WO2023063325A1 - Infection inhibitor for novel coronavirus (sars-cov-2) - Google Patents

Infection inhibitor for novel coronavirus (sars-cov-2) Download PDF

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WO2023063325A1
WO2023063325A1 PCT/JP2022/037941 JP2022037941W WO2023063325A1 WO 2023063325 A1 WO2023063325 A1 WO 2023063325A1 JP 2022037941 W JP2022037941 W JP 2022037941W WO 2023063325 A1 WO2023063325 A1 WO 2023063325A1
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cov
adam10
sars
cells
tmprss2
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French (fr)
Japanese (ja)
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純一郎 井上
寧 川口
仁 合田
徹 秋山
瑞生 山本
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国立大学法人 東京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K31/396—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having three-membered rings, e.g. aziridine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4704—2-Quinolinones, e.g. carbostyril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K31/54—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
    • A61K31/541—Non-condensed thiazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/20—Elemental chlorine; Inorganic compounds releasing chlorine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present invention relates to a therapeutic or prophylactic agent for novel coronavirus infectious disease (COVID-19, coronavirus disease 2019).
  • SARS-CoV-2 the virus that causes the new coronavirus infection, was recognized at the end of 2019 and became a global pandemic in 2020, posing a threat to civilization.
  • Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also capable of causing fatal pneumonia and systemic symptoms, but the infectious capacity of SARS-CoV-2 Pathogenicity is further enhanced.
  • SARS-CoV Severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 Middle East respiratory syndrome coronavirus
  • no drug has been developed that is sufficiently effective for the treatment of COVID-19, and although the vaccine currently inoculated has been successful in preventing the spread of infection, the emergence of virus mutations has reduced its effectiveness. has become uncertain. Further characterization of the virus and its interactions with host cells are needed to develop vaccines and therapeutics that reliably prevent SARS-CoV-2 infection.
  • the spike (S) protein present in the viral envelope also called viral membrane, outer membrane
  • ACE2 receptor binding domain
  • the S protein is then cleaved by the transmembrane serine protease 2 (TMPRSS2) present in the cell membrane, or by the endosomal protease cathepsin-B/L after the virus is endocytosed into the cell.
  • TMPRSS2 transmembrane serine protease 2
  • TMPRSS2 transmembrane serine protease 2
  • cathepsin-B/L endosomal protease cathepsin-B/L
  • cleavage exposes the fusion peptide within the S protein and attaches it to the cell or endosomal membrane.
  • fusion between the viral envelope and the cell membrane or endosomal membrane occurs, allowing viral RNA to enter the cytoplasm and establish infection.
  • Non-Patent Document 1 SARS-CoV-2 entry into cells can be inhibited by inhibiting TMPRSS2-dependent membrane fusion of SARS-CoV-2.
  • the purpose of the present invention is to provide therapeutic and preventive agents for COVID-19.
  • a therapeutic or preventive composition or a therapeutic or preventive agent for COVID-19 (coronavirus disease 2019), containing an ADAM10 inhibitor.
  • nucleic acid is siRNA.
  • nucleic acid is siRNA.
  • another agent preferably a therapeutic or preventive agent for COVID-19.
  • the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  • method of treatment or prevention of [12] The therapeutic or preventive method of [10] or [11] above, wherein the other drug is a TMPRSS2 inhibitor.
  • ADAM10 inhibitors for use in treating or preventing COVID-19 (coronavirus disease 2019).
  • the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine. combination.
  • the other drug is a TMPRSS2 inhibitor.
  • a therapeutic or preventive composition or therapeutic or preventive agent for COVID-19 comprising one or two compounds selected from the group consisting of marimastat and prinomastat.
  • the other drug is one or more compounds selected from the group consisting of E-64d, ammonium chloride, chloroquine and hydroxychloroquine. Or a therapeutic or prophylactic agent.
  • the other drug is one or more compounds selected from the group consisting of E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  • FIG. 1 shows TMPRSS2-independent membrane fusion induced by SARS-CoV-2 S protein (Example 1).
  • Each effector cell expressing SARS-CoV S, SARS-CoV-2 S or MERS-CoV S
  • target cells as control (Cont), receptor (Receptor) or receptor + TMPRSS2 (Receptor + TMPRSS2)
  • FIG. 10 is a graph showing quantitative results of cell fusion assay using expressing cells.
  • each effector cell expressing SARS-CoV S, SARS-CoV-2 S or MERS-CoV S
  • target cells as control (Cont), receptor (Receptor) or receptor + TMPRSS2 (Receptor + TMPRSS2)
  • Cont Cont
  • Receptor receptor + TMPRSS2
  • TMPRSS2 Receptor + TMPRSS2
  • effector cells SARS-CoV-2, SARS-CoV-2, ; is a graph showing the quantitative results of a cell fusion assay using ACE2- or TMPRSS2+ACE2-expressing cells as control (C), spike protein (S)) and target cells.
  • FIG. 2 shows the suppression of TMPRSS2-independent membrane fusion induced by SARS-CoV-2 S protein by metalloprotease inhibitors (Example 2).
  • the compounds in the dotted box selectively inhibited TMPRSS2-independent membrane fusion.
  • the upper panel shows the effect of metalloprotease inhibitors on cell fusion in co-cultures of cells expressing SARS-CoV-2 S protein and cells expressing ACE2 alone or in combination with TMPRSS2 (each compound (Ilomastat ( Effector cells (Control (Cont), SARS-CoV-2 Spike) and target cells in the presence of ilomastat, CTS-1027, marimastat, prinomastat Quantitative results of a cell fusion assay using control (Cont), ACE2, or TMPRSS2+ACE2-expressing cells).
  • the vertical axis indicates the relative cell fusion rate (Relative Cell fusion (%)).
  • the lower row shows effector cells (SARS-CoV-2 S) and target cells in the presence of each compound (ilomastat, CTS-1027, marimastat, prinomastat), DSP1-7 and
  • FIG. 10 is a graph showing quantitative results of DSP assay using DSP8-11 co-expressing cells.
  • FIG. The vertical axis indicates relative DSP activity (%).
  • Figure 3 shows the presence of a SARS-CoV-2-specific metalloprotease-dependent viral entry pathway (Example 3).
  • the vertical axis indicates pseudovirus entry (% of control).
  • FIG. 4 shows that the pattern of entry pathways is conserved in various mutants of SARS-CoV-2 (entry of SARS-CoV-2 mutants into cells via the metalloprotease-dependent pathway) (Example 4). ).
  • (a) A diagram showing the expression of the S protein of each SARS-CoV-2 strain (control (Cont), wild (WT), each mutant strain). The S protein was detected using an anti-Flag tag antibody that binds to the Flag tag at the C-terminus of the S protein (top). Vesicular stomatitis virus matrix protein (VSV M) served as a control (bottom). S0 indicates the uncleaved S protein, S2 indicates the truncated S2 domain of the S protein.
  • VSV M Vesicular stomatitis virus matrix protein
  • each SARS- Fig. 10 is a graph showing quantitative results of infection assay using CoV-2 strains (control (Cont), wild (WT), each mutant strain). The vertical axis shows pseudovirus entry (% of DMSO).
  • FIG. 5 shows involvement of ADAM-10 in the metalloprotease-dependent entry pathway of SARS-CoV-2 (Example 5).
  • Each strain control (Cont, S FIG. 10 is a graph showing the quantitative results of infection assays using protein-free pseudovirus-infected cells), SARS-CoV-2 S, VSV-G).
  • the vertical axis shows pseudovirus entry (% of DMSO).
  • Figure 6 shows the effect of drug on cell viability (cell viability is not affected by metalloprotease inhibitors) (Example 5).
  • Cell viability (% of DMSO) in the presence of each compound and without E-64d (Without E-64d) or 25 ⁇ M E-64d (With 25 ⁇ M E-64d) for each cell line (VeroE6, HEC50B, A704) (Cell viability (% of DMSO)), each value represents the mean ⁇ SD (n 3/group).
  • FIG. 4 is a diagram showing; (a) Two types of control and three types of siRNA with different sequences against ADAM10 (transfected for 48 hours) suppress the expression of ADAM10 (mock, control (Cont), precursor ( precursor), active ADAM10 (active ADAM10), Tublin). (b) Graph showing the effect of each siRNA on invasion of each pseudovirus (SARS-CoV-2, SARS-CoV, MERS-CoV, VSV).
  • FIG. 8 shows inhibition of SARS-CoV-2 live virus infection growth by metalloprotease inhibitors or ADAM10 knockdown (Example 6).
  • the vertical axis shows the relative SARS-CoV-2 N expression (/rpl13a) in logarithm (Log10 [Relative SARS-CoV-2 N expression (/rpl13a)]) (marimastat, marima), prinomastat ( prinomastat), nafamostat).
  • composition and agent of the present invention comprise an ADAM10 inhibitor as an active ingredient.
  • ADAM10 is an abbreviation for A disintegrin and metalloproteinase domain-containing protein 10, which is a kind of metalloprotease called ADAM family.
  • the human ADAM10 gene is based on the nucleotide sequence published in Genbank/NCBI Gene ID: 102. Also in the present invention, the human ADAM10 protein is referenced to the amino acid sequences published at NCBI Reference Sequence: NP_001101.1 and NP_001307499.1. Further, in the present invention, human ADAM10 mRNA is based on NCBI Reference Sequence: NM_001110.4 and NM_001320570.2.
  • an ADAM10 inhibitor is any substance that can inhibit ADAM10, and is used in the sense of including substances that inhibit the expression of ADAM10 and substances that inhibit the function of ADAM10.
  • ADAM10 inhibitors that specifically inhibit ADAM10 can be used in the present invention.
  • Substances that inhibit the expression of ADAM10 include nucleic acids against ADAM10 (eg, antisense nucleic acids such as antisense DNA, nucleic acids targeting ADAM10 such as siRNA, shRNA, microRNA, gRNA, and ribozymes).
  • Substances that inhibit the function of ADAM10 include substances that interact with ADAM10 to inhibit the function, and examples thereof include small molecules, antibodies, peptides, nucleic acids, and aptamers.
  • modified bases with in vivo stability or artificial bases can also be used as the bases that constitute the nucleic acids.
  • the nucleic acid sequence may contain not only sequences that perfectly match with the target nucleic acid, but also mismatch sequences that do not match with the target sequence as long as the expression inhibitory activity is maintained.
  • 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-forming sites, suppression of translocation from the nucleus to the cytoplasm by hybridization with mRNA, capping sites and poly(A) addition sites Suppression of splicing by hybridization with , Suppression of translation initiation by hybridization with the translation initiation factor binding site, Translation suppression by hybridization with the ribosome binding site near the initiation codon, and Hybridization with the translational region of mRNA and the polysome binding site
  • the expression of the target gene can be suppressed by inhibiting elongation of the
  • the antisense nucleic acid against ADAM10 is, for example, a single-stranded nucleic acid complementary to a partial nucleotide sequence selected from the ADAM10 gene sequence described above, the nucleotide sequence encoding the ADAM10 amino acid sequence described above, and the ADAM10 mRNA sequence described above.
  • Such nucleic acids may be naturally occurring or artificial nucleic acids, and may be based on both DNA and RNA.
  • the length of the antisense nucleic acid is usually about 15 bases to the same length as the full-length mRNA, preferably about 15 to about 30 bases.
  • the complementarity of the antisense nucleic acid does not necessarily have to be 100%, and may be such that it can complementarily bind to ADAM10-encoding DNA or RNA in vivo.
  • siRNA small interfering RNA
  • siRNA is an artificially synthesized small double-stranded RNA used for gene silencing by RNA interference (mRNA degradation), and the double-stranded RNA is supplied in vivo. It shall be used in the sense of including an siRNA expression vector capable of siRNAs introduced into cells bind to the RNA-induced silencing complex (risc). This complex binds to and cleaves mRNA having a sequence complementary to siRNA, thereby suppressing gene expression in a sequence-specific manner.
  • siRNA is prepared by synthesizing sense strand and antisense strand oligonucleotides with an automatic DNA/RNA synthesizer. can be prepared by annealing for about 1 to 8 hours at . The length of the siRNA is preferably 19-27 base pairs, more preferably 21-25 base pairs or 21-23 base pairs.
  • siRNA against ADAM10 can be designed based on its base sequence to cause degradation (RNA interference) of mRNA transcribed from the ADAM10 gene.
  • Examples of siRNAs that inhibit the expression of ADAM10 include siRNAs whose target sequence is the above-mentioned ADAM10 mRNA sequence.
  • shRNA short hairpin RNA
  • shRNA is an artificially synthesized hairpin-shaped RNA sequence used for gene silencing by RNA interference (mRNA degradation).
  • 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 a method similar to siRNA.
  • may be prepared by Hairpin structures of shRNAs introduced into cells are cleaved into siRNAs and bind to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA, thereby suppressing gene expression in a sequence-specific manner.
  • RISC RNA-induced silencing complex
  • the shRNA against ADAM10 can be designed based on its base sequence to cause degradation (RNA interference) of mRNA transcribed from the ADAM10 gene.
  • shRNAs that inhibit the expression of ADAM10 include shRNAs whose target sequence is the above-mentioned ADAM10 mRNA sequence.
  • miRNA is a functional nucleic acid that is encoded on the genome and eventually becomes a micro 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.
  • ADAM10 gene expression can be suppressed by introducing miRNA having a specific nucleotide sequence into cells using a vector and administering the miRNA to a living body.
  • gRNA guide RNA
  • gRNA is an RNA molecule used in genome editing technology.
  • gRNA specifically recognizes the target sequence, guides the binding of Cas9 protein to the target sequence, and enables gene knockout and knockin.
  • ADAM10 gene expression can be suppressed in vivo by administering gRNA targeting the ADAM10 gene in vivo.
  • gRNA shall be used in the meaning including sgRNA (single guide RNA).
  • the design method of gRNA in genome editing technology is widely known, for example, Benchmarking CRISPR on-target sgRNA design, Yan et al., Brief Bioinform, 15 Feb 2017.
  • a ribozyme is an RNA with catalytic activity. Although some ribozymes have various activities, studies on ribozymes as RNA-cleaving enzymes have made it possible to design ribozymes for the purpose of site-specific cleavage of RNA.
  • the ribozyme may be group I intron type, M1 RNA contained in RNaseP, etc., having a size of 400 nucleotides or more, or hammerhead type, hairpin type, etc. having about 40 nucleotides.
  • Aptamers include nucleic acid aptamers and peptide aptamers.
  • Nucleic acid aptamers and peptide aptamers used in the present invention are represented by the SELEX method (Systematic Evolution of Ligands by Exponential enrichment) and the mRNA display method. They can be obtained using in vitro molecular evolution techniques that are formed and then selected on the basis of affinity.
  • Antisense nucleic acids, siRNAs, shRNAs, miRNAs, ribozymes, and nucleic acid aptamers may contain various chemical modifications to improve their stability and activity.
  • phosphate residues may be substituted with chemically modified phosphate residues such as phosphorothioates (PS), methylphosphonates, phosphorodithionates, etc., to prevent degradation by hydrolases such as nucleases.
  • PS phosphorothioates
  • methylphosphonates methylphosphonates
  • phosphorodithionates etc.
  • at least a part thereof may be composed of a nucleic acid analogue such as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • An antibody against ADAM10 is an antibody that specifically binds to ADAM10 and inhibits the function of ADAM10 by binding.
  • antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, mouse antibodies, rat antibodies, camelid antibodies, antibody fragments (e.g., Fab, Fv, Fab', F (ab') 2 , scFv) and the like may be used, and these can be prepared according to known techniques by those skilled in the art.
  • Antibodies against ADAM10 can be produced according to known antibody or antiserum production methods using the ADAM10 protein or a portion thereof as an antigen.
  • ADAM10 protein or portions thereof can be prepared by known protein expression and purification methods. Examples of the ADAM10 protein include, but are not limited to, human ADAM10 defined by the ADAM10 sequence information described above. ADAM10 proteins from various organisms may be used as immunogens.
  • Antibodies to ADAM10 that can be used in the present invention can also be generated via phage display technology (see, eg, FEBS Letter, 441:20-24 (1998)).
  • compositions and agents of the present invention also contain, as active ingredients, one or two compounds selected from the group consisting of marimastat and prinomastat.
  • ADAM10 is involved in the metalloprotease-dependent entry pathway of SARS-CoV-2, and that SARS-CoV-2 infection can be inhibited by inhibiting ADAM10. Therefore, ADAM10 inhibitors can be used as active ingredients for the treatment or prevention of COVID-19.
  • SARS-CoV-2 exists not only in the originally discovered virus strain, but also in variants thereof (e.g. strain B.1.1.7 (alpha strain), B.1.351 strain (beta strain), P.1 strain (gamma strain), B.1.617.2 strain (Delta strain), B.1.1.529 strain (Omicron strain)).
  • SARS-CoV-2 is synonymous with severe acute respiratory syndrome coronavirus-2.
  • compositions and agents of the present invention can be provided as pharmaceuticals or pharmaceutical compositions.
  • the drug and pharmaceutical composition of the present invention contain the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • the medicaments and pharmaceutical compositions of the present invention also include medicaments and pharmaceutical compositions intended for gene therapy.
  • Such pharmaceuticals and pharmaceutical compositions contain ADAM10-targeted nucleic acids such as antisense nucleic acids, siRNA, shRNA, microRNA, gRNA, and ribozymes as active ingredients.
  • compositions and agents of the present invention may be used in combination with other agents other than the active ingredient of the present invention. That is, the compositions and agents of the present invention may further contain drugs other than the active ingredient of the present invention, and in this case, the dosage form may be integrated as a combination drug.
  • the composition and agent of the present invention may be administered together with other drugs other than the active ingredient of the present invention as different formulations, in which case they may be administered simultaneously or at different times. good.
  • a combination of active ingredients of the invention and other agents are provided.
  • drugs other than ADAM10 inhibitors include SARS-CoV-2 infection inhibitors (especially COVID-19 therapeutic or preventive agents).
  • SARS-CoV-2 infection inhibitors especially COVID-19 therapeutic or preventive agents.
  • Such other agents include, for example, metalloprotease inhibitors such as marimastat and prinomastat, cathepsin-B/L inhibitors such as E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  • Such other agents also include TMPRSS2 inhibitors such as, for example, nafamostat, camostat.
  • drugs other than these compounds include SARS-CoV-2 infection inhibitors (especially COVID-19 therapeutic or preventive agents).
  • SARS-CoV-2 infection inhibitors especially COVID-19 therapeutic or preventive agents.
  • agents include cathepsin-B/L inhibitors such as E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  • agents also include TMPRSS2 inhibitors such as, for example, nafamostat, camostat.
  • the route of administration is not particularly limited as long as the therapeutic or preventive effect of COVID-19 is obtained, but oral administration or parenteral administration (e.g., intravenous administration, subcutaneous administration, intraperitoneal administration) can be selected.
  • oral administration or parenteral administration e.g., intravenous administration, subcutaneous administration, intraperitoneal administration
  • Orally administered drugs include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, liquids, jellies, emulsions, and suspensions.
  • an appropriate dosage form can be selected according to the specific dosage form, and examples thereof include injections and suppositories.
  • These formulations can be formulated using a pharmaceutically acceptable carrier by a method commonly practiced in the art (for example, a known method described in the 18th revision of the Japanese Pharmacopoeia General Rules for Formulations, etc.). can.
  • Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, perfumes, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. is mentioned.
  • the dosage of the active ingredient in the present invention can be determined depending on the type of active ingredient, sex, age and body weight of the subject, symptoms, dosage form, route of administration, and the like.
  • the dosage per adult can be determined, for example, in the range of 0.0001 mg to 1000 mg / kg body weight. is not limited to
  • the above dosage of the active ingredient can be administered once a day or in 2 to 4 divided doses.
  • the compositions and agents of the present invention can be applied not only to humans in need thereof, but also to mammals other than humans (e.g., mice, rats, rabbits, dogs, cats, cows, horses, pigs, sheep, goats, monkeys). It can also be administered to
  • a method for treating or preventing COVID-19 comprising administering an ADAM10 inhibitor to a subject in need thereof.
  • the present invention also provides a method of treating or preventing COVID-19, comprising administering one or two compounds selected from the group consisting of marimastat and prinomastat to a subject in need thereof. be done.
  • the administration subject can typically be a COVID-19 patient or a person who may have COVID-19.
  • the therapeutic method and prophylactic method of the present invention can be carried out according to the description of the composition and agent of the present invention.
  • ADAM10 inhibitors for use in treating or preventing COVID-19 and combinations of ADAM10 inhibitors and other agents for use in treating or preventing COVID-19 are provided.
  • the present invention also provides one or two compounds selected from the group consisting of marimastat and prinomastat for use in the treatment or prevention of COVID-19 and Combinations of one or two compounds selected from the group consisting of , marimastat and prinomastat with other agents are provided.
  • ADAM10 inhibitors of the present invention, one or two compounds selected from the group consisting of marimastat and prinomastat, and combinations can be performed according to the description of the compositions and agents of the present invention.
  • an ADAM10 inhibitor for the manufacture of a composition for the treatment or prevention of COVID-19 or an agent for the treatment or prevention of COVID-19 and the composition for the treatment or prevention of COVID-19
  • a combination of an ADAM10 inhibitor and other agents for the manufacture of a therapeutic or prophylactic agent for COVID-19 is provided.
  • one or two compounds selected from the group consisting of marimastat and prinomastat for the manufacture of a composition for the treatment or prevention of COVID-19 or an agent for the treatment or prevention of COVID-19 and one or two compounds selected from the group consisting of marimastat and prinomastat for the manufacture of a composition for the treatment or prevention of COVID-19 or an agent for the treatment or prevention of COVID-19 , in combination with other agents is provided.
  • the uses of the invention can be carried out according to the description of the compositions and agents of the invention.
  • the present invention includes the following inventions.
  • a composition for treating COVID-19 coronavirus disease 2019
  • comprising an ADAM10 inhibitor comprising an ADAM10 inhibitor.
  • the therapeutic composition of claim 1 wherein the ADAM10 inhibitor is a nucleic acid.
  • the therapeutic composition of claim 2, wherein the nucleic acid is siRNA.
  • the therapeutic composition of any one of the above [101] to [103] which is used in combination with any one or a combination of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine .
  • the therapeutic composition of any one of the above [101] to [104] which is used in combination with a TMPRSS2 inhibitor.
  • the ADAM10 inhibitor is administered in combination with one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine, the above [ 106] to [108].
  • siRNAs and primers used in the Examples were as shown in Table 2.
  • the compounds (inhibitors) used in the examples were as shown in Table 3.
  • S spike protein
  • ACE2 ACE2, CD26, or TMPRSS2
  • pseudolentiviruses expressing one of the proteins were used as previously described (Yamamoto M. et al., 2020, Viruses, 12:629).
  • SARS-CoV-2 isolate (UT-NCGM02/Human/2020/Tokyo) (Imai M et al., 2020, Proc Natl Acad Sci USA 117:16587-16595) contains 5% fetal bovine serum (FBS) VeroE6-TMPRSS2 (JCRB1819) cells were grown in Dulbecco's Modified Eagle Medium (DMEM).
  • FBS fetal bovine serum
  • VeroE6-TMPRSS2 JCRB1819
  • siRNAs (Table 2) were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol. All protease inhibitors (Table 3) were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM.
  • DMSO dimethylsulfoxide
  • NC_045512.2) SARS-CoV-2 variants (B.1.1.7, EPI_ISL_601443; B.1.351 , MZ747297.1; B.1.617.1, EPI_ISL_1704611; B.1.617.2, EPI_ISL_3189054), SARS-CoV (NC_004718.3), WIV1-CoV (KF367457.1), HCoV-NL63 (NC_005831.2), Chimera S, and Flag-tagged 5′-GGA GGC GAT TAC AAG GAT GAC GAT GAC AAG TAA-3′ (underline indicates Flag tag at 3′ end) (SEQ ID NO: 10) are all from Integrated DNA Technologies (IA, USA) made by Synthesis with a Flag tag at the 3′ end corresponding to previously described codon-optimized MERS-CoV S (NC_019843.3) (Yamamoto M et al., 2016, Antimicrob Agents Chemother, 60:6532-65
  • DSP Assays to Monitor Membrane Fusion DSP assays were performed as previously described (Yamamoto M et al., 2020, Viruses, 12:629). Briefly, effector cells expressing S protein and target cells expressing CD26 or ACE2 alone or together with TMPRSS2 were seeded in 10 cm plates and incubated overnight. Cells were treated with 6 ⁇ M EnduRen (Promega), a substrate for Renilla luciferase (RL), for 2 hours. To test the effect of inhibitors, 0.25 ⁇ L of compound library or 1 ⁇ L of selected inhibitors dissolved in DMSO were added to 384-well plates (Greiner Bioscience, Frickenhausen, Germany).
  • VSV proteins pBS-N/pBS-P/pBS-L/pBS-G
  • Promoter-driven expression plasmids and p ⁇ G-Luci a plasmid lacking the G gene and encoding VSV genomic RNA encoding firefly luciferase
  • Supernatants were harvested 48 hours after transfection.
  • 293T cells were then transfected with S or VSV G expression plasmids by using calcium phosphate precipitation. Sixteen hours after transfection, cells were inoculated at a multiplicity of infection (MOI) of 1 with replication-deficient VSV. Two hours after infection, the cells were washed and incubated for an additional 16 hours before harvesting the pseudovirus-containing supernatant. For infection assays, cells were seeded in 96-well plates (2 x 104 cells/well) and incubated overnight. One hour prior to pseudovirus infection, cells were pretreated with inhibitors.
  • MOI multiplicity of infection
  • Luciferase activity was measured 16 hours after infection using the Bright-Glo luciferase assay system or the ONE-Glo luciferase assay system (Promega) and a Centro xS960 luminometer (Berthold).
  • RNA Cells were seeded in 96-well plates (5 x 104 cells/well) and incubated overnight. Cells were treated with inhibitors for 1 hour and SARS-CoV-2 was added at MOI. Cell lysis and cDNA synthesis were performed 24 h after infection using the SuperPrep II Cell Lysis and RT Kit for Quantitative PCR (qPCR) (SCQ-401; Toyobo, Osaka, Japan) according to the protocol.
  • qPCR Quantitative PCR
  • RT-PCR Quantitative real-time reverse transcription (RT)-PCR of SARS-CoV-2 N and ribosomal protein L13a (Rpl13a) was performed at 95 °C using the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA). 3 minutes followed by 50 cycles of 95°C for 10 seconds and 60°C for 1 minute. Data were normalized using the Rpl13a mRNA expression level of each sample.
  • Cytopathic Assay Cells were seeded in 24-well plates (1.5 ⁇ 10 5 cells/well) and incubated overnight. Cells were treated with inhibitors for 1 hour and then with MOI 1 of SARS-CoV-2. To maintain drug concentration, half of the culture supernatant was replaced daily with fresh medium containing drug. Three days after infection, cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. After washing with water four times, the wells were air-dried and crystal violet was dissolved in ethanol. Absorbance was measured at 595 nm using an iMark microplate reader (Bio-Rad).
  • Example 1 TMPRSS2-independent membrane fusion is induced by SARS-CoV-2 S protein.
  • a quantitative cell fusion assay between target cells co-expressing TMPRSS2 with a receptor such as SARS-CoV-2) or CD26 (for MERS-CoV) was used.
  • cell fusion kinetics induced by SARS-CoV, SARS-CoV-2, and MERS-CoV S proteins were determined using DSP assays.
  • Target cells expressing ACE2 alone or together with TMPRSS2 were used for co-culture with effector cells expressing SARS-CoV S and SARS-CoV-2 S, and cells expressing CD26 alone or together with TMPRSS2 were used for co-culture with MERS- Used for co-culture with effector cells expressing CoV S.
  • Relative cell fusion values were calculated by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both the receptor and TMPRSS2 at 240 min set at 100% (Fig. 1a).
  • target cells expressing ACE2 together with TMPRSS2 were also used for co-culture with effector cells expressing SARS-CoV S and SARS-CoV-2 S, and cells expressing CD26 together with TMPRSS2 were used for co-culture with MERS -used for co-culture with effector cells expressing CoV S.
  • Relative cell fusion values were calculated by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both the receptor and TMPRSS2 in the presence of DMSO set at 100% ( Fig. 1c).
  • target cells expressing ACE2 alone or together with TMPRSS2 were also used to co-culture with effector cells expressing SARS-CoV-2 S.
  • Relative cell fusion values were calculated by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both ACE2 and TMPRSS2 in the presence of DMSO set at 100% (Fig. 1d and Fig. 1e).
  • pepstatin A inhibitor of various aspartic protease
  • leupeptin leupeptin, inhibitor of various cysteine, serine, threonine protease
  • bestatin inhibitor of various amino acids peptidase inhibitor
  • Example 2 TMPRSS2-Independent Membrane Fusion Induced by SARS-CoV-2 S Protein is Suppressed by Various Metalloprotease Inhibitors
  • TMPRSS2-Independent Membrane Fusion Induced by SARS-CoV-2 S Protein is Suppressed by Various Metalloprotease Inhibitors
  • a validated compound library (1,630 compounds approved in clinical trials and 1,885 compounds with pharmacological activity) obtained from the University of Tokyo Drug Discovery Organization. ) were used to search for compounds that specifically inhibit TMPRSS2-independent membrane fusion but do not inhibit TMPRSS2-dependent membrane fusion.
  • relative cell fusion values were calculated by normalizing the RL activity of each compound to the RL activity of the control assay (DMSO alone; set at 100%). Each dot represents an individual compound. Dots within dotted boxes represent compounds that preferentially inhibit TMPRSS2-independent membrane fusion ( ⁇ 30% inhibition of relative cell fusion values using target cells expressing both TMPRSS2 and ACE2, and ACE2 only). >40% inhibition of relative cell fusion values using target cells expressing (Fig. 2a).
  • relative cell fusion values were obtained by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both ACE2 and TMPRSS2 in the presence of DMSO set at 100%. (Fig. 2b).
  • Example 3 The metalloprotease-dependent viral entry pathway is specific to SARS-CoV-2 and its presence depends on the cell type. Based on the findings from protein-mediated cell fusion experiments, we investigated whether a metalloprotease-dependent viral entry pathway to the cell surface exists in the SARS-CoV-2 S protein-enveloped vesicular stomatitis virus (VSV ) was confirmed using a pseudovirus (SARS-CoV-2 pseudovirus).
  • VSV vesicular stomatitis virus
  • VSV vesicular stomatitis virus
  • E-64d inhibited most of the entry routes in OVISE cells (Fig. 3c), suggesting that the endosomal route is almost exclusively in OVISE cells.
  • IGROV1 cells human ovary
  • OUMS-23 cells human colon
  • E-64d suppresses virus entry by about 80%, about 20% remains. This residual amount can be suppressed by combining E-64d with marimastat in IGROV1 cells and nafamostat in OUMS-23 cells (Fig. 3c).
  • IGROV1 cells the endosomal and metalloprotease-dependent invasion pathways coexist
  • OUMS-23 cells the endosomal and TMPRSS2-dependent pathways coexist.
  • HEC50B-TMPRSS2 cells HEC50B-TMPRSS2 cells
  • HEC50B-TMPRSS2 cells approximately 80% of the viral entry pathways were TMPRSS2-dependent, with most of the rest being marimastat-sensitive (Fig. 3e). This indicates the coexistence of metalloprotease-dependent and TMPRSS2-dependent invasion pathways. This result suggested the possibility that cells with both cell surface invasion pathways exist in vivo.
  • Example 4 Mutant strains of SARS-CoV-2 also enter cells via metalloprotease-dependent pathways like conventional strains. It is necessary to confirm whether the metalloprotease-dependent pathway is also utilized in mutant strains that continue to spread infection as the disease progresses. Therefore, we compared the effect of marimastat on the infection of pseudoviruses with the S protein of the conventional strain and the mutant strain.
  • ADAM-10 is involved in the SARS-CoV-2 metalloprotease-dependent entry pathway. Inhibition by metalloprotease inhibitors was analyzed.
  • VeroE6 a
  • HEC50B b
  • A704 c
  • G7570 CellTiter-Glo Luminescent Cell Viability Assay
  • Fig. 5 broad spectrum metalloprotease inhibitor.
  • VeroE6 cells Fig. 5a
  • HEC50B cells Fig. 5b
  • A704 cells Fig. 5c
  • SARS-CoV- 2 pseudovirus entry Fig. 5: broad spectrum metalloprotease inhibitor.
  • Fig. 5 selective metalloprotease inhibitor
  • VeroE6 and HEC50B cells have ⁇ 20-30% E-64d sensitive endosomal pathways (Fig. 3b)
  • Fig. 3b we selected in the presence of E-64d to readily recognize the reduction in metalloprotease-dependent pathways.
  • Fig. 5a, b we analyzed the effects of inhibitory agents (Fig. 5a, b).
  • A704 cells were mostly metalloprotease-dependent pathways (Fig. 3a, b), so selective inhibitors were used alone (Fig. 5c).
  • GW280264X (ADAM10/17 inhibitor) and GI1254023X (MMP9/ADAM10 inhibitor) significantly inhibited metalloprotease-dependent pathways
  • TAPI2 ADAM17 inhibitor
  • MMP2/9i MMP2/9 inhibitor
  • Fig. 5 A similar pattern of inhibition was observed in all three cell lines tested ( Figure 5), and cell viability was not affected by any of the metalloprotease inhibitors at the concentrations used in the experiments ( Figure 5). 6), metalloproteases involved in this pathway are common to the three cell lines, and the inhibitory effect of selective inhibitors strongly suggests the involvement of ADAM10.
  • ADAM10 expression was suppressed in HEC50B cells using 3 types of siRNA with different sequences.
  • HEC50B cells were transfected with two different control siRNAs or three different siRNAs against ADAM10 for 48 hours (Fig. 7a). HEC50B cells were transfected with siRNA for 48 hours and infected with pseudovirus. Relative pseudovirus entry was calculated by normalizing the FL activity of each condition to the FL activity of pseudovirus-infected cells in the absence of siRNA (mock), which was set at 100% (Fig. 7b).
  • SARS-CoV-2 virus metalloprotease-dependent entry pathway including ADAM10 could be a therapeutic target for COVID-19
  • pseudovirus it is necessary that the actual pathogenic SARS-CoV-2 virus infection is suppressed with metalloprotease inhibitors.
  • Marimastat and prinomastat have a blood concentration of approximately 600-900 nM in safe administration set in clinical trials, and in this experiment, they show an infection-inhibitory effect between 300 nM and 1000 nM. Therefore, these drugs have great potential to be used for the treatment of COVID-19. Furthermore, GW280264X (ADAM10/17 inhibitor) and GI1254023X (MMP9/ADAM10 inhibitor), but not TAPI2 (ADAM17 inhibitor), inhibited pathogenic SARS-CoV-2 virus infection in HEC50B cells ( Figure 8b). ).
  • ADAM10-targeted inhibitors suppress infection with the pathogenic SARS-CoV-2 virus and may be therapeutic agents for COVID-19.
  • the inhibitors that are actually administered for treatment include nucleic acid drugs similar to siRNA that suppress ADAM10 expression, as used in this study, ADAM10 enzymatic activity, and complex formation with proteins that functionally bind to ADAM10. A small molecule or antibody that inhibits is envisioned.
  • marimastat and E-64d Fig. 8d
  • marimastat and nafamostat Fig.
  • drugs that target the metalloprotease-dependent entry pathway and other drugs that can alleviate the symptoms of COVID-19 such as drugs that target the endosomal pathway and the TMPRSS2 pathway This is expected to lead to the development of more effective treatments.

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Abstract

The purpose of the present invention is to provide therapeutic and preventive agents for COVID-19. The present invention provides a therapeutic or preventive composition for COVID-19, said composition containing an ADAM10 inhibitor. The ADAM10 inhibitor is, for example, a small molecule, a nuclear acid, an siRNA, or the like. The composition of the present invention may be used in combination with a drug other than the ADAM10 inhibitor.

Description

新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）感染阻害剤Novel coronavirus (SARS-CoV-2) infection inhibitor 関連出願の参照Reference to Related Applications
　本願は、先行する米国出願である６３／２５４，２０２（出願日：２０２１年１０月１１日）の優先権の利益を享受するものであり、その開示内容全体は引用することにより本明細書の一部とされる。 This application claims the benefit of priority from prior U.S. application 63/254,202 (filed Oct. 11, 2021), the entire disclosure of which is hereby incorporated by reference. considered part.
　本発明は新型コロナウイルス感染症（COVID-19、coronavirus disease 2019）の治療または予防剤に関する。 The present invention relates to a therapeutic or prophylactic agent for novel coronavirus infectious disease (COVID-19, coronavirus disease 2019).
　新型コロナウイルス感染症の原因ウイルスであるSARS-CoV-2は、2019年末に認知され、2020年には世界的な感染拡大（パンデミック）となり人類の脅威となった。重症急性呼吸器症候群コロナウイルス（SARS-CoV）や中東呼吸器症候群コロナウイルス（MERS-CoV）も致死的な肺炎や全身症状を引き起こす能力を持っているが、SARS-CoV-2の感染能力と病原性はさらに強化されている。これまでにCOVID-19の治療に十分な効果を示す薬剤は開発されておらず、現在接種されているワクチンが感染拡大の防止に成果を上げているものの、ウイルス変異株の出現により、その効果が不確かになっている。SARS-CoV-2の感染を確実に防ぐワクチンや治療薬を開発するためには、ウイルスの特性や宿主細胞との相互作用をさらに明らかにすることが必要である。 SARS-CoV-2, the virus that causes the new coronavirus infection, was recognized at the end of 2019 and became a global pandemic in 2020, posing a threat to mankind. Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also capable of causing fatal pneumonia and systemic symptoms, but the infectious capacity of SARS-CoV-2 Pathogenicity is further enhanced. To date, no drug has been developed that is sufficiently effective for the treatment of COVID-19, and although the vaccine currently inoculated has been successful in preventing the spread of infection, the emergence of virus mutations has reduced its effectiveness. has become uncertain. Further characterization of the virus and its interactions with host cells are needed to develop vaccines and therapeutics that reliably prevent SARS-CoV-2 infection.
　SARS-CoV-2の細胞侵入には2つのステップがある。ウイルスエンベロープ（ウイルス膜、外膜とも呼ばれる）に存在するスパイク（S）タンパク質は、その受容体結合ドメイン（RBD）を介して、細胞膜に存在する受容体ACE2と結合する。次に，Sタンパク質は，細胞膜に存在する膜貫通型セリンプロテアーゼ2（TMPRSS2）で切断されるか、あるいはウイルスが細胞内にエンドサイトーシスで取り込まれた後にエンドソームのプロテアーゼであるカテプシン－B/L（cathepsin-B/L）によって切断される。いずれの場合でも切断によりSタンパク質内の融合ペプチドが露出して細胞膜やエンドソーム膜に付着する。その結果、ウイルスエンベロープと細胞膜あるいはエンドソーム膜との融合が起こることで、ウイルスRNAは細胞質に侵入し、感染が成立する。 There are two steps for SARS-CoV-2 cell entry. The spike (S) protein present in the viral envelope (also called viral membrane, outer membrane) binds to the receptor ACE2 present in the cell membrane through its receptor binding domain (RBD). The S protein is then cleaved by the transmembrane serine protease 2 (TMPRSS2) present in the cell membrane, or by the endosomal protease cathepsin-B/L after the virus is endocytosed into the cell. cleaved by (cathepsin-B/L). In either case, cleavage exposes the fusion peptide within the S protein and attaches it to the cell or endosomal membrane. As a result, fusion between the viral envelope and the cell membrane or endosomal membrane occurs, allowing viral RNA to enter the cytoplasm and establish infection.
　SARS-CoV-2のTMPRSS2依存的な膜融合を阻害することにより、SARS-CoV-2の細胞への侵入を阻害できることが確認されている（非特許文献１）。 It has been confirmed that SARS-CoV-2 entry into cells can be inhibited by inhibiting TMPRSS2-dependent membrane fusion of SARS-CoV-2 (Non-Patent Document 1).
　本発明は、COVID-19の治療および予防剤の提供を目的とする。 The purpose of the present invention is to provide therapeutic and preventive agents for COVID-19.
　今回、我々はSARS-CoV-2の新たな細胞膜からの侵入経路を同定した。この経路が、TMPRSS2に依存しないものの、様々なメタロプロテアーゼ阻害剤に感受性があることから、メタロプロテアーゼ依存性侵入経路と名付けた。さらに選択的なメタロプロテアーゼ阻害剤と遺伝子特異的なsiRNAを用いた実験により、ADAM10（A disintegrin and metalloproteinase domain-containing protein 10）がこの経路に関与していることを世界で初めて明らかにした。これらの結果から、SARS-CoV-2の生体内での感染拡大とCOVID-19の発症には、ADAM10を含むいくつかのメタロプロテアーゼの協調が重要であり、TMPRSS2やカテプシン－B/Lに加えて、これらのメタロプロテアーゼの触媒活性を阻害したり、発現を抑制したりすることが、COVID-19の治療に有効であることを提唱した。 This time, we identified a new entry route for SARS-CoV-2 through the cell membrane. Although this pathway is independent of TMPRSS2, it is sensitive to various metalloprotease inhibitors, hence we named it the metalloprotease-dependent entry pathway. Furthermore, experiments using selective metalloprotease inhibitors and gene-specific siRNA revealed for the first time in the world that ADAM10 (A disintegrin and metalloproteinase domain-containing protein 10) is involved in this pathway. These results suggest that coordination of several metalloproteases, including ADAM10, is important for the spread of SARS-CoV-2 in vivo and the onset of COVID-19. We proposed that inhibiting the catalytic activity or suppressing the expression of these metalloproteases would be effective for the treatment of COVID-19.
　本発明によれば以下の発明が提供される。
［１］ADAM10阻害剤を含む、COVID-19（coronavirus disease 2019）の治療または予防用組成物あるいは治療または予防剤。
［２］ADAM10阻害剤が核酸である、上記［１］に記載の治療または予防用組成物あるいは治療または予防剤。
［３］核酸がsiRNAである、上記［２］に記載の治療または予防用組成物あるいは治療または予防剤。
［４］他の薬剤（好ましくはCOVID-19治療または予防剤）と併用するための、上記［１］または［２］に記載の治療または予防用組成物あるいは治療または予防剤。
［５］他の薬剤が、マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、上記［４］に記載の治療または予防用組成物あるいは治療または予防剤。
［６］他の薬剤が、TMPRSS２阻害剤である、上記［４］または［５］に記載の治療または予防用組成物あるいは治療または予防剤。
［７］ADAM10阻害剤をそれを必要とする対象に投与する工程を含む、COVID-19（coronavirus disease 2019）の治療または予防方法。
［８］ADAM10阻害剤が核酸である、上記［６］に記載の治療または予防方法。
［９］核酸がsiRNAである、上記［７］に記載の治療または予防方法。
［１０］他の薬剤（好ましくはCOVID-19治療または予防剤）と併用して投与する、上記［７］または［８］に記載の治療または予防方法。
［１１］他の薬剤が、マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、上記［１０］に記載の治療または予防方法。
［１２］他の薬剤が、TMPRSS２阻害剤である、上記［１０］または［１１］に記載の治療または予防方法。
［１３］COVID-19（coronavirus disease 2019）の治療または予防に用いるための、ADAM10阻害剤。
［１４］COVID-19（coronavirus disease 2019）の治療または予防に用いるための、ADAM10阻害剤および他の薬剤（好ましくはCOVID-19治療または予防剤）の組合せ。
［１５］他の薬剤が、マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、上記［１４］に記載の組合せ。
［１６］他の薬剤が、TMPRSS２阻害剤である、上記［１４］または［１５］に記載の組合せ。
［１７］COVID-19（coronavirus disease 2019）の治療または予防用組成物の製造のためのADAM10阻害剤の使用。
［１８］COVID-19（coronavirus disease 2019）の治療または予防用組成物の製造のためのADAM10阻害剤および他の薬剤（好ましくはCOVID-19治療または予防剤）の組合せの使用。
［１９］他の薬剤が、マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、上記［１８］に記載の使用。
［２０］他の薬剤が、TMPRSS２阻害剤である、上記［１８］または［１９］に記載の使用。
［２１］マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物を含む、COVID-19（coronavirus disease 2019）の治療または予防用組成物あるいは治療または予防剤。
［２２］他の薬剤（好ましくはCOVID-19治療または予防剤）と併用するための、上記［２１］に記載の治療または予防用組成物あるいは治療または予防剤。
［２３］他の薬剤が、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、上記［２２］に記載の治療または予防用組成物あるいは治療または予防剤。
［２４］他の薬剤が、TMPRSS２阻害剤である、上記［２２］または［２３］に記載の治療または予防用組成物あるいは治療または予防剤。
［２５］マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物をそれを必要とする対象に投与する工程を含む、COVID-19（coronavirus disease 2019）の治療または予防方法。
［２６］他の薬剤（好ましくはCOVID-19治療または予防剤）と併用して投与する、上記［２５］に記載の治療または予防方法。
［２７］他の薬剤が、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、上記［２６］に記載の治療または予防方法。
［２７］他の薬剤が、TMPRSS２阻害剤である、上記［２６］または［２７］に記載の治療または予防方法。
［２８］COVID-19（coronavirus disease 2019）の治療または予防に用いるための、マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物。
［２９］COVID-19（coronavirus disease 2019）の治療または予防に用いるための、マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物と、他の薬剤（好ましくはCOVID-19治療または予防剤）との組合せ。
［３０］他の薬剤が、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、上記［２９］に記載の組合せ。
［３１］他の薬剤が、TMPRSS２阻害剤である、上記［２９］または［３０］に記載の組合せ。
［３２］COVID-19（coronavirus disease 2019）の治療または予防用組成物の製造のためのADAM10阻害剤の使用。
［３３］COVID-19（coronavirus disease 2019）の治療または予防用組成物の製造のための、マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物と、他の薬剤（好ましくはCOVID-19治療または予防剤）との組合せの使用。
［３４］他の薬剤が、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、上記［３３］に記載の使用。
［３５］他の薬剤が、TMPRSS２阻害剤である、上記［３３］または［３４］に記載の使用。 According to the present invention, the following inventions are provided.
[1] A therapeutic or preventive composition or a therapeutic or preventive agent for COVID-19 (coronavirus disease 2019), containing an ADAM10 inhibitor.
[2] The therapeutic or preventive composition or therapeutic or preventive agent according to [1] above, wherein the ADAM10 inhibitor is a nucleic acid.
[3] The therapeutic or preventive composition or therapeutic or preventive agent according to [2] above, wherein the nucleic acid is siRNA.
[4] The therapeutic or preventive composition or therapeutic or preventive agent according to [1] or [2] above, for use in combination with other drugs (preferably COVID-19 therapeutic or preventive agents).
[5] The above [4], wherein the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine. Therapeutic or preventive composition or therapeutic or preventive agent.
[6] The therapeutic or preventive composition or therapeutic or preventive agent according to [4] or [5] above, wherein the other drug is a TMPRSS2 inhibitor.
[7] A method for treating or preventing COVID-19 (coronavirus disease 2019), which comprises administering an ADAM10 inhibitor to a subject in need thereof.
[8] The therapeutic or preventive method of [6] above, wherein the ADAM10 inhibitor is a nucleic acid.
[9] The therapeutic or preventive method of [7] above, wherein the nucleic acid is siRNA.
[10] The method of treatment or prevention according to [7] or [8] above, which is administered in combination with another agent (preferably a therapeutic or preventive agent for COVID-19).
[11] The above [10], wherein the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine. method of treatment or prevention of
[12] The therapeutic or preventive method of [10] or [11] above, wherein the other drug is a TMPRSS2 inhibitor.
[13] ADAM10 inhibitors for use in treating or preventing COVID-19 (coronavirus disease 2019).
[14] Combinations of ADAM10 inhibitors and other agents (preferably COVID-19 therapeutic or prophylactic agents) for use in the treatment or prevention of COVID-19 (coronavirus disease 2019).
[15] The above [14], wherein the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine. combination.
[16] The combination of [14] or [15] above, wherein the other drug is a TMPRSS2 inhibitor.
[17] Use of an ADAM10 inhibitor for manufacturing a composition for treating or preventing COVID-19 (coronavirus disease 2019).
[18] Use of a combination of an ADAM10 inhibitor and another agent (preferably a COVID-19 therapeutic or prophylactic agent) for the manufacture of a therapeutic or prophylactic composition for COVID-19 (coronavirus disease 2019).
[19] The above [18], wherein the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine. Use of.
[20] The use of [18] or [19] above, wherein the other drug is a TMPRSS2 inhibitor.
[21] A therapeutic or preventive composition or therapeutic or preventive agent for COVID-19 (coronavirus disease 2019), comprising one or two compounds selected from the group consisting of marimastat and prinomastat.
[22] The therapeutic or preventive composition or therapeutic or preventive agent according to [21] above, for use in combination with other agents (preferably COVID-19 therapeutic or preventive agents).
[23] The therapeutic or preventive composition of [22] above, wherein the other drug is one or more compounds selected from the group consisting of E-64d, ammonium chloride, chloroquine and hydroxychloroquine. Or a therapeutic or prophylactic agent.
[24] The therapeutic or preventive composition or therapeutic or preventive agent of the above [22] or [23], wherein the other drug is a TMPRSS2 inhibitor.
[25] A method for treating or preventing COVID-19 (coronavirus disease 2019), which comprises the step of administering one or two compounds selected from the group consisting of marimastat and prinomastat to a subject in need thereof. .
[26] The method of treatment or prevention according to [25] above, which is administered in combination with other agents (preferably COVID-19 therapeutic or preventive agents).
[27] The method of treatment or prevention of [26] above, wherein the other drug is one or more compounds selected from the group consisting of E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
[27] The therapeutic or preventive method of [26] or [27] above, wherein the other drug is a TMPRSS2 inhibitor.
[28] One or two compounds selected from the group consisting of marimastat and prinomastat for use in treating or preventing COVID-19 (coronavirus disease 2019).
[29] One or two compounds selected from the group consisting of marimastat and prinomastat and other agents (preferably COVID- 19 therapeutic or prophylactic agents).
[30] The combination of [29] above, wherein the other drug is one or more compounds selected from the group consisting of E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
[31] The combination of [29] or [30] above, wherein the other agent is a TMPRSS2 inhibitor.
[32] Use of an ADAM10 inhibitor for manufacturing a composition for treating or preventing COVID-19 (coronavirus disease 2019).
[33] One or two compounds selected from the group consisting of marimastat and prinomastat and other drugs ( preferably in combination with COVID-19 therapeutic or prophylactic agents).
[34] The use of [33] above, wherein the other drug is one or more compounds selected from the group consisting of E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
[35] The use of [33] or [34] above, wherein the other drug is a TMPRSS2 inhibitor.
図1は、SARS-CoV-2のSタンパク質によって誘導されるTMPRSS2非依存性の膜融合（例１）を示す図である。（a）各エフェクター細胞（SARS-CoV S、SARS-CoV-2 SまたはMERS-CoV Sを発現）および標的細胞として、対照（Cont）、受容体（Receptor）または受容体＋TMPRSS2（Receptor + TMPRSS2）発現細胞を用いた細胞融合アッセイの定量結果を示すグラフである。縦軸は相対的細胞融合率（Relative Cell fusion (%)）、横軸は時間（分）（min）、各値は平均 ± 標準偏差（SD）（n = 3/グループ）、**はP < 0.01を示す。（b）各エフェクター細胞（SARS-CoV S、SARS-CoV-2 SまたはMERS-CoV Sを発現）および標的細胞として、対照（Cont）、受容体（Receptor）または受容体＋TMPRSS2（Receptor + TMPRSS2）発現細胞を用いた共培養の16時間後のSタンパク質を介した細胞融合の位相差画像である。矢印はシンシチウム形成、スケールバーは100μmを示す。（c）TMPRSS2発現細胞の膜融合に対するナファモスタットの効果（ナファモスタット（nafamostat）存在下における各エフェクター細胞（SARS-CoV S、SARS-CoV-2 SまたはMERS-CoV Sを発現；対照（Cont）、スパイクタンパク質（Spike））および標的細胞として、受容体＋TMPRSS2（Receptor + TMPRSS2）発現細胞を用いた細胞融合アッセイの定量結果）を示すグラフである。縦軸は相対的細胞融合率（Relative Cell fusion (%)）、各値は平均 ± SD（n = 3/グループ）を示す。（d）TMPRSS2を発現する細胞と発現しない細胞における膜融合に対するナファモスタットの効果（ナファモスタット（nafamo）存在下におけるエフェクター細胞（SARS-CoV-2；対照（C）、スパイクタンパク質（S））および標的細胞として、対照（C）、ACE2、またはACE2＋TMPRSS2発現細胞を用いた細胞融合アッセイの定量結果）を示すグラフである。縦軸は相対的細胞融合率（Relative Cell fusion (%)）、各値は平均 ± SD（n = 3/グループ）を示す。（e）ペプスタチンA（pep A）、ベスタチン（best）、ロイペプチン（leu）、E-64d、フーリン阻害剤II（furin II）またはナファモスタット（nafamo）存在下におけるエフェクター細胞（SARS-CoV-2、；対照（C）、スパイクタンパク質（S））および標的細胞として、ACE2、またはTMPRSS2＋ACE2発現細胞を用いた細胞融合アッセイの定量結果を示すグラフである。縦軸は相対的細胞融合率（Relative Cell fusion (%)）、各値は平均 ± SD（n = 3/グループ）を示す。FIG. 1 shows TMPRSS2-independent membrane fusion induced by SARS-CoV-2 S protein (Example 1). (a) Each effector cell (expressing SARS-CoV S, SARS-CoV-2 S or MERS-CoV S) and target cells as control (Cont), receptor (Receptor) or receptor + TMPRSS2 (Receptor + TMPRSS2) FIG. 10 is a graph showing quantitative results of cell fusion assay using expressing cells. FIG. Vertical axis is relative cell fusion (%), horizontal axis is time (minutes) (min), each value is mean ± standard deviation (SD) (n = 3/group), ** is P < 0.01. (b) each effector cell (expressing SARS-CoV S, SARS-CoV-2 S or MERS-CoV S) and target cells as control (Cont), receptor (Receptor) or receptor + TMPRSS2 (Receptor + TMPRSS2) Phase-contrast images of S protein-mediated cell fusion after 16 hours of co-culture with expressing cells. Arrows indicate syncytium formation, scale bar 100 μm. (c) Effect of nafamostat on membrane fusion of TMPRSS2-expressing cells (expressing each effector cell (SARS-CoV S, SARS-CoV-2 S or MERS-CoV S in the presence of nafamostat; control (Cont) , Spike) and quantification results of a cell fusion assay using receptor + TMPRSS2 (Receptor + TMPRSS2)-expressing cells as target cells). The vertical axis represents the relative cell fusion (%), and each value represents the mean ± SD (n = 3/group). (d) Effect of nafamostat on membrane fusion in cells expressing and not expressing TMPRSS2 (effector cells (SARS-CoV-2; control (C), spike protein (S) in the presence of nafamostat) and 2 is a graph showing quantitative results of a cell fusion assay using control (C), ACE2, or ACE2+TMPRSS2-expressing cells as target cells). The vertical axis represents the relative cell fusion (%), and each value represents the mean ± SD (n = 3/group). (e) effector cells (SARS-CoV-2, SARS-CoV-2, ; is a graph showing the quantitative results of a cell fusion assay using ACE2- or TMPRSS2+ACE2-expressing cells as control (C), spike protein (S)) and target cells. The vertical axis represents the relative cell fusion (%), and each value represents the mean ± SD (n = 3/group). 図2は、SARS-CoV-2のSタンパク質によって誘導されるTMPRSS2非依存性の膜融合のメタロプロテアーゼ阻害剤による抑制（例２）を示す図である。（a）各化合物存在下におけるエフェクター細胞（SARS-CoV-2 S）および標的細胞として、TMPRSS2＋ACE2発現細胞（X軸）またはACE2発現細胞（Y軸）を用いた細胞融合アッセイのスクリーニング結果の比較を示すグラフである。X軸は、各化合物（1μM）存在下でTMPRSS2とACE2の両方を発現する細胞を使用した相対的な細胞融合値（n =1）を示し、Y軸は、各化合物（1μM）存在下でACE2のみを発現する細胞を使用した相対的な細胞融合値（n =1）を示す。ナファモスタット（nafamostat）およびカモスタット（camostat）はTMPRSS2依存性の膜融合を特異的に抑制した。一方で、点線で囲った枠内の化合物はTMPRSS2非依存性の膜融合を選択的に阻害した。この中にイロマスタット（ilomastat）およびCTS-1027という2種類のメタロプロテアーゼ阻害剤が含まれていた。（b）上段は、SARS-CoV-2 Sタンパク質を発現する細胞とACE2を単独でまたはTMPRSS2と組み合わせて発現する細胞との共培養における細胞融合に対するメタロプロテアーゼ阻害剤の効果（各化合物（イロマスタット（ilomastat）、CTS-1027、マリマスタット（marimastat）、プリノマスタット（prinomastat））存在下におけるエフェクター細胞（対照（Cont）、SARS-CoV-2スパイクタンパク質（SARS-CoV-2 Spike））および標的細胞として、対照（Cont）、ACE2、またはTMPRSS2＋ACE2発現細胞を用いた細胞融合アッセイの定量結果）を示すグラフである。縦軸は相対的細胞融合率（Relative Cell fusion (%)）を示す。下段は、各化合物（イロマスタット（ilomastat）、CTS-1027、マリマスタット（marimastat）、プリノマスタット（prinomastat））存在下におけるエフェクター細胞（SARS-CoV-2 S）および標的細胞として、DSP1-7およびDSP8-11共発現細胞を用いたDSPアッセイの定量結果を示すグラフである。縦軸は相対的DSP活性（％）（Relative DSP activity (%)）を示す。FIG. 2 shows the suppression of TMPRSS2-independent membrane fusion induced by SARS-CoV-2 S protein by metalloprotease inhibitors (Example 2). (a) Comparison of screening results of cell fusion assay using TMPRSS2 + ACE2-expressing cells (X-axis) or ACE2-expressing cells (Y-axis) as effector cells (SARS-CoV-2 S) and target cells in the presence of each compound. It is a graph showing. The X-axis shows the relative cell fusion value (n = 1) using cells expressing both TMPRSS2 and ACE2 in the presence of each compound (1 µM), and the Y-axis shows the value in the presence of each compound (1 µM). Relative cell fusion values (n = 1) using cells expressing ACE2 only are shown. Nafamostat and camostat specifically inhibited TMPRSS2-dependent membrane fusion. On the other hand, the compounds in the dotted box selectively inhibited TMPRSS2-independent membrane fusion. These included two metalloprotease inhibitors, ilomastat and CTS-1027. (b) The upper panel shows the effect of metalloprotease inhibitors on cell fusion in co-cultures of cells expressing SARS-CoV-2 S protein and cells expressing ACE2 alone or in combination with TMPRSS2 (each compound (Ilomastat ( Effector cells (Control (Cont), SARS-CoV-2 Spike) and target cells in the presence of ilomastat, CTS-1027, marimastat, prinomastat Quantitative results of a cell fusion assay using control (Cont), ACE2, or TMPRSS2+ACE2-expressing cells). The vertical axis indicates the relative cell fusion rate (Relative Cell fusion (%)). The lower row shows effector cells (SARS-CoV-2 S) and target cells in the presence of each compound (ilomastat, CTS-1027, marimastat, prinomastat), DSP1-7 and FIG. 10 is a graph showing quantitative results of DSP assay using DSP8-11 co-expressing cells. FIG. The vertical axis indicates relative DSP activity (%). 図3は、SARS-CoV-2に特異的なメタロプロテアーゼ依存性のウイルス侵入経路の存在（例３）を示す図である。縦軸はシュードウイルス侵入（対照に対する％）（Pseudovirus entry (% of control）を示す。（a）各細胞株を用いた各化合物（マリマスタット（marimastat）、E-64d、ナファモスタット（nafamostat））存在下におけるシュードウイルス（対照（Cont）、SARS-CoV-2）を用いた感染アッセイの定量結果を示すグラフである。（b）～（e）各細胞株を用いた各化合物（E-64d、ナファモスタット（nafamostat、nafa、nafamo）、マリマスタット（marimastat、marima）、E-64dとマリマスタット（E-64d+marima））存在下におけるシュードウイルス（対照（Cont）、SARS-CoV-2）を用いた感染アッセイの定量結果を示すグラフである。Figure 3 shows the presence of a SARS-CoV-2-specific metalloprotease-dependent viral entry pathway (Example 3). The vertical axis indicates pseudovirus entry (% of control). (a) Each compound using each cell line (marimastat, E-64d, nafamostat) Graphs showing quantitative results of infection assays using pseudoviruses (control (Cont), SARS-CoV-2) in the presence of (b) to (e) each compound (E-64d) using each cell line , nafamostat, nafa, nafamo, marimastat, marima, E-64d and E-64d+marima) in the presence of pseudoviruses (Control (Cont), SARS-CoV-2) is a graph showing the quantitative results of an infection assay using . 図4は、侵入経路のパターンは、SARS-CoV-2の様々な変異体で保存されていること（SARS-CoV-2変異株のメタロプロテアーゼ依存性経路を介する細胞への侵入）（例４）を示す図である。（a）各SARS-CoV-2株（対照（Cont）、野生（WT）、各変異株）のSタンパク質の発現を示す図である。Sタンパク質は、Sタンパク質のC末端のFlagタグに結合する抗Flagタグ抗体を使用して検出した（上）。水疱性口内炎ウイルス マトリックスタンパク質(VSV M)は、対照とした（下）。S0は切断されていないSタンパク質、S2はSタンパク質の切断されたS2ドメインを示す。（b）HEC50B 細胞におけるSARS-CoV-2 Sを持つシュードウイルスの侵入に対するE-64dとマリマスタットの効果（HEC50Bでの各化合物（E-64d、マリマスタット（marimastat））存在下における各SARS-CoV-2株（対照（Cont）、野生（WT）、各変異株）を用いた感染アッセイの定量結果）を示すグラフである。縦軸はシュードウイルス侵入（DMSOに対する％）（Pseudovirus entry (% of DMSO）を示す。Figure 4 shows that the pattern of entry pathways is conserved in various mutants of SARS-CoV-2 (entry of SARS-CoV-2 mutants into cells via the metalloprotease-dependent pathway) (Example 4). ). (a) A diagram showing the expression of the S protein of each SARS-CoV-2 strain (control (Cont), wild (WT), each mutant strain). The S protein was detected using an anti-Flag tag antibody that binds to the Flag tag at the C-terminus of the S protein (top). Vesicular stomatitis virus matrix protein (VSV M) served as a control (bottom). S0 indicates the uncleaved S protein, S2 indicates the truncated S2 domain of the S protein. (b) Effects of E-64d and marimastat on the entry of pseudoviruses with SARS-CoV-2 S in HEC50B cells (each SARS- Fig. 10 is a graph showing quantitative results of infection assay using CoV-2 strains (control (Cont), wild (WT), each mutant strain). The vertical axis shows pseudovirus entry (% of DMSO). 図5は、SARS-CoV-2のメタロプロテアーゼ依存性侵入経路へのADAM-10の関与（例５）を示す図である。各細胞株（VeroE6、HEC50B、A704）での各化合物（広範囲メタロプロテアーゼ阻害剤（broad spectrum metalloprotease inhibitor）、選択的メタロプロテアーゼ阻害剤（selective metalloprotease inhibitor））存在下における各株（対照（Cont、Sタンパク質を含まないシュードウイルスに感染した細胞）、SARS-CoV-2 S、VSV-G）を用いた感染アッセイの定量結果を示すグラフである。縦軸はシュードウイルス侵入（DMSOに対する％）（Pseudovirus entry (% of DMSO）を示す。FIG. 5 shows involvement of ADAM-10 in the metalloprotease-dependent entry pathway of SARS-CoV-2 (Example 5). Each strain (control (Cont, S FIG. 10 is a graph showing the quantitative results of infection assays using protein-free pseudovirus-infected cells), SARS-CoV-2 S, VSV-G). The vertical axis shows pseudovirus entry (% of DMSO). 図6は、細胞生存率に対する薬物の効果（メタロプロテアーゼ阻害剤によっては細胞の生存率が影響を受けないこと）（例５）を示す図である。各細胞株（VeroE6、HEC50B、A704）での各化合物存在下およびE-64d非添加（Without E-64d）または25μMのE-64d（With 25μM E-64d）における細胞生存率（DMSOに対する％）（Cell viability（% of DMSO））、各値は平均 ± SD（n = 3/グループ）を示す。Figure 6 shows the effect of drug on cell viability (cell viability is not affected by metalloprotease inhibitors) (Example 5). Cell viability (% of DMSO) in the presence of each compound and without E-64d (Without E-64d) or 25 μM E-64d (With 25 μM E-64d) for each cell line (VeroE6, HEC50B, A704) (Cell viability (% of DMSO)), each value represents the mean ± SD (n = 3/group). 図7は、ACE2（上）、ADAM10（中央）およびチューブリン（下）発現に対するADAM10ノックダウンの効果（siRNAによる、ADAM10の発現抑制およびSARS-CoV-2シュードウイルス侵入抑制）（例５）を示す図である。（a）２種類の対照およびADAM10に対する３種類の配列の異なるsiRNA（48時間トランスフェクト）がADAM10の発現を抑制することを示す図である（モック（mock）、対照（Cont）、前駆体（precursor）、活性型ADAM10（active ADAM10）、チューブリン（Tublin））。（b）各siRNAが各シュードウイルス（SARS-CoV-2、SARS-CoV、MERS-CoV、VSV）の侵入に及ぼす影響を示すグラフである。縦軸はシュードウイルス侵入（モックに対する％）（Pseudovirus entry (% of mock）、各値は平均 ± SD（n = 3/グループ）を示す。Figure 7 shows the effect of ADAM10 knockdown on ACE2 (top), ADAM10 (middle) and tubulin (bottom) expression (suppression of ADAM10 expression and suppression of SARS-CoV-2 pseudovirus entry by siRNA) (Example 5). FIG. 4 is a diagram showing; (a) Two types of control and three types of siRNA with different sequences against ADAM10 (transfected for 48 hours) suppress the expression of ADAM10 (mock, control (Cont), precursor ( precursor), active ADAM10 (active ADAM10), Tublin). (b) Graph showing the effect of each siRNA on invasion of each pseudovirus (SARS-CoV-2, SARS-CoV, MERS-CoV, VSV). Pseudovirus entry (% of mock) is plotted on the vertical axis, and each value represents the mean ± SD (n = 3/group). 図8は、メタロプロテアーゼ阻害剤またはADAM10ノックダウンによるSARS-CoV-2生ウイルスの感染増殖の抑制（例６）を示す図である。縦軸は相対的SARS-CoV-2 N発現（/rpl13a）を対数（Log10[Relative SARS-CoV-2 N expression(/rpl13a)]）で示す（マリマスタット（marimastat、marima）、プリノマスタット（prinomastat）、ナファモスタット（nafamo））。FIG. 8 shows inhibition of SARS-CoV-2 live virus infection growth by metalloprotease inhibitors or ADAM10 knockdown (Example 6). The vertical axis shows the relative SARS-CoV-2 N expression (/rpl13a) in logarithm (Log10 [Relative SARS-CoV-2 N expression (/rpl13a)]) (marimastat, marima), prinomastat ( prinomastat), nafamostat).
発明の具体的説明Specific description of the invention
COVID-19の治療または予防剤
　本発明の組成物および剤は、ADAM10阻害剤を有効成分として含んでなるものである。ここで、「ADAM10」とはA disintegrin and metalloproteinase domain-containing protein 10の略語であり、ADAMファミリーと呼ばれるメタロプロテアーゼの一種である。 A therapeutic or preventive agent for COVID-19 The composition and agent of the present invention comprise an ADAM10 inhibitor as an active ingredient. Here, "ADAM10" is an abbreviation for A disintegrin and metalloproteinase domain-containing protein 10, which is a kind of metalloprotease called ADAM family.
　本発明においては、ヒトADAM10遺伝子は、Genbank/NCBI Gene ID: 102で公表された塩基配列を基準とする。本発明においてはまた、ヒトADAM10タンパク質は、NCBI Reference Sequence: NP_001101.1およびNP_001307499.1で公表されたアミノ酸配列を基準とする。本発明においてはさらに、ヒトADAM10のmRNAは、NCBI Reference Sequence: NM_001110.4およびNM_001320570.2を基準とする。 In the present invention, the human ADAM10 gene is based on the nucleotide sequence published in Genbank/NCBI Gene ID: 102. Also in the present invention, the human ADAM10 protein is referenced to the amino acid sequences published at NCBI Reference Sequence: NP_001101.1 and NP_001307499.1. Further, in the present invention, human ADAM10 mRNA is based on NCBI Reference Sequence: NM_001110.4 and NM_001320570.2.
　本発明においてADAM10阻害剤とは、ADAM10を阻害できるものであればよく、ADAM10の発現を阻害する物質およびADAM10の機能を阻害する物質を含む意味で用いられる。本発明においてADAM10阻害剤は、ADAM10を特異的に阻害するものを使用することができる。ADAM10の発現を阻害する物質としては、ADAM10に対する核酸（例えば、アンチセンスDNAのようなアンチセンス核酸、siRNA、shRNA、microRNA、gRNA、リボザイム等のADAM10を標的にした核酸）が挙げられる。ADAM10の機能を阻害する物質としては、ADAM10と相互作用して当該機能を阻害する物質が挙げられ、例えば、低分子、抗体、ペプチド、核酸、アプタマーが挙げられる。 In the present invention, an ADAM10 inhibitor is any substance that can inhibit ADAM10, and is used in the sense of including substances that inhibit the expression of ADAM10 and substances that inhibit the function of ADAM10. ADAM10 inhibitors that specifically inhibit ADAM10 can be used in the present invention. Substances that inhibit the expression of ADAM10 include nucleic acids against ADAM10 (eg, antisense nucleic acids such as antisense DNA, nucleic acids targeting ADAM10 such as siRNA, shRNA, microRNA, gRNA, and ribozymes). Substances that inhibit the function of ADAM10 include substances that interact with ADAM10 to inhibit the function, and examples thereof include small molecules, antibodies, peptides, nucleic acids, and aptamers.
　本発明において核酸を構成する塩基は、天然の塩基の他、生体内安定性を備えた修飾塩基または人工塩基を用いることもできる。また核酸配列は、ターゲット核酸と完全対合する配列だけではなく、発現阻害活性を保持する範囲でターゲット配列と対合しないミスマッチ配列が含まれていてもよい。 In the present invention, in addition to natural bases, modified bases with in vivo stability or artificial bases can also be used as the bases that constitute the nucleic acids. In addition, the nucleic acid sequence may contain not only sequences that perfectly match with the target nucleic acid, but also mismatch sequences that do not match with the target sequence as long as the expression inhibitory activity is maintained.
　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-forming sites, suppression of translocation from the nucleus to the cytoplasm by hybridization with mRNA, capping sites and poly(A) addition sites Suppression of splicing by hybridization with , Suppression of translation initiation by hybridization with the translation initiation factor binding site, Translation suppression by hybridization with the ribosome binding site near the initiation codon, and Hybridization with the translational region of mRNA and the polysome binding site 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.
　ADAM10に対するアンチセンス核酸は、例えば、前述のADAM10の遺伝子配列、前述のADAM10のアミノ酸配列をコードする塩基配列および前述のADAM10のmRNA配列から選ばれる一部の塩基配列と相補的な一本鎖核酸をいう。当該核酸は、天然由来の核酸でも人工核酸でもよく、ＤＮＡおよびＲＮＡのいずれに基づくものでもよい。アンチセンス核酸の長さは、通常約１５塩基からmRNAの全長と同程度の長さであり、約１５～約３０塩基長が好ましい。アンチセンス核酸の相補性は必ずしも１００％である必要はなく、生体内でADAM10をコードするＤＮＡまたはＲＮＡと相補的に結合しうる程度でよい。 The antisense nucleic acid against ADAM10 is, for example, a single-stranded nucleic acid complementary to a partial nucleotide sequence selected from the ADAM10 gene sequence described above, the nucleotide sequence encoding the ADAM10 amino acid sequence described above, and the ADAM10 mRNA sequence described above. Say. Such nucleic acids may be naturally occurring or artificial nucleic acids, and may be based on both DNA and RNA. The length of the antisense nucleic acid is usually about 15 bases to the same length as the full-length mRNA, preferably about 15 to about 30 bases. The complementarity of the antisense nucleic acid does not necessarily have to be 100%, and may be such that it can complementarily bind to ADAM10-encoding DNA or RNA in vivo.
　siRNA（small interfering RNA）は、ＲＮＡ干渉（mRNAの分解）による遺伝子サイレンシングのために用いられる、人工的に合成された低分子２本鎖ＲＮＡであり、当該二本鎖ＲＮＡを生体内で供給することのできるsiRNA発現ベクターを含む意味で用いられるものとする。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に結合し切断し、これにより、配列特異的に遺伝子の発現を抑制することができる。siRNAは、センス鎖およびアンチセンス鎖オリゴヌクレオチドをＤＮＡ／ＲＮＡ自動合成機でそれぞれ合成し、例えば、適当なアニーリング緩衝液中、９０～９５℃で約１分程度変性させた後、３０～７０℃で約１～８時間アニーリングさせることにより調製することができる。siRNAの長さは、１９～２７塩基対が好ましく、２１～２５塩基対または２１～２３塩基対がより好ましい。 siRNA (small interfering RNA) is an artificially synthesized small double-stranded RNA used for gene silencing by RNA interference (mRNA degradation), and the double-stranded RNA is supplied in vivo. It shall be used in the sense of including an siRNA expression vector capable of siRNAs introduced into cells bind to the RNA-induced silencing complex (risc). This complex binds to and cleaves mRNA having a sequence complementary to siRNA, thereby suppressing gene expression in a sequence-specific manner. siRNA is prepared by synthesizing sense strand and antisense strand oligonucleotides with an automatic DNA/RNA synthesizer. can be prepared by annealing for about 1 to 8 hours at . The length of the siRNA is preferably 19-27 base pairs, more preferably 21-25 base pairs or 21-23 base pairs.
　ADAM10に対するsiRNAは、ADAM10遺伝子から転写されるmRNAの分解（ＲＮＡ干渉）を引き起こすようにその塩基配列に基づいて設計することができる。ADAM10の発現を阻害するsiRNAとしては、例えば、前述のADAM10のmRNA配列を標的配列とするsiRNAが挙げられる。 siRNA against ADAM10 can be designed based on its base sequence to cause degradation (RNA interference) of mRNA transcribed from the ADAM10 gene. Examples of siRNAs that inhibit the expression of ADAM10 include siRNAs whose target sequence is the above-mentioned ADAM10 mRNA sequence.
　shRNA（short hairpin RNA）は、ＲＮＡ干渉（mRNAの分解）による遺伝子サイレンシングのために用いられる、人工的に合成されたヘアピン型の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に結合し切断し、これにより、配列特異的に遺伝子の発現を抑制することができる。 shRNA (short hairpin RNA) is an artificially synthesized hairpin-shaped RNA sequence used for gene silencing by RNA interference (mRNA degradation). 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 a method similar to siRNA. may be prepared by Hairpin structures of shRNAs introduced into cells are cleaved into siRNAs and bind to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA having a sequence complementary to siRNA, thereby suppressing gene expression in a sequence-specific manner.
　ADAM10に対するshRNAは、ADAM10遺伝子から転写されるmRNAの分解（ＲＮＡ干渉）を引き起こすようにその塩基配列に基づいて設計することができる。ADAM10の発現を阻害するshRNAとしては、例えば、前述のADAM10のmRNA配列を標的配列とするshRNAが挙げられる。 The shRNA against ADAM10 can be designed based on its base sequence to cause degradation (RNA interference) of mRNA transcribed from the ADAM10 gene. Examples of shRNAs that inhibit the expression of ADAM10 include shRNAs whose target sequence is the above-mentioned ADAM10 mRNA sequence.
　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。本発明においては特定の塩基配列を有するmiRNAをベクターによって細胞に導入し、生体に投与することにより、ADAM10遺伝子の発現を抑制することができる。 miRNA (microRNA, microRNA) is a functional nucleic acid that is encoded on the genome and eventually becomes a micro 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 In the present invention, ADAM10 gene expression can be suppressed by introducing miRNA having a specific nucleotide sequence into cells using a vector and administering the miRNA to a living body.
　gRNA（ガイドＲＮＡ）は、ゲノム編集技術に用いられるＲＮＡ分子である。ゲノム編集技術において、gRNAは標的配列を特異的に認識し、Ｃａｓ９タンパク質の標的配列への結合を導き、遺伝子のノックアウトやノックインを可能とする。本発明においてはADAM10遺伝子を標的とするgRNAを生体内に投与することにより、生体内でADAM10遺伝子の発現を抑制することができる。gRNAはsgRNA（シングルガイドＲＮＡ）を含む意味で用いられるものとする。ゲノム編集技術におけるgRNAの設計方法は広く知られており、例えば、Benchmarking CRISPR on-target sgRNA design, Yan et al., Brief Bioinform, 15 Feb 2017を参照することにより適宜設計することができる。 gRNA (guide RNA) is an RNA molecule used in genome editing technology. In genome editing technology, gRNA specifically recognizes the target sequence, guides the binding of Cas9 protein to the target sequence, and enables gene knockout and knockin. In the present invention, ADAM10 gene expression can be suppressed in vivo by administering gRNA targeting the ADAM10 gene in vivo. gRNA shall be used in the meaning including sgRNA (single guide RNA). The design method of gRNA in genome editing technology is widely known, for example, Benchmarking CRISPR on-target sgRNA design, Yan et al., Brief Bioinform, 15 Feb 2017.
　リボザイムは、触媒活性を有するＲＮＡである。リボザイムには種々の活性を有するものがあるが、ＲＮＡを切断する酵素としてのリボザイムの研究により、ＲＮＡの部位特異的な切断を目的とするリボザイムの設計が可能となっている。リボザイムは、グループＩイントロン型、ＲＮａｓｅＰに含まれるM1RNA等の４００ヌクレオチド以上の大きさのものであってもよく、ハンマーヘッド型、ヘアピン型等と呼ばれる４０ヌクレオチド程度のものであってもよい。 A ribozyme is an RNA with catalytic activity. Although some ribozymes have various activities, studies on ribozymes as RNA-cleaving enzymes have made it possible to design ribozymes for the purpose of site-specific cleavage of RNA. The ribozyme may be group I intron type, M1 RNA contained in RNaseP, etc., having a size of 400 nucleotides or more, or hammerhead type, hairpin type, etc. having about 40 nucleotides.
　アプタマーは、核酸アプタマーおよびペプチドアプタマーを含むものである。本発明において用いる核酸アプタマーおよびペプチドアプタマーは、ＳＥＬＥＸ法（Systematic Evolution of Ligands by Exponential enrichment）やmRNAディスプレイ（mRNA display）法などに代表される、ライブラリー分子と標的分子との複合体を試験管内で形成させた後にアフィニティーを基準に選抜する、試験管内分子進化法を用いて得ることができる。 Aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers and peptide aptamers used in the present invention are represented by the SELEX method (Systematic Evolution of Ligands by Exponential enrichment) and the mRNA display method. They can be obtained using in vitro molecular evolution techniques that are formed and then selected on the basis of affinity.
　アンチセンス核酸、siRNA、shRNA、miRNA、リボザイムおよび核酸アプタマーは、安定性や活性を向上させるために、種々の化学修飾を含んでいてもよい。例えば、ヌクレアーゼ等の加水分解酵素による分解を防ぐために、リン酸残基を、例えば、ホスホロチオエート（ＰＳ）、メチルホスホネート、ホスホロジチオネート等の化学修飾リン酸残基に置換してもよい。また、少なくとも一部をペプチド核酸（ＰＮＡ）等の核酸類似体により構成してもよい。 Antisense nucleic acids, siRNAs, shRNAs, miRNAs, ribozymes, and nucleic acid aptamers may contain various chemical modifications to improve their stability and activity. For example, phosphate residues may be substituted with chemically modified phosphate residues such as phosphorothioates (PS), methylphosphonates, phosphorodithionates, etc., to prevent degradation by hydrolases such as nucleases. Moreover, at least a part thereof may be composed of a nucleic acid analogue such as a peptide nucleic acid (PNA).
　ADAM10に対する抗体とは、ADAM10に特異的に結合する抗体であって、結合することによりADAM10の機能を阻害する抗体をいう。本発明では、抗体として、モノクローナル抗体、ポリクローナル抗体、キメラ抗体、ヒト化抗体、ヒト抗体、マウス抗体、ラット抗体、ラクダ抗体、抗体フラグメント（例えば、Fab、Fv、Fab’、F(ab’)２、scFv)等のいずれを使用してもよく、これらは当業者であれば公知の手法に従って調製することができる。 An antibody against ADAM10 is an antibody that specifically binds to ADAM10 and inhibits the function of ADAM10 by binding. In the present invention, antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, mouse antibodies, rat antibodies, camelid antibodies, antibody fragments (e.g., Fab, Fv, Fab', F (ab') 2 , scFv) and the like may be used, and these can be prepared according to known techniques by those skilled in the art.
　ADAM10に対する抗体は、ADAM10タンパク質またはその一部を抗原として、公知の抗体または抗血清の製造法に従って製造することができる。ADAM10タンパク質またはその一部は、公知のタンパク質発現法および精製法によって調製することができる。ADAM10タンパク質としては、例えば前述のADAM10の配列情報によって規定されるヒトADAM10等が挙げられるが、これに限定されるものではない。種々の生物由来のADAM10タンパク質を免疫原として用いてもよい。本発明に用いることができるADAM10に対する抗体はまた、ファージ・ディスプレー法（例えば、FEBS Letter, 441:20-24(1998)を参照）を介して作製することもできる。 Antibodies against ADAM10 can be produced according to known antibody or antiserum production methods using the ADAM10 protein or a portion thereof as an antigen. ADAM10 protein or portions thereof can be prepared by known protein expression and purification methods. Examples of the ADAM10 protein include, but are not limited to, human ADAM10 defined by the ADAM10 sequence information described above. ADAM10 proteins from various organisms may be used as immunogens. Antibodies to ADAM10 that can be used in the present invention can also be generated via phage display technology (see, eg, FEBS Letter, 441:20-24 (1998)).
　本発明の組成物および剤はまた、マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物を有効成分として含んでなるものである。 The compositions and agents of the present invention also contain, as active ingredients, one or two compounds selected from the group consisting of marimastat and prinomastat.
　後記実施例の通り、SARS-CoV-2のメタロプロテアーゼ依存性侵入経路にADAM10が関与すること、ADAM10を阻害することによりSARS-CoV-2の感染を阻害できることが示された。従って、ADAM10阻害剤はCOVID-19の治療または予防のための有効成分として使用することができる。 As shown in the examples below, it was shown that ADAM10 is involved in the metalloprotease-dependent entry pathway of SARS-CoV-2, and that SARS-CoV-2 infection can be inhibited by inhibiting ADAM10. Therefore, ADAM10 inhibitors can be used as active ingredients for the treatment or prevention of COVID-19.
　SARS-CoV-2は、最初に発見されたウイルス株のみならず、その変異株（例えば、B.1.1.7系統（アルファ株）、B.1.351系統（ベータ株）、P.1系統（ガンマ株）、B.1.617.2系統（デルタ株）、B.1.1.529系統（オミクロン株））を含む。なお、ＳＡＲＳ－ＣｏＶ－２は、重症急性呼吸器症候群コロナウイルス２（severe acute respiratory syndrome coronavirus-2）と同義である。 SARS-CoV-2 exists not only in the originally discovered virus strain, but also in variants thereof (e.g. strain B.1.1.7 (alpha strain), B.1.351 strain (beta strain), P.1 strain (gamma strain), B.1.617.2 strain (Delta strain), B.1.1.529 strain (Omicron strain)). SARS-CoV-2 is synonymous with severe acute respiratory syndrome coronavirus-2.
　本発明の組成物および剤は、医薬品または医薬組成物として提供することができる。本発明の医薬品および医薬組成物は、本発明の有効成分と、薬学的に許容される担体とを含有してなるものである。本発明の医薬品および医薬組成物には遺伝子治療を目的とした医薬品および医薬組成物も含まれる。このような医薬品および医薬組成物は、アンチセンス核酸、siRNA、shRNA、microRNA、gRNA、リボザイム等のADAM10を標的にした核酸を有効成分として含むものである。 The compositions and agents of the present invention can be provided as pharmaceuticals or pharmaceutical compositions. The drug and pharmaceutical composition of the present invention contain the active ingredient of the present invention and a pharmaceutically acceptable carrier. The medicaments and pharmaceutical compositions of the present invention also include medicaments and pharmaceutical compositions intended for gene therapy. Such pharmaceuticals and pharmaceutical compositions contain ADAM10-targeted nucleic acids such as antisense nucleic acids, siRNA, shRNA, microRNA, gRNA, and ribozymes as active ingredients.
　本発明の組成物および剤は、本発明の有効成分以外の他の薬剤と併用してもよい。すなわち、本発明の組成物および剤は、本発明の有効成分以外の他の薬剤をさらに含んでいてもよく、この場合、合剤として剤形を一体としてもよい。また本発明の組成物および剤は、本発明の有効成分以外の他の薬剤とともに、異なる製剤として投与してもよく、この場合、同時に投与しても、あるいは、時間をずらして投与してもよい。本発明の別の側面によると、本発明の有効成分およびそれ以外の他の薬剤の組合せが提供される。 The composition and agent of the present invention may be used in combination with other agents other than the active ingredient of the present invention. That is, the compositions and agents of the present invention may further contain drugs other than the active ingredient of the present invention, and in this case, the dosage form may be integrated as a combination drug. In addition, the composition and agent of the present invention may be administered together with other drugs other than the active ingredient of the present invention as different formulations, in which case they may be administered simultaneously or at different times. good. According to another aspect of the invention there is provided a combination of active ingredients of the invention and other agents.
　本発明の有効成分がADAM10阻害剤である場合、ADAM10阻害剤以外の他の薬剤としては、SARS-CoV-2感染阻害剤（特にCOVID-19治療または予防剤）が挙げられる。このような他の薬剤としては、例えば、マリマスタットおよびプリノマスタット等のメタロプロテアーゼ阻害剤、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキン等のカテプシン－B/L阻害剤が挙げられる。このような他の薬剤としてはまた、例えば、ナファモスタット、カモスタット等のTMPRSS２阻害剤が挙げられる。 When the active ingredient of the present invention is an ADAM10 inhibitor, drugs other than ADAM10 inhibitors include SARS-CoV-2 infection inhibitors (especially COVID-19 therapeutic or preventive agents). Such other agents include, for example, metalloprotease inhibitors such as marimastat and prinomastat, cathepsin-B/L inhibitors such as E-64d, ammonium chloride, chloroquine and hydroxychloroquine. Such other agents also include TMPRSS2 inhibitors such as, for example, nafamostat, camostat.
　本発明の有効成分がマリマスタットおよび／またはプリノマスタットである場合、これら化合物以外の他の薬剤としては、SARS-CoV-2感染阻害剤（特にCOVID-19治療または予防剤）が挙げられる。このような他の薬剤としては、例えば、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキン等のカテプシン－B/L阻害剤が挙げられる。このような他の薬剤としてはまた、例えば、ナファモスタット、カモスタット等のTMPRSS２阻害剤が挙げられる。 When the active ingredient of the present invention is marimastat and/or prinomastat, drugs other than these compounds include SARS-CoV-2 infection inhibitors (especially COVID-19 therapeutic or preventive agents). Such other agents include cathepsin-B/L inhibitors such as E-64d, ammonium chloride, chloroquine and hydroxychloroquine. Such other agents also include TMPRSS2 inhibitors such as, for example, nafamostat, camostat.
　本発明の有効成分を対象に投与する場合、COVID-19の治療または予防効果が得られる限り、投与経路は特に限定されるものではないが、経口投与または非経口投与（例えば、静脈内投与、皮下投与、腹腔内投与）を選択することができる。 When administering the active ingredient of the present invention to a subject, the route of administration is not particularly limited as long as the therapeutic or preventive effect of COVID-19 is obtained, but oral administration or parenteral administration (e.g., intravenous administration, subcutaneous administration, intraperitoneal administration) can be selected.
　経口投与剤としては、顆粒剤、散剤、錠剤（糖衣錠を含む）、丸剤、カプセル剤、シロップ剤、液剤、ゼリー剤、乳剤、懸濁剤が挙げられる。非経口投与剤としては、具体的な投与形態に応じて適切な剤形を選択することができ、例えば、注射剤、坐剤が挙げられる。これらの製剤は、当分野で通常行われている手法（例えば、第十八改正日本薬局方　製剤総則等に記載の公知の方法）により、薬学上許容される担体を用いて製剤化することができる。薬学上許容される担体としては、賦形剤、結合剤、希釈剤、添加剤、香料、緩衝剤、増粘剤、着色剤、安定剤、乳化剤、分散剤、懸濁化剤、防腐剤等が挙げられる。 Orally administered drugs include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, liquids, jellies, emulsions, and suspensions. For parenteral administration, an appropriate dosage form can be selected according to the specific dosage form, and examples thereof include injections and suppositories. These formulations can be formulated using a pharmaceutically acceptable carrier by a method commonly practiced in the art (for example, a known method described in the 18th revision of the Japanese Pharmacopoeia General Rules for Formulations, etc.). can. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, perfumes, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. is mentioned.
　本発明における有効成分の投与量は、有効成分の種類や、投与対象の性別、年齢および体重、症状、剤形および投与経路等に依存して決定できる。本発明において有効成分をCOVID-19の治療または予防を目的として投与する場合の成人１回あたりの投与量は、例えば、０．０００１ｍｇ～１０００ｍｇ／ｋｇ体重の範囲で決定することができるが、これに限定されるものではない。また、上記投与量の有効成分を１日１回でまたは２～４回に分けて投与することができる。本発明の組成物および剤は、それを必要とするヒトのみならず、ヒト以外の哺乳動物（例えば、マウス、ラット、ウサギ、イヌ、ネコ、ウシ、ウマ、ブタ、ヒツジ、ヤギ、サル）に対しても投与することができる。 The dosage of the active ingredient in the present invention can be determined depending on the type of active ingredient, sex, age and body weight of the subject, symptoms, dosage form, route of administration, and the like. When the active ingredient is administered for the purpose of treating or preventing COVID-19 in the present invention, the dosage per adult can be determined, for example, in the range of 0.0001 mg to 1000 mg / kg body weight. is not limited to In addition, the above dosage of the active ingredient can be administered once a day or in 2 to 4 divided doses. The compositions and agents of the present invention can be applied not only to humans in need thereof, but also to mammals other than humans (e.g., mice, rats, rabbits, dogs, cats, cows, horses, pigs, sheep, goats, monkeys). It can also be administered to
　本発明の別の側面によると、ADAM10阻害剤をそれを必要とする対象に投与する工程を含む、COVID-19の治療または予防方法が提供される。本発明によるとまた、マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物をそれを必要とする対象に投与する工程を含む、COVID-19の治療または予防方法が提供される。投与対象は典型的にはCOVID-19患者またはCOVID-19に罹患する可能性がある者とすることができる。本発明の治療方法および予防方法は本発明の組成物および剤の記載に従って実施することができる。 According to another aspect of the present invention, there is provided a method for treating or preventing COVID-19, comprising administering an ADAM10 inhibitor to a subject in need thereof. The present invention also provides a method of treating or preventing COVID-19, comprising administering one or two compounds selected from the group consisting of marimastat and prinomastat to a subject in need thereof. be done. The administration subject can typically be a COVID-19 patient or a person who may have COVID-19. The therapeutic method and prophylactic method of the present invention can be carried out according to the description of the composition and agent of the present invention.
　本発明の別の側面によると、COVID-19の治療または予防に用いるためのADAM10阻害剤と、COVID-19の治療または予防に用いるためのADAM10阻害剤および他の薬剤の組合せが提供される。本発明によるとまた、COVID-19の治療または予防に用いるためのマリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物と、COVID-19の治療または予防に用いるための、マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物と、他の薬剤との組合せが提供される。本発明のADAM10阻害剤、マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物、並びに組合せは本発明の組成物および剤の記載に従って実施することができる。 According to another aspect of the present invention, ADAM10 inhibitors for use in treating or preventing COVID-19 and combinations of ADAM10 inhibitors and other agents for use in treating or preventing COVID-19 are provided. The present invention also provides one or two compounds selected from the group consisting of marimastat and prinomastat for use in the treatment or prevention of COVID-19 and Combinations of one or two compounds selected from the group consisting of , marimastat and prinomastat with other agents are provided. ADAM10 inhibitors of the present invention, one or two compounds selected from the group consisting of marimastat and prinomastat, and combinations can be performed according to the description of the compositions and agents of the present invention.
　本発明の別の側面によると、COVID-19の治療または予防用組成物あるいはCOVID-19の治療または予防剤の製造のためのADAM10阻害剤の使用と、COVID-19の治療または予防用組成物あるいはCOVID-19の治療または予防剤の製造のためのADAM10阻害剤および他の薬剤の組合せの使用が提供される。本発明によるとまた、COVID-19の治療または予防用組成物あるいはCOVID-19の治療または予防剤の製造のためのマリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物の使用と、COVID-19の治療または予防用組成物あるいはCOVID-19の治療または予防剤の製造のための、マリマスタットおよびプリノマスタットからなる群から選択される１種または２種の化合物と、他の薬剤との組合せの使用が提供される。本発明の使用は本発明の組成物および剤の記載に従って実施することができる。 According to another aspect of the present invention, the use of an ADAM10 inhibitor for the manufacture of a composition for the treatment or prevention of COVID-19 or an agent for the treatment or prevention of COVID-19 and the composition for the treatment or prevention of COVID-19 Alternatively provided is the use of a combination of an ADAM10 inhibitor and other agents for the manufacture of a therapeutic or prophylactic agent for COVID-19. Also according to the present invention, one or two compounds selected from the group consisting of marimastat and prinomastat for the manufacture of a composition for the treatment or prevention of COVID-19 or an agent for the treatment or prevention of COVID-19 and one or two compounds selected from the group consisting of marimastat and prinomastat for the manufacture of a composition for the treatment or prevention of COVID-19 or an agent for the treatment or prevention of COVID-19 , in combination with other agents is provided. The uses of the invention can be carried out according to the description of the compositions and agents of the invention.
　本発明には以下の発明が含まれる。
［１０１］ADAM10阻害剤を含む、COVID-19（coronavirus disease 2019）治療用組成物。
［１０２］ADAM10阻害剤が核酸である、請求項１記載の治療用組成物。
［１０３］核酸がsiRNAである、請求項２記載の治療用組成物。
［１０４］マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキン及びヒドロキシクロロキンのいずれか一つ又は組み合わせにおいて併用する、上記［１０１］～［１０３］のいずれかに記載の治療用組成物。
［１０５］TMPRSS２阻害剤を併用する、上記［１０１］～［１０４］のいずれかに記載の治療用組成物。
［１０６］ADAM10阻害剤を投与する工程を含む、COVID-19（coronavirus disease 2019）の治療方法。
［１０７］ADAM10阻害剤が核酸である、上記［１０６］に記載の治療方法。
［１０８］核酸がsiRNAである、上記［１０７］に記載の治療方法。
［１０９］ADAM10阻害剤とともに、マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物を併用して投与する、上記［１０６］～［１０８］のいずれかに記載の治療方法。
［１１０］ADAM10阻害剤とともに、TMPRSS２阻害剤を併用して投与する、上記［１０６］～［１０９］のいずれかに記載の治療方法。
［１１１］COVID-19（coronavirus disease 2019）治療用組成物の製造のためのADAM10阻害剤の使用。 The present invention includes the following inventions.
[101] A composition for treating COVID-19 (coronavirus disease 2019), comprising an ADAM10 inhibitor.
[102] The therapeutic composition of claim 1, wherein the ADAM10 inhibitor is a nucleic acid.
[103] The therapeutic composition of claim 2, wherein the nucleic acid is siRNA.
[104] The therapeutic composition of any one of the above [101] to [103], which is used in combination with any one or a combination of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine .
[105] The therapeutic composition of any one of the above [101] to [104], which is used in combination with a TMPRSS2 inhibitor.
[106] A method of treating COVID-19 (coronavirus disease 2019), comprising the step of administering an ADAM10 inhibitor.
[107] The therapeutic method of [106] above, wherein the ADAM10 inhibitor is a nucleic acid.
[108] The therapeutic method of [107] above, wherein the nucleic acid is siRNA.
[109] The ADAM10 inhibitor is administered in combination with one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine, the above [ 106] to [108].
[110] The treatment method according to any one of [106] to [109] above, wherein a TMPRSS2 inhibitor is administered in combination with an ADAM10 inhibitor.
[111] Use of an ADAM10 inhibitor for the manufacture of a composition for treating COVID-19 (coronavirus disease 2019).
　以下の例に基づき本発明をより具体的に説明するが、本発明はこれらの例に限定されるものではない。 The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.
　実施例において使用した細胞株とその培養条件および入手先は表１に示される通りであった。
Figure JPOXMLDOC01-appb-T000001
 The cell lines used in the Examples, their culture conditions, and their sources of purchase are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
　実施例において使用したsiRNAおよびプライマーは表２に示される通りであった。
Figure JPOXMLDOC01-appb-T000002
 The siRNAs and primers used in the Examples were as shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
　実施例において使用した化合物（阻害剤）は表３に示される通りであった。
Figure JPOXMLDOC01-appb-T000003
 The compounds (inhibitors) used in the examples were as shown in Table 3.
Figure JPOXMLDOC01-appb-T000003
　スパイクタンパク質（S）、ACE2、CD26、またはTMPRSS2を発現する安定した細胞株を確立するために、タンパク質の1つを発現する組換えシュードレンチウイルスを以前に記載されたように使用した（Yamamoto M et al., 2020, Viruses, 12:629）。SARS-CoV-2分離株（UT-NCGM02/Human/2020/Tokyo）(Imai M et al., 2020, Proc Natl Acad Sci USA 117:16587-16595)は、5%ウシ胎児血清(FBS)を含むダルベッコ改変イーグル培地(DMEM)中のVeroE6-TMPRSS2(JCRB1819)細胞で増殖した。メーカーのプロトコルに従って、Lipofectamine RNAiMAX(Thermo Fisher Scientific、MA、米国）を使用して、siRNA(表２)をトランスフェクトした。すべてのプロテアーゼ阻害剤(表３)は、10mMの濃度でジメチルスルホキシド(DMSO)に溶解した。 To establish stable cell lines expressing spike protein (S), ACE2, CD26, or TMPRSS2, recombinant pseudolentiviruses expressing one of the proteins were used as previously described (Yamamoto M. et al., 2020, Viruses, 12:629). SARS-CoV-2 isolate (UT-NCGM02/Human/2020/Tokyo) (Imai M et al., 2020, Proc Natl Acad Sci USA 117:16587-16595) contains 5% fetal bovine serum (FBS) VeroE6-TMPRSS2 (JCRB1819) cells were grown in Dulbecco's Modified Eagle Medium (DMEM). siRNAs (Table 2) were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific, MA, USA) according to the manufacturer's protocol. All protease inhibitors (Table 3) were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mM.
発現ベクターの構築
　ACE2、CD26、TMPRSS2、またはスパイクタンパク質（S）の発現ベクターを構築するために、コード領域をレンチウイルストランスファープラスミド(CD500B-1; System Biosciences、CA、USA)にクローニングした。SARS-CoV-2のコドン最適化S遺伝子に対応する合成DNA(Wuhan-Hu-1; GenBank accession no. NC_045512.2)、SARS-CoV-2バリアント(B.1.1.7、EPI_ISL_601443; B.1.351、MZ747297.1; B.1.617.1、EPI_ISL_1704611; B.1.617.2、EPI_ISL_3189054)、SARS-CoV(NC_004718.3)、WIV1-CoV(KF367457.1)、HCoV-NL63 (NC_005831.2)、キメラS、およびFlagタグ付き5'-GGA GGC GAT TAC AAG GAT GAC GAT GAC AAG TAA-3 '（下線は3 '末端のFlagタグを示す）（配列番号１０）はすべてIntegrated DNA Technologies（IA、USA）によって作製された。以前に記載されたコドン最適化されたMERS-CoV S(NC_019843.3)(Yamamoto M et al., 2016, Antimicrob Agents Chemother, 60:6532-6539)に対応する3 '末端にFlagタグを持つ合成DNAを実施例において使用した。 Construction of expression vectors To construct expression vectors for ACE2, CD26, TMPRSS2, or spike protein (S), the coding regions were cloned into a lentiviral transfer plasmid (CD500B-1; System Biosciences, CA, USA). Synthetic DNA corresponding to the codon-optimized S gene of SARS-CoV-2 (Wuhan-Hu-1; GenBank accession no. NC_045512.2), SARS-CoV-2 variants (B.1.1.7, EPI_ISL_601443; B.1.351 , MZ747297.1; B.1.617.1, EPI_ISL_1704611; B.1.617.2, EPI_ISL_3189054), SARS-CoV (NC_004718.3), WIV1-CoV (KF367457.1), HCoV-NL63 (NC_005831.2), Chimera S, and Flag-tagged 5′-GGA GGC GAT TAC AAG GAT GAC GAT GAC AAG TAA-3′ (underline indicates Flag tag at 3′ end) (SEQ ID NO: 10) are all from Integrated DNA Technologies (IA, USA) made by Synthesis with a Flag tag at the 3′ end corresponding to previously described codon-optimized MERS-CoV S (NC_019843.3) (Yamamoto M et al., 2016, Antimicrob Agents Chemother, 60:6532-6539) DNA was used in the examples.
膜融合をモニターするためのDSPアッセイ
　DSPアッセイは、以前に記載されたように実施した（Yamamoto M et al., 2020, Viruses, 12:629）。簡単に説明すると、Sタンパク質を発現するエフェクター細胞と、CD26またはACE2を単独でまたはTMPRSS2とともに発現する標的細胞を10 cmプレートに播種し、一晩インキュベートした。細胞をウミシイタケルシフェラーゼ（RL)の基質である6 μM EnduRen(Promega)で2時間処理した。阻害剤の効果を試験するために、DMSOに溶解した0.25μLの化合物ライブラリーまたは1μLの選択した阻害剤を384ウェルプレート(Greiner Bioscience、Frickenhausen、Germany)に添加した。次に、Multidropディスペンサー(Thermo Fisher Scientific)を使用して、50 μLの各単細胞懸濁液(エフェクターおよび標的細胞)を384ウェルプレートに加えた。37°C、5% CO2で4時間インキュベートした後、Centro xS960ルミノメーター(Berthold、Bad Wildbad、Germany)を使用してRL活性を測定した。 DSP Assays to Monitor Membrane Fusion DSP assays were performed as previously described (Yamamoto M et al., 2020, Viruses, 12:629). Briefly, effector cells expressing S protein and target cells expressing CD26 or ACE2 alone or together with TMPRSS2 were seeded in 10 cm plates and incubated overnight. Cells were treated with 6 μM EnduRen (Promega), a substrate for Renilla luciferase (RL), for 2 hours. To test the effect of inhibitors, 0.25 μL of compound library or 1 μL of selected inhibitors dissolved in DMSO were added to 384-well plates (Greiner Bioscience, Frickenhausen, Germany). 50 μL of each single-cell suspension (effector and target cells) was then added to a 384-well plate using a Multidrop dispenser (Thermo Fisher Scientific). After incubation for 4 hours at 37°C, 5% CO2 , RL activity was measured using a Centro xS960 luminometer (Berthold, Bad Wildbad, Germany).
ウェスタンブロッティング
　ウエスタンブロット分析は、以前に記載されたように行った(Yamamoto M et al., 2019, Commun Biol, 2:292)。使用した抗体は、表４に示される通りであった。
Figure JPOXMLDOC01-appb-T000004
 Western Blotting Western blot analysis was performed as previously described (Yamamoto M et al., 2019, Commun Biol, 2:292). Antibodies used were as shown in Table 4.
Figure JPOXMLDOC01-appb-T000004
シュードタイプVSVウイルス粒子の調製と感染実験
　複製欠損VSVを作製するために、T7 RNAポリメラーゼを発現するBHK細胞を、VSVタンパク質（pBS-N/pBS-P/pBS-L/pBS-G）のT7プロモーター駆動発現プラスミドおよびpΔG-Luci（G遺伝子を欠き、ホタルルシフェラーゼをコードするVSVゲノムRNAをコードするプラスミド）で、以前に記載されているようにトランスフェクトした（Tani H et al., 2007, J Virol, 81:8601-8612、Tani H et al., 2010, J Virol, 84:2798-2807）。トランスフェクションの48時間後に、上清を回収した。次いで、293T細胞を、リン酸カルシウム沈殿を使用することにより、SまたはVSV Gの発現プラスミドでトランスフェクトした。トランスフェクションの16時間後に、細胞に複製欠損VSVを感染多重度（MOI）1で接種した。感染後2時間で、細胞を洗浄し、さらに16時間インキュベートしてから、シュードウイルスを含む上清を採取した。感染アッセイのために、細胞を96ウェルプレートに播種し（2 × 104細胞/ウェル）、一晩インキュベートした。シュードウイルス感染の1時間前に、細胞を阻害剤で前処理した。ルシフェラーゼ活性は、Bright-GloルシフェラーゼアッセイシステムまたはONE-Gloルシフェラーゼアッセイシステム（Promega）およびCentro xS960照度計（Berthold）を使用して、感染後16時間に測定した。 Preparation of pseudotyped VSV virus particles and infection experiments To generate replication-deficient VSV, BHK cells expressing T7 RNA polymerase were transfected with VSV proteins (pBS-N/pBS-P/pBS-L/pBS-G) into T7 cells. Promoter-driven expression plasmids and pΔG-Luci (a plasmid lacking the G gene and encoding VSV genomic RNA encoding firefly luciferase) were transfected as previously described (Tani H et al., 2007, J. Virol, 81:8601-8612, Tani H et al., 2010, J Virol, 84:2798-2807). Supernatants were harvested 48 hours after transfection. 293T cells were then transfected with S or VSV G expression plasmids by using calcium phosphate precipitation. Sixteen hours after transfection, cells were inoculated at a multiplicity of infection (MOI) of 1 with replication-deficient VSV. Two hours after infection, the cells were washed and incubated for an additional 16 hours before harvesting the pseudovirus-containing supernatant. For infection assays, cells were seeded in 96-well plates (2 x 104 cells/well) and incubated overnight. One hour prior to pseudovirus infection, cells were pretreated with inhibitors. Luciferase activity was measured 16 hours after infection using the Bright-Glo luciferase assay system or the ONE-Glo luciferase assay system (Promega) and a Centro xS960 luminometer (Berthold).
細胞内SARS-CoV-2 RNAの定量
　細胞を96ウェルプレートに播種し（5 × 104細胞/ウェル）、一晩インキュベートした。細胞を阻害剤で1時間処理し、MOIでSARS-CoV-2を添加した。プロトコルに従って、SuperPrep II細胞溶解および定量的PCR用RTキット（qPCR）（SCQ-401; Toyobo、大阪、日本）を使用して、感染後24時間で細胞溶解およびcDNA合成を行った。SARS-CoV-2 Nおよびリボソームタンパク質L13a（Rpl13a）の定量的リアルタイム逆転写(RT)-PCRは、CFX ConnectリアルタイムPCR検出システム（Bio-Rad、カリフォルニア州、米国）を用いて、95°Cで3分、続いて95°Cで10秒間の50サイクル、60°Cで1分により行った。各サンプルのRpl13a mRNA発現レベルを使用して、データを標準化した。 Quantification of intracellular SARS-CoV-2 RNA Cells were seeded in 96-well plates (5 x 104 cells/well) and incubated overnight. Cells were treated with inhibitors for 1 hour and SARS-CoV-2 was added at MOI. Cell lysis and cDNA synthesis were performed 24 h after infection using the SuperPrep II Cell Lysis and RT Kit for Quantitative PCR (qPCR) (SCQ-401; Toyobo, Osaka, Japan) according to the protocol. Quantitative real-time reverse transcription (RT)-PCR of SARS-CoV-2 N and ribosomal protein L13a (Rpl13a) was performed at 95 °C using the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA). 3 minutes followed by 50 cycles of 95°C for 10 seconds and 60°C for 1 minute. Data were normalized using the Rpl13a mRNA expression level of each sample.
細胞変性アッセイ
　細胞を24ウェルプレートに播種し（1.5 × 105細胞／ウェル）、一晩インキュベートした。細胞を阻害剤で1時間処理し、次にMOI 1のSARS-CoV-2で処理した。薬物濃度を維持するために、培養上清の半分を薬物を含む新鮮な培地と毎日交換した。感染後3日目に細胞を4%パラホルムアルデヒドで固定し、0.2%クリスタルバイオレットで染色した。水で４回洗浄した後、ウェルを風乾し、クリスタルバイオレットをエタノールで溶解した。iMarkマイクロプレートリーダー（Bio-Rad）を使用して、595 nmで吸光度を測定した。 Cytopathic Assay Cells were seeded in 24-well plates (1.5×10 5 cells/well) and incubated overnight. Cells were treated with inhibitors for 1 hour and then with MOI 1 of SARS-CoV-2. To maintain drug concentration, half of the culture supernatant was replaced daily with fresh medium containing drug. Three days after infection, cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. After washing with water four times, the wells were air-dried and crystal violet was dissolved in ethanol. Absorbance was measured at 595 nm using an iMark microplate reader (Bio-Rad).
統計分析
　平均値間の統計的に有意な差は、両側スチューデントのt検定を使用して決定した。Dunnettの検定とTukeyの検定は、多重比較に使用した。すべてのデータは3つの独立した実験を表し、値は平均 ± 標準偏差を表し、P <0.05 は統計的に有意と見なされる。 Statistical Analysis Statistically significant differences between means were determined using a two-tailed Student's t-test. Dunnett's test and Tukey's test were used for multiple comparisons. All data represent three independent experiments, values represent the mean ± standard deviation, P < 0.05 are considered statistically significant.
例１：SARS-CoV-2のSタンパク質によってTMPRSS2非依存性の膜融合が誘導される
　我々のコロナウイルス感染阻害剤のスクリーニングシステムでは、Sタンパク質を発現するエフェクター細胞と、ACE2（SARS-CoVおよびSARS-CoV-2の場合）またはCD26（MERS-CoVの場合）といった受容体とTMPRSS2とを共発現する標的細胞との間の定量的な細胞融合アッセイを用いた。 Example 1: TMPRSS2-independent membrane fusion is induced by SARS-CoV-2 S protein. A quantitative cell fusion assay between target cells co-expressing TMPRSS2 with a receptor such as SARS-CoV-2) or CD26 (for MERS-CoV) was used.
　具体的には、SARS-CoV、SARS-CoV-2、およびMERS-CoVのSタンパク質によって誘導される細胞融合動態は、DSPアッセイを使用して決定した。ACE2を単独でまたはTMPRSS2とともに発現する標的細胞を、SARS-CoV SおよびSARS-CoV-2 Sを発現するエフェクター細胞との共培養に使用し、CD26を単独またはTMPRSS2とともに発現する細胞を、MERS-CoV Sを発現するエフェクター細胞との共培養に使用した。相対細胞融合値は、各共培養のRL活性を、100%に設定された240分で受容体とTMPRSS2の両方を発現する細胞との共培養のRL活性に正規化することによって計算した（図1a）。 Specifically, cell fusion kinetics induced by SARS-CoV, SARS-CoV-2, and MERS-CoV S proteins were determined using DSP assays. Target cells expressing ACE2 alone or together with TMPRSS2 were used for co-culture with effector cells expressing SARS-CoV S and SARS-CoV-2 S, and cells expressing CD26 alone or together with TMPRSS2 were used for co-culture with MERS- Used for co-culture with effector cells expressing CoV S. Relative cell fusion values were calculated by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both the receptor and TMPRSS2 at 240 min set at 100% (Fig. 1a).
　具体的にはまた、TMPRSS2とともにACE2を発現する標的細胞は、SARS-CoV SおよびSARS-CoV-2 Sを発現するエフェクター細胞との共培養に使用し、TMPRSS2とともにCD26を発現する細胞は、MERS-CoV Sを発現するエフェクター細胞との共培養に使用した。相対細胞融合値は、各共培養のRL活性を、100%に設定されたDMSO存在下で受容体とTMPRSS2の両方を発現する細胞との共培養のRL活性に正規化することによって計算した（図1c）。 Specifically, target cells expressing ACE2 together with TMPRSS2 were also used for co-culture with effector cells expressing SARS-CoV S and SARS-CoV-2 S, and cells expressing CD26 together with TMPRSS2 were used for co-culture with MERS -used for co-culture with effector cells expressing CoV S. Relative cell fusion values were calculated by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both the receptor and TMPRSS2 in the presence of DMSO set at 100% ( Fig. 1c).
　具体的にはまた、ACE2を単独で、またはTMPRSS2とともに発現する標的細胞を、SARS-CoV-2 Sを発現するエフェクター細胞と共培養するために使用した。相対細胞融合値は、各共培養のRL活性を、100%に設定されたDMSO存在下でACE2とTMPRSS2の両方を発現する細胞との共培養のRL活性に正規化することによって計算した（図1dおよび図1e）。 Specifically, target cells expressing ACE2 alone or together with TMPRSS2 were also used to co-culture with effector cells expressing SARS-CoV-2 S. Relative cell fusion values were calculated by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both ACE2 and TMPRSS2 in the presence of DMSO set at 100% (Fig. 1d and Fig. 1e).
　結果は図１に示される通りであった。SARS-CoVやMERS-CoVのSタンパク質による細胞融合には受容体とともにTMPRSS2が必須であるが、SARS-CoV-2のSタンパク質では、TMPRSS2依存的な細胞融合とともにTMPRSS2に依存しない細胞融合が誘導された（図1aおよび図1b）。この結果と一致して、SARS-CoVおよびMERS-CoVのSタンパク質によって誘導されるTMPRSS2発現標的細胞との融合は、TMPRSS2を1μMのナファモスタット（nafamostat）で阻害することで完全に抑制されたが、SARS-CoV-2のSタンパク質による融合は、10μMのナファモスタットの存在下でも阻害剤なしの場合の20%残存した（図1c）。この残存融合量は、TMPRSS2非存在下（ACE2を発現するがTMPRSS2を発現しない標的細胞を使用する）でSARS-CoV-2のSタンパク質によって誘導された融合量とほぼ同じであった（図1d）。さらに、このSARS-CoV-2のSタンパク質によって誘導されるTMPRSS2非依存性融合は、E-64d（カテプシン－B/L（cathepsin-B/L）阻害剤）やフーリン（furin）阻害剤IIなど、SARS-CoV-2感染に対する阻害活性が知られているプロテアーゼの阻害剤では影響を受けなかった（図1e）。またペプスタチンA（pepstatin A、各種アスパラギン酸プロテアーゼ（aspartic protease）の阻害剤）、ロイペプチン（leupeptin、各種システイン、セリン、スレオニンプロテアーゼ（cysteine, serine, threonine protease）の阻害剤）、ベスタチン（bestatin、各種アミノペプチダーゼ（aminopeptidase）の阻害剤）は、SARS-CoV-2のSタンパク質によって誘導されるTMPRSS2非依存性の細胞融合に影響を与えなかった（図1e）。 The results were as shown in Figure 1. SARS-CoV and MERS-CoV S proteins require TMPRSS2 as well as receptors for cell fusion, but the SARS-CoV-2 S protein induces both TMPRSS2-dependent and TMPRSS2-independent cell fusion. (Figures 1a and 1b). Consistent with this result, SARS-CoV and MERS-CoV S protein-induced fusion with TMPRSS2-expressing target cells was completely suppressed by inhibiting TMPRSS2 with 1 μM nafamostat. , SARS-CoV-2 S protein-mediated fusion remained 20% of that in the absence of inhibitor even in the presence of 10 μM nafamostat (Fig. 1c). This amount of residual fusion was similar to that induced by SARS-CoV-2 S protein in the absence of TMPRSS2 (using target cells expressing ACE2 but not TMPRSS2) (Figure 1d). ). Moreover, this TMPRSS2-independent fusion induced by the SARS-CoV-2 S protein has been demonstrated by E-64d (cathepsin-B/L inhibitor) and furin inhibitor II. , was unaffected by protease inhibitors with known inhibitory activity against SARS-CoV-2 infection (Fig. 1e). In addition, pepstatin A (inhibitor of various aspartic protease), leupeptin (leupeptin, inhibitor of various cysteine, serine, threonine protease), bestatin (inhibitor of various amino acids) peptidase inhibitor) did not affect TMPRSS2-independent cell fusion induced by SARS-CoV-2 S protein (Fig. 1e).
例２：SARS-CoV-2のSタンパク質によって誘導されるTMPRSS2非依存性の膜融合は、様々なメタロプロテアーゼ阻害剤によって抑制される
　例１で示された、SARS-CoV-2のSタンパク質によって誘導されるTMPRSS2非依存性膜融合のメカニズムを解明するために，東京大学創薬機構から入手した検証済み化合物ライブラリー（Validated Compound Library、臨床試験で承認された1,630化合物および薬理活性を有する1,885化合物を含む）を用いて、TMPRSS2非依存性膜融合を特異的に阻害し，TMPRSS2依存性膜融合を阻害しない化合物を探索した。 Example 2: TMPRSS2-Independent Membrane Fusion Induced by SARS-CoV-2 S Protein is Suppressed by Various Metalloprotease Inhibitors To elucidate the mechanism of induced TMPRSS2-independent membrane fusion, we used a validated compound library (1,630 compounds approved in clinical trials and 1,885 compounds with pharmacological activity) obtained from the University of Tokyo Drug Discovery Organization. ) were used to search for compounds that specifically inhibit TMPRSS2-independent membrane fusion but do not inhibit TMPRSS2-dependent membrane fusion.
　具体的には、各化合物のＲＬ活性を対照アッセイ（ＤＭＳＯ単独；１００％に設定）のＲＬ活性に正規化することにより、相対細胞融合値を計算した。各ドットは個々の化合物を表す。点線のボックス内のドットは、TMPRSS2非依存性膜融合を優先的に阻害する化合物を表す（TMPRSS2とACE2の両方を発現する標的細胞を使用した相対細胞融合値の<30%阻害、およびACE2のみを発現する標的細胞を使用した相対細胞融合値の>40%阻害）（図２ａ）。 Specifically, relative cell fusion values were calculated by normalizing the RL activity of each compound to the RL activity of the control assay (DMSO alone; set at 100%). Each dot represents an individual compound. Dots within dotted boxes represent compounds that preferentially inhibit TMPRSS2-independent membrane fusion (<30% inhibition of relative cell fusion values using target cells expressing both TMPRSS2 and ACE2, and ACE2 only). >40% inhibition of relative cell fusion values using target cells expressing (Fig. 2a).
　結果は図２ａに示される通りであった。TMPRSS2とACE2の両方を発現している標的細胞を用いた細胞融合のスクリーニング結果（図２ａのX軸）と、TMPRSS2無しでACE2を単独で発現している標的細胞を用いたスクリーニング結果（図２ａのY軸）を比較することで、2種類のメタロプロテアーゼ阻害剤（イロマスタット（ilomastat）, CTS-1027）を候補として選択し、さらなる解析を行った。 The results were as shown in Figure 2a. Cell fusion screening results using target cells expressing both TMPRSS2 and ACE2 (X-axis in Fig. 2a) and screening results using target cells expressing ACE2 alone without TMPRSS2 (Fig. 2a ), two metalloprotease inhibitors (ilomastat, CTS-1027) were selected as candidates for further analysis.
　具体的には、相対細胞融合値は、各共培養のRL活性を、100%に設定されたDMSO存在下でACE2とTMPRSS2の両方を発現する細胞との共培養のRL活性に正規化することによって計算した（図２ｂ）。 Specifically, relative cell fusion values were obtained by normalizing the RL activity of each co-culture to that of co-cultures with cells expressing both ACE2 and TMPRSS2 in the presence of DMSO set at 100%. (Fig. 2b).
　結果は図２ｂに示される通りであった。イロマスタットとCTS-1027は、用量依存的にTMPRSS2非依存性細胞融合を優先的に阻害した（図２ｂ）。これらのデータは、死亡率の高いヒト病原性コロナウイルスの中でSARS-CoV-2に特異的なメタロプロテアーゼ依存性の細胞表面侵入経路が存在することを示唆している。さらに、メタロプロテアーゼ阻害剤をCOVID-19の予防・治療薬として用いる可能性を考え、CTS-1027と同様に抗がん剤の臨床試験で安全性が確認されているマリマスタット（marimastat）とプリノマスタット（prinomastat）が、SARS-CoV-2のSタンパク質によって誘導されるTMPRSS2非依存性細胞融合を特異的に阻害できることを示した（図2b）。 The results were as shown in Figure 2b. Ilomastat and CTS-1027 preferentially inhibited TMPRSS2-independent cell fusion in a dose-dependent manner (Fig. 2b). These data suggest that there is a metalloprotease-dependent cell surface entry pathway specific for SARS-CoV-2 among the high-mortality human pathogenic coronaviruses. In addition, considering the possibility of using metalloprotease inhibitors as preventive and therapeutic agents for COVID-19, we will combine marimastat, which has been confirmed to be safe in anticancer clinical trials, as well as CTS-1027. We showed that prinomastat can specifically inhibit TMPRSS2-independent cell fusion induced by the S protein of SARS-CoV-2 (Fig. 2b).
例３：メタロプロテアーゼ依存性のウイルス侵入経路は、SARS-CoV-2に特異的であり、その存在は細胞の種類に依存する
　例１および例２で示された、SARS-CoV-2のSタンパク質による細胞融合実験から得られた知見に基づき、メタロプロテアーゼ依存的な細胞表面へのウイルス侵入経路が存在するかどうかを、SARS-CoV-2のSタンパク質をエンベロープに持つ水胞性口内炎ウイルス（VSV）シュードウイルス（SARS-CoV-2シュードウイルス）を用いて確認した。 Example 3: The metalloprotease-dependent viral entry pathway is specific to SARS-CoV-2 and its presence depends on the cell type. Based on the findings from protein-mediated cell fusion experiments, we investigated whether a metalloprotease-dependent viral entry pathway to the cell surface exists in the SARS-CoV-2 S protein-enveloped vesicular stomatitis virus (VSV ) was confirmed using a pseudovirus (SARS-CoV-2 pseudovirus).
　具体的には、293T細胞によって産生されるSARS-CoV-2 Sを含む水疱性口内炎ウイルス（VSV）シュードウイルスの侵入に対する薬物の効果が解析した。相対的シュードウイルス侵入は、各条件のFL活性を、100%に設定したDMSOのみの存在下でSARS-CoV-2 S保有シュードウイルスに感染した細胞のFL活性に正規化することによって計算した（図3）。 Specifically, the effects of drugs on the invasion of vesicular stomatitis virus (VSV) pseudoviruses, including SARS-CoV-2 S produced by 293T cells, were analyzed. Relative pseudovirus entry was calculated by normalizing the FL activity of each condition to the FL activity of cells infected with SARS-CoV-2 S-carrying pseudoviruses in the presence of DMSO only, which was set at 100% ( Figure 3).
　結果は図3aに示される通りであった。A704細胞（ヒト腎臓）におけるSARS-CoV-2シュードウイルスの侵入は、1μMのマリマスタットによって完全に阻止された（図3a）。このことは、メタロプロテアーゼ依存性のSARS-CoV-2侵入経路が存在していることを示しており、他の細胞におけるメタロプロテアーゼ依存性侵入経路の存在は、1μMのマリマスタットで確認できることを示している。同様に、OVISE細胞（ヒト卵巣）では、25μMのE-64dで侵入経路の大半が遮断された（図3a）。このことは、他の細胞でカテプシン－B/L依存性のエンドソーム経路の存在を確認するには、25μMのE-64dで十分であることを示している。さらに、Calu-3細胞（ヒト肺）では、0.1μMのナファモスタットで侵入経路のほとんどが遮断された（図3a）ことから、他の細胞でTMPRSS2依存性の侵入経路の存在を確認するには、0.1μMあるいはそれ以上の濃度のナファモスタットで処理することが適切であることがわかった。そこで各細胞におけるメタロプロテアーゼ，TMPRSS2-，カテプシン－B/L経路の分布パターンを把握するため，マリマスタット1μM，E-64d 25μM，ナファモスタット10μMを用いて各侵入経路の存在を各種細胞で調べた。 The results were as shown in Figure 3a. SARS-CoV-2 pseudovirus entry in A704 cells (human kidney) was completely blocked by 1 μM marimastat (Fig. 3a). This indicates that a metalloprotease-dependent SARS-CoV-2 entry pathway exists, and that the presence of the metalloprotease-dependent entry pathway in other cells can be confirmed with 1 μM marimastat. ing. Similarly, in OVISE cells (human ovary), 25 μM E-64d blocked most of the entry pathways (Fig. 3a). This indicates that 25 μM E-64d is sufficient to confirm the presence of the cathepsin-B/L dependent endosomal pathway in other cells. Moreover, in Calu-3 cells (human lung), 0.1 μM nafamostat blocked most of the entry pathways (Fig. 3a), suggesting that TMPRSS2-dependent entry pathways could be confirmed in other cells. , it was found that treatment with nafamostat at a concentration of 0.1 μM or higher was appropriate. Therefore, in order to understand the distribution pattern of the metalloprotease, TMPRSS2-, and cathepsin-B/L pathways in each cell, the presence of each entry pathway was examined in various cells using marimastat 1 μM, E-64d 25 μM, and nafamostat 10 μM. .
　結果は図3b～3eに示される通りであった。マリマスタットは，VeroE6（アフリカミドリザル腎臓），HEC50B（ヒト子宮内膜），OVTOKO（ヒト卵巣），A704の各細胞のシュードウイルス侵入を有意に阻害した（図3b）。VeroE6細胞、HEC50B細胞、OVTOKO細胞では、メタロプロテアーゼ依存性の侵入経路に加えて、E-64dによってもウイルスの侵入が部分的に阻害され、マリマスタットとE-64dの併用は相加的な効果を示した（図3b）。これらの結果は、メタロプロテアーゼ依存性侵入経路と、カテプシン－B/L依存性のエンドソーム経路が共存し相互に独立していることを示している。E-64dがOVISE細胞の侵入経路の大半を阻害した（図3c）ことから、OVISE細胞ではほぼエンドソーム経路のみであると考えられる。一方、IGROV1細胞（ヒト卵巣）、OUMS-23細胞（ヒト結腸）では、E-64dが80%程度ウイルスの侵入を抑制するものの、20%程度が残存している。この残存した分は、IGROV1細胞ではマリマスタットを、OUMS-23細胞ではナファモスタットをE-64dと併用することで抑制することができる（図3c）。すなわちIGROV1細胞ではエンドソーム経路とメタロプロテアーゼ依存性侵入経路が共存し、OUMS-23細胞ではエンドソーム経路とTMPRSS2依存性経路が共存している。また、ナファモスタットがCalu-3細胞とCaco-2細胞（ヒト結腸）の侵入経路のほとんどを阻害した（図3d）ことから、これらの細胞ではほぼTMPRSS2依存性細胞表面経路のみであると考えられる。一方、メタロプロテアーゼ依存性とTMPRSS2依存性の両方の細胞表面侵入経路を同時に持つ細胞株が見当たらなかったため、TMPRSS2を異所的に発現するHEC50B細胞株（HEC50B-TMPRSS2細胞と呼ぶ）を作製した。HEC50B-TMPRSS2細胞では、ウイルス侵入経路の約80%がTMPRSS2依存性であり、残りのほとんどがマリマスタット感受性であった（図3e）。これは、メタロプロテアーゼ依存性の侵入経路とTMPRSS2依存性の侵入経路が共存していることを示している。この結果から、生体内には両方の細胞表面侵入経路を持つ細胞が存在する可能性が示唆された。 The results were as shown in Figures 3b to 3e. Marimastat significantly inhibited pseudovirus entry into VeroE6 (African green monkey kidney), HEC50B (human endometrium), OVTOKO (human ovary), and A704 cells (Fig. 3b). In VeroE6, HEC50B, and OVTOKO cells, in addition to the metalloprotease-dependent entry pathway, E-64d also partially inhibited viral entry, and the combination of marimastat and E-64d had an additive effect. (Fig. 3b). These results indicate that the metalloprotease-dependent entry pathway and the cathepsin-B/L-dependent endosomal pathway coexist and are independent of each other. E-64d inhibited most of the entry routes in OVISE cells (Fig. 3c), suggesting that the endosomal route is almost exclusively in OVISE cells. On the other hand, in IGROV1 cells (human ovary) and OUMS-23 cells (human colon), although E-64d suppresses virus entry by about 80%, about 20% remains. This residual amount can be suppressed by combining E-64d with marimastat in IGROV1 cells and nafamostat in OUMS-23 cells (Fig. 3c). In IGROV1 cells, the endosomal and metalloprotease-dependent invasion pathways coexist, and in OUMS-23 cells, the endosomal and TMPRSS2-dependent pathways coexist. In addition, nafamostat inhibited most of the entry pathways of Calu-3 and Caco-2 cells (human colon) (Fig. 3d), suggesting that the TMPRSS2-dependent cell surface pathway is almost exclusively present in these cells. . On the other hand, since no cell line with both metalloprotease-dependent and TMPRSS2-dependent cell surface entry pathways was found, we generated a HEC50B cell line that ectopically expresses TMPRSS2 (referred to as HEC50B-TMPRSS2 cells). In HEC50B-TMPRSS2 cells, approximately 80% of the viral entry pathways were TMPRSS2-dependent, with most of the rest being marimastat-sensitive (Fig. 3e). This indicates the coexistence of metalloprotease-dependent and TMPRSS2-dependent invasion pathways. This result suggested the possibility that cells with both cell surface invasion pathways exist in vivo.
例４：SARS-CoV-2の変異株も従来株と同様にメタロプロテアーゼ依存性経路を介して細胞に侵入する
　メタロプロテアーゼ依存性侵入経路を標的としたCOVID-19治療を考える上で、ワクチン接種が進む中で感染拡大し続ける変異株においてもメタロプロテアーゼ依存性経路が利用されるかどうかを確認する必要がある。このため、従来株と変異株のSタンパク質を持つシュードウイルスの感染におけるマリマスタットの効果を比較した。 Example 4: Mutant strains of SARS-CoV-2 also enter cells via metalloprotease-dependent pathways like conventional strains. It is necessary to confirm whether the metalloprotease-dependent pathway is also utilized in mutant strains that continue to spread infection as the disease progresses. Therefore, we compared the effect of marimastat on the infection of pseudoviruses with the S protein of the conventional strain and the mutant strain.
　結果は図4に示される通りであった。従来株と変異株のSタンパク質を持つシュードウイルスの感染におけるマリマスタットの効果は、α株（B.1.1.7）、β株（B.1.351）、δ株（B.1.617.2）、κ株（B.1.617.1）のいずれにおいても従来株と同程度にメタロプロテアーゼ依存性侵入経路を使うことがわかった（図4）。従ってメタロプロテアーゼ依存性経路を標的とした治療戦略は変異株に対しても有効である。 The results were as shown in Figure 4. The effects of marimastat on infection with conventional and mutant S protein-carrying pseudoviruses were: α (B.1.1.7), β (B.1.351), δ (B.1.617.2), κ Both strains (B.1.617.1) were found to use the metalloprotease-dependent invasion pathway to the same extent as the conventional strain (Fig. 4). Therefore, therapeutic strategies targeting metalloprotease-dependent pathways are also effective against mutant strains.
例５：SARS-CoV-2のメタロプロテアーゼ依存性侵入経路にADAM-10が関与する
　TMPRSS2依存性の侵入経路を持たないVeroE6細胞, HEC50B細胞, A704細胞においてSARS-CoV-2シュードウイルスの侵入に対するメタロプロテアーゼ阻害剤による抑制を解析した。 Example 5: ADAM-10 is involved in the SARS-CoV-2 metalloprotease-dependent entry pathway. Inhibition by metalloprotease inhibitors was analyzed.
　具体的には、E-64d存在下でのVeroE6およびHEC50B細胞、およびE-64d非存在下でのA704細胞におけるSARS-CoV-2 SまたはVSV Gを持つシュードウイルスの侵入に対するメタロプロテイナーゼ阻害剤の効果を解析した。各条件のFL活性を、１００％に設定されたDMSOのみの存在下でシュードウイルスに感染した細胞のFL活性に正規化することによって、相対的なシュードウイルス侵入を計算した。データは、HEC50BおよびVeroE6細胞についてはE-64d存在下、A704細胞についてはDMSOのみの存在下で、SARS-CoV-2 Sを持つシュードウイルスに感染した細胞から得られたデータと比較した（図5）。 Specifically, the effect of metalloproteinase inhibitors on entry of pseudoviruses with SARS-CoV-2 S or VSV G in VeroE6 and HEC50B cells in the presence of E-64d and in A704 cells in the absence of E-64d. We analyzed the effect. Relative pseudovirus entry was calculated by normalizing the FL activity of each condition to the FL activity of pseudovirus-infected cells in the presence of DMSO only, which was set at 100%. Data were compared to data obtained from cells infected with a pseudovirus carrying SARS-CoV-2 S in the presence of E-64d for HEC50B and VeroE6 cells and in the presence of DMSO alone for A704 cells (Fig. Five).
　具体的にはまた、VeroE6 (a)、HEC50B (b)、およびA704 (c)細胞をさまざまな薬剤で処理し、CellTiter-Glo発光細胞生存率アッセイ（G7570; Promega、WI、USA）を使用して製造元のプロトコルに従って処理の24時間後の細胞生存率を分析した。相対細胞生存率は、各条件のFL活性を、100%に設定したDMSOのみの存在下での細胞のFL活性に正規化することによって計算した（図6）。 Specifically, we also treated VeroE6 (a), HEC50B (b), and A704 (c) cells with various agents and used the CellTiter-Glo Luminescent Cell Viability Assay (G7570; Promega, WI, USA). We analyzed cell viability after 24 hours of treatment according to the manufacturer's protocol. Relative cell viability was calculated by normalizing the FL activity of each condition to the FL activity of cells in the presence of DMSO only, which was set at 100% (Fig. 6).
　結果は図5の広範囲メタロプロテアーゼ阻害剤（broad spectrum metalloprotease inhibitor）に示される通りであった。TMPRSS2依存性の侵入経路を持たないVeroE6細胞（図5a）, HEC50B細胞（図5b）, A704細胞（図5c）においてマリマスタットに加えて、プリノマスタット,イロマスタット, CTS-1027もSARS-CoV-2シュードウイルスの侵入を阻害した（図5：広範囲メタロプロテアーゼ阻害剤（broad spectrum metalloprotease inhibitor））。これらの阻害剤は特異性が低く多様なメタロプロテアーゼを阻害する。 The results were as shown in Fig. 5 broad spectrum metalloprotease inhibitor. In VeroE6 cells (Fig. 5a), HEC50B cells (Fig. 5b), and A704 cells (Fig. 5c), which do not have a TMPRSS2-dependent entry pathway, in addition to marimastat, prinomastat, ilomastat, and CTS-1027 also detected SARS-CoV- 2 pseudovirus entry (Fig. 5: broad spectrum metalloprotease inhibitor). These inhibitors inhibit a wide variety of metalloproteases with low specificity.
　次にメタロプロテアーゼ依存性経路に関与する酵素を特定するため、より選択性の高い阻害剤を用いて解析した（図5：selective metalloprotease inhibitor）。VeroE6細胞およびHEC50B細胞は、E-64d感受性エンドソーム経路を20-30%程度持っているので（図3b）、メタロプロテアーゼ依存性経路の減少を容易に認識するために、E-64d存在下で選択的阻害剤の効果を解析した（図5a, b）。一方A704細胞ではほとんどがメタロプロテアーゼ依存性経路なので（図3a, b）、選択的阻害剤を単独で使用した（図5c）。 Next, in order to identify enzymes involved in metalloprotease-dependent pathways, we analyzed using highly selective inhibitors (Fig. 5: selective metalloprotease inhibitor). Since VeroE6 and HEC50B cells have ~20-30% E-64d sensitive endosomal pathways (Fig. 3b), we selected in the presence of E-64d to readily recognize the reduction in metalloprotease-dependent pathways. We analyzed the effects of inhibitory agents (Fig. 5a, b). On the other hand, A704 cells were mostly metalloprotease-dependent pathways (Fig. 3a, b), so selective inhibitors were used alone (Fig. 5c).
　結果は図5の選択的メタロプロテアーゼ阻害剤（selective metalloprotease inhibitor）に示される通りであった。UK370106（MMP3/12阻害剤）はメタロプロテアーゼ依存性経路を有意に阻害したが、MMP408（MMP3/12/13阻害剤）は阻害しなかった（図5）。このことから、MMP3/12/13はメタロプロテアーゼ依存性経路に関与しないものの、本経路に関与する酵素はUK370106によって阻害される可能性が示唆された。MLN-4760（ACE2阻害剤）とBK-1361（ADAM8阻害剤）はメタロプロテアーゼ依存性経路にほとんど影響を与えなかった。さらに、GW280264X（ADAM10/17阻害剤）とGI1254023X（MMP9/ADAM10阻害剤）はメタロプロテアーゼ依存性経路を有意に阻害したが、TAPI2（ADAM17阻害剤）およびMMP2/9i（MMP2/9阻害剤）は阻害しなかった（図5）。試験を行った3つの細胞株すべてにおいて同様の阻害パターンが観察され（図5）、実験に用いた濃度ではいずれのメタロプロテアーゼ阻害剤によっても細胞の生存率が影響を受けなかったことから（図6）、この経路に関与するメタロプロテアーゼは3つの細胞株に共通しており、選択的阻害剤の抑制効果からADAM10の関与が強く推測された。 The results were as shown in Figure 5 for selective metalloprotease inhibitor. UK370106 (MMP3/12 inhibitor) significantly inhibited metalloprotease-dependent pathways, but MMP408 (MMP3/12/13 inhibitor) did not (Fig. 5). This suggests that although MMP3/12/13 is not involved in the metalloprotease-dependent pathway, UK370106 may inhibit enzymes involved in this pathway. MLN-4760 (ACE2 inhibitor) and BK-1361 (ADAM8 inhibitor) had little effect on metalloprotease-dependent pathways. Furthermore, GW280264X (ADAM10/17 inhibitor) and GI1254023X (MMP9/ADAM10 inhibitor) significantly inhibited metalloprotease-dependent pathways, whereas TAPI2 (ADAM17 inhibitor) and MMP2/9i (MMP2/9 inhibitor) did not inhibit (Fig. 5). A similar pattern of inhibition was observed in all three cell lines tested (Figure 5), and cell viability was not affected by any of the metalloprotease inhibitors at the concentrations used in the experiments (Figure 5). 6), metalloproteases involved in this pathway are common to the three cell lines, and the inhibitory effect of selective inhibitors strongly suggests the involvement of ADAM10.
　そこでHEC50B細胞において３種類の配列の異なるsiRNAを用いてADAM10の発現を抑制した。 Therefore, ADAM10 expression was suppressed in HEC50B cells using 3 types of siRNA with different sequences.
　具体的には、HEC50B細胞に、2つの異なる対照siRNAまたはADAM10に対する3つの異なるsiRNAを48時間トランスフェクトした（図7a）。HEC50B細胞にsiRNAを48時間トランスフェクトし、シュードウイルスに感染させた。相対的シュードウイルス侵入は、各条件のFL活性を、100%に設定したsiRNA非存在下でシュードウイルスに感染した細胞のFL活性（モック）に正規化することによって計算した（図7b）。 Specifically, HEC50B cells were transfected with two different control siRNAs or three different siRNAs against ADAM10 for 48 hours (Fig. 7a). HEC50B cells were transfected with siRNA for 48 hours and infected with pseudovirus. Relative pseudovirus entry was calculated by normalizing the FL activity of each condition to the FL activity of pseudovirus-infected cells in the absence of siRNA (mock), which was set at 100% (Fig. 7b).
　結果は図7に示される通りであった。いずれのsiRNAにおいてもSARS-CoV-2シュードウイルスの侵入が顕著に抑制されたが、SARS-CoV, MERS-CoV, VSVシュードウイルスの侵入には変化がなかった（図7）。以上の結果から、SARS-CoV-2特異的なメタロプロテアーゼ依存性経路にADAM10が関与することが明らかとなった。 The results were as shown in Figure 7. All siRNAs markedly suppressed SARS-CoV-2 pseudovirus entry, but did not change SARS-CoV, MERS-CoV, and VSV pseudovirus entry (Fig. 7). These results demonstrate that ADAM10 is involved in the SARS-CoV-2-specific metalloprotease-dependent pathway.
例６：SARS-CoV-2ウイルスのADAM10を含むメタロプロテアーゼ依存性侵入経路はCOVID-19の治療標的となり得る
　メタロプロテアーゼ依存性侵入経路がCOVID-19の治療標的となり得るためには、シュードウイルスに加えて実際の病原性SARS-CoV-２ウイルスの感染がメタロプロテアーゼ阻害剤で抑制されることが必要である。シュードウイルス実験からメタロプロテアーゼ依存性侵入経路を持つと考えられるVeroE6細胞, HEC50B細胞, A704細胞での生ウイルスの感染増殖をマリマスタットおよびプリノマスタット存在下またはADAM10に対するsiRNA存在下で解析した。 Example 6: SARS-CoV-2 virus metalloprotease-dependent entry pathway including ADAM10 could be a therapeutic target for COVID-19 For the metalloprotease-dependent entry pathway to be a therapeutic target for COVID-19, pseudovirus In addition, it is necessary that the actual pathogenic SARS-CoV-2 virus infection is suppressed with metalloprotease inhibitors. VeroE6 cells, HEC50B cells, and A704 cells, which are thought to have a metalloprotease-dependent entry pathway based on pseudovirus experiments, were infected and propagated with live virus in the presence of marimastat and prinomastat, or in the presence of siRNA against ADAM10.
　結果は図8に示される通りであった。いずれの細胞においても阻害剤の濃度依存的にウイルスの感染が抑制された（図8a）。マリマスタットおよびプリノマスタットの臨床試験で設定された安全性が確保された投与における血中濃度がおよそ600-900 nMであり、今回の実験において300nMから1000nMの間で感染阻害効果を示していることから、これらの薬剤はCOVID-19の治療に使える可能性が大きいと考えられる。さらに、HEC50B細胞においてGW280264X（ADAM10/17阻害剤）とGI1254023X（MMP9/ADAM10阻害剤）が病原性SARS-CoV-２ウイルスの感染を阻害しTAPI2（ADAM17阻害剤）が阻害しなかった（図8b）。また、siRNAによるADAM10の発現抑制は、病原性SARS-CoV-２ウイルスの感染を抑制した（図8c）。これらの結果はADAM10を標的とした阻害剤が病原性SARS-CoV-２ウイルスの感染を抑制しCOVID-19の治療薬となる可能性があることを示している。実際に治療で投与される阻害剤としては、この研究で用いたようなADAM10の発現を抑制するsiRNAに類似した核酸医薬やADAM10の酵素活性やADAM10と機能的に結合するタンパク質との複合体形成を抑制する低分子や抗体が想定される。また、マリマスタットとE-64dの併用（図8d）やマリマスタットとナファモスタットの併用（図8e）が病原性SARS-CoV-２ウイルスの感染を協調的に抑えた。またE-64dの代わりにエンドソームの酸性化を抑制する塩化アンモニウム（ammonium chloride (NH4Cl)）もマリマスタットとの併用で同程度の協調性を示したことから、E-64dや塩化アンモニウムに加えて塩化アンモニウムと同様にエンドソーム内の酸性化抑制効果が期待されるクロロキン（chloroquine）やヒドロキシクロロキン（hydroxychloroquine）なども併用効果が期待できる。 The results were as shown in FIG. In all cells, virus infection was suppressed in a concentration-dependent manner (Fig. 8a). Marimastat and prinomastat have a blood concentration of approximately 600-900 nM in safe administration set in clinical trials, and in this experiment, they show an infection-inhibitory effect between 300 nM and 1000 nM. Therefore, these drugs have great potential to be used for the treatment of COVID-19. Furthermore, GW280264X (ADAM10/17 inhibitor) and GI1254023X (MMP9/ADAM10 inhibitor), but not TAPI2 (ADAM17 inhibitor), inhibited pathogenic SARS-CoV-2 virus infection in HEC50B cells (Figure 8b). ). Moreover, suppression of ADAM10 expression by siRNA suppressed infection with the pathogenic SARS-CoV-2 virus (Fig. 8c). These results indicate that ADAM10-targeted inhibitors suppress infection with the pathogenic SARS-CoV-2 virus and may be therapeutic agents for COVID-19. The inhibitors that are actually administered for treatment include nucleic acid drugs similar to siRNA that suppress ADAM10 expression, as used in this study, ADAM10 enzymatic activity, and complex formation with proteins that functionally bind to ADAM10. A small molecule or antibody that inhibits is envisioned. In addition, the combination of marimastat and E-64d (Fig. 8d) and the combination of marimastat and nafamostat (Fig. 8e) cooperatively suppressed infection with the pathogenic SARS-CoV-2 virus. In addition, instead of E-64d, ammonium chloride (NH 4 Cl), which suppresses endosomal acidification, showed similar synergism when used in combination with marimastat. In addition, chloroquine and hydroxychloroquine, which are expected to have the same effect of suppressing acidification in endosomes as ammonium chloride, can be expected to have a combined effect.
　以上の成果から、COVID-19の治療においてもメタロプロテアーゼ依存性侵入経路を標的とする薬剤とエンドソーム経路やTMPRSS2経路を標的とする薬剤等の他のCOVID-19の症状を緩和できる薬剤とを併用することで、より有効な治療法の開発につながることが期待される。 Based on the above results, in the treatment of COVID-19, drugs that target the metalloprotease-dependent entry pathway and other drugs that can alleviate the symptoms of COVID-19, such as drugs that target the endosomal pathway and the TMPRSS2 pathway This is expected to lead to the development of more effective treatments.

Claims (20)

  1. 　ADAM10阻害剤を含む、COVID-19（coronavirus disease 2019）の治療または予防用組成物。 A composition for treating or preventing COVID-19 (coronavirus disease 2019) containing an ADAM10 inhibitor.
  2. 　ADAM10阻害剤が核酸である、請求項１に記載の治療または予防用組成物。 The therapeutic or preventive composition according to claim 1, wherein the ADAM10 inhibitor is a nucleic acid.
  3. 　核酸がsiRNAである、請求項２に記載の治療または予防用組成物。 The therapeutic or preventive composition according to claim 2, wherein the nucleic acid is siRNA.
  4. 　他の薬剤と併用するための、請求項１または２に記載の治療または予防用組成物。 The therapeutic or preventive composition according to claim 1 or 2, for use in combination with other drugs.
  5. 　他の薬剤が、マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、請求項４に記載の治療または予防用組成物。 5. Treatment or prevention according to claim 4, wherein the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine. composition.
  6. 　他の薬剤が、TMPRSS２阻害剤である、請求項４または５に記載の治療または予防用組成物。 The therapeutic or preventive composition according to claim 4 or 5, wherein the other drug is a TMPRSS2 inhibitor.
  7. 　ADAM10阻害剤をそれを必要とする対象に投与する工程を含む、COVID-19（coronavirus disease 2019）の治療または予防方法。 A method of treating or preventing COVID-19 (coronavirus disease 2019), which includes the step of administering an ADAM10 inhibitor to a subject in need thereof.
  8. 　ADAM10阻害剤が核酸である、請求項６に記載の治療または予防方法。 The therapeutic or preventive method according to claim 6, wherein the ADAM10 inhibitor is a nucleic acid.
  9. 　核酸がsiRNAである、請求項７に記載の治療または予防方法。 The therapeutic or preventive method according to claim 7, wherein the nucleic acid is siRNA.
  10. 　他の薬剤と併用して投与する、請求項７または８に記載の治療または予防方法。 The therapeutic or preventive method according to claim 7 or 8, which is administered in combination with other drugs.
  11. 　他の薬剤が、マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、請求項１０に記載の治療または予防方法。 11. Treatment or prevention according to claim 10, wherein the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine. Method.
  12. 　他の薬剤が、TMPRSS２阻害剤である、請求項１０または１１に記載の治療または予防方法。 The therapeutic or preventive method according to claim 10 or 11, wherein the other drug is a TMPRSS2 inhibitor.
  13. 　COVID-19（coronavirus disease 2019）の治療または予防に用いるための、ADAM10阻害剤。 ADAM10 inhibitors for use in the treatment or prevention of COVID-19 (coronavirus disease 2019).
  14. 　COVID-19（coronavirus disease 2019）の治療または予防に用いるための、ADAM10阻害剤および他の薬剤の組合せ。 A combination of an ADAM10 inhibitor and other agents for use in the treatment or prevention of COVID-19 (coronavirus disease 2019).
  15. 　他の薬剤が、マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、請求項１４に記載の組合せ。 The combination according to claim 14, wherein the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  16. 　他の薬剤が、TMPRSS２阻害剤である、請求項１４または１５に記載の組合せ。 The combination according to claim 14 or 15, wherein the other drug is a TMPRSS2 inhibitor.
  17. 　COVID-19（coronavirus disease 2019）の治療または予防用組成物の製造のためのADAM10阻害剤の使用。 　Use of an ADAM10 inhibitor for the manufacture of a composition for the treatment or prevention of COVID-19 (coronavirus disease 2019).
  18. 　COVID-19（coronavirus disease 2019）の治療または予防用組成物の製造のためのADAM10阻害剤および他の薬剤の組合せの使用。 　The use of a combination of an ADAM10 inhibitor and other agents for the manufacture of a composition for the treatment or prevention of COVID-19 (coronavirus disease 2019).
  19. 　他の薬剤が、マリマスタット、プリノマスタット、E-64d、塩化アンモニウム、クロロキンおよびヒドロキシクロロキンからなる群から選択される１種または２種以上の化合物である、請求項１８に記載の使用。 19. Use according to claim 18, wherein the other drug is one or more compounds selected from the group consisting of marimastat, prinomastat, E-64d, ammonium chloride, chloroquine and hydroxychloroquine.
  20. 　他の薬剤が、TMPRSS２阻害剤である、請求項１８または１９に記載の使用。

          20. Use according to claim 18 or 19, wherein the other agent is a TMPRSS2 inhibitor.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021076712A1 (en) * 2019-10-15 2021-04-22 Ophirex, Inc. Early management and prevention of sepsis and sepsis-like syndromes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021076712A1 (en) * 2019-10-15 2021-04-22 Ophirex, Inc. Early management and prevention of sepsis and sepsis-like syndromes

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ATHANASIOS DIDANGELOS: "ABSTRACT", MSPHERE, vol. 5, no. 3, XP055752095, DOI: 10.1128/mSphere.00367-20 *
IMBERT ISABELLE, GUILLEMOT JEAN-CLAUDE, BOURHIS JEAN-MARIE, BUSSETTA CÉCILE, COUTARD BRUNO, EGLOFF MARIE-PIERRE, FERRON FRANÇOIS, : "A second, non-canonical RNA-dependent RNA polymerase in SARS Coronavirus", THE EMBO JOURNAL / EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, IRL PRESS, OXFORD, vol. 25, no. 20, 18 October 2006 (2006-10-18), Oxford , pages 4933 - 4942, XP093055307, ISSN: 0261-4189, DOI: 10.1038/sj.emboj.7601368 *
JOCHER GEORG, GRASS VINCENT, TSCHIRNER SARAH K, RIEPLER LYDIA, BREIMANN STEPHAN, KAYA TUĞBERK, OELSNER MADLEN, HAMAD M SABRI, HOFM: "ADAM10 and ADAM17 promote SARS‐CoV‐2 cell entry and spike protein‐mediated lung cell fusion", EMBO REPORTS, NATURE PUBLISHING GROUP, LONDON, GB, vol. 23, no. 6, 7 June 2022 (2022-06-07), GB , XP093057297, ISSN: 1469-221X, DOI: 10.15252/embr.202154305 *
LIU TIANXIAO; LUO SONGYUAN; LIBBY PETER; SHI GUO-PING: "Cathepsin L-selective inhibitors: A potentially promising treatment for COVID-19 patients", PHARMACOLOGY & THERAPEUTICS, ELSEVIER., GB, vol. 213, 26 May 2020 (2020-05-26), GB , XP086249684, ISSN: 0163-7258, DOI: 10.1016/j.pharmthera.2020.107587 *
MA YUANCHEN, HUANG YINONG, WANG TAO, XIANG ANDY PENG, HUANG WEIJUN: "ACE2 Shedding and Furin Abundance in Target Organs may Influence the Efficiency of SARS-CoV-2 Entry", THE OPEN BIOINFORMATICS JOURNAL, vol. 14, no. 1, 22 March 2021 (2021-03-22), pages 1 - 12, XP093057298, ISSN: 1875-0362, DOI: 10.2174/1875036202114010001 *
NGUYEN HANH T., SHIJIAN ZHANG, QIAN WANG, SAUMYA ANANG, JIA WANG, HAITAO DING, JOHN C. KAPPES, JOSEPH SODROSKI: "Spike Glycoprotein and Host Cell Determinants of SARS-CoV-2 Entry and Cytopathic Effects", JOURNAL OF VIROLOGY, vol. 95, no. 5, pages 10.1128/jvi.02304 - 20, XP093057303, DOI: 10.1128/jvi.02304-20 *
SHEKHAWAT JYOTI; GAUBA KAVYA; GUPTA SHRUTI; PUROHIT PURVI; MITRA PRASENJIT; GARG MAHENDRA; MISRA SANJEEV; SHARMA PRAVEEN; BANERJEE: "Interleukin-6 Perpetrator of the COVID-19 Cytokine Storm", INDIAN JOURNAL OF CLINICAL BIOCHEMISTRY, SPRINGER PRIVATE LTD., IN, vol. 36, no. 4, 21 June 2021 (2021-06-21), IN , pages 440 - 450, XP037568810, ISSN: 0970-1915, DOI: 10.1007/s12291-021-00989-8 *
TOMITA YURIKO, MATSUYAMA SHUTOKU, FUKUHARA HIDEO, MAENAKA KATSUMI, KATAOKA HIROAKI, HASHIGUCHI TAKAO, TAKEDA MAKOTO: "The Physiological TMPRSS2 Inhibitor HAI-2 Alleviates SARS-CoV-2 Infection", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 95, no. 12, 24 May 2021 (2021-05-24), US , XP093057300, ISSN: 0022-538X, DOI: 10.1128/JVI.00434-21 *
YAMAMOTO MIZUKI, GOHDA JIN, KOBAYASHI AYAKO, TOMITA KEIKO, HIRAYAMA YOUKO, KOSHIKAWA NAOHIKO, SEIKI MOTOHARU, SEMBA KENTARO, AKIYA: "Metalloproteinase-Dependent and TMPRSS2-Independent Cell Surface Entry Pathway of SARS-CoV-2 Requires the Furin Cleavage Site and the S2 Domain of Spike Protein", MBIO, vol. 13, no. 4, 30 August 2022 (2022-08-30), XP093057295, DOI: 10.1128/mbio.00519-22 *

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