Salt form of peptide having antiviral activity

Abstract

Provided, as an anti-SARS-CoV-2 peptide drug, is an optimal salt form of a peptide that binds to a receptor-binding domain (RBD) in SARS-CoV-2.　A sodium or potassium salt of a peptide according to the present invention has, in a direction from an N-terminus to a C-terminus, a first region including a first helix and a second region including a second helix. The first region and the second region each include a site that binds to a receptor-binding domain (RBD) in SARS-CoV-2. A bond is formed between amino acid residues within five residues on the N-terminus side in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and amino acid residues within five residues on the C-terminus side in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, and the peptide binds to the receptor-binding domains (RBD) in SARS-CoV-2.

Description

Salt form of a peptide having antiviral activity

[0001] The present invention relates to an optimal salt form for a peptide that prevents viral infection and exhibits a therapeutic effect by binding to the Receptor Binding Domain (RBD) in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0002] Anti-SARS-CoV-2 peptides that strongly bind to the RBD of the spike of the virus SARS-CoV-2, which repeats various mutations, have been reported, and are expected to prevent and treat infections and diseases caused by the infection (see Patent Document 1).

[0003] International Publication No. 2023 / 282281

[0004] The anti-SARS-CoV-2 peptide that strongly binds to the RBD of the novel coronavirus including various mutant strains already reported is a peptide with a molecular weight of about 5000 consisting of 39 natural amino acids, and is designed to inhibit viral entry into human cells by strongly binding to the RBD of the spike of SARS-CoV-2. Although SARS-CoV-2 repeats mutations, a peptide Ce172 that also strongly binds to these mutant RBDs has been developed. For these peptides, it was an issue to determine an optimal salt form as an anti-SARS-CoV-2 peptide medicine for various physical properties such as solubility, solid stability, and hygroscopicity.

[0005] As a specific development issue, Trifluoroacetic acid: TFA, which is generally used in peptide synthesis, is classified as a "solvent for which no appropriate toxicity data can be found" other than categories 1 to 3 of residual solvents according to the "Residual Solvent Guidelines for Pharmaceuticals (https: / / www.pmda.go.jp / files / 000156502.pdf)" of PMDA. TFA is considered harmful to the human body, and it is not appropriate to use the TFA salt of the anti-SARS-CoV-2 peptide as a medicine. Therefore, it was an issue to determine a safe salt form to replace the TFA salt.

[0006] Furthermore, the TFA salts of the developed peptides, such as Ce172, have a maximum concentration of only about 6 mM (29 mg / mL) in pure water, and it is desirable to increase their solubility. Specifically, the goal was to achieve a solubility of 7 mM (35 mg / mL) or higher in physiological saline.

[0007] In order to develop an anti-SARS-CoV-2 peptide drug, the challenge was to achieve long-term stability in solid form, which is necessary for storage.

[0008] The inventors screened anti-SARS-CoV-2 peptides that strongly bind to the RBD of the novel coronavirus, including various previously reported mutant strains, and performed physical and chemical evaluations. As a result, they investigated the optimal salt form for anti-SARS-CoV-2 peptides that strongly bind to the RBD of the SARS-CoV-2 spike, and are expected to prevent and treat infection and infection-related diseases, primarily using Ce172, a naturally occurring peptide consisting of 39 amino acids, and Ce41, a peptide developed prior to Ce172. As a result, they discovered that the sodium (Na) salt, which achieves both high solubility (50 mg / mL or more) and solid stability (95% or more retained after 1 week at 70°C), is the most superior salt form. Based on these results, the sodium (Na) salt was selected as the optimal salt form for the anti-SARS-CoV-2 peptide pharmaceutical, and the potassium (K) salt, which has similar physical properties, was selected as a backup candidate salt form, thus completing the present invention.

[0009] In other words, the present invention is as follows: [1] A sodium or potassium salt of a peptide having a first region containing a first helix and a second region containing a second helix, wherein the first and second regions include a site that binds to the receptor-binding domain (RBD) of SARS-CoV-2, and a bond is formed between amino acid residues within the first five residues from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) of SARS-CoV-2 in the second region and amino acid residues within the first five residues from the C-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) of SARS-CoV-2, thereby binding to the receptor-binding domain (RBD) of SARS-CoV-2. [2] The sodium or potassium salt of the peptide in [1], wherein the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region comprises the amino acid sequence of (a), (b), (c), or (d) below, and the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region comprises the amino acid sequence of (e), (f), or (g) below; (a) the amino acid sequence of SEQ ID NO: 1, (b) the amino acid sequence of SEQ ID NO: 20, (c) the amino acid sequence of SEQ ID NO: 18, or (d) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 20, or 18; (e) the amino acid sequence of SEQ ID NO: 2, (f) the amino acid sequence of SEQ ID NO: 16, or (g) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 or 16. [3] A sodium or potassium salt of the peptide in [1], wherein the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 comprises the amino acid sequence of (b) or (d2) below, and the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 comprises the amino acid sequence of (f) or (g2) below; (b) the amino acid sequence of SEQ ID NO: 20, or (d2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 20; (f) the amino acid sequence of SEQ ID NO: 16, or (g2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 16.[4] A sodium or potassium salt of the peptide in [1], wherein the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region comprises the amino acid sequence of (c) or (d3) below, and the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region comprises the amino acid sequence of (f) or (g2) below; (c) the amino acid sequence of SEQ ID NO: 18, or (d3) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 18; (f) the amino acid sequence of SEQ ID NO: 16, or (g2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 16. [5] The sodium or potassium salt of any of the peptides [1] to [4], characterized in that the amino acid residues within the first five N-terminal residues and the amino acid residues within the first five C-terminal residues are a combination of a lysine residue and an aspartic acid residue, a lysine residue and a glutamic acid residue, an arginine residue and a glutamic acid residue, or an arginine residue and an aspartic acid residue. [6] The sodium or potassium salt of the peptide [2], characterized in that the amino acid sequence of (d) is an amino acid sequence in which at least one amino acid residue selected from the group consisting of the 1st, 8th, 11th, and 18th amino acid residues in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid residue. [7] The sodium or potassium salt of the peptide [2], characterized in that the amino acid sequence of (g) is an amino acid sequence in which at least one amino acid residue selected from the group consisting of the 2nd, 14th, and 18th amino acid residues in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid residue. [8] The sodium or potassium salt of the peptide in [2], characterized in that the amino acid sequence of (d) above is an amino acid sequence in which the 15th arginine residue and the 19th glutamic acid residue in the amino acid sequence of SEQ ID NO: 1 are replaced with a lysine residue and an aspartic acid residue, respectively. [9] The sodium or potassium salt of the peptide in [2], characterized in that the amino acid sequence of (g) above is an amino acid sequence in which the second glycine residue in the amino acid sequence of SEQ ID NO: 2 is replaced with an alanine residue.

[10] The sodium or potassium salt of the peptide [2], characterized in that the amino acid sequence of (g) above is an amino acid sequence in which the 18th arginine residue in the amino acid sequence of SEQ ID NO: 2 is replaced with a lysine residue.

[11] The sodium or potassium salt of the peptide [2], characterized in that the amino acid sequence of (d) above is an amino acid sequence in which the 10th tyrosine residue and the 13th methionine residue in the amino acid sequence of SEQ ID NO: 1 are further conserved.

[12] The sodium or potassium salt of the peptide [2], characterized in that the amino acid sequence of (g) above is an amino acid sequence in which the 1st histidine residue, the 9th serine residue, the 10th aspartic acid residue and the 13th tyrosine residue in the amino acid sequence of SEQ ID NO: 2 are further conserved.

[13] A sodium or potassium salt of any peptide from [1] to [4], characterized in that the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and / or the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region is 18 to 22 amino acid residues.

[14] A sodium or potassium salt of any peptide from [1] to [4], characterized in that it consists of the first region and the second region, and the total sequence length is 39 amino acid residues.

[15] A sodium or potassium salt of any peptide from [1] to [4], characterized in that it consists of one amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 14.

[16] A sodium or potassium salt of a peptide that binds to the receptor-binding domain (RBD) in SARS-CoV-2, having a first region and a second region in the direction from the N-terminus to the C-terminus, wherein the first region contains an amino acid sequence selected from the following (h), (i), (j), (k), or (l): (h) the amino acid sequence of SEQ ID NO: 15, (i) the amino acid sequence of SEQ ID NO: 18, (j) the amino acid sequence of SEQ ID NO: 20, (k) the amino acid sequence of SEQ ID NO: 22, or (l) the amino acid sequence of SEQ ID NO: 24, wherein the second region contains the amino acid sequence of SEQ ID NO: 16.

[17] A sodium or potassium salt of a peptide that binds to the receptor-binding domain (RBD) in SARS-CoV-2, consisting of the amino acid sequences of SEQ ID NOs: 17, 19, 21, 23, or 25.

[18] A sodium or potassium salt of any peptide from [1] to

[16] having a solubility of 35 mg / mL or more in physiological saline.

[19] A sodium or potassium salt of any peptide from [1] to

[16] having solid stability that maintains a purity of 85% or more after being stored at 70°C for one week.

[20] A sodium or potassium salt of any peptide from [1] to

[16] that forms a homodimer that forms a stable helical bundle structure and strongly binds to RBD on both sides of the dimer.

[21] A pharmaceutical composition containing a sodium or potassium salt of any peptide from [1] to

[16] as an active ingredient. This specification includes the disclosures of Japanese Patent Application No. 2024-192298, which forms the basis of the priority of this application.

[0010] We determined that the sodium (Na) salt is the optimal salt for developing the anti-SARS-CoV-2 peptide into a potent treatment for COVID-19. Specifically, it allows for the high solubility of the anti-SARS-CoV-2 peptide, enabling the creation of peptide formulations that can be administered at high concentrations as needed, in all possible dosage forms for peptide drugs, including nebulizers and DPI preparations. Furthermore, high solid stability and low hygroscopicity were confirmed. Based on these measurement results, we conclude that the sodium (Na) salt is the optimal salt form for developing peptide formulations such as Ce172 as peptide drugs for COVID-19, with the potassium (K) salt being the next best option.

[0011] This is a characteristic diagram showing the CD spectra of Ce4, Ce9, Ce41, Ce59, Ce113, Ce149, Ce172, Ce173, and Ce174 prepared in the examples. This is a characteristic diagram showing the interaction between the second lysine residue in the first region and the five C-terminal residues in the second region of the three-dimensional structural model of Ce41 prepared in the examples. This figure shows the results of measuring the dissolution rate of peptide Ce41 by preparing Na salt, K salt, Ca salt, Mg salt, and acetate salt, adding the peptide Ce41 to a D-PBS(-) solution under conditions where the final concentration of each salt was 1 mg / mL, and calculating the peptide concentration in the supernatant after 3, 10, and 30 minutes from the absorbance at 280 nm. The basic salts, Na salt, K salt, Ca salt, and Mg salt, dissolved completely within 3 minutes of being added to the solution. In this experiment, as shown in this figure, the Ca and Mg salts have lower peptide concentrations compared to the Na and K salts. However, this is thought to be due to differences in the peptide content in each weighed peptide salt and does not indicate differences in solubility in water or dissolution rate for each salt form. This figure shows the amino acid sequences of Ce172 (SEQ ID NO: 19) and Ce41 (SEQ ID NO: 13), which consist of 39 natural amino acids. This figure shows the HPLC analysis results for the TFA salt, Na salt, and K salt of Ce172. Almost identical patterns were observed, and no increase in the total amount of impurities was seen. However, some differences were observed around the retention time of 23-24 min. This figure shows the CD spectra of the TFA salt (A), Na salt (B), and K salt (C) of Ce172. In all salt forms, the 222 / 208 ratio of the CD spectrum is 1.1 or greater, indicating the formation of a typical helical bundle. This figure shows a schematic representation of the structure of Ce172 (A), and a figure showing the actual three-dimensional structure obtained through structural analysis (B). The design is intended to form a salt bridge near the N-terminus and C-terminus, and the dotted lines indicate that the bond is actually formed. This figure shows the result of X-ray crystallography analysis of the structure in which peptide Ce172 is bound to RBD. The actual structural analysis results show that peptide Ce172 forms a homodimer with the RBD-binding surface facing outwards and is bound to two RBDs.Figure (A) schematically shows that the peptide Ce172 forms a homodimer, and Figure (B) shows the actual three-dimensional structure obtained through structural analysis. Figure 1 shows the HPLC chromatogram in the solid stability test of the sodium salt of Ce172. The experimental conditions for each are shown in the right part of the figure. Figure 2 shows the HPLC chromatogram in the solid stability test of the potassium salt of Ce172. The experimental conditions for each are shown in the right part of the figure. Figure 3 shows the adsorption and desorption isotherms of the sodium salt (A) and potassium salt (B) of Ce172, plotted against the weight change of the sample (vertical axis) in the range of relative humidity from 0% to 95% (horizontal axis). The first adsorption curve is shown as a dark line.

[0012] The present invention will now be described in detail. 1. Peptide that binds to the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) The peptide according to this disclosure can bind to the receptor binding domain (RBD) of wild-type SARS-CoV-2 or mutant SARS-CoV-2. As a result, the peptide according to this disclosure can inhibit the binding of ACE2 (angiotensin-converting enzyme 2) expressed on cells to the receptor binding domain (RBD) of SARS-CoV-2, thereby preventing infection of cells with SARS-CoV-2. Regarding wild-type SARS-CoV-2, the structural analysis results of the Spike(S) protein present on the surface of the virus are shown in Wrapp, D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260-1263 (2020). The S protein forms a trimer and is known to have a basic amino acid sequence that is cleaved by the host protease furin. The S protein is cleaved by furin into the S1 subunit, which is responsible for receptor binding, and the S2 subunit, which is responsible for membrane fusion. The peptide according to this disclosure can bind to the receptor-binding domain (RBD) present in the S1 subunit. In this disclosure, "receptor-binding domain (RBD)" means the receptor-binding domain (RBD) present in the S1 subunit of the Spike protein.

[0013] The peptide according to this disclosure has two helical structures (for convenience, the N-terminal side will be referred to as the first helix and the C-terminal side as the second helix). The peptide according to this disclosure includes a region having the first helix at the N-terminus (for convenience, referred to as the first region) and a region having the second helix at the C-terminus (for convenience, referred to as the second region). A bond is formed between the amino acid residues within the first five residues at the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) of SARS-CoV-2 in the first region and the amino acid residues within the C-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) of SARS-CoV-2 in the second region. Thus, one of the features of the peptide according to this disclosure is that it can maintain a desired three-dimensional structure due to the bond formed between the amino acid residues within the first five residues at the N-terminus of the first region and the amino acid residues within the C-terminus of the second region. In this disclosure, the bond between the five or fewer amino acid residues at the N-terminus of the first region and the five or fewer amino acid residues at the C-terminus of the second region includes a salt bridge or a hydrogen bond. Hereinafter, the bond may be specifically referred to as a salt bridge or a hydrogen bond, but the term "bond" can refer to both a salt bridge and a hydrogen bond.

[0014] Note that "amino acid residues within the first five residues of the N-terminus" refers to amino acid residues located between the 1st and 5th amino acid residues counting from the N-terminus, and does not mean that the position of amino acid residues involved in the formation of the above bond is limited to the N-terminus. Similarly, "amino acid residues within the first five residues of the C-terminus" refers to amino acid residues located between the 1st and 5th amino acid residues counting from the C-terminus, and does not mean that the position of amino acid residues involved in the formation of the above bond is limited to the C-terminus.

[0015] Furthermore, the peptide relating to this disclosure is characterized by the introduction of hydrophilic amino acid residues in the first helix (specifically, for example, the arginine residue at position 15 and the glutamic acid residue at position 19 of SEQ ID NO: 1), which stabilizes and hydrophilizes the helix structure through the formation of salt bridges by these residues, thereby achieving extremely high solubility in ultrapure water or buffer solutions.

[0016] Furthermore, in the peptide relating to this disclosure, the first region and the second region each have an amino acid sequence capable of binding to the receptor-binding domain (RBD) of SARS-CoV-2 while in the three-dimensional structure described above, and can be defined by their respective amino acid sequences. Specifically, the sequence of the site in the first region that binds to the receptor-binding domain (RBD) of SARS-CoV-2 can be defined as the following amino acid sequences: (a) the amino acid sequence of SEQ ID NO: 1 (b) the amino acid sequence of SEQ ID NO: 20 (c) the amino acid sequence of SEQ ID NO: 18 (d) an amino acid sequence having 80% or more sequence identity with the amino acid sequences of SEQ ID NO: 1, 20, or 18

[0017] Furthermore, the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region can be defined as the following amino acid sequences: (e) the amino acid sequence of SEQ ID NO: 2; (f) the amino acid sequence of SEQ ID NO: 16; (g) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 or 16.

[0018] Furthermore, the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region can be defined as the following amino acid sequence: (a) the amino acid sequence of SEQ ID NO: 1; (d1) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0019] Furthermore, the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region can be defined as the following amino acid sequences: (e) the amino acid sequence of SEQ ID NO: 2; (g1) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0020] Furthermore, the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region can be defined as the following amino acid sequences: (b) the amino acid sequence of SEQ ID NO: 20; (d2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0021] Furthermore, the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region can be defined as the following amino acid sequences: (f) the amino acid sequence of SEQ ID NO: 16; (g2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0022] Furthermore, the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region can be defined as the following amino acid sequences: (c) the amino acid sequence of SEQ ID NO: 18; (d3) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 18.

[0023] Furthermore, the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region can be defined as the following amino acid sequences: (f) the amino acid sequence of SEQ ID NO: 16; (g2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0024] The amino acid sequences (a), (b), or (c) defining the first region have a lysine residue (the second amino acid residue from the N-terminus in SEQ ID NOs: 1, 18, or 20), and the amino acid sequences (e) or (f) defining the second region have an aspartic acid residue (the first amino acid residue from the C-terminus in SEQ ID NOs: 2 or 16). The peptides of this disclosure as defined in SEQ ID NOs: 1, 18, or 20 and SEQ ID NOs: 2 or 16 can be stabilized to a desired three-dimensional structure by the formation of the above-mentioned salt bridge between these lysine residues and aspartic acid residues.

[0025] However, the salt bridge formed between the first and second regions is not limited to those formed by the lysine residue and the aspartic acid residue. For example, even if the second amino acid residue from the N-terminus in SEQ ID NO: 1, 18, or 20 is an aspartic acid residue, and the first amino acid residue from the C-terminus in SEQ ID NO: 2 or 16 is a lysine residue, a salt bridge can be formed between these aspartic acid residues and lysine residues, stabilizing the desired three-dimensional structure.

[0026] Furthermore, the combinations of amino acid residues that form a salt bridge are not limited to the combination of lysine residue and aspartic acid residue described above. Examples include combinations of lysine residue and glutamic acid residue, arginine residue and glutamic acid residue, and arginine residue and aspartic acid residue. For example, even if the second position from the N-terminus in SEQ ID NO: 1, 18, or 20 is an arginine residue and the first position from the C-terminus in SEQ ID NO: 2 or 16 is a glutamic acid residue, a salt bridge can be formed between these arginine and glutamic acid residues, stabilizing the desired three-dimensional structure. Note that when forming a salt bridge between an arginine residue and a glutamic acid residue, the second position from the N-terminus in SEQ ID NO: 1, 18, or 20 may be a glutamic acid residue and the first position from the C-terminus in SEQ ID NO: 2 or 16 may be an arginine residue.

[0027] The amino acid sequences specified in (d), (d1), (d2), or (d3) above are amino acid sequences of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2, and can be amino acid sequences that have 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, or 95% or more sequence identity with the amino acid sequences of SEQ ID NOs. 1, 18, or 20, provided that the amino acid residues involved in the formation of the above binding between the first and second regions are preserved.

[0028] Furthermore, the amino acid sequences specified in (d), (d1), (d2), or (d3) above are amino acid sequences of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2, and can be amino acid sequences obtained by substituting 1 to 4 amino acid residues from the amino acid sequence of SEQ ID NO: 1, 18, or 20, provided that the amino acid residues involved in the formation of the above binding between the first and second regions are preserved. Note that an amino acid sequence obtained by substituting 1 amino acid residue from the amino acid sequence of SEQ ID NO: 1, 18, or 20 has 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, 18, or 20. An amino acid sequence obtained by substituting 2, 3, or 4 amino acid residues from the amino acid sequence of SEQ ID NO: 1, 18, or 20 has 90%, 85%, or 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, 18, or 20, respectively.

[0029] When substituting 1 to 4 amino acid residues into the amino acid sequence of SEQ ID NOs: 1, 18, or 20, the amino acid residues to be substituted are not particularly limited, but can be selected from the group consisting of the 1st amino acid residue (aspartic acid residue), the 8th amino acid residue (lysine residue), the 11th amino acid residue (glutamic acid residue), and the 18th amino acid residue (glutamic acid residue) in the amino acid sequence of SEQ ID NOs: 1, 18, or 20. These 1st amino acid residue (aspartic acid residue), 8th amino acid residue (lysine residue), 11th amino acid residue (glutamic acid residue), and 18th amino acid residue (glutamic acid residue) in the amino acid sequence of SEQ ID NOs: 1, 18, or 20 contribute to the stabilization of the helix structure, but their interaction with the receptor-binding domain (RBD) in SARS-CoV-2 is minimal, so substitution with amino acid residues with similar properties is possible.

[0030] The first aspartic acid residue in SEQ ID NOs: 1, 18, or 20 can be substituted with an asparagine residue or a glutamic acid residue, although this substitution is not particularly limited. Aspartic acid and asparagine, among others, have similar properties, as they both have the property of initiating a helix called an N-cap. Furthermore, aspartic acid and glutamic acid are both acidic amino acids and have similar properties. Therefore, even if the first aspartic acid residue in SEQ ID NOs: 1, 18, or 20 is substituted with an asparagine residue or a glutamic acid residue, the effect on the structure of the first helix and the interaction with the receptor-binding domain (RBD) in SARS-CoV-2 is minimal, and the function of the peptide according to this disclosure can be maintained.

[0031] Furthermore, the eighth lysine residue in SEQ ID NOs: 1, 18, or 20 can be substituted with an arginine or histidine residue, although this is not particularly limited. Lysine, arginine, and histidine are basic amino acids and have similar properties. Therefore, even if the eighth lysine residue in SEQ ID NOs: 1, 18, or 20 is substituted with an arginine or histidine residue, the effect on the structure of the first helix and the interaction with the receptor-binding domain (RBD) in SARS-CoV-2 is minimal, and the function of the peptide according to this disclosure can be maintained. In particular, since lysine and arginine do not have an aromatic ring, when the eighth lysine residue is substituted with an arginine residue, the effect on the structure of the first helix and the interaction with the receptor-binding domain (RBD) in SARS-CoV-2 is minimal, and the function of the peptide according to this disclosure can be maintained at a high level.

[0032] Furthermore, the 11th and 18th glutamic acid residues in SEQ ID NOs: 1, 18, or 20 can be substituted with aspartic acid residues, although this is not particularly limited. Both glutamic acid and aspartic acid are acidic amino acids and have similar properties. Therefore, even if the 11th and / or 18th glutamic acid residues in SEQ ID NOs: 1, 18, or 20 are substituted with aspartic acid residues, the effect on the structure of the first helix and the interaction with the receptor-binding domain (RBD) in SARS-CoV-2 is minimal, and the function of the peptide according to this disclosure can be maintained.

[0033] The amino acid sequences specified in (g), (g1), or (g2) above are amino acid sequences of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2, and can be amino acid sequences that have 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, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 or 16, provided that the amino acid residues involved in the formation of the above binding between the first and second regions are preserved.

[0034] Furthermore, the amino acid sequences specified in (g), (g1), or (g2) above are amino acid sequences of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2, and can be amino acid sequences obtained by substituting one to three amino acid residues from the amino acid sequence of SEQ ID NO: 2 or 16, as long as the amino acid residues involved in the formation of the above binding between the first and second regions are preserved. Note that an amino acid sequence obtained by substituting one amino acid residue from the amino acid sequence of SEQ ID NO: 2 or 16 has 94.7% sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 or 16. An amino acid sequence obtained by substituting two or three amino acid residues from the amino acid sequence of SEQ ID NO: 2 or 16 has 89.5% or 84.2% sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 or 16, respectively.

[0035] When substituting one to three amino acid residues into the amino acid sequence of SEQ ID NO: 2 or 16, the amino acid residues to be substituted are not particularly limited, but can be selected from the group consisting of the second amino acid residue (glycine residue), the 14th amino acid residue (glutamic acid residue), and the 18th amino acid residue (arginine residue) in the amino acid sequence of SEQ ID NO: 2 or 16.

[0036] The second glycine residue in SEQ ID NO: 2 or 16 can be substituted with an alanine residue. The second glycine residue in SEQ ID NO: 2 or 16 is the optimal amino acid residue for all receptor-binding domains (RBDs) in SARS-CoV-2 where the 501st asparagine residue in the RBD amino acid sequence is mutated to a tyrosine residue. However, for the receptor-binding domain (RBD) in wild-type SARS-CoV-2, substitution with an alanine residue results in even stronger binding.

[0037] In the peptide according to this disclosure, the first amino acid residue in SEQ ID NO: 2 or 16 is preferably a histidine residue. When the first amino acid residue in SEQ ID NO: 2 or 16 is a histidine residue, it can contribute to structural stabilization by interacting with the 19th amino acid residue in SEQ ID NO: 1, 18, or 20, for example, a glutamic acid residue. Furthermore, this histidine residue contributes to the stabilization of the helix bundle structure.

[0038] Furthermore, the 14th glutamic acid residue in SEQ ID NO: 2 or 16 can be substituted with an aspartic acid residue, although this is not particularly limited. Both glutamic acid and aspartic acid are acidic amino acids and have similar properties. Therefore, even if the 14th glutamic acid residue in SEQ ID NO: 2 or 16 is substituted with an aspartic acid residue, the effect on the structure of the second helix and the interaction with the receptor-binding domain (RBD) in SARS-CoV-2 is minimal, and the function of the peptide according to this disclosure can be maintained.

[0039] Furthermore, the 18th arginine residue in SEQ ID NO: 2 or 16 can be substituted with a lysine residue or a histidine residue, although this is not particularly limited. Arginine, lysine, and histidine are basic amino acids and have similar properties. Therefore, even if the 18th arginine residue in SEQ ID NO: 2 or 16 is substituted with a lysine residue or a histidine residue, the effect on the structure of the second helix and the interaction with the receptor-binding domain (RBD) in SARS-CoV-2 is minimal, and the function of the peptide according to this disclosure can be maintained. In particular, since lysine and arginine do not have an aromatic ring, when the 18th arginine residue is substituted with a lysine residue, the effect on the structure of the second helix and the interaction with the receptor-binding domain (RBD) in SARS-CoV-2 is minimal, and the function of the peptide according to this disclosure can be maintained at a high level.

[0040] As described above, examples of substituted amino acid residues have been explained for the sequences of the receptor-binding domain (RBD) sites in the first region and the second region, as defined in (d), (d1), (d2), or (d3) and (g), (g1), or (g2) above. However, the sequences of the receptor-binding domain (RBD) sites in the first region and the second region are not limited to those described above.

[0041] The sequence of the site in the first region of the peptide according to this disclosure that binds to the receptor-binding domain (RBD) of SARS-CoV-2 may be an amino acid sequence in which amino acid residues other than the first aspartic acid residue, the eighth lysine residue, the eleventh glutamic acid residue, and the eighth glutamic acid residue in SEQ ID NOs: 1, 18, or 20 are substituted with other amino acid residues, or it may be an amino acid sequence in which 1 to 4 amino acid residues are deleted and / or inserted from the amino acid sequence of SEQ ID NOs: 1, 18, or 20. In other words, the sequence of the site in the first region that binds to the receptor-binding domain (RBD) of SARS-CoV-2 is not limited to SEQ ID NOs: 1, 18, or 20, which consists of 20 amino acid residues, but can be an amino acid sequence consisting of 16 to 24 amino acid residues, preferably 18 to 22 amino acid residues.

[0042] Furthermore, the sequence of the site in the second region of the peptide according to this disclosure that binds to the receptor-binding domain (RBD) of SARS-CoV-2 may be an amino acid sequence in which amino acid residues other than the second glycine residue, the 14th glutamic acid residue, and the 18th arginine residue in SEQ ID NO: 2 or 16 are substituted with other amino acid residues, or it may be an amino acid sequence in which 1 to 3 amino acid residues are deleted and / or inserted from the amino acid sequence of SEQ ID NO: 2 or 16. In other words, the sequence of the site in the second region that binds to the receptor-binding domain (RBD) of SARS-CoV-2 is not limited to SEQ ID NO: 2 or 16, which consists of 19 amino acid residues, but can be an amino acid sequence consisting of 16 to 23 amino acid residues, preferably 18 to 22 amino acid residues.

[0043] The peptide according to this disclosure may consist of 36 to 44 amino acid residues in total, combining the first and second regions, but it is most preferable that it consists of 39 amino acid residues in total. By having 39 amino acid residues in total, a salt bridge can be formed between the N-terminal lysine residue in the first region and the C-terminal aspartate residue in the second region, and the size is suitable for binding to the receptor-binding domain (RBD) of SARS-CoV-2.

[0044] Thus, even when the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region consists of an amino acid sequence different from SEQ ID NO: 1, 18, or 20, and the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region consists of an amino acid sequence different from SEQ ID NO: 2 or 16, a salt bridge is formed between the amino acid residue within 5 residues on the N-terminal side (the second lysine residue from the N-terminal in SEQ ID NO: 1) in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and the amino acid residue within 5 residues on the C-terminal side (the first aspartic acid residue from the C-terminal in SEQ ID NO: 2) in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, whereby the desired three-dimensional structure can be maintained. Therefore, even when the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region consists of an amino acid sequence different from SEQ ID NO: 1, 18, or 20, and the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region consists of an amino acid sequence different from SEQ ID NO: 2 or 16, the peptide according to the present disclosure can bind to the receptor-binding domain (RBD) in SARS-CoV-2 in the same manner.

[0045] Here, regarding the first region consisting of the amino acid sequence of SEQ ID NO: 1 and the second region consisting of the amino acid sequence of SEQ ID NO: 2, the roles of each amino acid residue are summarized in Tables 1 and 2 below, respectively.

[0046]

[0047]

[0048] Here, regarding the first region consisting of the amino acid sequence of SEQ ID NO: 18 and the second region consisting of the amino acid sequence of SEQ ID NO: 16, the roles of each amino acid residue are summarized in Tables 3 and 4 below, respectively.

[0049]

[0050]

[0051] Here, the roles of each amino acid residue in the first region, which consists of the amino acid sequence of Sequence ID No. 20, are summarized in Table 5 below.

[0052]

[0053] In the table above, for example, "RBD S477" refers to the 477th serine residue in the amino acid sequence of the receptor-binding domain (RBD) in SARS-CoV-2 (the same meaning applies to other residues). Also, in the table above, "RBD N501Y with N501Y mutation" refers to the 501st tyrosine residue in the amino acid sequence of the receptor-binding domain (RBD) in SARS-CoV-2, which has a mutation that causes the 501st amino acid residue in the receptor-binding domain (RBD) to be a tyrosine residue.

[0054] As can be seen from the table above, the amino acid sequences of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region are not limited to SEQ ID NOs. 1, 18, 20, 2, or 16, respectively, but can be appropriately modified according to the role of each amino acid residue.

[0055] For example, the first aspartic acid residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region does not have a specific interaction with the RBD, but removing this amino acid residue reduces its binding ability. Therefore, it can be replaced with a glutamic acid residue, which has a chemical structure similar to aspartic acid. Also, the 15th arginine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region forms a salt bridge with the 19th glutamic acid residue that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region, contributing to the stabilization of the helix structure. Similar stability can be achieved by replacing these with lysine and aspartic acid residues, respectively. The second glycine residue from the N-terminus in the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in region 2 is extremely important for receptor-binding domains (RBDs) containing the N501Y mutation, including the receptor-binding domain (RBD) in the UK variant SARS-CoV-2 (B.1.1.7). However, for the receptor-binding domain (RBD) in wild-type SARS-CoV-2, substitution with an alanine residue results in even stronger binding ability. Furthermore, the third glutamate residue from the N-terminus in the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in region 2 can be replaced with a glutamine residue. In addition, the sixth leucine residue from the N-terminus in the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in region 2 can be replaced with a methionine residue. Furthermore, the 16th methionine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region can be a tryptophan residue.Furthermore, by simultaneously replacing the 3rd glutamate residue, 6th leucine residue, and 16th methionine residue from the N-terminus—which constitute the sequence of the receptor-binding domain (RBD) in SARS-CoV-2—with a glutamine residue, a methionine residue, and a tryptophan residue, a peptide can be created that strongly binds to mutant SARS-CoV-2 and exhibits antiviral activity.

[0056] However, it is preferable that the first amino acid residue from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region is an aspartic acid residue. By making the first amino acid residue from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 an aspartic acid residue, the binding activity to the receptor-binding domain (RBD) in SARS-CoV-2 can be improved.

[0057] Furthermore, it is preferable that the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region has an isoleucine residue as the fifth amino acid residue from the N-terminus. By making the fifth amino acid residue from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region an isoleucine residue, a stable interaction can be formed between the ninth isoleucine residue from the N-terminus that constitutes the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and the fifteenth phenylalanine residue from the N-terminus that constitutes the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region. By making the 9th residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region an isoleucine residue, a stable interaction can be formed between the 5th isoleucine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region, the 8th valine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, the 11th leucine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, the 12th isoleucine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, and the 15th phenylalanine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region. Furthermore, it is preferable that the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 has a tyrosine residue as the 10th amino acid residue from the N-terminus and a methionine residue as the 13th amino acid residue.By setting the 10th amino acid residue from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 as a tyrosine residue and the 13th as a methionine residue, high binding activity to the receptor-binding domain (RBD) in SARS-CoV-2 is achieved. If even one of these two residues is substituted with an amino acid residue other than these, the binding activity to the receptor-binding domain (RBD) in SARS-CoV-2 is significantly reduced. Furthermore, it is preferable that the 12th amino acid residue from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 is an isoleucine residue. By making the 12th amino acid residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region an isoleucine residue, a stable interaction can be formed between the 16th leucine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and the 8th valine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region. Furthermore, it is preferable that the 16th amino acid residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region is a leucine residue. By making the 16th residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region a leucine residue, a stable interaction can be formed between the isoleucine residue at the 12th position from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region, the alanine residue at the 4th position from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, and the valine residue at the 8th position from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region.

[0058] Regarding the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, it is preferable that the first residue from the N-terminus is a histidine residue. By making the first residue from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region a histidine residue, it is possible to stabilize the helix bundle structure and form a salt bridge with the 19th glutamate residue of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region, thereby stabilizing the turn structure.

[0059] Regarding the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2, it is preferable that the 10th amino acid residue from the N-terminus is an aspartic acid residue. By making the 10th amino acid residue from the N-terminus of the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 an aspartic acid residue, it becomes possible to form a salt bridge with the arginine residue at position 403 and the lysine residue at position 417 in the amino acid sequence of the receptor-binding domain (RBD) in SARS-CoV-2. If the 10th amino acid residue from the N-terminus of the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 is a glutamic acid residue, a salt bridge cannot be formed between the glutamic acid residue and the arginine residue at position 403 and the lysine residue at position 417 in the amino acid sequence of the receptor-binding domain (RBD) in SARS-CoV-2, and there is a risk that the binding activity to the receptor-binding domain (RBD) in SARS-CoV-2 will decrease.

[0060] Furthermore, it is preferable that the 13th amino acid residue from the N-terminus in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region is a tyrosine residue. By making the 13th amino acid residue from the N-terminus in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 a tyrosine residue, it becomes possible to form a hydrogen bond with the aspartic acid residue at position 420 in the amino acid sequence of the receptor-binding domain (RBD) in SARS-CoV-2.

[0061] Furthermore, it is preferable that the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 has an alanine residue as the fourth amino acid residue from the N-terminus. By making the fourth amino acid residue from the N-terminus of the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 an alanine residue, a stable interaction can be formed with the leucine residue at the 16th N-terminus that constitutes the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2. Furthermore, it is preferable that the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 has a valine residue as the eighth amino acid residue from the N-terminus. By making the 8th amino acid residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region a valine residue, a stable interaction can be formed between the 9th isoleucine residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region, the 12th isoleucine residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region, the 13th methionine residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region, and the 16th leucine residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region. Furthermore, it is preferable that the 11th amino acid residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region is a leucine residue. By making the 11th residue from the N-terminus of the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region a leucine residue, a stable interaction can be formed between the 9th isoleucine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and the 8th valine residue from the N-terminus that constitutes the sequence of the receptor-binding domain (RBD) in SARS-CoV-2 in the second region.Furthermore, it is preferable that the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 has a phenylalanine residue as the 15th amino acid residue from the N-terminus. By making the 15th amino acid residue from the N-terminus of the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 a phenylalanine residue, a stable interaction can be formed between the isoleucine residue at the 5th N-terminus of the sequence that constitutes the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2, the leucine residue at the 6th N-terminus of the sequence that constitutes the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2, and the isoleucine residue at the 9th N-terminus of the sequence that constitutes the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2.

[0062] In addition to these, the 14th amino acid residue from the N-terminus in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region can be any amino acid residue, but it is preferable that it be a glutamate residue. By making the 14th amino acid residue from the N-terminus in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region a glutamate residue, the hydrophilic properties of the peptide can be maintained to a high degree.

[0063] Furthermore, it is preferable that the 17th amino acid residue from the N-terminus in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region is a lysine residue. By making the 17th amino acid residue from the N-terminus in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 a lysine residue, a salt bridge can be formed with the 420th aspartic acid residue in the amino acid sequence of the receptor-binding domain (RBD) in SARS-CoV-2, and hydrogen bonds can be formed with the 421st tyrosine residue and the 460th asparagine residue.

[0064] Furthermore, the 18th amino acid residue from the N-terminus in the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region can be any amino acid residue, but it is preferable that it be an arginine residue. By making the 18th residue from the N-terminus of the second helix an arginine residue, the hydrophilic properties of the peptide can be maintained to a high degree, and it also forms a hydrogen bond with the 2nd lysine residue from the N-terminus of the first helix, contributing to the stabilization of the helix bundle structure.

[0065] Furthermore, substituting this 18th arginine residue with a lysine residue also allows the peptide to maintain a high degree of hydrophilicity.

[0066] Furthermore, one of the features of the peptides according to this disclosure is that the 15th amino acid residue in the amino acid sequence of SEQ ID NOs: 1, 18, or 20 is an arginine residue and the 19th amino acid residue is a glutamic acid residue. The formation of a salt bridge between the 15th arginine residue and the 19th glutamic acid residue in the amino acid sequence of SEQ ID NOs: 1, 18, or 20 can contribute to the structural stabilization of the first helix. This can improve the binding rate between the peptides according to this disclosure and the receptor-binding domain (RBD) in SARS-CoV-2.

[0067] In the peptides according to this disclosure, the 15th arginine residue and the 19th glutamic acid residue in the amino acid sequences of SEQ ID NOs: 1, 18, or 20 may be replaced with a lysine residue and an aspartic acid residue, respectively. In other words, in the peptides according to this disclosure, the 15th and 19th amino acid residues in the amino acid sequences of SEQ ID NOs: 1, 18, or 20 may be a combination of a lysine residue and an aspartic acid residue, or a combination of an arginine residue and a glutamic acid residue, but a combination of an arginine residue and a glutamic acid residue is particularly desirable. Even when the 15th position in SEQ ID NOs: 1, 18, or 20 is a lysine residue and the 19th position is an aspartic acid residue, a salt bridge can be formed between these lysine and aspartic acid residues, contributing to the structural stabilization of the first helix. In particular, when the 15th position of SEQ ID NO: 1, 18, or 20 is an arginine residue and the 19th position is a glutamic acid residue, the binding rate between the peptide according to this disclosure and the receptor-binding domain (RBD) in SARS-CoV-2 can be further improved compared to when the 15th position of SEQ ID NO: 1 is a lysine residue and the 19th position is an aspartic acid residue.

[0068] Furthermore, one of the features of the peptides relating to this disclosure is that the second amino acid in the amino acid sequence of SEQ ID NO: 2 or 16 is a glycine residue. Because the second amino acid in the amino acid sequence of SEQ ID NO: 2 or 16 is a glycine residue, the peptides relating to this disclosure have excellent binding activity to the receptor-binding domain (RBD) in both wild-type SARS-CoV-2 and mutant SARS-CoV-2.

[0069] Here, wild-type SARS-CoV-2 refers to the type of SARS-CoV-2 in which the amino acid sequence constituting the receptor-binding domain (RBD) has a glycine residue at position 339, a serine residue at position 371, a serine residue at position 373, a serine residue at position 375, a threonine residue at position 376, an aspartic acid residue at position 405, an arginine residue at position 408, a lysine residue at position 417, an asparagine residue at position 440, a glycine residue at position 446, a leucine residue at position 452, a serine residue at position 477, a threonine residue at position 478, a glutamic acid residue at position 484, a glutamine residue at position 493, a glycine residue at position 496, a glutamine residue at position 498, an asparagine residue at position 501, and a tyrosine residue at position 505. On the other hand, mutant SARS-CoV-2 is a mutation in the amino acid sequence that makes up the receptor-binding domain (RBD) in which the 339th amino acid is an aspartic acid residue, the 371st amino acid is a leucine or phenylalanine residue, the 373rd amino acid is a proline residue, the 375th amino acid is a phenylalanine residue, the 376th amino acid is an alanine residue, the 405th amino acid is an asparagine residue, the 408th amino acid is a serine residue, the 417th amino acid is an asparagine or threonine residue, and the 440th amino acid is a lysine residue. This refers to SARS-CoV-2 having at least one mutation selected from the group consisting of a mutation where the 446th position is a serine residue, a mutation where the 452nd position is an arginine residue, a mutation where the 477th position is an asparagine residue, a mutation where the 478th position is a lysine residue, a lysine residue, a glutamine residue, or an alanine residue at the 484th position, a mutation where the 493rd position is an arginine residue, a mutation where the 496th position is a serine residue, a mutation where the 498th position is an arginine residue, a mutation where the 501st position is a tyrosine residue, and a mutation where the 505th position is a histidine residue.More specifically, a variant of SARS-CoV-2 can be described as a type of SARS-CoV-2 that has a mutation in the amino acid sequence constituting the receptor-binding domain (RBD) where the 501st amino acid residue is a tyrosine residue, and the amino acid residues at positions 339, 371, 373, 375, 376, 405, 408, 417, 440, 446, 452, 477, 478, 484, 493, 496, 498, and 505 are wild-type (PANGO lineage name: B.1.1.7, sometimes commonly referred to as the British variant SARS-CoV-2). Furthermore, a variant of SARS-CoV-2 can be identified as a type of SARS-CoV-2 that has mutations in the amino acid sequence constituting the receptor-binding domain (RBD), such as a mutation where the 417th amino acid is an asparagine residue, a mutation where the 484th amino acid is a lysine residue, and a mutation where the 501st amino acid is a tyrosine residue, while the amino acid residues at positions 339, 371, 373, 375, 376, 405, 408, 440, 446, 452, 477, 478, 493, 496, 498, and 505 are wild-type (PANGO lineage name: B.1.351, sometimes commonly referred to as the South African variant of SARS-CoV-2). Furthermore, a variant of SARS-CoV-2 can be identified as a type of SARS-CoV-2 that has mutations in the amino acid sequence constituting the receptor-binding domain (RBD), such as a threonine residue at position 417, a lysine residue at position 484, and a tyrosine residue at position 501, while the amino acid residues at positions 339, 371, 373, 375, 376, 405, 408, 440, 446, 452, 477, 478, 493, 496, 498, and 505 are wild-type (PANGO lineage name: P.1, sometimes commonly referred to as the Brazilian variant of SARS-CoV-2).Furthermore, another variant of SARS-CoV-2 is a type of SARS-CoV-2 that has mutations in the amino acid sequence constituting the receptor-binding domain (RBD), specifically a mutation where the 452nd amino acid residue is an arginine residue and a mutation where the 478th amino acid residue is a lysine residue, with the amino acid residues at positions 339, 371, 373, 375, 376, 405, 408, 417, 440, 446, 477, 484, 493, 496, 498, 501, and 505 being wild-type (PANGO lineage name: B.1.617.2, sometimes commonly referred to as the Indian variant (δ type) SARS-CoV-2). Furthermore, another variant of SARS-CoV-2 is a type of SARS-CoV-2 that has mutations in the amino acid sequence constituting the receptor-binding domain (RBD), specifically a mutation where the 452nd amino acid residue is an arginine residue and a mutation where the 484th amino acid residue is a glutamine residue, with the amino acid residues at positions 339, 371, 373, 375, 376, 405, 408, 417, 440, 446, 477, 478, 493, 496, 498, 501, and 505 being wild-type (PANGO lineage name: B.1.617.1, sometimes commonly referred to as the Indian variant (κ type) SARS-CoV-2).Furthermore, another example of a mutant SARS-CoV-2 is a type of SARS-CoV-2 that has mutations in the amino acid sequence constituting the receptor-binding domain (RBD), such as a mutation where the 339th amino acid residue is aspartic acid, the 371st is leucine, the 373rd is proline, the 375th is phenylalanine, the 417th is asparagine, the 440th is lysine, the 446th is serine, the 477th is asparagine, the 478th is lysine, the 484th is alanine, the 493rd is arginine, the 496th is serine, the 498th is arginine, the 501st is tyrosine, and the 505th is histidine, while the amino acid residues at positions 376, 405, 408, and 452 are wild-type (PANGO systematic name: B.1.1.529 / BA.1, sometimes referred to as the omicron variant (BA.1 lineage) of SARS-CoV-2). Furthermore, variant SARS-CoV-2 includes mutations in the amino acid sequence constituting the receptor-binding domain (RBD), such as a mutation where the 339th amino acid is an aspartic acid residue, a mutation where the 371st amino acid is a phenylalanine residue, a mutation where the 373rd amino acid is a proline residue, a mutation where the 375th amino acid is a phenylalanine residue, a mutation where the 376th amino acid is an alanine residue, a mutation where the 405th amino acid is an asparagine residue, a mutation where the 408th amino acid is a serine residue, a mutation where the 417th amino acid is an asparagine residue, and a mutation where the 440th amino acid is an asparagine residue. One example of SARS-CoV-2 is a variant that has mutations resulting in a lysine residue at position 1, an asparagine residue at position 477, a lysine residue at position 478, an alanine residue at position 484, an arginine residue at position 493, an arginine residue at position 498, a tyrosine residue at position 501, and a histidine residue at position 505, with the amino acid residues at positions 446, 452, and 496 being wild-type (PANGO lineage name: B.1.1.529 / BA.2, sometimes referred to as the omicron variant (BA.2 lineage) SARS-CoV-2).

[0070] Furthermore, the sequence of the site in the second region of the peptide relating to this disclosure that binds to the receptor-binding domain (RBD) of SARS-CoV-2 may be an amino acid sequence in which the second glycine residue in the amino acid sequence of SEQ ID NO: 2 or 16 is replaced with an alanine residue. When the second amino acid residue in SEQ ID NO: 2 or 16 is replaced with an alanine residue, it exhibits superior binding activity to the receptor-binding domain (RBD) of SARS-CoV-2 of the type in which the 501st amino acid residue is wild-type, such as wild-type SARS-CoV-2, among the wild-type SARS-CoV-2 and mutant SARS-CoV-2 described above.

[0071] Furthermore, one of the features of the peptides according to this disclosure is that the 18th amino acid residue in the amino acid sequence of SEQ ID NO: 2 or 16 is an arginine residue. Because the 18th amino acid residue in the amino acid sequence of SEQ ID NO: 2 or 16 is an arginine residue, the peptides according to this disclosure have excellent binding activity to the receptor-binding domain (RBD) of wild-type SARS-CoV-2 and to the receptor-binding domains (RBDs) of all the mutant SARS-CoV-2 described above.

[0072] Herein, an example of the composition of the peptide relating to the present disclosure is shown in Table 6 below, but the technical scope of the present disclosure is not limited to peptides consisting of the following specific amino acid sequences.

[0073]

[0074] For example, the peptide of this disclosure is a peptide in which the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region includes the following amino acid sequences (h), (i), (j), (k), or (l): (h) the amino acid sequence of SEQ ID NO: 15, (i) the amino acid sequence of SEQ ID NO: 18, (j) the amino acid sequence of SEQ ID NO: 20, (k) the amino acid sequence of SEQ ID NO: 22, or (l) the amino acid sequence of SEQ ID NO: 24, and the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region includes the amino acid sequence of SEQ ID NO: 16.

[0075] Furthermore, the peptides of this disclosure include the following amino acid sequences: (m), (n), (o), or (p) in the first region, where the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 is (m) the amino acid sequence of SEQ ID NO: 4, (n) the amino acid sequence of SEQ ID NO: 6, (o) the amino acid sequence of SEQ ID NO: 7, or (p) the amino acid sequence of SEQ ID NO: 8, and the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region is the amino acid sequence of SEQ ID NO: 2, 3, 5, or 9.

[0076] Unlike the LCB1 peptide (SEQ ID NO: DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER (having three helices)) disclosed in L Cao et al., Science 370, 426-431 (2020), these specific peptides consist of two helices.

[0077] The full-length amino acid sequences of these specific peptides include peptides consisting of one amino acid sequence selected from the group consisting of SEQ ID NOs. 11-14, 17, 19, 21, 23, and 25. Sequence ID 11 (Ce4): DKEWILQKIYEIMRKLDEDGHAEASMRVSDLIYEFMKKD Sequence ID 12 (Ce9): DKEWILQKIYEIMRRLDEEGHAEASMRVSDLIYEFMKKD Sequence ID 13 (Ce41): DKEWILQKIYEIMRRLDEEGHGEASLRVSDLIYEFMKKD Sequence ID 14 (Ce59): DKEWILQKIYEIMRRLDEEGHGEASLRVSDLIYEFMKRD Sequence ID 17 (Ce113): DKEWILQKIYEIMQRLDEEGHGEASLMVSDLIYEFMKRD Sequence ID 19 (Ce172): DKLWILQKIYEIMVRLDEEGHGEASLMVSDLIYEFMKRD Sequence ID 21 (Ce149): DKEWILYKIYEIMVRLDEEGHGEASLMVSDLIYEFMKRD Sequence ID 23 (Ce173): DKLWILQKIYEIMQRLDEEGHGEASLMVSDLIYEFMKRD Sequence ID 25 (Ce174): DKEWILYKIYEIMQRLDEEGHGEASLMVSDLIYEFMKRD

[0078] Furthermore, these specific peptides are known to exhibit excellent binding activity to the receptor-binding domain (RBD) of SARS-CoV-2, as described in International Publication No. 2023 / 282281. Here, binding activity can be determined comprehensively from the binding rate and the degree of dissociation. While there are no particular limitations on the system for evaluating the binding activity of a specific peptide to the receptor-binding domain (RBD) of SARS-CoV-2, a system utilizing surface plasmon resonance (SPR) can be used. For example, the receptor-binding domain (RBD) of SARS-CoV-2 can be immobilized on the surface of a sensor chip, and a sample containing various analytes (peptides) can be brought into contact with the surface. The binding rate can then be measured based on the change in the SPR angle due to the binding of the receptor-binding domain (RBD) of SARS-CoV-2 to the peptide. Alternatively, a buffer without analytes can be flowed through the sensor chip surface with the receptor-binding domain (RBD) of SARS-CoV-2 bound to the peptide, and the degree of dissociation can be measured based on the change in the SPR angle due to the dissociation of the receptor-binding domain (RBD) of SARS-CoV-2 to the peptide.

[0079] Furthermore, the binding rate and dissociation degree between the peptide and the receptor-binding domain (RBD) in SARS-CoV-2 are not limited to systems utilizing surface plasmon resonance (SPR), but may also be measured using systems that analyze biomolecular interactions, such as biolayer interferometry (BLI).

[0080] Furthermore, the peptides relating to this disclosure may be peptides in which at least one amino acid is bound to at least one end of the N-terminus and C-terminus, as long as they have excellent binding activity to the receptor-binding domain (RBD) of SARS-CoV-2. They may also be in the form of fusion peptides formed by fusing with other peptides or proteins, peptides with inserted unnatural amino acids, peptides with tags or labels attached, and peptides with other modifications. For example, fusion peptides include, but are not limited to, those fused with membrane-permeable peptides (CPPs) so that they are introduced into cells upon administration. The peptides relating to this disclosure may be peptides in which at least one amino acid is bound to at least one end of the N-terminus and C-terminus, and fusion peptides formed by fusing with other peptides or proteins, peptides with inserted unnatural amino acids, peptides with tags or labels attached, and peptides with other modifications are also included in the peptides relating to this disclosure as long as they include the peptides relating to this disclosure.

[0081] Figure 1 shows the results of CD spectra measurements for Ce4, Ce9, Ce41, Ce59, Ce113, Ce149, Ce172, Ce173, and Ce174. Figure 1 shows that Ce4, Ce9, Ce41, Ce59, Ce113, Ce149, Ce172, Ce173, and Ce174 all form a helix bundle structure with the first helix in the first region and the second helix in the second region, thus acquiring a stable structure.

[0082] Figure 2 shows the results of the three-dimensional structural analysis of the complex of Ce41 and the receptor-binding domain (RBD) in the UK variant SARS-CoV-2 (B.1.1.7). The analysis revealed that the second lysine residue in the first region and the 15th phenylalanine residue (35th amino acid residue in the overall Ce41) and the 18th lysine residue (38th amino acid residue in the overall Ce41) in the second region form hydrogen bonds. Furthermore, it was found that the second lysine residue in the first region and the 19th aspartic acid residue in the second region (39th amino acid residue in the overall Ce41) form a salt bridge, and these interactions were found to contribute to the stabilization of the helix bundle structure of Ce41.

[0083] The peptide of the present invention described above can be defined as follows: [1] Having a first region containing a first helix and a second region containing a second helix in the direction from the N-terminus to the C-terminus, wherein the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 includes the following amino acid sequences: (a) the amino acid sequence of SEQ ID NO: 1, (b) the amino acid sequence of SEQ ID NO: 20, (c) the amino acid sequence of SEQ ID NO: 18, or (d) an amino acid sequence having 80% or more sequence identity with the amino acid sequences of SEQ ID NO: 1, 20, or 18. The sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 includes the following amino acid sequences: (e) the amino acid sequence of SEQ ID NO: 2, (f) the amino acid sequence of SEQ ID NO: 16, or (g) An amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 or 16, wherein a peptide forms a bond between the amino acid residues within the first five residues from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and the amino acid residues within the first five residues from the C-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, and binds to the receptor-binding domain (RBD) in SARS-CoV-2.

[0084] [2] Having a first region containing a first helix and a second region containing a second helix in the direction from the N-terminus to the C-terminus, the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 includes the following amino acid sequence (a) or (d1): (a) the amino acid sequence of SEQ ID NO: 1, or (d1) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 The sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 includes the following amino acid sequence (e) or (g1): (e) the amino acid sequence of SEQ ID NO: 2, or (g1) A peptide of [1] that has an amino acid sequence having 80% or more sequence identity with the amino acid sequence of Sequence ID No. 2, and which forms a bond between the amino acid residues within the first five residues from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and the amino acid residues within the first five residues from the C-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, thereby binding to the receptor-binding domain (RBD) in SARS-CoV-2.

[0085] [3] A first region containing a first helix and a second region containing a second helix, extending from the N-terminus to the C-terminus, wherein the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 includes the following amino acid sequence: (b) the amino acid sequence of SEQ ID NO: 20, or (d2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 20. The sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 includes the following amino acid sequence: (f) the amino acid sequence of SEQ ID NO: 16, or (g2) A peptide of [1] that has an amino acid sequence having 80% or more sequence identity with the amino acid sequence of Sequence ID No. 16, and which forms a bond between the amino acid residues within the first five residues from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and the amino acid residues within the first five residues from the C-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, thereby binding to the receptor-binding domain (RBD) in SARS-CoV-2.

[0086] [4] A first region containing a first helix and a second region containing a second helix, extending from the N-terminus to the C-terminus, wherein the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 includes the following amino acid sequence (c) or (d3): (c) the amino acid sequence of SEQ ID NO: 18, or (d3) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 18. The sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 includes the following amino acid sequence (f) or (g2): (f) the amino acid sequence of SEQ ID NO: 16, or (g2) A peptide of [1] that has an amino acid sequence having 80% or more sequence identity with the amino acid sequence of Sequence ID No. 16, and which forms a bond between the amino acid residues within the first five residues from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and the amino acid residues within the first five residues from the C-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region, thereby binding to the receptor-binding domain (RBD) in SARS-CoV-2.

[0087] [5] The peptide according to any one of [1] to [4], wherein the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 comprises the following amino acid sequences (h), (i), (j), (k), or (l): (h) the amino acid sequence of SEQ ID NO: 15, (i) the amino acid sequence of SEQ ID NO: 18, (j) the amino acid sequence of SEQ ID NO: 20, (k) the amino acid sequence of SEQ ID NO: 22, or (l) the amino acid sequence of SEQ ID NO: 24, and the sequence of the site in the second region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 comprises the amino acid sequence of SEQ ID NO: 16.

[0088] [6] The peptide according to [5], wherein the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region is the amino acid sequence of SEQ ID NO: 15.

[0089] [7] The peptide according to [5], wherein the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region is the amino acid sequence of SEQ ID NO: 18.

[0090] [8] The peptide according to [5], wherein the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region is the amino acid sequence of SEQ ID NO: 20.

[0091] [9] The peptide according to [5], wherein the sequence of the site in the first region that binds to the receptor-binding domain (RBD) in SARS-CoV-2 consists of the amino acid sequence of SEQ ID NO: 22.

[0092]

[10] The peptide according to [5], wherein the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region is the amino acid sequence of SEQ ID NO: 24.

[0093]

[11] The peptide according to any one of [1] to [4], characterized in that the amino acid residues within the first five N-terminal residues and the amino acid residues within the first five C-terminal residues are a combination of a lysine residue and an aspartic acid residue, a combination of a lysine residue and a glutamic acid residue, a combination of an arginine residue and a glutamic acid residue, or a combination of an arginine residue and an aspartic acid residue.

[0094]

[12] The peptide according to [1] or [2], characterized in that the amino acid sequence of (d) or (d1) is an amino acid sequence in which at least one amino acid residue selected from the group consisting of the 1st, 8th, 11th, and 18th amino acid residues in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid residue.

[0095]

[13] The peptide according to [1] or [2], characterized in that the amino acid sequence of (g) or (g1) is an amino acid sequence in which at least one amino acid residue selected from the group consisting of the second amino acid residue, the 14th amino acid residue, and the 18th amino acid residue in the amino acid sequence of Sequence ID No. 2 is substituted with another amino acid residue.

[0096]

[14] The peptide according to [1] or [2], characterized in that the amino acid sequence of (d) or (d1) is an amino acid sequence in which the 15th arginine residue and the 19th glutamic acid residue in the amino acid sequence of SEQ ID NO: 1 are replaced with a lysine residue and an aspartic acid residue, respectively.

[0097]

[15] The peptide according to [1] or [2], characterized in that the amino acid sequence of (g) or (g1) is an amino acid sequence in which the second glycine residue in the amino acid sequence of SEQ ID NO: 2 is replaced with an alanine residue.

[0098]

[16] The peptide according to [1] or [2], characterized in that the amino acid sequence of (g) or (g1) is an amino acid sequence in which the 18th arginine residue in the amino acid sequence of Sequence ID No. 2 is replaced with a lysine residue.

[0099]

[17] The peptide according to [1] or [2], characterized in that the amino acid sequence of (d) or (d1) is an amino acid sequence in which the 10th tyrosine residue and the 13th methionine residue of the amino acid sequence of Sequence ID No. 1 are further conserved.

[0100]

[18] The peptide according to [1] or [2], characterized in that the amino acid sequence of (g) or (g1) is an amino acid sequence in which the first histidine residue, the ninth serine residue, the tenth aspartic acid residue, and the thirteenth tyrosine residue of the amino acid sequence of Sequence ID No. 2 are further conserved.

[0101]

[19] The peptide according to any one of [1] to [4], characterized in that the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and / or the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region is 18 to 22 amino acid residues.

[0102]

[20] The peptide according to any one of [1] to [4], characterized in that it consists of the first region and the second region, and the total sequence length is 39 amino acid residues.

[0103]

[21] The peptide according to any one of [1] to [4], characterized in that it consists of one amino acid sequence selected from the group consisting of SEQ ID NOs: 11-14, 17, 19, 21, 23 and 25.

[0104]

[22] A peptide having a first region and a second region in the direction from the N-terminus to the C-terminus, wherein the first region contains an amino acid sequence selected from the following (h), (i), (j), (k) or (l): (h) the amino acid sequence of SEQ ID NO: 15, (i) the amino acid sequence of SEQ ID NO: 18, (j) the amino acid sequence of SEQ ID NO: 20, (k) the amino acid sequence of SEQ ID NO: 22, or (l) the amino acid sequence of SEQ ID NO: 24, and the second region contains the amino acid sequence of SEQ ID NO: 16, which binds to the receptor-binding domain (RBD) in SARS-CoV-2.

[0105]

[23] The peptide of

[22] , wherein the first region described above consists of the amino acid sequence of SEQ ID NO: 15.

[0106]

[24] The peptide of

[22] wherein the first region described above consists of the amino acid sequence of SEQ ID NO: 18.

[0107]

[25] The peptide of

[22] , wherein the first region described above consists of the amino acid sequence of SEQ ID NO: 20.

[0108]

[26] The peptide of

[22] , wherein the first region described above consists of the amino acid sequence of SEQ ID NO: 22.

[0109]

[27] The peptide of

[22] , wherein the first region described above consists of the amino acid sequence of SEQ ID NO: 24.

[0110]

[28] A peptide of any of

[22] to

[27] , wherein the second region described above consists of the amino acid sequence of SEQ ID NO: 16.

[0111]

[29] A peptide consisting of the amino acid sequence of Sequence ID No. 17, which binds to the receptor-binding domain (RBD) in SARS-CoV-2.

[0112]

[30] A peptide consisting of the amino acid sequence of Sequence ID No. 19, which binds to the receptor-binding domain (RBD) in SARS-CoV-2.

[0113]

[31] A peptide consisting of the amino acid sequence of Sequence ID No. 21, which binds to the receptor-binding domain (RBD) in SARS-CoV-2.

[0114]

[32] A peptide consisting of the amino acid sequence of Sequence ID No. 23, which binds to the receptor-binding domain (RBD) in SARS-CoV-2.

[0115]

[33] A peptide consisting of the amino acid sequence of Sequence ID No. 25, which binds to the receptor-binding domain (RBD) in SARS-CoV-2.

[0116]

[34] A pharmaceutical composition containing the peptide described in any of [1] to

[33] above as an active ingredient.

[0117]

[35] The pharmaceutical composition according to

[34] , comprising at least one pharmaceutically acceptable carrier.

[0118]

[36] The pharmaceutical composition described in

[34] or

[35] above, for the treatment of COVID-19.

[0119]

[37] A peptide according to any of [1] to

[33] above for use in the treatment of COVID-19.

[0120]

[38] A method for treating COVID-19, comprising administering a therapeutically effective amount of any of the peptides [1] to

[33] above to a patient in need.

[0121]

[39] Use of any of the peptides described in [1] to

[33] above for the manufacture of a pharmaceutical composition for the treatment of COVID-19.

[0122]

[40] In COVID-19, the 339th amino acid residue that constitutes the receptor-binding domain (RBD) is a glycine residue, the 371st amino acid residue is a serine residue, the 373rd amino acid residue is a serine residue, the 375th amino acid residue is a serine residue, the 376th amino acid residue is a threonine residue, the 405th amino acid residue is an aspartic acid residue, the 408th amino acid residue is an arginine residue, the 417th amino acid residue is a lysine residue, the 440th amino acid residue is an asparagine residue, the 446th amino acid residue is a glycine residue, the 452nd amino acid residue is a leucine residue, the 477th amino acid residue is a serine residue, the 478th amino acid residue is a threonine residue, the 484th amino acid residue is a glutamic acid residue, the 493rd amino acid residue is a glutamine residue, the 496th amino acid residue is a glycine residue, and the 498th amino acid residue is a glutamine residue. Wild-type SARS-CoV-2 has an asparagine residue as its first amino acid residue and a tyrosine residue as its 505th amino acid residue; the first mutant SARS-CoV-2 has a tyrosine residue as its 501st amino acid residue; the second mutant SARS-CoV-2 has a lysine residue as its 484th amino acid residue; and the third mutant SARS-CoV-2 has an asparagine residue as its 417th amino acid residue, a lysine residue as its 484th amino acid residue, and a tyrosine residue as its 501st amino acid residue. CoV-2; a fourth variant of SARS-CoV-2 in which the 417th amino acid residue is a threonine residue, the 484th amino acid residue is a lysine residue, and the 501st amino acid residue is a tyrosine residue; a fifth variant of SARS-CoV-2 in which the 452nd amino acid residue is an arginine residue and the 478th amino acid residue is a lysine residue; a sixth variant of SARS-CoV-2 in which the 452nd amino acid residue is an arginine residue and the 484th amino acid residue is a glutamine residue;The seventh variant of SARS-CoV-2 has aspartic acid at amino acid position 339, leucine at amino acid position 371, proline at amino acid position 373, phenylalanine at amino acid position 375, asparagine at amino acid position 417, lysine at amino acid position 440, serine at amino acid position 446, asparagine at amino acid position 477, lysine at amino acid position 478, alanine at amino acid position 484, arginine at amino acid position 493, serine at amino acid position 496, arginine at amino acid position 498, tyrosine at amino acid position 501, and histidine at amino acid position 505; the seventh variant of SARS-CoV-2 has aspartic acid at amino acid position 339 and phenylalanine at amino acid position 371. A pharmaceutical composition, peptide, therapeutic method, or use according to any of

[36] to

[39] above, characterized by being caused by infection with any of the eighth variant SARS-CoV-2, wherein the amino acid residue is a nin residue, the 373rd amino acid residue is a proline residue, the 375th amino acid residue is a phenylalanine residue, the 376th amino acid residue is an alanine residue, the 405th amino acid residue is an asparagine residue, the 408th amino acid residue is a serine residue, the 417th amino acid residue is an asparagine residue, the 440th amino acid residue is a lysine residue, the 477th amino acid residue is an asparagine residue, the 478th amino acid residue is a lysine residue, the 484th amino acid residue is an alanine residue, the 493rd amino acid residue is an arginine residue, the 498th amino acid residue is an arginine residue, the 501st amino acid residue is a tyrosine residue, and the 505th amino acid residue is a histidine residue.

[0123]

[41] The pharmaceutical composition for treating COVID-19 according to

[36] , characterized in that it is an aqueous solution in which the above peptide is dissolved.

[0124] 2. Method for producing the peptide that binds to the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) The peptide according to the present disclosure described above is approximately 39 amino acid residues in length and can be easily produced by general chemical synthesis methods. For example, examples of peptide synthesis methods include solid-phase synthesis and liquid-phase synthesis. Examples of solid-phase synthesis methods include using Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as the protecting group for the amino group. Furthermore, the above-mentioned peptide can be synthesized using a commercially available peptide synthesizer that applies solid-phase synthesis.

[0125] Furthermore, it is also possible to synthesize the DNA encoding the above-mentioned peptide and express the peptide in a host cell into which the DNA has been introduced. Host cells that can be used include yeast, bacteria such as Escherichia coli, insect cells, animal cells, and plant cells. Alternatively, the above-mentioned peptide can be synthesized in vitro using a cell-free protein synthesis system. However, it is preferable to produce the peptide according to this disclosure by chemical synthesis, for the following reasons: the peptide according to this disclosure is approximately 39 amino acid residues in length, making it easy to synthesize using general chemical synthesis methods; and manufacturing under GMP (Good Manufacturing Practice) compliant conditions is relatively easy with chemical synthesis.

[0126] The method for producing the peptide of the present invention is described in International Publication No. 2023 / 282281, and can be produced in accordance with said description.

[0127] The peptide of the present invention preferably forms a homodimer. The peptide forms a homodimer with the RBD-binding surface facing outward and binds to two RBDs. That is, the peptide of the present invention forms a homodimer that forms a stable helical bundle structure and binds strongly to RBDs on both sides of the dimer. Figure 8B shows the results of an actual analysis of the structure in which peptide Ce172 forms a homodimer with the RBD-binding surface facing outward and binds to two RBDs.

[0128] 3. Salt Forms of Peptides Binding to the Receptor Binding Domain (RBD) in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) of the Present Invention The salts of peptides binding to the receptor binding domain (RBD) in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) of the Present Invention are pharmaceutically acceptable salts, i.e., salts with pharmaceutically acceptable acids or bases. Examples of pharmaceutically acceptable salts include inorganic salts such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphate, hydrobromic acid, or nitric acid; organic salts such as tartaric acid, acetic acid, citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or p-toluenesulfonic acid; metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts; inorganic salts such as ammonium salts; and organic amine salts such as triethylamine salts and guanidine salts. Among these, sodium salts and potassium salts are preferred, with sodium salts being particularly preferred.

[0129] The sodium and potassium salts of the peptide that binds to the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) according to the present invention, particularly the sodium salt, exhibit the following excellent properties.

[0130] Solid Stability: It is resistant to decomposition even when stored for a long time. When comparing the peak area obtained by HPLC analysis before and after storage at 70°C for one week, the peak area after storage is 50% or more, preferably 55% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and especially preferably 95% or more of the peak area before storage. When comparing the peak area obtained by HPLC analysis before and after storage, the ratio of the peak area after storage to the peak area before storage is sometimes called purity. That is, when comparing the peak area obtained by HPLC analysis before and after storage, if the peak area after storage is 95% or more of the peak area before storage, it is said to have solid stability that maintains a purity of 955 or more even after storage.

[0131] The dissolution rate increases. When added to PBS solution at a concentration of 1 mg / mL, it dissolves completely within 10 minutes, preferably within 5 minutes, and more preferably within 3 minutes.

[0132] When the peptide concentration is calculated by adding physiological saline to a final solubility concentration of 50 mg / mL and measuring the absorbance of the supernatant, the peptide concentration is 30 mg / mL or higher, preferably 33 mg / mL or higher, more preferably 35 mg / mL or higher, and even more preferably 36 mg / mL or higher.

[0133] Peptide content: When the peptide concentration is measured by UV measurement at 280 nm, the peptide concentration is 70% or more, preferably 80% or more, and more preferably 85% or more.

[0134] Structural characteristics: It forms a helical bundle structure, and the 222 / 208 ratio of the CD spectrum is 1.1 or greater.

[0135] Furthermore, when the solution is dissolved in 20 mM Tris-HCl (pH 8.0) to a concentration of 0.1 mg / mL and the CD spectrum is measured, the proportion of helical structures analyzed from the CD spectrum measurement results is 80% or more, preferably 82% or more.

[0136] When evaluating the hygroscopicity measured using a hygroscopic dynamic water vapor adsorption measuring device, the weight increase due to moisture absorption at a relative humidity of 90% or less is 50% or less, preferably 45% or less, and more preferably 40% or less.

[0137] The peptide salt that binds to the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) according to the present invention can be produced by creating a trifluoroacetate (TFA) salt and then exchanging it with a sodium or potassium salt.

[0138] 4. Pharmaceutical Compositions, Therapeutic Drugs The peptide salts described above can be used as pharmaceutical compositions. As described above, the peptide salts described above have remarkably high solubility in ultrapure water or buffer solutions and have excellent binding activity to the receptor-binding domain (RBD) of SARS-CoV-2. Therefore, the peptide salts described above can be used as pharmaceutical compositions for the treatment and / or prevention of Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2. In other words, the present disclosure is the use of the peptide salts described above in the manufacture of pharmaceutical compositions for the treatment and / or prevention of Coronavirus Disease 2019 (COVID-19)

[0139] The target population for such pharmaceutical compositions is not particularly limited, but includes animals, such as mammals including humans, monkeys, mice, rats, hamsters, rabbits, guinea pigs, cattle, pigs, dogs, horses, cattle, goats, and sheep, and is preferably humans. The route of administration of the pharmaceutical compositions of this disclosure may be either oral or parenteral. The dosage of the pharmaceutical compositions of this disclosure can be formulated in various ways depending on factors such as the formulation method, administration method, administration time, age, weight, sex, pathological condition, excretion rate, and response sensitivity of the target population. The timing of administration of the pharmaceutical compositions of this disclosure is not particularly limited and may be arbitrary. It may be administered before, after, or between meals. Furthermore, the duration of administration / intake is not particularly limited. For example, the dosage varies depending on symptoms, age, weight, etc., but typically, for oral administration, it is about 0.01 mg to 1000 mg per day for adults, and these can be administered in one dose or in several divided doses. In addition, for parenteral administration, approximately 0.01 mg to 1000 mg can be administered by subcutaneous injection, intramuscular injection, or intravenous injection.

[0140] The pharmaceutical compositions of this disclosure can be manufactured in unit dose form by formulation using pharmaceutically acceptable carriers and / or excipients by methods normally practiced by persons with ordinary skill in the art, or by encapsulating them in multi-dose containers. Examples of carriers and excipients include lactose and magnesium stearate. As aqueous solutions for injection, physiological saline, isotonic solutions containing glucose or other adjuvants can be used, and may be used in combination with appropriate solubilizers, such as alcohol, polyalcohols such as propylene glycol, and nonionic surfactants. As oily solutions, sesame oil and soybean oil can be used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.

[0141] The pharmaceutical compositions of this disclosure can be formulated into any desired dosage form depending on the method of administration. The pharmaceutical compositions of the present invention can be administered by intravenous injection, intraperitoneal, oral, nasal, mucosal, intramuscular or subcutaneous, intranasal, intratracheal, skin, transdermal or intradermal routes. The administration form is not limited, but examples include tablets, granules, sprays, capsules, syrups, emulsions, suppositories, injections, ointments, tapes, etc.

[0142] The pharmaceutical compositions of this disclosure can be manufactured in accordance with known methods, such as those described in the Japanese Pharmacopoeia (JP), the United States Pharmacopeia (USP), or the European Pharmacopoeia (EP). Furthermore, they can be used in combination with known or future-discovered pharmaceuticals for the treatment and / or prevention of COVID-19 (including mRNA vaccines), or pharmaceuticals for the treatment and / or prevention of other diseases.

[0143] The present invention will be specifically described by the following embodiments, but the present invention is not limited to these embodiments.

[0144] Example 1: Salt Formations of Ce41 As a representative peptide that strongly binds to the receptor-binding domain (RBD) of the SARS-CoV-2 spike prior to the omicron-type mutation, which causes a great many mutations in the RBD, Ce41 was used to prepare sodium salt, potassium salt, calcium salt, magnesium salt, and acetate salt, and their dissolution rates and solid stability were evaluated. As a result, it was confirmed that the sodium salt was the most superior developed salt form in terms of dissolution rate and solid stability, as follows.

[0145] The dissolution rates of the peptide Ce41 were evaluated after preparing sodium, potassium, calcium, magnesium, and acetate salts. Ce41 peptide was added to D-PBS(-) solution under conditions where the final concentration of each salt was 1 mg / mL. The peptide concentration in the supernatant was calculated from the absorbance at 280 nm after 3, 10, and 30 minutes. The basic salts, sodium, potassium, calcium, and magnesium, dissolved completely within 3 minutes of addition to the solution. On the other hand, the acidic acetate salt dissolved significantly less efficiently than the basic metal salts, and did not completely dissolve even after 30 minutes. This is thought to be because Ce41 is an acidic peptide with an isoelectric point around pH 4, and the anionic form formed by the basic salts is advantageous for dissolution in aqueous solutions. The results of the dissolution rate evaluation were obtained (Figure 3).

[0146] The solid stability of the peptide Ce41 was evaluated by measuring the area ratio (ΔArea(%)) of the HPLC peaks before and after one week of storage at 70°C for one week. The results are shown in Table 7. These conditions were set to simulate the heat load when each salt is stored at room temperature for approximately two years.

[0147]

[0148] In Table 7, the differences in the initial concentrations of each salt reflect the different values ​​due to the raw materials used in the preparation of each salt, i.e., the purity of the TFA salts from different lots. From the results of the initial values ​​and the purity specifications after one week of storage, it was confirmed that the basic Na, K, Ca, and Mg salts have superior solid stability compared to the acidic acetate salts. Furthermore, when comparing the basic salts with each other, the Na salt was found to have the best stability.

[0149] Example 2: High activity of Ce41 has been confirmed against the salt forms of Ce172, including Wuhan variants and later mutant viruses. However, the binding ability of Ce41 tends to weaken against Omicron BA.1 and BA.2 variants and later, which have many mutations in the receptor-binding domain (RBD). Ce172, which strongly binds to the receptor-binding domain (RBD) of various Omicron variant viruses and exhibits strong efficacy against these variant viruses, has the same sequence as Ce41 except for four amino acids and has a similar isoelectric point. Therefore, it was predicted that Ce172 and Ce41 would have similar physical properties (Figure 4). For this reason, we prepared mainly the Na salt and K salt of Ce172 and performed various physical property measurements such as characterization, solubility evaluation, solid stability evaluation, and hygroscopicity evaluation of these salt forms.

[0150] After HPLC purification of the TFA salt of peptide Ce172, sodium (Na) and potassium (K) salts were prepared, respectively. HPLC analysis of each prepared salt revealed no increase in the total amount of impurities after salt exchange. However, it was confirmed that the sodium and potassium salts exhibited different impurity profiles (Figure 5). This is presumed to be because, in the case of the sodium salt, some of the impurities around the HPLC retention time of 23.0–24.0 min decreased during the salt exchange process.

[0151] Peptide concentrations were measured using 280 nm UV light, and volatile component content was determined by differential thermal analysis (TGA) and salt content by ion chromatography (IC). The content of each component in the Na salt and K salt was then calculated (Table 8).

[0152]

[0153] While the total mass balance (Total value in Table 8) for the Na salt was roughly in agreement with the weighed value at 98.1%, the K salt was 90.3%, suggesting the presence of an unknown impurity that does not absorb UV light.

[0154] Analysis of each salt after salt exchange using a circular dichroism spectroscopy (CD) device revealed that both the sodium and potassium salts of peptide Ce172 showed almost identical CD spectral results to the TFA salt, confirming that they form the same helical bundle structure under these salt conditions (Figure 6). The proportion of secondary structure was calculated from these CD spectral results (Table 9).

[0155]

[0156] Table 9 shows the results of CD spectroscopy measurements after dissolving three types of salts in 20 mM Tris-HCl (pH 8.0) at a concentration of 0.1 mg / mL. The proportions of secondary structures calculated from these CD spectra show good agreement with the structure of peptide Ce172 (Figure 7). The structure of Ce172 shown in Figure 7 was analyzed by X-ray crystallography.

[0157] This type of peptide has also been confirmed by analyzing the structure in which it forms a homodimer and binds to the receptor-binding domain (RBD) (Figure 8). Specifically, the 222 / 208 ratio in the CD spectrum of Figure 6 is greater than 1.1, and the high-resolution structural analysis confirmed that it adopts a helical bundle structure formed by four helices, which is in good agreement with the CD spectrum result indicating the formation of a typical helical bundle. Furthermore, it was structurally confirmed that by forming this homodimer, the dimer strongly binds to the receptor-binding domain (RBD) on both sides (Figure 8).

[0158] The sodium and potassium salts of Ce172 were added to physiological saline to a final concentration of 50 mg / mL, and the concentration of the dissolved peptide was calculated by measuring the absorbance of the supernatant (Table 10).

[0159]

[0160] Solubility evaluation of sodium and potassium salts in physiological saline: Both sodium and potassium salts showed similar saturation solubility of 36 mg / mL or higher in physiological saline, confirming that they both possess high solubility. The pH of the solutions was found to be more basic with potassium salt compared to sodium salt. This is likely due to the higher ionic content of potassium salt, as shown in Table 8.

[0161] The solid stability of the sodium and potassium salts of Ce172 was measured. HPLC measurements were performed one week after each measurement under conditions of 25°C, 25°C under 1000 lux of light irradiation, 40°C, and 70°C. The solid stability was then measured by comparing the results with the initial HPLC measurements.

[0162] Solid stability tests of the sodium salt of Ce172 were conducted, and HPLC measurements were taken at the initial stage and one week after each experimental condition (Figure 9). The results showed a slight increase in peaks near 27.3 min and 28.0 min under 25°C and 1000 lux of light irradiation, and under 70°C conditions. However, no significant changes were observed.

[0163] For the sodium salt of Ce172, the change in the area of ​​the main peak in HPLC was calculated after leaving it under each condition for one week (Table 11).

[0164]

[0165] Regarding the sodium salt of Ce172, a 4.4% reduction in the main peak was observed under conditions of light irradiation at 25°C for one week. However, even under the harsh condition of 70°C (a week at 70°C is equivalent to the heat load of storage at room temperature for two years), the reduction in the main peak was limited to 2.2%.

[0166] In solid stability experiments of the K salt of Ce172, HPLC measurements were taken at the initial stage and one week later under each experimental condition (Figure 10). The results showed no significant changes even under 1000 lux of light irradiation at 25°C. However, under 70°C conditions, a slight increase in peaks was observed in the ranges of 23.0 min–35.0 min and 27.0 min–28.6 min.

[0167] The change in the area of ​​the main peaks in HPLC was calculated for the potassium salt of Ce172 after being left under each condition for one week (Table 12). For the potassium salt of Ce172, even under the condition of being irradiated with light at a temperature of 25°C for one week, the decrease in the four main peaks was limited to 2.5%. However, under the harsh condition of 70°C (a period of one week under this condition is equivalent to the heat load of storage at room temperature for two years), a decrease in the main peaks of 6.2% was observed.

[0168]

[0169] The hygroscopic properties of Na salt and K salt were evaluated using a dynamic water vapor adsorption measuring device (Figure 11). As a result, the weight increase of Na salt at relative humidity below 90% was measured to be 36%, while that of K salt was measured to be 44%, confirming that Na salt is a less hygroscopic salt form.

[0170] When comparing the Na salt and K salt of Ce172 in terms of various physical properties, the Na salt is superior (Table 13).

[0171]

[0172] The peptide salt according to the present invention can be used as a composition to prevent infection of cells with wild-type SARS-CoV-2 and mutant SARS-CoV-2 in the body.

[0173] Sequence IDs 1-25 Synthetic Peptides All publications, patents and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A sodium or potassium salt of a peptide having a first region containing a first helix and a second region containing a second helix, wherein the first and second regions include a site that binds to the receptor-binding domain (RBD) of SARS-CoV-2, and a bond is formed between amino acid residues within the first five residues from the N-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) of SARS-CoV-2 in the second region and amino acid residues within the first five residues from the C-terminus of the sequence of the site that binds to the receptor-binding domain (RBD) of SARS-CoV-2.

2. The sodium or potassium salt of the peptide according to claim 1, wherein the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region comprises the amino acid sequence of (a), (b), (c), or (d) below, and the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region comprises the amino acid sequence of (e), (f), or (g) below; (a) the amino acid sequence of SEQ ID NO: 1, (b) the amino acid sequence of SEQ ID NO: 20, (c) the amino acid sequence of SEQ ID NO: 18, or (d) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, 20, or 18; (e) the amino acid sequence of SEQ ID NO: 2, (f) the amino acid sequence of SEQ ID NO: 16, or (g) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 or 16.

3. The sodium or potassium salt of the peptide according to claim 1, wherein the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region comprises the amino acid sequence of (b) or (d2) below, and the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region comprises the amino acid sequence of (f) or (g2) below; (b) the amino acid sequence of SEQ ID NO: 20, or (d2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 20; (f) the amino acid sequence of SEQ ID NO: 16, or (g2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:

16.

4. The sodium or potassium salt of the peptide according to claim 1, wherein the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region comprises the amino acid sequence of (c) or (d3) below, and the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region comprises the amino acid sequence of (f) or (g2) below; (c) the amino acid sequence of SEQ ID NO: 18, or (d3) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 18; (f) the amino acid sequence of SEQ ID NO: 16, or (g2) an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:

16.

5. The sodium or potassium salt of the peptide according to any one of claims 1 to 4, characterized in that the amino acid residues within the first five N-terminal residues and the amino acid residues within the first five C-terminal residues are a combination of a lysine residue and an aspartic acid residue, a combination of a lysine residue and a glutamic acid residue, a combination of an arginine residue and a glutamic acid residue, or a combination of an arginine residue and an aspartic acid residue.

6. The sodium or potassium salt of the peptide according to claim 2, characterized in that the amino acid sequence of (d) above is an amino acid sequence in which at least one amino acid residue selected from the group consisting of the 1st, 8th, 11th, and 18th amino acid residues in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid residue.

7. The sodium or potassium salt of the peptide according to claim 2, characterized in that the amino acid sequence of (g) above is an amino acid sequence in which at least one amino acid residue selected from the group consisting of the 2nd amino acid residue, the 14th amino acid residue, and the 18th amino acid residue in the amino acid sequence of SEQ ID NO: 2 is substituted with another amino acid residue.

8. The sodium or potassium salt of the peptide according to (d) above, characterized in that the amino acid sequence of the amino acid sequence of SEQ ID NO: 1 is an amino acid sequence in which the 15th arginine residue and the 19th glutamic acid residue are replaced with a lysine residue and an aspartic acid residue, respectively.

9. The sodium or potassium salt of the peptide according to claim 2, characterized in that the amino acid sequence of (g) above is an amino acid sequence in which the second glycine residue in the amino acid sequence of SEQ ID NO: 2 is replaced with an alanine residue.

10. The sodium or potassium salt of the peptide according to claim 2, characterized in that the amino acid sequence of (g) above is an amino acid sequence in which the 18th arginine residue in the amino acid sequence of SEQ ID NO: 2 is replaced with a lysine residue.

11. The sodium or potassium salt of the peptide according to claim 2, characterized in that the amino acid sequence of (d) above is an amino acid sequence in which the 10th tyrosine residue and the 13th methionine residue of the amino acid sequence of SEQ ID NO: 1 are further conserved.

12. The sodium or potassium salt of the peptide according to claim 2, characterized in that the amino acid sequence of (g) above is an amino acid sequence in which the first histidine residue, the ninth serine residue, the tenth aspartic acid residue, and the thirteenth tyrosine residue of the amino acid sequence of SEQ ID NO: 2 are further conserved.

13. The sodium or potassium salt of the peptide according to any one of claims 1 to 4, characterized in that the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the first region and / or the sequence of the site that binds to the receptor-binding domain (RBD) in SARS-CoV-2 in the second region is 18 to 22 amino acid residues.

14. A sodium or potassium salt of the peptide according to any one of claims 1 to 4, characterized in that it comprises the first region and the second region described above, and the total sequence length is 39 amino acid residues.

15. A sodium or potassium salt of the peptide according to any one of claims 1 to 4, characterized in that it consists of one amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 14.

16. A sodium or potassium salt of a peptide that binds to the receptor-binding domain (RBD) in SARS-CoV-2, having a first region and a second region in the direction from the N-terminus to the C-terminus, wherein the first region contains an amino acid sequence selected from the following (h), (i), (j), (k), or (l): (h) the amino acid sequence of SEQ ID NO: 15, (i) the amino acid sequence of SEQ ID NO: 18, (j) the amino acid sequence of SEQ ID NO: 20, (k) the amino acid sequence of SEQ ID NO: 22, or (l) the amino acid sequence of SEQ ID NO: 24, and the second region contains the amino acid sequence of SEQ ID NO:

16.

17. Sodium or potassium salt of a peptide that binds to the receptor-binding domain (RBD) of SARS-CoV-2, comprising the amino acid sequence of SEQ ID NOs. 17, 19, 21, 23, or 25.

18. The sodium or potassium salt of the peptide according to claim 1, 2, or 16, wherein its solubility in physiological saline is 35 mg / mL or more.

19. A sodium or potassium salt of the peptide according to claim 1, 2, or 16, which has solid stability that maintains a purity of 85% or more after being stored at 70°C for one week.

20. The sodium or potassium salt of the peptide according to claim 1, 2, or 16, which forms a homodimer that forms a stable helical bundle structure and strongly binds to RBD on both sides of the dimer.

21. A pharmaceutical composition containing a sodium or potassium salt of the peptide described in claim 1, 2, or 16 as an active ingredient.

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