cell” refers to the nucleolus that is the transcription site of ribosome RNA (rRNA) and the ribosome synthesis site in the biological cell of the present invention. It means a substance that affects and changes its form, specifically a substance that causes morphological abnormalities in the nucleolus.
  • morphological abnormalities of nucleolus in a biological cell are determined by the formation of a protein localized in the nucleolus and / or a change in localization in the present invention. Preferably, these substances are selected by the screening method of the present invention.
  • a substance that affects the morphology of nucleolus in a biological cell exhibits, in one embodiment, anti-flavivirus activity by inhibiting viral replication.
  • the pharmaceutical composition is preferably a pharmaceutical composition in which inhibition of viral replication is accompanied by a change in localization of the viral core protein in biological cells. This is because inhibition of viral replication, which ultimately shows anti-flavivirus activity, involves the localization of the flavivirus core protein.
  • compositions affecting the morphology of nucleolus in biological cells include substances having cyclin-dependent kinase (Cyclin-dependent kinase) inhibitory activity, substances having rRNA transcription inhibitory activity, DNA topoisomerase It is selected from substances having inhibitory activity or substances having glycogen synthase kinase-3 (GSK-3) inhibitory activity.
  • Cyclin-dependent kinase Cyclin-dependent kinase
  • rRNA transcription inhibitory activity substances having rRNA transcription inhibitory activity
  • DNA topoisomerase It is selected from substances having inhibitory activity or substances having glycogen synthase kinase-3 (GSK-3) inhibitory activity.
  • CDK in a substance having CDK inhibitory activity means a phosphorylase having activity only after binding to cyclin in a group of kinases that control the cell cycle, such as CDK1 (or cdc2), CDK2, CDK4, CDK6, etc. Each is activated at a different point in the cell cycle.
  • Cyclin is a protein that shifts the cell cycle in eukaryotic cells, and cyclins A, B, D, and E are involved in the rotation of the cell cycle. Cyclin A mainly binds to CDK1 and CDK2 to form a complex and controls the end of the DNA synthesis phase.
  • Cyclin B mainly binds to CDK2 to form a complex and controls progression to mitosis.
  • Cyclin D mainly binds to CDK4 and CDK6 to form a complex, and controls the transition between the DNA synthesis preparation phase / DNA synthesis phase.
  • Cyclin E mainly binds to CDK2 to form a complex and controls the transition to the DNA synthesis phase.
  • Substances having CDK inhibitory activity usually show the action of inhibiting cell growth by inhibiting CDK activity, and many of them have been developed as anticancer agents using the action, and among them, CDK4 / 6 inhibitor Ibrance (generic name: Parvocyclib, Pfizer Inc.).
  • substances that inhibit CDK1, CDK2, and CDK9 are preferred.
  • the substance having CDK inhibitory activity is 2-cyanoethylartelopaulone, 5-amino-3-((4- (aminosulfonyl) phenyl) amino) -N- (2,6-difluorophenyl).
  • a substance having a preferable CDK inhibitory activity is 2-cyanoethylarteropaulone, 5-amino-3-((4- (aminosulfonyl) phenyl) amino) -N- (2,6-difluorophenyl) -1H-1 , 2,4-triazole-1-carbothioamide, (4- (2-amino-4-methylthiazol-5-yl) pyrimidin-2-yl)-(3-nitrophenyl) amine, P276-00, A- Selected from 674563 and SU9516.
  • More preferable substances having CDK inhibitory activity are 2-cyanoethylarteropaulone, 5-amino-3-((4- (aminosulfonyl) phenyl) amino) -N- (2,6-difluorophenyl) -1H— Selected from 1,2,4-triazole-1-carbothioamide and (4- (2-amino-4-methylthiazol-5-yl) pyrimidin-2-yl)-(3-nitrophenyl) amine .
  • Flavopyridol (Kim KS, et al. 2000. J Med Chem, 43 (22), 4126-4134; Lu H, et al. 2005. J Med Chem, 48 (3), 737-743; Montagnoli A, et al. 2008. Nat Chem Biol, 4 (6), 357-365; Carlson BA, et al. 1996. Cancer Res, 56 (13), 2973-2978)
  • Flavopyridol hydrochloride (Senderowicz AM, 2002. Oncologist, 7 Suppl 3: 12-9; Carlson BA, et al, 1996. Cancer Res, 56 (13), 2973-2978; Parker BW, et al, 1998. Blood, 91 (2), 458-465; Drees M, et al, 1997. Clin Cancer Res, 3 (2), 273-279)
  • RRNA transcription means a reaction in which a transcription initiation complex containing RNA polymerase I is formed on the promoter of rDNA encoding ribosomal RNA (rRNA) and rRNA is synthesized.
  • a substance having rRNA transcription inhibitory activity usually suppresses or inhibits the transcription of rRNA such as RNA polymerase I, and as a result exhibits an action of inhibiting rRNA synthesis.
  • the substance having rRNA transcription inhibitory activity is typically selected from actinomycin D, doxorubicin, and aclarubicin, which will be described in detail below. It is known that actinomycin D treatment induces nucleolus decay (Scheer et al., 1975. 65: 163-179).
  • Actinomycin D Inhibiting rRNA transcriptional activity with actinomycin D results in nuclear segregation in which DFC and GC form a cap-like structure around the nucleolus (Scheer et al., 1975. 65: 163-179). (Waksman SA et al., 1940. Proc. Soc. Exp. Biol. Med. 45, 609-614; Goldberg IH et al., 1962. Science. 136, 315-316; Sobell HM et al., 1985.Proc Natl. Acad. Sci. USA. 82, 5328-5331)
  • DNA topoisomerase is an enzyme that temporarily cleaves one or both of double-stranded DNAs and binds the breakpoints again. It is roughly classified into two types (type I and type II). Type I cleaves only one strand of DNA double strands, and then recombines the cleaved DNA. Type II cleaves the double strand and recombines. Thereby, DNA topoisomerase eliminates the twist and distortion of the helical structure of DNA during replication. A substance that inhibits the activity of DNA topoisomerase usually inhibits the function of cleaving and reconnecting DNA, and also forms a bridge between DNAs to inhibit DNA replication.
  • Substances having DNA topoisomerase inhibitory activity are typically doxorubicin, aclarubicin, ellipticine, idarubicin, idarubicin hydrochloride, epirubicin hydrochloride, as detailed below.
  • Epirubicin HCl mitoxantrone hydrochloride
  • Mitoxantrone HCl mitoxantrone hydrochloride
  • daunorubicin hydrochloride Daunorubicin HCl
  • a preferred substance having DNA topoisomerase inhibitory activity is ellipticine.
  • Doxorubicin (same as above); Aclarubicin (same as above);
  • Daunorubicin hydrochloride (Fu Y, et al. Thromb Haemost, 2010, 104 (6), 1235-1241.)
  • GSK-3 was identified as a serine / threonine protein kinase that phosphorylates and inactivates glycogen synthase. In humans, it is classified into two isoforms, ⁇ and ⁇ , and is known to be involved in many cell function controls as well as glycogen metabolism. The substance that inhibits the activity of GSK-3 is typically 1-Azakenpaullone.
  • the “substance that affects the nucleolus morphology in biological cells” in the present invention includes various known biology such as CDK inhibition, rRNA transcription inhibition, DNA topoisomerase inhibition, and GSK-3 inhibition. And has various chemical structures as described above. All compounds with different known activities and different chemical structures all have the common effect of “influencing nucleolus morphology in biological cells”, thereby inhibiting flavivirus replication Has been proven. This means that all compounds that have the common effect of “influencing the nucleolus morphology in biological cells” inhibit flavivirus replication and have anti-flavivirus activity. It is proof of that.
  • flaviviruses utilize the nucleolus function of the host cell as a core protein as part of the viral replication function, and the morphological abnormalities in the nucleolus found for the first time in the present invention are: It is thought that it has the effect of reducing or destroying the normal nucleolus function that adversely affects the function of the core protein and promotes the synthesis of virus-derived proteins.
  • the substance or composition having anti-flavivirus activity of the present invention is administered in an effective amount, ie, an amount sufficient to meet the intended purpose.
  • an effective amount is an amount that affects the morphology of nucleoli in biological cells and inhibits or prevents flavivirus replication in susceptible cells of a subject, thereby preventing flavivirus infection. The amount to treat or prevent.
  • the substances and compositions having anti-flavivirus activity of the present invention may be used in various therapeutic or prophylactic methods.
  • the invention provides a method for treating or preventing a flavivirus-related disease or condition in a subject, which provides the subject with an effective amount that affects the nucleolar morphology of the biological cells of the invention.
  • the present invention administers a substance or composition having anti-flavivirus activity of the present invention alone.
  • the substance or composition having anti-flavivirus activity of the present invention is administered in combination with at least one additional therapeutic drug.
  • the substance or composition having anti-flavivirus activity of the present invention can be administered before administration of the therapeutic drug, simultaneously with the therapeutic drug, and / or after administration of the therapeutic drug.
  • a therapeutic drug that can be administered in combination with a substance or composition having anti-flavivirus activity of the present invention may have a beneficial effect in the treatment, management or prevention of flavivirus infection, or flavivirus-related diseases or conditions. It can be selected from a wide variety of biologically active compounds known in the art.
  • Such drugs include in particular antiviral drugs, such as interferons (eg, interferon alpha, pegylated interferon alpha), anti-flavivirus (monoclonal or polyclonal) antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, Includes helicase inhibitors, antisense compounds, ribozymes, and any combination thereof.
  • the substance having anti-flavivirus activity of the present invention is optionally formulated with one or more suitable pharmaceutically acceptable carriers or excipients to a subject in need thereof at the desired dosage.
  • Administration can be by any suitable route.
  • Various delivery systems are known and can be used to administer substances having anti-flavivirus activity of the invention, such as tablets, capsules, injections, capsules in liposomes, powders, Includes microcapsules.
  • Methods of administration include transdermal, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, intraocular and oral routes. Administration can be systemic or local.
  • the administration of a substance or composition having anti-flavivirus activity of the present invention can be such that the amount delivered is effective for the intended purpose.
  • the route of administration, formulation, and dosage will depend on the desired therapeutic effect, the severity of the flavivirus infection or flavivirus-related condition being treated (if already present), the presence of any other infection, the age of the patient, gender
  • the efficacy, bioavailability and in vivo half-life, use (or unused) of combination treatments and other clinical factors of the substance or composition with anti-flavivirus activity used, body weight, and overall health status Can depend. These factors can be easily determined by the attending physician in the course of treatment. As testing is performed using substances with anti-flavivirus activity of the present invention, further information regarding appropriate dosage levels and treatment times will be obtained.
  • the treatment of the present invention may consist of a single dose or multiple doses.
  • administration of a substance having anti-flavivirus activity of the present invention, or a composition thereof can be administered regularly or periodically over a specific period and at specific intervals, such as once every several hours, once a day, once a week Once, once a month (eg, time release form).
  • delivery can be continuous delivery over a period of time, for example intravenous delivery.
  • the amount of substance having anti-flavivirus activity administered is preferably about 1 ng / kg to about 100 mg / kg of the subject's body weight, such as about 100 ng / kg to about 50 mg / kg of the subject's body weight; or It can range from about 1 ⁇ g / kg to about 10 mg / kg of the subject's body weight.
  • Reference Example 1 Nucleolus localization of flavivirus core protein Flavivirus core protein was expressed in cells and its localization was examined.
  • Reference Example 1-1 Construction of transient culture cell expression plasmids Japanese encephalitis virus (JEV), dengue virus (DENV), and zika virus (ZIKV) core protein cDNAs are distributed by the National Institute of Infectious Diseases. I received it. West Nile virus (WNV) core protein cDNA was commissioned and synthesized by Integrated DNA Technologies (IDT, Inc., Illinois).
  • JEV Japanese encephalitis virus
  • DEV dengue virus
  • ZIKV zika virus
  • Each core protein cDNA was amplified by PCR reaction using the following primers, and each amplified cDNA was subjected to EcoRI of a pCAGGS vector containing a Flag-tag (Miyazaki et al., (1989) Gene. 79: 269-77). Incorporated into the site using In-Fusion HD Cloning Kit (Takara Bio Inc. Japan), pCAGGS / flag-JEVcore, pCAGGS / flag-DENVcore, pCAGGS / flag-ZIKAcore and pCAGGS / flag-WNVcore were constructed.
  • JEV core Fw GCTGTACAAGCTCGAGATGACTAAAAAACCAGGAGG (SEQ ID NO: 1) Rv ACAGGTTTTCCTCGAGTCTTTTGTTTTGCTTTCTGC (SEQ ID NO: 2)
  • DENV core Fw GCTGTACAAGCTCGAGATGAATAACCAACGGAAAAA (SEQ ID NO: 3) Rv ACAGGTTTTCCTCGAGTCTGCGTCTCCTATTCAAGA (SEQ ID NO: 4)
  • ZIKV core Fw GCTGTACAAGCTCGAGATGAAAAACCCAAAGAAGAA (SEQ ID NO: 5) Rv ACAGGTTTTCCTCGAGACGTCTCTTCCTCTCTTTCC (SEQ ID NO: 6)
  • WNV core Fw GCTGTACAAGCTCGAGATGTCTAAGAAACCAGGAGG (SEQ ID NO: 7) Rv ACAGGTTTTCCTCGAGTCTTTTCTTGTTTTGAGC (SEQ ID NO: 8)
  • Reference Example 1-2 Transient cultured cell expression Hepatocellular carcinoma Huh7 cells (distributed by National Institute of Infectious Diseases) are Dulbecco's modified Eagle medium (including 10% fetal bovine serum, 100 ⁇ g / mL penicillin, 100 ⁇ g) / mL streptomycin) at 37 ° C. under 5% CO 2.
  • Dulbecco's modified Eagle medium including 10% fetal bovine serum, 100 ⁇ g / mL penicillin, 100 ⁇ g) / mL streptomycin
  • Reference Example 1-3 Intracellular localization of baculovirus-derived core protein As in Reference Example 1-1, JEV, DENV, ZIKV and WNV core protein cDNAs were amplified by PCR, and AcGFP (Clontech) was added to the N-terminus. Is fused with the FLAG-One-STrEP (FOS) tag (Kouwaki T, et al.
  • FOS FLAG-One-STrEP
  • pFastBac1 (Thermo Fisher Scientific Inc, MA ) Using the In-Fusion HD Cloning Kit (Takara Bio Inc., Japan), pFastBac1 / AcGFP-JEVcore-FOS, pFastBac1 / AcGFP-DENVcore-FOS, pFastBac1 / AcGFP-ZIKAcore-FOS and pFastBac1 / AcGFP-WNVcore-FOS was constructed.
  • Transformable Escherichia coli (DH10DHBac (ThermorFisher Scientific Inc) was transformed into baculovirus expression plasmids pFastBac1 / AcGFP-JEVcore-FOS, pFastBac1 / AcGFP-DENVcore-FOS, pFastBac1 / AcGFP-ZIKAcore-FOS and pFastBac1 / AcGFP- Transformation was performed using WNVcore-FOS.
  • Sf9 insect cells After culturing the colonies, plasmid DNA was extracted and introduced into Sf9 insect cells (Thermo
  • transfection reagent Roche
  • Strep-Tactin-Sepharose was added to the supernatant and incubated at 4 ° C. for 12 hours.
  • Strep-Tactin® Sepharose was washed with buffer solution W (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM NaCl, EDTA) and then 2.4 mL Buffer E (100 mM Tris-HCl (pH 8.0), 150 The protein was eluted with mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin.
  • Each AcGFP fusion core protein purified from baculovirus was microinjected into the cytoplasm of Huh7 cells together with mouse IgG (Santa Cruz Biotechnology, Texas, USA) as an injection marker and incubated for 30 minutes.
  • Mouse IgG was detected with Alexa Fluor594 anti-mouse secondary antibody (Thermo Fisher Scientific Inc.). The obtained result is shown in FIG. Injection marker (bottom) shows intracellular localization of mouse IgG.
  • Mouse IgG injected into the cytoplasm remains in the cytoplasm because it cannot move into the nucleus. This shows that the sample has been accurately driven into the cytoplasm and that the nuclear membrane has not been physically destroyed by microinjection.
  • GFP (top) is an activity in which the core proteins micro-injected into mouse IgG and cytoplasm (GFP-JEV core, GFP-DENV core, GFP-ZIKV core, GFP-WNV core) are localized in the nucleus (nucleolus) It shows that there is.
  • Reference Example 1-4 Demonstration of nuclear translocation activity and nucleolar localization activity of JEV core protein purified from baculovirus AcGFP-JEV core and secondary anti-mouse antibody (Alexa Fluor Plus 555, Thermo Fisher) Scientific Inc.) and microinjected into the cytoplasm of Huh7 cells. After incubation for 30 minutes, the cells were fixed, and an immunofluorescent antibody method using an anti-necrophosmin antibody (anti-NPM1 antibody) (ab10530, Abcam, UK) was performed. The secondary antibody used was an anti-mouse secondary antibody (Alexa Fluor 645, Thermo Fisher Scientific Inc.), and genomic DNA (cell nucleus) was stained with DAPI. The obtained result is shown in FIG.
  • Injection marker represents that the fluorescence of Alexa Fluor Plus 555 mouse IgG, which is an injection marker, is present in the cytoplasm. This indicates that the injection marker remains in the implanted cytoplasm and does not move to the nucleus.
  • JEV core indicates that the JEV core protein microinjected into the cytoplasm together with Alexa Fluor Plus 555 mouse IgG co-localizes with NPM1, a nucleolar marker. This indicates that the JEV core protein is active in the nucleus and localizes in the nucleolus.
  • NPM1 and DAPI indicate the presence of nucleolus and genomic DNA (cell nucleus), respectively.
  • Example 1 Screening for substances with anti-flavivirus activity using changes in subcellular localization of JEV core as an index
  • a substance having activity was screened.
  • Example 1-1 Preparation of cell line stably expressing JEV core protein
  • a JEVcore gene cDNA amplified by PCR reaction was added to a lentiviral vector pLV- containing the superfolder GFP-11 (sfGFP11). Incorporated into the EcoRI site of CAG (Okada Y, et al. (2007) Nat.
  • the sfGFP1-10-SV40NLS gene cDNA was commissioned and synthesized by Integrated DNA Technologies (IDT, Inc., Illinois), and the lentiviral vector pFUGW (Addgene, plasmid # 14883) along with the neomycin resistance gene under the IRES (ribosome entry site inside mRNA) sequence.
  • the plasmid pFUGW / sfGFP1-10-SV40NLS was constructed using the In-Fusion HD Cloning Kit (Takara Bio Inc.).
  • pLV-CAG / sfGFP-JEVcore cDNA and pFUGW / sfGFP1-10-SV40NLS cDNA were introduced into Huh7 cells using TransIT-LT1 transfection reagent (Mirus Bio LLC), and 1 mg / mL G418 solution (nacalai tesque ) was added to selectively obtain introduced cells. Since G418-resistant Huh7 traces colonies, it was seeded at a low cell density, and single colonies with GFP fluorescence were collected under a fluorescence microscope to produce a cell line stably expressing JEV core protein.
  • Example 1-2 Screening of substance having anti-flavivirus activity 1 x 10 4 sfGFP-JEVcore stably expressing Huh7 cells prepared in Example 1-1 were seeded on a 96-well microplate (Greiner bio-one) After incubation in a 37 ° C, 5% CO 2 incubator for 24 hours, standard inhibitor kit 3 ver1.5 (Table 1: Kinase inhibitor kit, Ministry of Education, Culture, Sports, Science and Technology) Based on the support activities based on the "chemotherapy-based support activities (https://scads.jfcr.or.jp/kit/kit.html)", each low-molecular compound was added to the culture medium to a final concentration of 1 ⁇ M, and then 24 hours Cultured.
  • the cells were washed with PBS, then incubated with 3.7% formalin solution for 15 minutes to fix the cells, and genomic DNA (cell nuclei) was stained with (DOJINDO, Japan, final concentration 0.2 ⁇ g / mL).
  • the plate was subjected to a high-throughput cell function search system CV7000S (Yokogawa Electric Corporation), and three fluorescent photographs were taken per well to analyze the fluorescence intensity and intracellular localization of GFP.
  • FIG. Fig. 2 (A) is a graph of the average area of dots (green fluorescence) per cell (per nucleus) measured by measuring the GFP fluorescence (green) area present in the cell nucleus using DAPI staining as an index. is there.
  • the horizontal axis represents the well, and the vertical axis represents the average area of GFP fluorescence.
  • CDK inhibitors (2-cyanoethyl asterolone, 2-cyanoethl), Cdk1 / 2 inhibitor III (5-amino-3-((4- (aminosulfonyl) phenyl) amino)- N- (2,6-difluorophenyl) -1H-1,2,4-triazole-1-carbothioamide), Cdk2 / 9 inhibitor ((4- (2-amino-4-methylthiazol-5-yl) The wells administered with pyrimidin-2-yl)-(3-nitrophenyl) amine)) are shown, indicating that the fluorescence area is actually reduced.
  • FIG. 2 (B) shows fluorescence images when DMSO (control) and the above three CDK inhibitors were treated. It can be seen that sfGFP-JEVcore is localized in the nucleolus clearly present in the nucleus during DMSO treatment. On the other hand, in the CDK inhibitor-treated cells, the fluorescence of sfGFP-JEV core appears as small dots (dots). It can also be seen that the number of dots and the fluorescence brightness differ for each CDK inhibitor.
  • Example 1-3 Screening for CDK inhibitor that changes nucleolus localization of JEV core protein 1x10 5 sfGFP-JEVcore stably expressed in Example 1-1 were prepared in 15 mm round micro After culturing a 35 mm dish (IWAKI, Japan) with a cover glass (Matsunami, Japan) for 24 hours, the CDK inhibitor (Selleck Customized Library of 30 compounds: Cherry Pick Library (96- well) -L2000-Z237098-100uL (Selleck.co.jp, Japan, Table 2)) was added and further cultured for 24 hours.
  • CDK inhibitor Selleck Customized Library of 30 compounds: Cherry Pick Library (96- well) -L2000-Z237098-100uL (Selleck.co.jp, Japan, Table 2)
  • Table 2 shows CDK inhibitors with altered nucleolus localization of sfGFP-JEVcore.
  • PCAGGS / flag- prepared in Reference Example 1-1 after culturing a 35 mm dish (IWAKI, Japan) containing 1 x 10 5 Huh7 cells and 15 mm round micro cover glass (Matsunami, Japan) for 24 hours Introducing JEVcore using TransIT-LT1 Transfection Reagent (Mirus Bio LLC, WI), and further 24 hours later, CDK inhibitor (Selleck Customized Library of 30 compounds (Selleck.co.jp, Japan) And see Table 2)), and further cultured for 24 hours. After culturing, the cells were fixed and stained by the immunoantibody method performed in Reference Example 1-2. Table 2 shows CDK inhibitors with altered nucleolar localization of JEVcore (Flag-core).
  • Example 2 Nucleolus morphological change by Cdk1 / 2 inhibitor III
  • three CDK inhibitors, 2-cyanoethylartelopaulone, Cdk1 / 2 inhibitor III and Cdk2 / 9 inhibitor were actually fluorescent areas.
  • the cell used is an sfGFP-JEVcore stable expression cell line in which the core protein is already present in the nucleus (nucleolus).
  • a CDK inhibitor is added later to this cell line. .
  • the following experiment was performed.
  • Example 2-1 Nucleolar body abnormalities due to treatment with Cdk1 / 2 inhibitor III
  • Huh7 cells were cultured in the presence of Cdk1 / 2 inhibitor III at a final concentration of 1 ⁇ M for 24 hours, and then immunofluorescent antibody was used with anti-NPM1 antibody. It was.
  • the secondary antibody was an anti-mouse secondary antibody (Alexa Fluor 645), and the genomic DNA (cell nucleus) was stained with DAPI. The obtained result is shown in FIG.
  • Nucleotide discrimination can be seen from genomic DNA staining with DAPI (lower 4 images, showing the same cells as the upper NPM1 staining). Since NPM1 is a nucleolus-specific marker protein, a small structure in the nucleus can be said to be a nucleolus. From the above, it was found that nucleolus morphological abnormalities were caused by treatment with Cdk1 / 2 inhibitor III.
  • Example 2-2 Abnormal Nucleolar Morphology due to 2-Cyanoethyl Alstero Paulon Treatment 2 ⁇ 10 4 Huh7 cells were seeded in a 12-well plate containing 15 mm round micro cover glass (Matsunami), and 37 The cells were cultured for 24 hours in a 5% CO 2 incubator at 0 ° C. After culturing, 2-cyanoethylartelopoulos (standard inhibitor kit 3 ver1.5 (kinase inhibitor kit)) was added to the medium to a final concentration of 1 ⁇ M and cultured for 24 hours.
  • standard inhibitor kit 3 ver1.5 kinase inhibitor kit
  • AcGFP-JEV core recombinant protein (final concentration 100 ug / mL) purified from baculovirus is an injection marker goat anti-mouse IgG (H + L) high cross-adsorbed secondary antibody Alexa Fluor Plus 555 (Thermo Fisher Scientific Inc, final concentration 100 ug / mL), and microinjected into the cytoplasm of Huh7 using a glass needle. After culturing in a 5% CO 2 incubator at 37 ° C. for 30 minutes, the cells were fixed by incubation in 3.7% formalin solution for 15 minutes.
  • H + L high cross-adsorbed secondary antibody Alexa Fluor Plus 555
  • DAPI solution final concentration 0.2 ug / mL
  • TCS SP8 Leica
  • injection marker indicates that the fluorescence of Alexa Fluor PLUS Plus 555 mouse IgG, which is an injection marker, exists in the cytoplasm. This indicates that the injection marker remains in the implanted cytoplasm.
  • JEV core indicates that AcGFP-JEVcore exists in the nucleus and is localized in a small structure different from the normal nucleolus. This shows that since the JEV core protein is microinjected into the cytoplasm together with Alexa Fluor PLUS Plus 555 mouse IgG, the nuclear translocation of AcGFP-JEVcore is not inhibited by treatment with 2-cyanoethylarsteropaulone.
  • the JEV core protein present in the nucleus is localized in a small nucleolus that is different from the usual. This indicates that nucleolus morphological abnormalities are caused by treatment with 2-cyanoethylartelopaulone, and that JEV core protein is translocated to the nucleus and localizes to abnormally nucleated nucleolus.
  • DAPI stains genomic DNA (cell nuclei). merge is a combination of Injection marker, JEV core, and DAPI images.
  • JEV AT31 strain
  • Moi multiplicity of infection
  • the focus forming assay was performed by the following method. First, the culture supernatant of JEV-infected Huh7 cells was recovered and diluted 10-fold with PBS. On the day before the assay, a 24-well plate was seeded with 5 x 10 4 Vero cells (Mori Y., et al. (2005) Journal of virology 79: 3448-3458). After adding 100 ⁇ l / well, the cells were cultured for 2 hours.
  • the cells were washed with PBS, Dulbecco's modified Eagle's medium containing 10% methylcellulose (containing 10% fetal bovine serum, 100 ⁇ g / mL penicillin, 100 ⁇ g / mL streptomycin) was added, and the cells were further cultured for 2 days and fixed with 4% paraformaldehyde. did. Fixed cells were treated with 0.5% Triton-X100 for 5 minutes and incubated with anti-NS3 mouse antibody (Mori Y., et al. (2005) Journal of virology 79: 3448-3458) diluted 2000 times for 30 minutes.
  • FIG. A CDK inhibitor was added to JEV-infected Huh7 cells at the timings (a) and (b) shown in FIG. 4 (A) and incubated for 24 hours. Cell viability 48 hours after infection is shown in (B), and infection titer is shown in (C). It can be seen that treatment with a CDK inhibitor does not affect cell survival but suppresses viral replication.
  • Example 4 Nucleolus morphological changes caused by a substance having rRNA transcription inhibitory activity
  • the cells used are the sfGFP-JEVcore stable expression Huh7 cell line prepared in Example 1-1. 1 ⁇ 10 4 cells were seeded on a 96-well microplate (Greiner bio-one) and cultured at 37 ° C. After culturing in a 5% CO 2 incubator for 24 hours, rRNA transcription inhibitors actinomycin D, doxorubicin and aclarubicin were added to a final concentration of 1 ⁇ M and further cultured for 24 hours.
  • the cells were washed with PBS, then incubated with 3.7% formalin solution for 15 minutes to fix the cells, and genomic DNA (cell nuclei) was stained with (DOJINDO, Japan, final concentration 0.2 ⁇ g / mL).
  • the plate was subjected to a high-throughput cell function search system CV7000S (Yokogawa Electric Corporation), and three fluorescent photographs were taken per well to analyze the fluorescence intensity and intracellular localization of GFP.
  • FIG. The fluorescence image at the time of processing DMSO (control) and said three rRNA transcription inhibitor is shown. It can be seen that sfGFP-JEVcore is localized in the nucleolus clearly present in the nucleus during DMSO treatment. On the other hand, the rRNA transcription inhibitor actinomycin D, doxorubicin, and aclarubicin all appear to have small dots (dots) in the fluorescence of sfGFP-JEV core.
  • Example 5 Inhibition of Flavivirus Growth by Substances with GSK-3 Inhibitory Activity
  • Low molecular weight compounds obtained from the GSK-3 inhibitor library (https://www.selleck.co.jp/GSK-3.html) Screening was carried out substantially according to the procedure of Example 1 except that it was used as a candidate substance having viral activity. The obtained result is shown in FIG. Thereby, it was found that 1-azaken Paulon suppresses GFP fluorescence intensity.
  • Example 6 Further screening for substances with anti-flavivirus activity Low molecular weight compounds obtained from LOPAC1280 (distributed from graduate School of Pharmaceutical Sciences, Kyoto University) and FDA-approved Drug Library (distributed from graduate School of Pharmaceutical Sciences, Osaka University) Screening was carried out substantially according to the procedure of Example 1 except that it was used as a candidate substance having activity.
  • the cells used are the sfGFP-JEVcore stable expression Huh7 cell line prepared in Example 1-1. 1 ⁇ 10 4 cells were seeded on a 96-well microplate (Greiner bio-one) and cultured at 37 ° C.
  • FIG. The fluorescence image at the time of processing DMSO (control) and the said DNA topoisomerase inhibitor is shown. It can be seen that sfGFP-JEVcore is localized in the nucleolus clearly present in the nucleus during DMSO treatment. On the other hand, all of ellipticine, idarubicin, idarubicin hydrochloride, epirubicin hydrochloride, mitoxantrone hydrochloride, and daunorubicin hydrochloride have small sfGFP-JEV core fluorescence (dot-like) or disappear in the dispersal region in the nucleus. Exist.
  • the habitat of vector mosquitoes changes due to warming and the development of transportation means, and the distribution of flaviviruses may change in the future.
  • the West Nile virus first invaded the United States in 1999, many people were infected, and if the virus invades a new area, there is a high probability that many victims will be killed. Therefore, further progress is desired in the treatment of flavivirus not only in Japan but also in countries around the world.
  • the screening method of the present invention provides new knowledge about the replication mechanism and immune response / evasion mechanism, and leads to the development of new antiviral drugs.

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Abstract

L'invention concerne un procédé relatif à un effet sur la morphologie nucléolaire dans une cellule biologique en tant que cible potentielle d'un traitement d'une infection à flavivirus et de maladies associées au flavivirus chez des mammifères, y compris des êtres humains. L'invention concerne également une composition médicinale anti-flavivirus comprenant, en tant que principe actif, une substance qui est acquise par le procédé selon la présente invention et affecte la morphologie nucléolaire dans une cellule biologique.
PCT/JP2019/020916 2018-05-28 2019-05-27 Traitement antiviral d'une infection à flavivirus Ceased WO2019230654A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN114848634A (zh) * 2022-05-18 2022-08-05 西安医学院 Sb415286的应用及寨卡病毒抑制剂和药物
WO2022210644A1 (fr) * 2021-04-02 2022-10-06 国立研究開発法人医薬基盤・健康・栄養研究所 Médicament pour le traitement d'une large gamme d'infections virales, y compris le sars-cov -2
CN117110269A (zh) * 2023-10-20 2023-11-24 浙江迪福润丝生物科技有限公司 Jev蛋白酶抑制剂的筛选方法和抑制效果评价方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018017426A1 (fr) * 2016-07-16 2018-01-25 Florida State University Research Foundation, Inc. Composés et méthodes de traitement ou de prévention d'infections à favivirus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018017426A1 (fr) * 2016-07-16 2018-01-25 Florida State University Research Foundation, Inc. Composés et méthodes de traitement ou de prévention d'infections à favivirus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DRUGS IN JAPAN , ETHICAL DRUGS 2009, 2009, pages 12, 13 *
DRUGS IN JAPAN , ETHICAL DRUGS 2009, 2009, pages 1623 - 1628 *
SANGIAMBUT, S. ET AL.: "Multiple regions in dengue virus capsid protein contribute to nuclear localization during virus infection", J. GEN. VIROL., vol. 89, 2008, pages 1254 - 1264, XP055666415 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022210644A1 (fr) * 2021-04-02 2022-10-06 国立研究開発法人医薬基盤・健康・栄養研究所 Médicament pour le traitement d'une large gamme d'infections virales, y compris le sars-cov -2
CN114848634A (zh) * 2022-05-18 2022-08-05 西安医学院 Sb415286的应用及寨卡病毒抑制剂和药物
CN114848634B (zh) * 2022-05-18 2023-09-15 西安医学院 Sb415286的应用及寨卡病毒抑制剂和药物
CN117110269A (zh) * 2023-10-20 2023-11-24 浙江迪福润丝生物科技有限公司 Jev蛋白酶抑制剂的筛选方法和抑制效果评价方法
CN117110269B (zh) * 2023-10-20 2024-01-26 浙江迪福润丝生物科技有限公司 Jev蛋白酶抑制剂的筛选方法和抑制效果评价方法

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