Bifunctional peptide with mucous membrane adhesiveness and virus binding characteristic

Abstract

The present invention relates to a bifunctional peptide comprising a virus binding moiety (2) and a mucoprotein binding moiety (1) covalently bound to the virus binding moiety (2). The virus binding moiety is selected from peptides that bind to SARS-CoV-2, influenza A, influenza B, rhinoviruses and other enteroviruses, human parainfluenza viruses and / or metapneumoviruses. The mucoprotein binding moiety is selected from a lectin, such as trefoil factor 3.

Description

[0001] The present invention relates to: a bifunctional peptide according to the preamble of claim 1, the medical use of such a peptide according to the preamble of claim 9, a medicament comprising such a peptide according to the preamble of claim 11, and a method for preparing such a peptide according to the preamble of claim 13.

[0002] The ongoing SARS-CoV-2 pandemic shows that emerging and recurring viral pathogens are a persistent threat to the human population, requiring rapid and specific responses to curb widespread viral transmission. Despite the rapid implementation of hygiene concepts to slow viral transmission, vaccines and antiviral treatments remain the main measures to combat infectious diseases. However, the development and testing of vaccines usually takes several years. Antiviral therapy provides a rapid solution for treating infected individuals. Compared with macromolecules (such as proteins or peptides), small organic compounds dominate antiviral drugs. However, the development of new small molecule drugs is challenging because their chemical properties are usually complex, and therefore it is difficult to prepare screening objects with a large number of different compound analogs. In addition, the development of resistance is an increasingly serious problem, as demonstrated by antiviral drugs (such as oseltamivir or amantadine) against influenza.

[0003] Prophylactic treatment as an additional measure to combat the SARS-CoV-2 pandemic is currently not being used and is an unrealized opportunity that could offer several advantages.

[0004] Werner Hoffmann described heterodimers from trefoil factor (TFF3) and IgG-Fc-binding protein (FCGBP).

[0005] It is an object of the present invention to provide novel substances for the prophylactic treatment of viral diseases, ideally to avoid viral infections.

[0006] This object is achieved by a peptide having the claim elements of claim 1. Such a peptide is a bifunctional peptide, which comprises a virus binding part and a mucin binding part covalently bound to the virus binding part. The binding between the virus binding part and the mucin binding part can be direct covalent binding or binding through a linker.

[0007] The virus binding portion comprises or is a peptide having at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identity to:

[0008]

[0009] SEQ ID NO.2 to 13 and 23 to 43 relate to virus binding parts that are particularly suitable for binding to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While SEQ ID NO.2, 3 and 23 to 41 relate to linear virus binding peptides, SEQ ID NO.4 to 13, 42 and 43 relate to partially cyclized peptides, so-called stapled peptides. They contain at least one bridge between two amino acids that are not directly adjacent (but still nearby). The bridge acts like a staple and is used to stabilize the corresponding peptide. The bridge can be established by two side chains of the corresponding amino acid residues, or by a chemical part that is covalently bound to the corresponding amino acid residues in addition to their conventional side chains. In the case of SEQ ID NO.42 and 43, the bridge is a disulfide bridge formed between two cysteine ​​residues.

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

[0011] SEQ ID NOs. 49 and 50 describe peptides particularly suitable for binding to influenza A virus (particularly H5N1 strain 2.3.4 clade, bird strain). These peptides target the hemagglutinin (HA protein) of influenza A virus and are derived from the sequence of antibody 65C6 described by Zuo et al.

[0012] SEQ ID NO. 51 and 52 describe peptides particularly suitable for binding to influenza A virus (particularly H5N1 strain 2.3.2.1 branch, avian strain). These peptides target specific sequences of hemagglutinin (HA protein) commonly present in the HA protein of this strain variant, for example in the sequence of the HA protein described by Bui et al.

[0013] SEQ ID NOs. 53 and 54 describe peptides that are particularly suitable for binding to influenza B virus. These peptides target the neuraminidase of influenza B virus and are described by Madsen et al.

[0014] SEQ ID NOs. 55 and 56 describe peptides that are particularly suitable for binding to rhinovirus (RV) and other enteroviruses (EV). These peptides are derived from anti-RV antibodies and are described by Hrebik et al., Khan et al. and Smith et al.

[0015] SEQ ID NO. 57 describes a peptide particularly suitable for binding to human parainfluenza virus (HPIV), in particular HIPV1 and HIPV3 strains. The peptide targets the fusion protein (F protein) of HPIV and is derived from an anti-HPIV antibody described by Cabán et al.

[0016] SEQ ID NO. 58, 59 and 60 describe peptides that are particularly suitable for binding to metapneuvirus (MPV) and respiratory syncytial virus (RSV), in particular subtype A and / or subtype B. These peptides target the fusion protein (F protein) of MPV and RSV. They are derived from anti-HPIV antibodies and are described by Cabán et al.

[0017] SEQ ID NO. 61, 62 and 63 describe peptides that are particularly suitable for binding to RSV, in particular subtype A and / or subtype B. These peptides also target the fusion protein (F protein) of RSV and are described by Issmail et al, Zhu et al. and McLellan et al.

[0018] The mucin binding moiety comprises or is a peptide having at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identity 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 particularly suitable for targeting the intestinal mucosa of patients. TFF3 is a protein produced naturally in the airways (Thim et al. 2002).

[0019] The peptides claimed in the present invention are capable of enhancing the natural barrier function of respiratory mucus. Respiratory mucus is the first physical barrier encountered by respiratory pathogens approaching their respiratory epithelial target cells. Due to its high viscosity and microbial binding properties, mucus is an important part of our body's antimicrobial defense system and effectively removes adsorbed pathogens through ciliary movement. The peptides claimed in the present invention can be used to enhance mucus (mucus enhancement) by incorporating binding sites for viral pathogens to improve their antimicrobial properties. The retained pathogens can then be cleared before reaching the epithelial cell layer, thereby preventing infection. Therefore, the present method is to use modular biomimetic peptides to improve and support the antiviral efficacy of mucus.

[0020] Mucus enhancement is a new concept for protecting people from respiratory infections. The present approach can be combined with other (non)pharmacological measures to reduce the risk of infection in uninfected individuals and to reduce the risk of transmission and severe disease in already infected individuals.

[0021] The peptides claimed in the present invention form the core of an adjustable peptide-based system with molecules that can adhere to airway mucin and specifically adsorb viruses. Both parts of the peptide can be adapted to respond to rapidly changing conditions. This biomimetic approach enhances the natural antiviral properties of mucus, and since the peptide components are derived from human proteins, side effects are believed to be lower. The bifunctional peptides can also be used to treat infected patients because they will also capture viruses released from infected cells, thereby reducing viral titers and shedding into the environment. Finally, this method can be combined with established antiviral measures to help reduce the risk of infection and minimize the risk of disease.

[0022] Due to the modular composition of the claimed peptides, both functional parts of the peptide can be adapted to the desired application.

[0023] Suitable linkers for establishing a covalent bond between the mucin binding portion and the virus binding portion are polyethylene glycol (PEG) linkers or amino acid linkers comprising 1 to 20 amino acids, in particular 2 to 19 amino acids, in particular 3 to 18 amino acids, in particular 4 to 17 amino acids, in particular 5 to 16 amino acids, in particular 6 to 15 amino acids, in particular 7 to 14 amino acids, in particular 8 to 13 amino acids, in particular 9 to 12 amino acids, in particular 10 to 11 amino acids. A dipeptide, such as a GT dipeptide, is a particularly suitable linker. A linker comprising or consisting of an amino acid sequence having at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identity to SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46 or SEQ ID NO. 47 is also a particularly suitable linker. 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 connecting peptide produced by a chemical-enzymatic coupling reaction by Sortase A. SEQ ID NO.47 represents a connecting peptide produced by a chemical-enzymatic coupling reaction by transglutaminase. The virus binding portion or mucin binding portion peptide is coupled to a glutamyl residue (Q7) via a γ-glutamyl-ε-lysyl isopeptide bond.

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

[0025] In one embodiment, the virus binding moiety is covalently bound to the mucin binding moiety at amino acid position 57 of the mucin binding moiety. In one embodiment, the amino acid at position 57 is a cysteine ​​residue. In one embodiment, the amino acid at position 57 is a lysine residue. In one embodiment, the virus binding moiety is covalently bound to the mucin binding moiety at the C-terminus of the mucin binding moiety. In particular in the latter embodiment, which amino acid is present at position 57 (or at any other position) of the mucin binding moiety is of no particular importance for the ability to bind to the virus binding moiety.

[0026] In one embodiment, the peptide comprises at least two virus binding moieties. In this case, the mucin binding moiety is covalently bound to at least one of the at least two virus binding moieties, either directly or through a linker. The mucin binding moiety may also be bound to more than one virus binding moiety. For this purpose, in one embodiment, a bifurcated linker is particularly helpful. Such a bifurcated linker can be used to couple a single mucin binding moiety to multiple virus binding moieties that are arranged at approximately equal distances from the mucin binding moiety after coupling.

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

[0028] In one embodiment, the virus binding moiety is a peptide. In one embodiment, the virus binding moiety is an antimicrobial peptide, such as a defensin. Retrocyclin is a particularly suitable defensin. In another embodiment, the virus binding moiety is a non-peptide antiviral residue, such as a dendritic or linear polysulfate or polysaccharide.

[0029] In one embodiment, the virus binding moiety comprises or is a peptide that is at least 95%, particularly at least 96%, particularly at least 97%, particularly at least 98%, particularly at least 99%, particularly 100% identical to:

[0030]

[0031] These sequences relate to viral binding portions that are particularly suitable for binding to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0032] In one embodiment, the virus binding moiety is a peptide, wherein at least one residue of the peptide carries a replacement. In one embodiment, the C-terminal of the peptide is replaced by an amino group. In one embodiment, the N-terminal of the peptide is replaced by an acetyl group. In one embodiment, the C-terminal of the peptide is replaced by an amino group, and the N-terminal of the peptide is replaced by an acetyl group.

[0033] In one embodiment, the virus binding moiety is a peptide having at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identity to SEQ ID NO.2, and the mucin binding moiety (1) is a trefoil factor 3 peptide having at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identity to SEQ ID NO.1. SEQ ID NO.2 describes a peptide denoted p4, which has been shown in the past to bind to severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) with high affinity (Han et al., 2006). Despite the structural differences between SARS-CoV-1 and SARS-CoV-2, it was unexpectedly found that the p4 peptide is also very suitable for binding to SARS-CoV-2.

[0034] In one embodiment, the virus binding moiety is a peptide having at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identity to SEQ ID NO.3, and the mucin binding moiety (1) is a trefoil factor 3 peptide having at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identity to SEQ ID NO.1. SEQ ID NO.3 describes a peptide denoted p6, which has been shown in the past to bind to severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) with high affinity (Han et al., 2006). Despite the structural differences between SARS-CoV-1 and SARS-CoV-2, it was unexpectedly found that the p6 peptide is also very suitable for binding to SARS-CoV-2. Thus, the binding affinity of the p6 peptide is approximately twice as high as the affinity of the p4 peptide for SARS-CoV-2. Interestingly, p6 can also bind to SARS-CoV-2 variants α and δ with even higher affinity than the SARS-CoV-2 wild type (see Table 1).

[0035] It was observed that stapled variants of the p6 peptide had an even higher affinity for SARS-CoV-2 than the linear p6 peptide. In one embodiment, the virus-binding portion is a peptide that is at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identical to SEQ ID NO. 10. The peptide is a stapled variant of the p6 peptide. In the following, it is also referred to as p6-cyc or p6(R8)15 (S5) 22 .

[0036] The binding affinities (expressed as dissociation constants Kd) of the different virus binding moieties for SARS-CoV-2 are summarized in Table 1 below. They were determined by microscale thermophoresis (MST). It can be clearly seen that the p4 and p6 peptides (the latter in both their linear and cyclized forms) bind to several receptor binding domains of the currently circulating SARS-CoV-2 variants with nanomolar binding constants.

[0037] Table 1: Properties of different virus binding moieties.

[0038]

[0039] *1 At position 15 of p6-cyc, X(X 15 ) is an olefinated (R)-Ala residue; X (X 22 ) is an olefinic (S)-Ala residue; the peptide is olefinic through a 7-undecenyl residue in X 15 and X 22 There is cyclization between them.

[0040] *2 PBS = phosphate-buffered saline

[0041] *3 TFE = trifluoroethanol

[0042] In one embodiment, the peptide has at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99%, in particular 100% identity to SEQ ID NO. 18 or SEQ ID NO. 19. These SEQ ID NOs. relate to synthetic and recombinant TFF-3-p6 peptides which have been shown to have properties very suitable for solving the purposes of the present invention.

[0043] In one aspect, the invention relates to a peptide according to the previous description for use as a medicament.

[0044] In one aspect, the invention relates to the medical use of a peptide according to the previous description for the prevention or treatment of a viral infection.

[0045] In one embodiment, the viral infection to be treated or prevented is a SARS-CoV-2 infection.

[0046] In one aspect, the present invention relates to a medical method for treating or preventing viral infection in a patient in need of such treatment. The term "patient" as used herein refers to a human or animal patient. Mammals are particularly suitable animal patients. The method includes the step of administering a peptide according to the previous description to the patient (e.g., in the form of a medicament comprising the peptide). In one embodiment, this administration is accomplished by providing the peptide to the patient's nose or mouth. This can be particularly easily achieved by providing the peptide in the form of a spray. The peptide can then be administered, for example, as an aerosol such as a nasal spray or an inhalation spray. After administration, it will then contact the patient's mucosa and will allow the peptide to bind to the mucosa through the mucin binding portion. Afterwards, the peptide will remain in its bound form on the patient's mucosa and will be able to "filter" the virus out of the air inhaled by the patient due to its virus binding portion.

[0047] In one aspect, the present invention relates to a medical method for enhancing mucus in a patient in need thereof. The method comprises the step of administering to the patient a peptide according to the previous description (eg in the form of a medicament comprising the peptide).

[0048] In one aspect, the present invention relates to a medicament comprising a peptide according to the previous description as a pharmaceutically active ingredient. The medicament may comprise other pharmaceutically active ingredients. In one embodiment, the peptide is the only pharmaceutically active ingredient.

[0049] In one embodiment, the drug is formulated so that it can be administered as an aerosol (e.g., a nasal spray or inhalation spray). The drug (and the peptides therewith as the active pharmaceutical ingredient) then directly contacts the mucosa of the nasopharyngeal region after its administration. This in turn leads to mucus enhancement as described above. As a result, the patient's mucus is better prepared to bind and subsequently eliminate the virus that the patient is facing (either by inhaling the virus from the environment, or by exhaling the virus from the virus-infected cells of the patient's body).

[0050] In one aspect, the present invention relates to a method for preparing a peptide according to the previous description. The method comprises the steps described below. First, a virus binding moiety precursor is reacted with a first mucin binding moiety precursor. In this case, the first mucin binding moiety precursor comprises the first part of the final mucin binding moiety. The reaction results in the formation of a peptide precursor.

[0051] Afterwards, the peptide precursor is reacted with a second mucin binding moiety precursor. The second mucin binding moiety precursor comprises the second part of the mucin binding moiety. As a result of this second reaction step, the final peptide is formed. Of course, other intermediate reaction steps may also be completed. As an example, the mucin binding moiety may be divided into more than two mucin binding moiety precursors. The individual mucin binding moiety precursors are then reacted one after another with the formed peptide precursor to finally produce the peptide.

[0052] In one embodiment, the virus binding moiety precursor comprises a 4-pentynyl moiety that establishes a covalent bond with the mucin binding moiety of the final peptide. The 4-pentynyl moiety may still be present (in whole or in part) in the final peptide. In one embodiment, it may serve as a linker between the virus binding moiety and the mucin binding moiety.

[0053] In one embodiment, the peptide comprises a triazole residue that acts as a linker between the virus binding portion and the mucin binding portion. In one embodiment, the peptide comprises a polyethylene glycol residue that acts as a linker between the virus binding portion and the mucin binding portion. In one embodiment, such a polyethylene glycol residue is combined with a triazole residue to form a linker.

[0054] In one embodiment, the first mucin binding moiety precursor comprises less than half of all the amino acids of the mucin binding moiety. If only two mucin binding moiety precursors are used, the second mucin binding moiety precursor comprises more than half of all the amino acids of the mucin binding moiety. As a specific example, the first mucin binding moiety precursor comprises amino acids 36 to 59 of the mucin binding moiety, which in this embodiment is a trefoil factor 3 peptide. In addition, the second mucin binding moiety precursor comprises amino acids 1 to 35 of the mucin bend. After the first mucin binding moiety precursor (corresponding to the C-terminal portion of the mucin binding moiety) is reacted with the virus binding moiety, the second mucin binding moiety precursor (corresponding to the N-terminal portion of the mucin binding moiety) is reacted with the already formed first mucin binding moiety precursor and the construct of the virus binding moiety (i.e., the peptide precursor). As a result, the final peptide is formed.

[0055] All embodiments of the peptides can be combined in any desired manner and can be transferred to uses, medicines and different methods individually or in any arbitrary manner. Likewise, all embodiments of different uses can be combined in any desired manner and can be transferred to peptides, corresponding other uses, medicines and different methods individually or in any arbitrary manner. Likewise, all embodiments of medicines can be combined in any desired manner and can be transferred to peptides, different uses and different methods individually or in any arbitrary manner. In addition, all embodiments of different methods can be combined in any desired manner and can be transferred to peptides, different uses and corresponding other methods individually or in any arbitrary manner.

[0056] Further details of aspects of the present invention will be described below with reference to exemplary embodiments and accompanying drawings. In the drawings:

[0057] Figure 1A The structure of a first embodiment of a bifunctional peptide is schematically shown;

[0058] Figure 1BThe structure of a second embodiment of a bifunctional peptide is schematically shown;

[0059] Figure 2A The primary structure of one embodiment of the first mucin binding moiety precursor corresponding to SEQ ID NO. 15 is shown;

[0060] Figure 2B The primary structure of one embodiment of the second mucin binding moiety precursor corresponding to SEQ ID NO. 16 is shown;

[0061] Figure 2C The primary structure of one embodiment of the virus binding moiety precursor corresponding to SEQ ID NO. 17 is shown;

[0062] Figure 3 The primary structure of one embodiment of a peptide corresponding to SEQ ID NO. 18 is shown, the peptide comprising Figure 2A and 2B The mucin binding moiety is formed by the first and second mucin binding moiety precursors of Figure 2C The virus binding part formed by the virus binding part precursor;

[0063] Figure 4 The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 4 is shown;

[0064] Figure 5 The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 5 is shown;

[0065] Figure 6 The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 6 is shown;

[0066] Figure 7 The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 7 is shown;

[0067] Figure 8 The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 8 is shown;

[0068] Fig. 9 The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 9 is shown;

[0069] Fig.10 The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 10 is shown;

[0070] Fig.11The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 11 is shown;

[0071] Fig.12 The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 12 is shown;

[0072] Fig.13 The primary structure of one embodiment of the virus binding portion corresponding to SEQ ID NO. 13 is shown;

[0073] Fig.14 A graph illustrating the binding affinity between the p6 peptide and different SARS-CoV-2 variants is shown;

[0074] Fig.15 A graph illustrating the binding affinity between the bifunctional synthetic TFF3-p6 peptide corresponding to SEQ ID NO. 18 and different SARS-CoV-2 variants is shown;

[0075] Fig.16 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 23 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0076] Fig.17 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 24 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0077] Fig.18 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 25 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0078] Fig.19 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 26 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0079] Fig. 20 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 28 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0080] Fig.21A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 29 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0081] Fig. 22 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 30 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0082] Fig.23 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 31 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0083] Fig.24 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 32 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0084] Fig.25 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 33 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0085] Fig.26 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 34 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0086] Fig. 27 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 35 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0087] Fig.28 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 36 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0088] Fig.29 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 37 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0089] Fig.30A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 38 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0090] Fig.31 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 39 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0091] Fig.32 A graph illustrating the binding affinity between the p4 peptide variant corresponding to SEQ ID NO. 40 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0092] Fig.33 A graph illustrating the binding affinity between the p6 peptide variant corresponding to SEQ ID NO. 41 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0093] Fig.34 A graph illustrating the binding affinity between the p6 peptide variant corresponding to SEQ ID NO. 42 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown;

[0094] Fig.35 A graph illustrating the binding affinity between the p6 peptide variant corresponding to SEQ ID NO. 43 and the receptor binding domain of SARS-CoV-2 (2019-nCoV) is shown; and

[0095] Fig.36 A graph illustrating the binding affinity between the bifunctional recombinant TFF3-p6 peptide corresponding to SEQ ID NO. 19 and different SARS-CoV-2 variants is shown.

[0096] Figure 1A The structure of a first embodiment of a bifunctional peptide, i.e., TFF3-p6, is schematically illustrated. This is a peptide in which trefoil factor 3 (TFF3) peptide 1 is the mucin binding portion and in which p6 peptide 2 is the virus binding portion. 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 bound to lysine 57 of the TFF3 peptide.

[0097] Figure 1AAn alternative embodiment of a bifunctional peptide is shown. In this and all subsequent figures, similar elements will be indicated by the same reference numerals. Here, p6 peptide 2 is covalently bound to the C-terminus of TFF3 peptide 1.

[0098] The synthesis of TFF3-p6 will be described in more detail below.

[0099] For the production of TFF3, the protocol of Braga Emidio et al. 2021 was followed. Relative to the synthetic route published by Braga Emidio et al., azidolysine was introduced into TFF3 by replacing cysteine ​​at position 57.

[0100] TFF3(36-59) (C57AzK), which represents one embodiment of the first virus-binding moiety precursor, was prepared entirely by SPPS on Rink amide resin using the corresponding building block of azidolysine (AzK) at position 57.

[0101] TFF3 (36至59) C57AzK[M+H] + =2825.31 g / mol, [M+2H] 3+ / 3=943.7g / mol. The primary structure is as follows Figure 2A as shown in .

[0102] TFF3 (1 to 35) -Nbz, representing one embodiment of a second viral binding moiety precursor, was synthesized on Rink amide resin, which was initially functionalized by conventional coupling methods with 3-fluorenylmethoxycarbonyl-4-diaminobenzoic acid (3-fluorenylmethoxycarbonyl-4-diaminobenzoic acid, Fmoc-Dbz) (5 equivalents (eq) Fmoc-Dbz, 4.9 equivalents hexafluorophosphate azabenzotriazoletetramethyl uronium (HATU), 10 equivalents N,N-diisopropylethylamine (Dipea), sufficient to prepare a 0.2M solution of 3-Fmoc-4-diaminobenzoic acid (Fmoc-Dbz) in dimethylformamide (DMF). Then, conventional solid phase peptide synthesis (SPPS) was performed. The final residue was added as a Boc-protected amino acid. After the peptide was assembled on the resin, the C-terminus was activated by acylation with p-nitrophenyl chloroformate (5 equiv., CH2Cl2, 45 min) followed by the addition of base (Dipea, 0.5 M in DMF for 20 min) to promote intramolecular attack of the aniline to form the resin-bound benzimidazolinone. Cleavage from the resin was performed using 94:2.5:2.5:1.0 trifluoroacetic acid (TFA):1,2-Ethanedithiol (EDT):H2O:Triisopropylsilane (TIPS), and the crude was dissolved in 3 mL of 30% acetonitrile (MeCN) / H2O containing 0.1% TFA, filtered, and injected on a preparative high performance liquid chromatography (HPLC) system for purification. TFF3(1 to 35)-Nbz.3980.4:M + , 996.1: (M+4H) 4+ / 4The primary structure is as follows Figure 2B as shown in .

[0103] Pentynyl-(PEG)2-p6, representing one embodiment of a virus binding moiety, was formed by SPPS on Rink amide resin up to two PEG units. 4-pentynoic acid was then coupled manually (5 equivalents of 4-pentynoic acid, 4.9 equivalents of HATU and 10 equivalents of Dipea in DMF sufficient to make a 0.2 M solution of 4-pentynoic acid). The coupling was performed twice for 1.5 hours. The peptide was then cleaved with 95:2.5:2.5 TFA:H2O:TIPS and purified by preparative HPLC. Penty-p6[M+H] + =3975.90 g / mol, [M+2H] 3+ / 3=994.9g / mol. The primary structure is as follows Figure 2C as shown in .

[0104] In a vial, CuSO4 (10 eq.) was added from a 1 mM solution and mixed with THPTA (50 eq.) The organometallic complex was allowed to form while the other reagents were mixed.

[0105] In order to form a peptide precursor by click chemistry between azidolysine at position 57 and the alkynyl group of pentynyl-(PEG)2-p6, pentynyl-(PEG)2-p6 (1.5 equivalents) and TFF3 (36 to 59) (C57AzK) (1.0 equivalents) were dissolved in a reaction buffer (10 mM NH4HCO3) in a 1.5 mL Eppendorf tube. Subsequently, aminoguanidine (100 equivalents) was added from a 200 mM stock solution. Then, a mixture of CuSO4 and THPTA was added to the reaction mixture. Finally, sodium ascorbate (100 equivalents) was added to the reaction and the pH was adjusted to 8.0 with HCl / KOH. Nitrogen was allowed to flow into the reaction tube. The reaction tube was then sealed immediately. The incubation was then stirred at 37°C for 12 hours. A control was formed by dissolving 1 μL of the reaction crude in 20 μL 30% MeCN / H2O and then adding tris(2-carboxyethyl)phosphine (TCEP) to a concentration of 30 mM. After incubation at 37°C for 5 minutes, the samples were analyzed by HPLC-mass spectrometry (HPLC-MS).

[0106] For native chemical connection between TFF3 (1 to 35) -Nbz and TFF3 (36 to 59) (C57K-Pentyp6), TFF3 (1 to 35) -Nbz (1.0 equivalent) and TFF3 (36 to 59) (C57K-Pentyp6) (0.8 equivalent) were dissolved in reaction buffer (6M guanidine hydrochloride, 100mM NaHPO and 30mM TCEP, pH adjusted to 7.0 with HCl / NaOH). Then 2-mercaptoethanesulfonate (MeSNa) (100 equivalent) was added, and the reaction was stirred continuously at room temperature for 4 hours. By dissolving 1 μL of the reaction crude in 20 μL 30% acetonitrile (MeCN) / H2O, and then adding TCEP to reach a 30mM concentration to form a control. After incubation at 37°C for 5 minutes, the sample was analyzed by HPLC-MS. When no trace of thioester remained, the reaction volume was increased to 3 mL by adding 30% MeCN / H2O containing 0.1% TFA, filtered, and injected onto a preparative HPLC system for purification. TFF3-C57K (Penty-p6) was obtained, which has

[0107] [M+H] + =10600.96 g / mol, [M+8H] 8+ / 8=1326.1g / mol,[M+9H] 9+ / 9=1178.8g / mol[M+10H] 10+ / 10=1061.1 g / mol.

[0108] The primary structure is Figure 3 as shown in .

[0109] Native TFF3 was also produced recombinantly. To achieve this, the et al., 2020.

[0110] The TFF3 sequence was then extended at the 5' end to introduce a purification tag. Afterwards, the product was integrated into the plasmid pET28b for bacterial expression using sequence-and ligation-independent cloning (SLIC). The final plasmid was approved by sequencing and transformed into BL21 CodonPlus (DE3) RIPL (R = arginine, I = isoleucine, P = proline, L = leucine) cells for bacterial expression. This will be described in more detail below.

[0111] Preparation of exponentially growing stock culture of transformed Escherichia coli BL21 CodonPlus(DE3)RIPL

[0112] Escherichia coli BL21 Codon Plus (DE3) RIPL cells are transformed with the corresponding plasmids. The cells are streaked on lysogeny broth (LB) agar plates containing 50 μg / mL streptomycin, 25 μg / μL chloramphenicol and 50 μg / mL kanamycin, and incubated overnight in an incubator at 37 ° C. Pick a colony and inoculate LB culture medium containing 50 μg / mL streptomycin, 25 μg / μL chloramphenicol and 50 μg / mL kanamycin, and incubate overnight at 37 ° C in a rotary shaking incubator at 250rpm. Terrific broth (TB) culture medium is measured at an initial optical density (OD 600 ) was inoculated with LB medium at 0.02 and incubated in a rotary shaking incubator at 250 rpm and 37°C. The culture was grown to an OD of 600 7.527% DMSO was added to the culture, and the mixture (preculture) was snap frozen in liquid nitrogen and stored at -80°C for further use.

[0113] Protein expression and washing

[0114] The TB medium was diluted with OD 600 The pre-culture inoculation of 0.04. The culture was incubated at 26 ° C at 250 rpm in a rotary shaking incubator for 24 hours. After 24 hours, the bacterial culture was cooled on ice for 15 minutes and centrifuged at 3000 g for 15 minutes. The supernatant was discarded and the cells were resuspended in washing buffer (based on HEPES, pH 7) + DNA enzyme I + phenylmethylsulfonyl fluoride (phenylmethylsulfonylfluorid, PMSF). The suspension was centrifuged at 3000 g for 15 minutes and the supernatant was discarded. The washing step was repeated.

[0115] The target protein was purified in a tandem affinity purification using a His-tag and a Strep-tag (binding motif for streptavidin). A protease cleavage site allowed the protein to be eluted from the column. The eluted protein was dialyzed and verified on 5728 SDS-PAGE.

[0116] Preparation of lactam-stapled peptides

[0117] All lactam stapled peptides are prepared on Rink amide resin. The structural units for isopeptide bond formation are N-α-Fmoc-N-ε-4-methyltrityl-L-lysine (hereinafter referred to as MttK) and N-α-Fmoc-L-aspartic acid β-2-phenylisopropyl ester (hereinafter referred to as PhiPr). After SPPS, the peptide bound to the resin is treated with 2% TFA in dichloromethane (DCM) to induce deprotection of 4-methyltrityl and 2-phenylisopropyl. Then, isopeptide bond formation is allowed to occur, followed by the addition of 0.9 equivalents of HATU and 2 equivalents of DIPEA in DMF. After thorough rinsing in DMF and DCM, a second addition of the coupling mixture is performed. The peptide is cut from the resin using 95:2.5:2.5 TFA:H2O:TIPS and precipitated with cold ether. The peptide was then filtered, dried, and redissolved in 30% MeCN / H2O containing 0.1% TFA to a final volume of 3 mL. After filtration, the sample was injected into a preparative HPLC system for purification.

[0118] The resulting structure of the lactam-stapled peptide is as follows Figures 4 to 9 as shown in .

[0119] The binding affinities (expressed as dissociation constants, Kd) of different circular virus binding moieties to SARS-CoV-2 are summarized in Table 2 below. They were determined by microscale thermophoresis (MST). It can be clearly seen that the cyclic p6 peptide binds to SARS-CoV-2 with a micromolar binding constant.

[0120] Table 2: Properties of different circovirus binding moieties.

[0121]

[0122] nd = not determined

[0123] Preparation of stapled virus binding moieties by ring closing metatesis (RCM)

[0124] For the RCM reaction, the following atypical amino acids were used:

[0125] S5Fmoc=FmoC-(S)-2-(4-pentenyl)Ala-OH

[0126] R8FmoC=Fmoc-(R)-2-(7-octenyl)Ala-OH

[0127] The coupling conditions for these amino acids (AA) were: 3.0 equivalents AA, 2.9 equivalents HATU, 6 equivalents DIPEA. DMF was added to reach a 0.2 M concentration relative to AA.

[0128] For all natural amino acids, coupling conditions were: 5.0 eq. AA, 4.9 eq. HATU, 10 eq. DIPEA. DMF was added to reach a 0.2 M concentration relative to AA.

[0129] After confirming the quality of the non-stapled peptide by test cutting, RCM was performed. The resin was washed with 1mL DCM (3×1 minute) and then washed with 1mL of 1,2-dichloroethane (1,2-Dichloroethane, DCE) (3×1 minute). The resin (50μmol) was treated with a 6mM solution of 1mL of Grubbs first generation catalyst in DCE (20mol% relative to resin replacement). The resulting suspension was stirred for 2 hours at 20 to 25°C under nitrogen by continuous bubbling. Afterwards, the solvent was discharged. Then a double addition of Grubbs catalyst was performed. Subsequently, the resin was washed with 1mL of DCE (3×1 minute) and then washed with 1mL of DCM (3×1 minute) and then dried under a stream of nitrogen. The peptide was cut from the resin using 95:2.5:2.5 of TFA:H2O:TIPS. The crude was precipitated with cold ether, filtered and dried. The product was redissolved in 30% MeCN / H2O containing 0.1% TFA to a final volume of 3 mL. After filtration, the sample was injected into a preparative HPLC system for purification.

[0130] By swelling the resin of Fmoc deprotection in 1mL N-methyl-2-pyrrolidone (N-methyl-2-pyrrolidone, NMP) for 10 minutes and discharging solvent, RCM product is acetylated.Then, prepare 2mL of acetic anhydride and DIPEA solution in NMP (volume ratio is 85:315:1,600) and add in resin.Mixture is stirred for 45 minutes under nitrogen with bubbling gently, and discharging solvent subsequently.Fully wash with DCM and DMF subsequently.Finally, cut and purify as described above.

[0131] After deprotection, the peptide contains i) an S5 residue (alkenylated S-alanine) and an R8 residue (alkenylated R-alanine) or ii) two S5 residues. An alkenyl bridge is formed between i) an S5 residue and an R8 residue or between ii) two S5 residues. In the case of an S5 residue and an R8 residue, the alkenyl bridge is a 7-undecenyl residue. In addition, in the case of two S5 residues, the alkenyl bridge is a 4-octenyl residue.

[0132] The resulting structure of the stapled peptide produced by RCM is shown in Figures 10 to 13 as shown in .

[0133] The properties of the different circovirus binding moieties, including the binding affinity for SARS-CoV-2 (expressed as the dissociation constant Kd), are summarized in Table 3 below. The binding affinity was determined by microscale thermophoresis (MST). It can be clearly seen that the circovirus binding moieties p6(R8) 15 (S5) 22 Binds to SARS-CoV-2 with a nanomolar binding constant.

[0134] Table 3: Properties of different circovirus binding moieties.

[0135]

[0136] nd = not determined

[0137] Peptide binding affinity measurements

[0138] Affinity measurements are performed using microthermophoresis (MST). Here, the ratio of chemical and thermal diffusion (Soret effect) affects the mobility of the labeled compound (here: RBD) depending on the number of bound ligands (here: peptides). In MST, an infrared laser induces local hot spots and subsequently induces a temperature gradient. The molecules move along this gradient at the water-protein interface according to their properties. The binding of ligands leads to changes in the observed ordered water shell of the protein and can also change or increase the effective surface potential. This leads to changes in the thermophoretic behavior, which is manifested as an increase or decrease in the thermophoretic amplitude. After adding different concentrations of ligand, the changes in fluorescence (y-axis) can be plotted against the ligand concentration. From the half-maximum change in fluorescence (inflection point of the saturation curve), the Kd of the protein-ligand interaction can be derived according to the application of the mass-action law (Jerabek-Willemsen et al. 2014).

[0139] RBD labeling and purification were performed using the 2nd generation NHS-Red labeling kit (NanoTemper). The RBD solution was diluted to 10 μM with water, rebuffered in an aqueous sodium carbonate solution at pH 8.0, and incubated in the dark in 300 μM red dye. Size exclusion chromatography allows the obtained labeled RBD free of unreacted dye. From the spectral measurement analysis, the labeling efficiency was approximately 1:1 (protein: dye). Affinity measurements were performed in Dulbecco's Phosphate Buffered Saline (DPBS) supplemented with 0.05% (v / v) Tween 20 (Ca-free). 2+ and Mg 2+), and high-quality capillaries (NanoTemper).

[0140] MST was then measured by making 16 serial 1:1 dilutions of each peptide (final volume of each dilution was 10 μL) using PBS + 0.05% Tween 20 as diluent. Each diluted sample was then incubated with 10 μL of 10 nM labeled RBD stock solution, thus always maintaining a 5 nM concentration of labeled target protein in each sample.

[0141] The MST signal was then acquired in a NanoTemper Technologies Monolith NT.115Pico instrument at 20% excitation power and 40% MST power. The signal was analyzed 1.5 seconds after the IR laser was turned on, and the acquired data were fitted as previously shown (Bhatia et al. 2017). These conditions were kept the same for all measured samples.

[0142] Affinity measurement of p6 for RBD (SARS-CoV-2, o B.1.1.529)

[0143] In addition, the chemically synthesized p6 peptide (according to SEQ ID NO.3) was titrated against a constant concentration (5 nM) of the RBD of the fluorescently labeled SARS-CoV-2o variant (strain B.1.1.529). The dissociation constant (Kd) was determined to be 280 nM. Therefore, the p6 peptide also showed high binding affinity to the SARS-CoV-2o variant.

[0144] Fig.14 The results of the previously described affinity characterization of the p6 peptide (according to SEQ ID NO. 3) against the fluorescently labeled receptor binding domain (RBD) of wild type (filled circles), α variant (open circles), δ variant (triangle) and o variant (square) from SARS-CoV-2 using MST technology are shown (wild type, α variant and δ variant: N=3, error bars show SEM). It can be clearly seen that the p6 peptide binds to all SARS-CoV-2 variants / strains with high affinity (see also Table 1 for the quantitative results of these experiments).

[0145] p4 and p6 derivatives

[0146] A number of additional p4 and p6 derivatives were synthesized and tested for affinity to the receptor binding domain in an MST experiment as described above for the p6 peptide.

[0147] All p4 derivatives were synthesized as described in Sarto et al. 2022. The p6 derivatives p6-rigid, p6-dis, and p6-rigid-dis were ordered as TFA salts from PSL (Peptide Specialty Laboratories GmbH, Heidelberg). Upon arrival, the peptides were reconstituted in PBS, snap-frozen in liquid nitrogen, and stored at -80°C until further use.

[0148] Table 4 lists the different tested p4 derivatives and the dissociation constants obtained from the affinity measurements. The cysteine ​​residues of all peptides were in their normal, reduced state. The results of the affinity measurements are also given in Figures 16 to 32 Shown in.

[0149] Table 4: Properties of p4 derivatives (virus binding moiety).

[0150]

[0151] *4 CI = Confidence Interval

[0152] nd = not determined

[0153] All p4 derivatives showing a dissociation constant against SARS-CoV-2 RBD of less than 0.3, in particular less than 0.2, in particular less than 0.1, in particular less than 0.05, in particular less than 0.04, in particular less than 0.03, in particular less than 0.02 are particularly suitable virus-binding moieties.

[0154] Table 5 lists the different tested p6 derivatives and the dissociation constants obtained from the affinity measurements. The cysteine ​​residues of all peptides were in their oxidized state so that they can form intramolecular disulfide bonds. The results of the affinity measurements are also given in Figures 33 to 35 Shown in.

[0155] Table 5: Properties of p6 derivatives (virus binding moiety).

[0156]

[0157] Recombinant production of TFF3-p6

[0158] TFF3-p6 was also produced recombinantly. To achieve this, the et al., 2020.

[0159] 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). Afterwards, the product was integrated into the plasmid pET28b for bacterial expression using sequence-independent and ligated cloning (SLIC). The final plasmid was approved by sequencing and transformed into BL21 Codon Plus (DE3) RIPL cells for bacterial expression. This was performed in accordance with the detailed description in the paragraph "Preparation of exponentially growing stock cultures of transformed E. coli BL21 COdon Plus (DE3) RIPL" above.

[0160] Protein expression and washing

[0161] Use OD 600 The TB culture medium was inoculated with a pre-culture of 0.04. The culture was incubated at 26 ° C for 24 hours at 250 rpm in a rotary shaking incubator. After 24 hours, the bacterial culture was cooled on ice for 15 minutes and centrifuged at 3000 g for 15 minutes. The supernatant was discarded and the cells were resuspended in a wash buffer (based on HEPES, pH 8). The suspension was centrifuged at 3000 g for 15 minutes and the supernatant was discarded. The washing step was repeated. The bacterial cells were lysed in a lysis buffer (based on HEPES, pH 8) by ultrasound. The lysate was centrifuged at 12.000 g for 45 minutes, the precipitation was discarded, and the supernatant was filtered through a 0.2 μm filter.

[0162] Protein purification

[0163] Purification is performed when the target protein is purified using a His-tag and a Strep-tag (binding motif for streptavidin) for tandem affinity purification. Imidazole (ImH) is added to the filtered bacterial lysate to a final concentration of 40 mM. Pure 25 (Cytiva) fast protein liquid chromatography (fast protein liquid chromatography, FPLC) device applies lysate to HisTrap column (Cytiva). Column wash buffer (300mM NaCl, 50mM Tris-base, 40mM ImH, pH 7.5) is washed, protein elution buffer (300mM NaCl, 50mM Tris-base, 400mM ImH, pH 7.5) is eluted, and elution is collected in fractions. The fraction containing the target product is identified and merged by SDS-PAGE.

[0164] The combined fractions were applied to The Strep-Tactin XT 4Flow column (IBA Lifesciences) of Pure 25. The column is washed with washing buffer (100mM Tris-HCl, 150mM NaCl, 1mM ETDA, pH 8), and the protein is eluted with elution buffer (100mM Tris-HCl, 150mM NaCl, 1mM ETDA, 50mM biotin, pH 8), and the eluate is collected in fractions. The fraction containing the target product is identified by SDS-PAGE, and merged before being dialyzed against PBS (pH 7.4). NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) is used to determine the protein content by the absorption spectrum at 280nM.

[0165] To remove the tandem affinity purification tag, the purified product was incubated with ProTEV Plus (Promega) at 30°C overnight using 1 U ProTEV Plus per 15 μg of purified product according to the manufacturer's recommendations.

[0166] ImH was added to the reaction mixture to a final concentration of 40 mM, which was then applied to the The HisTrap column of Pure 25, and the column is washed with washing buffer (300mM NaCl, 50mM Tris-base, 40mM ImH, pH 7.5). The final product (TFF3-p6) flowing through is collected in fractions, and the purification tag and ProTEV Plus (also containing His-tag) of cutting are retained in the column. The fraction containing the final product is identified by SDS-PAGE, merged and dialyzed against PBS (pH7.4). Vivaspin PES centrifugal concentrator (Sartorius) is used to reduce the volume to a final protein concentration of 1mg / ml to 3mg / ml determined by NanoDrop2000. The solution is quick-frozen in liquid nitrogen, and stored at -20°C until further use. The correct final product and purity are controlled by SDS-PAGE and ultra-high performance liquid chromatography-mass spectrometry (Ultra-Performance-Liquid-Chromatography-Mass-Spectrometry, UPLC / MS).

[0167] Affinity measurement of TFF-p6 for the RBD of SARS-CoV-2

[0168] In these experiments, chemically synthesized TFF3-p6 (TFF3-C57K (Penty-p6) or TFF3-p6syn; the primary structure of which is shown in Figure 3 The recombinant TFF3-p6 according to SEQ ID NO. 19 (TFF3-p6 rec) was titrated against a constant concentration (5 nM) of fluorescently labeled wild-type SARS-CoV2 (also denoted nCov2019) and the delta variant of SARS-CoV2 (also denoted delta (B.1.617.2)). The resulting dissociation constants (Kd) are listed in Table 6. The affinity measurements of the TFF3-p6 syn are shown in Table 6. Fig.15 As shown in , and the affinity measurement results of TFF3-p6 rec are shown in Fig.36 as shown in .

[0169] Table 6: Dissociation constants measured for the affinity of TFF-p6 for the RBD of SARS-CoV-2.

[0170]

[0171] Cytotoxicity assay

[0172] Cell viability was determined using a cell counting kit (Hycultec, HY-K0301) according to the manufacturer's instructions.

[0173] A549, HBE and Calu-3 cells were cultured in DMEM supplemented with 10% (v / v) FBS, 100 U / mL penicillin and 100 μg / mL streptomycin. 4 Cells were seeded at a density of 10 cells / mL in 96-well plates in 90 μL DMEM medium per well at 37° C. and 5% CO 2 overnight.

[0174] 10 μL of recombinant TFF3-p6 or tag-TFF3-p6 (still containing the affinity tag) dissolved in deionized water was added to a dilution series (6.6×10 -6 M to 6.6×10 -10 M), and incubated 48 hours in addition at 37 ℃ and 5% CO2.For background subtraction, wells containing only samples without cells were also used.After incubation for 48 hours, CCK8 solution (10 μL / well) was added, and the absorbance (450nm / 650nm) after dye incubation for about 3 hours was measured using a Tecan plate reader (SPARK, TECAN-reader Tecan Group Ltd.).The cell viability was calculated by setting the untreated control to 100% after subtracting the background signal.

[0175] Neither recombinant TFF3-p6 nor tag-TFF3-p6 showed significant differences from the negative control. Instead, in all cases, cell viability was approximately 100%. In contrast, the positive control showed cell viability of 0% or close to 0%.

[0176] Antiviral efficacy

[0177] To test the antiviral efficacy of TFF3-p6, a pseudovirus infection system based on vesicular stomatitis virus (VSV) lacking VSV envelope glycoproteins but containing eGFP and the SARS-CoV-2 spike protein (VSVΔG-CoV-2; Markus Hoffmann et al.) was used.

[0178] TFF3-p6 or PBS (control) was mixed with VSVΔG-CoV-2 at 50 μM TFF3-p6 and 2.625×10 4 The mixture was added to VetoE6 cells and incubated at 37°C in a cell culture incubator for 24 hours. The number of infected cells was determined using a fluorescence microscope.

[0179] TFF3-p6 reduced the infection of VetoE6 cells by VSVΔG-CoV2 pseudovirus by approximately 40% compared with the PBS control.

[0180] List of references cited in previous chapters

[0181] 1.Bhatia, Sumati, et al. "Linear polysialoside outperforms dendriticanalogs for inhibition of influenza virus infection in vitro and in vivo." Biomaterials 138 (2017): 22-34.

[0182] 2. Braga Emidio, Nayara, et al. "Chemical Synthesis of TFF3 Reveals NovelMechanistic Insights and a Gut-Stable Metabolite." Joumal of MedicinalChemistry 64.13 (2021): 9484-9495.

[0183] 3.Bui,Vuong N.,et al.″Pathogenicity of an H5N1 avian influenza virusisolated in Vietnam in 2012and reliability of conjunctival samples fordiagnosis of infection.″Virus Research 179(2014):125-132.

[0184] 4.CabAn,Madelyn,et al.″Cross-protective antibodies against commonendemic respiratory viruses."NaCre Communications 14.1(2023):798.

[0185] 5.Han,Dong P.,Adam Penn-Nicholson,and Michael W.Cho.″Identificationof critical determinants on ACE2 for SARS-CoV entry and development of apotent entry inhibitor.″Virology 350.1(2006):15-25.

[0186] 6.Hoffmann,Markus,et a1.″SARS-CoV-2cell entry depends on ACE2 andTMPRSS2 and is blocked by a clinically proven protease inhibitor.″Cell 181.2(2020):271-280.

[0187] 7.Hoffmann,Miriam,Nicole L.Snyder,and Laura Hartmann.″Polymerslnspired by Heparin and Heparan Sulfate for Viral Targeting.″Macromolecules55.18(2022):7957-7973.

[0188] 8.Hoffmann,Werner.″Trefoil factor family(TFF)peptides and theirdifferent roles in the mucosal innate immune defense and more:An update.″Current Medicinal Chemistry 28.36(2021):7387-7399.

[0189] 9.Hrebik,Dominik,et al.″ICAM-1induced rearrangements of capsid andgenome prime rhinovirus 14 for activation and uncoating.″Proceedings of theNational Academy of Sciences 118.19(2021):e2024251118.

[0190] 10.Issmail,Leila,et al.″Prefusion-specific antibody-derived peptidestrivalently presented on DNA-nanoscaffolds as an innovative strategy againstRSV entry.″Frontiers in Virology 2(2022).

[0191] 11. Michael A.,et al.″Trefoil factors share a lectin activitythat defines their role in mucus.″Nature Communications 11.1(2020):1-9.

[0192] 12.Jerabek-Willemsen M.,et al.“MicroScale Thermophoresis:lnteractionanalysis and beyond.”Journal of Molecular Structure 1077(2014):101-113.

[0193] 13.Khan,Abdul Ghafoor,et al.″Human rhinovirus type 54 infection viaheparan sulfate is less efficient and strictly dependent on loW endosomalpH.″Journa / ofVirology 81.9(2007):4625-4632.

[0194] 14.Madsen,Anders,et al.″Human antibodies targeting influenza B virusneuraminidase active site are broadly protective.″lmmunity 53.4(2020):852-863.

[0195] 15.McLellan,Jason S.,et al.″Structure-based design of a fusionglycoprotein vaccine for respiratory syncytial virus.″science 342.6158(2013):592-598.

[0196] 16.Memczak,Henry,et al.″Anti-hemagglutinin antibody derived leadpeptides for inhibitors of influenza virus binding.″PLoS One 11.7(2016):e0159074.

[0197] 17.Sarto,Carolina,et al.″Atomistic insight into the essential bindingevent of ACE2-derived peptides to the SARS-CoV-2 spike protein.″BiologicalChemistry 403.5-6(2022):615-624.

[0198] 18.Smith,Thomas J.,et al.″Neutralizing antibody to human rhinovirus14 penetrates the receptor-binding canyon.″Nature 383.6598(1996):350-354.

[0199] 19.Thim,L.,F.Madsen,and S.S.Poulsen.″Effect of trefoil factors on theviscoelastic properties of mucus gels.″European Journal of ClinicalInvestigation32.7(2002):519-527.

[0200] 20.Zhu,Qing,et al.″A highly potent extended half-life antibody as apotential RSV vaccine surrogate for all infants.″ScienceTranslationalMedicine 9.388(2017):eaaj1928.

[0201] 21.Zuo,Teng,et al.″Comprehensive analysis of antibody recognition inconvalescent humans from highly pathogenic avian influenza H5N1 infection.″Nature Communications 6.1(2015):8855.

Claims

1. A peptide comprising a virus binding portion (2) and a mucin binding portion (1) covalently bound to the virus binding portion (2), Features: The virus binding portion (2) comprises or is a peptide having at least 95% identity with: 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; as well as The mucin binding moiety (1) comprises or is a peptide having at least 95% identity with SEQ ID NO.1, SEQ ID NO.14, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.

22.

2. The peptide according to claim 1, characterized in that The mucin binding moiety (1) comprises or is a trefoil factor 3 peptide having at least 95% identity to SEQ ID NO.

1.

3. A peptide according to any one of the preceding claims, characterized in that The peptide comprises at least two virus binding moieties (2), wherein the mucin binding moiety (1) is covalently bound to at least one of the at least two virus binding moieties (2).

4. A peptide according to any one of the preceding claims, characterized in that The virus binding portion (2) comprises or is a peptide having at least 95% identity with: 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. The peptide according to any one of the preceding claims, characterized in that: The virus binding part (2) is a peptide having at least 95% identity with SEQ ID NO.2, and the mucin binding part (1) is a trefoil factor 3 peptide having at least 95% identity with SEQ ID NO.

1.

6. A peptide according to any one of the preceding claims, characterized in that: The virus binding part (2) is a peptide having at least 95% identity with SEQ ID NO.3, and the mucin binding part (1) is a trefoil factor 3 peptide having at least 95% identity with SEQ ID NO.

1.

7. A peptide according to any one of the preceding claims, characterized in that The peptide has at least 95% identity with SEQ ID NO.

18.

8. The peptide according to any one of claims 1 to 6, characterized in that The peptide has at least 95% identity with SEQ ID NO.

19.

9. A peptide according to any preceding claim for use in the prevention or treatment of a viral infection.

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

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

12. The drug according to claim 11, characterized in that The drug is designed and formulated to be administered as a nasal spray or as an inhalation spray.

13. A method for preparing a peptide according to any one of claims 1 to 7, comprising the following steps: a) reacting the virus binding moiety precursor with a first mucin binding moiety precursor, said first mucin binding moiety precursor comprising the first part of the mucin binding moiety (1), thereby obtaining a peptide precursor; as well as b) reacting the peptide precursor with a second mucin binding moiety precursor comprising the second part of the mucin binding moiety (1).

14. The method according to claim 13, characterized in that The virus-binding moiety precursor comprises a 4-pentynyl moiety covalently bound to the virus-binding moiety (2).

15. The method according to claim 13 or 14, characterized in that The first mucin binding moiety precursor comprises less than half of all the amino acids of the mucin binding moiety (1).

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