WO2015050170A1 - Médicament d'inhibition du virus de la grippe - Google Patents

Médicament d'inhibition du virus de la grippe Download PDF

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WO2015050170A1
WO2015050170A1 PCT/JP2014/076317 JP2014076317W WO2015050170A1 WO 2015050170 A1 WO2015050170 A1 WO 2015050170A1 JP 2014076317 W JP2014076317 W JP 2014076317W WO 2015050170 A1 WO2015050170 A1 WO 2015050170A1
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subunit
amino acid
fragment
acid sequence
subunit fragment
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Japanese (ja)
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勇作 上村
柏木　孝仁
好勇 原
渡邊　浩
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学校法人 久留米大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16133—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the present invention relates to an influenza virus-derived PA subunit fragment and an influenza virus inhibitor containing the fragment.
  • Influenza virus is an RNA virus having minus-strand RNA as a genome. Influenza virus phenotypes or genomic base sequences are frequently mutated, and sometimes infect across species walls.
  • Influenza viruses are classified into three genera, type A, type B, and type C, based on the antigenicity of the proteins that make up the virus particles.
  • a type and B type are classified into three genera, type A, type B, and type C, based on the antigenicity of the proteins that make up the virus particles.
  • Non-Patent Document 1 The majority of current influenza drugs target neuraminidase (NA) present on the virus surface.
  • NA neuraminidase
  • the type of influenza virus (for example, H1N1, H3N2, H5N1, etc.) is determined by the combination of the subtypes.
  • oseltamivir commercially available as “Tamiflu”
  • zanamivir Relenza
  • Influenza virus RNA polymerase plays an important role in virus growth after human infection, and can therefore be a target for anti-influenza drugs.
  • the only drug therapy targeting viral RNA polymerase is T705 (generic name: favipiravir: Toyama Chemical) (Non-patent Document 8).
  • Viral RNA polymerase plays a broad role for virus replication (Non-patent Document 9).
  • This polymerase consists of a heterotrimeric complex, has a total molecular weight of approximately 250 kDa, and is composed of three subunits of PA, PB1 and PB2 (Non-patent Document 10). All three of these subunits are required for both transcription and replication (Non-Patent Document 11). So far, information on the structure of RNA polymerase is very limited (Non-Patent Documents 12 to 14).
  • NA inhibitor neuraminidase inhibitor
  • the present inventors have the effect that a fragment of the PA subunit, which is a protein of influenza virus itself, stops gene replication of influenza virus and suppresses the proliferation of influenza virus itself. As a result, the present invention has been completed.
  • the present invention is an influenza virus inhibitor using the PA subunit, which is a protein of the influenza virus itself, and the mechanism of action is completely different from existing NA inhibitors. Since the mechanism of action of NA inhibitors is inhibition of virus release, influenza virus growth cannot be inhibited. On the other hand, since the inhibitor according to the present invention has an action of stopping gene replication of influenza virus, it has an action of stopping the proliferation of influenza virus itself. In addition, while the vaccine is a countermeasure against a specific strain, the inhibitor of the present invention is uniformly effective against many strains.
  • the present invention includes the following.
  • Influenza virus-derived PA subunit fragment [2] Influenza viruses are A / Vietnam / 1194/2004 (H5N1) strain, A / WSN / 33 strain (Soviet H1N1), A / NT / 60/68 strain (Hong Kong type H3N2), A / HongKong / 156 / The PA subunit fragment according to [1], which is strain 97 (1997 avian H5N1) or A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1). [3] The PA subunit fragment according to [1] or [2], which is an N-terminal fragment of the PA subunit.
  • the PA subunit fragment contains 28th proline (P), 86th methionine (M), 91st valine (V) and 100th valine (from the N-terminal of the amino acid sequence of the natural PA subunit (
  • the PA subunit fragment contains 28th proline (P), 86th methionine (M), 91th valine (V) and 100th valine (N) from the N-terminal of the amino acid sequence of the natural PA subunit.
  • the PA subunit fragment is a PA subunit fragment containing the 187th leucine (L) and / or the 188th tryptophan (W) from the N-terminal of the amino acid sequence of the natural PA subunit, [1] Thru
  • the PA subunit fragment according to any one of [1] to [9] which comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4.
  • PA subunit fragment is a PA subunit fragment comprising the amino acid sequence from the N-terminal to the 187th amino acid sequence (SEQ ID NO: 5) of the natural PA subunit amino acid sequence A PA subunit fragment according to claim 1.
  • PA subunit fragment is a PA subunit fragment comprising the amino acid sequence from the N-terminal to the 188th amino acid sequence (SEQ ID NO: 6) of the natural PA subunit amino acid sequence A PA subunit fragment according to claim 1.
  • PA subunit fragment is a PA subunit fragment comprising the amino acid sequence from the N-terminal to the 192nd amino acid sequence (SEQ ID NO: 7) of the natural PA subunit amino acid sequence
  • PA subunit fragment according to claim 1 A nucleic acid encoding the PA subunit fragment according to any one of [1] to [13].
  • a recombinant vector comprising the nucleic acid according to [14].
  • a transformed cell comprising the vector according to [15].
  • An influenza virus inhibitor comprising an influenza virus-derived PA subunit fragment.
  • Influenza viruses derived from PA subunit fragments are A / Vietnam / 1194/2004 (H5N1) strain, A / WSN / 33 strain (Soviet H1N1), A / NT / 60/68 strain (Hong Kong type H3N2)
  • the influenza virus inhibitor according to [17] which is A / Hong Kong / 156/97 strain (1997 avian H5N1) or A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1).
  • the PA subunit fragment contains 28th proline (P), 86th methionine (M), 91th valine (V) and 100th valine (N) from the amino acid sequence of the natural PA subunit.
  • the PA subunit fragment contains 28th proline (P), 86th methionine (M), 91th valine (V) and 100th valine (N) from the amino acid sequence of the natural PA subunit.
  • the PA subunit fragment is a PA subunit fragment containing the 187th leucine (L) and / or the 188th tryptophan (W) from the N-terminus of the amino acid sequence of the natural PA subunit, [17] Thru
  • PA subunit fragment comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4.
  • Virus inhibitor Any of [17] to [22], wherein the PA subunit fragment is a PA subunit fragment comprising the amino acid sequence from the N-terminal to the 187th amino acid sequence (SEQ ID NO: 5) of the natural PA subunit amino acid sequence
  • PA subunit fragment is a PA subunit fragment comprising the amino acid sequence from the N-terminal to the 188th amino acid sequence (SEQ ID NO: 6) of the natural PA subunit amino acid sequence A PA subunit fragment according to claim 1.
  • PA subunit fragment is a PA subunit fragment comprising the amino acid sequence from the N-terminal to the 192nd amino acid sequence (SEQ ID NO: 7) of the natural PA subunit amino acid sequence A PA subunit fragment according to claim 1.
  • the influenza virus inhibitor according to any one of [17] to [29], which inhibits virus growth by inhibiting gene replication of influenza virus.
  • the present invention is an influenza virus inhibitor using the PA subunit, which is a protein of the influenza virus itself, and is completely different from the action of an influenza inhibitor such as Tamiflu, and is an epoch-making that suppresses the growth of the influenza virus itself.
  • an influenza inhibitor such as Tamiflu
  • the inhibitory effect is much superior to that of conventional Tamiflu and the like, and proliferation is suppressed to 1/1000 or less by administration on the microgram order.
  • it does not target HA and NA existing on the virus surface like conventional influenza drugs, but acts on RNP (nucleic acid protein complex) of influenza virus containing gene replication enzyme.
  • Any influenza virus strain selected from the group consisting of (1997 avian H5N1) and A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1) has an extremely excellent inhibitory action and has a wide range of applications independent of virus strain Is possible.
  • FIG. 1 is a diagram showing the construction of an influenza virus replicon applying the influenza virus reverse genetics method (virus artificial synthesis method).
  • FIG. 2 is a diagram showing an outline of the experimental system of the present invention.
  • mRNA viral mRNA
  • vRNA viral gene (viral genomic RNA);
  • cRNA complementary strand of viral gene
  • FIG. 3 is a schematic diagram showing an outline of the present invention.
  • FIG. 4 is a graph showing that VN PA N212 of the present invention has significant inhibitory activity from the low concentration stage on the activity of WSN RNP.
  • FIG. 5 is a diagram showing that PA subunit fragments derived from strains other than VN PA N212 also have the same inhibitory activity.
  • FIG. 5 shows the measurement by luciferase activity, and the right panel of FIG. 5 shows the measurement by Primer Extension Extension.
  • WSN A / WSN / 33 strain (Soviet H1N1); NT: A / NT / 60/68 strain (Hong Kong type H3N2); HK: A / HongKong / 156/97 strain (1997 avian H5N1); VN: A / Vietnam / 1194/2004 (H5N1) strain; SW: A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1)
  • FIG. 6 is a diagram showing the results of measurement using Renilla luciferase as a control in order to further verify the specificity.
  • FIG. 6 is a diagram showing the results of measurement using Renilla luciferase as a control in order to further verify the specificity.
  • FIG. 6 is a diagram showing the results of measurement using Renilla luciferase as a control in order
  • FIG. 7 is a diagram showing that VN PA N212 of the present invention has a significant inhibitory effect on other strains other than WSN (H1N1) as well.
  • WSN A / WSN / 33 strain (Soviet H1N1); NT: A / NT / 60/68 strain (Hong Kong type H3N2); HK: A / HongKong / 156/97 strain (1997 avian H5N1); VN: A / Vietnam / 1194/2004 (H5N1) strain; SW: A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1)
  • FIG. 8 is a diagram showing the results of confirming intracellular expression of each subunit constituting the RNP of influenza virus.
  • FIG. 8 is a diagram showing the results of confirming intracellular expression of each subunit constituting the RNP of influenza virus.
  • FIG. 9 is a diagram showing that endonuclease is involved in the loss (inhibition) of RNP in VN PA N212 of the present invention.
  • FIG. 10 is a diagram showing that endonuclease is involved in the loss (inhibition) of RNP in VN PA N212 of the present invention.
  • FIG. 11 is a diagram showing that the action of reducing the RNP activity of influenza virus by VN PA N212 of the present invention is caused by the loss of RNP itself.
  • FIG. 12 is a diagram showing the results of examining the inhibitory activity of various VN PA N212 mutants in which amino acids were deleted near the N-terminus of VN PA ⁇ N212 or amino acids were omitted in the middle or near the C-terminus.
  • FIG. 13 is a diagram showing the results of examining the inhibitory activity of various VN PA N212 mutants in which amino acids were deleted near the C-terminus of VN PA N212.
  • FIG. 14 is a graph showing that the inhibitory activity decreases when 187th leucine (L) and 188th tryptophan (W) are substituted from the N-terminus of VNVPA N212.
  • FIG. 15 shows that the PA subunit fragment expressed from the plasmid, not the plasmid itself, is the main body of inhibitory activity.
  • FIG. 16 is a diagram showing the results of confirming intracellular expression of each subunit constituting RNP of influenza virus (A, C), and in the VN PA N212 of the present invention, endonuclease is responsible for the loss (inhibition) of RNP. It is a figure (B) which shows having been involved.
  • the present invention is an influenza virus inhibitor using the PA subunit, which is a protein of the influenza virus itself, and has a completely different mechanism of action from existing NA inhibitors. Since the mechanism of action of NA inhibitors is inhibition of virus release, influenza virus growth cannot be inhibited. On the other hand, since the inhibitor according to the present invention has an action of stopping gene replication of influenza virus, it has an action of stopping the proliferation of influenza virus itself. In addition, while the vaccine is a countermeasure against a specific strain, the inhibitor of the present invention is uniformly effective against many strains.
  • FIG. 1 shows the construction of an influenza virus replicon applying the influenza virus reverse genetics method (virus artificial synthesis method).
  • PB1 subunit RNA polymerase PB2 subunit
  • PA RNA polymerase PA subunit
  • NP protein nuclear protein constituting the RNP (nucleic acid / protein complex) of influenza virus NP
  • vLUC modified with an influenza virus gene introducing a firefly luciferase gene in place of the influenza virus gene
  • PB1, PB2, PA and NP generated from each plasmid are expressed in the cell as proteins, and vLUC is expressed in the cells as virus-like RNA.
  • Each component assembles in 293T cells to form the influenza virus RNP (where the gene is firefly luciferase).
  • This RNP is a replicon in which gene replication and transcription can be autonomously performed in a cell, that is, gene replication and transcription of influenza virus are artificially constructed in the cell.
  • the gene is replaced with firefly luciferase, the protein generated from the transcript of this replicon is not an influenza virus protein but firefly luciferase.
  • the intracellular activity of influenza virus RNP can be evaluated.
  • how a gene replication / transcription activity is changed by adding a plasmid expressing a VN PA N212 fragment is measured.
  • the RNP can autonomously perform gene replication and transcription in the cell (influenza virus replicon). Extract replication / transcription products from cells and use specific primers for each nucleic acid (mRNA (viral mRNA), vRNA (viral gene; viral genomic RNA), cRNA (viral complementary strand))
  • mRNA viral mRNA
  • vRNA viral gene; viral genomic RNA
  • cRNA viral complementary strand
  • the present inventors based on the above RNP activity measurement system, the N-terminal fragment of the PA subunit of the VN strain (fragment from the N-terminal to the 212th amino acid of the PA subunit; hereinafter also referred to as “VN PA N212”) ) was added to the RNP of the WSN strain influenza virus, it was found that a significant decrease in RNP activity was observed (see FIGS. 3 and 4). On the other hand, almost no decrease in activity was observed in the C-terminal fragment of the PA subunit (the remaining fragment after VNVPA N212 was cleaved from the PA subunit; the fragment from the N-terminal position to the C-terminal position of the PA subunit). There wasn't.
  • the present invention is based on the knowledge that a partial fragment of the PA subunit isolated from a specific strain has a significant gene replication enzyme inhibitory effect. That is, it is the knowledge that influenza virus can be inhibited with influenza virus. The mechanism of action still remains unclear, but it acts on RNP (nucleic acid protein complex) of influenza virus containing gene replication enzyme, and has the effect of losing gene replication activity and transcription activity of influenza virus. Confirmed.
  • RNP nucleic acid protein complex
  • the endonuclease site contained in the PA subunit fragment is involved in the action, and it is speculated that it specifically acts on the influenza virus RNP. This fragment has a remarkable inhibitory activity and has a completely different mechanism of action from existing NA inhibitors, so it is considered promising as a next-generation influenza virus inhibitor.
  • the PA subunit fragment of the present invention may be derived from any strain of influenza virus.
  • the PA subunit fragment of the present invention can be obtained from the A / Vietnam / 1194/2004 (H5N1) strain.
  • Other influenza virus strains such as A / WSN / 33 strain (Soviet H1N1), A / NT / 60/68 strain (Hong Kong type H3N2), A / Hong Kong / 156/97 strain (1997 avian H5N1) or
  • the PA subunit fragment of the present invention can also be obtained from the A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1).
  • the A / WSN / 33 strain (Soviet type H1N1) is a common strain used in influenza research, and is in the same series as the seasonal Soviet type that was prevalent in the past.
  • the A / NT / 60/68 strain (Hong Kong type H3N2) is also a common strain used in influenza research and is in the same series as the seasonal Hong Kong type that is still prevalent.
  • the A / Hong Kong / 156/97 strain (1997 avian H5N1) is a highly pathogenic avian influenza that infects humans in 1997.
  • the A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1) is a pandemic strain (separated from Kurume University School of Medicine, Department of Infectious Medicine, Clinical Infectious Medicine Division) that was developed in 2009.
  • the PA subunit fragment of the present invention is preferably an N-terminal fragment of the PA subunit.
  • Examples of such an N-terminal fragment include a fragment having an amino acid sequence from the N-terminal to 212 amino acids of the PA subunit.
  • the specific amino acid sequence is as follows. (SEQ ID NO: 4)
  • Another preferred PA subunit fragment of the present invention is a fragment having an amino acid sequence from the N terminus of the PA subunit to at least 187 amino acids (SEQ ID NO: 5). Yet another preferred PA subunit fragment of the present invention is a fragment having an amino acid sequence from the N-terminus of the PA subunit to at least 188 amino acids (SEQ ID NO: 6). Yet another preferred PA subunit fragment of the present invention is a fragment having an amino acid sequence from the N-terminus of the PA subunit to at least 192 amino acids (SEQ ID NO: 7).
  • PA N-terminal The functions of PA N-terminal reported so far include endonuclease and protease. Endonucleases target cellular mRNAs and have the effect of cleaving cellular mRNA caps. In addition, the protease substrate is still unknown. Since active centers have been reported for any of these functions (endonuclease active center: D108 position; protease active center: T157 position), the present inventors have created mutants in which the amino acids at the respective active centers are substituted. The inhibitory effect was confirmed.
  • the PA subunit fragment of the present invention may be any fragment from the PA subunit as long as its endonuclease activity is retained.
  • the PA subunit fragment of the present invention is not limited in its amino acid length as long as its endonuclease activity is retained.
  • the PA subunit fragment of It may be a fragment consisting of amino acids up to position 190, 200, 210, 220, or 230.
  • the endonuclease activity of various N-terminal fragments of the PA subunit was examined in more detail.
  • the amino acids from the N-terminal to 2 to 10 amino acids N Amino acids from the end to 2-22, amino acids from the N end to 2 to 40, amino acids from the N end to 2 to 60, amino acids from the N end to 2 to 80, amino acids from the N end to 2 to 100, and It was found that the inhibitory activity was lost when amino acids from N-terminal to 2 to 107 were deleted respectively. From this, it was found that the amino acid at the N-terminal side of the fragment having the amino acid sequence from the N-terminal to 212 amino acids of the PA subunit is particularly important for the endonuclease activity.
  • the amino acids from the N-terminal to 7-107, the amino acids from the N-terminal to 27-107, the amino acids from the N-terminal to 47-107 Amino acids from N-terminal to 67-107, amino acids from N-terminal to 87-107, amino acids from N-terminal to 16-26, amino acids from N-terminal to 29-85, amino acids from N-terminal to 52-75 Inhibitory activity is lost when amino acids from N-terminal to 52-83, amino acids from N-terminal to 110-132, amino acids from N-terminal to 137-164 and amino acids from N-terminal to 136-186 are deleted.
  • the inhibitory activity is lost even if any part of the fragment near the N-terminal, central part, or C-terminal is omitted. Since the bets has been found, it was found that the three-dimensional structure for endonuclease activity is important.
  • the inhibitory activity of the PA subunit fragment includes the 28th proline (P), 86 counted from the N-terminal of the amino acid sequence of the natural PA subunit. It was found that it was essential to contain at least one amino acid selected from the group consisting of the th methionine (M), the 91 st valine (V) and the 100 th valine (V). That is, these four amino acid residues can be said to be essential amino acids that affect the endonuclease activity of PA. Therefore, the PA subunit fragment of the present invention needs to contain at least one amino acid of the above four amino acid residues in order to retain its endonuclease activity.
  • P 28th proline
  • V 91 st valine
  • V 100 th valine
  • the amino acid sequence from the N-terminal to at least 187 amino acids of the amino acid sequence of the natural PA subunit is essential for the inhibitory activity of the PA subunit fragment as described above
  • the 187th leucine ( L) and 188th tryptophan (W) are speculated to be important amino acids for endonuclease activity. Therefore, when these amino acid residues were substituted to examine whether the inhibitory activity was affected, it was found that the alanine-substituted products of these amino acid residues significantly reduced the inhibitory activity (Example 11). Therefore, the 187th leucine (L) and the 188th tryptophan (W) were found to be essential to retain endonuclease activity.
  • the PA subunit fragment of the present invention also includes an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4 or in the various PA subunit fragments described above. Good.
  • the total number and position of amino acids to be deleted, substituted or added are not particularly limited as long as the obtained PA subunit fragment retains endonuclease activity.
  • the total number of amino acids to be deleted, substituted or added is 1 or more, preferably 1 or several, and the specific range thereof is usually 1 to 10, preferably 1 to 5 for deletion, More preferably, the number is 1 to 2, the substitution is usually 1 to 20, preferably 1 to 10, more preferably 1 to 3, and the addition is usually 1 to 10, preferably 1 to 5.
  • the number is more preferably 1-2.
  • a mutation introduction kit using a site-directed mutagenesis method such as Kunkel method or Gapped duplex method, for example, QuikChange TM Site-Directed Mutagenesis Use Kit (Stratagene), GeneTailor TM Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: Takara Bio) Can do.
  • a vector in which a gene encoding PA is appropriately incorporated into an expression vector in a form suitable for the expression of the protein is prepared, and an animal cell, plant cell, insect cell, or yeast is prepared.
  • a method of preparing a transformant introduced into any one of microorganisms such as Escherichia coli and Escherichia coli, and culturing the transformant is also possible to employ a production method based on cell-free protein synthesis.
  • Cell-free protein synthesis can be performed using commercially available kits. Examples of such kits include reagent kits PROTEIOSTM (Toyobo), TNTTMSystem (Promega), synthesizer PG-Mate TM (Toyobo), RTS (Roche Diagnostics).
  • Such a transformant or PA produced by cell-free protein synthesis can be separated and purified by various separation operations utilizing its physical properties, chemical properties, etc., if desired.
  • the purification method include various types of liquid chromatography such as normal salting-out, centrifugation, ultrasonic crushing, ultrafiltration, gel filtration, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and dialysis. And combinations thereof.
  • PA is produced by transformant or cell-free protein synthesis so that PA is fused with an affinity tag, and PA is separated and purified.
  • the present invention also provides a nucleic acid encoding the PA subunit fragment.
  • a nucleic acid includes a nucleic acid having a nucleotide sequence encoding an amino acid sequence from the N-terminal to 212 amino acids of the PA subunit.
  • the specific nucleotide sequence is as follows. (SEQ ID NO: 3)
  • the present invention also provides a recombinant vector containing the nucleic acid.
  • a DNA fragment of an appropriate length containing the coding region of the target polypeptide is prepared. Nucleotides may be substituted in the nucleotide sequence of the coding region of the polypeptide of interest so as to be the optimal codon for expression in the host cell. This DNA fragment can then be inserted downstream of the promoter of an appropriate expression vector to produce a recombinant vector (eg, Molecular® Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). See).
  • plasmids derived from E. coli pBR322, pBR325, pUC12, pUC13, etc.
  • plasmids derived from Bacillus subtilis pUB110, pTP5, pC194, etc.
  • plasmids derived from yeast pSH19, pSH15, etc.
  • bacteriophages such as ⁇ phage, etc.
  • animal viruses such as retrovirus and vaccinia virus, and insect pathogenic viruses such as baculovirus can be used.
  • a promoter, enhancer, ribosome binding site, various signal sequences (splicing signal, poly A addition signal, etc.), cloning site, translation / transcription terminator, selection marker, SV40 replication origin, etc. may be added to the expression vector.
  • Examples of such vectors include pGEX series (Amersham Pharmacia Biotech), pET® Expression® System (Novagen), and the like.
  • the present invention also provides a transformed cell containing the above vector.
  • a method described in Molecular® Cloning® 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 for example, calcium phosphate method, DEAE-dextran method, transfection method
  • Microinjection method, lipofection method, electroporation method, transduction method, scrape loading method, shotgun method, etc. or infection.
  • Host cells include bacterial cells (eg, Escherichia, Bacillus, Bacillus, etc.), fungal cells (eg, yeast, Aspergillus, etc.), insect cells (eg, S2 cells, Sf cells, etc.), animal cells ( Examples thereof include CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells), plant cells, and the like. In the examples of the present invention, HEK293T cells were used.
  • the present invention also provides an influenza virus inhibitor containing the influenza virus-derived PA subunit fragment of the present invention.
  • the said influenza virus inhibitor contains at least 1 sort (s) or more of the influenza virus origin PA subunit fragment of this invention as an active ingredient, and may contain the pharmaceutically acceptable additive as needed.
  • the influenza virus inhibitor of the present invention can be administered by any of oral, parenteral or topical routes.
  • the dose varies depending on the species of the animal to be treated (mammals, particularly humans) and the individual's responsiveness to the drug, the dosage form of the selected preparation, and the administration time and interval.
  • the influenza virus-derived PA subunit fragment can be administered at a dose ranging from about 10 mg to about 200 mg / day, preferably from about 15 mg to about 150 mg / day, more preferably from about 20 mg to about 100 mg / day.
  • influenza virus inhibitor of the present invention in addition to the above-mentioned influenza virus-derived PA subunit fragment, optionally in combination with a known pharmaceutically acceptable carrier or diluent, orally, parenterally or topically It can be administered in a single dose or multiple doses depending on any route.
  • the influenza virus inhibitor of the present invention has various different dosage forms such as tablets, capsules, lozenges, troches, hard candy, powders, sprays, creams, ointments, suppositories, jelly, gels Preparations, pastes, lotions, ointments, aqueous suspensions, solutions for injection, elixirs, syrups, and the like.
  • E-MEM 10% FBS (Nissui) medium
  • HEK293T cells Human Embryonic Kidney Cell
  • the culture was performed in the presence of 5% CO 2 .
  • the medium of 293T cells was carefully removed and treated with 1 ml of 0.25% trypsin (containing 1 mM EDTA) (Nakarai), and 30 ml of E-MEM (containing 10% FBS) was added and suspended. 2 ml of each suspended cell solution was spread on a 6-well plate.
  • Influenza virus RNP construction plasmids were prepared at 0.2 ⁇ g / ⁇ l, respectively.
  • As the template RNA the NA gene derived from A / WSN / 33 (H1N1), or one obtained by replacing the NA gene with a luciferase gene was used.
  • the nucleic acid encoding each constituent protein was inserted into pcDNA3.1 (+) (Invitrogen).
  • the template RNA used was inserted into pPOLI (FodorFE, et al. J Virol 76: 8989-9001. 2002).
  • a plasmid for expression of the PA fragment used as an inhibitor was also prepared at 0.2 ⁇ g / ⁇ l.
  • the N-terminal 212 amino acids (“VN PA N212”) of PA derived from the target site strain having an inhibitory effect (A / Vietnam / 1194/2004 (H5N1)) were transferred to pcDNA3.1 (+) (Invitrogen). It was what was inserted.
  • C-terminal 505 amino acids of PA (PA C716) were used as a comparison target of the inhibitory effect.
  • Opti-MEM Opti-MEM
  • Opti-MEM Opti-MEM
  • 100 ⁇ l of Opti-MEM 250 ⁇ l was added, 24 ⁇ l of Lipofectamine2000 (Invitrogen) was added, and lightly suspended.
  • the above-mentioned plasmid solution was added to this, lightly suspended, and incubated at room temperature for 20 minutes. 300 ⁇ l (total volume) of this suspension was inoculated into the suspension cell solution on the plate. Cells were cultured for 30 hours at 37 ° C.
  • influenza virus strain-derived PA subunit fragments About PA subunit fragments derived from other influenza virus strains other than A / Vietnam / 1194/2004 (H5N1) by the same procedure as in Example 1.
  • the growth inhibitory effect of influenza virus was examined.
  • Other influenza virus strains include A / WSN / 33 strain (Soviet H1N1), A / NT / 60/68 strain (Hong Kong type H3N2), A / Hong Kong / 156/97 strain (1997 avian H5N1) and A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1) was used.
  • confirmation was also made in an experimental system (Primer Extension Assay) that can specifically detect gene replication and transcriptional activity of influenza virus.
  • Renilla luciferase In order to further verify the measurement specificity of the inhibitory activity using Renilla luciferase, measurement using Renilla luciferase as a control was performed.
  • the same procedure as in Example 1 was followed except that pRL-TK was used as a plasmid expressing Renilla luciferase.
  • Firefly luciferase was generated from intracellular artificial influenza virus RNP, whereas Renilla luciferase was expressed in cells completely independently of influenza virus RNP (internal control).
  • These two luciferases have different chromogenic substrates, and by using Promega's Dual luciferase assay kit, the activities of the two luciferases could be distinguished and measured. The measurement method was performed according to the manual attached to Promega.
  • VN PAN 212 The action of VN PAN 212 on other influenza virus strains was confirmed.
  • a / WSN / 33 (H1N1) used in Example 1 A / NT / 60/68 strain (Hong Kong type H3N2), A / Hong Kong / 156/97 strain (1997 avian type H5N1), A / The same procedure as in Example 1 was followed except that the WSN / 33 strain (Soviet H1N1) and A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1) were used.
  • VN PA N212 is A / NT / 60/68 strain (Hong Kong type H3N2), A / Hong Kong / 156/97 strain (1997 avian type H5N1), A / WSN / 33. Both the strain (Soviet type H1N1) and the A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1) showed a remarkable inhibitory activity similar to that of A / WSN / 33 (H1N1). From this, it was found that VN212PA ⁇ ⁇ N212 has a high inhibitory effect on any strain that would mainly affect humans.
  • PA subunit fragment mutant 1 In order to examine the inhibition mechanism by the PA subunit fragment, mutation was introduced into the PA subunit fragment and the inhibitory activity was measured.
  • the N-terminal functions of PA reported so far include endonucleases and proteases. Endonucleases target cellular mRNAs and have the effect of cleaving cellular mRNA caps. In addition, the protease substrate is still unknown. Since active centers have been reported for any of these functions, each mutant was prepared and its inhibitory effect was confirmed.
  • VN PA N212 mutant was used instead of VN PA N212.
  • VN PA N212 mutant QuikChange TM Site-Directed Mutagenesis Kit (Stratagene) was used by site-directed mutagenesis method (DpnI treatment method).
  • N212 / K134A lost its inhibitory activity similarly to N212 / D108A. This further confirmed that the endonuclease activity of VN PA N212 is involved in the loss (inhibition) of RNP.
  • RNP RNP using PA subunit fragment mutant
  • the expression of RNP was confirmed using the VN PA N212 mutant.
  • the amount of protein in the cell extract used in Example 7 was measured using SDS-PAGE method (12% gelling agent) and Western blot method (20% methanol, 100 V, 1 hour, Millipore PVDF membrane). Except for this, the same procedure as in Example 5 was followed.
  • VN PA N212 deletion mutant was used instead of VN PA N212.
  • VN PA N212 mutant QuikChange TM Site-Directed Mutagenesis Kit (Stratagene) was used by site-directed mutagenesis method (DpnI treatment method).
  • VN PA N212 amino acids from N-terminal to 7-107 (d2-107), amino acids from N-terminal to 27-107 (d27-107), amino acids from N-terminal to 47-107 (d47-107) ), Amino acids from N-terminal to 67-107 (d67-107), amino acids from N-terminal to 87-107 (d87-107), amino acids from N-terminal to 16-26 (d16-26), from N-terminal Amino acids from 29 to 85 (d29-85), Amino acids from N-terminal to 52-75 (d52-75), Amino acids from N-terminal to 52-83 (d52-83), N-terminal to 110-132 Amino acids (d110-132), amino acids from N-terminal to 137-164 (d137-164), and amino acids from N-terminal to 136-186 (d1 It was found that the deletion of each of 36-186) lost the inhibitory activity.
  • the three-dimensional structure is important for the endonuclease activity because the inhibitory activity is lost even if any part of the VNNPA ⁇ N212 near the N-terminal, middle part, and C-terminal is omitted. It was.
  • VN PA N212 deletion mutant was used instead of VN PA N212.
  • VN PA N212 mutant QuikChange TM Site-Directed Mutagenesis Kit (Stratagene) was used by site-directed mutagenesis method (DpnI treatment method).
  • Example 10 Measurement of inhibitory activity by PA subunit fragment mutant (5)
  • the results of Example 10 showed that the amino acid sequence from the N-terminal of the amino acid sequence of the natural PA subunit to at least 187 amino acids is essential for the inhibitory activity of the PA subunit fragment. From this, it was speculated that the 187th leucine (L) and the 188th tryptophan (W) counted from the N-terminal are amino acids important for endonuclease activity. Therefore, it was investigated whether substitution of these amino acid residues would affect the inhibitory activity. Specifically, fragments in which these amino acid residues were substituted with alanine were prepared and examined for inhibitory activity (N212 / L187A and N212 / W188A; FIG. 14).
  • VN PA N212 mutant was used instead of VN PA N212.
  • VN PA N212 mutant QuikChange TM Site-Directed Mutagenesis Kit (Stratagene) was used by site-directed mutagenesis method (DpnI treatment method).
  • a plasmid is used as an expression vector to express the PA subunit fragment, so that the inhibitory activity of the present invention is not due to the PA subunit fragment but to the plasmid itself or the act of introducing the plasmid. The suspicion remains. Therefore, the following experiment was conducted to eliminate such doubts.
  • VN PAN 212 As a plasmid for expression of the PA fragment, VN PAN 212 was inserted into pcDNA3.1 (+), and as a comparison target of the inhibitory effect, PA C-terminal 504 amino acids (VN / PA / C504) were pcDNA3.1 ( The procedure was the same as in Example 1, except that the plasmid inserted in (+) and the plasmid alone pcDNA3.1 (+) were used.
  • Example 5 when the PA subunit fragment of the present invention is allowed to act, components of influenza virus RNP (PB1, PB2, PA, NP) are lost from the cell (see FIG. 8).
  • RNP influenza virus
  • intracellular expression of each subunit constituting the RNP of influenza virus was confirmed for VN PA N212 and VN / PA / C504. Two concentrations were tested. The results are shown in FIG. 16 (FIG. 16A). The expression of VN PA N212 was confirmed, and all components of RNP were lost depending on the concentration (FIG. 16A). From this result and the result of the above Example 12 (result of the plasmid alone), it was confirmed that the PA subunit fragment expressed from the plasmid exhibited an inhibitory effect.
  • N212 / D108A, N212 / K134A and N212 / T157A were expressed in the same manner as in Example 5. These mutants were examined for inhibitory activity in the same manner as in Example 6. The results are shown in FIG. 16 (FIGS. 16B and C). Expression was confirmed in all variants of N212 / T157A, N212 / K134A, and N212 / D108A.
  • the PA subunit fragment of the present invention has been confirmed to act on RNP (nucleic acid protein complex) of influenza virus and to lose gene replication activity and transcription activity of influenza virus.
  • RNP nucleic acid protein complex
  • the endonuclease site contained in the PA subunit fragment is involved in the action, and specifically acts on the influenza virus RNP.
  • the PA subunit fragment of the present invention has remarkable influenza virus growth inhibitory activity and is quite promising as a next-generation influenza virus inhibitor because it has a completely different mechanism of action from existing NA inhibitors.
  • VN PA N212 was A / NT / 60/68 strain (Hong Kong type H3N2), A / Hong Kong / 156/97 strain (1997 avian type H5N1), A / WSN. / 33 strain (Soviet H1N1) and A / Kurume / K0910 / 2009 strain (2009 pandemic H1N1) showed the same significant inhibitory activity as A / WSN / 33 (H1N1), depending on the strain A wide range of adaptation is possible.

Abstract

La présente invention concerne un médicament anti-grippal qui a un mécanisme d'action entièrement nouveau et est exempt de défauts observés dans des médicaments anti-grippaux classiques tels que des vaccins et des médicaments d'inhibition des neuraminidases (NA). L'invention concerne aussi un fragment de sous-unité de PA dérivé du virus de la grippe et un médicament d'inhibition du virus de la grippe qui comprend le fragment de sous-unité de PA dérivé du virus de la grippe.
PCT/JP2014/076317 2013-10-02 2014-10-01 Médicament d'inhibition du virus de la grippe WO2015050170A1 (fr)

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