Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • 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
• • 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
• • 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
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • 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
  • A61P31/20—Antivirals for DNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes

Definitions

• the present invention relates to a pharmaceutical composition for treating or diagnosing a viral infection containing an active peptide that binds to a virus and inhibiting its infection, particularly for treating or diagnosing a viral infection containing Sugitake lectin and/or its mutant peptide.
• the present invention relates to diagnostic pharmaceutical compositions.
• the invention also relates to variants of the active peptide.
• sugar chains of complex carbohydrates such as glycoproteins and glycolipids that exist on cell surfaces and in body fluids function as a type of information element and are deeply involved in important life phenomena such as development, immunity, cancer, and infection.
• lectins which are sugar chain-binding proteins, function as sugar chain recognition molecules and, like sugar chains, play a biologically important role.
• Pholiota squarrosa lectin is a peptide consisting of 40 amino acid residues that specifically binds to ⁇ 1-6 fucose (Patent Document 1, Non-Patent Document 1), and is useful for the detection of AFP-L3. , used to determine the malignancy of colorectal cancer, detect pancreatic cancer, etc.
• Non-Patent Document 2 Lectins derived from hairy vetch (Vicia villosa), Wisteria floribunda, Aleuria aurantia, or Aspergillus orizae are known to be applied to anti-influenza virus agents (Patent Document 2).
• Griffithsin (GRFT) isolated from the red alga Griffithsia is being developed as a component of a vaginal gel for HIV prevention (Non-Patent Document 2).
• Flt3 receptor-binding lectin derived from Fuji pea (Lablab purpureus) has an infection-inhibiting effect on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza (Non-patent Document 3); CD209 antigen C (CD209C) and C-type lectin family 4 member G (CLEC4G) are known to significantly inhibit SARS-CoV-2 infection (Non-Patent Document 4).
• Viruses such as SARS-CoV-2 infect host cells through the binding between the spike protein (S protein) on the virus surface and angiotensin-converting enzyme 2 (ACE2) on the host cell membrane (Hoffmann et al., Cell 181:271 -280 e8; Monteil et al., Cell 181:905-913 e7; Wang et al., Cell 181:281-292 e6).
• S protein spike protein
• ACE2 angiotensin-converting enzyme 2
• CLEC4G and CD209C bind to the N-linked sugar chains of the spike protein near the S protein receptor binding domain (RBD)-ACE2 interface, thereby interfering with the binding of the S protein to the host cell surface. It has been reported that.
• the S protein is highly glycosylated with N-linked sugar chains attached to each of the 22 asparagine residues per monomer, forming a "sugar chain shield" on the surface of the S protein that prevents other molecules from forming. Access is restricted. As a result, the non-N-glycosylated open region is very narrow, but it corresponds to the contact interface with ACE2, and many neutralizing antibodies also bind to this region. Furthermore, most of the "variant of concern" viruses also have mutations in the same region, which can reduce the effectiveness of antibodies. In contrast, N-linked sugar chains are highly conserved among virus variants and are thought to be less susceptible to mutation. Therefore, antiviral agents that can specifically bind to N-linked sugar chains are expected to exhibit the ability to inhibit infection regardless of virus mutations.
• neutralizing antibodies are the most effective therapeutic agents for infections caused by SARS-CoV-2.
• the antibody's recognition site and the problematic mutation site overlap, making it less effective against mutant strains.
• neutralizing antibodies are biological preparations, manufacturing and storage costs are unavoidably high.
• sugar chain species themselves exist in various cells, etc. there is a problem in that lectins also recognize endogenous sugar chains other than the target virus. Therefore, in order to actually utilize a lectin as an antiviral agent, it is required that the lectin has specific binding ability to the N-linked sugar chain of S protein. To this end, it is important for the lectin to come into contact with the N-linked sugar chain as well as the underlying protein portion and acquire affinity.
• the above-mentioned lectins such as CLEC4G recognize a position away from the S protein in the sugar chain, and therefore have the disadvantage that they are difficult to contact with the protein portion.
• the object of the present invention is to have a strong binding property to N-linked sugar chains in S protein, which is useful for the treatment and/or diagnosis of infectious diseases caused by viruses such as SARS-CoV-2, and to be easily and inexpensively produced.
• the purpose is to provide lectins.
• Pholiota squarrosa lectin which is an ⁇ 1-6 fucose sugar chain-binding lectin, and its mutant peptides are capable of binding to N-links of viral proteins.
• the fucose ( ⁇ 1-6) [GlcNAc ( ⁇ 1-4)] GlcNAc structure (hereinafter sometimes referred to as "core fucose" in this specification), which is composed of fucose and two GlcNAcs, is located at the base of the type sugar chain.
• the present invention was completed based on the discovery that PhoSL and/or its mutant peptides can be effectively used for the treatment or diagnosis of viral infections.
• a pharmaceutical composition for treating or diagnosing a viral infection comprising an active peptide that binds to a virus and inhibits its infection, wherein the active peptide has an amino acid sequence set forth in SEQ ID NOs: 1 to 4.
• Pholiota squarrosa lectin Pholiota squarrosa lectin (PhoSL), which is an ⁇ 1-6 fucose sugar chain-binding lectin, and substitution of 1 to 4 amino acids in each of the amino acid sequences of PhoSL set forth in SEQ ID NOs: 1 to 4
• a pharmaceutical composition for treating or diagnosing a viral infection which is selected from mutant peptides having a modification selected from deletion, insertion, and addition.
• the active peptide does not substantially bind to high mannose sugar chains that do not contain ⁇ 1-6 fucose sugar chains and/or glycolipid sugar chains that do not contain ⁇ 1-6 fucose sugar chains; (1) The pharmaceutical composition according to any one of (4) to (4).
• the viral infection is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MARS-CoV) Any of (1) to (5), which is an infection caused by influenza virus, human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), or Ebola virus.
• the pharmaceutical composition according to any one of (1) to (5), wherein the viral infection is an infection caused by SARS-CoV-2, SARS-CoV, or MARS-CoV.
• the pharmaceutical composition according to any one of (1) to (5), wherein the viral infection is an infection caused by SARS-CoV-2.
• the pharmaceutical composition according to any one of (1) to (8), wherein the active peptide has a length of 38 to 40 mer.
• the modification includes substitution of Ala at position 1 with an aliphatic amino acid, aromatic amino acid, acidic amino acid, or basic amino acid in the amino acid sequence of PhoSL set forth in SEQ ID NO: 1, and a substituent group of Tyr at position 23.
• substitution of Gly at position 24 with a D-type amino acid substitution of Asp at position 25 with a D-type amino acid, substitution of Gly at position 26 with an amino acid having a side chain, His at position 38
• the pharmaceutical composition according to any one of (1) to (9), wherein the pharmaceutical composition is selected from the group consisting of substitution of , with an aromatic amino acid, and substitution of Thr at position 40 with an acidic amino acid.
• the modification includes He, Glu, Lys, Leu, Val, Phe, Tyr, norleucine (Nle) or L-tert-Leu (Tle) of Ala at position 1 in the amino acid sequence of PhoSL set forth in SEQ ID NO: 1.
• the mutant peptide is APVPVTKLVCDGDTYKCTAYLDXGDGKWVAQWDTAVFHTE, where X is 4-amino-L-Phe (SEQ ID NO: 6), IPVPVTKLVCDGDTYKCTAYLDYXDGKWVAQWDTAVFHTT, where X is D-Asn (SEQ ID NO: 7), YPVPVTKLVCDGDTYKCTAYLDYXDGKWVAQWDTAVFHTT, where X is D-Arg (SEQ ID NO: 8), APVPVTKLVCDGDTYKCTAYLDYGXGKWVAQWDTAVFHTD, where X is D-Asp (SEQ ID NO: 9), APVPVTKLVCDGDTYKCTAYLDXGDGKWVAQWDTAVFHTT, where X is 4-carboxy-L-Phe (SEQ ID NO: 10), EPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 11
• Pholiota squarrosa lectin having the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid sequence corresponds to Ala at position 1 in the amino acid sequence of PhoSL set forth in SEQ ID NO: 1.
• APVPVTKLVCDGDTYKCTAYLDXGDGKWVAQWDTAVFHTE where X is 4-amino-L-Phe (SEQ ID NO: 6), IPVPVTKLVCDGDTYKCTAYLDYXDGKWVAQWDTAVFHTT, where X is D-Asn (SEQ ID NO: 7), YPVPVTKLVCDGDTYKCTAYLDYXDGKWVAQWDTAVFHTT, where X is D-Arg (SEQ ID NO: 8), APVPVTKLVCDGDTYKCTAYLDYGXGKWVAQWDTAVFHTD, where X is D-Asp (SEQ ID NO: 9), APVPVTKLVCDGDTYKCTAYLDXGDGKWVAQWDTAVFHTT, where X is 4-carboxy-L-Phe (SEQ ID NO: 10), EPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 11), LPVPVTK
• PhoSL and its mutant peptides in the present invention strongly recognize the core fucose closest to the base of N-linked sugar chains, and as a result, they can strongly inhibit infection by viruses, especially SARS-CoV-2. Furthermore, the PhoSL and its mutant peptides can be produced by chemical synthesis, which can reduce production and storage costs.
• FIG. 1 shows that the PhoSL of the present invention recognizes the core fucose moiety near the root of a complex N-linked sugar chain consisting of N-acetylglucosamine (GlcNAc), mannose, and fucose.
• GlcNAc N-acetylglucosamine
• FRIL Non-Patent Document 3
• CD209C and CLEC4G Non-Patent Document 4
• GlcNAc terminal GlcNAc located far from the base of the N-linked sugar chain ( top panel).
• GRFT Non-Patent Document 2 is shown to recognize high mannose-type sugar chains (lower panel).
• FIG. 1 shows binding of PhoSL to S protein.
• A is a surface plasmon resonance (SPR) sensorgram showing the binding of PhoSL to S protein immobilized on a sensor chip using the amine coupling method. The concentration of each PhoSL as a monomer is shown in ⁇ M.
• the small vertical arrow indicates the start point of sample injection (300 s), and the large vertical arrow indicates the case where PhoSL and S protein (both trimers) bind in a 1:1 ratio, calculated from the fixed amount and molecular weight.
• the response value (231RU) is shown. It has been shown that a maximum of 6 to 7 PhoSLs bind to S protein.
• B shows the relationship between the amount of change in the equilibrium response value and the PhoSL concentration.
• C) shows binding inhibition by core fucose trisaccharides at a fixed concentration of PhoSL (0.1 ⁇ M). Includes experiments at a concentration of 0 ⁇ M (1558 RU).
• D shows the binding of PhoSL to the S protein derived from the Omicron mutant strain. In FIG.
• PhoSL refers to a peptide having the amino acid sequence set forth in SEQ ID NO: 1, unless otherwise specified. Same in the following figures.
• E shows binding of mutant PhoSL (SEQ ID NO: 10) to (wild type) S protein.
• Figure 3 shows inhibition of infection by SARS-CoV-2 by PhoSL.
• SARS-CoV-2 TY-WK-521/2022 strain (conventional strain) and TY38-873 (Omicron strain, BA.1)
• plaque assay in primate cell Vero E6 culture wells are shown. The concentrations shown are the concentration of PhoSL peptide as a monomer and the control peptide (46mer) synthesized in Reference Example 1.
• (B) shows the inhibition rate as a function of peptide concentration.
• Black circles indicate the effect of PhoSL
• white circles indicate the inhibitory effect of the control peptide (46mer) synthesized in Reference Example 1 on the conventional strain
• black circles indicate the inhibitory effect of PhoSL on the conventional strain
• open squares indicate the control synthesized in Reference Example 1.
• the black squares indicate the inhibitory effect of the peptide on the Omicron strain
• the black squares indicate the inhibitory effect of PhoSL on the Omicron strain. Means and deviations of duplicate experiments are shown.
• Figure 4 shows S protein aggregation induced by PhoSL.
• (A) shows a cryo-EM image of S protein alone (left panel) and a cryo-EM image of S protein-PhoSL (right panel).
• (B) shows a schematic model of aggregation of S protein (circles) and PhoSL (triangles) formed on a grid tip.
• (A) and (B) of FIG. 5 show a structural model (representative structure) of a complex of PhoSL and S protein obtained by MD simulation.
• PhoSL binds to an N-linked sugar chain containing a core fucose structure added to Asn331 (A) and Asn343 (B) of the S protein receptor binding domain (RBD).
• (C) is part of the cryo-EM structure (RBD) of the complex of the Fab fragment of S309 antibody and S protein (PDB registration code: 6wpt) (Pinto et al., Nature 583: 290-295, 2020 ).
• RBD cryo-EM structure
• fucose is shown as a sphere, and other sugars are shown as stick models.
• the hinge region between the two subdomains of the RBD is also shown.
• (D) and (E) show details of the molecular surface of the PhoSL-S protein complex formed by Asn331 (D) and Asn343 (E).
• FIG. 6 shows the binding of wild type PhoSL (A) and Ala1Lys mutant PhoSL (B) to S protein RBD.
• A wild type PhoSL
• B Ala1Lys mutant PhoSL
• This is a sensorgram showing the binding of PhoSL to RBD immobilized on a sensor chip by the Ni-NTA chelation method, and shows the difference (difference sensorgram) from that for a flow cell in which RBD is not immobilized.
• the concentration of each PhoSL as a monomer is shown in ⁇ M.
• the vertical axis shows the ratio of the response value when the PhoSL trimer binds to the immobilized RBD in a 1:1 ratio.
• the dotted line is a fitting curve obtained by the simultaneous kinetic analysis method.
• the "active peptide” that binds to a virus and inhibits its infection contained in the pharmaceutical composition for treating or diagnosing a viral infection of the present invention has the function of binding to a virus and inhibiting the infection of the virus into a host cell.
• Pholiota squarrosa lectin has the following amino acid sequences as shown in SEQ ID NOs: 1 to 4.
• APVPVTKLVCDGDTYKCTAYLD Y GDG K WVAQWDT A VFHT T (Sequence number 1) APVPV S KLVCDGDTYKCTA T LDFGDG H WVAQW SA N I FH Q G (SEQ ID NO: 2) APVPVTKLVCDGDTYKCTA T LD Y GD SN WVAQW G T S VFHT S (Sequence number 3) APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG (SEQ ID NO: 4)
• the amino acid sequence described in SEQ ID NO: 4 is the amino acid sequence of a lectin isolated from Sugitake mushroom by Kobayashi et al. (Non-Patent Document 1).
• the amino acid sequences shown in SEQ ID NOs: 1 to 3 are amino acid sequences encoded by the PhoSL gene (Patent No. 5531290).
• the underlined amino acid residues in the amino acid sequences set forth in SEQ ID NOs: 1 to 3 are amino acid residues that differ from the amino acid sequence set forth in SEQ ID NO: 4.
• Each of the amino acid sequences consists of 40 amino acid residues.
• the active peptide may be a mutant peptide of PhoSL.
• variant peptide refers to 1 to 4, or 1 to 3, or 1 or 2, or 1 amino acid for each of the amino acid sequences set forth in SEQ ID NOS: 1 to 4. It refers to a peptide having an amino acid sequence into which a modification selected from substitution, deletion, insertion, and addition (hereinafter sometimes referred to as "mutation") has been introduced. By introducing mutations into PhoSL, stronger binding with S protein is expected. The mode of mutation will be described later.
• the active peptide preferably binds to ⁇ 1-6 fucose sugar chains on the outer membrane of the virus, or on the outer shell of viruses without an outer membrane.
• the "outer membrane” of a virus refers to a lipid bilayer membrane called an envelope located at the outermost side of a virus particle.
• the envelope contains transmembrane proteins such as S protein.
• the "outer shell” of a virus refers to the capsid of the virus particle.
• ⁇ 1-6 fucose sugar chain refers to a structure in which fucose is bound to N-acetylglucosamine at the reducing end of an N-type sugar chain through an ⁇ 1-6 bond, that is, fucose ( ⁇ 1-6) [GlcNAc ( ⁇ 1-4)] GlcNAc structure.
• ⁇ 1-6 fucose sugar chain binding property refers to N This means that N-acetylglucosamine at the reducing end of the type sugar chain is not substantially bound.
• Substantially not bound means that high mannose sugar chains that do not contain ⁇ 1-6 fucose sugar chains and/or glycolipid sugar chains that do not contain ⁇ 1-6 fucose sugar chains, and the reducing terminal of N-type sugar chains. It means that the dissociation constant (25°C) with N-acetylglucosamine is, for example, 1.0 ⁇ 10 ⁇ 4 M or more.
• FIG 1 schematically shows that PhoSL of the present invention recognizes the sugar chain (core fucose) closest to the Asn residue (Asn331 or Asn343) of the N-linked sugar chain of S protein ( top panel).
• both lectins CLEC4G and CD209C have strong affinity for terminal acetylglucosamine (GlcNAC) of hybrid N-glycans, which is different from core fucose ( Figure 5A of Non-Patent Document 4).
• the active peptide has 60% or more amino acid sequence identity or homology with each of the amino acid sequences set forth in SEQ ID NOs: 1-4. Specifically, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, Sequence of 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more Preferably, they have identity or homology.
• Identity of two amino acid sequences refers to the proportion of identical amino acid residues that appear at each corresponding position when both amino acid sequences are aligned
• homoology refers to the proportion of identical amino acid residues that appear at each corresponding position when both amino acid sequences are aligned. Refers to the ratio at which the same or similar amino acid residues appear at each corresponding position when ):403-10) etc.
• the pharmaceutical composition of the present invention may contain one or more peptides selected from peptides having the amino acid sequences set forth in SEQ ID NOs: 1 to 4 and variant peptides thereof.
• virus as used herein is not particularly limited as long as it has a core fucose in its N-linked sugar chain, but examples include Coronaviridae, Orthomyxoviridae, Togaviridae, Paramyxoviridae, Examples include RNA viruses such as Rhabdoviridae, Bunyaviridae, and Foroviridae; DNA viruses such as Boxviridae and Hepadnaviridae; and retroviruses such as Retroviridae.
• SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
• SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
• SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
• MERS-CoV Middle East Respiratory Syndrome Coronavirus
• influenza virus e.g. , Wu et al., 2016, BBRC 473, 524-529; Li et al., 2021, Appl. Microbiol. Biotechnol. May 3, 1-14
• human immunodeficiency virus type 1 HAV-1 (e.g. Montfort et al. 2011, J. Immunol.
• HBV hepatitis B virus
• HCV hepatitis C virus
• Ebola virus e.g., Ritchie et al., 2010, Rapid Commun. Mass Spectrom. 24, 571).
• SARS-CoV-2, SARS-CoV, or MARS-CoV is more preferred, and SARS-CoV-2 is particularly preferred.
• Mutations include substitutions, deletions, insertions, and/or additions of 1 to 4 amino acids to each of the amino acid sequences set forth in SEQ ID NOs: 1 to 4.
• a specific example of a mutation herein is an amino acid substitution.
• Such substitutions include substitutions with aliphatic amino acids, aromatic amino acids, D-type amino acids, acidic amino acids, basic amino acids, amino acids having side chains, and the like.
• Examples of aliphatic amino acids include He, Leu, Val, Phe, Tyr, norleucine (Nle), and L-tert-Leu.
• aromatic amino acids include Tyr, Phe, and the like.
• D-type amino acids examples include D-Gln, D-Asn, D-Thr, D-Arg, D-Lys, and D-allylglycine.
• amino acids having side chains and acidic amino acids examples include L-Asp and L-Glu.
• basic amino acids examples include L-Lys, L-Arg, and L-His.
• the aromatic ring of the aromatic amino acid may be further substituted, and examples of such substituents include halogen atoms such as Cl, F, and Br; amino groups; carboxy groups; hydroxy groups; methyl groups, ethyl groups. Examples include lower alkyl groups such as.
• a mutant peptide can be designed, for example, mainly targeting amino acid residues of PhoSL that are involved in interaction with a part of the receptor binding motif (RBM) of S protein.
• RBM receptor binding motif
• the amino acid residues of PhoSL that interact with Asn331, Pro337, Asn343, Thr345, and Asn440 shown in FIG. 5(E) can be targeted.
• mutant peptides of PhoSL shown in SEQ ID NO: 1 include substitution of Ala at position 1 with an aliphatic amino acid, an aromatic amino acid, an acidic amino acid, or a basic amino acid in the amino acid sequence of PhoSL shown in SEQ ID NO: 1. , substitution of Tyr at position 23 with an aromatic amino acid having a substituent, substitution of Gly at position 24 with a D-type amino acid, substitution of Asp at position 25 with a D-type amino acid, and substitution of Gly at position 26 with a side chain. It is a peptide containing a substitution of an amino acid, a substitution of His at position 38 with an aromatic amino acid, and/or a substitution of Thr at position 40 with an acidic amino acid. The number of substitutions is 1-4, 1-3, 1 or 2, or 1.
• a preferable mutant peptide of PhoSL of SEQ ID NO: 1 for the amino acid sequence of PhoSL set forth in SEQ ID NO: 1, Ile, Glu, Lys, Leu, Val, Phe, Tyr, norleucine (Substitution of Tyr at position 23 with 3-chloro-L-Tyr (Cly), 4-amino-L-Phe (Paf) or 4-carboxy-L-Phe ( Pcf), substitution of Gly at position 24 with D-Gln, D-Asn, D-Thr, D-Arg, D-Lys or D-allylglycine, substitution of Asp at position 25 with D-Asp or D- Peptide containing substitution with Glu, substitution of Gly at position 26 with L-Asp or L-Glu, substitution of His at position 38 with Phe or Tyr, and/or substitution of Thr at position 40 with Asp or Glu It is.
• the number of substitutions is 1-4, 1-3, 1 or 2, or 1.
• Examples of combinations of mutations include, for example, Ala1Ile/Gly24D-Gln, Ala1Val/Gly24D-Gln/Thr40Glu, Ala1Ile/Gly24D-Asn/Tyr23Paf/Thr40Glu, Ala1Leu/Thr2 for the amino acid sequence of PhoSL set forth in SEQ ID NO: 1.
• the strong affinity between PhoSL and S protein is presumed to be due to the formation of favorable interactions such as hydrogen bonds and hydrophobic interactions between PhoSL and the amino acids of S protein.
• substitution of Ala at position 1 with an aliphatic amino acid is expected to strengthen the hydrophobic interaction between Ala at position 1 and the S protein of the virus.
• the substitution with an amino acid is expected to strengthen the hydrogen bond in addition to strengthening the hydrophobic interaction between Tyr at position 23 and the S protein.
• Substitution of Gly at position 24 with a D-type amino acid is expected to result in the formation of hydrogen bonds, hydrophobic interactions, and electrostatic interactions between the corresponding amino acid and the S protein.
• Substitution of Asp at position 25 with a D-type amino acid is expected to form an electrostatic interaction between the corresponding amino acid and the S protein.
• Substitution of His at position 38 with an aromatic amino acid is expected to dissolve the bond with Asp at position 25 and enable electrostatic interaction between Asp at position 25 and S protein.
• Substitution of Thr at position 40 with an acidic amino acid is expected to form an electrostatic interaction between the corresponding amino acid and the S protein.
• mutant peptides include substitution of Ala at position 1 with an aliphatic or aromatic amino acid, and substitution of Phe at position 23 with respect to the amino acid sequence of PhoSL set forth in SEQ ID NO: 2. , substitution of Gly at position 24 with a D-type amino acid, substitution of Asp at position 25 with a D-type amino acid, substitution of Gly at position 26 with an amino acid having a side chain, substitution of His at position 38 with an aromatic amino acid and/or the substitution of Gly at position 40 with an acidic amino acid.
• the number of substitutions is 1-4, 1-3, 1 or 2, or 1.
• mutant peptides include substitution of Ala at position 1 with an aliphatic or aromatic amino acid, and substitution of Tyr at position 23 with respect to the amino acid sequence of PhoSL set forth in SEQ ID NO: 3.
• substitution of Gly at position 24 with a D-type amino acid substitution of Asp at position 25 with a D-type amino acid, substitution of His at position 38 with an aromatic amino acid, and/or substitution of Ser at position 40 with an acidic amino acid.
• substitution of substitutions is 1-4, 1-3, 1 or 2, or 1.
• mutant peptides include substitution of Ala at position 1 with an aliphatic or aromatic amino acid, and substitution of Phe at position 23 with an amino acid having a substituent in the amino acid sequence of PhoSL set forth in SEQ ID NO: 4.
• substitution, substitution of Gly at position 24 with a D-type amino acid, substitution of Asp at position 25 with a D-type amino acid, substitution of Gly at position 26 with an amino acid having a side chain, substitution of His at position 38 with an aromatic amino acid This is a peptide containing a substitution and/or a substitution of Gly at position 40 with an acidic amino acid.
• the number of substitutions is 1-4, 1-3, 1 or 2, or 1.
• mutant peptides have the amino acid sequences set forth in SEQ ID NOs: 6-14 below: APVPVTKLVCDGDTYKCTAYLDXGDGKWVAQWDTAVFHTE, where X is 4-amino-L-Phe (SEQ ID NO: 6); IPVPVTKLVCDGDTYKCTAYLDYXDGKWVAQWDTAVFHTT, where X is D-Asn (SEQ ID NO: 7); YPVPVTKLVCDGDTYKCTAYLDYXDGKWVAQWDTAVFHTT, where X is D-Arg (SEQ ID NO: 8); APVPVTKLVCDGDTYKCTAYLDYGXGKWVAQWDTAVFHTD, where X is D-Asp (SEQ ID NO: 9); APVPVTKLVCDGDTYKCTAYLDXGDGKWVAQWDTAVFHTT, where X is 4-carboxy-L-Phe (SEQ ID NO: 10); EPVPVTKLVCDGDTYKCTAYLDY
• the active peptides (naturally derived) having the amino acid sequences set forth in SEQ ID NOs: 1 to 4 in the present invention can be isolated from Sugitake mushrooms by appropriately combining known extraction methods, separation methods, purification methods, etc.
• an aqueous medium extract of Sugitake lectin may be obtained using an aqueous medium as an extraction solvent, and a peptide having a molecular weight of about 4,000 to about 40,000 may be obtained from this extract by SDS electrophoresis.
• the active peptide and its mutant peptides may be produced by using a nucleic acid encoding the amino acid sequence of the naturally-derived active peptide and artificially expressing it in a known host cell different from the naturally-derived active peptide.
• the active peptide and its variant peptides may be chemically synthesized based on the amino acid sequence of the naturally occurring active peptide.
• Chemical synthesis can be performed by methods well known in the art, such as the peptide solid phase method (Merrifield, J Am Chem Soc 85:2149-2154, 1963). Specifically, a peptide solid-phase method using microwaves (for example, Japanese Patent No. 4773695) can be preferably used because the desired peptide can be synthesized rapidly (in a short period of time) and with high yield .
• Chemical synthesis allows easy introduction of unnatural amino acids, and can expand the degree of freedom in producing mutant peptides. Moreover, it can be manufactured easily and inexpensively, and storage costs can be suppressed. From these points, it is preferable to manufacture by chemical synthesis.
• the above-mentioned PhoSL and its mutant peptides both have an affinity for S protein as indicated by a dissociation constant K D (approximately 25°C) in the range of 100 nM or less, preferably 10 pM to 100 nM, more preferably 10 pM to 10 nM. have The affinity can be determined, for example, by surface plasmon resonance (SPR) method.
• the above dissociation constants assume that the PhoSL trimers bind to the S protein independently with similar affinities to each other.
• the affinity with S protein is 500 ⁇ 500 compared to the affinity with various core fucosylated glycans (K D : 2 ⁇ 10 ⁇ M; concentration as a monomer) (Non-patent Document 1) measured by frontal affinity chromatography. 2500 times higher.
• K D 2 ⁇ 10 ⁇ M; concentration as a monomer
• Non-patent Document 1 measured by frontal affinity chromatography. 2500 times higher.
• the therapeutic pharmaceutical composition according to the present invention may be either an oral formulation or a parenteral formulation.
• the dosage form is not particularly limited, and it can be formulated into tablets, granules, powders, capsules, elixirs, syrups, microcapsules, suspensions, etc. according to conventional methods.
• a solution containing the polypeptide of the present invention can be administered as a nasal spray or as an injection.
• a solution containing the polypeptide of the present invention can be administered as a nasal spray or as an injection.
• it may be administered before meals, after meals, or between meals.
• the therapeutic pharmaceutical composition according to the present invention contains materials such as carriers, excipients, binders, leavening agents, lubricants, sweeteners, flavoring agents, preservatives, stabilizers, and coating agents, as necessary. can do.
• specific ingredients that can be contained in tablets, capsules, etc. include binders such as tragacanth, gum arabic, corn starch, and gelatin; microcrystalline cellulose, crystalline excipients such as cellulose; leavening agents such as corn starch, pre-gelatinized starch, alginic acid, dextrin; lubricants such as magnesium stearate; flow improvers such as finely divided silicon dioxide; Lubricants; sweetening agents such as sucrose, lactose and aspartame; flavoring agents such as peppermint, vanilla flavoring and cherry, and the like.
• binders such as tragacanth, gum arabic, corn starch, and gelatin
• microcrystalline cellulose, crystalline excipients such as cellulose
• leavening agents such as corn starch, pre-gelatinized starch, alginic acid, dextrin
• lubricants such as magnesium stearate
• flow improvers such as finely divided silicon dioxide
• Lubricants such as
• Tablet coatings include, for example, shellac, sugar, or both.
• a syrup or elixir may contain, for example, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and cherry or orange flavor, and the like.
• various vitamins and various amino acids may be contained.
• the dosage of the therapeutic pharmaceutical composition according to the present invention may be set so as to ensure the amount required by the subject to which it is applied, and it may be used in the form of a formulation, or may be blended into foods, drinks, or disinfectants to administer the above-mentioned pharmaceutical composition.
• Compositions can be used.
• the diagnostic pharmaceutical composition according to the present invention can be used to diagnose viral infections in a subject by utilizing the strong binding characteristics between PhoSL and viral proteins.
• PhoSL and mutants without terminal modification The following wild type PhoSL and mutant PhoSL 40mer wild type PhoSL: APVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 1); Mutant PhoSL: EPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 11); LPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 12); KPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 13); YPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 14). was chemically synthesized as follows.
• Fmoc-Thr(tBu)-Alko-PEG resin 150 mg, 0.21 mmol/mg was swollen in DMF for several hours. 20% piperidine in DMF (approximately 1.5 ml) was added to the filtered resin and the mixture was shaken for 3 minutes at 50° C. under microwave irradiation, followed by washing with DMF. Then, under microwave irradiation at 50°C for 5-10 minutes, the corresponding amino acid synthons (5 equivalents for amino acid synthons or 1.5 equivalents for dipeptide synthons) in DMF, COMU (for synthons), 1 eq.) and DIEA (1 eq.
• the resin was then treated with the corresponding amino acid synthon (5 equivalents for amino acid synthon), COMU (1 equivalent for synthon), and DIEA (2 equivalents for synthon) in DMF (approximately 1 ml) at 50 °C for 510 min under microwave irradiation. ) and then washed with DMF. Subsequently, an acetyl capping reaction was performed for 1 minute at room temperature using 13 mM HOBt of Ac 2 O/DIEA/DMF (4.75/2.25/93.0 v/v/v) (approximately 2 ml), followed by and washed with DMF. These three reactions, namely deprotection of the Fmoc group, condensation of the amino acid synthon, and acetyl capping, were repeated successively to form a peptide chain.
• the obtained peptidyl resin was subjected to the same treatment as synthetic PhoSL.
• RP-HPLC reverse phase high performance liquid chromatography
• PhoSL mutants (SEQ ID NOs: 6 to 10) with purity >95% were purchased from BEX.
• SPR Surface plasmon resonance
• proline substitutions Phe817Pro, Ala892Pro, Ala899Pro, Ala942Pro, Lys986Pro, and Val987Pro
• arginine substitutions Arg683Ala, Arg685Ala
• the proline substitution stabilizes the three-dimensional structure of the S protein
• the arginine substitution Ze Grants resistance to.
• HBS-EP+buffer (10mM HEPES (pH 7.4), 150mM NaCl, 3mM EDTA, and 0.05% surfactant P20) was used as the running buffer.
• Example 3 SARS-CoV-2 Inhibition Assay A plaque reduction neutralization test (PRNT) was conducted under biosafety level 3 laboratory (BSL-3) conditions to evaluate neutralizing activity.
• the PhoSL peptide having the amino acid sequence described in SEQ ID NO: 1 synthesized in Example 1 and the control peptide synthesized in Reference Example 1 were dissolved in 20 mM Tris buffer (pH 7.5) containing 1 mM DTT. Serial dilutions were made 10-fold (from 10 ⁇ M to 1 nM) in minimum essential medium (MEM) supplemented with % fetal calf serum (FCS).
• MEM minimum essential medium
• FCS % fetal calf serum
• Equal volumes of SARS-CoV-2 of TY-WK-521/2022 strain (conventional strain) and TY38-873 strain (Omicron strain, BA.1) were mixed with the diluted peptide and incubated at 37° C. for 60 minutes. Each mixture was seeded onto a monolayer of primate Vero-E6 cells in the wells of a 24-well plate. After incubation for 60 minutes at 37°C, infected cells were overlaid with 1.25% methylcellulose 4000 in 2% FCS in MEM and incubated for 5 days. Plates were washed with PBS(-) to remove methylcellulose, fixed with 4% paraformaldehyde solution overnight at room temperature, rinsed, and stained with crystal violet. Experiments at all peptide concentrations were duplicated to estimate the uncertainty level.
• the IC 50 of PhoSL as a monomer was 0.37 ⁇ 0.17 ⁇ M (conventional strain) and 0.36 ⁇ 0.05 ⁇ M (Omicron strain) ( Figure 3 (B)) . This indicates that the infection inhibiting function of PhoSL is not easily affected by virus mutations.
• the inhibitory activity of PhoSL having the amino acid sequence set forth in SEQ ID NO: 1 above is 1.6 ⁇ g/mL in terms of weight concentration, which is higher than the inhibitory activity of 0.71 ⁇ g/mL by FRIL using PRNT assay (Non-Patent Document 3).
• PhoSL having the amino acid sequence set forth in SEQ ID NO: 1 was determined by the representative neutralizing antibody S309 (0.08 ⁇ g/mL by immunoassay) (Pinto, et al., Nature 586:290-295). , 2020) and S2X259 (0.14 ⁇ g/mL by luciferase assay) (Tortorici, et al., Nature 597:103-108, 2021), it was only about 1/10 to 1/20.
• Example 4 Cryo-EM analysis
• 3 ⁇ L of S protein sample and S protein/PhoSL complex sample concentration 3.2 ⁇ M of S protein and 138 ⁇ M of PhoSL, both concentrations as monomers
• a carbon grid Quantifoil, Cu, R1.2/1.3, 300 mesh
• micrographs were acquired on a Talos Arctica G2 (Thermo Fisher Scientific) microscope operating in nanoprobe mode at 200 kV and using EPU software. Micrographs were collected on a 4k x 4k using a Falcon 3EC direct electron detector (electron counting mode) with an apparent magnification of 120,000 (0.88 ⁇ /pixel).
• Example 5 Molecular Modeling Study Since it was difficult to determine the structure of the PhoSL-S protein complex due to aggregation, computer modeling was performed to obtain information regarding the binding mode. As modeling sites for binding to PhoSL, two glycosylation sites Asn331 and Asn343 in the RBD of S protein having a complex N-linked sugar chain were selected. This is because 98% of the N-linked sugar chains of both are core fucosylated, and the glycosylation site Asn343 has been shown to be particularly important for infection (Li et al., Cell 182:1284 -1294, 2020).
• the structural model of PhoSL-S protein interaction at Asn331 or Asn343 is basically based on Yamasaki, et al. , Glycobiology 29:576-587, 2019, by molecular dynamics (MD) simulation.
• the initial model consists of the crystal structure of PhoSL in complex with fucose ( ⁇ 1-6)[GlcNAc( ⁇ 1-4)]GlcNAc-OH (PDB entry code: 7VU9) and two GlcNAc at Asn331 and Asn343 (Fig. 1) was created by docking with the cryo-EM structure of the glycosylated S protein (PDB entry code: 6xkl; A chain, upper conformer) showing GlcNAc numbered "1" and "2". .
• GlcNAc1 was created in duplicate under the condition that the S protein does not cause steric conflicts. After docking, three mannoses were attached to GlcNAc2 to complete the core structure of the N-linked sugar chain.
• the AMBER2018 program package for molecular dynamics allows AMBERffl4SB (Maier et al. al., J Chem Theory Comput 11:3696-371, 2015) and GLYCAM_06j-1 In a force field (Kirschneret al., J. Comput Chem 29:622-655, 2008), 1) hydrogen atoms are attached to proteins and glycans, and 2) the net charge of the entire system is neutralized by the addition of sodium ions. , and 3) setting the TIP3P water molecule box to a thickness of 15 ⁇ , we created parameter and coordinate fills and performed a 10 ns MD simulation at 300 K. A representative structure was selected that was closest to the average coordinates of non-hydrogen atoms during the simulation.
• PhoSL Since the coordinates of PhoSL that recognize the core fucose of the N-linked sugar chain at Asn331 or Asn343 overlap with each other, it is unlikely that two PhoSL molecules bind at both sites simultaneously. Rather, one PhoSL molecule interacts with two N-glycans on the RBD, and the one bound to the core fucose of the N-glycan at Asn331 hydrogen bonds with the mannose moiety of the N-glycan at Asn343. form. The opposite is also true (FIGS. 5(D) and (E)). Furthermore, of great importance, PhoSL interacts with the amino acids of the S protein through hydrogen bonds or hydrophobic contacts. Interactions with these N-linked sugar chains and amino acids were observed throughout the simulations, and compared to the affinity with isolated N-linked sugar chains (Non-Patent Document 1), this interaction with S protein This fully explains the high affinity of (Fig. 2).
• Example 6 Analysis of binding of PhoSL to S protein derived from Omicron mutant strain by SPR By SPR, binding of PhoSL to S protein derived from Omicron mutant strain was observed using the same method and conditions as in Example 2 ( Figure 2 ( D)).
• Omicron mutant-derived S protein was purchased from AcroBiosystems, and in addition to the proline substitution and arginine substitution described in Example 2, it contains 34 omicron mutations (Cui et al., Cell 185, 860-871). , 2022).
• the 34 specific mutations are Ala67Val, His69-Val70 (deletion), Thr95Ile, Gly142Asp, Val143-Tyr145 (deletion), Asn211 (deletion), Leu212Ile, Arg214-Glu-Pro-Glu-Asp215 (insertion), Gl y339Asp , Ser371Leu, Ser373Pro, Ser375Phe, Lys417Asn, Asn440Lys, Gly446Ser, Ser477Asn, Thr478Lys, Glu484Ala, Gln493Arg, Gly496Ser, Gln498Arg, Asn501Tyr, Tyr505His, Thr547Lys, Asp614Gly, His655Tyr, Asn679Lys, Pro681His, Asn764Lys, Asp796Tyr, Asn856Lys, Gln954His, Asn9
• Example 7 Analysis of binding of mutant PhoSL to S protein by SPR Using the same method and conditions as in Example 2, binding of mutant PhoSL shown by SEQ ID NO: 10 (Tyr23Pcf) to S protein was observed by SPR. ( Figure 2(E)). As a result of the analysis, a dissociation constant of 8.4 nM was obtained, which is slightly weaker than that of wild-type PhoSL. This indicates that although this mutation alone does not lead to stronger binding, the introduction of the mutation can change binding.
• Example 8 Analysis of binding of wild type and mutant PhoSL to S protein RBD by SPR
• S protein RBD was purchased from AcroBiosystems, and was obtained by cutting out the Arg319-Phe451 portion from the protein described in Example 2. Val367Phe was replaced.
• the mutant PhoSLs shown by SEQ ID NOs: 6 to 14 were designed based on the complex model of PhoSL and RBD shown in Example 5, taking into consideration the possibility of enhanced binding.
• Surface plasmon resonance (SPR) measurements were performed using a Biacore X instrument (Cytiva) at 298K.
• S protein RBD was immobilized on Sensor Chip NTA (Cytiva) by Ni-NTA chelation method.
• Buffer (10mM HEPES (pH 7.4), 150mM NaCl, 0.05mM EDTA, and 0.005% surfactant P20) was used as the running buffer.
• the same buffer containing wild-type or mutant PhoSL at concentrations of 10 nM to 100 nM was injected into the flow cell at 20 ⁇ L/min for 5 minutes. Two flow cells were enabled, S protein RBD was immobilized on only one of them, and the influence of drift was minimized by taking the difference between the sensorgrams. Data analysis was performed by applying a simultaneous kinetic analysis method and using the attached software (BiaEvaluation3).
• FIG. 6 shows the difference sensorgram of wild type and mutant PhoSL (SEQ ID NO: 13).
• the dissociation constant K D of wild-type PhoSL was 0.37 nM (association constant K A was 2.7 ⁇ 10 9 M ⁇ 1 ), and the K D of the mutant represented by SEQ ID NO: 13 was 0.24 nM (binding The constant K A was 4.2 ⁇ 10 9 M ⁇ 1 ), and it was found that the mutant represented by SEQ ID NO: 13 had about 1.5 times the S protein RBD binding strength compared to wild-type PhoSL.
• Table 1 shows the dissociation constants for other mutants. In addition to the mutant shown by SEQ ID NO: 13, two mutant PhoSL (SEQ ID NO: 12, 14) showed slightly stronger binding than the wild type.
• the pharmaceutical composition according to the present invention is useful for treating and/or diagnosing infectious diseases caused by viruses, especially SARS-CoV-2.

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