WO2022181219A1 - Therapeutic drug for novel virus infectious disease - Google Patents

Therapeutic drug for novel virus infectious disease Download PDF

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WO2022181219A1
WO2022181219A1 PCT/JP2022/003631 JP2022003631W WO2022181219A1 WO 2022181219 A1 WO2022181219 A1 WO 2022181219A1 JP 2022003631 W JP2022003631 W JP 2022003631W WO 2022181219 A1 WO2022181219 A1 WO 2022181219A1
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protein
ace2
trpc3
nox2
clomipramine
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PCT/JP2022/003631
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French (fr)
Japanese (ja)
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基宏 西田
百合 加藤
和宏 西山
泰成 諫田
宏 朝倉
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国立大学法人九州大学
国立医薬品食品衛生研究所長が代表する日本国
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    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/27—Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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Definitions

  • the purpose of the present invention is to treat emerging viral infections and the like.
  • the present invention provides pharmaceutical compositions, related screening methods and biomarkers for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. etc.
  • Non-Patent Documents 1 and 2 The spread of the infectious disease (COVID-19) caused by the novel coronavirus (SARS-CoV-2) has become a major problem worldwide (Non-Patent Documents 1 and 2), and the development of effective medicines is urgently needed. (Non-Patent Documents 1-3).
  • SARS-CoV-2 the spike protein on the virus surface is activated by the transmembrane serine protease TMPRSS2, which is one of the proteases (Non-Patent Document 4), and angiotensin converting enzyme 2 (ACE2) on the host cell membrane is activated. binds to , is taken up into cells through endocytosis, and as a result, infects via RNA replication (Non-Patent Document 4).
  • Non-Patent Documents 5 and 6 smoke, cancer, diabetes, obesity, heart disease, hypertension, etc. have been reported as risk factors that cause aggravation of infection.
  • Non-Patent Documents 7-9 vasculitis, thrombosis, pulmonary fibrosis and heart failure have been reported as serious sequelae (Non-Patent Documents 7-9).
  • therapeutic drugs such as remdesivir, an RNA replication inhibitor that targets RNA-dependent RNA polymerase activity, and dexamethasone, an anti-inflammatory drug that targets inflammation, have been approved one after another.
  • Non-Patent Document 10 drugs that inhibit binding have been developed using peptides that mimic the binding site sequences of SARS-CoV-2 and ACE2, but peptide preparations are expensive and difficult to supply in large quantities.
  • Non-Patent Document 11 when targeting the binding of SARS-CoV-2 and ACE2, the emergence of SARS-CoV-2 mutant strains has been reported (Non-Patent Document 11), so there is a risk that the drug efficacy will be lost.
  • NADPH oxidase 2 (Nox2) protein is expressed in the mammalian heart, and Nox2 protein is activated by angiotensin II (AngII), TNF- ⁇ , endothelin-1, mechanical stimulation, etc. (Non-Patent Document 12).
  • Nox2-dependent reactive oxygen species (ROS) production is known to cause heart failure (Non-Patent Document 13).
  • the receptor-gated cation channel TRPC3 transient receptor potential cation channel, subfamily C, member 3 protein is expressed in many tissues, including heart, muscle, and endothelial cells, and transports cations such as Na + and Ca 2+ .
  • Non-Patent Document 14 Reported that ROS production is significantly suppressed by
  • Non-Patent Document 15 ROS are involved in the inflammation and tissue fibrosis seen in the aggravation and/or sequelae of COVID-19. Furthermore, there is a report that the ACE2 expression level is elevated in severe COVID-19 patients (Non-Patent Document 16).
  • RNA replication inhibitors and anti-inflammatory drugs have already been approved as COVID-19 therapeutic agents, but their therapeutic effects are by no means sufficient, and there is an urgent need to develop more effective drugs. .
  • the present invention aims to discover new pharmaceutical compositions for preventing and/or treating emerging viral infections such as COVID-19.
  • the present invention includes the following aspects.
  • ⁇ Pharmaceutical composition for preventing or treating emerging viral infections, etc.> [1] Prevent and/or treat emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome containing substances that inhibit TRPC3-Nox2 complex formation and suppress ACE2 internalization
  • a pharmaceutical composition for [2] The pharmaceutical composition according to [1], wherein the substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization is a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein and suppresses ACE2 internalization. thing.
  • ⁇ Screening method> A method of screening for substances for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, which inhibit TRPC3-Nox2 complex formation and The screening method for selecting a substance that suppresses ACE2 internalization.
  • a method for determining the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome comprising: (a10) measuring the amount of reactive oxygen species (ROS) produced (amount of biomarker to be tested) mediated by complex formation between TRPC3 protein and Nox2 protein in cardiomyocytes of the subject; (b10) comparing the amount of biomarker to be tested and the amount of ROS produced by reference cardiomyocytes (amount of control biomarker), and (c10) when the amount of biomarker to be tested is greater than the amount of control biomarker, A method of determining a subject as having increased severity of emerging viral infections, inflammatory diseases, sequelae and complications associated therewith, and acute respiratory distress syndrome.
  • ROS reactive oxygen species
  • control biomarker amount is the amount of ROS produced by a healthy subject.
  • control biomarker amount is the amount of ROS produced by a healthy subject.
  • amount of ROS produced through complex formation of TRPC3 protein and Nox2 protein in cardiomyocytes is replaced by the amount of oxidative stress markers in blood or urine.
  • amount of oxidative stress marker is the amount of 8-OH-dG, nitrotyrosine, malondialdehyde, and 4-hydroxy-2-nonenal.
  • ROS production via TRPC3 and Nox2 protein complex formation in cardiomyocytes to determine the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome Use as a biomarker.
  • FIG. 1 is a series of figures showing that clomipramine potently inhibits SARS-CoV-2 pseudoinfection.
  • FIG. 1A A schematic diagram showing the principle of measuring ACE2 internalization using BRET in HEK293 cells expressing KRAS-Rluc and human ACE2-Venus.
  • FIG. 1B is a graph showing the rate of inhibition of ACE2 internalization when pretreated for 1 hour with 13 candidate compounds (1 ⁇ M) that inhibit ACE2 internalization among compounds that inhibit TRPC3-Nox2 complex formation.
  • FIG. 1C A graph showing the inhibition rate of ACE2 internalization when 11 compounds (1 ⁇ M) reported to have therapeutic effects against COVID-19 were pretreated for 1 hour.
  • FIG. 1A A schematic diagram showing the principle of measuring ACE2 internalization using BRET in HEK293 cells expressing KRAS-Rluc and human ACE2-Venus.
  • FIG. 1B is a graph showing the rate of inhibition of ACE2 internalization when pretreated for 1 hour with 13 candidate compounds
  • FIG. 1D A graph showing the rate of inhibition of ACE2 internalization when seven tricyclic antidepressants (1 ⁇ M) including clomipramine were pretreated for 1 hour.
  • FIG. 1E A graph examining the effect of clomipramine on ACE2 enzymatic activity. DX600 is an ACE2 inhibitor and was used as a positive control.
  • FIG. 1F A photograph showing uptake of fluorescence-labeled purified S protein into human iPS cell-derived cardiomyocytes. The right figure shows that uptake of fluorescence-labeled purified S protein that has been taken up into cells is inhibited by treatment with clomipramine. The middle panel represents fluorescently labeled purified S protein taken up by cells. Dotted line represents cell membrane, N represents nucleus, scale bar is 10 ⁇ m.
  • FIG. 2 shows the inhibitory effect of clomipramine on SARS-CoV-2 infection and proliferation of VeroE6 cells stably expressing TMPRSS2 or human iPS cell-derived cardiomyocytes.
  • FIG. 2A is a graph showing the effect of treatment time of clomipramine or ibudilast on VeroE6 cells stably expressing TMPRSS2 and the effect of suppressing infection. Pre; 1 hour pretreatment for infection, Co-admin; simultaneous treatment for infection, Post; 1 hour post-infection treatment. Data are presented as mean ⁇ s.e.m. *P ⁇ 0.05.
  • FIG. 2B A graph showing the results of clomipramine or ibudilast treatment and virus infection titer measured by plaque assay.
  • FIG. 2C Graph showing intracellular viral copy number 6 hours after SARS-CoV-2 infection with clomipramine or ibudilast.
  • Figure 2D Graph showing intracellular viral copy number 18 hours after SARS-CoV-2 infection with clomipramine or ibudilast (D).
  • FIG. 2E Graph showing concentration-dependent changes in viral copy number for clomipramine (filled circles) or ibudilast (filled squares).
  • FIG. 2F A graph showing the results of examination of SARS-CoV-2 infection conditions in human iPS cell-derived cardiomyocytes.
  • FIG. 2G A graph showing the effect of clomipramine or ibudilast on the infection-suppressing effect of human iPS cell-derived cardiomyocytes 1 hour before (Pre) and 1 hour after infection with SARS-CoV-2 (Post). Data are presented as mean ⁇ s.e.m. **P ⁇ 0.01.
  • FIG. 2H A photograph showing an antibody-stained image of SARS-CoV-2 taken up by human iPS cell-derived cardiomyocytes.
  • FIG. 3 is a graph showing the inhibitory effects of clomipramine and remdesivir on SARS-CoV-2 infection and growth in VeroE6 cells stably expressing TMPRSS2.
  • Remdesivir monotherapy (filled circles) treatment 1 hour after infection, combined use of clomipramine (10 ⁇ M) and remdesivir (black squares), dashed line indicates infection-suppressing effect of 10 ⁇ M clomipramine.
  • Data are presented as mean ⁇ s.e.m.
  • the vertical axis is a relative value with the intracellular SARS-CoV-2 viral mRNA copy number in the solvent-treated group taken as 100%.
  • FIG. 4 shows that clomipramine suppresses SARS-CoV-2 variant infection.
  • Figure 4A Concentration-dependent ACE2 internalization by S protein mutants: N501Y, D614G, N501Y/D614G, and K417N/E484K/N501Y/D614G.
  • FIG. 4B A graph showing the inhibition rate of ACE2 internalization derived from S protein mutants in clomipramine (1 ⁇ M).
  • FIG. 4C A graph showing clomipramine concentration-dependent inhibitory effects on SARS-CoV-2 (JPN/TY/WT-521, hCov-19/Japan/QHN002/2020) infection on VeroE6 cells stably expressing TMPRSS2.
  • FIG. 5 shows that clomipramine suppresses mitochondrial metabolic disturbance by S protein.
  • FIG. 5A A graph showing oxygen consumption in human iPS cell-derived cardiomyocytes 24 hours after addition of S protein.
  • FIG. 5B A graph showing the results of quantitative analysis of basal respiration, ATP production, proton leak, and maximum respiration calculated from the trace in FIG. 5A. .
  • FIG. 6 shows that clomipramine suppresses the inflammatory response by S protein.
  • FIG. 6A A graph showing the expression level of TNF- ⁇ mRNA in primary cultured rat cardiomyocytes 3 hours after addition of S protein.
  • FIG. 6B A graph showing the expression level of IL-1 ⁇ mRNA in primary cultured rat cardiomyocytes 3 hours after addition of S protein.
  • FIG. 6C A graph showing the expression level of IL-6 mRNA in primary cultured rat cardiomyocytes 3 hours after addition of S protein.
  • FIG. 6D A graph showing the amount of TNF- ⁇ released from RAW264.7 cells upon exposure to S protein.
  • FIG. 7 shows that clomipramine inhibits endocytic pathways involving drebrin.
  • FIG. 7A shows an electropherogram (silver staining) of HEK293T cell lysate stably expressing ACE2-EGFP treated with S protein for 1.5 hours.
  • FIG. 7B-D Graphs showing mRNA expression levels of ⁇ -SMA (B), drebrin (C), and ACE2 (D) in primary cultured rat cardiac fibroblasts treated with TGF- ⁇ (20 ng/mL).
  • FIG. 7E Graph showing the fluorescence-labeled S protein internalization rate in primary rat cardiac fibroblasts treated with TGF- ⁇ (20 ng/mL).
  • FIG. 8 is a graph showing the improvement effect of clomipramine on functional deterioration of human iPS cell-derived cardiomyocytes due to SARS-CoV-2 infection.
  • CL clomipramine
  • BPM heart rate
  • Fig. 8A Contraction velocities in control (left), SARS-CoV-2 infection (middle) and SARS-CoV-2 infection + clomipramine addition (right) (first high peak) and relaxation rate (late low peak).
  • Figure 8B BPM in control, SARS-CoV-2-infected and SARS-CoV-2-infected plus clomipramine; heart rate (left), contraction rate (middle) and relaxation rate (right) graphs.
  • a substance that inhibits complex formation between TRPC3 protein and Nox2 protein (TRPC3-Nox2 complex formation) and suppresses ACE2 internalization, specifically TRPC3 protein and/or Nox2 protein Emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiration, containing substances that inhibit increased expression, preferably clomipramine, ibudilast, trifluoperazine, nifedipine, or tolnaftate, more preferably clomipramine
  • Pharmaceutical compositions for preventing and/or treating distress syndrome are provided.
  • the substance contained in the pharmaceutical composition of the present invention is a substance that has a viral uptake inhibitory effect in terms of suppressing ACE2 internalization, and the pharmaceutical composition of the present invention can also be referred to as a viral uptake inhibitor.
  • substances that suppress increased expression of TRPC3 protein and Nox2 protein include, for example, siRNA, shRNA, miRNA, ribozyme, antisense, etc. corresponding to TRPC3 gene and Nox2 gene.
  • siRNAs small interfering RNAs
  • siRNAs introduced into cells bind to the RNA-induced silencing complex (RISC). This complex binds and cleaves mRNAs with sequences complementary to the siRNA. This suppresses gene expression in a sequence-specific manner.
  • RISC RNA-induced silencing complex
  • shRNA short hairpin RNA
  • shRNA short hairpin RNA is a hairpin-shaped RNA sequence used for gene silencing by RNA interference.
  • shRNA may be introduced into cells by a vector and expressed with a U6 promoter or H1 promoter, or an oligonucleotide having a shRNA sequence may be synthesized by an automatic DNA/RNA synthesizer and self-annealed by the same method as siRNA.
  • miRNA microRNA, microRNA
  • miRNA is a functional nucleic acid that is encoded on the genome and finally becomes a minute RNA of about 20 bases through a multistep production process.
  • miRNAs are classified as functional ncRNAs (non-coding RNAs: a generic term for RNAs that are not translated into proteins), and play an important role in life phenomena by regulating the expression of other genes.
  • TRPC3 or Nox2 can be inhibited by administering miRNA having a specific nucleotide sequence to a living body.
  • the ribozyme of the present invention means an RNA molecule that specifically cleaves other single-stranded RNA molecules by a mechanism similar to DNA restriction endonuclease.
  • a ribozyme that recognizes and cleaves a specific base sequence in a single RNA strand can be produced by appropriately modifying the nucleic acid sequence of RNA by a known technique (Science, 239, p.1412-1416, 1988). .
  • An antisense nucleic acid is a nucleic acid complementary to a target sequence.
  • the antisense nucleic acid inhibits transcription initiation by triplex formation, inhibits transcription by hybridization with a site where an open loop structure is locally formed by RNA polymerase, inhibits transcription by hybridization with RNA that is being synthesized, Suppression of splicing by hybridization at junctions between introns and exons, suppression of splicing by hybridization with spliceosome formation sites, suppression of translocation from the nucleus to the cytoplasm by hybridization with mRNA, capping sites and poly(A) addition sites Splicing suppression by hybridization with, translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, translational region of mRNA and polysome binding site by hybridization with
  • the expression of the target gene can be suppressed by inhibiting elongation of the peptide chain, suppressing gene expression by hybridization with the
  • the "substance that inhibits ACE2 internalization” in the “substance that inhibits TRPC3-Nox2 complex formation and inhibits ACE2 internalization” is serine protease (TMPRSS2) or cathepsin enzyme-dependent endocytosis. is a substance that inhibits clathrin-dependent endocytosis. In the case of enzyme-dependent endocytosis, multiple intracellular backup functions (redundancy) exist, so it may be difficult to suppress all ACE2 internalization with substances that target the relevant enzymes. Furthermore, cathepsin L and TMPRSS2 enzymes, which are involved in ACE2 internalization in coronaviruses such as SARS-CoV-2, are expressed at different levels in different tissues and cell types.
  • clathrin-dependent endocytosis is a ubiquitous mechanism in all cell types and may exert an inhibitory effect on ACE2 internalization in many cell types. Therefore, a substance that inhibits clathrin-dependent endocytosis can be expected to have a therapeutic effect through a mechanism of action that is different from that of the past, and thus highly original pharmaceuticals can be provided.
  • the "substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization” is an existing drug of 1271 types of Prestwick's approved drug library, as detailed in the following examples.
  • the top 13 compounds (non-patent Reference 13) was identified, and a compound having an inhibitory effect on ACE2 internalization was searched using the bioluminescence resonance energy transfer (BRET) method (Fig. 1A).
  • BRET bioluminescence resonance energy transfer
  • FIG. 1A is a schematic diagram showing the principle of measuring ACE2 internalization using BRET in HEK293 cells expressing KRAS-Rluc and human ACE2-Venus.
  • BRET is used to quantitatively measure the protein-protein interaction between KRAS-Rluc protein, which is a cell membrane-associated protein KRAS fused to the fluorescent enzyme luciferase (Rluc), and ACE2-Venus, which is a human ACE2 protein fused to the fluorescent protein Venus.
  • Fig. 1A Spike protein (S protein) exposure internalizes ACE2, increasing the distance between the proteins and a concomitant decrease in BRET intensity.
  • the top 13 compounds with TRPC3-Nox2 complex formation inhibitory effect are as follows: Clomipramine, Ibudilast, Voriconazole, Lynestrenol, Pyrilamine, Clopidogrel, Trifluoperazine, Fipexide, Androsterone, Nifedipine, Tolnaftate, Mifepristone, Ticlopidine.
  • Clomipramine is a tricyclic antidepressant drug developed by Geigy (now Novartis Pharma K.K.) in Switzerland in the 1960s. The pharmacological action is to inhibit the uptake of serotonin and noradrenaline in the brain into nerve terminals. Indications in Japan are depression/depressive state, enuresis, and cataplexy associated with narcolepsy.
  • hydrochloride salt It is typically the hydrochloride salt and has the formula: and its chemical name (nomenclature) is 3-(3-chloro-10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropylamine monohydrochloride Salt (IUPAC).
  • Target diseases to be prevented and/or treated by the pharmaceutical composition of the present invention are typically emerging viral infections, inflammatory diseases associated therewith, sequelae and complications, and acute respiratory distress syndrome.
  • Emerging viral infections are newly recognized viral infections that have not been previously known and that pose a local or international public health concern, including, for example, the 2003 Severe Acute Respiratory Syndrome (SARS), which emerged in February 2012 and spread rapidly around the world, mainly in Asia, became a global threat.
  • SARS Severe Acute Respiratory Syndrome
  • MERS Middle East Respiratory Syndrome
  • COVID-19 novel coronavirus infection
  • Inflammatory diseases associated with emerging viral infections include cardiovascular inflammation and pneumonia, and sequelae associated with emerging viral infections include vasculitis, thrombosis, pulmonary fibrosis and heart failure, as well as headache, anxiety and depression. Symptoms include loss of taste and smell. Complications associated with emerging viral infections include respiratory failure; cardiovascular, including acute arrhythmias, acute cardiac injury, shock, cardiac arrest, and postrecovery myocarditis; Inflammatory complications, such as persistent fever, Guillain-Barré syndrome, Kawasaki disease-like symptoms, etc.; Secondary infections; Metabolic diseases, such as dyslipidemia and diabetic complications; Examples include convulsive seizures, disturbance of consciousness, and cerebrovascular disorders.
  • ARDS Acute respiratory distress syndrome
  • Etiologies are broadly classified into those that directly or indirectly damage the lungs. Examples of direct lung damage include pneumonia, aspiration (swallowing food or foreign objects into the trachea), pulmonary contusion, and drowning. trauma, massive blood transfusion, etc.
  • components in the blood may migrate into the alveolar space and cause pulmonary edema. These conditions are also included in ARDS.
  • prevention means preventing the onset of emerging viral infections, inflammatory diseases, sequelae and complications associated therewith, and acute respiratory distress syndrome.
  • treatment means (1) delaying the onset of emerging viral infections, inflammatory diseases, sequelae and complications associated therewith, and acute respiratory distress syndrome; (2) emerging viral infections, and slows or halts the progression, exacerbation or exacerbation of associated inflammatory diseases, sequelae and complications, and symptoms of acute respiratory distress syndrome; (3) emerging viral infections, their associated inflammatory diseases, sequelae and complications; and provide amelioration of symptoms of acute respiratory distress syndrome; or (4) a method or process intended to cure emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome.
  • Treatment may be administered prior to the onset of the disease or condition as a prophylactic measure, or alternatively, treatment may be administered after the onset of the disease.
  • a pharmaceutical composition usually means an agent for prevention or treatment of disease, or examination/diagnosis.
  • the substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization in the pharmaceutical composition of the present invention is preferably selected from clomipramine, ibudilast, trifluoperazine, nifedipine, tolnaftate, especially Clomipramine is preferred.
  • clomipramine suppresses RNA proliferation more strongly than the effect of remdesivir or clomipramine alone when co-added with remdesivir, an RNA-dependent RNA polymerase inhibitor. This result indicates that clomipramine and remdesivir combination therapy may be an effective treatment for COVID-19.
  • Clomipramine shows an infection-suppressing effect not only against the SARS-CoV-2 wild type, but also against the mutant strains N501Y, D614G, N501Y/D614G, and K417N/E484K/N501Y/D614G.
  • Clomipramine is known to inhibit clathrin-dependent endocytosis, but other compounds, including tricyclic antidepressants and endocytosis inhibitors, do not inhibit ACE2 internalization, thus clomipramine Speculating that there may be other targets, experiments were performed suggesting that ACE2-mediated S protein internalization is involved in drebrin-mediated endocytosis, which clomipramine inhibits. From this, we can expect the possibility that drebrin-dependent internalization can be suppressed even if the virus entry route is mediated by another receptor that does not depend on ACE2.
  • the pharmaceutical composition of the present invention can be formulated by methods known to those skilled in the art.
  • the pharmaceutical composition of the present invention can be administered both parenterally and orally.
  • parenteral administration for example, the composition can be injectable, nasal, pulmonary, or percutaneous administration.
  • it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
  • oral administration compositions such as tablets, capsules, pills, granules, powders, and syrups can be used.
  • Commercially available clomipramine is sold as tablets for oral administration.
  • the administration method can be appropriately selected according to the patient's age and symptoms.
  • the dose of a pharmaceutical composition containing a polypeptide can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, doses of 0.001 to 100000 mg per patient can be used, although the present invention is not necessarily limited to these figures.
  • the dosage and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can consider these conditions and set an appropriate dosage and administration method.
  • Another aspect of the present invention is a method for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, comprising complex formation (TRPC3-Nox2 complex formation) and suppresses ACE2 internalization, specifically a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein, preferably clomipramine, ibudilast, triflu
  • TRPC3-Nox2 complex formation complex formation
  • ACE2 internalization specifically a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein, preferably clomipramine, ibudilast, triflu
  • TRPC3-Nox2 complex formation complex formation
  • ACE2 internalization specifically a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein, preferably clomipramine, ibudilast, triflu
  • a method comprising administering an effective amount of operazine, nifedipine or tolnaftate, more preferably
  • the present invention provides TRPC3 and Nox2 proteins of the present invention for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome.
  • a substance that inhibits complex formation with (TRPC3-Nox2 complex formation) and suppresses ACE2 internalization specifically a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein, preferably clomipramine, ibudilast, It relates to trifluoperazine, nifedipine or tolnaftate, more preferably clomipramine.
  • the present invention provides a method for manufacturing a medicament for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome.
  • a substance that inhibits complex formation between TRPC3 protein and Nox2 protein (TRPC3-Nox2 complex formation) and suppresses ACE2 internalization, specifically a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein preferably relates to the use of clomipramine, ibudilast, trifluoperazine, nifedipine or tolnaftate, more preferably clomipramine.
  • the present invention provides methods of screening for substances that inhibit TRPC3-Nox2 complex formation and suppress ACE2 internalization.
  • One aspect of the screening method of the present invention is a method of screening for a substance that suppresses increased expression of TRPC3 protein and Nox2 protein and suppresses ACE2 internalization.
  • the step of measuring the expression level of TRPC3 or Nox2 in cells, and comparing the expression level with the expression level in the absence of the test substance determining that the test substance is a candidate substance that suppresses the increase in the expression of TRPC3 protein and Nox2 protein if the test substance is reduced in ACE2 internalization.
  • a screening method for a candidate substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization comprising the step of confirming
  • substances that inhibit TRPC3-Nox2 complex formation and suppress ACE2 internalization are effective in preventing or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. Therefore, the screening method of the present invention represents a screening method for candidate substances effective in the prevention or treatment of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome.
  • Cells are not limited as long as they are cultured cells.
  • they when using mouse or rat cultured myocardial cells, they are preferably isolated from the heart and then primary cultured.
  • macrophage cell line RAW264.7 was used, and TRPC3-Nox2 complex formation-dependent ROS generation can be observed.
  • a screening method including a step of confirming that the candidate substance does not affect the expression levels of TRPC3 or Nox2 in normal cells is more preferable.
  • test substance is a substance that can be evaluated as to whether it is a candidate substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization, and is not particularly limited. Examples include compounds, proteins, peptides, nucleic acids, lipids, carbohydrates, glycolipids, glycoproteins, metals and the like. The administration method of the test substance is also not particularly limited.
  • Measurements that reflect the expression level of TRPC3 or Nox2 are not particularly limited. For example, it can be the measured value of the amount of mRNA expressed from the TRPC3 or Nox2 gene (copy number or read number, etc.), the measured value of protein, the chemiluminescence intensity by reporter assay, and the like.
  • mRNA measurements can be obtained using known methods such as microarray, quantitative RT-PCR, or RNA-Seq.
  • Protein values can be measured by protein chip, ELISA method, Western blotting method, etc.
  • a substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization is an effective prophylactic or therapeutic agent for emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. becomes.
  • Another aspect of the present invention is a method for determining the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, comprising: (a10) measuring the amount of reactive oxygen species (ROS) produced (amount of biomarker to be tested) mediated by complex formation between TRPC3 protein and Nox2 protein in cardiomyocytes of the subject; (b10) comparing the amount of biomarker to be tested and the amount of ROS produced by reference cardiomyocytes (amount of control biomarker), and (c10) when the amount of biomarker to be tested is greater than the amount of control biomarker, Methods are provided for determining a subject to have high severity of emerging viral infections, inflammatory diseases, sequelae and complications associated therewith, and acute respiratory distress syndrome.
  • ROS reactive oxygen species
  • the amount of ROS produced through the formation of the TRPC3 protein and Nox2 protein complex in the reference cardiomyocytes is the same as the TRPC3 protein and Nox2 protein complex formation in the same subject's cardiomyocytes measured before step (a10). It may be the amount of ROS produced via complex formation between TRPC3 protein and Nox2 protein in cardiomyocytes of healthy subjects, and may be the average of the amount of ROS produced via complex formation.
  • the amount of ROS produced through complex formation between TRPC3 protein and Nox2 protein can be measured as described in Non-Patent Document 13. For example, by treating with dihydroethidium (DHE), the amount of superoxide produced from Nox2 can be measured indirectly by fluorescence methods.
  • DHE dihydroethidium
  • the present invention provides, as a further aspect, TRPC3 in cardiomyocytes capable of determining the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome.
  • Use as a biomarker of ROS production via protein-Nox2 protein complex formation is provided.
  • the present invention provides complex formation of TRPC3 and Nox2 proteins in cardiomyocytes that can determine the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. provide a biomarker that is the amount of ROS produced through
  • TRPC3 and Nox2 proteins have been found that ROS production via complex formation of TRPC3 and Nox2 proteins in cardiomyocytes is associated with emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. I found out what I was doing. The formation of a complex between TRPC3 and Nox2 proteins inhibits Nox2 protein degradation, resulting in increased ROS generation on the myocardial cell membrane. As a result, oxidative stress is induced by excessive ROS production, and various cells and tissues are thought to be damaged.
  • Cardiomyocytes of a subject are collected by biopsy, and the amount of ROS produced through complex formation between TRPC3 protein and Nox2 protein can be measured.
  • Cardiomyocytes are preferably human cardiomyocytes.
  • ROS production mediated by TRPC3 protein and Nox2 protein complex formation in cardiomyocytes can also be indirectly evaluated by measuring the amount of oxidative stress markers (such as 8-OH-dG) in blood or urine. This embodiment also forms part of the invention.
  • the present invention provides a method for determining the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, comprising: (a20) measuring the amount of oxidative stress markers (test biomarker amount) in the blood or urine of the subject; (b20) comparing the test biomarker amount with the reference oxidative stress marker amount in blood or urine (control biomarker amount), and (c20) the test biomarker amount is higher than the control biomarker amount
  • test biomarker amount the amount of oxidative stress markers in the blood or urine
  • control biomarker amount control biomarker amount
  • Methods are provided for determining a subject, in many cases, to have high severity of emerging viral infections, inflammatory diseases associated therewith, sequelae and complications, and acute respiratory distress syndrome.
  • Oxidative stress markers in blood or urine herein include, but are not limited to, 8-OH-dG, nitrotyrosine, malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE). These markers can be measured, for example, by the method described in Pediatric Infectious Immunity, Vol. 24, No. 2 (jspid.jp).
  • HEK293 cells ATCC
  • VeroE6 cells stably expressing TMPRSS2 JCRB 1819: JCRB Cell Bank
  • DMEM Dulbecco's modified Eagle's medium
  • Human iPS cell-derived cardiomyocytes were purchased from FUJIFILM Cellular Dynamics, Inc. and cultured according to the manufacturer's instructions.
  • Lipofectamine 3000 Thermo Fisher Scientific
  • Viafect transfection reagent was used for plasmid DNA introduction into HEK293 cells.
  • Reference example 2 Preparation of Purified S Protein and Fluorescent Labeled Purified S Protein
  • S protein spike protein
  • SARS-CoV-2 The spike protein (S protein) on the surface of SARS-CoV-2 was purified using the baculovirus-silkworm system (Non-Patent Document 15). Purified S protein was fluorescently labeled using HiLyte Fluor® 555 labeling kit-NH2 (Dojindo). The labeling method followed the instructions attached to the kit.
  • Example 1 Identification of clomipramine that strongly inhibits SARS-CoV-2 pseudoinfection Top 13 compounds with TRPC3-Nox2 complex formation inhibitory effect among 1271 existing drugs (Prestwick Chemical) in Prestwick's approved drug library Among them (Non-Patent Document 13), compounds with ACE2 internalization inhibitory effects were searched using the BRET method (Figs. 1A and 1B).
  • HEK293 cells were seeded in a 12-well plate (3.0 ⁇ 10 5 cells/well) and cultured for 24 hours at 37° C., 5% CO 2 .
  • KRAS-Rluc and an empty vector (pC-DNA3.1) were transfected as a negative control, and KRAS-Rluc and human ACE2-Venus were transfected as a positive control, and cultured for 24 hours at 37°C and 5% CO 2 .
  • Cells were then harvested, seeded (5.0 ⁇ 10 4 cells/well) on collagen-coated 96-well plates, and cultured for 24 hours at 37° C., 5% CO 2 .
  • the cell supernatant was replaced with a live cell imaging solution (Thermo Fisher Scientific), and 13 candidate compounds (clomipramine, ibudilast, voriconazole, linestrenol, pyrilamine, clopidogrel, trifluoperazine, fipexide, androsterone, Nifedipine, tolnaftate, mifepristone, ticlopidine) and 50 nM purified S protein were added simultaneously and incubated for 3 hours at 37° C., 5% CO 2 .
  • 13 candidate compounds clomipramine, ibudilast, voriconazole, linestrenol, pyrilamine, clopidogrel, trifluoperazine, fipexide, androsterone, Nifedipine, tolnaftate, mifepristone, ticlopidine
  • 50 nM purified S protein were added simultaneously and incubated for 3 hours at 37° C., 5% CO 2 .
  • Luminescence and fluorescence were measured in a plate reader (Nivo, Perkinelmer) and the BRET ratio was calculated by dividing the fluorescence signal (531/22 filter) by the luminescence signal (485/20 filter).
  • the internalization inhibition rate was determined by setting the internalization in the control (DMSO) as 100%.
  • DMSO control
  • clomipramine, ibudilast, trifluoperazine, nifedipine, and tolnaftate were found to have relatively high internalization inhibition rates (Fig. 1B).
  • clomipramine had an internalization inhibition rate of 40%, showing the most potent inhibitory effect (Fig. 1B).
  • 11 compounds Clomipramine, Cepharanthine, Ciclesonide, Nafamostat, Clolpromazine, Nelfinavir, Diclofenac, Ibuprofen, Indometacin, Loxoprofen (Loxoprofen), Nintedanib, Pilfenidon.
  • Clomipramine showed the most inhibitory effect (Fig. 1C).
  • Clomipramine is one of the tricyclic antidepressants, so other tricyclic antidepressants: Amitriptyline, Desipramine, Doxepin, Imipramine, Mirtazapine and Clozapine (Clozapine) were compared, and clomipramine was the most potent (Fig. 1D).
  • ACE2 enzyme activity was measured using an ACE2 inhibitor screening assay kit (BPS Bioscience #79923) according to the instructions attached to the kit. Fluorescence was measured with a plate reader (Nivo, Perkinelmer). 1 ⁇ M DX600, an ACE2 inhibitor, was added as a positive control. As a result, no inhibition of ACE2 enzyme activity was observed in the presence of clomipramine (Fig. 1E). From these results, 1) clomipramine does not target the enzyme activity center of ACE2, and 2) clomipramine inhibits ACE2 internalization due to viral infection, thereby contributing to the maintenance of ACE2 enzyme activity in the myocardial cell membrane. , may also act to suppress the action of angiotensin II and enhance the angiotensin 1-7/Mas receptor signal.
  • clomipramine was visualized to suppress intracellular uptake of S protein.
  • Human iPS cell-derived cardiomyocytes were seeded onto Matrigel-coated wells (1.7 ⁇ 10 4 cells/well) and cultured at 37° C., 5% CO 2 for at least 4 days.
  • 100 nM clomipramine was added thereto per well and cultured for 1 hour at 37° C. and 5% CO 2 .
  • 50 nM purified S protein was then added and incubated for 3 hours at 37° C., 5% CO 2 .
  • Example 2 Inhibitory effect of clomipramine on SARS-CoV-2 infection and growth of VeroE6 cells stably expressing TMPRSS2 or human iPS-CMs. The inhibitory effect was confirmed.
  • SARS-CoV-2 was obtained from the National Institute of Infectious Diseases, and experiments were conducted at the National Institute of Health Sciences.
  • VeroE6 cells stably expressing TMPRSS2 prepared in Reference Example 1 were seeded in a 96-well plate (1.5 ⁇ 10 4 cells/well), cultured for 24 hours, and then infected with SARS-CoV-2. 10 ⁇ M clomipramine and ibudilast compounds were added 1 hour before, at the same time and 1 hour after infection.
  • SARS-CoV-2 was infected at a multiplicity of infection (MOI) of 0.1. After infection, intracellular RNA was collected (CellAmp Direct RNA Prep Kit, TAKARA) and RT-qPCR was performed (TaqMan Fast Virus 1-Step Master Mix, Thermo Fisher Scientific).
  • MOI multiplicity of infection
  • Human iPS cell-derived cardiomyocytes were seeded (3 ⁇ 10 4 cells/well) on fibronectin-coated 96-well plates and cultured in the presence of serum for at least several days. After replacement with serum-free medium, the cells were infected with SARS-CoV-2 at an MOI of 2.5 and cultured at 37°C, 5% CO 2 for 48 hours. After infection, intracellular RNA was collected (CellAmp Direct RNA Prep Kit, TAKARA) and RT-qPCR was performed (TaqMan Fast Virus 1-Step Master Mix, Thermo Fisher Scientific).
  • a plaque assay was performed.
  • the supernatant of VeroE6 cells stably expressing TMPRSS2 was diluted with DMEM medium supplemented with 2% FBS and 1% penicillin/streptomycin, added to a separate tissue culture plate seeded with VeroE6 cells stably expressing TMPRSS2, and infected for 1 hour.
  • a DMEM medium containing 1% methylcellulose and 2% FBS was added and cultured at 37°C for 3 days. After the cells were fixed with formalin (Fujifilm Wako Pure Chemical Industries, Ltd.), they were stained with methylene blue, and the number of plaques formed was counted.
  • clomipramine (10 ⁇ M)-treated cells showed a marked decrease in the number of plaques compared to the control and ibudilast (10 ⁇ M)-treated groups (Fig. 2B).
  • Human iPS cell-derived cardiomyocytes were seeded (3 ⁇ 10 4 cells/well) on fibronectin-coated 96-well plates and cultured in the presence of serum for at least several days. After replacement with serum-free medium, the cells were infected with SARS-CoV-2 at an MOI of 2.5 and cultured at 37°C, 5% CO 2 for 48 hours. Viruses taken up into cells by antibodies that recognize SARS-CoV-2 S protein (anti-SARS spike glycoprotein, ab273433, abcam) and N protein (anti-SARS-CoV-2 nucleocapsid antibody, GTX135357, GeneTex) was detected with a confocal microscope. As a result, SARS-CoV-2 uptake into human iPS cell-derived cardiomyocytes was actually confirmed as a result of staining using a SARS-CoV-2 antibody (Fig. 2H).
  • Example 3 Combined effect of clomipramine and remdesivir on SARS-CoV-2 infection The combined effect of clomipramine and remdesivir on SARS-CoV-2 infection was investigated.
  • SARS-CoV-2 infection of VeroE6 cells stably expressing TMPRSS2 showed an 84% growth inhibitory effect with clomipramine pre-administration for 1 hour (Fig. 2E). is 100%, the drug administration reduces the RNA copy number to 16%, expressing an inhibitory effect of 84%).
  • No inhibitory effect was observed at low concentrations (1-3 ⁇ M) (Fig. 3, remdesivir single agent (black circles)).
  • clomipramine may synergistically enhance the antiviral activity of remdesivir. This means that clomipramine combined with remdesivir suppressed SARS-CoV-2 infection more strongly than clomipramine alone.
  • Example 4 Suppressive effect of clomipramine on SARS-CoV-2 mutant infection It has been reported that SARS-CoV-2 mutant infection is mediated by endocytosis and ACE2, similar to wild-type infection. Therefore, clomipramine, which suppresses ACE2-dependent virus internalization (infection), is thought to have a suppressive effect on mutant strain infection as well. Whether clomipramine is effective using S protein variants N501Y (from ⁇ strain), D614G (from ⁇ strain), N501Y/D614G (from ⁇ strain), and K417N/E484K/N501Y/D614G (from ⁇ strain) verified. At present, the above mutation is also found in the Omicron strain currently in fashion. Based on the S protein prepared according to Reference Example 2, an S protein mutant was created.
  • HEK293 cells were seeded in a 12-well plate (3.0 ⁇ 10 5 cells/well) and cultured for 24 hours at 37° C. and 5% CO 2 .
  • KRAS-Rluc and an empty vector (pC-DNA3.1) were transfected as a negative control, and KRAS-Rluc and human ACE2-Venus were transfected as a positive control, and cultured for 24 hours at 37°C and 5% CO 2 .
  • Cells were then harvested, seeded (5.0 ⁇ 10 4 cells/well) on collagen-coated 96-well plates, and cultured for 24 hours at 37° C., 5% CO 2 .
  • the cell supernatant was replaced with a live cell imaging solution (Thermo Fisher Scientific), purified S protein variants were added at various concentrations, and cultured for 3 hours at 37°C and 5% CO2.
  • 5 ⁇ M coelenterazine h (Coelemterazine h: wako) was added, and measurement was performed 10 minutes later.
  • Luminescence and fluorescence were measured in a plate reader (Nivo, Perkinelmer) and the BRET ratio was calculated by dividing the fluorescence signal (531/22 filter) by the luminescence signal (485/20 filter).
  • the internalization inhibition rate was determined by setting the internalization in the control (DMSO) as 100%. The results obtained are shown in FIG. 4A.
  • HEK293 cells were seeded in a 12-well plate (3.0 ⁇ 10 5 cells/well) and cultured for 24 hours at 37° C. and 5% CO 2 .
  • KRAS-Rluc and an empty vector (pC-DNA3.1) were transfected as a negative control, and KRAS-Rluc and human ACE2-Venus were transfected as a positive control, and cultured for 24 hours at 37°C and 5% CO 2 .
  • Cells were then harvested, seeded (5.0 ⁇ 10 4 cells/well) on collagen-coated 96-well plates, and cultured for 24 hours at 37° C., 5% CO 2 .
  • VeroE6 cells stably expressing TMPRSS2 were seeded in a 96-well plate (1.5 ⁇ 10 4 cells/well) and cultured for 24 hours. 2020). Each concentration of clomipramine was added 1 hour before infection. Viruses were infected at a multiplicity of infection (MOI) of 0.1. After infection, intracellular RNA was collected (CellAmp Direct RNA Prep Kit, TAKARA) and RT-qPCR was performed (TaqMan Fast Virus 1-Step Master Mix, Thermo Fisher Scientific). The results obtained are shown in FIG. 4C.
  • MOI multiplicity of infection
  • Example 5 Suppressive effect of clomipramine on mitochondrial metabolic disorders caused by S protein Heart failure has been reported as a sequela of SARS-CoV-2 infection. Therefore, we exposed the S protein and measured the oxygen consumption rate (OCR), one of the indicators of mitochondrial metabolic function. Specifically, oxygen consumption rate (OCR) was assessed using an XFp extracellular flux analyzer (Seahorse Bioscience) according to the accompanying instructions.
  • OCR oxygen consumption rate
  • Human iPS cell-derived cardiomyocytes (FUJIFILM Cellular Dynamics, Inc.) were plated at 10,000 cells/well, clomipramine (100 nM) was added, after 1 hour, S protein (50 nM) was added and cultured for 24 hours. did.
  • Cells were cultured in XF medium supplemented with 25 mM d-glucose, 1 mM pyruvate and 2 mM glutamine for 1 hour before analysis. After measuring basal respiration, the final concentration was 10 ⁇ M oligomycin (inhibitor of mitochondrial ATP synthase), 2 ⁇ M carbonyl cyanide p-[trifluoromethoxy]-ohenylhydrazone (FCCP) (uncoupler), 10 ⁇ M rotenone (electron transport OCR was measured after injection of 10 ⁇ M antimycin A (electron transport chain complex inhibitor) and 10 ⁇ M antimycin A (electron transport chain complex inhibitor).
  • Example 6 Effect of clomipramine to suppress inflammatory response by S protein It has been reported that cytokine storm is involved in the exacerbation of SARS-CoV-2, and that inflammatory response occurs in epithelial cells of various tissues. It was verified whether clomipramine is effective also in these. The mRNA expression levels of TNF- ⁇ , IL-1 ⁇ , and IL-6, which are inflammatory markers, were used as indices.
  • TNF- ⁇ , IL-1 ⁇ and IL-6 mRNA expression levels were measured by quantitative real-time PCR. Specifically, first, total RNA was extracted from cells using TRI Reagent (Sigma). It was reverse transcribed using ReverTra Ace qPCR RT master remix (Toyobo) to create cDNA. Quantitative real-time PCR was performed using the KAPA SYBR FAST qPCR kit (Roche). TNF- ⁇ release was also measured. Specifically, RAW264.7 cells (3 ⁇ 10 4 cells/well) were seeded in 96-well plates one day before stimulation. After exposing to S protein and culturing for 24 hours, the cell supernatant was collected. Cytokine concentrations were measured using Mouse Duo-Set ELISA kits (R&D Systems).
  • Example 7 Clomipramine's inhibitory effect on endocytosis Clomipramine is known to inhibit clathrin-dependent endocytosis, whereas other compounds, including tricyclic antidepressants and endocytosis inhibitors, inhibit ACE2 internalization. Since it does not inhibit, we speculated that clomipramine may have other targets. Therefore, HEK293T cells expressing ACE2-EGFP exposed to S protein in the presence or absence of clomipramine were subjected to silver staining after electrophoresis. MS) was analyzed. Specifically, the S protein-induced ACE2 internalization assay was performed as follows.
  • HEK293 cells expressing ACE2-EGFP purchased from ATCC (American Type Culture Collection) (1.5 ⁇ 10 4 cells/well) were treated with S protein (50 nM), and after 1.5 hours, the cells were harvested and treated with agarose. Immunoprecipitation was performed using a bead-labeled GFP antibody (Cosmo Bio). Clomipramine (1 ⁇ M) was added 1 hour prior to S protein treatment.
  • ACE2-EGFP-expressing HEK293 cells were treated with S protein, cells were harvested 1.5 hours later, and immunoprecipitation was performed using an agarose bead-labeled GFP antibody (Cosmo Bio). Clomipramine (1 ⁇ M) was added 1 hour prior to S protein treatment. The collected samples were electrophoresed by SDS-PAGE and silver-stained (antegral). LC-MS/MS analysis was performed using the protein extracted from the band where the intensity of the detected band changed, comparing the lane added with S protein only and the lane stimulated with S protein in the presence of clomipramine. .
  • drebrin is known to play a role as a suppressor against cell morphological changes and rotavirus intestinal membrane endocytosis, and it has been reported that its expression increases with myogenic differentiation (fibrosis) of cardiac fibroblasts. ing. Therefore, as a result of TGF- ⁇ stimulation of rat neonatal cardiac fibroblasts to induce fibrosis, ⁇ -SMA (fibrosis marker) mRNA increased approximately 2-fold and drebrin mRNA increased approximately 1.5-fold (Fig. 7B, C). . At this time, no change was observed in the ACE2 mRNA expression level (Fig. 7D).
  • Example 8 Effect of clomipramine to improve contractile dysfunction due to SARS-CoV-2 infection Heart failure has been reported as a sequela of SARS-CoV-2 infection. We examined the effect of clomipramine on
  • the shrinkage evaluation was performed as follows. Human iPS cell-derived cardiomyocytes were seeded at 50,000 cells/well in a 96-well plate, clomipramine (1 ⁇ M) was added for 1 hour, and SARS-CoV-2 (JPN/TY/WT-521) was added for 48 hours. cultured. Myocardial pulsation was photographed under a microscope, and BPM, contraction rate, and relaxation rate were analyzed by MUSCLEMOTION software.

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Abstract

Provided is a technology that, by identifying a cardiomyocyte marker, prevents or treats novel virus infectious diseases, as well as inflammatory diseases, delayed effects and complications, and also acute respiratory distress syndrome associated with the novel virus infectious diseases.　The present invention pertains to a pharmaceutical composition for preventing or treating novel virus infectious diseases, as well as inflammatory diseases, delayed effects and complications, and also acute respiratory distress syndrome associated with the novel virus infectious diseases. The pharmaceutical composition contains a substance that inhibits formation of TRPC3-Nox2 complex and suppressing ACE2 internalization. The present invention also pertains to a screening method and a biomarker related to the pharmaceutical composition.

Description

新興ウイルス感染症治療薬Emerging viral infectious disease drug
　本発明は、新興ウイルス感染症等の治療等を目的とする。詳細には、本発明は、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防および／または処置するための医薬組成物、関連するスクリーニング方法およびバイオマーカー等に関する。 The purpose of the present invention is to treat emerging viral infections and the like. In particular, the present invention provides pharmaceutical compositions, related screening methods and biomarkers for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. etc.
　新型コロナウイルス（SARS-CoV-2）が原因である感染症(COVID-19)の拡大が世界中で大きな問題となっており（非特許文献１、２）、有効な医薬品の開発が急務とされている（非特許文献１－３）。SARS-CoV-2は、プロテアーゼの一つである膜貫通型セリンプロテアーゼTMPRSS2により、ウイルス表面上のスパイクタンパク質が活性化され（非特許文献４）、宿主細胞膜上にあるアンジオテンシン転換酵素2（ACE2）に結合し、エンドサイトーシスを経て、細胞内に取り込まれ、結果、RNA複製を介して感染する（非特許文献４）。 The spread of the infectious disease (COVID-19) caused by the novel coronavirus (SARS-CoV-2) has become a major problem worldwide (Non-Patent Documents 1 and 2), and the development of effective medicines is urgently needed. (Non-Patent Documents 1-3). In SARS-CoV-2, the spike protein on the virus surface is activated by the transmembrane serine protease TMPRSS2, which is one of the proteases (Non-Patent Document 4), and angiotensin converting enzyme 2 (ACE2) on the host cell membrane is activated. binds to , is taken up into cells through endocytosis, and as a result, infects via RNA replication (Non-Patent Document 4).
　COVID-19の深刻な問題は重症化である。感染重症化を引き起こすリスク因子として、喫煙、がん、糖尿病、肥満、心疾患、高血圧などが報告されている（非特許文献５、６）。最近では、重篤な後遺症として血管炎、血栓、肺線維症や心不全が報告されている（非特許文献７－９）。SARS-CoV-2によるウイルス感染・重症化のメカニズムが明らかとなり、RNA依存性RNAポリメラーゼ活性を標的とするRNA複製阻害薬レムデシビルや炎症を標的とする抗炎症薬デキサメサゾンなどの治療薬が次々と承認されている。しかし、その治療効果は十分とは言えず、未だ有効な治療法は確立できていない（非特許文献１０）。最近では、SARS-CoV-2とACE2の結合部位の配列を模したペプチドを用いて結合を阻害する医薬品が開発されているものの、ペプチド製剤は高価であり、大量供給が難しい。また、SARS-CoV-2とACE2との結合を標的とする場合、SARS-CoV-2変異株の出現が報告されているため（非特許文献１１）、薬効が見られなくなる恐れもある。 A serious problem with COVID-19 is its exacerbation. Smoking, cancer, diabetes, obesity, heart disease, hypertension, etc. have been reported as risk factors that cause aggravation of infection (Non-Patent Documents 5 and 6). Recently, vasculitis, thrombosis, pulmonary fibrosis and heart failure have been reported as serious sequelae (Non-Patent Documents 7-9). The mechanism of viral infection and aggravation by SARS-CoV-2 has been clarified, and therapeutic drugs such as remdesivir, an RNA replication inhibitor that targets RNA-dependent RNA polymerase activity, and dexamethasone, an anti-inflammatory drug that targets inflammation, have been approved one after another. It is However, its therapeutic effect cannot be said to be sufficient, and an effective therapeutic method has not yet been established (Non-Patent Document 10). Recently, drugs that inhibit binding have been developed using peptides that mimic the binding site sequences of SARS-CoV-2 and ACE2, but peptide preparations are expensive and difficult to supply in large quantities. In addition, when targeting the binding of SARS-CoV-2 and ACE2, the emergence of SARS-CoV-2 mutant strains has been reported (Non-Patent Document 11), so there is a risk that the drug efficacy will be lost.
　哺乳類の心臓には、NADPHオキシダーゼ2（Nox2）タンパク質が発現しており、Nox2タンパク質は、アンジオテンシンII（AngII）、TNF-α、エンドセリン-1や機械刺激などにより活性化される（非特許文献１２）。Nox2依存的な活性酸素種（ROS）産生は、心不全を引き起こすことが知られている（非特許文献１３）。受容体作動性カチオンチャネルTRPC3（transient receptor potential cation channel, subfamily C, member 3）タンパク質は、心臓、筋肉、内皮細胞など多くの組織に発現しており、Na+、Ca2+などのカチオンを輸送するチャネルである（非特許文献１３）。本発明者らは以前、抗がん剤誘発性心不全モデルマウスにおいて、TRPC3タンパク質とNox2タンパク質の複合体形成による過剰なROS産生が心筋萎縮を誘導し、TRPC3-Nox2複合体形成の薬理学的阻害により、ROS産生が有意に抑制されることを報告した（非特許文献１４）。 NADPH oxidase 2 (Nox2) protein is expressed in the mammalian heart, and Nox2 protein is activated by angiotensin II (AngII), TNF-α, endothelin-1, mechanical stimulation, etc. (Non-Patent Document 12). ). Nox2-dependent reactive oxygen species (ROS) production is known to cause heart failure (Non-Patent Document 13). The receptor-gated cation channel TRPC3 (transient receptor potential cation channel, subfamily C, member 3) protein is expressed in many tissues, including heart, muscle, and endothelial cells, and transports cations such as Na + and Ca 2+ . (Non-Patent Document 13). The present inventors previously reported that in anticancer drug-induced heart failure model mice, excessive ROS production by complex formation of TRPC3 and Nox2 proteins induces myocardial atrophy, and pharmacological inhibition of TRPC3-Nox2 complex formation was reported. Reported that ROS production is significantly suppressed by (Non-Patent Document 14).
　他方、COVID-19の重症化および／または後遺症に見られる炎症や組織の線維化には、ROSが関与していることが示されている（非特許文献１５）。さらに、COVID-19重症患者では、ACE2発現量が上昇しているという報告がある（非特許文献１６）。 On the other hand, it has been shown that ROS are involved in the inflammation and tissue fibrosis seen in the aggravation and/or sequelae of COVID-19 (Non-Patent Document 15). Furthermore, there is a report that the ACE2 expression level is elevated in severe COVID-19 patients (Non-Patent Document 16).
　上記の通り、COVID-19治療薬として、既にRNA複製阻害薬や抗炎症薬が承認されているが、その治療効果は決して十分とは言えず、より有効な医薬品の開発が急務とされている。この状況に鑑み、本発明は、COVID-19をはじめとする新興ウイルス感染症等を予防および／または処置するための新たな医薬組成物の発見を目的とする。 As mentioned above, RNA replication inhibitors and anti-inflammatory drugs have already been approved as COVID-19 therapeutic agents, but their therapeutic effects are by no means sufficient, and there is an urgent need to develop more effective drugs. . In view of this situation, the present invention aims to discover new pharmaceutical compositions for preventing and/or treating emerging viral infections such as COVID-19.
　COVID-19重症化のリスク要因として、喫煙や高血糖、抗がん剤等の薬物などが報告されている。本発明者らは以前、抗がん剤誘発性心不全モデルマウスにおいて、受容体作動性カチオンチャネルTRPC3タンパク質とNADPH オキシダーゼ2Nox2タンパク質との複合体形成を介したNox2安定化による過剰なROS産生が、心不全発症に関与することを報告した。興味深いことに、今回、抗がん剤をはじめとするリスク因子の曝露を受けたマウス心臓では、SARS-CoV-2受容体であるACE2の発現量が有意に増加しており、この心筋ACE2発現量増加は、TRPC3タンパク質とNox2タンパク質との複合体形成（以下、「TRPC3-Nox2複合体形成」と称する。）を阻害することで抑制されること、を新たに見出した。 Smoking, hyperglycemia, drugs such as anticancer drugs, etc. have been reported as risk factors for the aggravation of COVID-19. The present inventors previously reported that excessive ROS production by Nox2 stabilization via complex formation between the receptor-gated cation channel TRPC3 protein and NADPH oxidase 2Nox2 protein was associated with heart failure in a mouse model of anticancer drug-induced heart failure. reported to be involved in the onset. Interestingly, in mouse hearts exposed to anticancer drugs and other risk factors, the expression of ACE2, a SARS-CoV-2 receptor, was significantly increased. It was newly found that the increase in amount is suppressed by inhibiting complex formation between TRPC3 protein and Nox2 protein (hereinafter referred to as "TRPC3-Nox2 complex formation").
　SARS-CoV-2とACE2の結合を標的とした場合、上記の通り、SARS-CoV-2変異株の出現が報告されているため、薬効が見られなくなる恐れがある。そこで、我々はより普遍的な標的としてSARS-CoV-2と結合するACE2の内在化に着目した。 　When targeting the binding of SARS-CoV-2 and ACE2, as mentioned above, the emergence of SARS-CoV-2 mutants has been reported, so there is a risk that the drug efficacy will be lost. Therefore, we focused on the internalization of ACE2, which binds to SARS-CoV-2, as a more universal target.
　TRPC3-Nox2複合体形成の阻害活性をもつ既承認薬の中から、SARS-CoV-2ウイルス感染の初期過程であるACE2内在化を強く抑制する薬を、スパイクタンパク質による疑似感染評価系を用いて探索した。その結果、三環系抗うつ薬であるクロミプラミンがスパイクタンパク質によるACE2内在化を最も強く抑制することを見出し、本発明を完成させた。 Among the approved drugs with inhibitory activity on TRPC3-Nox2 complex formation, drugs that strongly suppress ACE2 internalization, which is the initial process of SARS-CoV-2 virus infection, were tested using a spike protein pseudo-infection evaluation system. explored. As a result, they found that clomipramine, a tricyclic antidepressant, most strongly suppresses ACE2 internalization by spike protein, and completed the present invention.
　したがって、本発明は、以下の態様を含む。
＜新興ウイルス感染症等を予防または処置するための医薬組成物＞
［１］
　TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質を含有する、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防および／または処置するための医薬組成物。
［２］
　TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質が、TRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制しかつACE2内在化を抑制する物質である、［１］記載の医薬組成物。
［３］
　ACE2内在化を抑制する物質が、クラスリン依存的なエンドサイトーシスを阻害する物質である、［１］または［２］記載の医薬組成物。
［４］
　TRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制しかつACE2内在化を抑制する物質が、クロミプラミン、イブジラスト、トリフルオペラジン、ニフェジピン、トルナフテートの中から選ばれる、［１］から［３］までのいずれか記載の医薬組成物。
［５］
　TRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制しかつACE2内在化を抑制する物質が、クロミプラミンである、［４］記載の医薬組成物。
［６］
　新興ウイルス感染症を予防および／または処置するための、［１］から［５］までのいずれか記載の医薬組成物。
［７］
　新興ウイルス感染症がCOVID-19である、［５］記載の医薬組成物。 Accordingly, the present invention includes the following aspects.
<Pharmaceutical composition for preventing or treating emerging viral infections, etc.>
[1]
Prevent and/or treat emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome containing substances that inhibit TRPC3-Nox2 complex formation and suppress ACE2 internalization A pharmaceutical composition for
[2]
The pharmaceutical composition according to [1], wherein the substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization is a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein and suppresses ACE2 internalization. thing.
[3]
The pharmaceutical composition of [1] or [2], wherein the substance that suppresses ACE2 internalization is a substance that inhibits clathrin-dependent endocytosis.
[4]
Any of [1] to [3], wherein the substance that suppresses the increased expression of TRPC3 protein and/or Nox2 protein and suppresses ACE2 internalization is selected from clomipramine, ibudilast, trifluoperazine, nifedipine, and tolnaftate A pharmaceutical composition according to
[5]
The pharmaceutical composition of [4], wherein the substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein and suppresses ACE2 internalization is clomipramine.
[6]
The pharmaceutical composition according to any one of [1] to [5] for preventing and/or treating emerging viral infections.
[7]
The pharmaceutical composition of [5], wherein the emerging viral infection is COVID-19.
＜スクリーニング方法＞
［８］
　新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防および／または処置するための物質をスクリーニングする方法であって、TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質を選別する、該スクリーニング方法。 <Screening method>
[8]
A method of screening for substances for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, which inhibit TRPC3-Nox2 complex formation and The screening method for selecting a substance that suppresses ACE2 internalization.
＜バイオマーカー等＞
［９］
　新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化を判定する方法であって、
 (a10) 被検者の心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介した活性酸素（ROS）産生量 (被検バイオマーカー量) を測定する工程、
 (b10) 被検バイオマーカー量と、基準の心筋細胞のROS産生量 (対照バイオマーカー量) とを比較する工程、および
 (c10) 被検バイオマーカー量が対照バイオマーカー量よりも多い場合に、被検者を、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化が高いと判定する方法。
［１０］
　対照バイオマーカー量が、健常者のROS産生量である、［９］に記載の方法。
［１１］
　心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量が、血液中または尿中における酸化ストレスマーカー量で代替される、［９］または［１０］記載の方法。
［１２］
　酸化ストレスマーカー量が、8-OH-dG、ニトロチロシン、マロンジアルデヒド、4-ハイドロキシ-2-ノネナールの量である、［１１］記載の方法。
［１３］
　心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量の、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化を判定するためのバイオマーカーとしての使用。 <Biomarkers, etc.>
[9]
A method for determining the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, comprising:
(a10) measuring the amount of reactive oxygen species (ROS) produced (amount of biomarker to be tested) mediated by complex formation between TRPC3 protein and Nox2 protein in cardiomyocytes of the subject;
(b10) comparing the amount of biomarker to be tested and the amount of ROS produced by reference cardiomyocytes (amount of control biomarker), and (c10) when the amount of biomarker to be tested is greater than the amount of control biomarker, A method of determining a subject as having increased severity of emerging viral infections, inflammatory diseases, sequelae and complications associated therewith, and acute respiratory distress syndrome.
[10]
The method of [9], wherein the control biomarker amount is the amount of ROS produced by a healthy subject.
[11]
The method according to [9] or [10], wherein the amount of ROS produced through complex formation of TRPC3 protein and Nox2 protein in cardiomyocytes is replaced by the amount of oxidative stress markers in blood or urine.
[12]
The method according to [11], wherein the amount of oxidative stress marker is the amount of 8-OH-dG, nitrotyrosine, malondialdehyde, and 4-hydroxy-2-nonenal.
[13]
ROS production via TRPC3 and Nox2 protein complex formation in cardiomyocytes to determine the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome Use as a biomarker.
　TRPC3-Nox2複合体形成を標的とした、COVID-19をはじめとする新興ウイルス感染症などの疾患に対する画期的な医薬組成物を提供することができる。また、TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する候補物質をスクリーニングする方法を提供することができる。 It is possible to provide epoch-making pharmaceutical compositions for diseases such as COVID-19 and other emerging viral infections that target TRPC3-Nox2 complex formation. Also, a method of screening for a candidate substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization can be provided.
図１は、クロミプラミンがSARS-CoV-2偽感染を強力に阻害することを示す一連の図である。図１Ａ：KRAS-RlucとヒトACE2-Venus を発現させたHEK293細胞のBRETを用いたACE2内在化の測定原理を示す模式図である。図１Ｂは、TRPC3-Nox2複合体形成を阻害する化合物のうち、ACE2内在化を抑制する13候補化合物（1μM）を、1時間前処置した際のACE2内在化抑制率を示すグラフである。図１Ｃ：COVID-19治療効果が報告されている11化合物（1μM）を、1時間前処置した際のACE2内在化抑制率を示すグラフである。図１Ｄ：クロミプラミンを含む7つの三環系抗うつ薬（1μM）を、1時間前処置した際のACE2内在化抑制率を示すグラフである。図１Ｅ：クロミプラミンのACE2酵素活性への影響を調べたグラフである。DX600は、ACE2阻害剤であり、ポジティブコントロールとして使用した。図１F：ヒトiPS細胞由来心筋細胞への蛍光標識精製Sタンパク質の取り込みを示す写真である。右図は、細胞内に取り込まれた蛍光標識精製Sタンパク質が、クロミプラミン処置により取り込み抑制されていることを示す。中央図は、細胞に取り込まれた蛍光標識精製Sタンパク質を表す。点線は細胞膜、Nは核を表し、スケールバーは10μmである。FIG. 1 is a series of figures showing that clomipramine potently inhibits SARS-CoV-2 pseudoinfection. FIG. 1A: A schematic diagram showing the principle of measuring ACE2 internalization using BRET in HEK293 cells expressing KRAS-Rluc and human ACE2-Venus. FIG. 1B is a graph showing the rate of inhibition of ACE2 internalization when pretreated for 1 hour with 13 candidate compounds (1 μM) that inhibit ACE2 internalization among compounds that inhibit TRPC3-Nox2 complex formation. FIG. 1C: A graph showing the inhibition rate of ACE2 internalization when 11 compounds (1 μM) reported to have therapeutic effects against COVID-19 were pretreated for 1 hour. FIG. 1D: A graph showing the rate of inhibition of ACE2 internalization when seven tricyclic antidepressants (1 μM) including clomipramine were pretreated for 1 hour. FIG. 1E: A graph examining the effect of clomipramine on ACE2 enzymatic activity. DX600 is an ACE2 inhibitor and was used as a positive control. FIG. 1F: A photograph showing uptake of fluorescence-labeled purified S protein into human iPS cell-derived cardiomyocytes. The right figure shows that uptake of fluorescence-labeled purified S protein that has been taken up into cells is inhibited by treatment with clomipramine. The middle panel represents fluorescently labeled purified S protein taken up by cells. Dotted line represents cell membrane, N represents nucleus, scale bar is 10 μm.
図２は、TMPRSS2安定発現VeroE6細胞またはヒトiPS細胞由来心筋細胞へのSARS-CoV-2感染および増殖に対するクロミプラミンの抑制作用を示す。図２Ａ：TMPRSS2安定発現VeroE6細胞におけるクロミプラミンまたはイブジラストの処置時間と感染抑制効果への影響を示すグラフである。Pre; 感染1時間前処置、Co-admin; 感染同時処置、Post; 感染１時間後処置を表す。データは平均値±標準誤差で示す。*P<0.05。図２Ｂ：クロミプラミンまたはイブジラスト処理し、ウイルスの感染力価をプラークアッセイにより測定した結果を示すグラフである。図２Ｃ：クロミプラミンまたはイブジラスト処理し、SARS-CoV-2感染の6時間後における細胞内ウイルスコピー数を示すグラフである。図２Ｄ：クロミプラミンまたはイブジラスト処理し、SARS-CoV-2感染の18時間後(D)における細胞内ウイルスコピー数を示すグラフである。図２Ｅ：クロミプラミン（黒丸）またはイブジラスト（黒四角）における濃度依存的なウイルスコピー数変化を示すグラフである。図２Ｆ：ヒトiPS細胞由来心筋細胞におけるSARS-CoV-2感染条件の検討結果を示すグラフである。図２Ｇ：ヒトiPS細胞由来心筋細胞へのSARS-CoV-2感染1時間前（Pre）、感染1時間後（Post）におけるクロミプラミンまたはイブジラストの感染抑制効果への影響を示すグラフである。データは平均値±標準誤差で示す。**P<0.01.図２Ｈ：ヒトiPS細胞由来心筋細胞に取り込まれたSARS-CoV-2の抗体染色像を示す写真である。FIG. 2 shows the inhibitory effect of clomipramine on SARS-CoV-2 infection and proliferation of VeroE6 cells stably expressing TMPRSS2 or human iPS cell-derived cardiomyocytes. FIG. 2A is a graph showing the effect of treatment time of clomipramine or ibudilast on VeroE6 cells stably expressing TMPRSS2 and the effect of suppressing infection. Pre; 1 hour pretreatment for infection, Co-admin; simultaneous treatment for infection, Post; 1 hour post-infection treatment. Data are presented as mean ± s.e.m. *P<0.05. FIG. 2B: A graph showing the results of clomipramine or ibudilast treatment and virus infection titer measured by plaque assay. Figure 2C: Graph showing intracellular viral copy number 6 hours after SARS-CoV-2 infection with clomipramine or ibudilast. Figure 2D: Graph showing intracellular viral copy number 18 hours after SARS-CoV-2 infection with clomipramine or ibudilast (D). FIG. 2E: Graph showing concentration-dependent changes in viral copy number for clomipramine (filled circles) or ibudilast (filled squares). FIG. 2F: A graph showing the results of examination of SARS-CoV-2 infection conditions in human iPS cell-derived cardiomyocytes. FIG. 2G: A graph showing the effect of clomipramine or ibudilast on the infection-suppressing effect of human iPS cell-derived cardiomyocytes 1 hour before (Pre) and 1 hour after infection with SARS-CoV-2 (Post). Data are presented as mean ± s.e.m. **P<0.01. FIG. 2H: A photograph showing an antibody-stained image of SARS-CoV-2 taken up by human iPS cell-derived cardiomyocytes.
図３は、クロミプラミンおよびレムデシビルの併用におけるTMPRSS2安定発現VeroE6細胞内でのSARS-CoV-2感染・増殖抑制効果を示すグラフである。レムデシビル単剤（黒丸）感染1時間後処置、クロミプラミン（10μM）とレムデシビル併用（黒四角）、破線は10μMクロミプラミンの感染抑制効果を示す。データは平均値±標準誤差で示す。縦軸は、溶媒処置群での細胞内のSARS-CoV-2ウイルスmRNAコピー数を100%とした相対値である。FIG. 3 is a graph showing the inhibitory effects of clomipramine and remdesivir on SARS-CoV-2 infection and growth in VeroE6 cells stably expressing TMPRSS2. Remdesivir monotherapy (filled circles) treatment 1 hour after infection, combined use of clomipramine (10 μM) and remdesivir (black squares), dashed line indicates infection-suppressing effect of 10 μM clomipramine. Data are presented as mean ± s.e.m. The vertical axis is a relative value with the intracellular SARS-CoV-2 viral mRNA copy number in the solvent-treated group taken as 100%.
図４は、クロミプラミンが、SARS-CoV-2変異株の感染を抑制することを示す。図４Ａ：Sタンパク質変異株：N501Y、D614G、N501Y/D614G、およびK417N/E484K/N501Y/D614Gによる濃度依存的なACE2内在化を示すグラフである。図４Ｂ：クロミプラミン(1μM)におけるSタンパク質変異株由来のACE2内在化抑制率を示すグラフである。図４Ｃ：クロミプラミン濃度依存的なTMPRSS2安定発現VeroE6細胞へのSARS-CoV-2（JPN/TY/WT-521、hCov-19/Japan/QHN002/2020）感染抑制効果を示すグラフである。FIG. 4 shows that clomipramine suppresses SARS-CoV-2 variant infection. Figure 4A: Concentration-dependent ACE2 internalization by S protein mutants: N501Y, D614G, N501Y/D614G, and K417N/E484K/N501Y/D614G. FIG. 4B: A graph showing the inhibition rate of ACE2 internalization derived from S protein mutants in clomipramine (1 μM). FIG. 4C: A graph showing clomipramine concentration-dependent inhibitory effects on SARS-CoV-2 (JPN/TY/WT-521, hCov-19/Japan/QHN002/2020) infection on VeroE6 cells stably expressing TMPRSS2.
図５は、クロミプラミンが、Sタンパク質によるミトコンドリア代謝障害を抑制することを示す。図５Ａ：Sタンパク質を添加24時間後のヒトiPS細胞由来心筋細胞における酸素消費量を示すグラフである。細胞に下記のミトコンドリア電子伝達阻害薬を順に処置することで、基礎呼吸量（薬物処置前の呼吸量－ROY/ANT処置後の最小呼吸量）、ATP産生（OL処置による減少差分）、プロトンリーク（OL処置後の呼吸量－ROY/ANT処置後の最小呼吸量）、最大呼吸量（FCCP処置後の最大呼吸量－ROY/ANT処置後の最小呼吸量）を導出した。OCR：酸素消費量、OL：オリゴマイシン（ミトコンドリアATP合成酵素の阻害剤）、FCCP：カルボニルシアニド-p-トリフルオロメトキシフェニルヒドラゾン（脱共役剤）、ROT：ロテノン（電子伝達系複合体阻害剤）、ANT：アンチマイシンA（電子伝達系複合体阻害剤）図５Ｂ：図５Ａのトレースから算出された基礎呼吸量、ATP産生、プロトンリーク、最大呼吸量を定量解析した結果を示すグラフである。FIG. 5 shows that clomipramine suppresses mitochondrial metabolic disturbance by S protein. FIG. 5A: A graph showing oxygen consumption in human iPS cell-derived cardiomyocytes 24 hours after addition of S protein. By treating cells with the following mitochondrial electron transfer inhibitors in order, basal respiratory rate (respiratory rate before drug treatment - minimum respiratory rate after ROY/ANT treatment), ATP production (difference in decrease due to OL treatment), proton leak (respiratory volume after OL treatment - minimum respiratory volume after ROY/ANT treatment) and maximum respiratory volume (maximal respiratory volume after FCCP treatment - minimum respiratory volume after ROY/ANT treatment) were derived. OCR: oxygen consumption, OL: oligomycin (inhibitor of mitochondrial ATP synthase), FCCP: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (uncoupler), ROT: rotenone (electron transport chain complex inhibitor) ), ANT: antimycin A (electron transport chain complex inhibitor) FIG. 5B: A graph showing the results of quantitative analysis of basal respiration, ATP production, proton leak, and maximum respiration calculated from the trace in FIG. 5A. .
図６は、クロミプラミンが、Sタンパク質による炎症応答を抑制することを示す。図６Ａ：Sタンパク質を添加し3時間後のラット初代培養心筋細胞におけるTNF-αのmRNA発現量を示すグラフである。図６Ｂ：Sタンパク質を添加し3時間後のラット初代培養心筋細胞におけるIL-1βのmRNA発現量を示すグラフである。図６Ｃ：Sタンパク質を添加し3時間後のラット初代培養心筋細胞におけるIL-6のmRNA発現量を示すグラフである。図６Ｄ：Sタンパク質曝露によるRAW264.7細胞からのTNF-α放出量を示すグラフである。FIG. 6 shows that clomipramine suppresses the inflammatory response by S protein. FIG. 6A: A graph showing the expression level of TNF-α mRNA in primary cultured rat cardiomyocytes 3 hours after addition of S protein. FIG. 6B: A graph showing the expression level of IL-1β mRNA in primary cultured rat cardiomyocytes 3 hours after addition of S protein. FIG. 6C: A graph showing the expression level of IL-6 mRNA in primary cultured rat cardiomyocytes 3 hours after addition of S protein. FIG. 6D: A graph showing the amount of TNF-α released from RAW264.7 cells upon exposure to S protein.
図７は、クロミプラミンが、ドレブリンを含むエンドサイトーシス経路を抑制することを示す。図７Ａ：1.5時間Sタンパク質を処置したACE2-EGFP安定発現HEK293T細胞破砕物の電気泳動像（銀染色）を示す。図７Ｂ－Ｄ：TGF-β(20 ng/mL)処置したラット初代培養心線維芽細胞におけるα-SMA (B)、ドレブリン (C)、ACE2 (D)のmRNA発現量示すグラフである。図７Ｅ：TGF-β(20 ng/mL)処置したラット初代培養心線維芽細胞における蛍光標識したSタンパク質内在化率を示すグラフである。FIG. 7 shows that clomipramine inhibits endocytic pathways involving drebrin. FIG. 7A shows an electropherogram (silver staining) of HEK293T cell lysate stably expressing ACE2-EGFP treated with S protein for 1.5 hours. FIG. 7B-D: Graphs showing mRNA expression levels of α-SMA (B), drebrin (C), and ACE2 (D) in primary cultured rat cardiac fibroblasts treated with TGF-β (20 ng/mL). FIG. 7E: Graph showing the fluorescence-labeled S protein internalization rate in primary rat cardiac fibroblasts treated with TGF-β (20 ng/mL).
図８は、SARS-CoV-2感染によるヒトiPS細胞由来心筋細胞の機能低下に対するクロミプラミンの改善効果を示すグラフである。CL; クロミプラミン, BPM; 心拍数図８Ａ：対照（左）、SARS-CoV-2感染（中央）およびSARS-CoV-2感染＋クロミプラミン添加（右）におけるそれぞれの収縮速度（Ｍ型波形の最初の高いピーク）および弛緩速度（後半の低いピーク）を示す。図８Ｂ：対照、SARS-CoV-2感染およびSARS-CoV-2感染＋クロミプラミン添加におけるBPM; 心拍数（左）、収縮速度（中央）および弛緩速度（右）のグラフである。FIG. 8 is a graph showing the improvement effect of clomipramine on functional deterioration of human iPS cell-derived cardiomyocytes due to SARS-CoV-2 infection. CL; clomipramine, BPM; heart rate Fig. 8A: Contraction velocities in control (left), SARS-CoV-2 infection (middle) and SARS-CoV-2 infection + clomipramine addition (right) (first high peak) and relaxation rate (late low peak). Figure 8B: BPM in control, SARS-CoV-2-infected and SARS-CoV-2-infected plus clomipramine; heart rate (left), contraction rate (middle) and relaxation rate (right) graphs.
＜医薬組成物＞
　本発明は一実施態様において、TRPC3タンパク質とNox2タンパク質との複合体形成（TRPC3-Nox2複合体形成）を阻害しかつACE2内在化を抑制する物質、具体的にはTRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制する物質、好ましくはクロミプラミン、イブジラスト、トリフルオペラジン、ニフェジピン、またはトルナフテート、さらに好ましくはクロミプラミンを含有する、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防および／または処置するための医薬組成物を提供する。本発明の医薬組成物に含まれる物質は、ACE2内在化を抑制する点において、ウイルス取り込み阻害作用を有する物質であり、本発明の医薬組成物は、ウイルス取り込み阻害薬と称することもできる。 <Pharmaceutical composition>
In one embodiment of the present invention, a substance that inhibits complex formation between TRPC3 protein and Nox2 protein (TRPC3-Nox2 complex formation) and suppresses ACE2 internalization, specifically TRPC3 protein and/or Nox2 protein Emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiration, containing substances that inhibit increased expression, preferably clomipramine, ibudilast, trifluoperazine, nifedipine, or tolnaftate, more preferably clomipramine Pharmaceutical compositions for preventing and/or treating distress syndrome are provided. The substance contained in the pharmaceutical composition of the present invention is a substance that has a viral uptake inhibitory effect in terms of suppressing ACE2 internalization, and the pharmaceutical composition of the present invention can also be referred to as a viral uptake inhibitor.
　COVID-19は、2019年暮れに中国・武漢にて、世界で初めてヒト患者が確認されてから、2ヶ月あまりで世界152カ国に拡散し、世界保健機構（WHO）が2020年3月11日にパンデミックを宣言した感染症である。日本では、感染者の多くが無症候性キャリアもしくは軽症であるものの、重症化しさらに高齢者や基礎疾患がある人の場合には死に至ることがある。従って、COVID-19に対して有効に作用する治療薬の開発は急務である。既に全世界的にSARS-CoV-2の感染が拡大している現状を鑑みると、安全性が確認された既存の薬から治療薬を探す、いわゆるドラッグリポジショニングは、早期開発の点から極めて有効である。 COVID-19 spread to 152 countries in just over two months after the world's first human patient was confirmed in Wuhan, China at the end of 2019, and the World Health Organization (WHO) announced on March 11, 2020 It is an infectious disease that has been declared a pandemic. In Japan, most infected people are asymptomatic carriers or have mild symptoms, but the disease can become severe and even fatal in the elderly or those with underlying medical conditions. Therefore, there is an urgent need to develop therapeutic agents that act effectively against COVID-19. Considering the current situation where SARS-CoV-2 infection is already spreading worldwide, so-called drug repositioning, which searches for therapeutic drugs from existing drugs whose safety has been confirmed, is extremely effective in terms of early development. is.
　そこで、本発明者らは、抗がん剤誘発性心不全モデルマウスでは、TRPC3タンパク質およびNox2タンパク質の複合体形成を介したNox2安定化による過剰なROS産生が関与し、そして、このマウス心臓においてSARS-CoV-2受容体であるACE2発現量が有意に増加していること、および心筋のTRPC3-Nox2複合体形成を阻害することで薬物（抗がん剤）をはじめとするCOVID-19重症化リスク因子曝露による心筋ACE2発現増加が抑制されることに着目し、TRPC3-Nox2複合体形成阻害効果をもつ化合物を同定することにより、かつ、普遍的な標的としてSARS-CoV-2と結合するACE2の細胞内在化に着目し、スパイクタンパク質による疑似感染評価系を用いて探索することにより、三環系抗うつ薬であるクロミプラミンが、細胞内ウイルスRNA増殖を顕著に抑制することを見出した。 Therefore, the present inventors have found that in anticancer drug-induced heart failure model mice, excessive ROS production by Nox2 stabilization mediated by complex formation of TRPC3 and Nox2 proteins is involved, and that SARS is involved in this mouse heart. - Significantly increased expression of ACE2, a CoV-2 receptor, and aggravation of COVID-19 by drugs (anticancer drugs) by inhibiting TRPC3-Nox2 complex formation in myocardium Focusing on the fact that myocardial ACE2 expression is suppressed by exposure to risk factors, we identified compounds that inhibit TRPC3-Nox2 complex formation, and ACE2 that binds to SARS-CoV-2 as a universal target. Focusing on the cellular internalization of viral RNA, we found that clomipramine, a tricyclic antidepressant, markedly inhibited intracellular viral RNA replication.
　本発明において、「TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質」における「TRPC3-Nox2複合体形成を阻害する物質」には、ストレス環境下（＝病態時）のTRPC3タンパク質およびNox2タンパク質の発現増加を抑制する物質であり、好ましくは、正常細胞におけるTRPC3やNox2の発現量に影響しない物質である。これには、例えば、Prestwick社の既承認薬ライブラリー1271種の既存薬 (Prestwick Chemical) の中から同定された、TRPC3-Nox2複合体形成阻害効果をもつ化合物の上位13種が挙げられる（非特許文献１３）。 In the present invention, the "substance that inhibits TRPC3-Nox2 complex formation" in the "substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization" includes TRPC3 protein under a stress environment (=during pathology). and Nox2 protein expression increase, preferably a substance that does not affect the expression levels of TRPC3 and Nox2 in normal cells. This includes, for example, the top 13 compounds with TRPC3-Nox2 complex formation inhibitory effects identified from among 1271 existing drugs (Prestwick Chemical) in Prestwick's approved drug library (non- Patent document 13).
　本発明の一実施態様では、TRPC3タンパク質およびNox2タンパク質の発現増加を抑制する物質として、例えば、TRPC3遺伝子およびNox2遺伝子に対応するsiRNA、shRNA、miRNA、リボザイム、アンチセンス等が挙げられる。
　siRNA (small interfering RNA) は、RNA干渉による遺伝子サイレンシングのために用いられる21～23塩基対の低分子２本鎖RNAである。細胞内に導入されたsiRNAは、RNA誘導サイレンシング複合体 (RISC) と結合する。この複合体はsiRNAと相補的な配列を持つmRNAに結合し切断する。これにより、配列特異的に遺伝子の発現を抑制する。
　shRNA (short hairpin RNA) は、RNA干渉による遺伝子サイレンシングのために用いられるヘアピン型のRNA配列である。shRNAは、ベクターによって細胞に導入し、U6プロモーターまたはH1プロモーターで発現させてもよいし、shRNA配列を有するオリゴヌクレオチドをDNA/RNA自動合成機で合成し、siRNAと同様の方法によりセルフアニーリングさせることによって調製してもよい。細胞内に導入されたshRNAのヘアピン構造は、siRNAへと切断され、RNA誘導サイレンシング複合体 (RISC) と結合する。この複合体はsiRNAと相補的な配列を持つmRNAに結合し切断する。これにより、配列特異的に遺伝子の発現を抑制する。
　miRNA (microRNA、マイクロRNA) は、ゲノム上にコードされ、多段階的な生成過程を経て最終的に約２０塩基の微小ＲＮＡとなる機能性核酸である。miRNAは、機能性のncRNA (non-coding RNA、非コードRNA：タンパク質に翻訳されないRNAの総称) に分類されており、他の遺伝子の発現を調節するという、生命現象において重要な役割を担っている。特定の塩基配列を有するmiRNAを生体に投与することにより、TRPC3またはNox2の発現を阻害することができる。 In one embodiment of the present invention, substances that suppress increased expression of TRPC3 protein and Nox2 protein include, for example, siRNA, shRNA, miRNA, ribozyme, antisense, etc. corresponding to TRPC3 gene and Nox2 gene.
siRNAs (small interfering RNAs) are small double-stranded RNAs of 21-23 base pairs that are used for gene silencing by RNA interference. siRNAs introduced into cells bind to the RNA-induced silencing complex (RISC). This complex binds and cleaves mRNAs with sequences complementary to the siRNA. This suppresses gene expression in a sequence-specific manner.
shRNA (short hairpin RNA) is a hairpin-shaped RNA sequence used for gene silencing by RNA interference. shRNA may be introduced into cells by a vector and expressed with a U6 promoter or H1 promoter, or an oligonucleotide having a shRNA sequence may be synthesized by an automatic DNA/RNA synthesizer and self-annealed by the same method as siRNA. may be prepared by Hairpin structures of shRNAs introduced into cells are cleaved into siRNAs and bound to the RNA-induced silencing complex (RISC). This complex binds and cleaves mRNAs with sequences complementary to the siRNA. This suppresses gene expression in a sequence-specific manner.
miRNA (microRNA, microRNA) is a functional nucleic acid that is encoded on the genome and finally becomes a minute RNA of about 20 bases through a multistep production process. miRNAs are classified as functional ncRNAs (non-coding RNAs: a generic term for RNAs that are not translated into proteins), and play an important role in life phenomena by regulating the expression of other genes. there is The expression of TRPC3 or Nox2 can be inhibited by administering miRNA having a specific nucleotide sequence to a living body.
　本発明のリボザイムとはDNA制限エンドヌクレアーゼと類似の機構により他の一本鎖RNA分子を特異的に切断するRNA分子を意味する。既知の手法によりRNAの核酸配列を適宜修飾することにより、RNA一本鎖中の特定の塩基配列を認識し切断するリボザイムを作製することができる (Science，239，p．１412-1416，1988) 。 The ribozyme of the present invention means an RNA molecule that specifically cleaves other single-stranded RNA molecules by a mechanism similar to DNA restriction endonuclease. A ribozyme that recognizes and cleaves a specific base sequence in a single RNA strand can be produced by appropriately modifying the nucleic acid sequence of RNA by a known technique (Science, 239, p.1412-1416, 1988). .
　アンチセンス核酸は、標的配列に相補的な核酸である。アンチセンス核酸は、三重鎖形成による転写開始阻害、RNAポリメラーゼによって局部的に開状ループ構造が形成された部位とのハイブリッド形成による転写抑制、合成の進みつつあるＲＮＡとのハイブリッド形成による転写阻害、イントロンとエクソンとの接合点でのハイブリッド形成によるスプライシング抑制、スプライソソーム形成部位とのハイブリッド形成によるスプライシング抑制、mRNAとのハイブリッド形成による核から細胞質への移行抑制、キャッピング部位やポリ (A) 付加部位とのハイブリッド形成によるスプライシング抑制、翻訳開始因子結合部位とのハイブリッド形成による翻訳開始抑制、開始コドン近傍のリボソーム結合部位とのハイブリッド形成による翻訳抑制、ｍＲＮＡの翻訳領域やポリソーム結合部位とのハイブリッド形成によるペプチド鎖の伸長阻止、核酸とタンパク質との相互作用部位とのハイブリッド形成による遺伝子発現抑制等により、標的遺伝子の発現を抑制することができる。 An antisense nucleic acid is a nucleic acid complementary to a target sequence. The antisense nucleic acid inhibits transcription initiation by triplex formation, inhibits transcription by hybridization with a site where an open loop structure is locally formed by RNA polymerase, inhibits transcription by hybridization with RNA that is being synthesized, Suppression of splicing by hybridization at junctions between introns and exons, suppression of splicing by hybridization with spliceosome formation sites, suppression of translocation from the nucleus to the cytoplasm by hybridization with mRNA, capping sites and poly(A) addition sites Splicing suppression by hybridization with, translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, translational region of mRNA and polysome binding site by hybridization with The expression of the target gene can be suppressed by inhibiting elongation of the peptide chain, suppressing gene expression by hybridization with the site of interaction between the nucleic acid and the protein, and the like.
　本発明において、「TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質」における「ACE2内在化を抑制する物質」とは、セリンプロテアーゼ（TMPRSS2）やカテプシン酵素依存的なエンドサイトーシスとは異なるクラスリン依存的なエンドサイトーシスを阻害する物質をいう。酵素依存的なエンドサイトーシスの場合、細胞内には複数のバックアップ機能（redundancy）が存在するため、該当する酵素を標的とする物質ではACE2内在化の全てを抑制するのは難しいと考えられる。さらに、SARS-CoV-2などのコロナウイルスにおけるACE2内在化に関与するカテプシンLやTMPRSS2酵素は、組織・細胞種ごとで発現量が異なる。他方、クラスリン依存的なエンドサイトーシスは全ての細胞種において普遍的な機構であり、多くの細胞種に対してACE2内在化を抑制する効果を発揮できる可能性がある。よって、クラスリン依存的なエンドサイトーシスを阻害する物質は、これまでとは異なる作用機序での治療効果が期待できるため、オリジナリティの高い医薬を提供できる。 In the present invention, the "substance that inhibits ACE2 internalization" in the "substance that inhibits TRPC3-Nox2 complex formation and inhibits ACE2 internalization" is serine protease (TMPRSS2) or cathepsin enzyme-dependent endocytosis. is a substance that inhibits clathrin-dependent endocytosis. In the case of enzyme-dependent endocytosis, multiple intracellular backup functions (redundancy) exist, so it may be difficult to suppress all ACE2 internalization with substances that target the relevant enzymes. Furthermore, cathepsin L and TMPRSS2 enzymes, which are involved in ACE2 internalization in coronaviruses such as SARS-CoV-2, are expressed at different levels in different tissues and cell types. On the other hand, clathrin-dependent endocytosis is a ubiquitous mechanism in all cell types and may exert an inhibitory effect on ACE2 internalization in many cell types. Therefore, a substance that inhibits clathrin-dependent endocytosis can be expected to have a therapeutic effect through a mechanism of action that is different from that of the past, and thus highly original pharmaceuticals can be provided.
　本発明において、「TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質」は、以下の実施例において詳述している通り、Prestwick社の既承認薬ライブラリー1271種の既存薬 (Prestwick Chemical、 https://www.namiki-s.co.jp/supply/common.php?id=Prestwick_Chemical) の中から、TRPC3-Nox2複合体形成阻害効果をもつ化合物の上位13種（非特許文献１３）を同定し、生物発光共鳴エネルギー移動（Bioluminescence Resonance Energy Transfer; BRET)法を用いてACE2内在化抑制効果をもつ化合物を探索した(図１A)。図１Ａは、KRAS-RlucとヒトACE2-Venus を発現させたHEK293細胞のBRETを用いたACE2内在化の測定原理を示す模式図である。細胞膜結合タンパク質KRASに蛍発光酵素ルシフェラーゼ（Rluc）を融合させたKRAS-Rlucタンパク質とヒトACE2タンパク質に蛍光タンパク質Venusを融合させたACE2-Venusとのタンパク質間相互作用を、BRETにより定量的に測定する（図１A）。スパイクタンパク質（Sタンパク質）曝露により、ACE2が内在化することで、両タンパク質の距離が離れ、それに伴ってBRET強度が低下する。
　1271種の既存薬 (Prestwick Chemical) のうち、TRPC3-Nox2複合体形成阻害効果をもつ化合物の上位13種は次の通りである：
クロミプラミン（Clomipramine）、イブジラスト（Ibudilast）、ボリコナゾール（Voriconazole）、リネストレノール（Lynestrenol）、ピリラミン（Pyrilamine）、クロピドグレル（Clopidogrel）、トリフルオペラジン（Trifluoperazine）、フィペキシド（Fipexide）、アンドロステロン（Androsterone）、ニフェジピン（Nifedipine）、トルナフテート（Tolnaftate）、ミフェプリストン（Mifepristone）、チクロピジン（Ticlopidine）。 In the present invention, the "substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization" is an existing drug of 1271 types of Prestwick's approved drug library, as detailed in the following examples. (Prestwick Chemical, https://www.namiki-s.co.jp/supply/common.php?id=Prestwick_Chemical), the top 13 compounds (non-patent Reference 13) was identified, and a compound having an inhibitory effect on ACE2 internalization was searched using the bioluminescence resonance energy transfer (BRET) method (Fig. 1A). FIG. 1A is a schematic diagram showing the principle of measuring ACE2 internalization using BRET in HEK293 cells expressing KRAS-Rluc and human ACE2-Venus. BRET is used to quantitatively measure the protein-protein interaction between KRAS-Rluc protein, which is a cell membrane-associated protein KRAS fused to the fluorescent enzyme luciferase (Rluc), and ACE2-Venus, which is a human ACE2 protein fused to the fluorescent protein Venus. (Fig. 1A). Spike protein (S protein) exposure internalizes ACE2, increasing the distance between the proteins and a concomitant decrease in BRET intensity.
Among 1271 existing drugs (Prestwick Chemical), the top 13 compounds with TRPC3-Nox2 complex formation inhibitory effect are as follows:
Clomipramine, Ibudilast, Voriconazole, Lynestrenol, Pyrilamine, Clopidogrel, Trifluoperazine, Fipexide, Androsterone, Nifedipine, Tolnaftate, Mifepristone, Ticlopidine.
　その中で、比較的ACE2内在化抑制率が高い化合物は、クロミプラミン、イブジラスト、トリフルオペラジン、ニフェジピン、およびトルナフテートであることが判明した(図１B)。最も強力な抑制効果を有する、好ましい化合物は、クロミプラミンである。クロミプラミンは、1960年代にスイスのガイギー社（現ノバルティスファーマ株式会社）によって開発された三環系抗うつ薬の一種である。薬理作用は、脳内のセロトニンおよびノルアドレナリンの神経終末への取り込みを阻害する。日本における適応症は、うつ病・うつ状態、遺尿症、ナルコレプシーに伴う情動脱力発作である。典型的には、塩酸塩であり、式：
Figure JPOXMLDOC01-appb-C000001
 で示され、その化学名（命名法）は、3-(3-クロロ-10,11-ジヒドロ-5H-ジベンゾ[b,f]アゼピン-5-イル)-N,N-ジメチルプロピルアミン 一塩酸塩（IUPAC）である。 Among them, clomipramine, ibudilast, trifluoperazine, nifedipine, and tolnaftate were found to have a relatively high rate of inhibition of ACE2 internalization (Fig. 1B). A preferred compound with the strongest inhibitory effect is clomipramine. Clomipramine is a tricyclic antidepressant drug developed by Geigy (now Novartis Pharma K.K.) in Switzerland in the 1960s. The pharmacological action is to inhibit the uptake of serotonin and noradrenaline in the brain into nerve terminals. Indications in Japan are depression/depressive state, enuresis, and cataplexy associated with narcolepsy. It is typically the hydrochloride salt and has the formula:
Figure JPOXMLDOC01-appb-C000001
 and its chemical name (nomenclature) is 3-(3-chloro-10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropylamine monohydrochloride Salt (IUPAC).
　本発明の医薬組成物が予防および／または処置する対象疾患は、典型的には、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群である。 Target diseases to be prevented and/or treated by the pharmaceutical composition of the present invention are typically emerging viral infections, inflammatory diseases associated therewith, sequelae and complications, and acute respiratory distress syndrome.
　新興ウイルス感染症とは、従来知られていなかった、新しく認識されたウイルス感染症であり、局地的あるいは国際的に公衆衛生上問題となる感染症を意味し、これには、例えば、2003年2月に発生し、アジア地域を中心にとして瞬く間に世界各地に広がり、世界的な脅威となった重症急性呼吸器症候群（SARS）、2012年アラビア半島の国々を中心として発生し、その後ヨーロッパ地域などにも感染が拡大した中東呼吸器症候群(MERS)、そして、中国の武漢から発生し、パンデミックを引き起こしている新型コロナウイルス感染症（COVID-19）が挙げられ、これらはすべて、コロナウイルスによる感染症である。 Emerging viral infections are newly recognized viral infections that have not been previously known and that pose a local or international public health concern, including, for example, the 2003 Severe Acute Respiratory Syndrome (SARS), which emerged in February 2012 and spread rapidly around the world, mainly in Asia, became a global threat. The Middle East Respiratory Syndrome (MERS), which has spread to Europe and other regions, and the novel coronavirus infection (COVID-19), which originated in Wuhan, China and is causing a pandemic, are all of these. It is an infection caused by a virus.
　新興ウイルス感染症に関連する炎症性疾患には、心血管炎や肺炎などがあり、新興ウイルス感染症に関連する後遺症には、血管炎、血栓、肺線維症や心不全、ならびに頭痛、不安・うつ症状、味覚・嗅覚障害などが挙げられる。新興ウイルス感染症に関連する合併症は、呼吸不全；心血管系、例えば急性期の不整脈、急性心障害、ショック、心停止、回復後の心筋炎など；血栓塞栓症、例えば肺塞栓症や急性期脳卒中など；炎症性合併症、例えば持続的な発熱、ギラン・バレー症候群、川崎病様症状など；二次性感染症；代謝疾患、例えば脂質異常症や糖尿病合併症；神経・精神系異常、例えば痙攣発作や意識障害、脳血管障害など、を挙げることができる。 Inflammatory diseases associated with emerging viral infections include cardiovascular inflammation and pneumonia, and sequelae associated with emerging viral infections include vasculitis, thrombosis, pulmonary fibrosis and heart failure, as well as headache, anxiety and depression. Symptoms include loss of taste and smell. Complications associated with emerging viral infections include respiratory failure; cardiovascular, including acute arrhythmias, acute cardiac injury, shock, cardiac arrest, and postrecovery myocarditis; Inflammatory complications, such as persistent fever, Guillain-Barré syndrome, Kawasaki disease-like symptoms, etc.; Secondary infections; Metabolic diseases, such as dyslipidemia and diabetic complications; Examples include convulsive seizures, disturbance of consciousness, and cerebrovascular disorders.
　急性呼吸窮迫症候群は、ARDSと称され、重症の呼吸不全をきたす疾患である。病因は、直接的または間接的に肺を障害するものに大別される。直接的に肺を障害するものとして、肺炎や誤嚥(食べ物や異物を気管内に飲み込んでしまうこと)、肺挫傷、溺水などが挙げられ、間接的に肺を障害するものとして、敗血症や重症な外傷、大量輸血などが挙げられる。また、種々の原因によって肺の血管透過性が進行した結果、血液中の成分が肺胞腔内に移動し、肺水腫を起こすことがある。これらの病態も、ARDSに包含される。 Acute respiratory distress syndrome, called ARDS, is a disease that causes severe respiratory failure. Etiologies are broadly classified into those that directly or indirectly damage the lungs. Examples of direct lung damage include pneumonia, aspiration (swallowing food or foreign objects into the trachea), pulmonary contusion, and drowning. trauma, massive blood transfusion, etc. In addition, as a result of progression of pulmonary vascular permeability due to various causes, components in the blood may migrate into the alveolar space and cause pulmonary edema. These conditions are also included in ARDS.
　本発明において、「予防」とは、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の発症を事前に防ぐことを意味する。 In the present invention, "prevention" means preventing the onset of emerging viral infections, inflammatory diseases, sequelae and complications associated therewith, and acute respiratory distress syndrome.
　本発明において、「処置」とは、 (１) 新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の発症を遅延させる； (２) 新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の症状の進行、増悪または悪化を減速または停止させる； (３) 新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の症状の寛解をもたらす；あるいは (４) 新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を治癒させることを目的とする方法またはプロセスを意味する。処置は、予防的措置として疾患または状態の発症前に施してもよいし、あるいはまた、処置は、疾患の発症後に施すことができる。 In the present invention, "treatment" means (1) delaying the onset of emerging viral infections, inflammatory diseases, sequelae and complications associated therewith, and acute respiratory distress syndrome; (2) emerging viral infections, and slows or halts the progression, exacerbation or exacerbation of associated inflammatory diseases, sequelae and complications, and symptoms of acute respiratory distress syndrome; (3) emerging viral infections, their associated inflammatory diseases, sequelae and complications; and provide amelioration of symptoms of acute respiratory distress syndrome; or (4) a method or process intended to cure emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. means. Treatment may be administered prior to the onset of the disease or condition as a prophylactic measure, or alternatively, treatment may be administered after the onset of the disease.
　本発明において医薬組成物とは、通常、疾患の予防もしくは処置、または検査・診断のための薬剤を意味する。 In the present invention, a pharmaceutical composition usually means an agent for prevention or treatment of disease, or examination/diagnosis.
　本明細書の実施例において、TRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制しかつACE2内在化を抑制する物は、新興ウイルス感染症、特にCOVID-19における後遺症の処置に有効であることが、ミトコンドリア代謝障害の抑制効果、炎症応答の抑制効果、および心筋細胞の収縮障害の改善効果を有することを示すことにより、証明されている。 In the examples of the present specification, substances that suppress increased expression of TRPC3 protein and/or Nox2 protein and suppress ACE2 internalization were found to be effective in treating the sequelae of emerging viral infections, particularly COVID-19. , by showing that it has inhibitory effects on mitochondrial metabolic disorders, inhibitory effects on inflammatory responses, and ameliorating effects on cardiomyocyte contractile disorders.
　上記の通り、本発明の医薬組成物におけるTRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質は好ましくは、クロミプラミン、イブジラスト、トリフルオペラジン、ニフェジピン、トルナフテートの中から選ばれ、特に好ましくはクロミプラミンである。 As described above, the substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization in the pharmaceutical composition of the present invention is preferably selected from clomipramine, ibudilast, trifluoperazine, nifedipine, tolnaftate, especially Clomipramine is preferred.
　本明細書の実施例において、クロミプラミンは、RNA依存的RNAポリメラーゼ阻害剤であるレムデシビルとの同時添加により、レムデシビルまたはクロミプラミン単剤の効果よりもより強くRNA増殖を抑制することを証明した。この結果は、クロミプラミンとレムデシビルの併用療法がCOVID-19の有効な治療薬となる可能性を示している。 In the examples of this specification, it was demonstrated that clomipramine suppresses RNA proliferation more strongly than the effect of remdesivir or clomipramine alone when co-added with remdesivir, an RNA-dependent RNA polymerase inhibitor. This result indicates that clomipramine and remdesivir combination therapy may be an effective treatment for COVID-19.
　クロミプラミンは、SARS-CoV-2野生株のみならず、変異株N501Y、D614G、N501Y/D614G、およびK417N/E484K/N501Y/D614Gに対しても、感染抑制効果を示す。クロミプラミンは、クラスリン依存性エンドサイトーシスを阻害することが知られているが、三環系抗うつ薬やエンドサイトーシス阻害薬を含む他の化合物ではACE2内在化を抑制しないため、クロミプラミンには他の標的があるのではないかと推測し、実験を行い、ACE2を介したSタンパク質内在化がドレブリンを介したエンドサイトーシスに関与しており、クロミプラミンはそこを阻害することが示唆された。このことから、ACE2に依存しない別の受容体を介したウイルス侵入経路であっても、ドレブリン依存的な内在化であれば抑制できるという可能性が期待できる。 Clomipramine shows an infection-suppressing effect not only against the SARS-CoV-2 wild type, but also against the mutant strains N501Y, D614G, N501Y/D614G, and K417N/E484K/N501Y/D614G. Clomipramine is known to inhibit clathrin-dependent endocytosis, but other compounds, including tricyclic antidepressants and endocytosis inhibitors, do not inhibit ACE2 internalization, thus clomipramine Speculating that there may be other targets, experiments were performed suggesting that ACE2-mediated S protein internalization is involved in drebrin-mediated endocytosis, which clomipramine inhibits. From this, we can expect the possibility that drebrin-dependent internalization can be suppressed even if the virus entry route is mediated by another receptor that does not depend on ACE2.
　本発明の医薬組成物は、当業者に公知の方法で製剤化することが可能である。本発明の医薬組成物は、非経口投与および経口投与のいずれによっても投与することができる。非経口投与の場合、例えば、注射剤型、経鼻投与剤型、経肺投与剤型、経皮投与型の組成物とすることができる。例えば、静脈内注射、筋肉内注射、腹腔内注射、皮下注射などにより全身または局部的に投与することができる。経口投与の場合、錠剤、カプセル剤、丸剤、顆粒剤、散剤、シロップ剤等の組成物とすることができる。市販のクロミプラミンは、経口投与用の錠剤として販売されている。 The pharmaceutical composition of the present invention can be formulated by methods known to those skilled in the art. The pharmaceutical composition of the present invention can be administered both parenterally and orally. For parenteral administration, for example, the composition can be injectable, nasal, pulmonary, or percutaneous administration. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like. For oral administration, compositions such as tablets, capsules, pills, granules, powders, and syrups can be used. Commercially available clomipramine is sold as tablets for oral administration.
　投与方法は、患者の年齢、症状により適宜選択することができる。ポリペプチドを含有する医薬組成物の投与量は、例えば、１回につき体重1kgあたり0.0001mgから1000mgの範囲に設定することが可能である。または、例えば、患者あたり0.001～100000mgの投与量とすることもできるが、本発明はこれらの数値に必ずしも制限されるものではない。投与量および投与方法は、患者の体重、年齢、症状などにより変動するが、当業者であればそれらの条件を考慮し適当な投与量および投与方法を設定することが可能である。 The administration method can be appropriately selected according to the patient's age and symptoms. The dose of a pharmaceutical composition containing a polypeptide can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, doses of 0.001 to 100000 mg per patient can be used, although the present invention is not necessarily limited to these figures. The dosage and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can consider these conditions and set an appropriate dosage and administration method.
　本発明は別の態様として、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防および／または処置するための方法であって、TRPC3タンパク質とNox2タンパク質との複合体形成（TRPC3-Nox2複合体形成）を阻害しかつACE2内在化を抑制する物質、具体的にはTRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制する物質、好ましくはクロミプラミン、イブジラスト、トリフルオペラジン、ニフェジピン、またはトルナフテート、さらに好ましくはクロミプラミンを、そのような処置等を必要としている患者に、その有効量を投与することを含む方法に関する。 Another aspect of the present invention is a method for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, comprising complex formation (TRPC3-Nox2 complex formation) and suppresses ACE2 internalization, specifically a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein, preferably clomipramine, ibudilast, triflu A method comprising administering an effective amount of operazine, nifedipine or tolnaftate, more preferably clomipramine, to a patient in need of such treatment or the like.
　さらに、本発明は別の態様として、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防および／または処置するための、本発明のTRPC3タンパク質とNox2タンパク質との複合体形成（TRPC3-Nox2複合体形成）を阻害しかつACE2内在化を抑制する物質、具体的にはTRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制する物質、好ましくはクロミプラミン、イブジラスト、トリフルオペラジン、ニフェジピン、またはトルナフテート、さらに好ましくはクロミプラミンに関する。 Furthermore, in another aspect, the present invention provides TRPC3 and Nox2 proteins of the present invention for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. A substance that inhibits complex formation with (TRPC3-Nox2 complex formation) and suppresses ACE2 internalization, specifically a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein, preferably clomipramine, ibudilast, It relates to trifluoperazine, nifedipine or tolnaftate, more preferably clomipramine.
　本発明はさらなる別の態様として、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防および／または処置するための医薬を製造するための、本発明のTRPC3タンパク質とNox2タンパク質との複合体形成（TRPC3-Nox2複合体形成）を阻害しかつACE2内在化を抑制する物質、具体的にはTRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制する物質、好ましくはクロミプラミン、イブジラスト、トリフルオペラジン、ニフェジピン、またはトルナフテート、さらに好ましくはクロミプラミンの使用に関する。 As still another aspect, the present invention provides a method for manufacturing a medicament for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. A substance that inhibits complex formation between TRPC3 protein and Nox2 protein (TRPC3-Nox2 complex formation) and suppresses ACE2 internalization, specifically a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein, preferably relates to the use of clomipramine, ibudilast, trifluoperazine, nifedipine or tolnaftate, more preferably clomipramine.
＜スクリーニング方法＞
　本発明は別の実施態様において、TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質をスクリーニングする方法を提供する。本発明のスクリーニング方法における一つの形態は、TRPC3タンパク質およびNox2タンパク質の発現増加を抑制しかつACE2内在化を抑制する物質をスクリーニングする方法である。この形態においては、具体的には、被験物質の存在下で、細胞中のTRPC3またはNox2の発現量を測定する工程と、前記発現量が、前記被験物質の非存在下における発現量と比較して低下していた場合に、前記被験物質はTRPC3タンパク質およびNox2タンパク質の発現増加を抑制する候補物質であると判断する工程とを備え、さらに得られた被験物質が、ACE2内在化を抑制することを確認する工程を備える、TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質の候補物質のスクリーニング方法を提供する。また、TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質は新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防または処置に有効であることから、本発明のスクリーニング方法は、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の予防または処置に有効な候補物質のスクリーニング方法に相当する。 <Screening method>
In another embodiment, the present invention provides methods of screening for substances that inhibit TRPC3-Nox2 complex formation and suppress ACE2 internalization. One aspect of the screening method of the present invention is a method of screening for a substance that suppresses increased expression of TRPC3 protein and Nox2 protein and suppresses ACE2 internalization. In this embodiment, specifically, in the presence of a test substance, the step of measuring the expression level of TRPC3 or Nox2 in cells, and comparing the expression level with the expression level in the absence of the test substance determining that the test substance is a candidate substance that suppresses the increase in the expression of TRPC3 protein and Nox2 protein if the test substance is reduced in ACE2 internalization. A screening method for a candidate substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization, comprising the step of confirming In addition, substances that inhibit TRPC3-Nox2 complex formation and suppress ACE2 internalization are effective in preventing or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. Therefore, the screening method of the present invention represents a screening method for candidate substances effective in the prevention or treatment of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome.
　細胞は、培養細胞である限り制限されない。例えば、マウスまたはラットの培養心筋細胞を利用する場合、心臓から単離して初代培養することが好ましい。市販の細胞株としては、マクロファージ細胞株RAW264.7を使用し、TRPC3-Nox2複合体形成依存的なROS生成を見ることができる。細胞は好ましくは、ヒトの心筋細胞であり、より好ましくはストレス環境下（＝病態時）の心筋細胞である。ここに、候補物質が、正常細胞のTRPC3やNox2発現量には影響を与えないことを確認する工程を包含するスクリーニング方法は、より好ましい。 Cells are not limited as long as they are cultured cells. For example, when using mouse or rat cultured myocardial cells, they are preferably isolated from the heart and then primary cultured. As a commercially available cell line, macrophage cell line RAW264.7 was used, and TRPC3-Nox2 complex formation-dependent ROS generation can be observed. The cells are preferably human myocardial cells, more preferably myocardial cells under stress (=pathological condition). Here, a screening method including a step of confirming that the candidate substance does not affect the expression levels of TRPC3 or Nox2 in normal cells is more preferable.
　被験物質とは、TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質の候補物質であるか否かを評価する対象となりうる物質であり、特に制限されない。例えば、化合物、タンパク質、ペプチド、核酸、脂質、糖質、糖脂質、糖タンパク、金属等を挙げることができる。被験物質の投与方法も特に制限されない。 A test substance is a substance that can be evaluated as to whether it is a candidate substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization, and is not particularly limited. Examples include compounds, proteins, peptides, nucleic acids, lipids, carbohydrates, glycolipids, glycoproteins, metals and the like. The administration method of the test substance is also not particularly limited.
　TRPC3またはNox2の発現量を反映する測定値は、特に制限されない。例えば、TRPC3またはNox2遺伝子から発現されるmRNA量 (コピー数またはリード数等) の測定値、タンパク質の測定値、およびレポーターアッセイによる化学発光強度等であり得る。 　Measurements that reflect the expression level of TRPC3 or Nox2 are not particularly limited. For example, it can be the measured value of the amount of mRNA expressed from the TRPC3 or Nox2 gene (copy number or read number, etc.), the measured value of protein, the chemiluminescence intensity by reporter assay, and the like.
　mRNAの測定値は、マイクロアレイ、定量的RT-PCR法、またはRNA-Seq法等の公知の方法を使用して取得することができる。 　mRNA measurements can be obtained using known methods such as microarray, quantitative RT-PCR, or RNA-Seq.
　タンパク質の測定値は、プロテインチップ、ELISA法、ウエスタンブロッティング法等によって測定することができる。 Protein values can be measured by protein chip, ELISA method, Western blotting method, etc.
　TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質は、効果的な新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の予防剤又は治療剤となる。 A substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization is an effective prophylactic or therapeutic agent for emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. becomes.
＜バイオマーカー等＞
　本発明は別の態様として、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化を判定する方法であって、
 (a10) 被検者の心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介した活性酸素（ROS）産生量 (被検バイオマーカー量) を測定する工程、
 (b10) 被検バイオマーカー量と、基準の心筋細胞のROS産生量 (対照バイオマーカー量) とを比較する工程、および
 (c10) 被検バイオマーカー量が対照バイオマーカー量よりも多い場合に、被検者を、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化が高いと判定する方法、を提供する。 <Biomarkers, etc.>
Another aspect of the present invention is a method for determining the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, comprising:
(a10) measuring the amount of reactive oxygen species (ROS) produced (amount of biomarker to be tested) mediated by complex formation between TRPC3 protein and Nox2 protein in cardiomyocytes of the subject;
(b10) comparing the amount of biomarker to be tested and the amount of ROS produced by reference cardiomyocytes (amount of control biomarker), and (c10) when the amount of biomarker to be tested is greater than the amount of control biomarker, Methods are provided for determining a subject to have high severity of emerging viral infections, inflammatory diseases, sequelae and complications associated therewith, and acute respiratory distress syndrome.
　基準の心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量は、 (a10) 工程より前に測定された同一被検者の心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量であってもよく、健常者の心筋細胞のTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量の平均であってもよい。TRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量は非特許文献１３に記載されているようにして測定できる。例えば、ジヒドロエチジウム (DHE) を処置することで、Nox2から生成されるスーパーオキシド量を蛍光法により間接的に、測定することができる。 The amount of ROS produced through the formation of the TRPC3 protein and Nox2 protein complex in the reference cardiomyocytes is the same as the TRPC3 protein and Nox2 protein complex formation in the same subject's cardiomyocytes measured before step (a10). It may be the amount of ROS produced via complex formation between TRPC3 protein and Nox2 protein in cardiomyocytes of healthy subjects, and may be the average of the amount of ROS produced via complex formation. The amount of ROS produced through complex formation between TRPC3 protein and Nox2 protein can be measured as described in Non-Patent Document 13. For example, by treating with dihydroethidium (DHE), the amount of superoxide produced from Nox2 can be measured indirectly by fluorescence methods.
　これに関連し、本発明はさらに別の態様として、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化を判定することができる、心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量のバイオマーカーとしての使用を提供する。さらに、本発明は、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化を判定することができる、心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量であるバイオマーカーを提供する。 In this regard, the present invention provides, as a further aspect, TRPC3 in cardiomyocytes capable of determining the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. Use as a biomarker of ROS production via protein-Nox2 protein complex formation is provided. Furthermore, the present invention provides complex formation of TRPC3 and Nox2 proteins in cardiomyocytes that can determine the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. provide a biomarker that is the amount of ROS produced through
　本発明者らは、心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量が、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群に関連していることを見出した。TRPC3タンパク質とNox2タンパク質が複合体を形成することで、Nox2タンパク質の分解が抑制され、その結果、心筋細胞膜上でのROS生成が増大する。それにより、過剰なROS産生による酸化ストレスが惹起され、様々な細胞・組織が傷害されると考えられる。 We found that ROS production via complex formation of TRPC3 and Nox2 proteins in cardiomyocytes is associated with emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome. I found out what I was doing. The formation of a complex between TRPC3 and Nox2 proteins inhibits Nox2 protein degradation, resulting in increased ROS generation on the myocardial cell membrane. As a result, oxidative stress is induced by excessive ROS production, and various cells and tissues are thought to be damaged.
　被検者の心筋細胞は生検 (バイオプシー) によって採取し、TRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量を測定することができる。心筋細胞は好ましくはヒトの心筋細胞である。心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量はまた、血液中または尿中における酸化ストレスマーカー（8-OH-dGなど）量を測定することで間接的に評価できる。この実施態様も、本発明の一部を構成する。すなわち、本発明は、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化を判定する方法であって、
 (a20) 被検者の血液中または尿中における酸化ストレスマーカー量 (被検バイオマーカー量) を測定する工程、
 (b20) 被検バイオマーカー量と、基準の血液中または尿中における酸化ストレスマーカー量 (対照バイオマーカー量) とを比較する工程、および
 (c20) 被検バイオマーカー量が対照バイオマーカー量よりも多い場合に、被検者を、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化が高いと判定する方法、を提供する。ここに、血液中または尿中における酸化ストレスマーカーとして、8-OH-dG、ニトロチロシン、マロンジアルデヒド（MDA）、4－ハイドロキシ－２－ノネナール（HNE）が挙げられるが、これらに限定されない。これらマーカーは、例えば、小児感染免疫第24巻第2号 (jspid.jp)に記載されている手法により、測定することができる。 Cardiomyocytes of a subject are collected by biopsy, and the amount of ROS produced through complex formation between TRPC3 protein and Nox2 protein can be measured. Cardiomyocytes are preferably human cardiomyocytes. ROS production mediated by TRPC3 protein and Nox2 protein complex formation in cardiomyocytes can also be indirectly evaluated by measuring the amount of oxidative stress markers (such as 8-OH-dG) in blood or urine. This embodiment also forms part of the invention. That is, the present invention provides a method for determining the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, comprising:
(a20) measuring the amount of oxidative stress markers (test biomarker amount) in the blood or urine of the subject;
(b20) comparing the test biomarker amount with the reference oxidative stress marker amount in blood or urine (control biomarker amount), and (c20) the test biomarker amount is higher than the control biomarker amount Methods are provided for determining a subject, in many cases, to have high severity of emerging viral infections, inflammatory diseases associated therewith, sequelae and complications, and acute respiratory distress syndrome. Oxidative stress markers in blood or urine herein include, but are not limited to, 8-OH-dG, nitrotyrosine, malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE). These markers can be measured, for example, by the method described in Pediatric Infectious Immunity, Vol. 24, No. 2 (jspid.jp).
　以下、本発明を参考例および実施例により、詳細に説明するが、これらは本発明の範囲を限定するものでなく、単なる例示であることに留意すべきである。 Hereinafter, the present invention will be described in detail by way of reference examples and examples, but it should be noted that these are merely examples and do not limit the scope of the present invention.
参考例１
細胞の培養
　実施例では、HEK293細胞(ATCC)、TMPRSS2安定発現VeroE6細胞 (JCRB 1819：JCRB細胞バンク)を使用した。これらの細胞は、Dulbecco’s 改変イーグル培地 (DMEM)に10% FBSと1% ペニシリン/ストレプトマイシン加えたDMEM培地にて培養した。また、ヒトiPS細胞由来心筋細胞はFUJIFILM Cellular Dynamics, Inc.から購入し、企業の説明書に従い培養した。HEK293細胞へのプラスミドDNA導入には、リポフェクタミン3000(Thermo fisher Scientific)、バイアフェクト・トランスフェクション試薬(Viafect transfection reagent；Promega)を使用した。 Reference example 1
Cultivation of Cells In the examples, HEK293 cells (ATCC) and VeroE6 cells stably expressing TMPRSS2 (JCRB 1819: JCRB Cell Bank) were used. These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. Human iPS cell-derived cardiomyocytes were purchased from FUJIFILM Cellular Dynamics, Inc. and cultured according to the manufacturer's instructions. Lipofectamine 3000 (Thermo Fisher Scientific), Viafect transfection reagent (Promega) was used for plasmid DNA introduction into HEK293 cells.
参考例２
精製Sタンパク質と蛍光標識精製Sタンパク質の調製
　SARS-CoV-2の表面のスパイクタンパク質(Sタンパク質)はバキュロウイルス-シルクワーム　システムを用いて精製した（非特許文献１５）。精製したSタンパク質をHiLyte Fluor（登録商標） 555 ラベリングキット-NH2 (Dojindo)を用いて蛍光標識した。標識方法はキット付属の説明書に従った。 Reference example 2
Preparation of Purified S Protein and Fluorescent Labeled Purified S Protein The spike protein (S protein) on the surface of SARS-CoV-2 was purified using the baculovirus-silkworm system (Non-Patent Document 15). Purified S protein was fluorescently labeled using HiLyte Fluor® 555 labeling kit-NH2 (Dojindo). The labeling method followed the instructions attached to the kit.
実施例１
SARS-CoV-2偽感染を強力に阻害するクロミプラミンを同定
　Prestwick社の既承認薬ライブラリー1271種の既存薬 (Prestwick Chemical) のうち、TRPC3-Nox2複合体形成阻害効果をもつ化合物の上位13種の中から（非特許文献１３）、BRET法を用いてACE2内在化抑制効果をもつ化合物を探索した(図１A,B)。 Example 1
Identification of clomipramine that strongly inhibits SARS-CoV-2 pseudoinfection Top 13 compounds with TRPC3-Nox2 complex formation inhibitory effect among 1271 existing drugs (Prestwick Chemical) in Prestwick's approved drug library Among them (Non-Patent Document 13), compounds with ACE2 internalization inhibitory effects were searched using the BRET method (Figs. 1A and 1B).
　詳細には、HEK293細胞を12穴プレートに播種（3.0×105細胞/ウエル）し、24時間、37℃、5% CO2条件下で培養した。ネガティブコントロールとしてKRAS-Rlucと空ベクター(pC-DNA3.1)を、ポジティブコントロールとしてKRAS-RlucとヒトACE2-Venusをトランスフェクションし、24時間、37℃、5% CO2で培養した。その後、細胞を回収し、コラーゲンコーティングした96穴プレートに播種（5.0×104細胞/ウエル）し、24時間、37℃、5% CO2で培養した。細胞上清を生細胞イメージング溶液（Live cell imaging solution；Thermo Fisher Scientific）に置換し、候補化合物13種（クロミプラミン、イブジラスト、ボリコナゾール、リネストレノール、ピリラミン、クロピドグレル、トリフルオペラジン、フィペキシド、アンドロステロン、ニフェジピン、トルナフテート、ミフェプリストン、チクロピジン）と50 nM精製Sタンパク質を同時に添加し、3時間、37℃、5% CO2で培養した。 Specifically, HEK293 cells were seeded in a 12-well plate (3.0×10 5 cells/well) and cultured for 24 hours at 37° C., 5% CO 2 . KRAS-Rluc and an empty vector (pC-DNA3.1) were transfected as a negative control, and KRAS-Rluc and human ACE2-Venus were transfected as a positive control, and cultured for 24 hours at 37°C and 5% CO 2 . Cells were then harvested, seeded (5.0×10 4 cells/well) on collagen-coated 96-well plates, and cultured for 24 hours at 37° C., 5% CO 2 . The cell supernatant was replaced with a live cell imaging solution (Thermo Fisher Scientific), and 13 candidate compounds (clomipramine, ibudilast, voriconazole, linestrenol, pyrilamine, clopidogrel, trifluoperazine, fipexide, androsterone, Nifedipine, tolnaftate, mifepristone, ticlopidine) and 50 nM purified S protein were added simultaneously and incubated for 3 hours at 37° C., 5% CO 2 .
　次いで、5μM セレンテラジンh（Coelemterazine h：wako）を添加し、10分後に測定を行った。発光および蛍光はプレートリーダー (Nivo, Perkinelmer)にて測定し、BRET比は蛍光シグナル(531/22フィルター)を発光シグナル(485/20 フィルター)で割り、算出した。対照 (DMSO)での内在化を100％として内在化抑制率を求めた。
　その結果、比較的内在化抑制率が高い化合物は、クロミプラミン、イブジラスト、トリフルオペラジン、ニフェジピン、およびトルナフテートであることが判明した(図１B)。その中で、クロミプラミンは、内在化抑制率が40%であり、最も強力に抑制効果を示した(図１B)。 Next, 5 μM coelenterazine h (Coelemterazine h: wako) was added, and measurement was performed 10 minutes later. Luminescence and fluorescence were measured in a plate reader (Nivo, Perkinelmer) and the BRET ratio was calculated by dividing the fluorescence signal (531/22 filter) by the luminescence signal (485/20 filter). The internalization inhibition rate was determined by setting the internalization in the control (DMSO) as 100%.
As a result, clomipramine, ibudilast, trifluoperazine, nifedipine, and tolnaftate were found to have relatively high internalization inhibition rates (Fig. 1B). Among them, clomipramine had an internalization inhibition rate of 40%, showing the most potent inhibitory effect (Fig. 1B).
　次に、現在コロナ治療薬として承認、もしくは文献的にコロナ治療効果が報告されている11化合物において、内在化抑制効果を検討した。
11化合物：クロミプラミン（Clomipramine）、セファランチン（Cepharanthine）、シクレソニド（ciclesonide）、ナファモスタット（Nafamostat）、クロルプロマジン（Clolpromazine）、ネルフィナビル（Nelfinavir）、ジクロフェナク（Dicrofenac）、イブプロフェン（Ibuprofen）、インドメタシン（Indometacine）、ロキソプロフェン（Loxoprofen）、ニンテダニブ（Nintedanib）、ピルフェニドン（Pilfenidon）。 Next, we examined the internalization-suppressing effect of 11 compounds that are currently approved as corona therapeutic agents or have been reported in the literature to be effective in treating corona.
11 compounds: Clomipramine, Cepharanthine, Ciclesonide, Nafamostat, Clolpromazine, Nelfinavir, Diclofenac, Ibuprofen, Indometacin, Loxoprofen (Loxoprofen), Nintedanib, Pilfenidon.
　その結果、クロミプラミンが一番抑制効果を示した(図１C)。クロミプラミンは三環系抗うつ薬の一つであるため、他の三環系抗うつ薬：アミトリプチリン（Amitriptyline）、デシプラミン（Desipramine）、ドキセピン（Doxepin）、イミプラミン（Imipramine）、ミルタザピン（Mirtazapine）およびクロザピン（Clozapine）におけるACE2内在化抑制効果を比較したが、クロミプラミンが一番強力であった(図１D)。 As a result, clomipramine showed the most inhibitory effect (Fig. 1C). Clomipramine is one of the tricyclic antidepressants, so other tricyclic antidepressants: Amitriptyline, Desipramine, Doxepin, Imipramine, Mirtazapine and Clozapine (Clozapine) were compared, and clomipramine was the most potent (Fig. 1D).
　次に、クロミプラミン存在下におけるACE2酵素活性を測定した。ACE2酵素活性は、ACE2阻害剤スクリーニングアッセイキット（ACE2 inhibitor screening assay kit：BPS Bioscience #79923）を用い、キット付属の説明書に従って測定した。蛍光はプレートリーダー (Nivo, Perkinelmer)にて測定した。ポジティブコントロールとしてACE2阻害剤である1μM DX600を添加した。結果、クロミプラミン存在下において、ACE2酵素活性に阻害は見られなかった(図１E)。この結果から、１）クロミプラミンはACE2の酵素活性中心を分子標的としないこと、２）クロミプラミンはウイルス感染によるACE2内在化を阻害することで、心筋細胞膜におけるACE2酵素活性の維持にも寄与することから、アンジオテンシンII作用の抑制や、アンジオテンシン1-7/Mas受容体シグナルの増強にも働く可能性が示された。 Next, the ACE2 enzymatic activity was measured in the presence of clomipramine. ACE2 enzyme activity was measured using an ACE2 inhibitor screening assay kit (BPS Bioscience #79923) according to the instructions attached to the kit. Fluorescence was measured with a plate reader (Nivo, Perkinelmer). 1 μM DX600, an ACE2 inhibitor, was added as a positive control. As a result, no inhibition of ACE2 enzyme activity was observed in the presence of clomipramine (Fig. 1E). From these results, 1) clomipramine does not target the enzyme activity center of ACE2, and 2) clomipramine inhibits ACE2 internalization due to viral infection, thereby contributing to the maintenance of ACE2 enzyme activity in the myocardial cell membrane. , may also act to suppress the action of angiotensin II and enhance the angiotensin 1-7/Mas receptor signal.
　次いで、免疫染色により、クロミプラミンが、Sタンパク質の細胞内取込みを抑制する状態を可視化した。
　ヒトiPS細胞由来心筋細胞をマトリゲルコーティングされたウエルに播種（1.7×104細胞/ウエル）し、少なくとも4日間は、37℃、5% CO2条件下で培養した。そこへ1ウエルあたり100nMクロミプラミンを添加し、1時間、37℃、5% CO2で培養した。次いで、50 nM精製Sタンパク質を添加し、3時間、37℃、5% CO2で培養した。反応後、培地を除去し、4% パラホルムアルデヒド (wako)にて細胞を固定し、PBSに0.1% トリトン X-100と1% BSAを添加した可溶化・ブロッキング溶液にて室温、1時間、反応させた。洗浄後、Alexa Fluor 488 ファロイジン (1:500, Thermo Fisher Scientific)とDAPI (1:2000, Thermo Fisher Scientific)で細胞と核を染色し、Fluoro-KEEPER antifade reagent(nacalai)にて包埋し、共焦点顕微鏡 (LSM700, Zeiss)にて観察した。結果、ヒトiPS細胞由来心筋細胞に蛍光標識精製Sタンパク質を添加すると細胞内に蛍光標識精製Sタンパク質が取り込まれ（図１F中央、矢印：核周辺の白色のドット）、これはクロミプラミンを1時間前処理することで抑制された(図１F右)。 Then, by immunostaining, clomipramine was visualized to suppress intracellular uptake of S protein.
Human iPS cell-derived cardiomyocytes were seeded onto Matrigel-coated wells (1.7×10 4 cells/well) and cultured at 37° C., 5% CO 2 for at least 4 days. 100 nM clomipramine was added thereto per well and cultured for 1 hour at 37° C. and 5% CO 2 . 50 nM purified S protein was then added and incubated for 3 hours at 37° C., 5% CO 2 . After the reaction, remove the medium, fix the cells with 4% paraformaldehyde (wako), and react with a solubilization/blocking solution consisting of 0.1% Triton X-100 and 1% BSA in PBS for 1 hour at room temperature. let me After washing, cells and nuclei were stained with Alexa Fluor 488 phalloidin (1:500, Thermo Fisher Scientific) and DAPI (1:2000, Thermo Fisher Scientific), embedded in Fluoro-KEEPER antifade reagent (nacalai), and co-cultured. Observation was made with a focusing microscope (LSM700, Zeiss). As a result, when fluorescence-labeled purified S protein was added to human iPS cell-derived cardiomyocytes, the fluorescence-labeled purified S protein was taken up into the cells (Fig. 1F center, arrow: white dot around the nucleus). It was suppressed by treatment (Fig. 1F right).
実施例２
TMPRSS2安定発現VeroE6細胞またはヒトiPS-CMsへのSARS-CoV-2感染および増殖に対するクロミプラミンの抑制作用
　TMPRSS2安定発現VeroE6細胞を用いたSARS-CoV-2感染実験により、クロミプラミンによるウイルスの感染および増殖に対する抑制効果を確認した。なお、SARS-CoV-2は、国立感染症研究所より入手し、実験は国立医薬品食品衛生研究所にて行った。
　参考例１にて調製したTMPRSS2安定発現VeroE6細胞を96穴プレートに播種 (1.5×104 細胞/ウエル)し、24時間培養した後、SARS-CoV-2を感染させた。10μMのクロミプラミンおよびイブジラストの各化合物を感染1時間前、同時、1時間後に添加した。SARS-CoV-2は、感染多重度 (M.O.I) 0.1で感染させた。感染後、細胞内RNAを採取 (CellAmp Direct RNA Prep Kit, TAKARA)し、RT-qPCRを実施した(TaqMan Fast Virus 1-Step Master Mix, Thermo Fisher Scientific)。 Example 2
Inhibitory effect of clomipramine on SARS-CoV-2 infection and growth of VeroE6 cells stably expressing TMPRSS2 or human iPS-CMs. The inhibitory effect was confirmed. SARS-CoV-2 was obtained from the National Institute of Infectious Diseases, and experiments were conducted at the National Institute of Health Sciences.
VeroE6 cells stably expressing TMPRSS2 prepared in Reference Example 1 were seeded in a 96-well plate (1.5×10 4 cells/well), cultured for 24 hours, and then infected with SARS-CoV-2. 10 μM clomipramine and ibudilast compounds were added 1 hour before, at the same time and 1 hour after infection. SARS-CoV-2 was infected at a multiplicity of infection (MOI) of 0.1. After infection, intracellular RNA was collected (CellAmp Direct RNA Prep Kit, TAKARA) and RT-qPCR was performed (TaqMan Fast Virus 1-Step Master Mix, Thermo Fisher Scientific).
　ヒトiPS細胞由来心筋細胞をフィブロネクチンでコートした96穴プレートに播種 (3×104 細胞/ウエル)し、少なくとも数日間、血清存在下で培養した。無血清培地に置換後、SARS-CoV-2をM.O.Iが2.5になるように感染させ、48時間、37℃、5% CO2で培養した。感染後、細胞内RNAを採取 (CellAmp Direct RNA Prep Kit, TAKARA)し、RT-qPCRを実施した(TaqMan Fast Virus 1-Step Master Mix, Thermo Fisher Scientific)。
　結果、TMPRSS2安定発現VeroE6細胞へのSARS-CoV-2感染1時間前、同時、感染1時間後にクロミプラミンを添加すると、細胞内のウイルス増殖が抑制された(図２A)。対照化合物としてTRPC3-Nox2複合体形成を最も強く阻害するイブジラストを使用したが、いずれの条件においてもウイルス増殖は抑制されなかった(図２A)。 Human iPS cell-derived cardiomyocytes were seeded (3×10 4 cells/well) on fibronectin-coated 96-well plates and cultured in the presence of serum for at least several days. After replacement with serum-free medium, the cells were infected with SARS-CoV-2 at an MOI of 2.5 and cultured at 37°C, 5% CO 2 for 48 hours. After infection, intracellular RNA was collected (CellAmp Direct RNA Prep Kit, TAKARA) and RT-qPCR was performed (TaqMan Fast Virus 1-Step Master Mix, Thermo Fisher Scientific).
As a result, the addition of clomipramine to VeroE6 cells stably expressing TMPRSS2 1 hour before, at the same time, and 1 hour after infection with SARS-CoV-2 inhibited viral proliferation in the cells (Fig. 2A). Ibudilast, which most strongly inhibits TRPC3-Nox2 complex formation, was used as a control compound, but virus growth was not suppressed under any conditions (Fig. 2A).
　次に、プラークアッセイを行った。TMPRSS2安定発現VeroE6細胞の上清を2%FBS、1%ペニシリン/ストレプトマイシンを加えたDMEM培地で希釈し、別にTMPRSS2安定発現VeroE6細胞を播種した組織培養用プレートに加え、1時間感染させた。その後、1%メチルセルロースと2%FBSを含むDMEM培地を添加し、3日間、37℃にて培養した。細胞をホルマリン（富士フィルム和光純薬）にて固定後、メチレンブルーにより染色し、形成されたプラーク数を計測した。培養上清を用いてプラークアッセイを行った結果、クロミプラミン（10μM）処理した細胞群では対照やイブジラスト（10μM）処理群と比較して、著しくプラーク数が減少していた(図２B)。 Next, a plaque assay was performed. The supernatant of VeroE6 cells stably expressing TMPRSS2 was diluted with DMEM medium supplemented with 2% FBS and 1% penicillin/streptomycin, added to a separate tissue culture plate seeded with VeroE6 cells stably expressing TMPRSS2, and infected for 1 hour. Then, a DMEM medium containing 1% methylcellulose and 2% FBS was added and cultured at 37°C for 3 days. After the cells were fixed with formalin (Fujifilm Wako Pure Chemical Industries, Ltd.), they were stained with methylene blue, and the number of plaques formed was counted. As a result of plaque assay using the culture supernatant, clomipramine (10 μM)-treated cells showed a marked decrease in the number of plaques compared to the control and ibudilast (10 μM)-treated groups (Fig. 2B).
　また、クロミプラミン（10μM）やイブジラスト（10μM）を1時間前処理しておき、SARS-CoV-2を感染させてから6時間後(図２C)、および18時間後 (図２D)の細胞内ウイルスmRNA量を測定したところ、どちらの時間でもクロミプラミン処理によりウイルス増殖が強力に抑制され、イブジラストでは抑制されなかった(図２C,D)。次いで、濃度依存性を測定した結果、クロミプラミンのED50は、GraphPad Prizmソフトウエアで用量作用曲線のフィッティングを行い算定したところ、4.4μMであった(図２E)。イブジラストは濃度を上げても、ウイルス増殖抑制効果が見られなかった(図２E)。 In addition, after pretreatment with clomipramine (10 μM) or ibudilast (10 μM) for 1 hour, intracellular virus was observed 6 hours (Fig. 2C) and 18 hours (Fig. 2D) after SARS-CoV-2 infection. Measurement of mRNA levels revealed that clomipramine treatment strongly suppressed viral growth at both time points, but ibudilast did not (Fig. 2C,D). Concentration dependence was then measured and the ED 50 of clomipramine was calculated to be 4.4 μM by dose-response curve fitting with GraphPad Prizm software (FIG. 2E). Even if ibudilast was increased in concentration, no viral growth inhibitory effect was observed (Fig. 2E).
　次いで、ヒトiPS細胞由来心筋細胞を用い、SARS-CoV-2感染および増殖に対するクロミプラミンの作用を調べた。
　SARS-CoV-2を血清有り(+)、無し(-)の培地中で培養したヒトiPS細胞由来心筋細胞に感染させたところ、24、48、72時間後の細胞内mRNAからウイルスmRNAが検出されたことから、SARS-CoV-2は、ヒトiPS細胞由来心筋細胞にも感染することが確認された(図２F)。TMPRSS2安定発現VeroE6細胞での薬効評価と同様に、ヒトiPS細胞由来心筋細胞へのSARS-CoV-2感染1時間前、感染1時間後にクロミプラミンを処理したところ、ウイルス増殖が有意に抑制された(図２G)。他方、イブジラスト処理では、抑制されなかった(図２G)。 Next, we investigated the effect of clomipramine on SARS-CoV-2 infection and proliferation using human iPS cell-derived cardiomyocytes.
Human iPS cell-derived cardiomyocytes cultured in media with (+) and without (-) serum were infected with SARS-CoV-2, and viral mRNA was detected from intracellular mRNA 24, 48, and 72 hours later. It was confirmed that SARS-CoV-2 also infects human iPS cell-derived cardiomyocytes (Fig. 2F). Similar to the drug efficacy evaluation using VeroE6 cells stably expressing TMPRSS2, when human iPS cell-derived cardiomyocytes were treated with clomipramine 1 hour before and 1 hour after SARS-CoV-2 infection, viral proliferation was significantly suppressed ( Figure 2G). On the other hand, ibudilast treatment was not inhibited (Fig. 2G).
　次に、免疫染色を行った。ヒトiPS細胞由来心筋細胞をフィブロネクチンでコートした96穴プレートに播種 (3×104細胞/ウエル)し、少なくとも数日間、血清存在下で培養した。無血清培地に置換後、SARS-CoV-2をM.O.Iが2.5になるように感染させ、48時間、37℃、5% CO2で培養した。SARS-CoV-2のSタンパク質（anti-SARS spike glycoprotein, ab273433, abcam）、Nタンパク質（anti-SARS-CoV-2 nucleocapsid antibody, GTX135357, GeneTex）を認識する抗体にて細胞内に取り込まれたウイルスを共焦点顕微鏡にて検出した。これにより、実際、SARS-CoV-2抗体を用いて染色した結果、ヒトiPS細胞由来心筋細胞へのSARS-CoV-2取り込みが確認された(図２H)。 Next, immunostaining was performed. Human iPS cell-derived cardiomyocytes were seeded (3×10 4 cells/well) on fibronectin-coated 96-well plates and cultured in the presence of serum for at least several days. After replacement with serum-free medium, the cells were infected with SARS-CoV-2 at an MOI of 2.5 and cultured at 37°C, 5% CO 2 for 48 hours. Viruses taken up into cells by antibodies that recognize SARS-CoV-2 S protein (anti-SARS spike glycoprotein, ab273433, abcam) and N protein (anti-SARS-CoV-2 nucleocapsid antibody, GTX135357, GeneTex) was detected with a confocal microscope. As a result, SARS-CoV-2 uptake into human iPS cell-derived cardiomyocytes was actually confirmed as a result of staining using a SARS-CoV-2 antibody (Fig. 2H).
実施例３
SARS-CoV-2感染に対するクロミプラミンおよびレムデシビルの併用効果
　SARS-CoV-2感染に対するクロミプラミンおよびレムデシビルの併用効果を調べた。TMPRSS2安定発現VeroE6細胞へのSARS-CoV-2感染はクロミプラミンの1時間前投与で84%の増殖抑制効果が見られたが(図２E)（抑制率％は、ウイルス感染対照細胞のRNAコピー数を100％とした場合に、薬物投与によりRNAコピー数が16％に減少し、84％の抑制効果と表現）、既にCOVID-19治療薬として承認されているレムデシビルは、感染1時間後処理において、低濃度（1-3μM）では抑制効果が見られなかった(図３、レムデシビル単剤（黒丸）)。細胞種や組織の違いによりレムデシビルの感受性が異なる可能性が考えられるため、より高濃度、かつ、クロミプラミンと併用して同様の実験を行った。その結果、レムデシビル単独処理細胞において、10μM処理では28%、30μM処理では55%のウイルス増殖抑制効果が確認された（抑制率％は同上）。次に、クロミプラミンとレムデシビルをそれぞれ10μMずつ併用処理したところ、99%もの抑制効果が確認された(図３)（GraphPad Prizmソフトウエア）。レムデシビル単独のEC50値が9μMであったのに対し、クロミプラミンの併用はレムデシビルのEC50値を4μMと、2倍以上低濃度側にシフトさせた。このことから、クロミプラミンはレムデシビルの抗ウイルス活性を相乗的に高める可能性が示された。このことは、クロミプラミン単独より、レムデシビルと併用した方がSARS-CoV-2感染を強く抑制したことを意味する。 Example 3
Combined effect of clomipramine and remdesivir on SARS-CoV-2 infection The combined effect of clomipramine and remdesivir on SARS-CoV-2 infection was investigated. SARS-CoV-2 infection of VeroE6 cells stably expressing TMPRSS2 showed an 84% growth inhibitory effect with clomipramine pre-administration for 1 hour (Fig. 2E). is 100%, the drug administration reduces the RNA copy number to 16%, expressing an inhibitory effect of 84%). , No inhibitory effect was observed at low concentrations (1-3 μM) (Fig. 3, remdesivir single agent (black circles)). Since the sensitivity of remdesivir may differ depending on the cell type and tissue, similar experiments were conducted using a higher concentration of remdesivir in combination with clomipramine. As a result, in cells treated with remdesivir alone, 28% of viral growth inhibitory effect was confirmed with 10 μM treatment, and 55% with 30 μM treatment (inhibition rate % is the same as above). Next, when 10 μM each of clomipramine and remdesivir were co-treated, a 99% inhibitory effect was confirmed (Fig. 3) (GraphPad Prizm software). While the EC50 value of remdesivir alone was 9 μM, the concomitant use of clomipramine shifted the EC50 value of remdesivir to 4 μM, more than 2-fold to the lower concentration side. This suggests that clomipramine may synergistically enhance the antiviral activity of remdesivir. This means that clomipramine combined with remdesivir suppressed SARS-CoV-2 infection more strongly than clomipramine alone.
実施例４
SARS-CoV-2変異株感染に対するクロミプラミンの抑制効果
　SARS-CoV-2変異株の感染は、野生型と同様に、エンドサイトーシスを介し、ACE2を媒介して行われると報告されている。そのため、ACE2依存的なウイルス内在化（感染）を抑制するクロミプラミンは、変異株感染に対しても抑制効果があると考えられる。Sタンパク質変異体N501Y（α株由来）、D614G（β株由来）、N501Y/D614G（β株由来）、およびK417N/E484K/N501Y/D614G（δ株由来）を用い、クロミプラミンが有効であるかどうかを検証した。現在、流行中のオミクロン株にも上記の変異は認められている。参考例２に従って調製したSタンパク質を基にSタンパク質変異体を作成した。 Example 4
Suppressive effect of clomipramine on SARS-CoV-2 mutant infection It has been reported that SARS-CoV-2 mutant infection is mediated by endocytosis and ACE2, similar to wild-type infection. Therefore, clomipramine, which suppresses ACE2-dependent virus internalization (infection), is thought to have a suppressive effect on mutant strain infection as well. Whether clomipramine is effective using S protein variants N501Y (from α strain), D614G (from β strain), N501Y/D614G (from β strain), and K417N/E484K/N501Y/D614G (from δ strain) verified. At present, the above mutation is also found in the Omicron strain currently in fashion. Based on the S protein prepared according to Reference Example 2, an S protein mutant was created.
　HEK293細胞を12穴プレートに播種（3.0×105細胞/ウエル）し、24時間、37℃、5% CO2条件下で培養した。ネガティブコントロールとしてKRAS-Rlucと空ベクター(pC-DNA3.1)を、ポジティブコントロールとしてKRAS-RlucとヒトACE2-Venusをトランスフェクションし、24時間、37℃、5% CO2で培養した。その後、細胞を回収し、コラーゲンコーティングした96穴プレートに播種（5.0×104細胞/ウエル）し、24時間、37℃、5% CO2で培養した。細胞上清を生細胞イメージング溶液（Live cell imaging solution；Thermo Fisher Scientific）に置換し、各濃度で精製Sタンパク質変異体を添加し、3時間、37℃、5% CO2で培養した。
　次いで、5μM セレンテラジンh（Coelemterazine h：wako）を添加し、10分後に測定を行った。発光および蛍光はプレートリーダー (Nivo, Perkinelmer)にて測定し、BRET比は蛍光シグナル(531/22フィルター)を発光シグナル(485/20 フィルター)で割り、算出した。対照 (DMSO)での内在化を100％として内在化抑制率を求めた。得られた結果を図４Aに示す。 HEK293 cells were seeded in a 12-well plate (3.0×10 5 cells/well) and cultured for 24 hours at 37° C. and 5% CO 2 . KRAS-Rluc and an empty vector (pC-DNA3.1) were transfected as a negative control, and KRAS-Rluc and human ACE2-Venus were transfected as a positive control, and cultured for 24 hours at 37°C and 5% CO 2 . Cells were then harvested, seeded (5.0×10 4 cells/well) on collagen-coated 96-well plates, and cultured for 24 hours at 37° C., 5% CO 2 . The cell supernatant was replaced with a live cell imaging solution (Thermo Fisher Scientific), purified S protein variants were added at various concentrations, and cultured for 3 hours at 37°C and 5% CO2.
Next, 5 μM coelenterazine h (Coelemterazine h: wako) was added, and measurement was performed 10 minutes later. Luminescence and fluorescence were measured in a plate reader (Nivo, Perkinelmer) and the BRET ratio was calculated by dividing the fluorescence signal (531/22 filter) by the luminescence signal (485/20 filter). The internalization inhibition rate was determined by setting the internalization in the control (DMSO) as 100%. The results obtained are shown in FIG. 4A.
　HEK293細胞を12穴プレートに播種（3.0×105細胞/ウエル）し、24時間、37℃、5% CO2条件下で培養した。ネガティブコントロールとしてKRAS-Rlucと空ベクター(pC-DNA3.1)を、ポジティブコントロールとしてKRAS-RlucとヒトACE2-Venusをトランスフェクションし、24時間、37℃、5% CO2で培養した。その後、細胞を回収し、コラーゲンコーティングした96穴プレートに播種（5.0×104細胞/ウエル）し、24時間、37℃、5% CO2で培養した。細胞上清を生細胞イメージング溶液（Live cell imaging solution；Thermo Fisher Scientific）に置換し、クロミプラミン（1μM）と50 nM各精製Sタンパク質変異株を同時に添加し、3時間、37℃、5% CO2で培養した。
　次いで、5μM セレンテラジンh（Coelemterazine h：wako）を添加し、10分後に測定を行った。発光および蛍光はプレートリーダー (Nivo, Perkinelmer)にて測定し、BRET比は蛍光シグナル(531/22フィルター)を発光シグナル(485/20 フィルター)で割り、算出した。対照 (DMSO)での内在化を100％として内在化抑制率を求めた。得られた結果を図４BAに示す。 HEK293 cells were seeded in a 12-well plate (3.0×10 5 cells/well) and cultured for 24 hours at 37° C. and 5% CO 2 . KRAS-Rluc and an empty vector (pC-DNA3.1) were transfected as a negative control, and KRAS-Rluc and human ACE2-Venus were transfected as a positive control, and cultured for 24 hours at 37°C and 5% CO 2 . Cells were then harvested, seeded (5.0×10 4 cells/well) on collagen-coated 96-well plates, and cultured for 24 hours at 37° C., 5% CO 2 . Replace the cell supernatant with a live cell imaging solution (Thermo Fisher Scientific), add clomipramine (1 μM) and 50 nM of each purified S protein mutant at the same time, and incubate for 3 hours at 37°C, 5% CO 2 . cultured in
Next, 5 μM coelenterazine h (Coelemterazine h: wako) was added, and measurement was performed 10 minutes later. Luminescence and fluorescence were measured in a plate reader (Nivo, Perkinelmer) and the BRET ratio was calculated by dividing the fluorescence signal (531/22 filter) by the luminescence signal (485/20 filter). The internalization inhibition rate was determined by setting the internalization in the control (DMSO) as 100%. The results obtained are shown in FIG. 4BA.
　TMPRSS2安定発現VeroE6細胞を96穴プレートに播種 (1.5×104 細胞/ウエル)し、24時間培養した後、SARS-CoV-2 （JPN/TY/WT-521、hCov-19/Japan/QHN002/2020）を感染させた。各濃度のクロミプラミンを感染1時間前に添加した。ウイルスは、感染多重度 (M.O.I) 0.1で感染させた。感染後、細胞内RNAを採取 (CellAmp Direct RNA Prep Kit, TAKARA)し、RT-qPCRを実施した(TaqMan Fast Virus 1-Step Master Mix, Thermo Fisher Scientific)。得られた結果を図４Cに示す。 VeroE6 cells stably expressing TMPRSS2 were seeded in a 96-well plate (1.5 × 10 4 cells/well) and cultured for 24 hours. 2020). Each concentration of clomipramine was added 1 hour before infection. Viruses were infected at a multiplicity of infection (MOI) of 0.1. After infection, intracellular RNA was collected (CellAmp Direct RNA Prep Kit, TAKARA) and RT-qPCR was performed (TaqMan Fast Virus 1-Step Master Mix, Thermo Fisher Scientific). The results obtained are shown in FIG. 4C.
　結果、ACE2を介してSタンパク質変異株は内在化されること（図４A）、クロミプラミンがACE2依存的な内在化を野生株であるJPN/TY/WT-521と同様に阻害することが明らかとなった（図４B）。実際に、英国由来のSARS-CoV-2変異株（hCov-19/Japan/QHN002/2020）のTMPRSS2安定発現VeroE6細胞への感染は、クロミプラミンによって抑制された（図４C）。これらの結果は、クロミプラミンがACE2を介したSARS-CoV-2野生株および変異株の感染を防ぐことを示唆している。 As a result, it was found that the mutant S protein was internalized via ACE2 (Fig. 4A), and that clomipramine inhibited ACE2-dependent internalization in the same way as the wild type JPN/TY/WT-521. became (Fig. 4B). Indeed, the infection of the UK-derived SARS-CoV-2 mutant strain (hCov-19/Japan/QHN002/2020) to VeroE6 cells stably expressing TMPRSS2 was suppressed by clomipramine (Fig. 4C). These results suggest that clomipramine prevents ACE2-mediated infection of SARS-CoV-2 wild-type and mutant strains.
実施例５
クロミプラミンにおけるSタンパク質によるミトコンドリア代謝障害の抑制効果
　SARS-CoV-2感染の後遺症として心不全が報告されているため、偽感染モデルを用いてヒトiPS細胞由来心筋細胞におけるSタンパク質の及ぼす影響を検証した。
　そのため、Sタンパク質を曝露し、ミトコンドリア代謝機能の指標の一つである酸素消費率（OCR）を測定した。詳細には、酸素消費率（OCR）を、XFp細胞外フラックスアナライザー（Seahorse Bioscience）を使用して付属の説明書に従い評価した。ヒトiPS細胞由来心筋細胞（FUJIFILM Cellular Dynamics, Inc.）を10,000細胞/ウエルでプレートに播種し、クロミプラミン（100 nM）を添加し、1時間後に、Sタンパク質（50 nM）を添加し24時間培養した。分析前に、細胞を25mM d-グルコース、1mMピルビン酸および2mMグルタミンを添加したXF培地で1時間培養した。基礎呼吸を測定後、終濃度10μMオリゴマイシン（ミトコンドリアATP合成酵素の阻害剤）、2μMカルボニルシアニドp-[トリフルオロメトキシ]-オヘニルヒドラゾン（FCCP）（脱共役剤）、10μMロテノン（電子伝達系複合体阻害剤）および10μMアンチマイシンA（電子伝達系複合体阻害剤）を注入後にOCRを測定した。 Example 5
Suppressive effect of clomipramine on mitochondrial metabolic disorders caused by S protein Heart failure has been reported as a sequela of SARS-CoV-2 infection.
Therefore, we exposed the S protein and measured the oxygen consumption rate (OCR), one of the indicators of mitochondrial metabolic function. Specifically, oxygen consumption rate (OCR) was assessed using an XFp extracellular flux analyzer (Seahorse Bioscience) according to the accompanying instructions. Human iPS cell-derived cardiomyocytes (FUJIFILM Cellular Dynamics, Inc.) were plated at 10,000 cells/well, clomipramine (100 nM) was added, after 1 hour, S protein (50 nM) was added and cultured for 24 hours. did. Cells were cultured in XF medium supplemented with 25 mM d-glucose, 1 mM pyruvate and 2 mM glutamine for 1 hour before analysis. After measuring basal respiration, the final concentration was 10 μM oligomycin (inhibitor of mitochondrial ATP synthase), 2 μM carbonyl cyanide p-[trifluoromethoxy]-ohenylhydrazone (FCCP) (uncoupler), 10 μM rotenone (electron transport OCR was measured after injection of 10 μM antimycin A (electron transport chain complex inhibitor) and 10 μM antimycin A (electron transport chain complex inhibitor).
　結果、基礎呼吸、ATP産生、プロトンリーク、最大呼吸が有意に減少した（図５A,B）。クロミプラミンを予め処置すると、Sタンパク質による上記の項目の減少傾向が抑制された（図５A,B）。これらのことから、SARS-CoV-2感染における心筋細胞でのミトコンドリア代謝機能障害が示唆され、これをクロミプラミンが抑制できることが見出された。 As a result, basal respiration, ATP production, proton leak, and maximal respiration decreased significantly (Fig. 5A,B). Pre-treatment with clomipramine inhibited the decrease in the above items by S protein (Fig. 5A,B). These findings suggest mitochondrial metabolic dysfunction in cardiomyocytes in SARS-CoV-2 infection, and it was found that clomipramine could suppress this.
実施例６
クロミプラミンにおけるSタンパク質による炎症応答の抑制効果
　SARS-CoV-2重症化にはサイトカインストームが関与していること、また様々な組織の上皮細胞において炎症応答が生じていることが報告されているため、これらにおいてもクロミプラミンが有効であるかを検証した。炎症性マーカーであるTNF-α、IL-1β、IL-6のmRNA発現量を指標とした。 Example 6
Effect of clomipramine to suppress inflammatory response by S protein It has been reported that cytokine storm is involved in the exacerbation of SARS-CoV-2, and that inflammatory response occurs in epithelial cells of various tissues. It was verified whether clomipramine is effective also in these. The mRNA expression levels of TNF-α, IL-1β, and IL-6, which are inflammatory markers, were used as indices.
　TNF-α、IL-1β、IL-6の各mRNA発現量は、定量的リアルタイムPCRによって測定した。詳細には、まず、TRI 試薬（TRI Reagent；Sigma）を使用して細胞から全RNAを抽出した。それをReverTra Ace qPCR RT master remix（東洋紡）を使用して逆転写し、cDNAを作成した。定量的リアルタイムPCRは、KAPA SYBR FAST qPCRキット（Roche）を使用して行った。また、TNF-α放出量も測定した。詳細には、RAW264.7細胞（3×104細胞/ウエル）を、刺激1日前に96ウエルプレートに播種した。Sタンパク質を曝露し、24時間培養後、細胞上清を回収した。サイトカイン濃度は、Mouse Duo-Set ELISAキット（R＆Dシステム）を使用して測定した。 TNF-α, IL-1β and IL-6 mRNA expression levels were measured by quantitative real-time PCR. Specifically, first, total RNA was extracted from cells using TRI Reagent (Sigma). It was reverse transcribed using ReverTra Ace qPCR RT master remix (Toyobo) to create cDNA. Quantitative real-time PCR was performed using the KAPA SYBR FAST qPCR kit (Roche). TNF-α release was also measured. Specifically, RAW264.7 cells (3×10 4 cells/well) were seeded in 96-well plates one day before stimulation. After exposing to S protein and culturing for 24 hours, the cell supernatant was collected. Cytokine concentrations were measured using Mouse Duo-Set ELISA kits (R&D Systems).
　結果、ラット初代培養心筋細胞にSタンパク質を3時間曝露すると炎症性マーカーであるTNF-α、IL-1β、IL-6のmRNA発現量が上昇した（図６A、B、C）。マクロファージのライン化細胞であるRAW264.7細胞をSタンパク質で24時間刺激するとTNF-αが放出され、クロミプラミンはそれを抑制した（図６D）。このことから、Sタンパク質が内在化することにより炎症性サイトカインの上昇が引き起こされ、これをクロミプラミンが抑制することが明らかになった。そして、イブジラスト処置でも同様にTNF-α放出が抑制されたことから、クロミプラミンのマクロファージ炎症抑制作用はTRPC3-Nox2複合体形成の阻害によることが示唆された。 As a result, when rat primary cultured cardiomyocytes were exposed to S protein for 3 hours, the mRNA expression levels of the inflammatory markers TNF-α, IL-1β, and IL-6 increased (Fig. 6A, B, C). When RAW264.7 cells, which are macrophage lineage cells, were stimulated with S protein for 24 hours, TNF-α was released, and clomipramine suppressed it (Fig. 6D). From this, it was clarified that the internalization of S protein induced an increase in inflammatory cytokines, and that clomipramine suppressed this. Ibudilast treatment also inhibited TNF-α release, suggesting that clomipramine's inhibitory effect on macrophage inflammation is due to inhibition of TRPC3-Nox2 complex formation.
実施例７
クロミプラミンにおけるエンドサイトーシスの阻害効果
　クロミプラミンはクラスリン依存性エンドサイトーシスを阻害することが知られているが、三環系抗うつ薬やエンドサイトーシス阻害薬を含む他の化合物ではACE2内在化を抑制しないため、クロミプラミンには他の標的があるのではないかと推測した。そこで、クロミプラミン有り無しの条件下でSタンパク質を曝露したACE2-EGFP発現HEK293T細胞を使用し電気泳動後銀染色し、濃さに変動があったバンドに含まれるタンパク質を質量分析（LC-MS/MS）にて解析した。詳細には、Sタンパク質誘導性ACE2内在化アッセイは次のようにして行った。 Example 7
Clomipramine's inhibitory effect on endocytosis Clomipramine is known to inhibit clathrin-dependent endocytosis, whereas other compounds, including tricyclic antidepressants and endocytosis inhibitors, inhibit ACE2 internalization. Since it does not inhibit, we speculated that clomipramine may have other targets. Therefore, HEK293T cells expressing ACE2-EGFP exposed to S protein in the presence or absence of clomipramine were subjected to silver staining after electrophoresis. MS) was analyzed. Specifically, the S protein-induced ACE2 internalization assay was performed as follows.
　ACE2-EGFPを発現させたHEK293細胞（ATCC（American Type Culture Collection）から購入）（1.5×104細胞/ウエル）にSタンパク質（50 nM）を処置し、1.5時間後に細胞を回収して、アガロースビーズ標識GFP抗体 (コスモバイオ)を使用して免疫沈降を行った。クロミプラミン（1μM）はSタンパク質処置する1時間前に添加した。 HEK293 cells expressing ACE2-EGFP (purchased from ATCC (American Type Culture Collection)) (1.5×10 4 cells/well) were treated with S protein (50 nM), and after 1.5 hours, the cells were harvested and treated with agarose. Immunoprecipitation was performed using a bead-labeled GFP antibody (Cosmo Bio). Clomipramine (1 μM) was added 1 hour prior to S protein treatment.
　また、銀染色と質量分析は次のようにして行った。
　ACE2-EGFP発現HEK293細胞にSタンパク質を処置し、1.5時間後に細胞を回収して、アガロースビーズ標識GFP抗体 (コスモバイオ)を使用して免疫沈降を行った。クロミプラミン（1μM）はSタンパク質処置する1時間前に添加した。回収したサンプルをSDS-PAGEにて泳動し、銀染色（アンテグラル）した。Sタンパク質のみを添加したレーンとクロミプラミン存在下でSタンパク質刺激したレーンを比較し、検出されたバンドの濃さに変化があったバンドから抽出したタンパク質を用いてLC-MS/MS解析を行った。 In addition, silver staining and mass spectrometry were performed as follows.
ACE2-EGFP-expressing HEK293 cells were treated with S protein, cells were harvested 1.5 hours later, and immunoprecipitation was performed using an agarose bead-labeled GFP antibody (Cosmo Bio). Clomipramine (1 μM) was added 1 hour prior to S protein treatment. The collected samples were electrophoresed by SDS-PAGE and silver-stained (antegral). LC-MS/MS analysis was performed using the protein extracted from the band where the intensity of the detected band changed, comparing the lane added with S protein only and the lane stimulated with S protein in the presence of clomipramine. .
　結果、アクチン結合タンパク質である7つのタンパク質が同定された（図７A,表1）。
表１：クロミプラミンの標的候補タンパク質
Figure JPOXMLDOC01-appb-T000002
 As a result, seven proteins were identified as actin-binding proteins (Fig. 7A, Table 1).
Table 1: Candidate target proteins for clomipramine
Figure JPOXMLDOC01-appb-T000002
　そのうち、ドレブリンは細胞の形態変化やロタウイルスの腸管膜エンドサイトーシスに対する抑制因子としての役割が知られており、心線維芽細胞の筋分化（線維化）に伴って発現増加することが報告されている。そこで、ラット新生児心線維芽細胞をTGF-β刺激し線維化させた結果、α-SMA（線維化マーカー）は約2倍、ドレブリンは約1.5倍mRNAが上昇していた（図７B、C）。このとき、ACE2 mRNA発現量に変動は見られなかった（図７D）。次に、線維化を促進させた心筋線維芽細胞に蛍光標識したSタンパク質を取り込ませた結果、陽性細胞が増加しクロミプラミンはこの増加を抑制した（図７E）。線維化により増加したSタンパク質内在化細胞の割合とドレブリン発現量の増加の割合は同程度であったことから、ACE2を介したSタンパク質内在化がドレブリンを介したエンドサイトーシスに関与しており、クロミプラミンはそこを阻害することが示唆された。 Among them, drebrin is known to play a role as a suppressor against cell morphological changes and rotavirus intestinal membrane endocytosis, and it has been reported that its expression increases with myogenic differentiation (fibrosis) of cardiac fibroblasts. ing. Therefore, as a result of TGF-β stimulation of rat neonatal cardiac fibroblasts to induce fibrosis, α-SMA (fibrosis marker) mRNA increased approximately 2-fold and drebrin mRNA increased approximately 1.5-fold (Fig. 7B, C). . At this time, no change was observed in the ACE2 mRNA expression level (Fig. 7D). Next, as a result of incorporating fluorescence-labeled S protein into myocardial fibroblasts with accelerated fibrosis, the number of positive cells increased, and clomipramine suppressed this increase (Fig. 7E). ACE2-mediated S protein internalization is involved in drebrin-mediated endocytosis, as the percentage of S protein-internalized cells increased by fibrosis was similar to the increase in drebrin expression. , clomipramine was suggested to inhibit it.
実施例８
クロミプラミンにおけるSARS-CoV-2感染による収縮障害の改善効果
　SARS-CoV-2感染の後遺症として心不全が報告されているため、ヒトiPS細胞由来心筋細胞にSARS-CoV-2を感染させ、収縮・弛緩に対するクロミプラミンの影響を検証した。 Example 8
Effect of clomipramine to improve contractile dysfunction due to SARS-CoV-2 infection Heart failure has been reported as a sequela of SARS-CoV-2 infection. We examined the effect of clomipramine on
　詳細には、収縮評価は次のようにして行った。ヒトiPS細胞由来心筋細胞を50,000細胞 /ウエルで96穴プレートに播種し、クロミプラミン（1μM）を添加し1時間後に、SARS-CoV-2（JPN/TY/WT-521）を添加して48時間培養した。心筋の拍動を顕微鏡下で撮影し、MUSCLEMOTIONソフトウエアによりBPM、収縮速度、弛緩速度を解析した。 Specifically, the shrinkage evaluation was performed as follows. Human iPS cell-derived cardiomyocytes were seeded at 50,000 cells/well in a 96-well plate, clomipramine (1 μM) was added for 1 hour, and SARS-CoV-2 (JPN/TY/WT-521) was added for 48 hours. cultured. Myocardial pulsation was photographed under a microscope, and BPM, contraction rate, and relaxation rate were analyzed by MUSCLEMOTION software.
　結果、SARS-CoV-2感染（JPN/TY/WT-521, moi=1）の48時間後に、拍動数が下がり、収縮および弛緩が抑制された。クロミプラミン（1μM, 前処理）により、いずれのパラメーターも改善されることが明らかになった（図8）。これらの結果から、クロミプラミンがACE2を介したSARS-CoV-2の感染による心機能の重症化を防ぐことが示唆された。 As a result, 48 hours after SARS-CoV-2 infection (JPN/TY/WT-521, moi=1), the heart rate decreased and contraction and relaxation were suppressed. It was found that clomipramine (1 μM, pretreatment) improved all parameters (Fig. 8). These results suggested that clomipramine prevented ACE2-mediated SARS-CoV-2 infection from aggravating cardiac function.

Claims (13)

  1. 　TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質を含有する、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防および／または処置するための医薬組成物。 Prevent and/or treat emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome containing substances that inhibit TRPC3-Nox2 complex formation and suppress ACE2 internalization A pharmaceutical composition for
  2. 　TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質が、TRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制しかつACE2内在化を抑制する物質である、請求項１記載の医薬組成物。 2. The pharmaceutical composition according to claim 1, wherein the substance that inhibits TRPC3-Nox2 complex formation and suppresses ACE2 internalization is a substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein and suppresses ACE2 internalization. thing.
  3. 　ACE2内在化を抑制する物質が、クラスリン依存的なエンドサイトーシスを阻害する物質である、請求項１または２記載の医薬組成物。 The pharmaceutical composition according to claim 1 or 2, wherein the substance that suppresses ACE2 internalization is a substance that inhibits clathrin-dependent endocytosis.
  4. 　TRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制しかつACE2内在化を抑制する物質が、クロミプラミン、イブジラスト、トリフルオペラジン、ニフェジピン、トルナフテートの中から選ばれる、請求項１から３までのいずれか記載の医薬組成物。 4. Any one of claims 1 to 3, wherein the substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein and suppresses ACE2 internalization is selected from clomipramine, ibudilast, trifluoperazine, nifedipine, and tolnaftate. pharmaceutical composition of
  5. 　TRPC3タンパク質および／またはNox2タンパク質の発現増加を抑制しかつACE2内在化を抑制する物質が、クロミプラミンである、請求項４記載の医薬組成物。 The pharmaceutical composition according to claim 4, wherein the substance that suppresses increased expression of TRPC3 protein and/or Nox2 protein and suppresses ACE2 internalization is clomipramine.
  6. 　新興ウイルス感染症を予防および／または処置するための、請求項１から５までのいずれか記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 5 for preventing and/or treating emerging viral infections.
  7. 　新興ウイルス感染症がCOVID-19である、請求項５記載の医薬組成物。 The pharmaceutical composition according to claim 5, wherein the emerging viral infection is COVID-19.
  8. 　新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群を予防および／または処置するための物質をスクリーニングする方法であって、TRPC3-Nox2複合体形成を阻害しかつACE2内在化を抑制する物質を選別する、該スクリーニング方法。 A method of screening for substances for preventing and/or treating emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, which inhibit TRPC3-Nox2 complex formation and The screening method for selecting a substance that suppresses ACE2 internalization.
  9. 　新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化を判定する方法であって、
     (a10) 被検者の心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介した活性酸素（ROS）産生量 (被検バイオマーカー量) を測定する工程、
     (b10) 被検バイオマーカー量と、基準の心筋細胞のROS産生量 (対照バイオマーカー量) とを比較する工程、および
     (c10) 被検バイオマーカー量が対照バイオマーカー量よりも多い場合に、被検者を、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化が高いと判定する方法。     A method for determining the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome, comprising:
    (a10) measuring the amount of reactive oxygen species (ROS) produced (amount of biomarker to be tested) mediated by complex formation between TRPC3 protein and Nox2 protein in cardiomyocytes of the subject;
    (b10) comparing the amount of biomarker to be tested and the amount of ROS produced by reference cardiomyocytes (amount of control biomarker), and (c10) when the amount of biomarker to be tested is greater than the amount of control biomarker, A method of determining a subject as having increased severity of emerging viral infections, inflammatory diseases, sequelae and complications associated therewith, and acute respiratory distress syndrome.
  10. 　対照バイオマーカー量が、健常者のROS産生量である、請求項９に記載の方法。 The method according to claim 9, wherein the control biomarker amount is the amount of ROS produced by healthy subjects.
  11. 　心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量が、血液中または尿中における酸化ストレスマーカー量で代替される、請求項９または１０記載の方法。 The method according to claim 9 or 10, wherein the amount of ROS produced through complex formation of TRPC3 protein and Nox2 protein in cardiomyocytes is replaced by the amount of oxidative stress markers in blood or urine.
  12. 　酸化ストレスマーカー量が、8-OH-dG、ニトロチロシン、マロンジアルデヒド、4-ハイドロキシ-2-ノネナールの量である、請求項１１記載の方法。 The method according to claim 11, wherein the amount of oxidative stress marker is the amount of 8-OH-dG, nitrotyrosine, malondialdehyde, and 4-hydroxy-2-nonenal.
  13. 　心筋細胞におけるTRPC3タンパク質とNox2タンパク質の複合体形成を介したROS産生量の、新興ウイルス感染症、それに関連する炎症性疾患、後遺症および合併症、ならびに急性呼吸窮迫症候群の重症化を判定するためのバイオマーカーとしての使用。 ROS production via TRPC3 and Nox2 protein complex formation in cardiomyocytes to determine the severity of emerging viral infections, their associated inflammatory diseases, sequelae and complications, and acute respiratory distress syndrome Use as a biomarker.
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