Bifunctional peptides with mucoadhesive and virus-binding properties

Abstract

The present invention provides novel substances that are useful for the preventive treatment of viral diseases, and ideally, for avoiding viral infections. The bifunctional peptide of the present invention has a virus-binding site 2 and a mucin-binding site 1 covalently linked to the virus-binding site 2. The virus-binding site 2 is selected from peptides that bind to SARS-CoV-2, influenza A, influenza B, rhinovirus, or other enteroviruses, human parainfluenza viruses, and / or metaneuviruses. The mucin-binding site 1 is selected from lectins such as trefoil factor 3.

Description

[Technical field]

[0001] The present invention relates to a bifunctional peptide as described in the preamble of claim 1, to the medical use of said peptide as described in the preamble of claim 9, to a medicament containing said peptide as described in the preamble of claim 11 and to a method for producing said peptide as described in the preamble of claim 13. [Background technology]

[0002] The ongoing SARS-CoV-2 pandemic has illustrated that new and frequently emerging viral pathogens are an ever-present threat to humanity, and that rapid and specific measures are required to contain the widespread spread of viruses. Even with the rapid promotion of hygiene measures to blunt viral transmission, vaccines and antiviral therapies are the main countermeasures against infectious diseases. However, vaccines can take years to develop and test. Antiviral therapies offer a quick solution to treat already infected individuals. The group of antiviral drugs is dominated by small organic compounds rather than large molecules such as proteins and peptides. However, the development of novel small molecule drugs is often chemically complex and challenging. This makes it difficult to prepare a wide variety of different compound analogs for screening. Moreover, the emergence of resistance is an increasingly big problem, as is evident with antiviral drugs against influenza such as oseltamivir and amantadine.

[0003] To date, preventive treatment is not available as an additional measure against the SARS-CoV-2 pandemic, and many potential benefits are being missed.

[0004] Werner Hoffmann (Non-Patent Document 1) describes a heterodimer of trefoil factor (TFF3) and IgG-Fc binding protein (FCGBP). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hoffmann, Werner. “Trefoil factor family(TFF) peptides and their different roles in the mucosal innate immune defense and more: An update.” Current Medicinal Chemistry 28.36 (2021): 7387-7399. [Non-Patent Document 2] Bhatia, Sumati, et al. “Linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection in vitro and in vivo.” Biomaterials 138 (2017): 22-34.2. [Non-Patent Document 3] Braga Emidio, Nayara, et al. “Chemical Synthesis of TFF3 Reveals Novel Mechanistic Insights and a Gut-Stable Metabolite.” Journal of Medicinal Chemistry 64.13 (2021): 9484-9495. [Non-Patent Document 4] Bui, Vuong N., et al. “Pathogenicity of an H5N1 avian influenza virus isolated in Vietnam in 2012 and reliability of conjunctival samples for diagnosis of infection.” Virus Research 179 (2014): 125-132. [Non-Patent Document 5] Caban, Madelyn, et al. “Cross-protective antibodies against common endemic respiratory viruses.” Nature Communications 14.1 (2023): 798. Non-Patent Document 6 Han, Dong P., Adam Penn-Nicholson, and Michael W. Cho. “Identification of critical determinants on ACE2 for SARS-CoV entry and development of a potent entry inhibitor.” Virology 350.1 (2006): 15-25. Non-Patent Document 7 Hoffmann, Markus, et al. “SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.” Cell 181.2 (2020): 271-280. Non-Patent Document 8 Hoffmann, Miriam, Nicole L. Snyder, and Laura Hartmann. “Polymers Inspired by Heparin and Heparan Sulfate for Viral Targeting.” Macromolecules 55.18 (2022): 7957-7973. Non-Patent Document 9 Hoffmann, Werner. “Trefoil factor family (TFF) peptides and their different roles in the mucosal innate immune defense and more: An update.” Current Medicinal Chemistry 28.36 (2021): 7387-7399. [Non-Patent Document 10] Hrebik, Dominik, et al. “ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating.” Proceedings of the National Academy of Sciences 118.19 (2021): e2024251118. [Non-Patent Document 11] Issmail, Leila, et al. “Prefusion-specific antibody-derived peptides trivalently presented on DNA-nanoscaffolds as an innovative strategy against RSV entry.” Frontiers in Virology 2 (2022). [Non-Patent Document 12] Jarva, Michael A., et al. “Trefoil factors share a lectin activity that defines their role in mucus.” Nature Communications 11.1 (2020): 1-9. [Non-Patent Document 13] Jerabek-Willemsen M., et al. “MicroScale Thermophoresis: Interaction analysis and beyond.” Journal of Molecular Structure 1077 (2014): 101-113. [Non-Patent Document 14] Khan, Abdul Ghafoor, et al. “Human rhinovirus type 54 infection via heparan sulfate is less efficient and strictly dependent on low endosomal pH.” Journal of Virology 81.9 (2007): 4625-4632. [Non-Patent Document 15] Madsen, Anders, et al. “Human antibodies targeting influenza B virus neuraminidase active site are broadly protective.” Immunity 53.4 (2020): 852-863. [Non-Patent Document 16] McLellan, Jason S., et al. “Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus.” science 342.6158 (2013): 592-598. [Non-Patent Document 17] Memczak, Henry, et al. “Anti-hemagglutinin antibody derived lead peptides for inhibitors of influenza virus binding.” PLoS One 11.7 (2016): e0159074. [Non-Patent Document 18] Sarto, Carolina, et al. “Atomistic insight into the essential binding event of ACE2-derived peptides to the SARS-CoV-2 spike protein.” Biological Chemistry 403.5-6 (2022): 615-624. [Non-Patent Document 19] Smith, Thomas J., et al. “Neutralizing antibody to human rhinovirus 14 penetrates the RECEPTOR-binding canyon.” Nature 383.6598 (1996): 350-354. [Non-Patent Document 20] Thim, L., F. Madsen, and SS Poulsen. “Effect of trefoil factors on the viscoelastic properties of mucus gels.” European Journal of Clinical Investigation 32.7 (2002): 519-527. [Non-Patent Document 21] Zhu, Qing, et al. “A highly potent extended half-life antibody as a potential RSV vaccine surrogate for all infants.” Science Translational Medicine 9.388 (2017): eaaj1928. [Non-Patent Document 22] Zuo, Teng, et al. “Comprehensive analysis of antibody recognition in convalescent humans from highly pathogenic avian influenza H5N1 infection.” Nature Communications 6.1 (2015): 8855. Summary of the Invention [Problem to be solved by the invention]

[0006] One object of the present invention is to provide novel agents useful in the prophylactic treatment of viral diseases, and ideally, to prevent viral infection. [Means for solving the problem]

[0007] This object is achieved by a peptide with the claimed components of claim 1. Such a peptide is a bifunctional peptide having a virus binding site and a mucin binding site covalently linked to said virus binding site. The bond between said virus binding site and said mucin binding site may be a direct covalent bond or may be linked via a linker.

[0008] The virus binding sites are SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63, or is the peptide which is 95% or more identical, particularly 96% or more identical, particularly 97% or more identical, particularly 98% or more identical, particularly 99% or more identical, and particularly 100% identical.

[0009] SEQ ID NOs: 2-13 and 23-43 relate to virus-binding sites highly suitable for binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SEQ ID NOs: 2, 3 and 23-41 relate to linear virus-binding peptides, whereas SEQ ID NOs: 4-13, 42 and 43 relate to partially cyclized peptides, so-called stapled peptides. Stapled peptides have at least one bridge between two amino acids that are not directly consecutive (but are adjacent). The bridge serves to stabilize the respective peptide by acting like a clasp. The bridge may be established by two side chains at the respective amino acid residue or by a chemical moiety that is covalently linked to the respective amino acid residue in addition to the normal side chain of the respective amino acid residue. In the case of SEQ ID NOs: 42 and 43, the bridge is a disulfide bridge formed between two cysteine ​​residues.

[0010] SEQ ID NO: 48 describes a peptide highly suitable for binding to influenza A viruses, particularly strains H1, H3 and H7. This peptide targets the hemagglutinin (HA protein) of influenza A viruses and is described in the papers by Memczak et al. and Miriam Hoffmann et al.

[0011] SEQ ID NOs: 49 and 50 describe peptides highly suitable for binding to influenza A viruses (specifically H5N1 strain clade 2.3.4, avian strains), which target the hemagglutinin (HA protein) of influenza A viruses and are derived from the sequence of the antibody 65C6 described in Zuo et al.

[0012] SEQ ID NOs: 51 and 52 describe peptides highly suitable for binding to influenza A viruses (specifically H5N1 strain clade 2.3.2.1, avian strains) that target a specific sequence of the hemagglutinin (HA protein) commonly found in the HA protein of variants of this strain, such as the sequence of the HA protein described in Bui et al.

[0013] SEQ ID NOs: 53 and 54 describe peptides highly suitable for binding to influenza B virus, which target the neuraminidase of influenza B virus and are described in Madsen et al.

[0014] SEQ ID NOs: 55 and 56 describe peptides highly suitable for binding to rhinoviruses (RV) and other enteroviruses (EV), derived from anti-RV antibodies described in Hrebik et al., Khan et al. and Smith et al.

[0015] SEQ ID NO:57 describes a peptide highly suitable for binding to human parainfluenza viruses (HPIV), particularly HIPV1 and HIPV3 strains, which targets the fusion protein (F protein) of HPIV and was derived from the anti-HPIV antibody described in Caban et al.

[0016] SEQ ID NOs: 58, 59 and 60 describe peptides highly suitable for binding to metaneuvirus (MPV) and respiratory syncytial virus (RSV), particularly subtype A and / or subtype B. These peptides target the fusion protein (F protein) of MPV and RSV. These peptides were derived from anti-HPIV antibodies and are described in Caban et al.

[0017] SEQ ID NOs: 61, 62 and 63 describe peptides highly suitable for binding to RSV, particularly subtype A and / or subtype B. These peptides also target the fusion protein (F protein) of RSV and are described in Issmail et al., Zhu et al. and McLellan et al.

[0018] The mucin binding site contains or is a peptide that is 95% or more identical, particularly 96% or more identical, particularly 97% or more identical, particularly 98% or more identical, particularly 99% or more identical, particularly 100% identical to SEQ ID NO:1 (modified trefoil factor 3, TFF3(C57K)), SEQ ID NO:14 (native TFF3), SEQ ID NO:20 (native TFF1), SEQ ID NO:21 (native TFF2) or SEQ ID NO:22 (Jacalin). These lectins are highly suitable for targeting the intestinal mucosa of patients. TFF3 is a protein that is naturally produced in the airways (Thim et al. 2002).

[0019] The claimed peptides are capable of enhancing the natural barrier function of respiratory mucus. Respiratory mucus is the first physical barrier encountered by respiratory pathogens approaching their target cells in the respiratory epithelium. Mucus has high viscosity and bacterial binding properties and is an important part of the human body's bacterial defense system, effectively removing adsorbed pathogens via ciliary movement. Using the claimed peptides, it is possible to enhance mucus by increasing its antibacterial properties through the introduction of binding sites for viral pathogens (mucus strengthening). This allows residual pathogens to be removed before they reach the epithelial cell layer, thus preventing infection. Thus, the present approach is to enhance and support the antiviral effect of mucus with modular biomimetic peptides.

[0020] Mucus strengthening is a novel concept for protecting humans against respiratory infections. This approach, combined with other (non-)pharmacological countermeasures, can reduce the risk of infection in uninfected individuals and the risk of transmission and worsening of disease in already infected individuals.

[0021] The claimed peptide constitutes the core of a tunable peptide-based system of molecules capable of attaching to airway mucins and specifically absorbing viruses. Both parts of the peptide can be adapted to respond to rapidly changing conditions. The biomimetic approach reinforces the inherent antiviral properties of mucosa and is likely to have low side effects since the peptide components are derived from human proteins. The bifunctional peptide also captures viruses released from infected cells, reducing viral titers and shedding into the environment, making it therapeutically useful in infected patients. Finally, the approach can be used in conjunction with established antiviral countermeasures to synergistically help reduce the risk of infection and disease.

[0022] The modular composition of the claimed peptides allows both functional sites of the peptide to be tailored for a desired application.

[0023] As a linker for establishing a covalent bond between the mucin binding site and the virus binding site, a polyethylene glycol (PEG) linker or an amino acid linker containing 1 to 20 amino acids, particularly 2 to 19 amino acids, particularly 3 to 18 amino acids, particularly 4 to 17 amino acids, particularly 5 to 16 amino acids, particularly 6 to 15 amino acids, particularly 7 to 14 amino acids, particularly 8 to 13 amino acids, particularly 9 to 12 amino acids, particularly 10 to 11 amino acids, is suitable. Dipeptides such as GT dipeptide are very suitable linkers. Linkers containing an amino acid sequence that is 95% or more identical to SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ ID NO: 47, particularly 96% or more identical, particularly 97% or more identical, particularly 98% or more identical, particularly 99% or more identical, particularly 100% identical, or consisting of only the amino acid sequence, are also very suitable linkers. SEQ ID NO: 44 is an example of a flexible linker. SEQ ID NO: 45 is an example of a rigid linker. SEQ ID NO: 46 represents a linking peptide derived from a chemical-enzymatic coupling reaction by sortase A. SEQ ID NO: 47 represents a linking peptide derived from a chemical-enzymatic coupling reaction by transglutaminase. The peptide of the virus binding site or the mucin binding site is bound to a glutamic acid residue (Q7) by a γ-glutamic acid-ε-lysine isopeptide bond.

[0024] In one embodiment, the mucin-binding site comprises a glycotope-binding domain and binds to N-acetylglucosamine-α-1,4-galactose (GlcNAc-α-1,4-Gal) disaccharide with an affinity of 150 μM or more, particularly 100 μM or more, particularly 75 μM or more, particularly 50 μM or more, particularly 25 μM or more, particularly within the range of 25 μM to 150 μM, particularly 50 μM to 125 μM, particularly 75 μM to 100 μM. The GlcNAc-α-1,4-Gal disaccharide is a glycotope that is specifically present in mucins such as mucin 2 (Muc2), mucin 5AC (Muc5AC), and mucin 6 (Muc6). As an example, native trefoil factor 3 (TFF3) has a binding affinity for the GlcNAc-α-1,4-Gal disaccharide of 56 μM (Jarva et al., 2020).

[0025] In one embodiment, the virus binding site is covalently attached to the mucin binding moiety at amino acid 57 of the mucin binding site. In one embodiment, amino acid 57 is a cysteine ​​residue. In one embodiment, amino acid 57 is a lysine residue. In one embodiment, the virus binding site is covalently attached to the mucin binding moiety at the C-terminus of the mucin binding site. Notably, in the latter embodiment, the presence of an amino acid at position 57 (or any other position) of the mucin binding site is not particularly important to the ability to bind to the virus binding site.

[0026] In one embodiment, the peptide has two or more virus binding sites. In this case, the mucin binding site is covalently bound to at least one of the two or more virus binding sites directly or via a linker. The mucin binding site may also be bound to multiple virus binding sites. For this purpose, in one embodiment, a branched linker is extremely useful. Such a branched linker can be used to bind to a single mucin binding site and multiple virus binding sites that are approximately equidistant from the mucin binding site after binding.

[0027] In one embodiment, the viral binding site is a site suitable for binding to a viral spike protein.

[0028] In one embodiment, the viral binding site is a peptide. In one embodiment, the viral binding site is an antimicrobial peptide, such as a defensin. Retrocyclins are highly suitable defensins. In another embodiment, the viral binding site is a non-peptide antiviral moiety, such as a dendritic or linear polysulfate or polycarbohydrate.

[0029] In one embodiment, the virus binding site contains or is a peptide that is 95% or more identical, particularly 96% or more identical, particularly 97% or more identical, particularly 98% or more identical, particularly 99% or more identical, particularly 100% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 or SEQ ID NO:43. These sequences relate to virus binding sites that are highly suitable for binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

[0030] In one embodiment, the virus binding site is a peptide in which at least one residue of the peptide has a substitution. In one embodiment, the C-terminus of the peptide is substituted with an amino group. In one embodiment, the N-terminus of the peptide is substituted with an acetyl group. In one embodiment, the C-terminus of the peptide is substituted with an amino group and the N-terminus of the peptide is substituted with an acetyl group.

[0031] In one embodiment, the virus binding site is a peptide that is 95% or more identical to SEQ ID NO:2, particularly 96% or more identical, particularly 97% or more identical, particularly 98% or more identical, particularly 99% or more identical, particularly 100% identical, and the mucin binding site (1) is a trefoil factor 3 peptide that is 95% or more identical to SEQ ID NO:1, particularly 96% or more identical, particularly 97% or more identical, particularly 98% or more identical, particularly 99% or more identical, particularly 100% identical. SEQ ID NO:2 describes a peptide designated p4 that has already been previously identified as binding with high affinity to severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) (Han et al., 2006). Despite the structural differences between SARS-CoV-1 and SARS-CoV-2, it was surprisingly found that the p4 peptide is also highly suitable for binding to SARS-CoV-2.

[0032] In one embodiment, the virus binding site is a peptide that is 95% or more identical to SEQ ID NO: 3, particularly 96% or more identical, particularly 97% or more identical, particularly 98% or more identical, particularly 99% or more identical, particularly 100% identical, and the mucin binding site (1) is a trefoil factor 3 peptide that is 95% or more identical to SEQ ID NO: 1, particularly 96% or more identical, particularly 97% or more identical, particularly 98% or more identical, particularly 99% or more identical, particularly 100% identical. SEQ ID NO: 3 describes a peptide designated p6 that has already been previously identified as binding with high affinity to severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) (Han et al., 2006). Despite the structural differences between SARS-CoV-1 and SARS-CoV-2, it has surprisingly been found that the p6 peptide is also highly suitable for binding to SARS-CoV-2. This gives the p6 peptide approximately twice the binding affinity to SARS-CoV-2 as the p4 peptide. Interestingly, p6 is also able to bind to the alpha and delta variants of SARS-CoV-2 with even higher affinity than the Wuhan wild type of SARS-CoV-2 (see Table 1).

[0033] An even higher affinity for SARS-CoV-2 than that of the linear p6 peptide was observed for the stapled variant of the p6 peptide. In one embodiment, the virus binding site is a peptide that is 95% or more identical to SEQ ID NO: 10, particularly 96% or more identical, particularly 97% or more identical, particularly 98% or more identical, particularly 99% or more identical, particularly 100% identical. The peptide is a stapled variant of the p6 peptide. Hereinafter, it is referred to as p6-cyc or p6(R8). 15 (S5) 22 It is also called.

[0034] Table 1 below summarizes the binding affinities (listed as dissociation constants Kd) of the different viral binding sites to SARS-CoV-2, measured by microscale thermophoresis (MST). It is clear that the p4 and p6 peptides (the latter in both linear and cyclized forms) bind to multiple receptor binding domains of currently circulating SARS-CoV-2 variants with nanomolar binding constants.

[0035] [Table 1]

[0036] In one embodiment, the peptide is at least 95% identical, in particular at least 96% identical, in particular at least 97% identical, in particular at least 98% identical, in particular at least 99% identical, in particular 100% identical to SEQ ID NO: 18 or SEQ ID NO: 19. These SEQ ID NOs relate to synthetic and recombinant TFF3-p6 peptides that have been shown to have properties highly suitable for solving the problem in question.

[0037] In one aspect, the present invention relates to the use of the aforementioned peptides as medicaments.

[0038] In one aspect, the present invention relates to the medical use of the aforementioned peptides in the prevention or treatment of viral infections.

[0039] In one embodiment, the viral infection to be treated or prevented is infection with SARS-CoV-2.

[0040] In one aspect, the present invention relates to a medical method for treating or preventing a viral infection in a patient in need of such treatment. As used herein, "patient" refers to a human or animal patient. Mammals are particularly suitable animal patients. The method comprises administering to the patient the peptide as described above (e.g. in the form of a medicament containing the peptide). In one embodiment, the administration is performed by providing the peptide to the nose or mouth of the patient. This can be most easily achieved by providing the peptide in a spray dosage form. That is, the peptide can be administered as an aerosol, for example a nasal spray or an inhalant. The administered peptide can come into contact with the mucous membrane of the patient and bind to the mucous membrane via the mucin binding site. The peptide can then remain in a form bound to the mucous membrane of the patient and "filter" the virus from the air inhaled by the patient by its virus binding site.

[0041] In one aspect, the present invention relates to a medical method for mucosal strengthening in a patient in need of such treatment, the method comprising administering to said patient a peptide as described above (e.g. in the form of a medicament containing the peptide).

[0042] In one aspect, the present invention relates to a pharmaceutical composition comprising the above-mentioned peptide as a pharmaceutical active ingredient. The pharmaceutical composition may contain other pharmaceutical active ingredients. In one embodiment, the peptide is the only pharmaceutical active ingredient.

[0043] In one embodiment, the medicament is formulated to be administered as an aerosol, such as a nasal spray or inhalation. That is, the medicament (with the peptide as the medicament active ingredient) is administered to directly contact the mucous membrane of the nasopharyngeal region. This leads to the aforementioned mucous membrane strengthening. As a result, the mucous membrane of the patient is well prepared to bind and remove the virus that the patient encounters (either inhaled air from outside the body or exhaled air from virus-infected cells of the patient's body).

[0044] In one aspect, the present invention relates to a method for producing the aforementioned peptide, comprising the steps of: first reacting a first mucin-binding site precursor with a virus-binding site precursor, the first mucin-binding site precursor having a first site in the final mucin-binding site; this reaction results in the formation of a peptide precursor.

[0045] The peptide precursor is then reacted with a second mucin-binding site precursor, which has a second site in the mucin-binding site. As a result of this second reaction step, the final peptide is formed. Of course, other intermediate reaction steps may be performed. As an example, the mucin-binding site can be divided into three or more mucin-binding site precursors. Then, the individual mucin-binding site precursors are reacted in turn with the already formed peptide precursors to finally obtain the peptide.

[0046] In one embodiment, the virus binding site precursor has a 4-pentynoyl moiety that establishes a covalent bond with the mucin binding site in the final peptide. The 4-pentynoyl moiety may remain (in whole or in part) in the final peptide. In one embodiment, the 4-pentynoyl moiety may function as a linker between the virus binding site and the mucin binding site.

[0047] In one embodiment, the peptide comprises a triazole residue that functions as a linker between the virus binding site and the mucin binding site. In one embodiment, the peptide comprises a polyethylene glycol residue that functions as a linker between the virus binding site and the mucin binding site. In one embodiment, such a polyethylene glycol residue and a triazole residue are combined to form the linker.

[0048] In one embodiment, the first mucin-binding site precursor has less than half of the total amino acids of the mucin-binding site. When only two mucin-binding site precursors are used, the second mucin-binding site precursor has more than half of the total amino acids of the mucin-binding site. As a specific example, the first mucin-binding site precursor has amino acids 36-59 of the mucin-binding site, which is the trefoil factor 3 peptide in this embodiment. In addition, the second mucin-binding site precursor has amino acids 1-35 of the mucin-binding site. After reacting the first mucin-binding site precursor (corresponding to the C-terminal portion of the mucin-binding site) with the virus-binding site, the second mucin-binding site precursor (corresponding to the N-terminal portion of the mucin-binding site) is reacted with the construct already formed by the first mucin-binding site precursor and the virus-binding site (i.e., peptide precursor). As a result, the final peptide is formed.

[0049] Any of the embodiments of the peptides can be combined with each other in any desired manner and can be incorporated into the uses, the medicines, or the different methods, either individually or randomly. Similarly, any of the different uses can be combined with each other in any desired manner and can be incorporated into the peptides, other uses, the medicines, or the different methods, either individually or randomly. Similarly, any of the embodiments of the medicines can be combined with each other in any desired manner and can be incorporated into the peptides, other uses, or the different methods, either individually or randomly. Furthermore, any of the different methods can be combined with each other in any desired manner and can be incorporated into the peptides, other uses, or the different methods, either individually or randomly.

[0050] Further details of each aspect of the invention will now be described with reference to exemplary embodiments and the accompanying drawings. [Brief description of the drawings]

[0051] [Figure 1A] FIG. 1 is a schematic diagram showing the structure of a first embodiment of a bifunctional peptide. [Figure 1B] FIG. 2 is a schematic diagram showing the structure of a second embodiment of a bifunctional peptide. [Figure 2A] FIG. 1 shows the primary structure of one embodiment of the first mucin binding site precursor corresponding to SEQ ID NO:15. [Figure 2B] FIG. 1 shows the primary structure of one embodiment of a second mucin binding site precursor corresponding to SEQ ID NO:16. [Figure 2C] FIG. 1 shows the primary structure of one embodiment of a viral binding site precursor corresponding to SEQ ID NO:17. [Diagram 3] FIG. 2 shows the primary structure of one embodiment of a peptide corresponding to SEQ ID NO: 18, having a mucin binding site made from the first and second mucin binding site precursors of FIG. 2A and FIG. 2B, and a virus binding site made from the virus binding site precursor of FIG. 2C. [Figure 4] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:4. [Diagram 5] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:5. [Figure 6] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:6. [Figure 7] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:7. [Figure 8] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:8. [Figure 9] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:9. [Figure 10] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:10. [Figure 11] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:11. [Figure 12] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:12. [Figure 13] FIG. 1 shows the primary structure of one embodiment of the viral binding site corresponding to SEQ ID NO:13. [Figure 14] A plot showing the binding affinity between the p6 peptide and each SARS-CoV-2 variant. [Figure 15] 1 is a plot showing the binding affinity of the synthetic bifunctional TFF3-p6 peptide corresponding to SEQ ID NO: 18 with each SARS-CoV-2 mutant strain. [Figure 16] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 23 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 17] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO:24 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 18]1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 25 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 19] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 26 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 20] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 28 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 21] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO:29 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 22] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 30 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 23] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 31 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 24] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 32 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Diagram 25] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 33 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 26] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 34 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 27] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 35 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 28]1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 36 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Figure 29] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 37 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Diagram 30] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 38 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Diagram 31] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 39 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Diagram 32] 1 is a plot showing the binding affinity between the p4 peptide variants corresponding to SEQ ID NO: 40 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Diagram 33] 1 is a plot showing the binding affinity between the p6 peptide variants corresponding to SEQ ID NO: 41 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Diagram 34] 1 is a plot showing the binding affinity between the p6 peptide variants corresponding to SEQ ID NO: 42 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Diagram 35] 1 is a plot showing the binding affinity between the p6 peptide variants corresponding to SEQ ID NO: 43 and the receptor binding domain of SARS-CoV-2 (2019-nCoV). [Diagram 36] 1 is a plot showing the binding affinity between the recombinant bifunctional TFF3-p6 peptide corresponding to SEQ ID NO: 19 and each SARS-CoV-2 mutant strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] 1A is a schematic diagram showing the structure of a first embodiment of a bifunctional peptide, namely, TFF3-p6, in which trefoil factor 3 (TFF3) peptide 1 is the mucin binding site and p6 peptide 2 is the virus binding site. TFF3 peptide 1 has a primary sequence corresponding to SEQ ID NO: 1. p6 peptide 2 has a primary sequence corresponding to SEQ ID NO: 3. p6 peptide 2 is covalently attached to lysine 57 of the TFF3 peptide.

[0053] FIG. 1B shows another embodiment of a bifunctional peptide, in which like features / components are labeled with like reference numbers in this and subsequent figures, in which p6 peptide 2 is covalently attached to the C-terminus of TFF3 peptide 1.

[0054] The synthesis of TFF3-p6 is described in detail below.

[0055] For the preparation of TFF3, we followed the protocol of Braga Emidio et al. 2021. Deviating from the synthetic route published by Braga Emidio et al., the cysteine ​​at position 57 of TFF3 was replaced by an azidolysine.

[0056] One embodiment of the first mucin binding site precursor, TFF3(36-59)(C57AzK), was synthesized by SPPS on rink amide resin using a building block corresponding to the azidolysine (AzK) at position 57 (TFF3 (36~59) C57AzK[M+H] + = 2825.31 g / mol, [M+2H] 3+ / 3=943.7 g / mol). Figure 2A shows its primary structure.

[0057] TFF3(1-35)-Nbz, representing one embodiment of a second mucin binding site precursor, was synthesized on rink amide resin, which was first functionalized with 3-fluorenylmethoxycarbonyl-4-diaminobenzoic acid (Fmoc-Dbz) by standard coupling methods (5 equivalents (eq) of Fmoc-Dbz, 4.9 eq of azabenzotriazole tetramethyluronium hexafluorophosphate (HATU), 10 eq of N,N-diisopropylethylamine (Dipea) in sufficient dimethylformamide (DMF) to make a 0.2 M solution for 3-Fmoc-Dbz). General solid phase peptide synthesis (SPPS) methods were then performed. The last residue was added as a Boc-protected amino acid. After assembly of the peptide on the resin, the C-terminus was capped with p-nitrophenyl chloroformate (5 eq of CH 2 Cl 2 After activation by acylation with 1,2-ethanedithiol (EDT):H, the resin-bound benzimidazolinone was formed by addition of base (Dipea (0.5 M) in DMF; 20 min) to promote intramolecular attack of the anilide. 2 Cleavage from the resin was performed with O:triisopropylsilane (TIPS) (94:2.5:2.5:1.0), and the crude reaction product was dissolved in 3 mL of 30% acetonitrile (MeCN) / H2O containing 0.1% TFA. 2 The product was dissolved in 200 mL of DMSO, filtered, and purified by injection into a preparative high performance liquid chromatography (HPLC) system (TFF3(1-35)-Nbz.3980.4:M + , 996.1:(M+4H) 4+ / 4). Figure 2B shows its primary structure.

[0058] Pentinoic acid-(PEG) showing one embodiment of a viral binding site 2-p6 was prepared by SPPS method up to two PEG units on Rink Amide resin. It was then manually coupled with 4-pentynoic acid (5 eq 4-pentynoic acid, 4.9 eq HATU, 10 eq Dipea, and enough DMF to prepare a 0.2 M solution of 4-pentynoic acid). The coupling was carried out twice over 1.5 hours. It was then eluted with TFA:H 2 The peptide was cleaved with O:TIPS (95:2.5:2.5) and purified by preparative HPLC (Pentinoyl-p6[M+H] + = 3975.90 g / mol, [M+2H] 3+ / 3=994.9 g / mol). Figure 2C shows its primary structure.

[0059] In a vial, prepare a 1 mM solution of CuSO 4 (10 eq) was charged and mixed with THPTA (50 eq). During the mixing of the remaining reagents, they formed an organometallic complex.

[0060] Azidolysine 57 and pentanoyl-(PEG) 2 To form the peptide precursor by click chemistry between the alkyne group of -p6, add reaction buffer (10 mM NHCl) in a 1.5 mL Eppendorf tube. 4 HCO 3 ) to pentanoyl-(PEG) 2 -p6 (1.5 eq) and TFF3(36-59)(C57AzK) (1.0 eq) were dissolved in the solution. Aminoguanidine (100 eq) was then added from a 200 mM stock solution. This was followed by the addition of CuSO 4 A mixture of 20 μL of 30% MeCN / H2O was added to the reaction mixture. Finally, sodium ascorbate (100 eq) was added to the reaction mixture and the pH was adjusted to 8.0 with HCl / KOH. The reaction tube was flushed with nitrogen, then immediately sealed and left at 37° C. for 12 hours with stirring. 20 μL of 30% MeCN / H2O was added to the reaction mixture. 2 1 μL of the reaction crude solution was dissolved in O, and tris(2-carboxyethyl)phosphine (TCEP) was added to reach a concentration of 30 mM. After standing at 37° C. for 5 min, the sample was analyzed by HPLC-mass spectrometry (HPLC-MS).

[0061] To perform native chemical ligation between TFF3(1–35)-Nbz and TFF3(36–59) (C57K-pentynoyl p6), use reaction buffer (6 M guanidinium hydrochloride, 100 mM NaCl, 2 HPO 4 TFF3(1-35)-Nbz (1.0 eq) and TFF3(36-59)(C57K-pentynoyl p6) (0.8 eq) were dissolved in 10 mM NaCl (100 eq) and 30 mM TCEP; pH adjusted to 7.0 with HCl / NaOH. 2-Mercaptoethanesulfonate (MeSNa) (100 eq) was then added and the reaction was stirred at room temperature for 4 hours. 1 μL of the crude reaction solution was dissolved in 20 μL of 30% acetonitrile (MeCN) / H2O. 2 After dissolving in 200, TCEP was added until the concentration reached 30 mM. After standing at 37°C for 5 min, the sample was analyzed by HPLC-MS. The thioester was reduced to less than trace amounts, and the thioester was dissolved in 30% MeCN / H2O containing 0.1% TFA. 2 The reaction volume was increased to 3 mL by addition of O, filtered, and purified by injection into a preparative HPLC system to give TFF3-C57K(pentynoyl-p6) ([M+H] + =10600.96g / mol, [M+8H] 8+ / 8=1326.1g / mol, [M+9H] 9+ / 9=1178.8g / mol[M+10H] 10+ The primary structure of the compound was shown in Figure 3.

[0062] Additionally, native TFF3 was prepared recombinantly. This was performed according to and adapted from the protocol in Jarva et al., 2020.

[0063] Briefly, the 5' end of the TFF3 sequence was extended to introduce a purification tag. The product was then incorporated into the plasmid pET28b for bacterial expression using sequence- and ligation-independent cloning (SLIC). The final plasmid was verified by sequencing and transformed into BL21CodonPlus(DE3)RIPL (R=arginine, I=isoleucine (Ile), P=proline, L=leucine) cells for bacterial expression, as described in detail below.

[0064] (Preparation of exponentially growing stock cultures of transformed E. coli BL21CodonPlus(DE3)RIPL) Such plasmids were transformed into E. coli BL21CodonPlus(DE3)RIPL cells. The cells were spread onto lysogeny broth (LB) agar plates containing 50 μg / mL streptomycin, 25 μg / mL chloramphenicol, and 50 μg / mL kanamycin and grown overnight in an incubator at 37°C. One colony was picked and inoculated into LB medium containing 50 μg / mL streptomycin, 25 μg / mL chloramphenicol, and 50 μg / mL kanamycin and grown overnight in an orbital shaker at 250 rpm and 37°C. The initial optical density at 600 nm (OD 600 Terrific Broth (TB) medium was inoculated with an LB culture with an OD of 0.02 and grown in a rotary shaker at 250 rpm and 37°C. The culture reached an OD 600 >2.867. 7.527% DMSO was added to the culture and the mixture (preculture) was flash frozen in liquid nitrogen and stored at -80°C for future use.

[0065] Protein Expression and Washing The above preculture was grown on TB medium at OD 600=0.04. The culture was grown for 24 hours at 250 rpm and 26°C in a rotary shaker incubator. After 24 hours, the bacterial culture was cooled on ice for 15 minutes and centrifuged at 3000g for 15 minutes in a centrifuge. The supernatant was removed and the cells were resuspended in washing buffer (HEPES-based; pH=7) + DNaseI + phenylmethylsulfonyl fluoride (PMSF). The suspension was centrifuged at 3000g for 15 minutes and the supernatant was removed. This washing process was repeated.

[0066] The target protein was purified by tandem affinity purification using His tag and Strep tag (streptavidin binding motif). The protein was eluted from the column at the protease cleavage site. The eluted protein was dialyzed and confirmed by SDS-PAGE.

[0067] Preparation of Lactam Stapled Peptides All lactam-stapled peptides were prepared on rink amide resin. The building blocks for isopeptide bond formation were N-α-Fmoc-N-ε-4-methyltrityl-L-lysine (hereafter referred to as MttK) and (N-α-Fmoc-L-aspartic acid) β-2-phenylisopropyl ester (hereafter referred to as PhiPr). The peptides bound to the resin via SPPS were treated with 2% TFA in dichloromethane (DCM) for 4-methyltrityl and 2-phenylisopropyl deprotection. After isopeptide bond formation, a solution of 0.9 equivalents of HATU and 2 equivalents of DIPEA in DMF was added. After extensive washing with DMF and DCM, the subsequent coupling cocktail was applied. TFA:H 2 The peptide was cleaved from the resin with O:TIPS (95:2.5:2.5) and precipitated with cold ether. The peptide was then filtered, dried, and diluted with 30% MeCN / H2O containing 0.1% TFA to a final volume of 3 mL. 2 The solution was redissolved in O. After filtration, the sample was injected into a preparative HPLC system for purification.

[0068] 4 to 9 show the structures of the resulting lactam-stapled peptides.

[0069] Table 2 below summarizes the binding affinity (listed as dissociation constant Kd) of each cyclic viral binding site to SARS-CoV-2, as measured by microscale thermophoresis (MST). It can be seen that each cyclic p6 peptide binds to SARS-CoV-2 with a binding constant in the micromolar range.

[0070] [Table 2]

[0071] Preparation of stapled viral binding sites by ring-closing metathesis (RCM) The following non-canonical amino acids were used in the RCM reactions: S5Fmoc = Fmoc-(S)-2-(4-pentenyl)Ala-OH R8Fmoc = Fmoc-(R)-2-(7-octenyl)Ala-OH

[0072] For these amino acids (AA), the coupling conditions were 3.0 equivalents of AA, 2.9 eq of HATU, and 6 eq of DIPEA. DMF was added to reach a concentration of 0.2 M relative to AA.

[0073] For all natural amino acids, the coupling conditions were 5.0 equivalents of AA, 4.9 eq of HATU, and 10 eq of DIPEA. DMF was added to reach a concentration of 0.2 M relative to AA.

[0074] After confirming the mass of the peptide before stapled by trial cleavage, RCM was performed. The resin was washed with 1 mL DCM (3 times × 1 min) followed by 1 mL 1,2-dichloroethane (DCE) (3 times × 1 min). The resin (50 μmol) was treated with 1 mL Grubbs 1st generation catalyst (6 mM) in DCM (resin substitution = 20 mol %). The resulting suspension was stirred at 20-25 °C while continuously bubbling with nitrogen for 2 h. The solvent was then removed. Grubbs catalyst was then added in duplicate. The resin was then washed with 1 mL DCM (3 times × 1 min) followed by 1 mL DCE (3 times × 1 min) and dried under a nitrogen stream. TFA:H 2 The peptide was cleaved from the resin with O:TIPS (95:2.5:2.5). The reaction crude was precipitated with cold ether, filtered, and dried. The product was diluted with 30% MeCN / H2O containing 0.1% TFA to a final volume of 3 mL. 2 The mixture was redissolved in O. After filtration, the sample was injected onto a preparative HPLC system for purification.

[0075] For acetylation of the RCM product, the Fmoc-deprotected resin was swollen in 1 mL of N-methyl-2-pyrrolidone (NMP) for 10 min, the solvent was removed, and 2 mL of a solution of acetic anhydride and DIPEA in NMP (volume ratio = 85:315:1,600) was added to the resin. The mixture was gently stirred for 45 min while bubbling with nitrogen, after which the solvent was removed. It was then thoroughly washed with DCM and DMF. Finally, cleavage and purification were carried out as described above.

[0076] After deprotection, the peptide had: i) an S5 residue (alkenylated S-alanine) and an R8 residue (alkenylated R-alanine); or ii) two S5 residues. An alkenyl bridge was formed between i) the S5 residue and the R8 residue, or ii) between two S5 residues. In the case of the S5 and R8 residues, the alkenyl bridge is a 7-undecenyl residue, and in the case of two S5 residues, the alkenyl bridge is a 4-octenyl residue.

[0077] 10-13 show the resulting structures of stapled peptides prepared by RCM.

[0078] Table 3 below summarizes the individual properties of each cyclic viral binding site, including their binding affinity to SARS-CoV-2 (listed as the dissociation constant Kd). The binding affinity was measured by microscale thermophoresis (MST). Each cyclic p6 peptide p6(R8) 15 (S5) 22 It is clear that it binds to SARS-CoV-2 with a nanomolar binding constant.

[0079] [Table 3]

[0080] (Measurement of peptide binding affinity) The affinity was measured by microscale thermophoresis (MST), where the ratio of chemical and thermal diffusion (Soret effect) depending on the number of bound ligands (peptides in this case) influences the mobility of the labeled compound (RBD in this case). In MST, an infrared laser creates a localized heat spot, followed by a temperature gradient. Molecules move along this gradient depending on their properties at the water-protein interface. Bound ligands can cause changes in the ordered water shell of the observed protein as well as changes or additions to its effective surface potential. This leads to a change in the thermophoretic behavior by increasing or decreasing the thermophoretic amplitude. By adding different ligand concentrations, it is possible to plot the change in fluorescence (on the y-axis) versus the ligand concentration. From the half-maximum change in fluorescence (the inflection point from the saturation curve), the law of mass action can be applied to derive the Kd of the protein-ligand interaction (Jerabek-Willemsen et al. 2014).

[0081] Labeling and purification of RBD was performed using a second generation NHS-Red labeling kit (NanoTemper). The RBD solution was diluted with water to 10 μM, rebuffered to pH = 8.0 with aqueous sodium carbonate, and incubated in the dark with 300 μM red dye. Size exclusion chromatography yielded labeled RBD free of unreacted dye. Spectroscopic measurements showed that the labeling efficiency was approximately 1:1 (protein:dye). For affinity measurements, the RBD was diluted with Dulbecco's phosphate buffered saline (DPBS) containing 0.05% (v / v) Tween 20 (Ca 2+ , Mg 2+ The (free) solution was placed in a high quality capillary (NanoTemper).

[0082] Next, 16 serial 1:1 dilutions of each peptide were made and the MSTs were measured in the same manner, using PBS + 0.05% Tween 20 as the diluent, each with a final volume of 10 μL. Each diluted sample was then placed with 10 μL of the labeled RBD (10 nM) stock solution, so that the concentration of the labeled target protein in each sample was kept at 5 nM.

[0083] The MST signal was acquired with a Monolith NT.115Pico instrument (NanoTemper Technologies) at 20% excitation power and 40% MST power. Signal analysis was performed 1.5 s after the start of the IR laser, and the fitting of the acquired data was performed as previously described (Bhatia et al. 2017). These conditions were the same for all samples measured.

[0084] (Measurement of affinity of p6 for RBD (SARS-CoV-2, Omicron B.1.1.529)) Furthermore, the chemically synthesized p6 peptide (according to SEQ ID NO:3) was titrated against a fixed concentration (5 nM) of fluorescently labeled RBD of the Omicron variant of SARS-CoV-2 (B.1.1.529 strain). The dissociation constant (Kd) was measured to be 280 nM. Thus, the p6 peptide also exhibited high binding affinity to the Omicron variant of SARS-CoV-2.

[0085] Figure 14 shows the results of the previously described affinity characterization of the p6 peptide (of SEQ ID NO: 3) using MST method against fluorescently labeled receptor binding domains (RBDs) from wild-type (filled circles), alpha mutant (open circles), delta mutant (triangles) and omicron mutant (squares) of SARS-CoV-2 (wild-type, alpha and delta mutants: (N=3), error bars indicate SEM). It can be clearly seen that the p6 peptide binds with high affinity to all SARS-CoV-2 mutants / strains (see also Table 1 for quantitative results of these experiments).

[0086] (p4 and p6 derivatives) Additionally, several additional p4 and p6 derivatives were synthesized and their affinity for the receptor binding domain was tested in the MST experiments described above for the p6 peptide.

[0087] Each derivative of p4 was synthesized as described in Sarto et al. 2022. For p6 derivatives, p6-rigid, p6-dis, and p6-rigid-dis in TFA salt form were ordered from PSL (Peptide Specialty Laboratories GmbH, Heidelberg). Upon arrival, each peptide was reconstituted in PBS, flash frozen in liquid nitrogen, and stored at -80 °C until further use.

[0088] Table 4 lists each of the p4 derivatives tested and the dissociation constants obtained from the affinity measurements. The cysteine ​​residues in each peptide are in their normal reduced state. The results of each affinity measurement are also shown in Figures 16-32.

[0089] [Table 4]

[0090] p4 derivatives having a dissociation constant for the RBD of SARS-CoV-2 of less than 0.3, particularly less than 0.2, particularly less than 0.1, particularly less than 0.05, particularly less than 0.04, particularly less than 0.03, particularly less than 0.02 are highly suitable virus binding sites.

[0091] Table 5 lists each p6 derivative tested and the dissociation constants obtained from affinity measurements. The cysteine ​​residues in each peptide form intramolecular disulfide bonds in their oxidized state. The results of each affinity measurement are also shown in Figures 33-35.

[0092] [Table 5]

[0093] (Genetically recombinant preparation of TFF3-p6) TFF3-p6 was also prepared recombinantly. To achieve this, the protocol of Jarva et al., 2020 was followed.

[0094] The TFF3 sequence was then extended to introduce a purification tag and a TEV (Tobacco Etch Virus) protease cleavage site at the 5' end and p6 at the 3' end. The product was then integrated into the pET28b plasmid for bacterial expression using sequence- and ligation-independent cloning (SLIC). The final plasmid was sequenced and transformed into BL21CodonPlus(DE3)RIPL cells for bacterial expression, as previously described in detail in the section "Preparation of exponentially growing stock cultures of transformed E. coli BL21CodonPlus(DE3)RIPL".

[0095] Protein Expression and Washing The resulting preculture was grown on TB medium at OD600 = 0.04. The culture was grown for 24 hours at 250 rpm and 26°C in a rotary shaker. After 24 hours, the bacterial culture was cooled on ice for 15 minutes and centrifuged at 3000g for 15 minutes in a centrifuge. The supernatant was removed and the cells were resuspended in washing buffer (HEPES-based; pH = 8). The suspension was centrifuged at 3000g for 15 minutes and the supernatant was removed. This washing process was repeated. The bacterial cells were lysed by sonication in lysis buffer (HEPES-based; pH = 8). The lysate was centrifuged at 12.000g for 45 minutes, the pellet was removed and the supernatant was filtered through a 0.2 μm filter.

[0096] (Protein purification) The protein of interest was purified by tandem affinity purification using His-tag and Strep-tag (streptavidin binding motif) purification. Imidazole (ImH) was added to the filtered cell lysate to a final concentration of 40 mM. The lysate was applied to a HisTrap column (Cytiva) using a fast protein liquid chromatography (FPLC) device (Akta Pure 25 (Cytiva)). The column was washed with wash buffer (300 mM NaCl, 50 mM Tris base, 40 mM ImH; pH = 7.5) and the protein was eluted with elution buffer (300 mM NaCl, 50 mM Tris base, 400 mM ImH; pH = 7.5) and the eluate was collected in fractions. Fractions containing the product of interest were identified by SDS-PAGE and collected.

[0097] The collected fractions were applied to a Strep-Tactin XT 4Flow column (IBA Lifesciences) using an AktaPure 25. The column was washed with wash buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM ETDA; pH = 8) and the protein was eluted with elution buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM ETDM, 50 mM biotin; pH = 8) and the eluate was collected in fractions. Fractions containing the desired product were identified by SDS-PAGE, collected, and then dialyzed against PBS (pH = 7.4). Protein content was determined by absorbance at 280 nM using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific).

[0098] To remove the tandem affinity purification tags, the purified product was incubated overnight at 30° C. with ProTEV Plus (Promega) using 1 U of ProTEV Plus per 15 μg of purified product according to the manufacturer's recommendations.

[0099] ImH was added to the reaction mixture to a final concentration of 40 mM, and the mixture was applied to a HisTrap column using an Äkta Pure 25 and the column was washed with wash buffer (300 mM NaCl, 50 mM Tris base, 40 mM ImH; pH = 7.5). The cleaved purification tag and ProTEV Plus (which also contained the His tag) remained in the column, while the flow-through containing the final product (TFF3-p6) was fractionated. The fractions containing the final product were identified by SDS-PAGE, collected, and dialyzed against PBS (pH = 7.4). The volume was reduced using Vivaspin PES centrifugal concentrators (Sartorius) to a final protein concentration of 1 mg / mL to 3 mg / mL as measured by NanoDrop2000. The solution was flash frozen in liquid nitrogen and stored at -20 °C until further use. The exact final product and its purity were controlled by SDS-PAGE and ultra-performance liquid chromatography mass spectrometry (UPLC / MS) methods.

[0100] (Measuring the affinity of TFF-p6 to the RBD of SARS-CoV-2) In these experiments, chemically synthesized TFF3-p6 (TFF3-C57K (pentynoyl-p6), i.e., TFF3-p6 syn (the primary structure of which is depicted in FIG. 3 and reproduced in SEQ ID NO: 18) and recombinant TFF3-p6 (TFF3-p6 rec; SEQ ID NO: 19) were titrated against a fixed concentration (5 nM) of fluorescently labeled RBD of wild-type SARS-CoV2 (also referred to as nCov2019) and the delta mutant strain of SARS-CoV2 (also referred to as Delta (B.1.617.2)). The resulting dissociation constants (Kd) are listed in Table 6. Figure 15 shows the results of this affinity measurement for TFF3-p6 syn, and Figure 36 shows the results of this affinity measurement for TFF3-p6 rec.

[0101] [Table 6]

[0102] Cytotoxicity Assay Cell viability was measured using a viable cell count kit (HY-K0301, Hycultec) according to the manufacturer's instructions.

[0103] A549 cells, HBE cells, and Calu-3 cells were cultured in DMEM supplemented with 10% (v / v) FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. 5 × 10 cells were cultured in 90 μL of DMEM medium per well of a 96-well plate. 4 Culture the cells at 37°C and 5% CO at a concentration of 100 cells / mL. 2 The seeds were sown overnight at 4°C.

[0104] 10 μL of recombinant TFF3-p6 or tag-TFF3-p6 (with the affinity tag remaining) dissolved in deionized water was serially diluted (6.6 × 10 -6 M~6.6×10 -10 M) and positive control (1% SDS) and negative control (liquid vehicle; neutral, H 2O) was added and incubated at 37°C and 5% CO 2 The wells were incubated for another 48 hours at 4°C for 10 min. For background subtraction, wells containing only the sample without cells were also used. After 48 hours of incubation, CCK8 solution (10 μL / well) was added and incubated with the dye for approximately 3 hours, after which absorbance measurements (450 nm / 650 nm) were performed using a Tecan plate reader (SPARK, TECAN-reader (Tecan Group)). After subtracting the background signal, cell viability was calculated with the untreated control set at 100%.

[0105] Neither recombinant TFF3-p6 nor tag-TFF3-p6 showed significant differences from the negative control. Rather, cell viability was approximately 100% in both cases. In contrast, the positive control showed 0% or close to 0% cell viability.

[0106] (Antiviral effect) To test the antiviral effect of TFF3-p6, we used a pseudovirus infection system based on vesicular stomatitis virus (VSV) containing eGFP and the SARS-CoV-2 spike protein instead of the VSV envelope protein (VSVΔG-CoV-2; Markus Hoffmann et al.).

[0107] TFF3-p6 or PSB (control) was incubated with VSVΔG-CoV-2 at final concentrations of TFF3-p6 = 50 μM and virus = 2.625 × 10 4 The mixture was incubated for 30 minutes to obtain a 100 μL solution of ffu / mL. This mixture was added to VeroE6 cells and incubated at 37° C. for 24 hours in a cell culture incubator. Images were taken using a fluorescent microscope to determine the number of infected cells.

[0108] TFF3-p6 was able to reduce infection of VeroE6 cells by VSVΔG-CoV2 pseudovirus by approximately 40% compared to the PBS control.

Claims

1. A peptide having a virus-binding site (2) and a mucin-binding site (1) covalently bonded to the virus-binding site (2), the virus binding site (2) contains a peptide that is 95% or more identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 or SEQ ID NO:63, or is the peptide; A peptide, characterized in that the mucin binding site (1) contains or is a peptide that is 95% or more identical to SEQ ID NO:1, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:

22.

2. 2. The peptide according to claim 1, characterized in that the mucin binding site (1) contains or is a trefoil factor 3 peptide that is 95% or more identical to SEQ ID NO:

1.

3. 3. The peptide according to claim 1 or 2, characterized in that the peptide has two or more virus binding sites (2), and the mucin binding site (1) is covalently bonded to at least one of the two or more virus binding sites (2).

4. A peptide according to any one of claims 1 to 3, characterized in that the virus binding site (2) contains or is a peptide that is 95% or more identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 or SEQ ID NO:

43.

5. 5. A peptide according to any one of claims 1 to 4, characterized in that the virus binding site (2) is a peptide that is 95% or more identical to SEQ ID NO:2, and the mucin binding site (1) is a trefoil factor 3 peptide that is 95% or more identical to SEQ ID NO:

1.

6. 6. A peptide according to any one of claims 1 to 5, characterized in that the virus binding site (2) is a peptide that is 95% or more identical to SEQ ID NO:3, and the mucin binding site (1) is a trefoil factor 3 peptide that is 95% or more identical to SEQ ID NO:

1.

7. 7. The peptide according to any one of claims 1 to 6, characterized in that the peptide is 95% or more identical to SEQ ID NO:

18.

8. 7. The peptide according to any one of claims 1 to 6, characterized in that the peptide is 95% or more identical to SEQ ID NO:

19.

9. A peptide according to any one of claims 1 to 8 for the prevention or treatment of a viral infection.

10. A peptide according to any one of claims 1 to 7 for the use according to claim 9, characterized in that the viral infection is an infection with SARS-CoV-2.

11. A pharmaceutical comprising the peptide according to any one of claims 1 to 7 as a medicament active ingredient.

12. 12. The medicament of claim 11, wherein the medicament is designed and configured to be administered as a nasal spray or as an inhalant.

13. A method for producing a peptide according to any one of claims 1 to 7, comprising the steps of: a) reacting a virus-binding site precursor with a first mucin-binding site precursor having a first portion of the mucin-binding site (1) to obtain a peptide precursor; b) reacting the peptide precursor with a second mucin-binding site precursor having a second portion of the mucin-binding site (1); A method comprising:

14. 14. The method of claim 13, wherein the viral binding site precursor has a 4-pentynoyl moiety covalently attached to the viral binding site (2).

15. 15. The method of claim 13 or 14, wherein the first mucin binding site precursor has less than half of the total amino acids of the mucin binding site (1).

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