Pharmaceutical composition for treating or diagnosing virus infectious disease and containing pholiota squarrosa lectin and / or mutant peptide thereof

Abstract

Provided is a lectin that is useful for treatment and / or diagnosis of infectious diseases from viruses such as SARS-CoV-2, the lectin binding strongly to N-linked sugar chain in S protein, and being producible in a simple manner and at a low cost.　The present invention is a pharmaceutical composition for treatment or diagnosis of virus infectious diseases, the composition containing an active peptide that binds to a virus to inhibit the infection therewith. The active peptide is selected from: Pholiota squarrosa lectin (PhoSL), which is a lectin that binds to an α1-6 fucose sugar chain and has amino acid sequences represented by SEQ ID NOs: 1-4; and a mutant peptide which has PhoSL amino acid sequences represented by SEQ ID NOs: 1-4 with a modification introduced to each of said sequences, the modification being selected from substitution, deletion, insertion and addition of 1 to 4 amino acids.

Description

Pharmaceutical composition for treating or diagnosing viral infections, comprising Sugitake lectin and / or its mutant peptide

[0001] The present invention relates to a pharmaceutical composition for treating or diagnosing a viral infection, which comprises an active peptide that binds to a virus and inhibits its infection, particularly to a pharmaceutical composition for treating or diagnosing a viral infection, which comprises Sugitake mushroom lectin and / or a mutant peptide thereof. The present invention also relates to a mutant of the active peptide.

[0002] Sugar chains of glycoconjugates such as glycoproteins and glycolipids present on cell surfaces and in body fluids function as a kind of information element and are deeply involved in important life phenomena such as development, immunity, cancer, infection, etc. On the other hand, lectins, which are carbohydrate-binding proteins, function as carbohydrate-recognition molecules and play important biological roles similar to those of carbohydrates.

[0003] Many types of lectins have been isolated from plants, fungi, seaweed, etc., and their biochemical properties have been elucidated. For example, Sugitake mushroom lectin (Pholiota squarrosa lectin) (PhoSL) is a peptide consisting of 40 amino acid residues that specifically binds to α1-6 fucose (Patent Document 1, Non-Patent Document 1), and has been used for detecting AFP-L3, determining the malignancy of colon cancer, detecting pancreatic cancer, etc. Lectins derived from hairy vetch (Vicia villosa), wisteria (Wisteria floribunda), white chanterelle (Aleuria aurantia), or koji mold (Aspergillus oryzae) are known to be used as 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).

[0004] It is known that Flt3 receptor-binding lectin (FRIL) derived from Lablab purpureus has an inhibitory effect on infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza (Non-patent Document 3), and that CD209 antigen C (CD209C) and C-type lectin family 4 member G (CLEC4G) significantly inhibit SARS-CoV-2 infection (Non-patent Document 4). Viruses such as SARS-CoV-2 infect host cells through binding of the spike protein (S protein) on the viral surface to 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). The two lectins mentioned above, CLEC4G and CD209C, have been reported to bind to the N-linked glycans 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.

[0005] The S protein is highly glycosylated with N-linked glycans attached to each of the 22 asparagine residues per monomer, forming a "glycan shield" on the surface of the S protein that restricts access by other molecules. As a result, the open, N-glycosylated region is very narrow, but this corresponds to the contact interface with ACE2, and many neutralizing antibodies also bind to this region. Furthermore, most "variants of concern" viruses also have mutations in this region, which may reduce the efficacy of antibodies. In contrast, N-linked glycans are highly conserved among viral variants and are thought to be less susceptible to mutation. Therefore, antiviral agents that can specifically bind to N-linked glycans are expected to be able to inhibit infection regardless of viral mutations.

[0006] Currently, neutralizing antibodies are the most effective therapeutic agents for SARS-CoV-2 infection. However, the overlap between the antibody recognition site and the problematic mutation-prone site reduces their effectiveness against mutant strains. Furthermore, because neutralizing antibodies are biological products, the production and storage costs are inevitably high. Furthermore, because glycan species themselves are present in various cells, lectins can also recognize endogenous glycans other than those of the target virus, creating a problem. Therefore, for lectins to actually be used as antiviral agents, they must have specific binding affinity for the N-linked glycans of the S protein. To achieve this, it is important for the lectin to contact both the N-linked glycans and the underlying protein moiety, thereby acquiring affinity. However, the above-mentioned lectins, such as CLEC4G, have the disadvantage of recognizing glycans at positions distant from the S protein, making them less likely to contact the protein moiety.

[0007] Japanese Patent No. 4514163 Japanese Patent Application Laid-Open No. 2014-201587

[0008] Kobayashi et al., 2012, J. Biol. Chem. 287, 33973-33982 Lee, 2019, Mar. Drugs, 17, 567 Liu et al., 2020, Cell Rep. 32, 108016 Hoffmann et al., 2021, EMBO J. e108375

[0009] An object of the present invention is to provide a lectin that has strong binding affinity to N-linked glycans in S proteins, is useful for the treatment and / or diagnosis of infectious diseases caused by viruses such as SARS-CoV-2, and can be produced simply and inexpensively.

[0010] As a result of intensive research to achieve the above object, the present inventors have found that Sugitake lectin (Pholiota squarrosa lectin) (PhoSL), an α1-6 fucose glycan-binding lectin, and its mutant peptides recognize and strongly bind to a fucose(α1-6)[GlcNAc(β1-4)]GlcNAc structure (hereinafter sometimes referred to as "core fucose") consisting of a fucose and two GlcNAc residues, which is located at the base of the N-linked glycan of a viral protein, and that such PhoSL and / or its mutant peptides can be effectively used in the treatment or diagnosis of viral infections, thereby completing the present invention.

[0011] That is, the present invention provides the following: (1) A pharmaceutical composition for the treatment or diagnosis of a viral infection, comprising an active peptide that binds to a virus and inhibits its infection, wherein the active peptide is selected from Sugitake lectin (Pholiota squarrosa lectin) (PhoSL), an α1-6 fucose glycan-binding lectin having the amino acid sequences set forth in SEQ ID NOS: 1 to 4, and mutant peptides obtained by introducing modifications selected from substitution, deletion, insertion, and addition of 1 to 4 amino acids into each of the amino acid sequences of PhoSL set forth in SEQ ID NOS: 1 to 4. (2) The pharmaceutical composition according to (1), wherein the active peptide has an amino acid sequence identity of 90% or more with the amino acid sequence of PhoSL set forth in SEQ ID NOS: 1. (3) The pharmaceutical composition according to (1) or (2), wherein the active peptide binds to an α1-6 fucose glycan on the outer membrane or shell of a virus. (4) The pharmaceutical composition according to any one of (1) to (3), wherein the active peptide binds to a fucose(α1-6)[GlcNAc(β1-4)]GlcNAc structure in the sugar chain of a viral spike protein (S protein). (5) The pharmaceutical composition according to any one of (1) to (4), wherein the active peptide does not substantially bind to a high mannose sugar chain not containing an α1-6 fucose sugar chain and / or a glycolipid sugar chain not containing an α1-6 fucose sugar chain. (6) The pharmaceutical composition according to any one of (1) to (5), wherein the viral infection is an infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MARS-CoV), influenza virus, human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), or Ebola virus. (7) 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. (8) The pharmaceutical composition according to any one of (1) to (5), wherein the viral infection is an infection caused by SARS-CoV-2.(9) The pharmaceutical composition according to any one of (1) to (8), wherein the active peptide has a length of 38 to 40 mer. (10) The pharmaceutical composition according to any one of (1) to (9), wherein the modification is selected from the group consisting of 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 with an aromatic amino acid having a substituent for Tyr at position 23, substitution of Gly at position 24 with a D-amino acid, substitution of Asp at position 25 with a D-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 substitution of Thr at position 40 with an acidic amino acid. (11) The modification is a substitution of Ala at position 1 in the amino acid sequence of PhoSL set forth in SEQ ID NO: 1 with Ile, Glu, Lys, Leu, Val, Phe, Tyr, norleucine (Nle), or L-tert-Leu (Tle), a substitution of Tyr at position 23 with 3-chloro-L-Tyr or 4-carboxy-L-Phe, a substitution of Gly at position 24 with D-Gln, D-Asn, or a substitution of Gly at position 25 with D-Asn. , substitution of Asp at position 25 with D-Asp or D-Glu, substitution of Gly at position 26 with L-Asp or L-Glu, substitution of His at position 38 with Phe or Tyr, and substitution of Thr at position 40 with Asp or Glu. (12) The pharmaceutical composition according to any one of (1) to (9), wherein the active peptide has a dissociation constant K of 10 pM to 100 nM with respect to the S protein or a partial region thereof. Ｄ(12) The pharmaceutical composition according to any one of (1) to (11), having an affinity shown by (approximately 25°C). (13) The mutant peptide is selected from the group consisting of 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), LPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 12), KPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 13), and (14) The pharmaceutical composition according to any one of (1) to (12), wherein the lectin is selected from the group consisting of Sugitake lectin (Pholiota squarrosa) having the amino acid sequence set forth in SEQ ID NO: 1. a mutant of PhoSL (phosphoryl lectin) (PhoSL), the amino acid sequence of which contains 1 to 4 substitutions selected from the group consisting of: substitution of Ala at position 1 with Ile, Glu, Lys, Leu, Val, Phe, Tyr, norleucine (Nle), or L-tert-Leu (Tle); substitution of Tyr at position 23 with 4-amino-L-Phe or 4-carboxy-L-Phe; 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-Glu; substitution of His at position 38 with Phe or Tyr; and substitution of Thr at position 40 with Asp or Glu in the amino acid sequence of PhoSL set forth in SEQ ID NO: 1.(15) 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), LPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 12), KPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 13), and The mutant according to (14), selected from the group consisting of YPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 14).

[0012] PhoSL and its mutant peptides of the present invention strongly recognize the core fucose closest to the base of the N-linked glycan, and as a result, can potently inhibit infection by viruses, particularly SARS-CoV-2. Furthermore, PhoSL and its mutant peptides can be produced by chemical synthesis, which reduces the costs of production and storage.

[0013] Figure 1 shows that PhoSL of the present invention recognizes the core fucose portion near the base of complex N-linked glycans consisting of N-acetylglucosamine (GlcNAc), mannose, and fucose. On the other hand, FRIL (Non-Patent Document 3) recognizes the mannose portion, while CD209C and CLEC4G (Non-Patent Document 4) recognize the GlcNAc (terminal GlcNAc) located far from the base of the N-linked glycan (upper panel). GRFT (Non-Patent Document 2) also recognizes high-mannose glycans (lower panel). In the figure, triangles represent fucose, squares represent GlcNAc, and circles represent mannose. "α6," "β4," and other designations indicate the linkage mode. Specifically, "α6" indicates that the 1st carbon of GlcNAc is α-glycosidically linked to the 6th carbon of fucose. Figure 2 shows the 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 by amine coupling. The concentration of each PhoSL monomer is shown in μM. The small vertical arrow indicates the start of sample injection (300 s). The large vertical arrow indicates the response value (231 RU) calculated from the immobilized amount and molecular weight when PhoSL and S protein (both trimers) bind at a 1:1 ratio. This shows that a maximum of 6-7 PhoSL molecules bind to S protein. (B) shows the relationship between the change in equilibrium response value and PhoSL concentration. (C) shows the inhibition of binding by the core fucose trisaccharide at a fixed PhoSL concentration (0.1 μM). This includes an experiment at a concentration of 0 μM (1558 RU). (D) shows the binding of PhoSL to S protein from an Omicron mutant strain. In Figure 2, "PhoSL" refers to a peptide having the amino acid sequence set forth in SEQ ID NO: 1, unless otherwise specified. The same applies to the following figures. (E) shows the binding of mutant PhoSL (SEQ ID NO: 10) to the (wild-type) S protein. Figure 3 shows the inhibition of infection by SARS-CoV-2 by PhoSL. (A) shows wells from a plaque assay of SARS-CoV-2 (TY-WK-521 / 2022 strain (conventional strain) and TY38-873 (Omicron strain, BA.1)) in primate cell (Vero E6) culture.The indicated concentrations are the concentrations of the PhoSL peptide as a monomer and the control peptide (46-mer) 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 (46-mer) synthesized in Reference Example 1 on the conventional strain, black circles indicate the inhibitory effect of PhoSL on the conventional strain, white squares indicate the inhibitory effect of the control peptide synthesized in Reference Example 1 on the Omicron strain, and black squares indicate the inhibitory effect of PhoSL on the Omicron strain. The mean and deviation of duplicate experiments are shown. Figure 4 shows the aggregation of S protein 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 the aggregation of S protein (circles) and PhoSL (triangles) formed on a grid chip. Figure 5 (A) and (B) show structural models (representative structures) of the complex between PhoSL and S protein, based on MD simulation. PhoSL is bound to an N-linked glycan containing a core fucose structure attached to Asn331 (A) and Asn343 (B) of the S protein receptor-binding domain (RBD). (C) shows a portion of the cryo-EM structure (RBD) of the complex between the Fab fragment of the S309 antibody and S protein (PDB accession code: 6wpt) (Pinto et al., Nature 583:290-295, 2020). In (A) to (C), 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 at Asn331 (D) and Asn343 (E). In (D) and (E), dashed and thick lines indicate hydrogen bonds (donor-acceptor distance <3.5 Å) and hydrophobic interactions (C-C distance <4.5 Å), respectively. However, contacts involved in core fucose recognition by PhoSL are excluded. Figure 6 shows the binding of wild-type PhoSL (A) and the Ala1Lys mutant PhoSL (B) to the S protein RBD.This is a sensorgram showing the binding of PhoSL to RBD immobilized on a sensor chip by Ni-NTA chelation, showing the difference (difference sensorgram) from that for a flow cell without immobilized RBD. The concentrations of PhoSL as a monomer are shown in μM. On the horizontal axis, 0 to 300 s represents the binding phase, and 300 to 1500 s represents the dissociation phase. The vertical axis represents the ratio of the response value when a PhoSL trimer binds to the immobilized RBD at a 1:1 ratio. The dotted line is a fitting curve obtained by simultaneous kinetic analysis.

[0014] Hereinafter, embodiments of the present invention will be described in detail.

[0015] The "active peptide" contained in the pharmaceutical composition for the treatment or diagnosis of viral infections of the present invention, which binds to a virus and inhibits infection, is Pholiota squarrosa lectin (PhoSL), which has the function of binding to a virus and inhibiting infection of host cells by the virus and has the amino acid sequence set forth in SEQ ID NOs: 1 to 4 below.

[0016] APVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 1) APVPVSKLVCDGDTYKCTATLDFGDGHWVAQWSANIFHQG (SEQ ID NO: 2) APVPVTKLVCDGDTYKCTATLDYGDSNWVAQWGTSVFHTS (SEQ ID NO: 3) APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG (SEQ ID NO: 4)

[0017] The amino acid sequence of SEQ ID NO: 4 is the amino acid sequence of a lectin isolated from Sugitake mushroom by Kobayashi et al. (Non-Patent Document 1). On the other hand, the amino acid sequences of SEQ ID NOs: 1 to 3 are amino acid sequences encoded by the PhoSL gene (Japanese Patent No. 5531290). The underlined amino acid residues in the amino acid sequences of SEQ ID NOs: 1 to 3 are amino acid residues that differ from those in the amino acid sequence of SEQ ID NO: 4. Each of these amino acid sequences consists of 40 amino acid residues.

[0018] The active peptide may also be a mutant peptide of PhoSL. As used herein, a "mutant peptide" refers to a peptide having an amino acid sequence in which a modification (hereinafter sometimes referred to as a "mutation") selected from substitution, deletion, insertion, and addition of 1 to 4, 1 to 3, 1 or 2, or 1 amino acid is introduced into each of the amino acid sequences set forth in SEQ ID NOs: 1 to 4. By introducing a mutation into PhoSL, stronger binding to S protein is expected. The form of the mutation will be described below.

[0019] The active peptide preferably binds to the α1-6 fucose sugar chain on the outer membrane of the virus, or on the outer shell of a virus that does not have an outer membrane. In this specification, the "outer membrane" of a virus refers to the lipid bilayer membrane called the envelope that is located on 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.

[0020] As used herein, "α1-6 fucose sugar chain" refers to a structure in which fucose is bound to N-acetylglucosamine at the reducing end of an N-glycan via an α1-6 bond, i.e., a fucose(α1-6)[GlcNAc(β1-4)]GlcNAc structure. Furthermore, "α1-6 fucose sugar chain binding ability" refers to the fact that N-acetylglucosamine at the reducing end of an N-glycan is not substantially bound to a high mannose sugar chain not containing an α1-6 fucose sugar chain and / or a glycolipid sugar chain not containing an α1-6 fucose sugar chain. The term "not substantially bound" refers to the fact that the dissociation constant (25°C) between a high mannose sugar chain not containing an α1-6 fucose sugar chain and / or a glycolipid sugar chain not containing an α1-6 fucose sugar chain and the N-acetylglucosamine at the reducing end of an N-glycan is, for example, 1.0 x 10 －４ It means that it is M or above.

[0021] Figure 1 shows a schematic diagram of PhoSL of the present invention recognizing the glycan (core fucose) located closest to the Asn residue (Asn331 or Asn343) of the N-linked glycan of S protein (upper panel). In contrast, both lectins CLEC4G and CD209C have strong affinity for the terminal acetylglucosamine (GlcNAC) of hybrid-type N-glycans, which is distinct from core fucose (Figure 5A of Non-Patent Document 4). On the other hand, the lectin FRIL from Lablab bean has strong affinity for hybrid-type and complex-type N-glycans, but has almost no affinity for trimannose-deficient glycans. Therefore, FRIL is believed to strongly recognize trimannose (Figure 3F of Non-Patent Document 3).

[0022] The active peptide preferably has 60% or more amino acid sequence identity or homology with each of the amino acid sequences set forth in SEQ ID NOs: 1 to 4. Specifically, it preferably has 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 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 sequence identity or homology with each of the amino acid sequences set forth in SEQ ID NOs: 1 to 4. The "identity" of two amino acid sequences refers to the ratio of identical amino acid residues appearing at corresponding positions when the two amino acid sequences are aligned, and the "homology" of two amino acid sequences refers to the ratio of identical or similar amino acid residues appearing at corresponding positions when the two amino acid sequences are aligned. This can be determined, for example, using the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10).

[0023] 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 mutant peptides thereof.

[0024] The "virus" as used herein is not particularly limited as long as it has core fucose in its N-linked sugar chain, and examples include RNA viruses such as Coronaviridae, Orthomyxoviridae, Togaviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, and Foroviridae; DNA viruses such as Bacillusviridae and Hepadnaviridae; and retroviruses such as Retroviridae. Among these, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV) (e.g., Ritchie et al., 2010, Virology 399, 257-269), Middle East respiratory syndrome coronavirus (MARS-CoV) (e.g., Cho et al., 2021, J. Proteome Res. https: / / doi.org / 10.1021 / acs.jproteome.1c00323), 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 (HIV-1) (e.g., Montfort et al. 2011, J. Immunol. 187, 4676-4685), hepatitis B virus (HBV) (e.g., Hyakumura et al., 2015, J. Virol. 89, 11312-11322), hepatitis C virus (HCV) (e.g., Guo et al., 2018, Medicine 97, e0208), or Ebola virus (e.g., Ritchie et al., 2010, Rapid Commun. Mass Spectrom. 24, 571-585). More preferred are SARS-CoV-2, SARS-CoV, or MARS-CoV, with SARS-CoV-2 being particularly preferred.

[0025] Mutations include substitutions, deletions, insertions, and / or additions of 1 to 4 amino acids in each of the amino acid sequences set forth in SEQ ID NOS: 1 to 4. A specific example of a mutation herein is amino acid substitution. Examples of such substitutions include substitutions with aliphatic amino acids, aromatic amino acids, D-amino acids, acidic amino acids, basic amino acids, and amino acids having side chains. Examples of aliphatic amino acids include Ile, Leu, Val, Phe, Tyr, norleucine (Nle), and L-tert-Leu. Examples of aromatic amino acids include Tyr and Phe. Examples of D-amino acids include D-Gln, D-Asn, D-Thr, D-Arg, D-Lys, and D-allylglycine. Examples of amino acids having side chains and acidic amino acids include L-Asp and L-Glu. Examples of basic amino acids 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; and lower alkyl groups such as methyl and ethyl groups.

[0026] As described above, one to four mutations can be introduced into PhoSL having the amino acid sequences set forth in SEQ ID NOs: 1 to 4, and the length of the resulting mutant peptide is preferably 38 to 40 mer.

[0027] For example, mutant peptides can be designed to target amino acid residues in PhoSL that interact with a portion of the receptor binding motif (RBM) of the S protein, such as Asn331, Pro337, Asn343, Thr345, and Asn440 shown in Figure 5(E).

[0028] Examples of mutant peptides of PhoSL of SEQ ID NO: 1 include peptides that, relative to the amino acid sequence of PhoSL set forth in SEQ ID NO: 1, include substitution of Ala at position 1 with an aliphatic amino acid, aromatic amino acid, acidic amino acid, or basic amino acid, substitution of Tyr at position 23 with an aromatic amino acid having a substituent, substitution of Gly at position 24 with a D-amino acid, substitution of Asp at position 25 with a D-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 substitution of Thr at position 40 with an acidic amino acid. The number of substitutions may be 1 to 4, 1 to 3, 1 or 2, or 1.

[0029] Specific examples of preferred mutant peptides of PhoSL of SEQ ID NO: 1 include those in which Ala at position 1 is substituted with Ile, Glu, Lys, Leu, Val, Phe, Tyr, norleucine (Nle), or L-tert-Leu (Tle) in the amino acid sequence of PhoSL of SEQ ID NO: 1, and Tyr at position 23 is substituted with 3-chloro-L-Tyr (Cly), 4-amino-L-Phe (Paf), or 4-carboxy. and / or a peptide comprising a substitution of Gly at position 24 with D-Gln, D-Asn, D-Thr, D-Arg, D-Lys, or D-allylglycine, a substitution of Asp at position 25 with D-Asp or D-Glu, a substitution of Gly at position 26 with L-Asp or L-Glu, a substitution of His at position 38 with Phe or Tyr, and / or a substitution of Thr at position 40 with Asp or Glu. The number of substitutions is 1 to 4, 1 to 3, 1 or 2, or 1.

[0030] Examples of combinations of mutations include Ala1Ile / Gly24D-Gln, Ala1Val / Gly24D-Gln / Thr40Glu, Ala1Ile / Gly24D-Asn / Tyr23Paf / Thr40Glu, and Ala1Leu / Thr23Cly / His38Ph in the amino acid sequence of PhoSL set forth in SEQ ID NO: 1. e, Ala1Nle / Gly24D-Thr / Thr40Asp, Ala1Tyr / Gly24D-Arg, Asp25D-Asp / Thr40Asp, Ala1Tle / Tyr23Pcf / His38Tyr / Thr40Asp, Tyr23Pcf / Gly24D-Lys, Gly26Asp / Thr40Gly, etc.

[0031] 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 amino acids of the S protein. Specifically, 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 viral S protein, while substitution of Tyr at position 23 with an aromatic amino acid having a substituent 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-amino acid is expected to form 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-amino acid is expected to form electrostatic interactions between the corresponding amino acid and the S protein. Substitution of His at position 38 with an aromatic amino acid is expected to eliminate the bond with Asp at position 25 and enable electrostatic interaction between Asp at position 25 and the S protein. Substitution of Thr at position 40 with an acidic amino acid is expected to result in the formation of electrostatic interactions between the corresponding amino acid and the S protein.

[0032] Other examples of mutant peptides include peptides that, relative to the amino acid sequence of PhoSL set forth in SEQ ID NO: 2, include: substitution of Ala at position 1 with an aliphatic or aromatic amino acid, substitution of Phe at position 23 with an aromatic amino acid having a substituent, substitution of Gly at position 24 with a D-amino acid, substitution of Asp at position 25 with a D-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 substitution of Gly at position 40 with an acidic amino acid. The number of substitutions may be 1 to 4, 1 to 3, 1 or 2, or 1.

[0033] Other examples of mutant peptides include peptides that, relative to the amino acid sequence of PhoSL set forth in SEQ ID NO: 3, include the following: substitution of Ala at position 1 with an aliphatic or aromatic amino acid, substitution of Tyr at position 23 with an aromatic amino acid having a substituent, substitution of Gly at position 24 with a D-amino acid, substitution of Asp at position 25 with a D-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. The number of substitutions may be 1 to 4, 1 to 3, 1 or 2, or 1.

[0034] Other examples of mutant peptides include peptides that, relative to the amino acid sequence of PhoSL set forth in SEQ ID NO: 4, include the following: substitution of Ala at position 1 with an aliphatic or aromatic amino acid, substitution of Phe at position 23 with an amino acid having a substituent, substitution of Gly at position 24 with a D-amino acid, substitution of Asp at position 25 with a D-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 substitution of Gly at position 40 with an acidic amino acid. The number of substitutions may be 1 to 4, 1 to 3, 1 or 2, or 1.

[0035] Representative 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); EPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 11); LPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 12); KPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 13); YPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 14).

[0036] The active peptides (naturally occurring) having the amino acid sequences set forth in SEQ ID NOS: 1 to 4 in the present invention can be isolated from Sugitake mushroom by appropriately combining known extraction methods, separation methods, purification methods, etc. For example, an aqueous medium extract of Sugitake mushroom lectin can be obtained using an aqueous medium as an extraction solvent, and peptides having a molecular weight of about 4,000 to about 40,000 can be obtained from this extract by SDS electrophoresis.

[0037] The active peptide and its mutant peptide may be prepared by artificially expressing a nucleic acid encoding the amino acid sequence of a naturally occurring active peptide in a known host cell different from the naturally occurring one.

[0038] Alternatively, active peptides and their mutant peptides may be chemically synthesized based on the amino acid sequence of a 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, the peptide solid-phase method using microwaves (e.g., Japanese Patent No. 4773695) is preferred because it allows the desired peptide to be synthesized quickly (in a short time) and in high yield. Chemical synthesis allows for the easy introduction of unnatural amino acids, thereby expanding the flexibility in the production of mutant peptides. Furthermore, production is simple and inexpensive, and storage costs can be reduced. From these perspectives, production by chemical synthesis is preferred.

[0039] The above-mentioned PhoSL and its mutant peptides have a dissociation constant K of 100 nM or less, preferably 10 pM to 100 nM, more preferably 10 pM to 10 nM, relative to S protein. Ｄ (approximately 25°C). The affinity can be determined, for example, by surface plasmon resonance (SPR). The above dissociation constants assume that PhoSL trimers bind independently to S protein with similar affinities.

[0040] The affinity of various core fucosylated glycans (K) to S protein was measured by frontal affinity chromatography. Ｄ The affinity of the PhoSL-S protein is 500 to 2,500 times higher than that of the PhoSL-S protein (2 to 10 μM; concentration as a monomer) (Non-Patent Document 1). This is thought to be due to the fact that, in the present invention, multiple N-glycans cooperatively participate in the binding to one PhoSL trimer and that a favorable interaction is formed between PhoSL and the S protein.

[0041] There are several possible mechanisms by which PhoSL inhibits viral infection, and no specific mechanism has been identified. One possible mechanism is S protein aggregation. Specifically, S protein molecules gather on the viral surface, or S proteins on the same or different viral particles bind to each other, causing the virus to aggregate. This results in limited access of the S protein or viral particles to host cell receptors. Figure 4 shows that a mixture of PhoSL and S protein does not result in a nearly uniform distribution of S protein, but rather forms aggregates of PhoSL and S protein due to the multivalency of both trimeric PhoSL and S protein.

[0042] The pharmaceutical composition for treatment according to the present invention may be either an oral or parenteral preparation, and the dosage form is not particularly limited, and it may be formulated into tablets, granules, powders, capsules, elixirs, syrups, microcapsules, suspensions, etc. according to conventional methods.

[0043] When administered parenterally, for example, a solution containing the polypeptide of the present invention can be administered as a nasal spray or an injection, and when administered orally, it can be administered before, after, or between meals.

[0044] The therapeutic pharmaceutical composition according to the present invention may contain materials such as carriers, excipients, binders, swelling agents, lubricants, sweeteners, flavoring agents, preservatives, stabilizers, and coating agents, as needed.

[0045] Specific ingredients that can be contained in, for example, tablets, capsules, etc. in the pharmaceutical composition for treatment according to the present invention include binders such as tragacanth, gum arabic, corn starch, and gelatin; excipients such as microcrystalline cellulose and crystalline cellulose; leavening agents such as corn starch, pregelatinized starch, alginic acid, and dextrin; lubricants such as magnesium stearate; flow improvers such as fine silicon dioxide; glidants such as glycerin fatty acid esters; sweeteners such as sucrose, lactose, and aspartame; and flavorings such as peppermint, vanilla flavor, and cherry flavor.

[0046] Various other materials may also be included as coatings or to modify the physical form of the dosage unit. Tablet coatings include, for example, shellac, sugar, or both. Syrups or elixirs may contain, for example, sucrose as a sweetener, methylparaben and propylparaben as preservatives, colorings, and cherry or orange flavoring. In addition, various vitamins and amino acids may be included.

[0047] The dosage of the therapeutic pharmaceutical composition of the present invention may be set so as to ensure the amount required by the subject, and the pharmaceutical composition may be formulated and used, or may be incorporated into food, beverages, or disinfectants.

[0048] Furthermore, the diagnostic pharmaceutical composition according to the present invention can be used to diagnose viral infections in subjects by taking advantage of the strong binding properties of PhoSL to viral proteins.

[0049] EXAMPLES Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to these examples in any way.

[0050] [material] Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(Ot Bu)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-(Dmb)Gly-OH, and Fmoc-Asp(OtBu)-Thr(psiMe, Mepro)-OH were purchased from Merck (Darmstadt, Germany) or Watanabe Chemical Industry Co., Ltd. and used as monoamino acid or dipeptide synthons. Fmoc-Thr(tBu)-Alko-PEG resin and Fmoc-Val-Alko-PEG resin were purchased from Watanabe Chemical Industry Co., Ltd. Other reagents were purchased from Tokyo Chemical Industry Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., and Sigma-Aldrich (St. Louis, MO, USA). [Peptide Synthesis] Peptides were synthesized by microwave-assisted solid-phase peptide synthesis (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963) using a microwave reactor MWS-1000 (Tokyo Rikakikai).

[0051] Example 1 Synthesis of PhoSL and mutants without terminal modification Wild-type PhoSL and mutant PhoSL as follows: 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.

[0052] Fmoc-Thr(tBu)-Alko-PEG resin (150 mg, 0.21 mmol / mg) was swollen in DMF for several hours. Approximately 1.5 ml of 20% piperidine in DMF was added to the filtered resin, and the mixture was shaken under microwave irradiation at 50°C for 3 minutes, followed by washing with DMF. The resin was then treated with the corresponding amino acid synthon (5 equivalents per amino acid synthon or 1.5 equivalents per dipeptide synthon), COMU (1 equivalent per synthon), and DIEA (1 equivalent per synthon) in DMF (approximately 1 ml) under microwave irradiation at 50°C for 5-10 minutes, followed by washing with DMF. Acetaminophen was then added to the resin. ２ The acetyl-capping reaction was carried out at room temperature for 3 minutes using 13 mM HOBt in O / DIEA / DMF (4.75 / 2.25 / 93.0 v / v / v, approximately 1.5 ml), followed by washing with DMF. These three reactions, i.e., deprotection of the Fmoc group, condensation of the amino acid synthon, and acetyl-capping, were repeated consecutively to form the peptide chain.

[0053] The resulting peptidyl resin was washed with methylene chloride, dried, and then cleaved with a cleavage cocktail (TFA / H ２ Approximately 6 ml of HCl / EDT / TIS (94 / 2.5 / 2.5 / 1) was added. The mixture was shaken at room temperature for 2 hours. The peptide was released from the resin, and the peptide side chains were deprotected. The resin was filtered and washed three times with TFA. The combined filtrate was concentrated under a stream of nitrogen gas. The resulting residue was precipitated by adding ice-cold diethyl ether to give a white solid. The solid was washed twice with ice-cold diethyl ether, dissolved in 20% aqueous acetonitrile, and lyophilized.

[0054] PhoSL peptide (55% yield) was obtained by reversed-phase high-performance liquid chromatography (RP-HPLC) using an Inertsil ODS-3 column (250 x 20 mm) containing 0.1% TFA in water and 0.1% TFA in acetonitrile (A / B = 75 / 25) at a flow rate of 6.0 ml / min. The proportion of eluent B was increased linearly from 25% to 30% over 60 min. The column was heated to 40°C and monitored by UV absorption at 220 nm.

[0055] Reference Example 1: Synthesis of Hepatitis B Virus Surface Antigen Pre-S1 Peptide (Genotype C) with No Terminal Modification As a control for the infection assay, the 46-mer GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQV (SEQ ID NO: 5) was synthesized as follows. Fmoc-Val-Alko-PEG resin (250 mg, 0.24 mmol / mg) was swollen in DMF for several hours. To the filtered resin was added 20% piperidine in DMF (approximately 2 ml), and the mixture was shaken under microwave irradiation at 50°C for 2 minutes, followed by washing with DMF. The resin was then treated with the corresponding amino acid synthon (5 equivalents per amino acid synthon), COMU (1 equivalent per synthon), and DIEA (2 equivalents per synthon) in DMF (approximately 1 ml) under microwave irradiation at 50° C. for 510 minutes, followed by washing with DMF. ２ The acetyl-capping reaction was carried out at room temperature for 1 minute using approximately 2 ml of O / DIEA / DMF (4.75 / 2.25 / 93.0 v / v / v), followed by washing with DMF. These three reactions, i.e., deprotection of the Fmoc group, condensation of the amino acid synthon, and acetyl-capping, were repeated consecutively to form the peptide chain.

[0056] The resulting peptidyl resin was treated in the same manner as the synthetic PhoSL. The resulting material was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) [Conditions: Column: Inertsil WP300-C8 (250 x 10 mm); Eluent A: water containing 0.1% TFA; Eluent B: acetonitrile containing 0.1% TFA (A / B = 90 / 10); Flow rate: 4.0 ml / min; Column oven: 40°C; UV absorption at 220 nm was monitored while increasing the proportion of Eluent B linearly from 10% to over 40% over 40 min] to obtain the control peptide (yield: 21%).

[0057] Additionally, mutants of PhosL (SEQ ID NOs: 6-10) with purity >95% were purchased from BEX.

[0058] Example 2: Surface Plasmon Resonance (SPR) The binding of PhoSL to S protein was observed by surface plasmon resonance (SPR) ( Figure 2A ). Surface plasmon resonance (SPR) measurements were performed using a Biacore X instrument (Cytiva) at 298K. S protein (Super Stable Trimer, AcroBiosystems, Newark, DE, USA) was immobilized on a Sensor Chip CM5 (Cytiva) by the amine coupling method. The S protein is the extracellular domain of AA Val16-Pro1213AA, expressed in human 293 (HEK293) cells, and contains a T4 fibritin trimerization motif and a polyhistidine tag at the C-terminus. These include proline substitutions (Phe817Pro, Ala892Pro, Ala899Pro, Ala942Pro, Lys986Pro, and Val987Pro) and arginine substitutions (Arg683Ala, Arg685Ala), where the proline substitutions stabilize the three-dimensional structure of the S protein, and the arginine substitutions confer protease resistance.

[0059] HBS-EP+ buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) was used as the running buffer. Wild-type or mutant PhoSL at concentrations ranging from 10 nM to 1 μM in the same buffer was injected through the flow cell at 10–20 μL / min for 5 min. At lower concentrations, injections were repeated until the response reached a plateau level.

[0060] Inhibition of S protein-PhoSL binding by the core fucose trisaccharide (fucose(α1-6)[GlcNAc(β1-4)]GlcNAc-OH) (Tokyo Chemical Industry Co., Ltd.) was also analyzed in the same buffer system at a fixed concentration of PhoSL (100 nM) and 0 to 1 mM core fucose.

[0061] The relationship between the equilibrium response values ​​(y) at different concentrations of PhoSL (x) was determined using a custom FORTRAN 90 program, using the equation representing 1:1 binding: y = a / (l + b / x), where a and b are fitting variables and represent the maximum response value (Rmax) and dissociation constant (K), respectively. Ｄ )) yielding a dissociation constant (K) of 3.9 ± 0.6 nM. Ｄ ) (approximately 25°C) was obtained (Fig. 2(B); concentrations are values ​​as monomers). Similarly, the relationship between the equilibrium response values ​​(y) at different concentrations of core fucose trisaccharide (x) can be calculated using the Hill equation: y = a / (1 + (x / b) ｃ ) (where a, b, and c are all fitting variables, and represent the response value in the absence of inhibition, IC ５０ , Hill coefficient), and the core fucose trisaccharide is fitted with IC ５０ The binding of PhoSL to S protein was competitively inhibited with a value of 10.4±0.2 μM (FIG. 2(C)). These results indicated that PhoSL binds to S protein via recognition of the core fucose.

[0062] Example 3: SARS-CoV-2 Inhibition Assay A plaque reduction neutralization test (PRNT) was performed under Biosafety Level 3 laboratory (BSL-3) conditions to evaluate neutralizing activity. The PhoSL peptide having the amino acid sequence set forth 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 and serially diluted 10-fold (10 μM to 1 nM) in minimum essential medium (MEM) supplemented with 2% fetal calf serum (FCS). Equal volumes of SARS-CoV-2 strains TY-WK-521 / 2022 (conventional strain) and TY38-873 (Omicron strain, BA.1) were mixed with the diluted peptides and incubated at 37°C for 60 minutes. Each mixture was inoculated into wells of a 24-well plate onto a monolayer of primate Vero-E6 cells. After 60 minutes of incubation 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 the methylcellulose, fixed overnight at room temperature with 4% paraformaldehyde solution, rinsed, and stained with crystal violet. Experiments at all peptide concentrations were performed in duplicate to estimate the uncertainty level.

[0063] A home-written FORTRAN 90 program was used to calculate the number of plaques (y) as a function of the concentration of PhoSL (x) using the Hill equation: y = a / (l + (x / b) ｃ ) (where a, b, and c are the background plaque count (variable), IC ５０ (variable), and Hill coefficient (variable). Calculations were performed for all combinations of duplicate data to evaluate the error of the parameters. In the graph of Figure 3(B), the results are displayed as the percentage reduction (inhibition rate) relative to the background plaque count. In the presence of 10 μM PhoSL, no plaques were observed in both duplicate wells of the plate. In the concentration-dependent analysis, the IC of PhoSL as a monomer was ５０ The inhibitory activity of PhoSL against the infection was 0.37±0.17 μM (conventional strain) and 0.36±0.05 μM (Omicron strain) (FIG. 3(B)). This indicates that the infection-inhibiting function of PhoSL is not easily affected by viral mutations.

[0064] The inhibitory activity of PhoSL having the amino acid sequence set forth in SEQ ID NO: 1 was 1.6 μg / mL in terms of weight concentration, which was better than the inhibitory activity of FRIL (0.71 μg / mL) using a PRNT assay (Non-Patent Document 3) and slightly weaker than the inhibitory activity of GRFT (approximately 0.5 μg / mL) using a quantitative reverse transcription / real-time PCR (qRT-PCR) assay of GRFT (Ahan et al., ACS Infect. Dis. 8:1253-1264, 2022). It was also much stronger than the inhibitory activity of CLEC4G or CD209C (50-100 μg / mL) (Non-Patent Document 4). Furthermore, the inhibitory activity of PhoSL having the amino acid sequence set forth in SEQ ID NO: 1 was only about 1 / 10 to 1 / 20 of that of the representative neutralizing antibodies S309 (0.08 μg / mL by immunological assay) (Pinto, et al., Nature 586: 290-295, 2020) and S2X259 (0.14 μg / mL by luciferase assay) (Tortorici, et al., Nature 597: 103-108, 2021).

[0065] Example 4 Cryo-EM Analysis Cryo-grids were prepared using 50 mM NaCl and 0.02% NaN ３ Three microliters of the S protein sample and the S protein / PhoSL complex sample (3.2 μM S protein and 138 μM PhoSL, both concentrations as monomers) dissolved in 5 mM Tris (8.0) buffer containing HCl were placed on a carbon grid (Quantifoil, Cu, R1.2 / 1.3, 300 mesh) and flash-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA).

[0066] Micrographs were acquired on a Talos Arctica G2 (Thermo Fisher Scientific) microscope operating in nanoprobe mode at 200 kV using EPU software for automated data collection. Micrographs were collected on a 4k × 4k array using a Falcon 3EC direct electron detector (electron counting mode) at an apparent magnification of 120,000 (0.88 Å / pixel).

[0067] The S protein solution snap-frozen on the grid displayed an image containing well-distributed particles (Fig. 4(A), left panel). In contrast, the S protein solution containing PhoSL displayed an image lacking S protein particles and showing aggregation of PhoSL and S protein (Fig. 4(A), right panel). This aggregation should be due to the multivalency of both the trimeric PhoSL and S protein, as simply modeled in Fig. 4(B). Considering that 6–7 PhoSL trimers bind to a single protein trimer in the SPR experiment (Fig. 2(A)), the actual aggregation in solution is a complex, irregular, three-dimensional array. This result demonstrated that PhoSL aggregates S protein.

[0068] Example 5 Molecular Modeling Study Because it was difficult to determine the structure of the PhoSL-S protein complex due to aggregation, computer modeling was performed to obtain information about the binding mode. Two glycosylation sites, Asn331 and Asn343, in the RBD of the S protein, which has complex N-linked glycans, were selected as modeling sites for PhoSL binding. This is because 98% of the N-linked glycans on both proteins are core-fucosylated, and glycosylation site Asn343 in particular has been shown to be important for infection (Li et al., Cell 182:1284-1294, 2020).

[0069] A structural model of the PhoSL-S protein interaction at Asn331 or Asn343 was generated by molecular dynamics (MD) simulations, essentially following Yamasaki et al., Glycobiology 29:576-587, 2019. The initial model was generated by docking the crystal structure of PhoSL in complex with fucose(α1-6)[GlcNAc(β1-4)]GlcNAc-OH (PDB entry code: 7VU9) with the cryo-EM structure of glycosylated S protein (PDB entry code: 6xkl; A chain, upper conformer), which shows two GlcNAcs at Asn331 and Asn343 (GlcNAcs numbered "1" and "2" in Figure 1). Among them, GlcNAc1 was prepared in duplicate under the condition that S protein would not cause a steric clash. After docking, three mannoses were attached to GlcNAc2 to complete the core structure of the N-linked glycan.

[0070] The AMBER2018 program package for molecular dynamics (Case et al., AMBER2018, University of California, San Francisco, 2018) was used with AMBERffl4SB (Maier et al., J. Chem Theory Comput 11:3696-371, 2015) and GLYCAM_06j-1 (Kirschner et al., J. Comput Chem 29:622-655, 2008), parameters and coordinate filling were created by 1) attaching hydrogen atoms to the protein and glycan, 2) neutralizing the net charge of the entire system by adding sodium ions, and 3) setting the TIP3P water molecule box to a thickness of 15 Å, and MD simulations were performed for 10 ns at 300 K. Representative structures were selected based on the average coordinates of the non-hydrogen atoms in the simulation.

[0071] 10 ns MD simulations of the complex between PhoSL and the S protein receptor-binding domain (RBD) at Asn331 (Fig. 5(A)) and at Asn343 (Fig. 5(B)) show that PhoSL binds stably to the S protein without significant changes in its relative orientation. In contrast, the RBD exhibits relative fluctuations around the hinge between its two subdomains. Because Asn331 and Asn343 are located on different sides of the RBD from the molecular interface with ACE2 (RBM, Fig. 5(A) and (B)), PhoSL bound to these sites is less likely to compete with ACE2. This is similar to the neutralizing antibody S309, which recognizes the core fucose structure in the N-linked glycan of Asn343 (Fig. 5(C)).

[0072] Because the coordinates of PhoSL molecules recognizing the core fucose of the N-linked glycan at Asn331 or Asn343 overlap, it is unlikely that two PhoSL molecules would simultaneously bind to both sites. Rather, one PhoSL molecule interacts with the two N-linked glycans of the RBD, and the one bound to the core fucose of the N-linked glycan at Asn331 forms a hydrogen bond with the mannose moiety of the N-linked glycan at Asn343, and vice versa (Figure 5(D) and (E)). Furthermore, and very importantly, PhoSL interacts with amino acids of the S protein via hydrogen bonds or hydrophobic contacts. These interactions with N-linked glycans and amino acids were observed throughout the simulations, which fully explains the higher affinity for the S protein (Figure 2) compared to the affinity for isolated N-linked glycans (NPL 1).

[0073] Example 6 Analysis of Binding of PhoSL to S Protein Derived from an Omicron Mutant by SPR Binding of PhoSL to S Protein Derived from an Omicron Mutant was observed by SPR using the same method and conditions as in Example 2 ( FIG. 2(D) ). The S Protein derived from an Omicron mutant was purchased from AcroBiosystems and contains 34 Omicron mutations (Cui et al., Cell 185, 860-871, 2022) in addition to the proline and arginine substitutions described in Example 2. The specific 34 mutations are Ala67Val, His69-Val70 (deletion), Thr95Ile, Gly142Asp, Val143-Tyr145 (deletion), Asn211 (deletion), Leu212Ile, Arg214-Glu-Pro-Glu-Asp215 (insertion), Gly339Asp, Ser371Leu, Ser373Pro, Ser375Phe, Lys417Asn, Asn440Lys, and Gly446Ser. , Ser477Asn, Thr478Lys, Glu484Ala, Gln493Arg, Gly496Ser, Gln498Arg, Asn501Tyr, Tyr505His, Thr547Lys, Asp614Gly, His655Tyr, Asn679Lys, Pro681His, Asn764Lys, Asp796Tyr, Asn856Lys, Gln954His, Asn969Lys, and Leu981Phe. Analysis revealed a dissociation constant of 3.7 nM, almost the same as that of the wild-type. This suggests that the effect of PhoSL may be highly resistant to viral mutations.

[0074] Example 7 Analysis of Binding of Mutant PhoSL to S Protein by SPR Using the same method and conditions as in Example 2, the binding of mutant PhoSL (Tyr23Pcf) represented by SEQ ID NO: 10 to S protein was observed by SPR ( FIG. 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 an increase in binding strength, the introduction of the mutation can alter binding.

[0075] Example 8: Analysis of Binding of Wild-Type and Mutant PhoSL to S Protein RBD by SPR. S Protein RBD was purchased from AcroBiosystems. This was prepared by cleaving the Arg319-Phe451 portion of the protein described in Example 2 and substituting Val367Phe. Mutant PhoSLs represented by SEQ ID NOS: 6-14 were designed based on the PhoSL-RBD complex model shown in Example 5, with the possibility of enhanced binding in mind. Surface plasmon resonance (SPR) measurements were performed at 298 K using a Biacore X instrument (Cytiva). S Protein RBD was immobilized on a Sensor Chip NTA (Cytiva) by the Ni-NTA chelation method. A buffer solution (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.05 mM EDTA, and 0.005% surfactant P20) was used as the running buffer. The same buffer solution containing wild-type or mutant PhoSL at concentrations of 10 nM to 100 nM was injected into the flow cell at 20 μL / min for 5 minutes. Two flow cells were used, and the S protein RBD was immobilized on only one of them. The effect of drift was minimized by taking the difference in the sensorgram. Data analysis was performed using the simultaneous kinetic analysis method and the accompanying software (BiaEvaluation3). Figure 6 shows the difference sensorgrams of wild-type and mutant PhoSL (SEQ ID NO: 13). As a result of the analysis, the dissociation constant K Ｄ is 0.37 nM (binding constant K Ａ is 2.7 x 10 ９ M －１ ), K by the mutant shown in SEQ ID NO: 13 Ｄ is 0.24 nM (binding constant K Ａ is 4.2 x 10 ９ M －１ ), and it was found that the mutant shown in SEQ ID NO: 13 had approximately 1.5 times stronger binding affinity to the S protein RBD than the wild-type PhoSL. The dissociation constants of other mutants are shown in Table 1. In addition to the mutant shown in SEQ ID NO: 13, two mutant PhoSLs (SEQ ID NOs: 12 and 14) showed slightly stronger binding than the wild-type.

[0076]

[0077] The pharmaceutical compositions of the present invention are useful for the treatment and / or diagnosis of infections caused by viruses, particularly SARS-CoV-2.

Claims

1. A pharmaceutical composition for the treatment or diagnosis of a viral infection, comprising an active peptide that binds to a virus and inhibits infection thereof, wherein the active peptide is selected from Pholiota squarrosa lectin (PhoSL), an α1-6 fucose glycan-binding lectin having the amino acid sequence set forth in SEQ ID NOs: 1 to 4, and a mutant peptide obtained by introducing a modification selected from substitution, deletion, insertion, and addition of 1 to 4 amino acids into each of the amino acid sequences of PhoSL set forth in SEQ ID NOs: 1 to 4.

2. The pharmaceutical composition of claim 1 , wherein the active peptide has an amino acid sequence identity of 90% or more with the amino acid sequence of PhoSL set forth in SEQ ID NO:

1.

3. The pharmaceutical composition according to claim 1, wherein the active peptide binds to an α1-6 fucose glycan on the outer membrane or shell of a virus.

4. The pharmaceutical composition according to claim 1, wherein the active peptide binds to a fucose(α1-6)[GlcNAc(β1-4)]GlcNAc structure in the glycan of a viral spike protein (S protein).

5. The pharmaceutical composition according to claim 1, wherein the active peptide does not substantially bind to a high mannose sugar chain that does not contain an α1-6 fucose sugar chain and / or a glycolipid sugar chain that does not contain an α1-6 fucose sugar chain.

6. 2. The pharmaceutical composition of claim 1, wherein the viral infection is an infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MARS-CoV), influenza virus, human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), or Ebola virus.

7. 2. The pharmaceutical composition of claim 1, wherein the viral infection is an infection caused by SARS-CoV-2, SARS-CoV, or MARS-CoV.

8. The pharmaceutical composition of claim 1, wherein the viral infection is an infection caused by SARS-CoV-2.

9. The pharmaceutical composition of claim 1, wherein the active peptide has a length of 38-40 mer.

10. The pharmaceutical composition of claim 1, wherein the modification is selected from the group consisting of substitution of Ala at position 1 in the amino acid sequence of PhoSL set forth in SEQ ID NO:1 with an aliphatic amino acid, aromatic amino acid, acidic amino acid, or basic amino acid, substitution of Tyr at position 23 with an aromatic amino acid having a substituent, substitution of Gly at position 24 with a D-amino acid, substitution of Asp at position 25 with a D-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 substitution of Thr at position 40 with an acidic amino acid.

11. 2. The pharmaceutical composition according to claim 1, wherein the modification is selected from the group consisting of substitution of Ala at position 1 in the amino acid sequence of PhoSL set forth in SEQ ID NO:1 with Ile, Glu, Lys, Leu, Val, Phe, Tyr, norleucine (Nle) or L-tert-Leu (Tle), substitution of Tyr at position 23 with 3-chloro-L-Tyr or 4-carboxy-L-Phe, 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-Glu, substitution of Gly at position 26 with L-Asp or L-Glu, substitution of His at position 38 with Phe or Tyr, and substitution of Thr at position 40 with Asp or Glu.

12. The active peptide has a dissociation constant K of 10 pM to 100 nM with respect to the S protein. Ｄ The pharmaceutical composition of claim 1, having an affinity at about 25°C.

13. 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), LPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 12), KPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 13), and 2. The pharmaceutical composition of claim 1, wherein the amino acid sequence is selected from the group consisting of YPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 14).

14. Sugitake lectin (Pholiota squarrosa) having the amino acid sequence set forth in SEQ ID NO:1 1. A mutant of PhoSL (phosphoryl lectin), the amino acid sequence of which contains 1 to 4 substitutions selected from the group consisting of substitution of Ala at position 1 with Ile, Glu, Lys, Leu, Val, Phe, Tyr, norleucine (Nle) or L-tert-Leu (Tle), substitution of Tyr at position 23 with 3-chloro-L-Tyr or 4-carboxy-L-Phe, 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-Glu, substitution of His at position 38 with Phe or Tyr, and substitution of Thr at position 40 with Asp or Glu in the amino acid sequence of PhoSL as set forth in SEQ ID NO:

1.

15. 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), LPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 12), KPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 13), and 15. The mutant of claim 14, selected from the group consisting of YPVPVTKLVCDGDTYKCTAYLDYGDGKWVAQWDTAVFHTT (SEQ ID NO: 14).