Structurally stabilized antiviral SARS-CoV-2 peptide and its use

Abstract

Disclosed herein are cross-linked peptides useful for disrupting and inhibiting coronavirus infection (e.g., infection by SARS-CoV-2). Also disclosed are methods for treating and / or preventing coronavirus infection (e.g., COVID-19). The disclosure relates to structurally stabilized SARS-CoV-2 antiviral peptides and methods for using such peptides in the prevention and treatment of coronavirus infection.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Patent Application No. 62 / 985,100, filed on March 4, 2020, the entirety of which is incorporated herein by reference.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. The name of the ASCII copy, created on March 4, 2021, is 00530_0401WO1_2823_W01WO_SL.txt, and its size is 163,968 bytes.

[0003] technical field This disclosure relates to structurally stabilized SARS-CoV-2 antiviral peptides, as well as methods for using such peptides in the prevention and treatment of coronavirus infections. [Background technology]

[0004] background There are currently no antiviral drugs to prevent or treat infections caused by novel coronavirus (nCoV) pandemics, such as COVID-19 caused by Wuhan nCoV (also known as 2019-nCoV or SARS-CoV-2). COVID-19 has been declared a high-risk global health emergency by the World Health Organization (WHO) and, as of March 2021, has caused 114,857,764 cases of respiratory illness and 2,551,459 deaths worldwide.

[0005] SARS-CoV-2 contains a surface protein that undergoes a conformational change upon association with host cells, resulting in the formation of a 6-helix bundle that joins the host membrane and the viral membrane. Peptide-based inhibition of the viral fusion process is mechanistically viable and clinically effective (e.g., Fuzeon (i.e., enfurvirtide), approved by the FDA in 2003), but the biophysical and pharmacological tendencies of peptides, including loss of bioactive form and rapid in vivo proteolysis (e.g., 100 mg twice daily self-injection), limit the broader application of this validated approach. Therefore, new strategies for the prevention and / or treatment of COVID-19 infection are urgently needed to effectively mitigate the pandemic. [Overview of the Initiative] [Means for solving the problem]

[0006] overview This application discloses compositions and methods for peptide stabilization techniques (e.g., staples, stitches) that reproduce and enhance the structure of bioactive helices to produce targeted prophylactic and therapeutic agents for the prevention and / or treatment of coronavirus (e.g., betacoronaviruses such as SARS-CoV-2) infections. By inserting “staples” (e.g., total hydrocarbon staples) or “stitches” into natural peptides, bioactive helical structures can be restored, embedding otherwise unstable amide bonds into the core of the helical structure, and / or constraining the amide bonds in a manner that prevents their recognition and proteolysis by the body’s proteases, thereby conferring significant protease resistance. Herein, structurally stabilized peptide inhibitors of coronavirus (e.g., betacoronaviruses such as SARS-CoV-2) are disclosed. These structurally stabilized peptide inhibitors are used to prevent and / or treat coronavirus (e.g., betacoronaviruses such as SARS-CoV-2) infections such as COVID-19.

[0007] This disclosure provides structurally stabilized peptides of amino acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, or 100% identical to any one of the sequences of SEQ ID NOs: 10 or 258 (core template sequences for SARS-CoV-2 HR2 and EK1, respectively) or SEQ ID NOs: 133, 40, 136, 42, 30, 113, 34, 36, 134, 39, 135, 42, 137, 50, 52, 51, 31-33, 37, 41, 44-49, 177, and 179, wherein the structurally stabilized peptides have at least one (1, 2, 3, 4, 5, or 6) of the following properties: (i) binding to the 5-helix bundle of the SARS-CoV-2 S protein; (ii) binding to SARS-CoV-2 (iii) disrupts the interaction between the 5-helix bundle of the S protein and the peptide of SEQ ID NO: 10 or 258; (iii) is alpha-helical; (iv) is protease-resistant; (v) inhibits the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibits cell infection by SARS-CoV-2. This disclosure also provides structurally stabilized peptides of an amino acid sequence containing, in part, one of the sequences of SEQ ID NOs: 10 or 258, or SEQ ID NOs: 133, 40, 136, 42, 30, 113, 34, 36, 134, 39, 135, 42, 137, 50, 52, 51, 31-33, 37, 41, 44-49, 177, and 179 (having 0-10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, insertions, and / or deletions), wherein the structurally stabilized peptide has at least one (1, 2, 3, 4, 5, or 6) of the following properties: (i) binding to the 5-helix bundle of the SARS-CoV-2 S protein; (ii) SARS-CoV-2 (iii) disrupts the interaction between the 5-helix bundle of the S protein and the peptide of SEQ ID NO: 10 or 258; (iii) is alpha-helical; (iv) is protease-resistant; (v) inhibits the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibits cell infection by SARS-CoV-2.In some cases, one or more of positions 1, 3, 5, 6, 8, 10, 12, 13, 15, 17, and 19 of SEQ ID NO: 10 or 258 are either unsubstituted or substituted by a conserved amino acid substitution. In some cases, one or more (1, 2, 3, 4, 5, 6) of positions 2, 4, 7, 9, 11, 14, 16, or 18 of SEQ ID NO: 10 or 258 are substituted by an α,α-disubstituted unnatural amino acid with an olefin side chain. In certain cases, one or more of positions 4, 8, 10, 13, 15, 17, and 18 of SEQ ID NO: 10 or 258 are either unsubstituted or, if substituted, substituted by a conserved amino acid. In certain cases, one or more of positions 1, 5, 7, 11, or 12 of SEQ ID NO: 10 or 258 are substituted by a conserved amino acid if substituted. A guiding characteristic of altering the amino acid sequence of SEQ ID NO: 10 or 258 is that it should still bind to the 5-helix bundle of SARS-CoV-2 and inhibit or disrupt the association between the 5-helix bundle and the peptide of SEQ ID NO: 10 or 258. In some examples, structurally stabilized peptides contain one of the sequences of SEQ ID NOs: 133, 40, 136, 42, 30, 113, 34, 36, 134, 39, 135, 42, 137, 50, 52, 51, 31-33, 37, 41, 44-49, 177, and 179. The above peptides can have amino acid lengths of 19-100 (e.g., at least 19, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95), and these peptides can be lipid-modified. The peptides can also be modified to be conjugated to polyethylene glycol (PEG). Furthermore, these peptides can be modified to include additional N-terminal (e.g., either SEQ ID NO: 250 or 251) and / or C-terminal (e.g., any one of SEQ ID NOs: 252-255) sequences of the corresponding SARS-CoV-2 HR2 peptide. In some cases, these peptides can be modified to include the amino acid sequence GSGSGC (SEQ ID NO: 256) appended to the C-terminus of the amino acid sequence.In some cases, the amino acid sequence further includes a C-terminal peptide / PEG spacer conjugate cholesterol such as GSGSGC(SEQ ID NO: 256)-Ac-PEG4-cholesterol. In some cases, these peptides can be modified to include GSGSGC(SEQ ID NO: 256)-(PEG4-chol)-carboxamide added to the C-terminus of the amino acid sequence. These structurally stabilized peptides are useful for the treatment or prevention of coronavirus infection (e.g., COVID-19). This disclosure also relates to methods for producing the above structurally stabilized peptides. For example, one of the peptides SEQ ID NOs: 133, 40, 136, 42, 30, 113, 34, 36, 134, 39, 135, 42, 137, 50, 52, 51, 31-33, 37, 41, 44-49, 177, and 179 is crosslinked (e.g., by a ruthenium-mediated ring-closing metathesis reaction). This method may further include formulating cross-linked peptides as sterile pharmaceutical compositions useful for administration to human subjects requiring them (e.g., intravenously, subcutaneously, topically, or intranasally).

[0008] In one embodiment, the present disclosure features a structurally stabilized polypeptide comprising an amino acid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 94% identical to the sequence shown in SEQ ID NO: 10 (IQKEIDRLNEVAKNLNESL). In some examples, the sequence is as follows (position 1 is the N-terminal isoleucine of SEQ ID NO: 10, and position 19 is the C-terminal leucine of SEQ ID NO: 10): (i) 7th and 11th place; (ii) 10th and 14th place; (iii) 12th and 16th place; (iv) 14th and 18th place (v) 2nd and 9th place; (vi) 4th and 11th place; (vii) 9th and 16th place; (viii) 2nd and 6th place; (ix) 8th and 12th place; (x) 9th and 13th place; (xi) 11th and 15th place; (xii) 14th and 18th place; (xiii) 15th and 19th place; (xiv) 7th and 14th place; (xv) 3rd and 10th place; (xvi) 6th and 13th place; (xvii) 13th and 17th place; (xiii) 3rd and 7th place; (xix) 3rd, 7th, 13th, and 17th place; (xx) 3rd, 7th, 14th, and 18th place; (xxi) 2nd, 6th, 14th, and 18th place; (xxii) 2nd, 6th, 13th, and 17th place; (xxiii) 3rd, 10th, and 17th place; (xiv) 2nd, 9th, and 13th place; (xv) 3rd, 10th, and 14th place; (xvi) 6th, 13th, and 17th; or (xvii) 7th, 14th, and 18th, The amino acids at the selected positions are replaced by α,α-disubstituted non-natural amino acids having olefin side chains. In some examples, if the amino acid sequence contains further substitutions, those substitutions are (A) or (B): (A) Positions 4, 8, 10, 13, 15, 17 and 18 of SEQ ID NO: 10 are either not substituted with α,α-disubstituted unnatural amino acids having olefin side chains, or they are substituted with conservative amino acid substitutions; Positions 1, 5, 7, and 11, if substituted, are substituted with conservative amino acid substitutions or α,α-disubstituted unnatural amino acids having olefin side chains; and The remaining positions of SEQ ID NO: 10 may be substituted with any amino acid, or with α,α-disubstituted unnatural amino acids having olefin side chains; or (B) One or more of positions 1, 3, 5, 6, 8, 10, 12, 13, 15, 17, and 19 of SEQ ID NO: 10 are either unsubstituted or, if substituted, are replaced by a conservative amino acid substitution.

[0009] In some examples, a structurally stabilized polypeptide at one or more of positions 2, 4, 7, 9, 11, 14, 16, and 18 of SEQ ID NO: 10 can be replaced with any amino acid or α,α-disubstituted non-natural amino acid having an olefin side chain. In some examples, the structurally stabilized peptide is 15–100 amino acid long, and optionally 19–45 amino acid long. In some examples, the structurally stabilized peptide has one or more of the following properties: (i) binding to the recombinant 5-helix bundle of the SARS-CoV-2 S protein; (ii) disrupting the interaction between the 5-helix bundle and SEQ ID NO: 10; (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0010] In some cases, the amino acid sequence of the structurally stabilized polypeptide is at least 70% (70%, 75%, 80%, 85%, 90%, 95%) identical to the sequence shown in SEQ ID NO: 10. In some cases, the amino acid sequence of the structurally stabilized polypeptide is at least 80% (80%, 85%, 90%, 95%) identical to the sequence shown in SEQ ID NO: 10. In some cases, the amino acid sequence of the structurally stabilized polypeptide contains the sequence of SEQ ID NO: 50. In some cases, the amino acid sequence of the structurally stabilized polypeptide contains the sequence of SEQ ID NO: 52. In some cases, the amino acid sequence of the structurally stabilized polypeptide contains the sequence of SEQ ID NO: 51. In some cases, the amino acid sequence contains one of the sequences of SEQ ID NOs: 133, 40, 136, 42, 30, 113, 34, 36, 134, 39, 135, 42, and 137.

[0011] In some examples, structurally-stabilized further includes the amino acid sequence ISGINASVVN (SEQ ID NO: 250) added to the N-terminus of the amino acid sequence. In some examples, structurally-stabilized further includes the amino acid sequence DISGINASVVN (SEQ ID NO: 251) added to the N-terminus of the amino acid sequence. In some examples, structurally-stabilized further includes the amino acid sequence IDLQEL (SEQ ID NO: 252) added to the C-terminus of the amino acid sequence. In some examples, structurally-stabilized further includes the amino acid sequence IDLQELGKYEQYI (SEQ ID NO: 253) added to the C-terminus of the amino acid sequence. In some examples, structurally-stabilized further includes the amino acid sequence IDLQELGSGSGC (SEQ ID NO: 254) added to the C-terminus of the amino acid sequence. In some examples, structurally-stabilized further includes the amino acid sequence IDLQELGKYEQYIGSGSGC (SEQ ID NO: 255) added to the C-terminus of the amino acid sequence.

[0012] In some examples, the structurally stabilized further contains polyethylene glycol. In some examples, the structurally stabilized further contains cholesterol. In some examples, the structurally stabilized further contains GSGSGC(SEQ ID NO: 256)-(PEG4-chol)-carboxamide.

[0013] In another aspect, the present disclosure features a structurally stabilized polypeptide comprising an amino acid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 94% identical to the sequence described in SEQ ID NO: 258 (with N-terminal leucine at position 1 and C-terminal tyrosine at position 19): (i) 2nd place, 9th place, and 15th place; (ii) 3rd place, 10th place, and 16th place; (iii) 2nd, 6th, 13th, and 17th place; (iv) 3rd, 7th, 13th, and 17th place; (v) 2nd, 6th, 14th, and 18th place; or (vi) 3rd, 7th, 14th, and 18th, The amino acids at the selected positions are replaced by α,α-disubstituted non-natural amino acids having olefin side chains. In some cases, if the amino acid sequence has additional substitutions, they are as follows: one or more of positions 2, 4, 7, 9, 11, 14, 16, or 18 of SEQ ID NO: 258 are replaced by any amino acid if they are not replaced by α,α-disubstituted non-natural amino acids having olefin side chains; and one or more of positions 1, 3, 5, 6, 8, 10, 12, 13, 15, 17, and 19 of SEQ ID NO: 110 are replaced by conserved amino acid substitutions if they are not substituted or are substituted. In some cases, if the amino acid sequence has further substitutions, they are located at one or more of positions 2, 9, 11, 14, or 16 of SEQ ID NO: 258, and the substitutions may be for any amino acid, including conserved substitutions. In some cases, if the amino acid sequence has further substitutions, they are located at one or more of positions 1, 5, 7, 11, or 12 of SEQ ID NO: 258, in which case the substitutions are conserved amino acid substitutions. In some cases, the peptide is 19–100 amino acid long. Finally, structurally stabilized peptides have one or more of the following properties: (i) binding to the 5-helix bundle of the SARS-CoV-2 S protein; (ii) disrupting the interaction between the 5-helix bundle and SEQ ID NO: 258; (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0014] In another aspect, the present disclosure features a structurally stabilized polypeptide comprising an amino acid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 94% identical to the sequence described in SEQ ID NO: 110 (with the N-terminal serine at position 1 and the C-terminal leucine at position 36): (i) 13th, 20th, and 27th (ii) 14th, 21st, and 28th; (iii) 13th, 17th, 24th, and 28th place; (iv) 14th, 18th, 24th, and 28th; (v) 13th, 17th, 25th, and 29th; or (vi) 14th, 18th, 25th, and 29th, The amino acid at the selected position is replaced by an α,α-disubstituted unnatural amino acid having an olefin side chain; If the amino acid sequence has further substitutions (or more), they are (A) (A) If one or more of positions 4, 8, 10, 13, 15, 17, and 18 of SEQ ID NO: 110 are not substituted with α,α-disubstituted non-natural amino acids having an olefin side chain, they are either unsubstituted or substituted with a conservative amino acid substitution; If one or more of positions 1, 5, 7, and 11 of sequence number 110 are substituted, the substitutions are by conservative amino acid substitutions; Based on the fact that one or more of the remaining positions in SEQ ID NO: 110 can be substituted with any amino acid; and Peptides are 15-100 amino acid long; and The structurally stabilized peptide has one or more of the following properties: (i) binding to the recombinant 5-helix bundle of the SARS-CoV-2 S protein; (ii) disrupting the interaction between the 5-helix bundle and SEQ ID NO: 258; (iii) being an alpha helix; (iv) being protease resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0015] In some examples, the structurally stabilized polypeptide contains at least 70% (70%, 75%, 80%, 85%, 90%, 95%) the same amino acid sequence as the sequence shown in SEQ ID NO: 177. In some examples, the structurally stabilized polypeptide contains at least 70% (70%, 75%, 80%, 85%, 90%, 95%) the same amino acid sequence as the sequence shown in SEQ ID NO: 179. In some examples, the structurally stabilized polypeptide contains the same amino acid sequence as the sequence shown in SEQ ID NO: 179. In some examples, the structurally stabilized polypeptide further contains the amino acid sequence GSGSGC (SEQ ID NO: 256) appended to the C-terminus of the amino acid sequence.

[0016] In some examples, the structurally stabilized polypeptide further contains polyethylene glycol. In some examples, the structurally stabilized polypeptide further contains cholesterol.

[0017] In some examples, the structurally stabilized polypeptide further includes GSGSGC(SEQ ID NO: 256)-(PEG4-chol)-carboxamide.

[0018] In one embodiment, the present disclosure features a peptide comprising at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive amino acids of the amino acid sequence shown in Sequence ID No. 9, where at least two (e.g., 2, 3, 4, or 5) amino acids separated by 2, 3, or 6 amino acids are replaced by α,α-disubstituted non-natural amino acids having olefin side chains. In some examples, the SARS-CoV-2 HR2 peptide template sequence is 45 amino acids or less in length (e.g., 42, 43, 44, or 45), but it should be understood that the SARS-CoV-2 HR2 peptide template sequence can, of course, be extended at the N-terminus or C-terminus (with or without chemical derivatization) to maintain or optimize activity. The peptide binds to the recombinant SARS-CoV-2 5-helix bundle S protein. The peptide can also inhibit or disrupt the interaction between the SARS-CoV-2 HR2 sequence (e.g., SEQ ID NOs. 9, 10, 103, 104, 106, or 108) and the recombinant SARS-CoV-2 5-helix bundle S protein.

[0019] In some examples, the peptide contains or consists of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive amino acids from the amino acid sequence described in any one of SEQ ID NOs. 11-29. In some examples, the peptide contains or consists of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive amino acids from the amino acid sequence described in any one of SEQ ID NOs. 11-29, where permissible, one, two, three, four, or five amino acid substitutions on the non-interacting surface, or homologous substitutions on the interacting surface, in order to avoid disruption of the critical binding interaction between the staple peptide and the recombinant 5-helix bundle target of SARS-CoV-2. In some examples, the peptide contains or consists of the amino acid sequence described in any one of SEQ ID NOs. 11-29, where permissible, one, two, three, four, or five amino acid substitutions. These peptides possess one or more (e.g., 1, 2, 3, 4) of the following properties: (i) binding to recombinant SARS-CoV-2 5-helix bundle S protein; (ii) inhibiting or disrupting the interaction between SARS-CoV-2 HR2 sequences (e.g., SEQ ID NOs. 9, 10, 103, 104, 106, or 108) and recombinant SARS-CoV-2 5-helix bundle S protein; (iii) inhibiting fusion of SARS-CoV-2 with host cells; and / or (iv) inhibiting cell infection by SARS-CoV-2.

[0020] In another embodiment, the disclosure features a structurally stabilized peptide comprising at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive amino acids of the amino acid sequence shown in Sequence ID No. 9, where at least two (e.g., 2, 3, 4, or 5) amino acids separated by 2, 3, or 6 amino acids are replaced by α,α-disubstituted non-natural amino acids having olefin side chains. The side chains of the α,α-disubstituted non-natural amino acids having olefin side chains are crosslinked. In some examples, the SARS-CoV-2 HR2 peptide template sequence is 45 amino acids or less in length (e.g., 42, 43, 44, or 45), but can be extended at the N-terminus or C-terminus (with or without chemical derivatization) to maintain or optimize activity. Structurally stabilized peptides have one or more (e.g., 1, 2, 3, 4, 5, 6) of the following properties: (i) binding to recombinant SARS-CoV-2 5-helix bundle S protein; (ii) inhibiting or disrupting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (e.g., SEQ ID NOs. 9, 10, 103, 104, 106, or 108); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2. In some examples, structurally stabilized peptides have an amino acid length of 42-45 (e.g., 42, 43, 44, 45).

[0021] In some examples, structurally stabilized peptides contain or consist of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive amino acids from the amino acid sequence described in any one of SEQ ID NOs. 11-29, and the side chains of α,α-disubstituted unnatural amino acids having olefin side chains are cross-linked (e.g., stapled and / or stitched). In some examples, structurally stabilized peptides contain or consist of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive amino acids of the amino acid sequence described in any one of SEQ ID NOs: 11-29, which has 1, 2, 3, 4, or 5 amino acid substitutions, and the side chains of α,α-disubstituted unnatural amino acids having olefin side chains are crosslinked (e.g., stapled and / or stitched). In some examples, structurally stabilized peptides have an amino acid length of 42–45 (e.g., 42, 43, 44, 45).

[0022] In another embodiment, the Disclosure provides peptides comprising at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids of the amino acid sequence shown in Sequence ID No. 10, where at least two (e.g., 2, 3, 4, or 5) amino acids separated by 2, 3, or 6 amino acids are replaced by α,α-disubstituted unnatural amino acids having olefin side chains. In some examples, the peptide sequence template is up to 45 amino acid lengths (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45), but in some examples, it can be extended at the N-terminus or C-terminus (with or without chemical derivatization) to maintain or optimize activity. The peptide binds to the recombinant SARS-CoV-2 5-helix bundle S protein. The peptide can also inhibit or disrupt the interaction between the SARS-CoV-2 HR2 sequence (e.g., SEQ ID NOs. 9, 10, 103, 104, 106, or 108) and the recombinant SARS-CoV-2 5-helix bundle S protein.

[0023] In some examples, the peptide contains or consists of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids from the amino acid sequence described in any one of SEQ ID NOs.30-52. In some examples, the peptide contains or consists of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids from the amino acid sequence described in any one of SEQ ID NOs.30-52, which, if permissible, have either 1, 2, 3, 4, or 5 amino acid substitutions on the non-interacting surface or homologous substitutions on the interacting surface, in order to avoid disruption of the critical binding interaction between the staple peptide and the recombinant 5-helix bundle target of SARS-CoV-2.

[0024] In some examples, the peptides contain or consist of the amino acid sequences described in any one of Sequence IDs 30-52, which have one, two, three, four, or five amino acid substitutions. These peptides have one or more (e.g., one, two, three, or four) of the following properties: (i) binding to the recombinant SARS-CoV-2 5-helix bundle S protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (Sequence ID 9); (iii) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (iv) inhibiting cell infection by SARS-CoV-2.

[0025] In another embodiment, the disclosure features a structurally stabilized peptide comprising at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 10, where at least two (e.g., 2, 3, 4, or 5) amino acids separated by 2, 3, or 6 amino acids are replaced by α,α-disubstituted non-natural amino acids having olefin side chains. The side chains of the α,α-disubstituted non-natural amino acids having olefin side chains are crosslinked. The peptide is 45 amino acid length or less (e.g., 42, 43, 44, or 45), but can be elongated at the N-terminus or C-terminus (with or without chemical derivatization) to maintain or optimize activity. Structurally stabilized peptides have one or more (e.g., 1, 2, 3, 4, 5) of the following properties: (i) binding to recombinant SARS-CoV-2 5-helix bundle S protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2. In some examples, structurally stabilized peptides have an amino acid length of 19–45 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45).

[0026] In some examples, structurally stabilized peptides contain or consist of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids from the amino acid sequence described in any one of SEQ ID NOs. 30-52, and the side chains of α,α-disubstituted unnatural amino acids having olefin side chains are crosslinked (e.g., stapled and / or stitched). In some examples, structurally stabilized peptides contain or consist of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acids from the amino acid sequence described in any one of SEQ ID NOs. 30-52, having 1, 2, 3, 4, or 5 amino acid substitutions, and the side chains of α,α-disubstituted unnatural amino acids having olefin side chains are crosslinked (e.g., stapled and / or stitched). In some examples, structurally stabilized peptides contain or consist of the amino acid sequences described in any one of Sequence IDs 30-52, which have 1, 2, 3, 4, or 5 amino acid substitutions, and the side chains of α,α-disubstituted unnatural amino acids having olefin side chains are crosslinked (e.g., stapled and / or stitched). In some examples, structurally stabilized peptides have amino acid lengths of 19-45 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45).

[0027] In some examples, the peptides described above and in this disclosure, or structurally stabilized (e.g., stapled, stitched) peptides, have one, two, three, four, five, or all six of the following properties: (i) binding to recombinant SARS-CoV-2 5-helix bundle S protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0028] In one aspect, the present disclosure relates to a structurally stabilized peptide comprising or comprising the following formula: [ka] or relating to a pharmaceutically acceptable salt thereof.

[0029] In some examples, each R1 and R2 is H or C1~C 10 The elements are alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and any of these may be substituted or unsubstituted. In some examples, each R3 is independently alkylene, alkenylene, or alkynylene, and any of these may be substituted or unsubstituted. In some examples, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each [Xaa] w is one of sequence numbers 53;56, 59, 62, 65, 68, 74, 77, 80, 82, 86, or 87, or one of I or IQ; each [Xaa] x is one of sequence numbers 54, 57, 60, 63, 66, 69, 70, 72, 75, 78, 81 or 83, or one of KEI, EID, RLN, EVA, VAK, NLN or LNE; and each [Xaa] y It is one of SEQ ID NOs. 55; 58, 61, 64, 67, 71, 73, 76, 79, 84, 85, or one of YI, ESL, SL, or L. In some examples, the structurally stabilized peptide has one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to the recombinant SARS-CoV-2 5-helix bundle S protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0030] In some examples, R1 is an alkyl group or a methyl group. In some examples, R2 is alkenyl. In some examples, R3 is an alkyl group or a methyl group.

[0031] In one aspect, the present disclosure relates to a structurally stabilized peptide comprising or consisting of the following formula:

Chemical formula

[0032] In some examples, each of R1, R3, R4, and R6 is H or C1-C 10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl or heterocyclylalkyl, any of which may or may not be substituted. In some examples, each R3 is independently alkylene, alkenylene or alkynylene, any of which may or may not be substituted. In some examples, [Xaa] t is one of SEQ ID NO: 53, 56, 59 or one of I or IQ. In some examples, [Xaa] u is one of SEQ ID NO: 54, 57, 60 or one of KEI or EID. In some examples, [Xaa] v is one of SEQ ID NO: 88-100. In some examples, [Xaa] x is one of SEQ ID NO: 63, 66, 69 or one of NLN or LNE. In some examples, [Xaa] yIt is either one of SEQ ID NOs. 64 or 67, or one of YI, SL, or L. In some examples, the structurally stabilized peptide has one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to the recombinant SARS-CoV-2 5-helix bundle S protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO. 9); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0033] In one aspect, the present disclosure relates to a structurally stabilized peptide comprising the following formula: [ka] or characterized by a pharmaceutically acceptable salt thereof.

[0034] In some examples, [Xaa] w This is either one of sequence numbers 62, 74, or 77, or I or IQ. In some examples, [Xaa] x This is one of sequence numbers 63, 70, 72, 75, or 78. In some examples, [Xaa] y is one of sequence numbers 69, 81, or 83, or one of EVA, VAK, NLN, or LNE. In some examples, [Xaa] zR1 is either sequence number 76 or 79, or one of YI, ESL, SL, or L. In some examples, each R1 and R4 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and any of these may or may not be substituted. In some examples, each R2 and R3 is independently alkylene, alkenylene, or alkynylene, and any of these may or may not be substituted. In some examples, the structurally stabilized peptide has one or more (1, 2, 4, 5, 6) of the following properties: (i) binding to the recombinant SARS-CoV-2 5-helix bundle S protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2. In some examples, R1 is an alkyl group or methyl group. In some examples, R2 is an alkenyl group. In some examples, R3 is an alkenyl group. In some examples, R4 is an alkyl group or methyl group.

[0035] In one aspect, the present disclosure relates to a structurally stabilized peptide comprising the following formula: [ka] or characterized by a pharmaceutically acceptable salt thereof.

[0036] In some examples, [Xaa] u This is one of sequence numbers 53, 59, or 59. In some examples, [Xaa] v This is one of sequence numbers 54, 57, or 60. In some examples, [Xaa] w This is one of sequence numbers 88, 91, or 94. In some examples, [Xaa] yThis is sequence number 63. In some examples, [Xaa] y This is sequence number 69. In some examples, [Xaa] z is YI. In some examples, R1, R3, R4 and R7 are independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl or heterocyclylalkyl, any of which are substituted or unsubstituted; in some examples, R2, R5 and R6 are independently alkylene, alkenylene or alkynylene, any of which are substituted or unsubstituted. In some examples, the structurally stabilized peptide has one or more (1, 2, 4, 5, 6) of the following properties: (i) binding to recombinant SARS-CoV-2 5-helix bundle S protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0037] In some cases, the structurally stabilized peptides or pharmaceutically acceptable salts thereof disclosed herein are up to 45 amino acid lengths. In some cases, the structurally stabilized peptides are 19, 20, 21, 22, 3, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid lengths.

[0038] In one embodiment, the disclosure features a pharmaceutical composition comprising one of the peptides disclosed herein. In one embodiment, the disclosure features a pharmaceutical compound comprising one of the structurally stabilized peptides disclosed herein. In some examples, the pharmaceutical compound comprises a pharmaceutically acceptable carrier.

[0039] In one aspect, the Disclosure provides a method for treating a coronavirus infection (e.g., COVID-19) in a human subject requiring treatment for the coronavirus infection, comprising administering a therapeutically effective amount of any one of the peptides disclosed herein to the human subject. In another aspect, the Disclosure provides a method for treating a coronavirus infection (e.g., COVID-19) in a human subject requiring treatment for the coronavirus infection, comprising administering a therapeutically effective amount of any one of the structurally stabilized peptides disclosed herein to the human subject.

[0040] In one aspect, the Disclosure provides a method for preventing coronavirus infection (e.g., COVID-19) in a human subject requiring prevention of coronavirus infection, comprising administering a therapeutically effective amount of any one of the peptides disclosed herein to the human subject. In another aspect, the Disclosure provides a method for preventing coronavirus infection (e.g., COVID-19) in a human subject requiring prevention of coronavirus infection, comprising administering a therapeutically effective amount of any one of the structurally stabilized peptides disclosed herein to the human subject.

[0041] In some cases, the methods described herein are methods for treating or preventing coronavirus infection (e.g., COVID-19). In some cases, the coronavirus infection is caused by a beta-coronavirus. In some cases, the coronavirus infection is caused by infection with SARS-CoV-2.

[0042] In one embodiment, the present disclosure provides a method for preparing a structurally stabilized peptide, comprising (a) providing the peptides disclosed herein (e.g., SEQ ID NOs: 11-52 or 112-180), and (b) crosslinking the peptide. In some examples, the crosslinking of the peptide is performed by a ruthenium-catalyzed metathesis reaction.

[0043] In one embodiment, the disclosure features a nanoparticle-containing composition comprising one of the structurally stabilized peptides disclosed herein. In some examples, the peptide or structurally stabilized peptide comprises one or more of 8, 81, and 82. In some examples, 8, 81, and 82 are (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine. In some examples, the peptide or structurally stabilized peptide comprises one or more of X, X1, X2, X3, and X4. In some examples, X, X1, X2, X3, and X4 are (S)-α-(4'-pentenyl)alanine, respectively. In some examples, the peptide or structurally stabilized peptide comprises # which is α,α-bis(4'-pentenyl)glycine or α,α-bis(7'-octenyl)glycine. In some examples, the peptide or structurally stabilized peptide contains % of (S)-α-(7'-octenyl)alanine or (S)-α-(4'-pentenyl)alanine. In some examples, the nanoparticles are PLGA nanoparticles. In certain cases, the lactic acid:glycolic acid ratio of the PLGA nanoparticles is in the range of 2:98 to 100:0.

[0044] In one embodiment, the disclosure features structurally stabilized peptides, where 8, 81, and 82 = (R)-α(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine; X, X1, X2, X3, and X4 = (S)-α-(4'-pentenyl)alanine; # = α,α-bis(4'-pentenyl)glycine or α,α-bis(7'-octenyl)glycine; and % = (S)-α-(7'-octenyl)alanine or (S)-α-(4'-pentenyl)alanine. In another embodiment, the structurally stabilized peptides include 8, 81 and 82 = (R)-α-(7'-octenyl)alanine; X, X1, X2, X3 and X4 = (S)-α-(4'-pentenyl)alanine; # = α,α-bis(4'-pentenyl)glycine; and % = (S)-α-(7'-octenyl)alanine.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, but exemplary methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, the application containing the definitions shall prevail. The materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure.

[0046] Other features and advantages of this disclosure will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0047] [Figure 1A] Figures 1A and 1B show the amino acid sequence of the S protein (Sequence ID 1) (Figure 1A) and the three generated sequences of the SARS-CoV-2 helical bundle (Sequence IDs 261-263) (Figure 1B). The underlined sequence in Figure 1B represents the HR1 sequence, and the sequence enclosed in a square in Figure 1B represents the HR2 sequence. [Figure 1B] Figures 1A and 1B show the amino acid sequence of the S protein (Sequence ID 1) (Figure 1A) and the three generated sequences of the SARS-CoV-2 helical bundle (Sequence IDs 261-263) (Figure 1B). The underlined sequence in Figure 1B represents the HR1 sequence, and the sequence enclosed in a square in Figure 1B represents the HR2 sequence. [Figure 2] Figure 2 is a schematic diagram of the SARS-CoV-2 spike (S) protein, including the sequence composition of the heptad repeat domain 1 (HR1) (SEQ ID NO: 2) and the heptad repeat domain 2 (HR2) (SEQ ID NO: 3) fusion domain. [Figure 3] Figure 3 shows the mechanism of action of the SARS-CoV-2 S fusion inhibitor peptide. [Figure 4]Figure 4 shows a partial helical wheel of the amphiphilic alpha-helix (SEQ ID NOs: 4-6) of the SARS-CoV-2 S HR2(1169-1210) domain, primarily showing the hydrophobic binding interface, with adjacent charged or polar residues located around the binding interface and on the non-interacting surfaces. Arrows indicate hydrophobic moments. [Figure 5] Figure 5 shows a partial helical wheel of the amphiphilic alpha-helix (SEQ ID NO: 7) of the SARS-CoV-2 S HR2(1179-1197) domain, primarily showing the hydrophobic binding interface, with adjacent charged or polar residues located around the binding interface and on the non-interacting surface. Arrows indicate hydrophobic moments. [Figure 6] Figures 6A and 6B show the alignment of the HR1 and HR2 regions of SARS-CoV-2 and SARS-CoV-1 ("SARS") (Figure 6A), and the alignment of the HR2 sequences from SARS-CoV-2, MERS, and the surrogate HR2 type sequence ("EK1") (Figure 6B). In Figure 6A, the SARS HR1 sequence is shown as sequence number 8. The SARS-CoV-2 HR1 sequence is shown as sequence number 2. The SARS-CoV-1 and SARS-CoV-2 HR2 sequences are shown as sequence number 3. In Figure 6B, the SARS-CoV-2 sequence is shown as sequence number 108, the MERS sequence as sequence number 259, and the EK1 sequence as sequence number 110. The core template helical sequences of SARS-CoV-2 HR2 and its two homologs are underlined, and the core template sequences from SARS-CoV-2 HR2 and EK1 are shown as SEQ ID NO: 10 and SEQ ID NO: 258, respectively. The alignment in Figure 6B allows for the identification of possible residues and amino acids in the SARS-CoV-2 HR2 sequence that may be modified. [Figure 7] Figure 7 shows various non-natural amino acids containing olefin tethers that can be used to generate hydrocarbon stapled SARS-CoV-2 S peptides supporting staples across positions i, i+3; i, i+4, and i, i+7. A library of single-staple COVID-19-S peptides is generated using a single-staple scan. [Figure 8] Figure 8 shows various staple compositions in multi-staple peptides and a staple scan for generating a library of multi-staple SARS-CoV-2 S peptides. [Figure 9] Figure 9 shows various staple compositions in tandem stitch peptides for generating a library of stitched SARS-CoV-2 S peptides. [Figure 10] Figure 10 illustrates an exemplary approach for designing, synthesizing, and identifying optimal staple peptide constructs for targeting the SARS-CoV-2 fusion apparatus, including Ala scans, staple scans, and the generation of variable N-terminus and C-terminus deletions, additions, and derivatization libraries. Single and dual staple and stitch constructs, including alanine and staple and stitch scans, are used to identify the optimal staple peptide for in vitro and in vivo analysis. [Figure 11-1] Figure 11 shows exemplary structurally stabilized SARS-CoV-2 HR2 peptide sequences generated by i, i+4 and i, i+7 staple scans of the core template sequences (aa1169~1197), as well as their variations characterized by N-terminal and C-terminal staple-free sequence extension, terminal derivatization (e.g., PEG4-cholesterol), incorporation of double staples and stitches, and application of staples to alternative HR2 type sequences. The letter designation next to the sequence number is a key to the staple position in the sequence. [Figure 11-2] Figure 11 shows exemplary structurally stabilized SARS-CoV-2 HR2 peptide sequences generated by i, i+4 and i, i+7 staple scans of the core template sequences (aa1169~1197), as well as their variations characterized by N-terminal and C-terminal staple-free sequence extension, terminal derivatization (e.g., PEG4-cholesterol), incorporation of double staples and stitches, and application of staples to alternative HR2 type sequences. The letter designation next to the sequence number is a key to the staple position in the sequence. [Figure 11-3]Figure 11 shows exemplary structurally stabilized SARS-CoV-2 HR2 peptide sequences generated by i, i+4 and i, i+7 staple scans of the core template sequences (aa1169~1197), as well as their variations characterized by N-terminal and C-terminal staple-free sequence extension, terminal derivatization (e.g., PEG4-cholesterol), incorporation of double staples and stitches, and application of staples to alternative HR2 type sequences. The letter designation next to the sequence number is a key to the staple position in the sequence. [Figure 12] Figures 12A and 12B show that the insertion of staples into the core template sequence (aa1169-1197) confers a distinct alpha-helical structure compared to the unstapled sequence, and that this structural benefit is preserved by adding unstapled sequences to the N-terminus and / or C-terminus. Figure 12A compares the circular dichroism spectrum of the unstapled core template sequence (SEQ ID NO: 10) with that of sequences containing stitches J, S (SEQ ID NO: 47) or K, T (SEQ ID NO: 48), or double staples N, S (SEQ ID NO: 49) or N, T (SEQ ID NO: 51). Figure 12B compares the circular dichroism spectra of longer unstapled HR2 sequences (SEQ ID NOs: 9, 108, 110) with those containing double staples O, S (SEQ ID NO: 158) and N, S (SEQ ID NO: 177). [Figure 13]Figures 13A and 13B show that the insertion of double staples or stitches into the core template sequences (aa1169-1197) confers significant protease resistance compared to the unstapled sequences, depending on the sequence, staple type, and staple location. Figure 13A shows that both double staples and stitches (sequence numbers 48 and 52) confer significant resistance to proteinase K treatment (half-life > 1000 minutes), while the unstapled sequence (sequence number 10) is rapidly digested (half-life 35 minutes). Figure 13B shows that a longer, unstapled HR2 sequence (SEQ ID NO: 9) is rapidly digested by proteinase K (half-life 25 minutes), and insertion of double staples O, S (SEQ ID NO: 158) only slightly enhances proteolytic resistance (half-life 33 minutes), whereas insertion of double staples N, S (SEQ ID NO: 177) into another HR2-type sequence (SEQ ID NO: 110) confers significant proteolytic resistance to proteinase K (half-life 840 minutes). [Figure 14] Figures 14A and 14B show the mouse plasma stability (no degradation) of two double-staple peptides of the core template sequence (aa1169-1197), including sequence number 51 (staple N, T) in Figure 14A and sequence number 52 (staple O, T) in Figure 14B. [Figure 15]Figures 15A and 15B show the results of a direct fluorescence polarization coupling assay using an N-terminal FITC-derivativeized i,i+4 staple scan library of recombinant SARS-CoV-2 5-helix-binding protein and core template sequences (aa1179-1197, SEQ ID NO: 10). Figure 15A shows the different binding activities of staple peptides based on staple location, as reflected by the change in fluorescence polarization (ΔmP) at a 4 μM 5-HB protein concentration. Figure 15B shows the dose-response curves of the fluorescent i,i+4 staple scan library to 5-HB protein, highlighting that, depending on the specific staple location, the i,i+4 staple peptides bind better, similarly, or worse than the unstapled core template sequence. In both Figure 15A and Figure 15B, the sequence from top to bottom has sequence numbers 130, 36, 37, 131, 132, 38, 133, 134, 39, 40, 135, 136, 41, 42, 137, and 10. [Figure 16-1]Figures 16A–16D show the results of direct fluorescence polarized coupling assays using N-terminal FITC-derivativeized i,i+7 staple scan libraries of recombinant SARS-CoV-2 5-helix-binding protein and core template sequences (aa1179–1197, SEQ ID NO: 10). Figure 16A shows the different binding activities of staple peptides based on staple position, reflected by the change in fluorescence polarization (ΔmP) at a 4 μM 5-HB protein concentration. Figure 16B shows the dose-response curves of the fluorescent i,i+7 staple scan libraries to 5-HB protein, highlighting that, depending on the specific staple position, the i,i+7 staple peptides bind better, similarly, or worse than the unstapled core template sequences. In both Figures 16A and 16B, the sequences from top to bottom have SEQ ID NOs: 112, 30, 31, 113, 114, 32, 33, 115, 34, 35, 116, 117, and 10. Figure 16C shows a schematic diagram of a helical wheel indicating the residues involved in preferred (light gray), unpreferred (dark gray), and intermediate (medium gray) i, i+7 staples. The residues involved in the two staples are shown as bisected circles, with a left semicircle representing the incorporation of the residue at the N-terminal position of the staple and a right semicircle representing the incorporation of the residue at the C-terminal position of the staple. If the semicircles are colored white, the indicated residue position is not involved in either the N-terminal or C-terminal staple position. Staple positions located on the hydrophobic surface disrupt 5-HB binding activity, and unexpectedly, staple positions located on the hydrophilic surface opposite the 5-HB binding surface are also unpreferred (dark gray residues; marked with X). In contrast, selective staple positions at the boundary between the hydrophobic binding surface and the hydrophilic surface are preferred (light gray residues; marked with stars). Figure 16D shows the SARS-CoV-2 HR2 sequence, highlighting the roles of specific amino acids involved in the heptad repeat of HR1, and the degree of staple resistance or intolerance at specific positions, providing information on which residues are more or less suitable for amino acid substitutions. [Figure 16-2]Figures 16A–16D show the results of direct fluorescence polarized coupling assays using N-terminal FITC-derivativeized i,i+7 staple scan libraries of recombinant SARS-CoV-2 5-helix-binding protein and core template sequences (aa1179–1197, SEQ ID NO: 10). Figure 16A shows the different binding activities of staple peptides based on staple position, reflected by the change in fluorescence polarization (ΔmP) at a 4 μM 5-HB protein concentration. Figure 16B shows the dose-response curves of the fluorescent i,i+7 staple scan libraries to 5-HB protein, highlighting that, depending on the specific staple position, the i,i+7 staple peptides bind better, similarly, or worse than the unstapled core template sequences. In both Figures 16A and 16B, the sequences from top to bottom have SEQ ID NOs: 112, 30, 31, 113, 114, 32, 33, 115, 34, 35, 116, 117, and 10. Figure 16C shows a schematic diagram of a helical wheel indicating the residues involved in preferred (light gray), unpreferred (dark gray), and intermediate (medium gray) i, i+7 staples. The residues involved in the two staples are shown as bisected circles, with a left semicircle representing the incorporation of the residue at the N-terminal position of the staple and a right semicircle representing the incorporation of the residue at the C-terminal position of the staple. If the semicircles are colored white, the indicated residue position is not involved in either the N-terminal or C-terminal staple position. Staple positions located on the hydrophobic surface disrupt 5-HB binding activity, and unexpectedly, staple positions located on the hydrophilic surface opposite the 5-HB binding surface are also unpreferred (dark gray residues; marked with X). In contrast, selective staple positions at the boundary between the hydrophobic binding surface and the hydrophilic surface are preferred (light gray residues; marked with stars). Figure 16D shows the SARS-CoV-2 HR2 sequence, highlighting the roles of specific amino acids involved in the heptad repeat of HR1, and the degree of staple resistance or intolerance at specific positions, providing information on which residues are more or less suitable for amino acid substitutions. [Figure 16-3]Figures 16A–16D show the results of direct fluorescence polarized coupling assays using N-terminal FITC-derivativeized i,i+7 staple scan libraries of recombinant SARS-CoV-2 5-helix-binding protein and core template sequences (aa1179–1197, SEQ ID NO: 10). Figure 16A shows the different binding activities of staple peptides based on staple position, reflected by the change in fluorescence polarization (ΔmP) at a 4 μM 5-HB protein concentration. Figure 16B shows the dose-response curves of the fluorescent i,i+7 staple scan libraries to 5-HB protein, highlighting that, depending on the specific staple position, the i,i+7 staple peptides bind better, similarly, or worse than the unstapled core template sequences. In both Figures 16A and 16B, the sequences from top to bottom have SEQ ID NOs: 112, 30, 31, 113, 114, 32, 33, 115, 34, 35, 116, 117, and 10. Figure 16C shows a schematic diagram of a helical wheel indicating the residues involved in preferred (light gray), unpreferred (dark gray), and intermediate (medium gray) i, i+7 staples. The residues involved in the two staples are shown as bisected circles, with a left semicircle representing the incorporation of the residue at the N-terminal position of the staple and a right semicircle representing the incorporation of the residue at the C-terminal position of the staple. If the semicircles are colored white, the indicated residue position is not involved in either the N-terminal or C-terminal staple position. Staple positions located on the hydrophobic surface disrupt 5-HB binding activity, and unexpectedly, staple positions located on the hydrophilic surface opposite the 5-HB binding surface are also unpreferred (dark gray residues; marked with X). In contrast, selective staple positions at the boundary between the hydrophobic binding surface and the hydrophilic surface are preferred (light gray residues; marked with stars). Figure 16D shows the SARS-CoV-2 HR2 sequence, highlighting the roles of specific amino acids involved in the heptad repeat of HR1, and the degree of staple resistance or intolerance at specific positions, providing information on which residues are more or less suitable for amino acid substitutions. [Figure 17]Figures 17A and 17B show the results of direct fluorescence polarized coupling assays using N-terminal FITC-derivativeized double i, i+4 staple peptides (SEQ ID NO: 51(N,T) and SEQ ID NO: 52(O,T)) of recombinant SARS-CoV-2 5-helix-binding protein and core template sequences (aa1179-1197, SEQ ID NO: 10). Figure 17A shows the different binding activities of the staple peptides based on the double staple position, as reflected by the change in fluorescence polarization (ΔmP) at a 4 μM 5-HB protein concentration. Figure 17B shows the dose-response curves for the fluorescent double staple peptides to the 5-HB protein, highlighting in each example that the insertion of double staples results in enhanced binding activity compared to the core template sequence without staples. [Figure 18] Figure 18 shows the results of direct fluorescence polarized coupling assays using the core template sequence, SEQ ID NO: 10, or LEYEBKKLEEAIKKLEESY (SEQ ID NO: 258) recombinant SARS-CoV-2 5-helix-binding protein and N-terminal FITC-derivativeized double i, i+4 staple peptides (from top to bottom, SEQ ID NOs: 156, 158, 160, 162, 179, and 180) within the context of longer HR2 (SEQ ID NO: 9) and alternative HR2-type (SEQ ID NO: 110) sequences, respectively. The plots demonstrate the comparative dose-responsive binding activity of the double staple peptides to 5-HB of SARS-CoV-2. [Figure 19] Figures 19A–19C show the results of a competitive ELISA binding assay in which the interaction between the SARS-CoV-2 5-HB protein and the SARS-CoV-2 stapleless HR2 sequence corresponding to SEQ ID NO: 9 is competed for by serial dilutions of i, i+4 stapled scan libraries (SEQ ID NOs: 138–152, from top to bottom) of the core template sequence (SEQ ID NO: 10) with N-terminal extension (aa1169–1197). Figure 19A shows the complete dose-response competitive binding curve, and Figures 19B and 19C highlight the comparative competitive binding activity of each construct at doses of 3 μM and 10 μM, respectively. [Figure 20]Figure 20 shows the results of a competitive ELISA binding assay in which the interaction between the SARS-CoV-2 5-HB protein and the SARS-CoV-2 unstapled HR2 sequence corresponding to SEQ ID NO: 9 is competed for by double-stapled and stitch peptides (SEQ ID NOs: 10, 52, 51, 50, 49, 48, 47, 44, and 43, from top to bottom) of the core template SARS-CoV-2 HR2 sequence corresponding to SEQ ID NO: 10 at a fixed dose (10 μM). The unstapled core template sequence (SEQ ID NO: 10) cannot compete with the longer HR2 template sequence (SEQ ID NO: 9) for binding to 5-HB, but the selective double-staple peptides (staple combinations O, S, and K, T) and stitch peptides (staple combinations H, L) of the core template sequence can partially disrupt the binding interaction at a dose of 10 μM. [Figure 21] Figure 21 shows the results of a competitive ELISA binding assay in which the interaction between the SARS-CoV-2 5-HB protein and the SARS-CoV-2 stapleless HR2 sequence corresponding to SEQ ID NO: 9 is competed for by dose-responsive treatment with double-stapled and stitched peptides (SEQ ID NOs: 9, 153, 154, 156, 158, 160, and 162, from top to bottom) of the longer HR2 sequence corresponding to SEQ ID NO: 9. The effectiveness in disrupting the 5-HB / HR2 interaction depends on the type of staples and staple arrangement of the double-stapled and stitched peptides within the core template sequence (SEQ ID NO: 10) in the context of the longer HR2 peptide (SEQ ID NO: 9). [Figure 22]Figure 22 shows the results of a competitive ELISA binding assay in which the interaction between the SARS-CoV-2 5-HB protein and the SARS-CoV-2 stapleless HR2 sequence corresponding to SEQ ID NO: 9 is competed for by dose-responsive treatment with double-stapled and stitched peptides (SEQ ID NOs: 110 and 175-180, from top to bottom) of alternative HR2 sequences corresponding to SEQ ID NO: 110. The effectiveness in disrupting the 5-HB / HR2 interaction depends on the type of staples and staple arrangement of the double-stapled and stitched core template sequence in the context of the longer HR2-type peptide (SEQ ID NO: 110), with double-stapled N,S producing the most potent competitive inhibitors in this group. [Figure 23] Figure 23 shows the antiviral activity of exemplary double-staple and stitched peptides of the core template sequence of SEQ ID NO: 10 (SEQ ID NOs: 43, 49, 48, 52, and 22, from top to bottom) and the double-staple peptide of the longer HR2 sequence corresponding to SEQ ID NO: 9. The peptides were screened at 25 μM for their ability to inhibit infection of Vero E6 cells by live wild-type SARS-CoV-2 virus, and the percentage of infected cells was plotted. In each case, the staple peptide inhibited infection compared to treatment with vehicle control. [Figure 24] Figure 24 shows that hits from peptide screening in SARS-CoV-2 exposed Vero E6 cells subjected to SARS-CoV-2 infection were then subjected to further dose-response studies, as exemplified by a double-staple core template sequence (SEQ ID NO: 52) having staples O, T with an IC50 of less than 6 μM for inhibiting SARS-CoV-2 infection in the assay. [Figure 25] Figure 25 shows the different antiviral activities of double-stapled core template sequences (SEQ ID NO: 10) and stitch peptides (SEQ ID NOs: 10, 43, 44, 47-52, from left to right) as evaluated in high throughput by an antibody-based SARS-CoV-2 detection platform in infected Vero E6 cells. [Figure 26]Figure 26 shows that double i, i+7 stapling and stitching at the indicated positions outside the core template sequence (SEQ ID NO: 10) in the context of a longer HR2 peptide sequence (SEQ ID NO: 9), as evaluated in high throughput by an antibody-based SARS-CoV-2 detection platform in infected Vero E6 cells, did not yield compounds with antiviral activity. The sequences from top to bottom include SEQ ID NOs: 9, 26-28, 19, 22, 23, 25, and 24. [Figure 27] Figure 27 shows the different antiviral activities of exemplary double-staple and stitch peptides of the core template sequence (SEQ ID NO: 10) in the context of a longer HR2 peptide sequence corresponding to SEQ ID NO: 9, evaluated in high throughput by an antibody-based SARS-CoV-2 detection platform in infected Vero E6 cells. Constructs with double i, i+4 staples O, S exhibited the most potent antiviral activity, followed by compounds with O, T; I, R; and N, S staples, while N, T and H, L constructs showed no effect in this assay across the indicated dose range. The sequences from top to bottom are SEQ ID NOs: 9, 156, 158, 160, 162, 154, and 153. [Figure 28] Figure 28 shows the different antiviral activities of exemplary double-staple and stitch peptides of an alternative core template sequence (SEQ ID NO: 258) in the context of its longer HR2-type peptide sequence corresponding to SEQ ID NO: 110, as evaluated in high throughput by an antibody-based SARS-CoV-2 detection platform in infected Vero E6 cells. Constructs with double i, i+4 staples N, S exhibited the most potent antiviral activity, followed by peptides containing N, T staples; however, other compounds in this group did not show significant effect in this assay across the indicated dose range. The sequences from top to bottom have SEQ ID NOs: 110 and 175–180. [Figure 29]Figure 29 shows the different antiviral activities of double-stapled and stitched peptides of the core template sequence (SEQ ID NO: 10) compared to a staple-free core template sequence that shows no antiviral activity, as evaluated by a SARS-CoV-2 pseudovirus assay that counts the number of infected cells by IXM microscopy based on fluorescence of ACE2-expressing 293T cells infected with GFP-expressing pseudovirus. The sequences from top to bottom are SEQ ID NOs: 10, 43, 44, and 47-52. [Figure 30] Figure 30 shows the different antiviral activities of double-stapled and stitched peptides of the core template sequence (SEQ ID NO: 10) in the context of its longer HR2 sequence (SEQ ID NO: 9), as evaluated by a SARS-CoV-2 pseudovirus assay that counts the number of infected cells by IXM microscopy based on fluorescence from ACE2-expressing 293T cells infected with GFP-expressing pseudovirus. The sequences from top to bottom have SEQ ID NOs: 153, 154, 156, 158, 160, and 162. [Figure 31] Figure 31 shows the different antiviral activities of double-staple peptides of a core template sequence (SEQ ID NO: 10) with or without N-terminal peptide elongation (aa1168~1176) and C-terminal derivatization with GSGSGC (SEQ ID NO: 256)-(PEG4-chol)-carboxamide, as evaluated by a SARS-CoV-2 pseudovirus assay that counts the number of infected cells by IXM microscopy based on fluorescence from ACE2-expressing 293T cells infected with GFP-expressing pseudovirus. The sequences from top to bottom are SEQ ID NOs: 155, 159, 161, and 167~170. [Figure 32] Figure 32 shows the different antiviral activities of double-stapled and stitched peptides of an alternative core template sequence (SEQ ID NO: 258) in the context of its longer HR2 type sequence (SEQ ID NO: 110), as evaluated by a SARS-CoV-2 pseudovirus assay that counts the number of infected cells by IXM microscopy based on fluorescence from ACE2-expressing 293T cells infected with GFP-expressing pseudovirus. The sequences from top to bottom have SEQ ID NOs: 175-180. [Modes for carrying out the invention]

[0048] Detailed explanation This disclosure is based, in particular, on the discovery that stabilizing peptides (e.g., staple, double staple, stitch, staple and stitch) can be designed to selectively bind to one or more coronaviruses (e.g., beta-coronaviruses such as SARS-CoV-2). Accordingly, this disclosure provides novel methods and compositions (e.g., peptides, stabilizing peptides, combinations of peptides; combinations of stabilizing peptides; combinations of peptides and stabilizing peptides) for treating infection by one or more coronaviruses (e.g., beta-coronaviruses such as SARS-CoV-2), for developing treatments against said infection, and for preventing said infection. Accordingly, the peptides and compositions disclosed herein can be used to prevent and / or treat COVID-19.

[0049] Coronavirus peptide An exemplary amino acid sequence of the coronavirus surface glycoprotein is provided in Figure 1. (See also GenBank accession number QHD43416.1.) An exemplary amino acid sequence of the heptad repeat domain 1 (HR1) of SARS-CoV-2 S is shown in Figure 2. An exemplary amino acid sequence of the heptad repeat domain 2 (HR2) of SARS-CoV-2 S is also shown in Figure 2.

[0050] Other exemplary amino acid sequences of HR2 in SARS-CoV-2 S are provided in Table 1 as SEQ ID NOs. 9, 10, 103, 104, 106, 108, and 110 (alternative HR2 region (EK1)).

[0051] In certain examples, the SARS-CoV-2 HR1 or HR2 peptides described herein (e.g., SEQ ID NOs: 2, 3, 9, 10, 103, 104, 106, 108, and 110) may also include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (for the amino acid sequence described in any one of SEQ ID NOs: 2, 3, 9, 10, 103, 104, 106, 108, or 110), for example, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved and / or non-conserved amino acid substitutions. Furthermore, in some examples, at least two (e.g., 2, 3, 4, 5, or 6) amino acids in SEQ ID NOs: 2, 3, 9, 10, 103, 104, 106, 108, or 110 may be substituted with α,α-disubstituted unnatural amino acids having olefin side chains. The type of substitution performed can be guided, for example, by the alignment of the HR2-like regions of the SARS, MERS, and EK1 peptides (Figure 6B), and the guidelines provided by Figure 16D. The guidelines provided in the following section on structurally stabilized peptides regarding modifiable amino acids are equally applicable to the peptides described herein. In such alignments, residues that do not change between SARS, MERS, and EK1 are either unmodified or substituted with unnatural or conserved amino acids. Residues in alignments that are found to be substituted by a conservative substitution in the HR2-like region of MERS or EK1 (e.g., isoleucine in SARS is substituted with leucine or methionine) are either unsubstituted or substituted by a conserved amino acid substitution. Non-conserved residues between the HR2-like regions of SARS, MERS, and EK1 can be replaced by any amino acid.

[0052] "Conservative amino acid substitution" means replacing one amino acid with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), and amino acids with acidic side chains and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine).

[0053] In some examples, the SARS-CoV-2 HR1 or HR2 peptides described herein (e.g., SEQ ID NOs: 2, 3, 9, 10, 103, 104, 106, 108, or 110) may also contain at least one, at least two, at least three, at least four, or at least five amino acids attached to the N-terminus of the peptide. In some examples, the SARS-CoV-2 HR1 or HR2 peptides described herein (e.g., SEQ ID NOs: 2 or 3, 9, 10, 103, 104, 106, 108 or 110) may also contain at least one, at least two, at least three, at least four, or at least five amino acids deleted at the N-terminus of the peptide.

[0054] In some cases, the peptides are lipid-added. In some cases, the peptides are modified to contain polyethylene glycol and / or cholesterol. In some cases, the peptides (e.g., SEQ ID NOs. 3, 9, 10, 103, 104, 106, 108, or 110) contain the GSGSGC(SEQ ID NOs. 256) sequence added to the C-terminus of the peptide. In some cases, the peptides (e.g., SEQ ID NOs. 3, 9, 10, 103, 104, 106, 108, or 110) contain GSGSGC(SEQ ID NOs. 256)-(PEG4-chol)-carboxamide added to the C-terminus of the peptide. In some examples, the peptide is one of SEQ ID NOs. 102, 105, 107, and 109, or the peptide in SEQ ID NOs. 102, 105, 107, and 109 has positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 that differ from these sequences.

[0055] In some examples, peptides have an amino acid length of 19 to 100 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 345, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100).

[0056] In some cases, the peptides described above bind to the recombinant 5-helix bundle of the SARS-CoV-2 S protein and / or inhibit or disrupt the interaction between the recombinant 5-helix bundle and the SARS-CoV-2 HR2 peptide (e.g., one of SEQ ID NOs. 9, 10, 103, 104, 106, 108); and / or inhibit the fusion of SARS-CoV-2 with host cells; and / or inhibit the infection of cells by SARS-CoV-2.

[0057] Structurally stabilized peptides Staple or stitched SARS-CoV-2 peptides based on a portion or alternative HR2 region (EK1) of the HR2 region are disclosed herein. In some examples, the staple or stitched SARS-CoV-2 peptide is based on SARS-CoV-2 HR2 (1169-1210) It originates from (ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYI (Sequence ID 9)). In some cases, the staple or stitch SARS-CoV-2 peptide derived from Sequence ID 9 is as follows, as shown in Table 1 below: SAH-SARS-CoV-2-A;SAH-SARS-CoV-2-B;SAH-SARS-CoV-2-C;SAH-SARS-CoV-2-D;SAH-SARS-CoV-2-E;SAH-SARS-CoV-2-F;SAH-SARS-CoV-2-G;SAH-SARS-CoV-2-A,D;SAH-SARS-CoV-2-A ,E;SAH-SARS-CoV-2-A,F;SAH-SARS-CoV-2-A,G;SAH-SARS-CoV-2-B,D;SAH-SARS-CoV-2-B,E;SAH-SARS-CoV-2-B,F;SAH-SARS-C oV-2-B,G;SAH-SARS-CoV-2-C,D;SAH-SARS-CoV-2-C,E;SAH-SARS-CoV-2-C,F; or SAH-SARS-CoV-2-C,G (eg, SEQ ID NOS: 11-29). Additional sequences are shown in Table 1.

[0058] In some cases, staples or stitches of SARS-CoV-2 peptides are used to connect SARS-CoV-2 HR2 (1179-1197)It originates from (IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10)). In some cases, the staple or stitch SARS-CoV-2 peptide derived from SEQ ID NO: 10 is as follows: SAH-SARS-CoV-2-H;SAH-SARS-CoV-2-I;SAH-SARS-CoV-2-J;SAH-SARS-CoV-2-K;SAH-SARS-CoV-2-L;SAH-SARS-CoV-2-M;SAH-SARS-CoV-2-N;SAH-SARS-CoV-2-O;SAH-SARS-CoV-2-P;SAH-SARS-CoV-2-Q;SAH-SARS-CoV -2-R;SAH-SARS-CoV-2-S;SAH-SARS-CoV-2-T;SAH-SARS-CoV-2-HL;SAH-SARS-CoV-2-IM;SAH-SARS-CoV-2-HQ;SAH-SARS-CoV-2-IR;SAH-SARS-CoV SAH-SARS-CoV-2-N,T; Additional sequences are shown in Table 1.

[0059] In some cases, SARS-CoV-2 HR2 (1179-1197) (The staple or stitched SARS-CoV-2 peptide derived from IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10) further contains the amino acid sequence ISGINASVVN (SEQ ID NO: 250) added to the N-terminus of the amino acid sequence. In some examples, SARS-CoV-2 HR2 (1179-1197) (The staple or stitched SARS-CoV-2 peptide derived from IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10) further contains the amino acid sequence DISGINASVVN (SEQ ID NO: 251) added to the N-terminus of the amino acid sequence. In some examples, SARS-CoV-2 HR2 (1179-1197)(The staple or stitched SARS-CoV-2 peptide derived from IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10) further contains the amino acid sequence IDLQEL (SEQ ID NO: 252) added to the C-terminus of the amino acid sequence. In some examples, SARS-CoV-2 HR2 (1179-1197) (The staple or stitched SARS-CoV-2 peptide derived from IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10) further contains the amino acid sequence IDLQELGKYEQYI (SEQ ID NO: 253) added to the C-terminus of the amino acid sequence. In some examples, SARS-CoV-2 HR2 (1179-1197) (The staple or stitch SARS-CoV-2 peptide derived from IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10) further contains the amino acid sequence IDLQELGSGSGC (SEQ ID NO: 254) added to the C-terminus of the amino acid sequence. In some examples, SARS-CoV-2 HR2 (1179-1197) (The staple or stitch SARS-CoV-2 peptide derived from IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10) further contains the amino acid sequence IDLQELGKYEQYIGSGSGC (SEQ ID NO: 255) which is added to the C-terminus of the amino acid sequence.

[0060] In some cases, staples or stitches of SARS-CoV-2 peptides are used to connect SARS-CoV-2 HR2 (1179-1197)* (In formula IQKEIDRLNEVAKNLNESL* (SEQ ID NO: 102), * = GSGSGC (SEQ ID NO: 256)-(PEG4-chol)-carboxamide). In some cases, staple or stitch SARS-CoV-2 peptides are found in COVID-19 HR2 (1169-1197) Derived from (ISGINASVVNIQKEIDRLNEVAKNLNESL (SEQ ID NO: 103)). In some cases, staple or stitch SARS-CoV-2 peptides are found in COVID-19 HR2 (1179-1203) It originates from (IQKEIDRLNEVAKNLNESLIDLQEL (Sequence ID 104)). In some cases, staple or stitch SARS-CoV-2 peptides are found in COVID-19 HR2 (1179-1203)*(IQKEIDRLNEVAKNLNESLIDLQEL*(SEQ ID NO: 105)). In some examples, the staple or stitch SARS-CoV-2 peptide is COVID19 HR2 (1168-1197) (DISGINASVVNIQKEIDRLNEVAKNLNESL(SEQ ID NO: 106)). In some examples, the staple or stitch SARS-CoV-2 peptide is COVID19 HR2 (1168-1197)* (DISGINASVVNIQKEIDRLNEVAKNLNESL*(SEQ ID NO: 107)). In some examples, the staple or stitch SARS-CoV-2 peptide is COVID19 HR2 (1168-1203) (DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL(SEQ ID NO: 108)). In some examples, the staple or stitch SARS-CoV-2 peptide is COVID19 HR2 (1168-1203)* (DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL*(SEQ ID NO: 109)). In some examples, the staple or stitch SARS-CoV-2 peptide is EK1 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL(SEQ ID NO: 110)). In some examples, the staple or stitch SARS-CoV-2 peptide is EK1* (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL*(SEQ ID NO: 111)).

[0061] In some examples, the SARS-CoV-2 HR2 stabilizing peptide comprises any one of SEQ ID NOs: 11 - 52 or 112 - 180. In some examples, the SARS-CoV-2 HR2 stabilizing peptide consists of any one of SEQ ID NOs: 11 - 52 or 112 - 180. In some examples, the staple and / or stitch SARS-CoV-2 peptide is derived from SEQ ID NOs: 9, 10, 103, 104, 106, 108, and 110 and is listed in Table 1.

Table 1-1

[0062] In Table 1, "8" is 8, 81, and 82 = (R)-α(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine; X, X1, X2, X3, and X4 = (S)-α-(4'-pentenyl)alanine; # = α,α-bis(4'-pentenyl)glycine or α,α-bis(7'-octenyl)glycine; % = (S)-α-(7'-octenyl)alanine or (S)-α-(4'-pentenyl)alanine; B = norleucine; and * = GSGSGC (SEQ ID NO: 256)-(PEG4-chol)-carboxamide. It should be understood that the above peptides can be modified to include additional amino acids at the N-terminus and / or C-terminus (e.g., by adding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), and / or to have deletions at the N-terminus and / or C-terminus (e.g., by deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).

[0063] Note that the bold and underlined sequences used herein (e.g., Table 1) identify the N-terminal and C-terminal staple amino acids and interstitial sequences between staples for each peptide disclosed. In some cases (e.g., SEQ ID NOs. 11-16, 30-42, and 112-152), the structurally stabilized peptides are single-staple peptides. In some cases (e.g., SEQ ID NOs. 18-20, 22-24, 26-28, 49-52, 155-174, and 177-180), the structurally stabilized peptides are double-staple peptides. In some cases (e.g., SEQ ID NOs. 17, 43-48, 153, 154, 175, and 176), the structurally stabilized peptides are stitch peptides. In some cases (e.g., SEQ ID NOs. 21, 25, and 29), the structurally stabilized peptides are both staple and stitch peptides.

[0064] This disclosure encompasses all peptides and structurally stabilized peptides listed in Table 1, as well as their variants. In some examples, structurally stabilized peptides have amino acid lengths of 19 to 100 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 345, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100). In some examples, the structurally stabilized peptides described above possess one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to recombinant 5-helix bundle protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9 or 10); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0065] In some examples, peptides containing 0 to 10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions are disclosed herein compared to one of the single-staple peptides in Table 1 (e.g., SEQ ID NOs. 11-16, 30-42, and 112-152). In some examples, peptides that are at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical) to one of the single-staple peptides in Table 1 (e.g., SEQ ID NOs. 11-16, 30-42, and 112-152) are disclosed herein. In some examples, structurally stabilized peptides have an amino acid length of 19–100 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 345, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100). In some examples, the structurally stabilized peptides described above possess one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to recombinant 5-helix bundle protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9 or 10); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0066] In some examples, peptides containing 0 to 10 amino acid substitutions (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) compared to one of the double-staple peptides in Table 1 (e.g., SEQ ID NOs. 18-20, 22-24, 26-28, 49-52, 155-174, and 177-180) are disclosed herein. In some examples, peptides that are at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical) to one of the double-staple peptides in Table 1 (e.g., SEQ ID NOs. 18-20, 22-24, 26-28, 49-52, 155-174, and 177-180) are disclosed herein. In some examples, structurally stabilized peptides have an amino acid length of 19–100 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 345, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100). In some examples, the structurally stabilized peptides described above possess one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to recombinant 5-helix bundle protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9 or 10); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0067] In some examples, peptides containing 0 to 10 amino acid substitutions (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) compared to one of the stitch peptides in Table 1 (e.g., SEQ ID NOs. 17, 43-48, 153, 154, 175, and 176) are disclosed herein. In some examples, peptides that are at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical) to one of the stitch peptides in Table 1 (e.g., SEQ ID NOs. 17, 43-48, 153, 154, 175, and 176) are disclosed herein. In some examples, structurally stabilized peptides have an amino acid length of 19–100 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 345, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100). In some examples, the structurally stabilized peptides described above possess one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to recombinant 5-helix bundle protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9 or 10); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0068] In some examples, peptides are disclosed herein that contain 0 to 10 amino acid substitutions (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) compared to one of the peptides in Table 1 (e.g., SEQ ID NOs. 21, 25, and 29) and are both staples and stitches. In some examples, peptides are disclosed herein that are at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical) to one of the peptides in Table 1 (e.g., SEQ ID NOs. 21, 25, and 29) and are both staples and stitches. In some cases, these structurally stabilized peptides possess one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to recombinant 5-helix bundle protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9 or 10); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0069] In some examples, structurally stabilized peptides have an amino acid length of 19–100 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 345, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100). In some examples, the structurally stabilized peptides described above possess one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to recombinant 5-helix bundle protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0070] In some examples, a staple or stitch peptide is a peptide containing or consisting of any one of the amino acid sequences of SEQ ID NOs. 9, 10, 103, 104, 106, 108, and 110, except that at least two (e.g., 2, 3, 4, 5, 6) amino acids in SEQ ID NOs. 9, 10, 103, 104, 106, 108, and 110 are replaced with non-natural amino acids that can form a staple or stitch. In some examples, the non-natural amino acids are α,α-disubstituted non-natural amino acids having an olefin side chain. In some examples, a staple or stitch peptide is a peptide containing or consisting of any one of the amino acid sequences of SEQ ID NOs. 10, 103, 104, 106, 108, and 110, except that at least two (e.g., 2, 3, 4, 5, 6) amino acids in SEQ ID NOs. 10, 103, 104, 106, 108, and 110 are replaced with non-natural amino acids that can form a staple or stitch. In some cases, non-natural amino acids are α,α-disubstituted non-natural amino acids with olefin side chains. In some cases, structurally stabilized peptides have an amino acid length of 19 to 100 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 345, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100) amino acids. In some examples, the structurally stabilized peptides described above possess one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to recombinant 5-helix bundle protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9 or 10); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0071] In some examples, peptides containing 0 to 10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions are disclosed herein compared to one of the unmodified peptides in Table 1 (e.g., SEQ ID NOs. 9, 10, 103, 104, 106, 108, and 110). In some examples, peptides that are at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) identical to one of the unmodified peptides in Table 1 (e.g., SEQ ID NOs. 9, 10, 103, 104, 106, 108, and 110) are disclosed herein. In some examples, the substitutions described herein are conservative substitutions. In some examples, structurally stabilized peptides have an amino acid length of 19–100 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 345, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100). In some examples, the structurally stabilized peptides described above possess one or more (1, 2, 3, 4, 5, 6) of the following properties: (i) binding to recombinant 5-helix bundle protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (SEQ ID NO: 9 or 10); (iii) being alpha-helical; (iv) being protease-resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2.

[0072] In some examples, any substitution described herein may be a conservative substitution. In some examples, any substitution described herein may be a non-conservative substitution.

[0073] In some examples, in any of the peptides containing IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10) (i.e., any peptide disclosed herein containing IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10); for example, the peptides listed in Table 1), the following amino acid hydrophobic amino acid substitutions can be made (shown in bold and underline): [ka] . Therefore, for example, I1179, I1183, L1186, A1190, L1193, and L1197 can be substituted with valine, isoleucine, leucine, phenylalanine, tryptophan, or cysteine. In some cases, these positions may be substituted with alanine or histidine.

[0074] In some examples, amino acid substitutions can be made in any of the peptides containing IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10) (i.e., any peptide disclosed herein containing IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10); for example, the peptides listed in Table 1) at the following positions (shown in bold and underlined): [ka] . In some examples, any of these bolded and underlined positions (Q1180, E1182, R1185, N1187, V1189, N1192, N1194, or S1196) can be substituted with an α,α disubstituted unnatural amino acid having an olefin side chain. In some examples, substitutions at these positions are to nonpolar amino acids (e.g., G, A, P, V, L, IM, W, F, or C). In some examples, substitutions at these positions are to alanine. In some examples, substitutions at these positions are to improve peptide bonds (i.e., substitutions to the 5-helix bundle of SARS-CoV-2).

[0075] In some examples, substitutions do not occur at one or more of the following positions in any of the peptides containing IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10) (i.e., any peptide disclosed herein containing IQKEIDRLNEVAKNLNESL (SEQ ID NO: 10); for example, the peptides listed in Table 1) (indicated in bold and underlined): [ka] . In particular, these bold and underlined positions (i.e., K1181, D1184, E1188, K1191, E1195) are not substituted with staple amino acids (e.g., α,α disubstituted unnatural amino acids with olefin side chains).

[0076] In some examples, substitutions are made at one or more of the following positions. [ka] . In these examples, the substitution is to a charged amino acid or a polar amino acid (e.g., R, K, H, D, E, Q, Y, S, T, or N).

[0077] In some examples, with respect to sequence number 9, no substitutions are made at the following positions that directly contact HR1 (shown in bold and underlined; I1169, I1172, A1174, S1175, V1177, I1198, L1200, L1203), or if substitutions are made, one or more of these positions may be substituted with conserved amino acid substitutions for the following (e.g., for I, A, V, or L, the conserved substitution is one of G, A, V, L, or I; and for S, the conserved substitution is T, M, or C): [ka] . In some cases, if D1168 also exists as D1168 in the sequence, it should also not be substituted, or should only be substituted with a conserved amino acid substitution (e.g., E).

[0078] In some examples, with respect to SEQ ID NO: 9, one or more of the following positions exposed to the solvent (S1170, G1171, N1173, V1176, N1178, D1199, Q1201, or E1202) can be replaced with any amino acid substitution (shown in bold and underline): [ka] .

[0079] In some cases, unnatural amino acids that can be used as staple or stitch amino acids include: (R)-2-(2'-propenyl)alanine; (R)-2-(4'-pentenyl)alanine; (R)-α-(7'-octenyl)alanine; (S)-α-(2'-propenyl)alanine; (S)-α-(4'-pentenyl)alanine; (S)-2-(7'-octenyl)alanine; α,α-bis(4'-pentenyl)glycine; and α,α-bis(7'-octenyl)glycine.

[0080] In some embodiments, internal staples replace two amino acid side chains, i.e., each staple is between two amino acids separated by, for example, two, three, or six amino acids. In some embodiments, internal stitches replace three amino acid side chains, i.e., a stitch is a pair of crosslinks between three amino acids separated by, for example, two, three, or six amino acids. In some embodiments, the amino acids forming the staple or stitch are located at staple positions i and i+3, respectively. In some embodiments, the amino acids forming the staple or stitch are located at staple positions i and i+4, respectively. In some embodiments, the amino acids forming the staple or stitch are located at staple positions i and i+7, respectively. For example, if a peptide has the sequence ...X1, X2, X3, X4, X5, X6, X7, X8, X9..., then crosslinks between X1 and X4 (i and i+3), or between X1 and X5 (i and i+4), or between X1 and X8 (i and i+7) are useful hydrocarbon staple forms of that peptide. The use of multiple crosslinks (e.g., two, three, four, or more) is also envisioned. Further descriptions relating to the preparation and use of hydrocarbon staple peptides can be found, for example, in U.S. Patent Publications 2012 / 0172285, 2010 / 0286057, and 2005 / 0250680, all of which are incorporated herein by reference in their entirety.

[0081] "Peptide staple" is a term derived from synthetic methodologies in which two olefin-containing side chains (e.g., crosslinkable side chains) present in a peptide chain are covalently bonded (e.g., "stapled together") by a ring-closing metathesis (RCM) reaction to form a crosslinking ring (see, e.g., Blackwell et al., J. Org. Chem., 66:5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). Structural stabilization may be achieved, for example, by stapling the peptide (see, e.g., Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are incorporated herein by reference in their entirety). In some cases, the staples are hydrocarbon staples.

[0082] In some examples, structural stabilization is a stitch. As used herein, the term “peptide stitch” refers to multiple tandem-staple events in a single peptide chain to provide a “stitch” (e.g., tandem or multiple staple) peptide in which two staples are linked, for example, to a common residue. Peptide stitches are disclosed, for example, in International Publication No. 2008 / 121767 and International Publication No. 2010 / 068684, both of which are incorporated herein by reference in their entirety.

[0083] In some examples, the staples or stitches used herein are lactam staples or stitches; UV-additional cycloadjusted staples or stitches; oxime staples or stitches; thioether staples or stitches; double-click staples or stitches; bislactam staples or stitches; bisarylated staples or stitches; or any two or more combinations thereof. The stabilizing peptides described herein include staple peptides and stitch peptides, as well as peptides containing multiple stitches, multiple staples, or mixtures of staples and stitches, or any other chemical strategies for structural enhancement (e.g., Balaram P.Cur.Opin.Struct.Biol.1992;2:845;Kemp DS,et al.,J.Am.Chem.Soc.1996;118:4240;Orner BP,et al.,J.Am.Chem.Soc.2001;123:5382;Chin JW,et al.,Int.Ed.2001;40:3806;Chapman RN,et al.,J.Am.Chem.Soc.2004;126:12252;Horne WS,et al.,Chem.,Int.Ed.2008;47:2853;Madden et See also al., Chem Commun (Camb). 2009 Oct 7; (37): 5588-5590; Lau et al., Chem. Soc. Rev., 2015, 44: 91-102; and Gunnoo et al., Org. Biomol. Chem., 2016, 14: 8002-8013. Each of these is incorporated herein by reference in whole.

[0084] Peptides are "structurally stabilized" in the sense that they maintain their native secondary structure. For example, staples allow peptides that tend to have an α-helical secondary structure to maintain that native α-helical structure. This secondary structure can increase the peptide's resistance to proteolytic cleavage and heat, and can enhance target binding affinity, hydrophobicity, and cell permeability. Therefore, staple (crosslinked) peptides described herein have improved biological activity and pharmacology compared to their corresponding non-staple (non-crosslinked) peptides.

[0085] In certain cases, modifications(s) for introducing structural stabilization (e.g., internal crosslinking, e.g., staples, stitches) to the SARS-CoV-2 HR2 peptide described herein may be located on a face of the SARS-CoV-2 HR2 helix that does not interact with the recombinant 5-helix bundle of SARS-CoV-2. Alternatively, modifications(s) for introducing stabilization (e.g., internal crosslinking, e.g., staples or stitches) to the SARS-CoV-2 HR2 peptide described herein may be located on a face of the SARS-CoV-2 HR2 helix that interacts with the 5-helix bundle of SARS-CoV-2. In some cases, the SARS-CoV-2 HR2 peptide described herein is stabilized by introducing staples or stitches (e.g., hydrocarbon staples or stitches) at the interface between the interacting and non-interacting helical faces of the SARS-CoV-2 HR2 protein.

[0086] In some examples, modifications to introduce structural stabilization to the SARS-CoV-2 HR2 peptide described herein (e.g., internal crosslinking, e.g., stapling or stitching) are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to the following residues: (i) Sequence IDs 9-5 and 9-12; (ii) Sequence IDs 9-6 and 9-13; (iii) Sequence IDs 9-7 and 9-14; (iv) Sequence IDs 9-26 and 33; (v) Sequence IDs 9-27 and 34; (vi) Sequence IDs 9-33 and 40; (vii) Sequence IDs 9-26, 33 and 40; (viii) Sequence IDs 9-5, 12, 26 and 33; (ix) Sequence IDs 9-5, 12, 27 and 34; (x) Sequence numbers 5, 12, 33 and 40 of sequence 9; (xi) Sequence IDs 9-5, 12, 26, 33 and 40; (xii) Sequence IDs 9-6, 13, 26 and 33; (xiii) Sequence IDs 9-6, 13, 27 and 34; (xiv) Sequence IDs 9-6, 13, 33 and 40; (xv) Sequence numbers 9, 6, 13, 26, 33 and 40; (xvi) Sequence numbers 9, 7, 14, 26 and 33; (xvii) Sequence IDs 9-7, 14, 27 and 34; (xviii) Sequence IDs 9-7, 14, 33 and 40; or (xix) Sequence numbers 7, 14, 26, 33 and 40 of sequence number 9.

[0087] In some examples, modifications to introduce structural stabilization to the SARS-CoV-2 HR2 peptide described herein (e.g., internal crosslinking, e.g., stapling or stitching) are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to the following residues: (i) Sequence IDs 10-1 and 10-8; (ii) Sequence IDs 10-2 and 10-9; (iii) Sequence IDs 10-3 and 10; (iv) Sequence IDs 10-4 and 11; (v) Sequence IDs 10-5 and 12; (vi) Sequence IDs 10-6 and 13; (vii) Sequence IDs 10-7 and 14; (viii) Sequence IDs 10-8 and 15; (iv) Sequence IDs 10-9 and 16; (x) Sequence IDs 10-10 and 17; (xi) Sequence IDs 10-11 and 18; (xi) Sequence IDs 10-12 and 19; (xii) Sequence ID 10-1 and 5; (xiv) Sequence ID 10-2 and 6; (xv) Sequence ID 10, 3 and 7; (xvi) Sequence ID 10, 4 and 8; (xvii) Sequence IDs 10-5 and 9; (xviii) Sequence IDs 10-6 and 10; (xiv) Sequence IDs 10-7 and 11; (xx) Sequence IDs 10-8 and 12; (xxi) Sequence IDs 10-9 and 13; (xxii) Sequence IDs 10-10 and 14; (xxiii) Sequence IDs 10-11 and 15; (xxiv) Sequence IDs 10-12 and 16; (xxv) Sequence IDs 10-13 and 17; (xxvi) Sequence IDs 10-14 and 18; (xxvii) Sequence IDs 10-15 and 19; (xxviii) Sequence IDs 10-2, 9 and 16; (xxiv) Sequence IDs 10-3, 10 and 17; (xxx) Sequence IDs 10-2, 9, and 13; (xxxi) Sequence IDs 10-3, 10, and 14; (xxxxii) Sequence IDs 10-6, 13, and 17; (xxxiv) Sequence IDs 10-7, 14 and 18; (xxxv) Sequence numbers 10-2, 6, 13 and 17; (xxxvi) Sequence numbers 3, 7, 13 and 17 of sequence number 10; (xxxvii) Sequence IDs 10-2, 6, 14 and 18; or (xxxviii) Sequence numbers 3, 7, 14 and 18 of sequence number 10.

[0088] In certain examples, the SARS-CoV-2 HR2 peptides described herein (e.g., SEQ ID NOs. 11-52, 112-180, or 258) may also include one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions (for the amino acid sequence described in any one of SEQ ID NOs. 11-52, 112-180, or 258), such as one or more (e.g., 1, 2, 3, 4, or 5) conserved and / or non-conserved amino acid substitutions. In some examples, the SARS-CoV-2 HR2 peptides described herein (e.g., SEQ ID NOs. 11-52, 112-180, or 258) may also include at least one, at least two, at least three, at least four, or at least five amino acids added to the N-terminus of the peptide. In some examples, the SARS-CoV-2 HR2 peptides described herein (e.g., SEQ ID NOs. 11-52, 112-180, or 258) may also contain at least one, at least two, at least three, at least four, or at least five amino acids added to the C-terminus of the peptide.

[0089] In one embodiment, the structurally stabilized SARS-CoV-2 HR2 peptide is given by formula (I), [ka] or comprising a pharmaceutically acceptable salt thereof, in the formula, Each R1 and R2 is independently H or C1~C 10 Alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 is alkyl, alkenyl, or alkynyl; [R4-K-R4] n And each of them is replaced by 0 to 6 R5s; R4 is an alkyl, alkenyl, or alkynyl; R5 is a halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R6, fluorescent moiety, or radioisotope; K is O, S, SO, SO2, CO, CO2, CONR6, or [ka] And, R6 is H, alkyl, or therapeutic agent; n is an integer between 1 and 4; x is an integer between 2 and 10; Each y is an independent integer between 0 and 100; z is an integer between 1 and 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); Each Xaa is an amino acid independently; and The structurally stabilized peptide binds to the recombinant 5-helix bundle COVID-19 S protein.

[0090] In some embodiments, [Xaa] of formula (I) w [Xaa] of equation (I) x and [Xaa] of equation (I) y Each of these is as described for one of the structures 1 through 60 in Table 2. For example, [Xaa] for structure 1 in Table 2. w [Xaa] x and [Xaa] y For stabilized peptides containing [Xaa], [Xaa]w, [Xaa]x, and [Xaa]y are ISGI (SEQ ID NO: 53), ASVVNI (SEQ ID NO: 54), and KEIDRLNEVAKNLNESLIDLQELGKYEQYI (SEQ ID NO: 55), respectively. As another example, [Xaa] in construct 2 of Table 2 w [Xaa] x and [Xaa] y For stabilized peptides containing [Xaa]w, [Xaa]x, and [Xaa]y are ISGIN (SEQ ID NO: 56), SVVNIQ (SEQ ID NO: 57), and EIDRLNEVAKNLNESLIDLQELGKYEQYI (SEQ ID NO: 58), respectively. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0091] In certain examples, the sequences shown above in Table 2 may have at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid substitutions or deletions. The SARS-CoV-2 HR2 peptide may contain any of the amino acid sequences described herein.

[0092] In some examples, formula (I) containing the sequences shown in Table 2 above may have one or more of the following properties: (i) binding to recombinant SARS-CoV-2 5-helix bundle S protein; (ii) being an alpha helix; (iii) being protease resistant; (iv) inhibiting fusion of SARS-CoV-2 with host cells; and / or (v) inhibiting cell infection by SARS-CoV-2.

[0093] The tether of formula (I) is an alkyl, alkenyl, or alkynyl moiety (e.g., C5, C8, C 11 , or C 12 Alkyl, C5, C8, or C 11 Alkenyl, or C5, C8, C 11 , or C 12 It may contain alkynyl compounds. The tether amino acid may be alpha-disubstituted (e.g., C1-C3 or methyl).

[0094] In some examples of formula (I), x is 2, 3, or 6. In some examples of formula (I), each y is independently an integer between 0 and 15, or between 3 and 15. In some examples of formula (I), R1 and R2 are independently H or C1-C6 alkyl. In some examples of formula (I), R1 and R2 are independently C1-C3 alkyl. In some examples of formula (I), at least one of R1 and R2 is methyl. For example, both R1 and R2 could be methyl. In some examples of formula (I), R3 is alkyl (e.g., C8 alkyl) and x is 3. In some examples of formula (I), R3 is C 11 It is an alkyl group, and x is 6. In some examples of formula (I), R3 is an alkenyl (e.g., a C8 alkenyl), and x is 3. In some examples of formula (I), x is 6, and R3 is C 11 It is an alkenyl. In some examples, R3 is a linear alkyl, alkenyl, or alkynyl. In some examples, R3 is -CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-.

[0095] In one embodiment, the structurally stabilized COVID-19 HR2 peptide comprises formula (I) or a pharmaceutically acceptable salt thereof, wherein, Each R1 and R2 is H or C1-C 10 Alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which may be substituted or unsubstituted; Each R3 is independently alkylene, alkenylene, or alkynylene, and none of these may be substituted or unsubstituted; z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and (a) Each [Xaa] w ISGI (sequence number 53), and each [Xaa] x is ASVVNI (sequence number 54), and each [Xaa] yThis is KEIDRLNEVAKNLNESLIDLQELGKYEQYI (sequence number 55); (b) Each [Xaa] w This is ISGIN (sequence number 56), and each [Xaa] x SVVNIQ (Sequence ID 57), and each [Xaa] y This is EIDRLNEVAKNLNESLIDLQELGKYEQYI (sequence number 58); (c) Each [Xaa] w ISGINA (sequence number 59) is, and each [Xaa] x is VVNIQK (sequence number 60), and each [Xaa] y This is IDRLNEVAKNLNESLIDLQELGKYEQYI (sequence number 61); (d) Each [Xaa] w ISGINASVVNIQKEIDRLNEVAKNL (Sequence ID 62), and each [Xaa] x is ESLIDL (sequence number 63), and each [Xaa] y This is ELGKYEQYI (sequence number 64); (e) Each [Xaa] w ISGINASVVNIQKEIDRLNEVAKNLN (Sequence ID 65), and each [Xaa] x is SLIDLQ (sequence number 66), and each [Xaa] y This is LGKYEQYI (sequence number 67); (f) Each [Xaa] w ISGINASVVNIQKEIDRLNEVAKNLNESLIDL (Sequence ID 68), and each [Xaa] x is ELGKYE (sequence number 69), and each [Xaa] y is YI; (g) Each [Xaa] w is I, and each [Xaa] x is KEIDRL (sequence number 70), and each [Xaa] y This is EVAKNLNESL (sequence number 71); (h) Each [Xaa] w This is IQ, and each [Xaa] xis EIDRLN (SEQ ID NO: 72), and each [Xaa] y is VAKNLNESL (SEQ ID NO: 73); (i) Each [Xaa] w is IQKEI (SEQ ID NO: 74), each [Xaa] x is RLNEVA (SEQ ID NO: 75), and each [Xaa] y is NLNESL (SEQ ID NO: 76); (j) Each [Xaa] w is IQKEID (SEQ ID NO: 77), each [Xaa] x is LNEVAK (SEQ ID NO: 78), and each [Xaa] y is LNESL (SEQ ID NO: 79); (k) Each [Xaa] w is IQKEIDRL (SEQ ID NO: 80), each [Xaa] x is EVAKNL (SEQ ID NO: 81), and each [Xaa] y is ESL; (l) Each [Xaa] w is IQKEIDRLN (SEQ ID NO: 82), each [Xaa] x is VAKNLN (SEQ ID NO: 83), and each [Xaa] y is SL; (m) Each [Xaa] w is I, each [Xaa] x is KEI, and each [Xaa] y is RLNEVAKNLNESL (SEQ ID NO: 84); (n) Each [Xaa] w is IQ, each [Xaa] x is EID, and each [Xaa] y is LNEVAKNLNESL (SEQ ID NO: 85); (o) Each [Xaa] w is IQKEI (SEQ ID NO: 74), each [Xaa] x is RLN, and each [Xaa] y is VAKNLNESL (SEQ ID NO: 73); (p) Each [Xaa] w is IQKEIDRL (SEQ ID NO: 80), each [Xaa]x is EVA, and each [Xaa] y This is NLNESL (sequence number 76); (q) Each [Xaa] w This is IQKEIDRLN (sequence number 82), and each [Xaa] x is VAK, and each [Xaa] y This is LNESL (sequence number 79); (r) Each [Xaa] w This is IQKEIDRLNEVA (sequence number 86), and each [Xaa] x is NLN, and each [Xaa] y It is SL; (s) Each [Xaa] w This is IQKEIDRLNEVAK (sequence number 87), and each [Xaa] x is an LNE, and each [Xaa] y is L; (t) Each [Xaa]w is missing, each [Xaa]x is QKEIDR (sequence code 228), and each [Xaa]y is NEVAKNLNESL (sequence code 229); (u) Each [Xaa]w is IQK, each [Xaa]x is IDRLNE (sequence number 230), and each [Xaa]y is AKNLNESL (sequence number 231); (v) Each [Xaa]w is IQKE (sequence number 232), each [Xaa]x is DRLNEV (sequence number 181), and each [Xaa]y is KNLNESL (sequence number 182); (w) Each [Xaa]w is IQKEIDR (sequence code 183), each [Xaa]x is NEVAKN (sequence code 184), and each [Xaa]y is NESL (sequence code 185); (x) Each [Xaa]w is IQKEIDRLNE (sequence code 186), each [Xaa]x is AKNLNE (sequence code 187), and each [Xaa]y is L; (y) Each [Xaa]w is IQKEIDRLNEV (sequence code 188), each [Xaa]x is KNLNES (sequence code 189), and each [Xaa]y is missing; (z) Each [Xaa]w is QKE, each [Xaa]x is DRLNEVAKNLNESL (sequence number 190), and each [Xaa]y is missing; (aa) Each [Xaa]w is IQK, each [Xaa]x is IDR, and each [Xaa]y is NEVAKNLNESL (sequence number 229); (bb) Each [Xaa]w is IQK, each [Xaa]x is IDR, and each [Xaa]y is NEVAKNLNESL (sequence number 229); (cc) Each [Xaa]w is IQKE (sequence number 232), each [Xaa]x is DRL, and each [Xaa]y is EVAKNLNESL (sequence number 71); (dd) Each [Xaa]w is IQKEID (sequence number 77), each [Xaa]x is LNE, and each [Xaa]y is AKNLNESL (sequence number 231); (ee) Each [Xaa]w is IQKEIDR (sequence code 183), each [Xaa]x is NEV, and each [Xaa]y is KNLNESL (sequence code 182); (ff) Each [Xaa]w is IQKEIDRLNE (sequence code 186), each [Xaa]x is AKN, and each [Xaa]y is NESL (sequence code 185); (gg) Each [Xaa]w is IQKEIDRLNEV (sequence code 188), each [Xaa]x is KNL, and each [Xaa]y is ESL; (hh) Each [Xaa]w is IQKEIDRLNEVAKN (sequence number 191), each [Xaa]x is NES, and each [Xaa]y is missing; (ii) Each [Xaa]w is ISGINASVVN (sequence code 250), each [Xaa]x is QKEIDR (sequence code 228), and each [Xaa]y is NEVAKNLNESL (sequence code 229); (jj) Each [Xaa]w is ISGINASVVN (sequence code 193), each [Xaa]x is KEIDRL (sequence code 70), and each [Xaa]y is EVAKNLNESL (sequence code 71); (kk) Each [Xaa]w is ISGINASVVNIQ (sequence code 194), each [Xaa]x is EIDRLN (sequence code 72), and each [Xaa]y is VAKNLNESL (sequence code 73); (ll) Each [Xaa]w is ISGINASVVNIQK (sequence number 195), each [Xaa]x is IDRLNE (sequence number 230), and each [Xaa]y is AKNLNESL (sequence number 231); (mm) Each [Xaa]w is ISGINASVVNIQKE (sequence code 196), each [Xaa]x is DRLNEV (sequence code 181), and each [Xaa]y is KNLNESL (sequence code 182); (nn) Each [Xaa]w is ISGINASVVNIQKEI (sequence number 197), each [Xaa]x is RLNEVA (sequence number 75), and each [Xaa]y is NLNESL (sequence number 76); (oo) Each [Xaa]w is ISGINASVVNIQKEID (sequence code 198), each [Xaa]x is LNEVAK (sequence code 78), and each [Xaa]y is LNESL (sequence code 79); (pp) Each [Xaa]w is ISGINASVVNIQKEIDR (sequence code 199), each [Xaa]x is NEVAKN (sequence code 184), and each [Xaa]y is NESL (sequence code 185); (qq) Each [Xaa]w is ISGINASVVNIQKEIDRL (sequence code 200), each [Xaa]x is EVAKNL (sequence code 81), and each [Xaa]y is ESL; (rr) Each [Xaa]w is ISGINASVVNIQKEIDRLN (sequence code 201), each [Xaa]x is VAKNLN (sequence code 83), and each [Xaa]y is SL; (ss) Each [Xaa]w is ISGINASVVNIQKEIDRLNE (sequence code 202), each [Xaa]x is AKNLNE (sequence code 187), and each [Xaa]y is L; (tt) Each [Xaa]w is ISGINASVVNIQKEIDRLNEV (sequence code 203), each [Xaa]x is KNLNES (sequence code 189), and each [Xaa]y is missing; (uu) Each [Xaa]w is ISGINASVVN (sequence code 250), each [Xaa]x is QKE, and each [Xaa]y is DRLNEVAKNLNESL (sequence code 190); (vv) Each [Xaa]w is ISGINASVVNI (sequence code 193), each [Xaa]x is KEI, and each [Xaa]y is RLNEVAKNLNESL (sequence code 84); (ww) Each [Xaa]w is ISGINASVVNIQ (sequence number 194), each [Xaa]x is EID, and each [Xaa]y is LNEVAKNLNESL (sequence number 85); (xx) Each [Xaa]w is ISGINASVVNIQK (sequence number 195), each [Xaa]x is IDR, and each [Xaa]y is NEVAKNLNESL (sequence number 229); (yy) Each [Xaa]w is ISGINASVVNIQKE (sequence number 196), each [Xaa]x is DRL, and each [Xaa]y is EVAKNLNESL (sequence number 71); (zz) Each [Xaa]w is ISGINASVVNIQKEI (sequence number 197), each [Xaa]x is RLN, and each [Xaa]y is VAKNLNESL (sequence number 73); (aaa) Each [Xaa]w is ISGINASVVNIQKEID (sequence code 198), each [Xaa]x is LNE, and each [Xaa]y is AKNLNESL (sequence code 231); (bbb) Each [Xaa]w is ISGINASVVNIQKEIDR (sequence code 199), each [Xaa]x is NEV, and each [Xaa]y is KNLNESL (sequence code 182); (ccc) Each [Xaa]w is ISGINASVVNIQKEIDRL (sequence code 200), each [Xaa]x is EVA, and each [Xaa]y is NLNESL (sequence code 76); (ddd) Each [Xaa]w is ISGINASVVNIQKEIDRLN (sequence code 201), each [Xaa]x is VAK, and each [Xaa]y is LNESL (sequence code 79); (eee) Each [Xaa]w is ISGINASVVNIQKEIDRLNE (sequence code 202), each [Xaa]x is AKN, and each [Xaa]y is NESL (sequence code 185); (fff) Each [Xaa]w is ISGINASVVNIQKEIDRLNEV (sequence code 203), each [Xaa]x is KNL, and each [Xaa]y is ESL; (ggg) Each [Xaa]w is ISGINASVVNIQKEIDRLNEVA (sequence number 204), each [Xaa]x is NLN, and each [Xaa]y is SL; (hhh) Each [Xaa]w is ISGINASVVNIQKEIDRLNEVAK (sequence number 205), each [Xaa]x is LNE, and each [Xaa]y is L; or (iii) Each [Xaa]w is ISGINASVVNIQKEIDRLNEVAKN (sequence number 206), each [Xaa]x is NES, and each [Xaa]y is missing. The structurally stabilized SARS-CoV-2 HR2 peptide binds to the recombinant SARS-CoV-2 5-helix bundle S protein. In some cases, R1 is alkyl. In some cases, R1 is a methyl group. In some cases, R3 is alkyl. In some cases, R3 is a methyl group. In some cases, R2 is alkenyl. In some cases, Z is 1.

[0096] In another aspect of equation (I), both alpha, alpha disubstitution stereocenters are either in an R configuration or an S configuration (e.g., i, i+4 crosslink), or one stereocenter is R and the other is S (e.g., i, i+7 crosslink). Thus, when equation (I) is expressed as follows: [ka] Both the C' and C'' disubstituted centers can be in the R configuration, or both can be in the S configuration (for example, when x is 3). In equation (I), when x is 6, the C' disubstituted center is in the R configuration and the C'' disubstituted center is in the S configuration. The R3 double bond in equation (I) can have an E or Z stereochemical configuration.

[0097] In some examples of equation (I), R3 is [R4-K-R4] n R4 is a linear alkyl, alkenyl, or alkynyl.

[0098] In some cases, "z" in formula (I) is greater than 1. In some cases, z=2, as shown in formula (II). In these cases, the peptide contains more than one staple. In some cases, the peptide contains two staples (i.e., the peptide is double-stapled), as shown in formula (II). In some cases, a double-stapled peptide contains multiple staples in the same construct, [Xaa] t and [Xaa] u [Xaa] w [Xaa] x , and [Xaa] y Constructs containing the double-staple peptide are prepared. Double-staple peptides are provided as constructs 61-97 in Table 3.

[0099] Formula II provides the structure of a double-staple peptide. [ka]

[0100] For example, [Xaa] of structure 61 in Table 3 t [Xaa] u [Xaa] v [Xaa] x , and [Xaa] y For stabilized peptides containing [Xaa] t [Xaa] u [Xaa] v [Xaa]x , and [Xaa] y These are ISGI (sequence number 53), ASVVNI (sequence number 54), and KEIDRLNEVAKNL (sequence number 88), ESLIDL (sequence number 63), and ELGKYEQYI (sequence number 64), respectively. As another example, see [Xaa] in construct 62 of Table 3. t [Xaa] u [Xaa] v [Xaa] x , and [Xaa] y For stabilized peptides containing [Xaa] t [Xaa] u [Xaa] v [Xaa] x , and [Xaa] y These are ISGI (sequence number 53), ASVVNI (sequence number 54), and KEIDRLNEVAKNLN (sequence number 89), SLIDLQ (sequence number 66), and LGKYEQYI (sequence number 67), respectively. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0101] In one embodiment, the structurally stabilized (stitched) SARS-CoV-2 HR2 peptide is given by formula (III): [ka] or comprising a pharmaceutically acceptable salt thereof, in the formula, Each of R1 and R4 is independently H or C 1-10 Alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which may be substituted or unsubstituted; R2 and R3 each independently, C 5-20 Alkyl, alkenyl, alkynyl; [R4-K-R4] n And each of these is replaced by 0 to 6 R5s; R5 is a halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R6, fluorescent moiety, or radioisotope; K is O, S, SO, SO2, CO, CO2, CONR6, or [ka] And, R6 is H, alkyl, or therapeutic agent; n is an integer from 1 to 4; and [Xaa] w ;[Xaa] x ;[Xaa] y ; and [Xaa] z This is shown in Table 4.

[0102] In some embodiments, [Xaa] of formula (III) w [Xaa] of equation (III) x [Xaa] of equation (III) y [Xaa] of equation (III) z Each of these is as described for any one of the structures 98-108 in Table 4. For example, [Xaa]w, [Xaa]x, [Xaa]y, and [Xaa] in structure 98 of Table 4. z For stabilized peptides containing [Xaa]w, [Xaa]x, [Xaa]y, and [Xaa] zThese are ISGINASVVNIQKEIDRLNEVAKNL (sequence number 62), ESLIDL (sequence number 63), ELGKYE (sequence number 69), and YI, respectively. As another example, [Xaa]w, [Xaa]x, [Xaa]y, and [Xaa] in construct 99 of Table 4. z For stabilized peptides containing [Xaa]w, [Xaa]x, [Xaa]y, and [Xaa] z These are I, KEIDRL (sequence number 70), EVAKNL (sequence number 81), and ESL, respectively. [Table 4-1] [Table 4-2]

[0103] In some examples, formula (III) containing the sequences shown in Table 4 above may have one or more of the following properties: (i) binding to recombinant SARS-CoV-2 5-helix bundle S protein; (ii) being alpha-helical; (iii) being protease-resistant; (iv) inhibiting fusion of SARS-CoV-2 with host cells; and / or (v) inhibiting cell infection by SARS-CoV-2.

[0104] In some examples of formula (III), R1 and R4 are independently H or C1-C6 alkyl. In some examples of formula (III), R1 and R4 are independently C1-C3 alkyl. In some examples of formula (III), at least one of R1 and R4 is methyl. For example, both R1 and R4 may be methyl. In some examples of formula (III), R2 and R3 are independently alkyl (e.g., C 12 It is alkyl. In some examples of formula (III), R2 and R3 are, independently, C 12 It is alkyl. In some examples of formula (III), R2 and R3 are independently linear alkyl, alkenyl, or alkynyl (e.g., linear C12 (Alkyl, alkenyl, or alkynyl) In some examples of formula (III), R2 is -CH2-CH2-CH2-CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-CH2-. In some examples of formula (III), R3 is -CH2-CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-CH2-CH2-CH2-.

[0105] In some cases, the structurally stabilized SARS-CoV-2 HR2 peptide comprises formula (III) or a pharmaceutically acceptable salt thereof, in which, [Xaa] w ;[Xaa] x ;[Xaa] y ; and [Xaa] z This is shown in Table 4; Each R1 and R4 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and any of these may be substituted or unsubstituted; Each R2 and R3 is independently alkylene, alkenylene, or alkynylene, and none of these may be substituted or unsubstituted; the structurally stabilized SARS-CoV-2 HR2 peptide binds to the recombinant SARS-CoV-2 5-helix bundle S protein. In some examples, R1 is alkyl. In some examples, R1 is a methyl group. In some examples, R4 is alkyl. In some examples, R4 is a methyl group. In some examples, R2 is alkenyl. In some examples, R3 is alkenyl.

[0106] In another aspect of equation (III), of the three α,α disubstitution stereocenters: (i) two stereocenters are in the R configuration and one stereocenter is in the S configuration; or (ii) two stereocenters are in the S configuration and one stereocenter is in the R configuration. Thus, when equation (III) is expressed as follows: [ka] Both the C' and C''' disubstituted stereocenters can be in the R configuration, or both can be in the S configuration. If both C' and C''' are in the R configuration, then C'' is in the S configuration. If both C' and C''' are in the S configuration, then C'' is in the R configuration. The double bonds in R2 and R3 of equation (III) can be in the E or Z stereochemical configuration.

[0107] In some examples of equation (III), R3 is [R4-K-R4] n R4 is a linear alkyl, alkenyl, or alkynyl.

[0108] In another embodiment, the structurally stabilized peptide has the following structure: [ka] As shown, both staples and stitches, or a pharmaceutically acceptable salt thereof, in the formula, Each of R1, R3, R4, and R7 is independently an H or C1-10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and is either substituted or unsubstituted; Each of R2, R5, and R6 is independently a C5-20 alkyl, alkenyl, or alkynyl [R4-K-R4]n; each of these is substituted with 0 to 6 R5s; R5 is a halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R6, fluorescent moiety, or radioisotope; K is O, S, SO, SO2, CO, CO2, CONR6, or [ka] And, R6 is H, alkyl, or therapeutic agent; n is an integer from 1 to 4; and [Xaa]u [Xaa] v [Xaa] w [Xaa] x [Xaa] y , and [Xaa] z This is shown in Table 5.

[0109] In some embodiments, each of [Xaa]u, [Xaa]v, [Xaa]w, [Xaa]x, [Xaa]y, and [Xaa]z in formula (IV) is as described for any one of the structures 109 to 111 in Table 5. For example, [Xaa] in structure 109 in Table 5 u [Xaa] v [Xaa] w [Xaa] x [Xaa] y and [Xaa] z For stabilized peptides containing [Xaa]u, [Xaa] v [Xaa] w [Xaa] x [Xaa] y and [Xaa] z These are ISGI (sequence number 53); ASVVNI (sequence number 54); KEIDRLNEVAKNL (sequence number 88); ESLIDL (sequence number 63); ELGKYE (sequence number 69); and YI. As another example, [Xaa] in construct 110 of Table 5. u [Xaa] v [Xaa] w [Xaa] x [Xaa] y and [Xaa] z For stabilized peptides containing [Xaa] u [Xaa] v [Xaa] w [Xaa] x [Xaa] y and [Xaa] z These are ISGIN(sequence number 56);SVVNIQ(sequence number 57);EIDRLNEVAKNL(sequence number 91);ESLIDL(sequence number 63);ELGKYE(sequence number 69); and YI. [Table 5-1] [Table 5-2]

[0110] In some examples, formula (IV) containing the sequence shown in Table 5 above may have one or more of the following properties: (i) binding to recombinant SARS-CoV-2 5-helix bundle S protein; (ii) being an alpha helix; (iii) being protease resistant; (iv) inhibiting fusion of SARS-CoV-2 with host cells; and / or (v) inhibiting cell infection by SARS-CoV-2.

[0111] In some examples of formula (IV), R1, R3, R4, and R7 are each independently H or C1-C6 alkyl. In some examples of formula (IV), R2, R5, and R6 are each independently C1-C3 alkyl. In some examples of formula (IV), at least one of R1, R3, R4, and R7 is methyl. For example, R1, R3, R4, and R7 may all be methyl. In some examples of formula (IV), R2, R5, and R6 are each independently alkyl (e.g., C 12 It is alkyl. In some examples of formula (IV), R2, R5, and R6 are each independently C 12 It is alkyl. In some examples of formula (IV), R2, R5, and R6 are each independently linear alkyl, alkenyl, or alkynyl (e.g., linear C 12(Alkyl, alkenyl, or alkynyl) In some examples of formula (IV), R2 is -CH2-CH2-CH2-CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-CH2-. In some examples of formula (IV), R5 is -CH2-CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-CH2-CH2-CH2-. In some examples of formula (IV), R6 is -CH2-CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-CH2-CH2-CH2-.

[0112] In some cases, the structurally stabilized SARS-CoV-2 HR2 peptide comprises formula (IV) or a pharmaceutically acceptable salt thereof, where, [Xaa] u ;[Xaa] v ;[Xaa] w ;[Xaa] x ;[Xaa] y ; and [Xaa] z This is shown in Table 5; Each R1 and R4 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and any of these may be substituted or unsubstituted; Each R2 and R3 is independently alkylene, alkenylene, or alkynylene, and none of these may be substituted or unsubstituted; the structurally stabilized SARS-CoV-2 HR2 peptide binds to the recombinant SARS-CoV-2 5-helix bundle S protein. In some examples, R1 is alkyl. In some examples, R1 is a methyl group. In some examples, R4 is alkyl. In some examples, R4 is a methyl group. In some examples, R2 is alkenyl. In some examples, R3 is alkenyl. When used herein, "C i-j The term (where i and j are integers and are used in combination with a chemical group) indicates a range of carbon atoms in a chemical group, where ij defines the range. For example, C 1-6Alkyl refers to an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms.

[0113] As used herein, the term “alkyl,” used alone or in combination with other terms, refers to a saturated hydrocarbon group that may be linear or branched. In some embodiments, alkyl groups contain 1 to 7, 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and n-heptyl. In some embodiments, the alkyl group is methyl, ethyl, or propyl. The term “alkylene” refers to a linked alkyl group.

[0114] As used herein, “alkenyl,” when used alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon double bonds. In some embodiments, the alkenyl moiety contains 2 to 6 or 2 to 4 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, and sec-butenyl.

[0115] As used herein, “alkynyl,” when used alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propyne-1-yl, and propyne-2-yl. In some embodiments, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.

[0116] As used herein, “alkynyl,” when used alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propyne-1-yl, and propyne-2-yl. In some embodiments, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.

[0117] As used herein, the term "cycloalkylalkyl," when used alone or in combination with other terms, refers to a group of the formula cycloalkyl-alkyl-. In some embodiments, the alkyl moiety has 1 to 4, 1 to 3, 1 to 2, or 1 carbon atom. In some embodiments, the alkyl moiety is methylene. In some embodiments, the cycloalkyl moiety has 3 to 10 ring members or 3 to 7 ring members. In some embodiments, the cycloalkyl group is monocyclic or bicyclic. In some embodiments, the cycloalkyl moiety is monocyclic. In some embodiments, the cycloalkyl moiety is C 3-7 It is a monocyclic cycloalkyl group.

[0118] As used herein, the term “heteroarylalkyl,” when used alone or in combination with other terms, refers to a group of the formula heteroaryl-alkyl-. In some embodiments, the alkyl moiety has 1 to 4, 1 to 3, 1 to 2, or 1 carbon atom. In some embodiments, the alkyl moiety is methylene. In some embodiments, the heteroaryl moiety is a monocyclic or bicyclic group having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl moiety has 5 to 10 carbon atoms.

[0119] As used herein, the term “substituted” means that a hydrogen atom is replaced by a non-hydrogen group. It should be understood that substitutions in a given atom are limited by their valence.

[0120] As used herein, “halo” or “halogen,” used alone or in combination with other terms, includes fluoro, chloro, bromo, and iodine. In some embodiments, the halo is F or Cl.

[0121] In some embodiments, the disclosure features a structurally stabilized (e.g., stapled or stitched) peptide (or a modified version thereof) comprising any one amino acid sequence of SEQ ID NOs: 9, 10, 103, 104, 106, 108, or 110, wherein two amino acid side chains separated by 2, 3, or 6 amino acids are replaced by internal staples, three amino acid side chains are replaced by internal stitches, four amino acid side chains are replaced by two internal staples, or five amino acid side chains are replaced by a combination of internal staples and internal stitches. In some embodiments, the disclosure features a structurally stabilized (e.g., stapled or stitched) peptide (or a modified version thereof) comprising one of the amino acid sequences of SEQ ID NOs: 9, 10, 103, 104, 106, 108, or 110, wherein the two amino acid side chains separated by three amino acids are replaced by internal staples. In some embodiments, the disclosure features a structurally stabilized (e.g., stapled or stitched) peptide (or a modified version thereof) comprising one of the amino acid sequences of SEQ ID NOs: 9, 10, 103, 104, 106, 108, or 110, wherein the two amino acid side chains separated by six amino acids are replaced by internal staples. In some embodiments, the disclosure features a structurally stabilized (e.g., stapled or stitched) peptide (or a modified version thereof) comprising one of the amino acid sequences of SEQ ID NOs: 9, 10, 103, 104, 106, 108, or 110, wherein the three amino acid side chains are replaced by internal stitches.

[0122] Staples or stitch peptides can have an amino acid length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100. In certain embodiments, staples or stitch peptides have an amino acid length of 19 to 45 (i.e., 19, 20, 21, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). In certain embodiments, the staple or stitch peptide has an amino acid length of 19 to 35 amino acids (i.e., 19, 20, 21, 22, 23, 34, 235, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). In certain embodiments, the staple or stitch peptide has an amino acid length of 19 to 42 amino acids (i.e., 19, 20, 21, 22, 23, 34, 235, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42). In certain embodiments, the staple or stitch peptide has an amino acid length of 19 amino acids. In another particular embodiment, the staple or stitch peptide has an amino acid length of 42 amino acids. Exemplary COVID-19 HR2 staple or stitch peptides are shown in Tables 1 to 5 and described by formulas (I) to (IV). In one embodiment, the COVID-19 HR2 staple or stitch peptide comprises or comprises a staple or stitch version of any one amino acid sequence of SEQ ID NOs: 11-52 or 112-180 (e.g., the product of a ring-closing metathesis reaction performed on a peptide containing any one amino acid sequence of SEQ ID NOs: 11-52 or 112-180, respectively). In one embodiment, the SARS-CoV-2 HR2 staple or stitch peptide comprises or comprises a staple or stitch version of the amino acid sequence of SEQ ID NO: 9 (e.g., the product of a ring-closing metathesis reaction performed on a peptide containing the amino acid sequence of SEQ ID NO: 9).In one embodiment, the SARS-CoV-2 HR2 staple or stitch peptide comprises or consists of a staple or stitch version of the amino acid sequence of SEQ ID NO: 10 (e.g., the product of a ring-closing metathesis reaction performed on a peptide containing the amino acid sequence of SEQ ID NO: 10).

[0123] In certain embodiments, the staple peptide comprises or comprises a variant of the amino acid sequence described in any one of SEQ ID NOs: 9, 10, 103, 104, 106, 108, or 110, wherein two amino acids separated by three amino acids each (i.e., positions i and i+4) are modified to structurally stabilize the peptide (e.g., by substituting them with non-natural amino acids (i.e., staple amino acids) to enable hydrocarbon stapling). In certain embodiments, the staple peptide comprises or comprises a variant of the amino acid sequence described in any one of SEQ ID NOs: 9, 10, 103, 104, 106, 108, or 110, wherein two amino acids separated by six amino acids each (i.e., positions i and i+7) are modified to structurally stabilize the peptide (e.g., by substituting them with non-natural amino acids (i.e., staple amino acids) to enable hydrocarbon stapling).

[0124] In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 5 and 12 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 6 and 13 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 7 and 14 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 26 and 33 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 27 and 34 of SEQ ID NO: 9. In certain embodiments, two amino acids separated by six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 33 and 40 of SEQ ID NO: 9. In certain embodiments, three amino acids separated by six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 26, 33, and 40 of SEQ ID NO: 9. In certain embodiments, two amino acids separated by six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 5, 12, 26, and 33 of SEQ ID NO: 9. In certain embodiments, two amino acids separated by six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 5, 12, 27, and 34 of SEQ ID NO: 9. In certain embodiments, two amino acids separated by six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 5 and 12, 33, and 40 of SEQ ID NO: 9.In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 5, 12, 26, 33, and 40 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 6, 13, 26, and 33 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 6, 13, 27, and 34 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 6, 13, 33, and 40 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 6, 13, 26, 33, and 40 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 7, 14, 26, and 33 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 7, 14, 27, and 34 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 7, 14, 33, and 40 of SEQ ID NO: 9. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 7, 14, 26, 33, and 40 of SEQ ID NO: 9.

[0125] In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 2 and 9 of SEQ ID NO: 10. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 3 and 10 of SEQ ID NO: 10. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 6 and 13 of SEQ ID NO: 10. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 7 and 14 of SEQ ID NO: 10. In certain embodiments, the two amino acids separated by the six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 9 and 16 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 10 and 17 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by three amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 2 and 6 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by three amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 3 and 7 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by three amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 6 and 10 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by three amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 9 and 13 of SEQ ID NO: 10. In certain embodiments, the two amino acids separated by the three amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 10 and 14 of SEQ ID NO: 10.In certain embodiments, two amino acids separated by three amino acids each are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 13 and 17 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by three amino acids each are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 14 and 18 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by six amino acids each are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 2, 9, and 16 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by six amino acids each are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 3, 10, and 17 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by six or three amino acids each are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 2, 9, and 13 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by six or three amino acids each are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 3, 10, and 14 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by six or three amino acids each are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 6, 13, and 17 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by six or three amino acids each are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 7, 14, and 18 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by three or six amino acids each are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 2, 6, 13, and 17 of SEQ ID NO: 10. In certain embodiments, two amino acids separated by three or six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 3, 7, 13, and 17 of SEQ ID NO: 10.In certain embodiments, the two amino acids separated by three or six amino acids are located at the amino acid positions of the SARS-CoV-2 HR2 peptide corresponding to positions 2, 6, 14, and 18 of SEQ ID NO: 10.

[0126] In certain embodiments, the stitch peptide comprises or consists of a variant of the amino acid sequence described in any one of SEQ ID NOs: 9, 10, 103, 104, 106, 108, or 110, wherein two, three, four, or five amino acids at positions such as i, i+3, i, i+4, and i+7 are substituted (for example, by substituting them with non-natural amino acids (i.e., stitch amino acids) to enable hydrocarbon stitching) in order to structurally stabilize the peptide.

[0127] While hydrocarbon tethers are common, other tethers may also be used in the structurally stabilized SARS-CoV-2 HR2 peptides described herein. For example, a tether may contain one or more ethers, thioethers, esters, amines or amides, or triazole moieties. In some cases, naturally occurring amino acid side chains can be incorporated into the tether. For example, a tether can be coupled with functional groups such as hydroxyl in serine, thiol in cysteine, primary amine in lysine, acid in aspartic acid or glutamic acid, or amide in asparagine or glutamine. Thus, it is possible to construct a tether using naturally occurring amino acids rather than using a tether made by coupling two naturally occurring amino acids. It is also possible to use a single naturally occurring amino acid together with a natural amino acid. Triazole-containing crosslinks (e.g., 1,4-triazole or 1,5-triazole) can be used (see, for example, Kawamoto et al. 2012 Journal of Medicinal Chemistry 55:1137; WO2010 / 060112).Furthermore, other methods for performing different types of stapling are well known in the art and can be used in conjunction with the SARS-CoV-2 HR2 peptide described herein (e.g., Lactam stapling: Shepherd et al., J. Am. Chem. Soc., 127:2974-2983 (2005); UV-cycloaddition stapling: Madden et al., Bioorg. Med. Chem. Lett., 21:1472-1475 (2011); Disulfide stapling: Jackson et al., Am. Chem. Soc., 113:9391-9392 (1991); Oxime stapling: Haney et al., Chem. Commun., 47:10915-10917 (2011); Thioether stapling: Brunel and Dawson,Chem.Commun.,552-2554(2005);Photoswitchable stapling:JRKumita et al.,Proc.Natl.Acad.Sci.USA,97:3803-3808(2000);Double-click stapling:Lau et al.,Chem.Sci.,5:1804-1809(2014);Bis-lactam stapling:JCPhelan et al.,,J.Am.Chem.Soc.,119:455-460(1997);and Bis-arylation stapling:AMSpokoyny et al., J. Am. Chem. Soc., 135:5946-5949 (2013)).

[0128] It is also conceivable that the length of the tether can be varied. For example, a shorter tether can be used when it is desirable to provide a relatively high degree of constraint to the secondary alpha helical structure, but in some cases, it may be desirable not to provide much constraint to the secondary alpha helical structure, and therefore a longer tether may be preferable.

[0129] Furthermore, while tethers extending from amino acids i to i+3, i to i+4, and i to i+7 are common, primarily to provide tethers on a single plane of the alpha-helix, tethers can be synthesized to extend to any combination of amino acid numbers and can also be used in combination to incorporate multiple tethers.

[0130] In some cases, the hydrocarbon tethers (i.e., crosslinks) described herein can be further manipulated. In one example, the double bond of a hydrocarbon alkenyl tether (e.g., synthesized using ruthenium-catalyzed ring-closing metathesis (RCM)) can be oxidized (e.g., via epoxidation, aminohydroxylation, or dihydroxylation) to provide one of the following compounds. [ka]

[0131] Either the epoxide moiety or the free hydroxyl moiety can be further functionalized. For example, the epoxide can be treated with a nucleophile, which provides additional functional groups that can be used, for example, to attach therapeutic agents. Such derivatization can alternatively be achieved by synthetic operations of the amino or carboxyl terminus of the peptide, or via amino acid side chains. Other agents, such as those that facilitate the entry of the peptide into cells, can be attached to the functionalized tether.

[0132] In some cases, alpha-disubstituted amino acids are used in peptides to improve the stability of the alpha-helical secondary structure. However, there are also cases where alpha-disubstituted amino acids are not required, and mono-α substituents (e.g., in tethered amino acids) are used instead.

[0133] Structurally stabilized (e.g., stapled or stitched) peptides may include drugs, toxins, polyethylene glycol derivatives; secondary peptides; carbohydrates, etc. When polymers or other active ingredients are linked to structurally stabilized (e.g., stapled or stitched) peptides, it may be desirable for the composition to be substantially homogeneous.

[0134] The addition of polyethylene glycol (PEG) molecules can improve the pharmacokinetic and pharmacodynamic properties of peptides. For example, PEGylation can reduce renal clearance, potentially leading to more stable plasma concentrations. PEG is a water-soluble polymer and can be represented by the following formula when bound to a peptide:

[0135] XO--(CH2CH2O) n --CH2CH2--Y (wherein n is between 2 and 10,000, and X is H or a terminal modification, e.g., C) 1-4 Y is alkyl; and Y is an amide, carbamate, or urea linkage to the amine group of the peptide (including, but not limited to, the epsilonamine or N-terminus of lysine). Y may also be a maleimide linkage to a thiol group (including, but not limited to, the thiol group of cysteine). Other methods for linking PEG directly or indirectly to peptides are known to those skilled in the art. PEG may be linear or branched. Various forms of PEG, including various functionalized derivatives, are commercially available.

[0136] PEG having a biodegradable linkage in the main chain can be used. For example, PEG can be prepared using ester links that are susceptible to hydrolysis. Conjugates having biodegradable PEG links are described in International Publication No. 99 / 34833; International Publication No. 99 / 14259 and U.S. Patent No. 6,348,558.

[0137] In certain embodiments, a macromolecular polymer (e.g., PEG) is linked to a structurally stabilized (e.g., staple or stitch) peptide described herein via an intermediate linker. In certain embodiments, the linker consists of 1 to 20 amino acids linked by peptide bonds, selected from 20 naturally occurring amino acids. Some of these amino acids can be glycosylated, as is well understood by those skilled in the art. In other embodiments, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker consists mostly of sterically unhindered amino acids such as glycine and alanine. Non-peptide linkers are also possible, e.g., -NH(CH2) n Alkyl linkers such as C(O)- (wherein n=2 to 20) can be used. These alkyl linkers may be further substituted with any non-sterically hindered group such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. U.S. Patent No. 5,446,090 describes the use of bifunctional PEG linkers and their use in the formation of conjugates having peptides at each PEG linker terminus.

[0138] In some embodiments, structurally stabilized (e.g., stapled or stitched) peptides may also be modified, for example, to further enhance cellular uptake or increase in vivo stability. For example, acylation or PEGylation of structurally stabilized peptides can enhance cellular uptake, increase bioavailability, increase blood circulation, alter pharmacokinetics, reduce immunogenicity, and / or reduce the required administration frequency.

[0139] In some embodiments, the structurally stabilized (e.g., stapled or stitched) peptides disclosed herein have enhanced ability to permeate cell membranes (e.g., compared to unstabilized peptides). See, for example, International Publication 2017 / 147283, which is incorporated herein in its entirety by reference.

[0140] Treatment method This disclosure features a method of using any structurally stabilized (e.g., staple or stitch) peptide (or a pharmaceutical composition comprising such structurally stabilized peptide) described herein for the prevention and / or treatment of coronavirus (e.g., beta-coronavirus such as SARS-CoV-2) infection or coronavirus disease (e.g., COVID-19). As used herein, the terms “treat” or “treating” refer to alleviating, inhibiting, or improving a disease or infection that a subject (e.g., a human) is suffering from.

[0141] The structurally stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein may be useful for treating subjects (e.g., human subjects) having a coronavirus (e.g., beta-coronavirus) infection. The structurally stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein may also be useful for treating human subjects having coronavirus disease. In certain embodiments, the coronavirus infection is one of the following: 229E (alpha-coronavirus); NL63 (alpha-coronavirus); OC43 (beta-coronavirus); HKU1 (beta-coronavirus); Middle East Respiratory Syndrome (MERS); SARS-CoV; or SARS-CoV-2. In certain embodiments, the coronavirus disease is caused by a COVID-19 infection.

[0142] The structurally stabilized (e.g., stapled or stitched) peptides (or compositions containing the peptides) described herein may be useful in preventing coronavirus (e.g., beta-coronavirus) infection in human subjects. The peptides (or compositions containing the peptides) described herein may also be useful in preventing coronavirus disease in subjects (e.g., human subjects). In certain embodiments, coronavirus infection is infection with one of the following: 229E (alpha-coronavirus); NL63 (alpha-coronavirus); OC43 (beta-coronavirus); HKU1 (beta-coronavirus); Middle East Respiratory Syndrome (MERS); SARS-CoV; or SARS-COVID-19. In certain embodiments, coronavirus disease is caused by COVID-19 infection.

[0143] In certain embodiments, the peptides or variants described in Tables 1 to 5 are administered to human subjects requiring them. In certain embodiments, a stapled SARS-CoV-2 HR2 peptide containing or comprising SEQ ID NO: 9 or a modified version thereof is administered to human subjects requiring it. In certain embodiments, a stapled SARS-CoV-2 HR2 peptide containing or comprising SEQ ID NO: 10 or a modified version thereof is administered to human subjects requiring it.

[0144] In certain embodiments, one of the peptides having sequence numbers 11-52, 102, 105, 107, 109, or 111-180 as described in Table 1, or a variant thereof (as described herein), is administered to a human subject requiring it. Possible variations of these peptides are described in the section on structurally stabilized peptides. Further guidance is provided in Figures 6B and 16D. Variants of these sequences have at least one (e.g., 1, 2, 3, 4, 5) of the following properties: (i) bind to recombinant 5-helix bundle protein; (ii) be alpha-helical; (iii) be protease-resistant; (iv) inhibit fusion of SARS-CoV-2 with host cells; and / or (v) inhibit cell infection by SARS-CoV-2. In a particular embodiment, a peptide having at least 50%, 55%, 60%, 65%, 709%, 75%, 80%, 85%, 90%, 92%, 94%, or 95% identity with any one of the peptides having SEQ ID NOs: 11-52, 102, 105, 107, 109, or 111-180 is administered to a human subject in need. In a particular embodiment, a peptide having 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, insertions, and / or deletions is administered to a human subject in need.

[0145] In some embodiments, a human subject is infected with a coronavirus (e.g., beta-coronavirus). In some embodiments, a human subject is at risk of being infected with a coronavirus (e.g., beta-coronavirus). In some embodiments, a human subject is at risk of developing coronavirus disease (e.g., beta-coronavirus). In some examples, a human subject is at risk of being infected with a coronavirus or developing coronavirus disease if they live in an area (e.g., a city, state, country) experiencing an active coronavirus pandemic (e.g., an area where at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 20, at least 30, at least 40 or more people are diagnosed with coronavirus). In some embodiments, a human subject is at risk of contracting coronavirus or developing coronavirus disease if they live in an area near (e.g., a bordering city, state, or country) a second area (e.g., a city, state, or country) that is experiencing an active coronavirus pandemic (e.g., a bordering area) where at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 20, at least 30, at least 40, or more people have been diagnosed with coronavirus. In certain embodiments, coronavirus disease is caused by SARS-CoV-2 infection. In certain embodiments, a subject has or is at risk of developing COVID-19.

[0146] Generally, the method comprises selecting a subject and administering to the subject, for example, in or as part of a pharmaceutical composition, an effective amount of one or more of the structurally stabilized (e.g., stapled or stitched) peptides described herein, and, if necessary, repeating the administration as required for the prevention or treatment of coronavirus infection or coronavirus disease, which may be administered orally, intranasally, intravenously, subcutaneously, intramuscularly, or topically, including, for example, administration to the skin, nasal cavity, sinuses, respiratory tree, and lungs. In some examples, the administration is by topical respiratory application, including application to the nasal mucosa, sinus mucosa, or respiratory tree, including the lungs. In some examples, the topical application includes application to the skin. A subject may be selected for treatment, for example, based on the determination that the subject has a coronavirus infection (e.g., a beta-coronavirus such as SARS-CoV-2). The peptides of this disclosure can be used to determine whether a subject is infected with a coronavirus.

[0147] The specific dosage and treatment regimen for any particular patient depends on a variety of factors, including the activity of the specific compound used, age, weight, overall health, sex, diet, administration time, excretion rate, drug combinations, severity and course of the disease, condition or symptom, the patient's nature to the disease, condition or symptom, and the judgment of the treating physician.

[0148] An effective dose may be administered in one or more doses, applications, or applications. The therapeutic effective dose (i.e., effective dose) of a therapeutic compound depends on the therapeutic compound selected. The composition may be administered from one or more times per day to one or more times per week; including once every other day. Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's general health condition and / or age, and other pre-existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, the therapeutic effective dose of a subject with the therapeutic compound described herein may include a single treatment or a series of treatments. For example, an effective dose may be administered at least once.

[0149] Pharmaceutical composition Any one or more structurally stabilized (e.g., stapled or stitched) peptides described herein can be formulated as a pharmaceutical composition or for use in a pharmaceutical composition. The pharmaceutical composition may be used in the methods of treatment or prevention described herein (see above). In certain embodiments, the pharmaceutical composition comprises a structurally stabilized (e.g., stapled or stitched) peptide containing, or comprising, an amino acid sequence identical to, the amino acid sequences listed in Table 1, except for 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or one amino acid substitution, insertion, or deletion. These changes to the amino acid sequences can be made on the non-interacting alpha helix plane of these peptides (i.e., for amino acids that do not interact with the coronavirus 5-helix bundle) and / or on the interacting alpha helix plane (i.e., for amino acids that interact with the coronavirus 5-helix bundle). Such compositions can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA's CDER Data Standards Manual, version 004 (available at fda.gov / cder / dsm / DRG / drg00301.htm). For example, compositions can be formulated or adapted for administration by inhalation (e.g., oral and / or nasal inhalation (e.g., via nebulizer or spray)), injection (e.g., intravenous, intra-arterial, subdermal, intraperitoneal, intramuscular, and / or subcutaneous); and / or oral administration, transmucosal administration, and / or topical administration (including topical (e.g., nasal) sprays and / or solutions).

[0150] In some examples, a pharmaceutical composition may contain one or more effective amounts of a structurally stabilized (e.g., stapled or stitched) peptide. As used herein, “effective amount” and “effective to treat” refer to one or more structurally stabilized (e.g., stapled or stitched) peptide or a concentration of the pharmaceutical composition described herein that is effective in the context of its administration for a period of time (including acute or chronic administration and periodic or continuous administration) to produce an intended effect or physiological outcome (e.g., treatment of infection).

[0151] The pharmaceutical compositions of the present invention may include one or more structurally stabilized (e.g., staple or stitch) peptides as described herein, as well as any pharmaceutically acceptable carrier and / or vehicle. In some examples, the pharmaceuticals may further include one or more additional therapeutic agents in amounts effective to achieve the modulation of a disease or disease symptom.

[0152] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with the compound of the present invention, does not destroy its pharmacological activity, and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound.

[0153] The pharmaceutical compositions of the present invention may contain any conventional non-toxic, pharmaceutically acceptable carrier, adjuvant, or vehicle. In some cases, the pH of the formulation may be adjusted with a pharmaceutically acceptable acid, base, or buffer to enhance the stability of the formulated compound or its delivery form. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0154] In some cases, one or more structurally stabilized (e.g., staple or stitch) peptides disclosed herein can be conjugated to, for example, a carrier protein. Such a conjugated composition may be monovalent or polyvalent. For example, a conjugated composition may contain one structurally stabilized (e.g., staple or stitch) peptide disclosed herein conjugated to a carrier protein. Alternatively, a conjugated composition may contain two or more structurally stabilized (e.g., staple or stitch) peptides disclosed herein conjugated to a carrier.

[0155] As used herein, when two entities are “conjugated” to one another, they are linked by direct or indirect covalent or non-covalent interactions. In certain embodiments, the association is covalent. In other embodiments, the association is non-covalent. Non-covalent interactions include hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, and the like. Indirect covalent interactions occur when the two entities are covalently linked, optionally via linker groups.

[0156] Carrier proteins may include any protein that increases or enhances immunogenicity in a subject. Exemplary carrier proteins are described in the Art (see, for example, Fattom et al., Infect.Immun., 58:2309-2312, 1990; Devi et al., Proc.Natl.Acad.Sci.USA 88:7175-7179, 1991; Li et al., Infect.Immun. 57:3823-3827, 1989; Szu et al., Infect.Immun. 59:4555-4561, 1991; Szu et al., J.Exp.Med. 166:1510-1524, 1987; and Szu et al., Infect.Immun. 62:4440-4444, 1994). The polymer support may be a natural or synthetic material containing one or more primary and / or secondary amino groups, azide groups, or carboxyl groups. The support may be water-soluble.

[0157] Method for preparing staples or stitch peptides In one embodiment, the present disclosure features a method for producing structurally stabilized peptides. This method comprises (a) providing peptides comprising at least two non-natural amino acids having olefin side chains (e.g., SEQ ID NOs: 11-52 or 112-180), and (b) crosslinking the peptides. In some examples, the crosslinking of the peptides is performed by a ruthenium-catalyzed metathesis reaction.

[0158] Staple Peptide Synthesis: Using Fmoc-based solid-phase peptide synthesis, staple peptide fusion inhibitors were synthesized according to the method for producing total hydrocarbon staple peptides reported by the inventors (Bird et al., Curr. Protocol. Chem, Biol., 3(3):99-117 (2011; Bird et al., Methods) Enzymol., 446:369-86 (2008). To achieve various staple lengths, α-methyl and α-alkenyl amino acids were incorporated into specific pairings at separate positions, such as i and i+4, using two S-pentenylalanine residues (S5). For the stapling reaction, Grubbs' first-generation ruthenium catalyst dissolved in dichloroethane was added to the resin-bound peptide. To ensure maximum conversion, 3–5 stapling cycles were performed. The peptide was then cleaved from the resin using trifluoroacetic acid, precipitated with a hexane:ether (1:1) mixture, air-dried, and purified by LC-MS. All peptides were quantified by amino acid analysis.

[0159] Stitch Peptide Synthesis: Methods for synthesizing the stitch peptides described herein are known in the art. However, the following exemplary methods may be used. Useful synthetic chemical transformations and protecting group methodologies (protection and deprotection) for synthesizing the compounds described herein are known in the art, including, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TW. Greene and PG. MWuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0160] Derivatization of staple or stitch peptides with PEG4-cholesterol: Dissolve 200 mg of Boc-PEG4-COOH (www.biochempeg.com / product / Boc-NH-PEG4-COOH.html) in 10 mL of THF. Then, with stirring, add 400 mg of cholesterol (Sigma), followed by 0.1 mL of diisopropylcarbodiimide and 7 mg of dimethylaminopyridine. Monitor the reaction by LC-MS on a C3 column, which is typically completed in 1 hour. Add 10 mL of trifluoroacetic acid and stir for 15 minutes, again monitoring by LC-MS. Remove the solvent, dissolve the crude material in 5 mL of THF, and purify by preparative LC-MS. Pool the product fraction and lyophilize. Dissolve the dried product in 10 mL of THF, add 1.5 mL of diisopropylethylamine, followed by 0.36 mL of bromoacetyl bromide dropwise. Confirm that the reaction is complete using LC-MS, typically after 20 minutes. The product, bromoacetylated PEG-4 cholesterol, is purified by LC-MS. The reaction between BrAc-PEG4-chol and the cysteine-containing peptide is then carried out as follows: 5 mg of the peptide (e.g., DISGINASVVNIQXEIDXLNEVAKXLNEXLIDLQELGSGSGC) is dissolved in 350 μL of 5 mM DMF, then 350 μL of 10 mM BrAc-PEG4-Chol solution (in DMF) is added, followed by 35 μL of 50 mM TCEP (in water), and finally 3.2 μL of DIEA (10 equivalents relative to the peptide) is added with stirring. The reaction is monitored by LC-MS using a C3 column. The cholesterol peptide adduct is purified by preparative LC-MS after an overnight reaction.

[0161] The peptides of the present invention can be prepared by chemical synthesis methods well known to those skilled in the art. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, WH Freeman & Co., New York, NY, 1992, p. 77. Thus, the peptides can be synthesized, for example, using the Applied Biosystems Peptide Synthesizer Model 430A or 431, using side-chain protected amino acids and the automated Merrifield technique of solid-phase synthesis with α-NH2 protected by either t-Boc or Fmoc chemistry.

[0162] One method of preparing the peptides described herein is to use solid-phase peptide synthesis (SPPS). The C-terminal amino acid is bonded to a crosslinked polystyrene resin via an acid-unstable bond with a linker molecule. This resin is insoluble in the solvent used in the synthesis, making the washing away of excess reagents and by-products relatively easy and rapid. The N-terminus is protected with an Fmoc group, which is stable in acid but can be removed by a base. Any side-chain functional groups are protected with acid-unstable groups that are base-stable.

[0163] Longer peptides could be constructed by linking individual synthetic peptides using natural chemical ligation. Stitch amino acid insertion can be performed, for example, as described in Young and Schultz, J Biol Chem. 2010 Apr 9;285(15):11039-11044. Alternatively, longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct the gene encoding the peptide of the present invention, the amino acid sequence is back-translated to obtain a nucleic acid sequence encoding an amino acid sequence having codons optimal for the organism in which the gene is expressed, preferably. The synthetic gene is then constructed, typically by synthesizing oligonucleotides encoding the peptide and, if necessary, any regulatory elements. The synthetic gene is inserted into a suitable cloning vector and transfected into host cells. The peptide is then expressed under appropriate conditions suitable for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0164] Peptides can be prepared in a high-throughput combinatorial manner using high-throughput multi-channel combinatorial synthesizers, for example, available from Advanced Chemtech or Symphony X. Peptide bonds can be replaced by retroinverso bonds (C(O)-NH); reducing amide bonds (NH-CH2); thiomethylene bonds (S-CH2 or CH2-S); oxomethylene bonds (O-CH2 or CH2-O); ethylene bonds (CH2-CH2); thioamide bonds (C(S)-NH); trans-olefin bonds (CH=CH); fluorosubstituted trans-olefin bonds (CF=CH); ketomethylene bonds (C(O)-CHR or CHR-C(O) [wherein R is H or CH3]); and fluoro-ketomethylene bonds (C(O)-CFR or CFR-C(O) [wherein R is H or F or CH3]) to enhance the physiological stability of the peptide.

[0165] Peptides can be further modified by acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluorescence, formylation, myristoylation, palmitoylation, other lipidation (e.g., cholesterol), phosphorylation (Ser, Tyr, or Thr), stearoylation, succinylation, and sulfurylation. As described above, peptides can be conjugated with, for example, polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 linear or branched alkyl groups); fatty acid radicals; and combinations thereof. α,α-disubstituted unnatural amino acids containing olefin side chains of various lengths can be synthesized by known methods (Williams et al. J.Am.Chem.Soc., 113:9276, 1991; Schafmeister et al., J.Am.Chem Soc., 122:5891, 2000; and Bird et al., Methods Enzymol., 446:369, 2008; Bird et al, Current Protocols in Chemical Biology, 2011). In some examples, for peptides where i is linked to i+7 and i+7 is linked to i+14 stitches (stabilizing 4 turns of the helix): one R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine), one (one) bis-pentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine), and one R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine) are used. In some examples, for peptides where i is linked to i+7 and i+7 is linked to i+14 stitches (stabilizing 4 turns of the helix), one S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine), one (one) bis-pentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine), and one R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine) are used.In some examples, for peptides where i is linked to i+7 and i+7 is linked to i+14 stitches (stabilizing 4 turns of the helix): one S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine), one bis-pentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine), and one S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine) are used. In some examples, for peptides where i is linked to i+7 and i+7 is linked to i+14 stitches (stabilizing 4 turns of the helix), one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7'-octenyl)glycine), and one S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine) are used. In some examples, for peptides where i is linked to i+7 and i+7 is linked to i+14 stitches (stabilizing 4 turns of the helix): one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7'-octenyl)glycine), and one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine) are used. In some examples, for peptides where i is linked to i+7 and i+7 is linked to i+14 stitches (stabilizing 4 turns of the helix): one S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7'-octenyl)glycine), and one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine) are used.In some examples, for peptides where i is linked to i+7 and i+7 is linked to i+14 stitches (stabilizing 4 turns of the helix), one S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7'-octenyl)glycine), and one S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine) are used. R-octenylalanine is synthesized using the same route, except that the starting chiral auxiliary gives the R-alkyl stereoisomer. Also, 8-iodooctene is used instead of 5-iodopentene. Inhibitors are synthesized on solid supports using solid-phase peptide synthesis on MBHA resin (SPPS) (see, for example, International Publication 2010 / 148335).

[0166] Fmoc-protected α-amino acids (olefinic amino acids N-Fmoc-α,α-bis(4'-pentenyl)glycine, (S)-N-Fmoc-α-(4'-pentenyl)alanine, (R)-N-Fmoc-α-(7'-octenyl)alanine, (R)-N-Fmoc-α-(7'-octenyl)alanine, and (R)-N-Fmoc-α-(4'-pentenyl)alanine), 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) and Rink Amide MBHA are commercially available, for example, from Novabiochem (San Diego, California). Dimethylformamide (DMF), N-methyl-2-pyrrolidinone (NMP), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), fluorescein isothiocyanate (FITC), and piperidine are commercially available, for example, from Sigma-Aldrich. Olefin amino acid synthesis has been reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0167] Furthermore, suitable methods for obtaining (e.g., synthesizing), stitching, and purifying the peptides disclosed herein are also known in the art (see, for example, Bird et al., Methods in Enzymol., 446:369-386 (2008); Bird et al., Current Protocols in Chemical Biology, 2011; Walensky et al., Science, 305:1466-1470 (2004); Schafmeister et al., J.Am.Chem.Soc., 122:5891-5892 (2000); U.S. Patent Application No. 12 / 525,123 filed March 18, 2010; and U.S. Patent No. 7,723,468 issued May 25, 2010. Each of these is incorporated herein by reference in its entirety).

[0168] In some cases, the peptides are substantially free of or isolated from unstitched or unstapled peptide contaminants. Methods for purifying peptides include, for example, synthesizing the peptides on a solid support. After cyclization, the solid support can be isolated and suspended in a solution of a solvent such as DMSO, a DMSO / dichloromethane mixture, or a DMSO / NMP mixture. The DMSO / dichloromethane or DMSO / NMP mixture may contain about 30%, 40%, 50%, or 60% DMSO. In a specific example, a 50% / 50% DMSO / NMP solution is used. The solution may be incubated for 1, 6, 12, or 24 hours, after which the resin may be washed with, for example, dichloromethane or NMP. In one example, the resin is washed with NMP. Shaking and bubbling of an inert gas into the solution may be performed.

[0169] The properties of the stitch or staple peptides of this disclosure can be assayed, for example, using the methods described below and in the examples.

[0170] Assay for determining the properties and efficacy of stabilized peptides Assay for determining α-helicity: Dissolve the compound in an aqueous solution (e.g., 5 μM potassium phosphate solution (pH 7) or distilled H2O, up to a concentration of 25–50 μM). Circular dichroism (CD) spectra are obtained using a spectropolarimeter (e.g., Jasco J-710, Aviv) with standard measurement parameters (e.g., temperature 20°C; wavelength 190–260 nm; step resolution 0.5 nm; velocity 20 nm / sec; accumulation 10; response 1 sec; bandwidth 1 nm; path length 0.1 cm). The α-helical content of each peptide is calculated by dividing the average residue ellipticity by the reported value of a model helical decapeptide (Yang et al., Methods Enzymol., 1986).

[0171] Assay for determining the melting temperature (Tm): The crosslinked or unmodified template peptide is dissolved in distilled H2O or other buffer or solvent (to a final concentration of, e.g., 50 μM), and Tm is determined by measuring the change in ellipticity over a temperature range (e.g., 4–95°C) using a spectropolarimeter (e.g., Jasco J-710, Aviv) with standard parameters (e.g., wavelength 222 nm; step resolution 0.5 nm; rate 20 nm / sec; accumulation 10; response 1 sec; bandwidth 1 nm; heating rate: 1°C / min; path length 0.1 cm).

[0172] In vitro protease resistance assay: Amide bonds in peptide backbones are susceptible to hydrolysis by proteases, thereby making peptidolytic compounds vulnerable to rapid in vivo degradation. However, peptide helix formation can typically embed and / or twist and / or shield the amide backbones, thus preventing or substantially delaying proteolytic cleavage. The peptide-mimicking macrocyclic molecules of the present invention can be subjected to in vitro enzymatic proteolysis (e.g., trypsin, chymotrypsin, pepsin) to evaluate any changes in degradation rate compared to the corresponding uncrosslinked or alternatively stapled polypeptides. For example, peptide-mimicking macrocyclic molecules and the corresponding uncrosslinked polypeptides are incubated with trypsin agarose, and the reaction is quenched at various time points by centrifugation and subsequent HPLC injection, with residual substrate quantified by UV absorption at 280 nm. In short, a peptide-mimicking macrocyclic molecule and peptide-mimicking precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S / E approximately 125) for 0, 10, 20, 90, and 180 minutes. The reaction mixture is rapidly quenched by benchtop centrifugation; the remaining substrate in the isolated supernatant is quantified by peak detection based on HPLC at 280 nm. The proteolytic reaction exhibits first-order kinetics, and the rate constant k is determined from a plot of ln[S] against time.

[0173] Peptide-mimicking macrocyclic molecules and / or their corresponding uncrosslinked polypeptides can be incubated with fresh mouse, rat, and / or human serum (e.g., 1-2 mL) at 37°C for, for, e.g., 0, 1, 2, 4, 8, and 24 hours, respectively. Samples of different macrocyclic molecule concentrations can be prepared by serial dilution with serum. To determine the level of intact compound, the following procedure can be used: Extract the sample by, for example, transferring 100 μL of serum to a 2 ml centrifuge tube, followed by the addition of 10 μL of 50% formic acid and 500 μL of acetonitrile, and centrifugation at 14,000 RPM for 10 minutes at 4+ / -2°C. Then, transfer the supernatant to a new 2 ml tube and evaporate with Turbovap under N2 < 10 psi, 37°C. Reconstitute the sample with 100 μL of 50:50 acetonitrile:water and subject to LC-MS / MS analysis. Equivalent or similar procedures for testing ex vivo stability are known and can be used to determine the stability of macrocyclic molecules in serum.

[0174] Plasma Stability Assay: Staple peptide stability can be tested in freshly collected mouse plasma gathered in lithium heparin tubes. A triple incubation is set up using 500 μl of plasma supplemented with 10 μM of individual peptides. The samples are gently shaken in an orbital shaker at 37°C, and 25 μl aliquots are taken at 0, 5, 15, 30, 60, 240, 360, and 480 minutes. These are added to a 100 μl mixture containing 10% methanol, 10% water, and 80% acetonitrile to halt further peptide degradation. The samples are allowed to stand on ice for the duration of the assay and then transferred to a MultiScreen Solvinert 0.45 μm low-binding hydrophilic PTFE plate (Millipore). The filtrate is analyzed directly by LC-MS / MS. Peptides are detected as bi- or tri-charged ions using a Sciex 5500 mass spectrometer. The percentage of residual peptide is determined by the decrease in peak area in chromatography, and the half-life is calculated by logarithmically transforming the result.

[0175] In vivo protease resistance assays: A key advantage of peptide staples is that they translate in vitro protease resistance into significantly improved pharmacokinetics in vivo.

[0176] SAH-SARS-CoV-2 levels in plasma are detected and quantified using an analytical assay based on liquid chromatography / mass spectrometry. For pharmacokinetic analysis, the peptide is dissolved in sterile 5% dextrose aqueous solution (1 mg / mL) and administered to C57BL / 6 mice (Jackson Laboratory) by bolus tail vein or intraperitoneal injection (e.g., 5, 10, 25, 50 mg / kg). Blood is collected by posterior orbital puncture 5, 30, 60, 120, and 240 minutes after administration to 5 animals at each time point. Plasma is collected after centrifugation (2,500 × g, 5 min, 4°C) and stored at -70°C until assay. The peptide concentration in plasma is determined by reverse-phase high-performance liquid chromatography using electrospray ionization mass spectrometry detection (Aristoteli et al., Journal of Proteome Res., 2007; Walden et al., Analytical and Bioanalytical Chem., 2004). Test samples are assayed with a series of seven calibration standards of plasma peptide concentrations ranging from 1.0 μg / mL to 50.0 μg / mL, drug-free plasma assayed with or without the addition of an internal standard, and three quality control samples (e.g., 3.75, 15.0, and 45.0 μg / mL). Standard curves are constructed by plotting the analyte / internal standard chromatographic peak area ratio against known drug concentrations at each calibration standard. Linear least-squares regression is performed with weights proportional to the reciprocal of the analyte concentration normalized to the number of calibration standards. The drug concentration in the test sample is calculated using the slope and y-intercept values ​​of the best-fit curve. Plasma concentration-time curves are analyzed using a standard non-compartmental method with WinNonlin Professional 5.0 software (Pharsight Corp., Cary, NC) to obtain pharmacokinetic parameters such as early and late plasma half-lives, peak plasma levels, total plasma clearance, and apparent volume of distribution.

[0177] The persistence of the stabilizing alpha-helix of the COVID-19 (SAH-SARS-CoV-2) peptide in the nasal mucosa after topical administration (i.e., nasal spray) and in the respiratory mucosa after spraying was investigated in relation to pre- and post-infection blockade of viral fusion and dissemination. Mice were exposed to single SAH-SARS-CoV-2 treatment with nasal spray or nebulizer at a series of intervals prior to intransitive infection with rgCOVID-19, and the relative mucosal stability and prophylactic efficacy of the SAH-SARS-CoV-2 construct were measured using the period of protection from mucosal infection (assessed histologically as above or by PCR as below).

[0178] In vitro binding assays: To evaluate the binding and affinity of peptide-mimicking macrocyclic molecules and peptide-mimicking precursors to acceptor proteins, fluorescence polarization assays (FPAs), for example, can be used. FPA techniques use polarization and fluorescence tracers to measure molecular orientation and mobility. When excited by polarization, fluorescence tracers bound to molecules or peptides, and then to proteins with high apparent molecular weights (e.g., FITCs) (e.g., FITC-labeled peptides bound to large proteins), emit higher levels of polarized fluorescence compared to fluorescence tracers bound to smaller molecules or peptides alone (e.g., FITC-labeled peptides free in solution) because of their slower rotational speed upon protein binding.

[0179] In vitro substitution assays to characterize peptide-protein interaction antagonists: To evaluate the binding and affinity of compounds that antagonize the interaction between a peptide and an acceptor protein, fluorescence polarization assays (FPAs) are used, for example, utilizing fluorescent peptides derived from a template peptide sequence or macrocyclic peptide-mimicking molecules. FPA techniques measure molecular orientation and mobility using polarization and fluorescence tracers. When excited by polarization, a fluorescence tracer bound to a molecule with a high apparent molecular weight (e.g., FITC) (e.g., a FITC-labeled peptide bound to a large protein) emits a higher level of polarized fluorescence because it rotates more slowly compared to the FITC-derivative molecule alone (e.g., a free FITC-labeled peptide in solution). Compounds that antagonize the interaction between a fluorescent peptide and an acceptor protein can be detected in competitive binding FPA experiments, allowing for the quantification and comparison of the different potencies of the compounds in disrupting the interaction.

[0180] Production and fluorescence polarization assay of 5-helix bundle protein: Recombinant 5-helix bundle (5HB) protein with a C-terminal hexa-His (SEQ ID NO: 101) tag containing 5 of the 6 helices constituting the core of a hairpin SARS-CoV-2 S trimer linked by a short peptide linker was designed according to the gp41 5-HB design (Root et al. Science, 291(5505):884-8(2001); Bird et al., J Clin Invest. 2014 May;124(5):2113-24). Plasmids were transformed into E. coli BL21(DE3), cultured in Luria broth, and induced overnight at 37°C with 0.1 M isopropyl β-D-thiogalactoside. Cells were collected by centrifugation at 5,000 g for 20 minutes, resuspended in buffer A (100 mM NaH2PO4, 20 mM Tris, 8 M urea; pH 7.4), and dissolved by stirring overnight at 4°C. The mixture was clarified by centrifugation (35,000 g for 30 minutes) and then conjugated to a nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) column at room temperature. The conjugated 5-HB was washed with buffer A (pH 6.3), eluted with buffer A (pH 4.5), regenerated by dilution (1:2) with PBS (50 mM sodium phosphate, 100 mM NaCl; pH 7.5), concentrated on a 10 kDa Amicon Centricon (7 dilutions and reconcentrations) to obtain a protein solution of approximately 1 mg / ml. The protein purity was evaluated by SDS-PAGE and determined to be over 90%. The fluorescent peptide of SARS-CoV-2 S HR2 (25 nM) is incubated with the 5-HB protein at the indicated concentration in room-temperature binding buffer (50 mM sodium phosphate, 100 mM NaCl; pH 7.5). Direct binding activity at equilibrium (e.g., 10 minutes) is measured by fluorescence polarization using a SpectraMax M5 microplate reader (BMG Labtech). For competitive binding assays, fixed concentrations of the FITC peptide and 5-HB protein reflecting the EC90 for direct binding are then incubated with serial dilutions of the acetylated SAH-SARS-CoV-2 peptide to create competitive curves for comparative analysis.The binding assay was performed in a triple series, and Kis was calculated using nonlinear regression analysis of competitively bound isotherms with Prism software (GraphPad).

[0181] Assay for screening binding activity to SARS-CoV-2 5-helix bundle: In some examples, the methods disclosed herein include direct and competitive screening assays. For example, a method may include determining whether an active agent alters (e.g., reduces) the binding of one or more peptides disclosed herein to SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle). In some examples, the method includes (i) determining the binding level between one or more peptides disclosed herein and SARS-CoV-2 (e.g., to SARS-CoV-2 5-helix bundles) (e.g., in the absence of an active agent); (ii) detecting the binding level between one or more peptides (e.g., one or more peptides of (i)) and SARS-CoV-2 (e.g., to SARS-CoV-2 5-helix bundles) in the presence of an active agent, wherein a change (e.g., reduction) in the binding level between one or more peptides and SARS-CoV-2 (e.g., to SARS-CoV-2 5-helix bundles) indicates that the active agent is a candidate active agent that binds to SARS-CoV-2; and (iii) selecting a candidate active agent. In some examples, step (i) includes contacting one or more peptides with SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle) and detecting the binding level of one or more peptides to SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle). In some examples, step (ii) includes contacting one or more peptides and an active agent with SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle) and detecting the binding level of one or more peptides to SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle).SARS-CoV-2 (e.g., in the SARS-CoV-2 5-helix bundle) can be brought into contact with one or more peptides and activators simultaneously or at different times (e.g., one or more peptides can be brought into contact with SARS-CoV-2 (e.g., in the SARS-CoV-2 5-helix bundle) before or after the activator). In some embodiments, candidate activators are administered to a suitable animal model (e.g., an animal model of COVID-19) to determine whether the activator reduces the level of COVID-19 infection in the animals.

[0182] In some examples, one or both of the peptide and / or SARS-CoV-2 helix bundle may contain a label, enabling the detection of the peptide and / or the SARS-CoV-2 helix bundle. In some examples, the peptide contains a label. In some examples, the SARS-CoV-2 helix bundle contains a label. In some examples, both the peptide and the SARS-CoV-2 helix bundle contain a label. The label may be any label known in the art, including but not limited to fluorescent labels, radioisotope labels, or enzyme labels. In some examples, the label is directly detectable by itself (e.g., radioisotope labels or fluorescent labels). In some examples (e.g., in the case of enzyme labels), the label may be indirectly detectable, for example, by catalyzing a chemical change in a chemical substrate compound or composition, and the chemical substrate compound or composition may be directly detectable.

[0183] Competitive SARS-CoV-2 5-HB binding assay by ELISA: Microwells are coated overnight at 4°C with 50 μl of PBS containing nutraavidin (4 μg / ml). Wells are washed twice with PBS (PBS-T) containing 0.05% Tween® 20, and blocked with 4% BSA in PBS-T at 37°C for 45 minutes. Then, 50 μl of 250 nM biotinylated PEG is added. 2-SARS-CoV-2 HR2 (SEQ ID NO: 9) was added to PBS-T containing 1% BSA, incubated for 1 hour with shaking, and then washed four times with 300 μl of PBS-T. Next, 1:2 serial dilutions of SARS-CoV-2 peptide starting at 10 μM containing 50 nM recombinant 5-HB in 50 μL of PBS-T containing 1% BSA were added to the plate, shaken at room temperature for 2 hours, and then washed four times with 300 μL of PBS-T. Finally, 50 μL of a 1:5000 dilution of 6×His-tagged-HRP conjugated goat polyclonal was added. After incubation at room temperature for 40 minutes, the wells were washed five times and colored by adding 50 μl of tetramethylbenzidine (TMB) solution. After 20 minutes, the wells containing the TMB solution are stopped by adding 50 μl of H2SO4 (2M), and the absorbance at 450 nm is read using a microplate reader (Molecular Devices). Using Prism software (Graphpad), the concentration of the competing peptide corresponding to the maximum half-volume signal (IC50) is determined by interpolation of the resulting binding curves. Each peptide competitor is tested in triplicate in at least two separate experiments.

[0184] Cell Peripherality Assay: To measure the cell peripherality of peptides or cross-linked polypeptides, intact cells are incubated with fluorescent cross-linked polypeptide (10 μM) in serum-free medium or medium supplemented with human serum at 37°C for 4 hours, washed twice with medium, and incubated with trypsin (0.25%) at 37°C for 10 minutes. Cells are washed again and resuspended in PBS. Cell fluorescence can be measured using, for example, a FACSCalibur flow cytometer or Cellomics KineticScan. RTM The analysis is performed using one of the HCS readers.

[0185] Antiviral efficacy assay: Evaluate the efficacy of SAH-SARS-CoV-2 peptide in the prevention and treatment of COVID-19 infection in monolayer cell cultures. The virus detection platform was developed for SARS-CoV-2 based on previous screening for Ebola virus (see Anantpadma M. et al., Antimicrob Agents Chemother. 2016;60(8):4471-81. Epub 2016 / 05 / 11. doi:10.1128 / AAC.00543-16. PubMed PMID:27161622; PMCID:PMC4958205). Vero E6 cells plated in a 384-well format were treated with serial dilutions of staple peptide (e.g., a starting dose of 10 μM) for 1 hour in a triple-chain configuration, followed by a 4-hour challenge with SARS-CoV-2 to achieve control infection of 10%–20% of cells (a predetermined optimal infectivity to evaluate the dynamic range of the test compound in the assay). Infected cells were then washed, fixed with 4% paraformaldehyde, rewashed with PBS, immunostained with anti-SARS-CoV-2 nucleocapsid monoclonal antibody, followed by anti-Ig secondary antibody (Alexa Fluor 488; Life Technologies), and counterstained with HCS CellMask blue. Cells were imaged across the z-plane using a Nikon Ti Eclipse automated microscope and analyzed using CellProfiler software, and infection efficiency was calculated by dividing the infected cells by the total cells. Control cytotoxicity assays were performed using the Cell-Titer Glo (Promega) assay and the LDH release (Roche) assay.

[0186] Another approach uses qPCR-based virus detection in naturally susceptible human-derived Huh770 and Calu-371 cells expressing ACE2, as well as in MatTek Life Sciences primary lung epithelial and alveolar cell models infected with SARS-CoV-2 virus (e.g., USA-WA1 / 2020; Hong Kong VM20001061). Cultured cells are treated with serial dilutions of staple peptide for 1 hour, followed by challenge with SARS-CoV-2. Culture supernatant is sampled, the virus is lysed in the presence of an RNAse inhibitor, and RT and qPCR are performed as described. See Suzuki et al. J Vis Exp.2018(141).Epub 2018 / 11 / 20.doi:10.3791 / 58407. BHQ quench dye vs primers validated by the CDC are purchased from IDT, and genomic equivalents are calculated from the Ct values.

[0187] Another approach evaluates the antiviral activity of the SAH-SARS-CoV-2 staple peptide using pseudotyped viruses. A 293T-hsACE2 stable cell line (catalog number C-HA101) with a GFP (catalog number RVP-701G, lot number CG-113A) reporter and pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain) particles are used (Integral Molecular). The neutralization assay is performed according to the manufacturer's protocol. Briefly, a single dose of 5 μL of the peptide (5 μM final dose) is incubated with 5 μL of pseudotyped SARS-CoV-2-GFP at 37°C for 1 hour in a 384-well black clear-bottom plate, followed by the addition of 30 μL of 1,000 293T-hsACE2 cells (in 10% FBS DMEM, phenol red-free medium) and incubation in a humidified incubator for 48 or 72 hours. Hoechst 33342 and DRAQ7 dyes were added, and the plates were imaged at 10x magnification on a Molecular Devices ImageXpress Micro Confocal Laser. GFP(+) cells were counted and plotted using Prism software (Graphpad).

[0188] To evaluate the ability of lead staple peptides to prevent SARS-CoV-2 infection, K18-hACE2 (Jackson Laboratory) mice (n=10 / arm; 5 males, 5 females) were administered the staple peptide or vehicle via intranasal or oropharyngeal route, and 24 hours later, 10 4 The viral dose of PFU was administered intranasally. For evaluation by autopsy, mice were euthanized 4 days later (peak of viremia), and the viral load was quantified by qPCR from supernatant samples of lung homogenate prepared as described using a tissue lysator (Qiagen). See Bao L et al. Nature. 2020. Epub 2020 / 05 / 08; doi:10.1038 / s41586-020-2312-y. To evaluate the ability of the lead staple peptide to treat or mitigate established SARS-CoV-2 infection, K18-hACE2 mice (n=10 / arm; 5 males, 5 females) were given 10 doses on day 1. 4 Intranasal inoculation with a viral dose of PFU is followed by daily oropharyngeal or intraperitoneal treatment with staple peptide or vehicle for 10 days (days 2-12). In an alternative design, administration is delayed to 3-5 days post-inoculation to simulate treatment initiation based on symptoms or positive tests. Mice are continuously monitored to record body weight and clinical signs, and disease progression is scored as weight loss greater than 10%, respiratory distress, and / or stunted growth. The doses and routes of the most effective compounds are then investigated in both preventive and treatment studies to determine the minimum dose to protect the mice. The same experimental design is used, except that four treatment groups (n=10; 5 males, 5 females) are administered the original dose, followed by three tapering doses in 4-fold increments.

[0189] Clinical Trials: Clinical trials can be conducted to determine the suitability of the cross-linked polypeptide of the present invention for human treatment. For example, patients exposed to or diagnosed with SARS-CoV-2 infection are selected and divided into a treatment group and one or more control groups, with the treatment group receiving the cross-linked polypeptide of the present invention and the control groups receiving a placebo or a known antiviral drug. Thus, the treatment safety and efficacy of the cross-linked polypeptide of the present invention can be evaluated by comparing the patient groups with respect to factors such as prevention of symptoms, time to symptom resolution, and / or overall infection severity. In another example, uninfected patients are identified and given either the cross-linked polypeptide or a placebo. After treatment, the patients are followed up. In both examples, the group of SARS-CoV-2 exposed patients treated with the cross-linked polypeptide avoids developing infection, or the group of patients with SARS-CoV-2 infection shows symptom resolution or reduction compared to the patient control group treated with placebo. [Examples]

[0190] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. Where specific materials are mentioned, they are merely for illustrative purposes and not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without demonstrating the capabilities of the invention and without departing from its scope.

[0191] Example 1: Design and synthesis of SARS-CoV-2 HR2 staple peptide To design peptides that can inhibit the fusion of coronaviruses into host cells, a series of staple peptides with differentially localized chemical staples were designed. Native residues were replaced at the (i,i+4) or (i,i+7) positions in the form of double staples or stitches by α,α-disubstituted unnatural olefin residues ("X") and combinations thereof, followed by ruthenium-catalyzed olefin metathesis to position the differentially localized chemical staples within the SARS-CoV-2 HR2 domain (i.e., amino acids 1169-1210 or 1179-1197) of the surface glycoprotein [Severe Acute Respiratory Syndrome Coronavirus 2] sequence (see Figure 1) (see Table 1). Several designs incorporated staples on the non-interacting amphiphilic surface of the helix or at the boundary between the hydrophobic interacting surface and the amphiphilic surface of the helix (Figures 4 and 5).

[0192] The SAH-SARS-CoV-2 construct was designed by replacing two naturally occurring amino acids with the unnatural S-2-(4'-pentenyl)alanine (S5) amino acid at positions i,i+4 (i.e., three adjacent amino acids) to create a staple across one α-helical turn, or by replacing (R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-deca-9-enoic acid (R8) and S5 at positions i,i+7 to create a staple across two α-helical turns. The asymmetric synthesis of the α,α-disubstituted amino acids was carried out as previously described in detail (Schafmeister et al., J.Am.Chem.Soc., 2000; Walensky et al., Science, 2004; Bird et al. Current Protocols in Chemical Biology, 2011 (the whole of which is incorporated by reference)).

[0193] A "staple scan" was performed to identify interaction-critical residues and binding surfaces, respectively, to determine the design of optimized constructs and negative control mutants. Depending on the experimental application, the N-terminus of the SAH was capped with acetyl or fluorophores (e.g., FITC, rhodamine).

[0194] Double-staple peptides are produced by incorporating two S5-S5, two -R8-S5, or other combinations of cross-linked non-natural amino acids. Multiple-staple or stitch peptides are produced using a similar principle.

[0195] The SAH-SARS-CoV-2 peptides shown in Table 1 were synthesized using solid-phase Fmoc chemistry and ruthenium-catalyzed olefin metathesis. Subsequently, the peptides were deprotected and cleaved, purified by reverse-phase high-performance liquid chromatography / mass spectrometry (LC / MS), and quantified by amino acid analysis (AAA) (Bird et al., Methods Enzymol., 2008).

[0196] Example 2: Evaluation of alpha-helical stabilization of SARS-CoV-2 HR2 staple peptide Generally, short peptides do not exhibit a significant α-helical structure in solution. This is because the entropy cost of maintaining a sterically constrained structure is not overcome by the enthalpy gain from hydrogen bonding in the peptide backbone. To demonstrate secondary structure improvement of hydrocarbon staple peptides, circular dichroism (CD) spectra were recorded and analyzed using a Model 410 Aviv Biomedical spectrometer. Each spectrum was obtained by scanning 190–260 nm five times in 0.5 nm increments using a 1 mm path length cell and averaging the results together with an averaging time of 0.5 seconds. Target peptide concentrations for the CD studies were 25–50 μM in 50 mM potassium phosphate (pH 7.5) or Milli-Q deionized water, and the exact concentrations were confirmed by quantitative AAA of two CD sample dilutions. The CD spectra were first plotted as wavelength versus millidegrees. Once the precise peptide concentration was confirmed, the average residue ellipticity [θ] in degrees·cm²·dmol-1·residue-1 units was calculated using the formula: [θ] = millidegrees / molar concentration / number of amino acid residues. After conversion to average residue ellipticity, the percentage of α-helicity was calculated using the equation helicity (%) = 100 x [θ]²²² / maximum [θ]²²² (where maximum [θ]²²² = -40,000 x [1 - (2.5 / number of amino acid residues)]). Staple constructs that reinforced the α-helical structure were then subjected to protease resistance testing, binding analysis, and antiviral activity assays. Figures 12A and 12B show that the HR2 peptides without staples corresponding to SEQ ID NOs. 10, 9, 106, and 110 showed little to no alpha-helical structure in solution by circular dichroism analysis, while insertion of double staples (i.e., SEQ ID NOs. 49, 51, 158, and 177) and stitches (i.e., SEQ ID NOs. 47 and 48) into such sequences effectively induced alpha-helicity, as evidenced by the gradual increase in absorption at [θ]222. Such stapled constructs with enhanced α-helical structure were then subjected to protease resistance testing, binding analysis, and antiviral activity assays.

[0197] Example 3: Determination of protease resistance of SARS-CoV-2 HR2 staple peptide Linear peptides are susceptible to rapid proteolysis in vitro and in vivo, limiting the application of native peptides for mechanistic analysis and therapeutic use. In contrast, amide bonds involved in the hydrogen bonding network of structured peptide helices are poorly enzyme substrates, as are residues shielded by hydrocarbon staples themselves (Bird et al, PNAS, 2010). To assess the relative protease resistance mediated by hydrocarbon staples, in vitro proteolysis was measured by LC / MS (Agilent 1200) using the following parameters: 20 μL injection, 0.6 mL flow rate, 15-minute runtime consisting of a gradient from water (0.1% formic acid) to 20–80% acetonitrile (0.075% formic acid) over 10 minutes, 4-minute wash to return to initial gradient conditions, and 0.5-minute after-time. The DAD signal was set to 280 nm with a bandwidth of 8 nm, and the MSD was set to scan mode on one channel with (M+2H) / 2, + / -1 mass units and the other channel with (M+3H) / 3, + / -1 mass units. By integrating each MSD signal, >10 8The area under the curve for counts was obtained. The reaction sample consisted of 5 μL of peptide (1 mM stock) in DMSO and 195 μL of buffer consisting of 50 mM Tris HCl (pH 7.4). After injecting the sample at time 0, 2 μL of 100 ng / μL proteinase K (New England Biolabs) was added, and the amount of intact peptide was quantified by continuous injection over time. Each MSD data point was normalized using an internal control of acetylated tryptophan carboxamide at a concentration of 100 μM. The MSD area versus time plot gave an exponential decay curve, and the half-life was determined by nonlinear regression analysis using Prism software (GraphPad). Figures 13 and 13B show how the insertion of double staples or stitches into the core template sequence (aa1169~1197) conferred significant protease stability compared to the unstapled sequence, depending on the sequence, staple type, and staple location. Figure 13A shows that both double-stapled and stitched sequences (SEQ ID NOs. 48 and 52) conferred significant resistance to proteinase K treatment (half-life > 1000 mins), while the unstapled sequence (SEQ ID NOs. 10) was rapidly digested (half-life 35 mins). Figure 13B shows that a longer unstapled HR2 sequence (SEQ ID NOs. 9) was rapidly digested by proteinase K (half-life 25 mins), and insertion of double-stapled O,S (SEQ ID NOs. 158) only slightly enhanced proteolytic resistance (half-life 33 mins), while insertion of double-stapled N,S (SEQ ID NOs. 177) into another HR2-type sequence (SEQ ID NOs. 111) conferred significant proteolytic resistance to proteinase K (half-life 840 mins). Protease resistance and stability of the staple peptides were also measured using a mouse plasma stability assay. Staple peptide stability was tested in freshly collected mouse plasma collected in lithium heparin tubes. A triple incubation cycle was set up using 500 μl of plasma to which 10 μM of individual peptides were added.The samples were gently shaken in an orbital shaker at 37°C, and 25 μl aliquots were taken at 0, 5, 15, 30, 60, 240, 360, and 480 minutes. These were added to a 100 μl mixture containing 10% methanol, 10% water, and 80% acetonitrile to halt further peptide degradation. The samples were allowed to stand on ice for the duration of the assay and then transferred to a MultiScreen Solvinert 0.45 μm low-binding hydrophilic PTFE plate (Millipore). The filtrate was analyzed directly by LC-MS / MS. The peptides were detected as bi- or tri-charged ions using a Sciex 5500 mass spectrometer. The percentage of residual peptide was determined by the decrease in peak area on the chromatogram, and the half-life was calculated by logarithmically transforming the data. Figures 14A and 14B show that two double-staple peptides of the core template sequence (aa1169-1197), including sequence number 51 (staple N, T) in Figure 14A and sequence number 52 (staple O, T) in Figure 14B, showed no degradation over time during incubation with mouse plasma.

[0198] Example 4: Investigation of SARS-CoV-2 binding activity of SAH-SARS-CoV-2 peptide To measure the direct binding affinity to the SARS-CoV-2 fusion bundle, a direct fluorescence polarization assay (FPA) was performed using recombinant 5-helix bundle protein and fluorescent SARS-CoV-2 HR2 peptide (with excitation wavelength 488 nm and emission wavelength 522 nm) by adding FITC-bAla to the N-terminus of the sequences shown in Table 1. More specifically, a recombinant 5-helix bundle protein (SEQ ID NO: 263) was designed containing five of the six helices that constitute the core of a hairpin SARS-CoV-2 trimer, linked by a short peptide linker, according to the design of the SARS-CoV-2 5-helix bundle. The recombinant 5-helix bundle lacks the third HR2 helix, but is otherwise soluble, stable, and helical, thus supporting FITC-SARS-CoV-2 HR2 (1179-1197) or -SARS-CoV-2 HR2 (1169-1210)The incorporation of the sixth HR2 peptide in peptide and derivative forms results in a stable complex, which can be monitored by FPA to directly measure binding affinity. The relative binding activity of different SARS-CoV-2 HR2 constructs to the 5-helix fusion bundle was measured and compared using the FPA assay. FITC-SARS-CoV-2 HR2 peptides were mixed with serial dilutions of recombinant 5-helix bundle proteins to generate binding isotherms. Fluorescence polarization (in mP units) was measured using a SpectraMax fluorometer, and EC50 values ​​were calculated by nonlinear regression analysis of the competition curves using Prism software (Graphpad).

[0199] Figures 15A and 15B show the results of a direct fluorescence polarization coupling assay using an N-terminal FITC-derivativeized i,i+4 staple scan library of recombinant SARS-CoV-2 5-helix-binding protein and core template sequences (aa1179-1197, SEQ ID NO: 10). Figure 15A shows the different binding activities of the staple peptides based on the i,i+4 staple position, as reflected by the change in fluorescence polarization (ΔmP) at a 4 μM 5-HB protein concentration. Figure 15B shows the dose-response curve of the fluorescent i,i+4 staple scan library to the 5-HB protein, highlighting that, depending on the specific staple position, the i,i+4 staple peptides bind better, similarly, or worse than the unstapled core template sequence. Figures 16A and 16B show the results of a direct fluorescence polarization coupling assay using an N-terminal FITC-derivativeated i,i+7 staple scan library of recombinant SARS-CoV-2 5-helix-binding protein and core template sequences (aa1179-1197, SEQ ID NO: 10). Figure 16A shows the different binding activities of the staple peptide based on the i,i+7 staple position, as reflected by the change in fluorescence polarization (ΔmP) at a 4 μM 5-HB protein concentration. Figure 16B shows the dose-response curve of the fluorescent i,i+7 staple scan library to the 5-HB protein, highlighting that, depending on the specific staple position, the i,i+7 staple peptide binds better, similarly, or worse than the unstapled core template sequence. Figure 16C shows a schematic diagram of a helical wheel indicating the residues involved in preferred (light gray), unpreferred (dark gray), and intermediate (medium gray) i,i+7 stabling. The residues involved in the two staples are shown as bisected circles, with a left semicircle colored to represent the activity of the staple when the residue is incorporated at the N-terminal position of the staple, and a right semicircle colored to represent the activity of the staple when the residue is incorporated at the C-terminal position of the staple. If the semicircle is colored white, the indicated residue position is not involved in either the N-terminal or C-terminal position of the staple.Staple locations on the hydrophobic surface disrupted 5-HB binding activity, and unexpectedly, staple locations on the hydrophilic surface opposite the 5-HB binding surface were also unfavorable (marked with X). In contrast, selected staple locations at the boundary between the hydrophobic and hydrophilic surfaces were favorable (marked with a star). Figures 17A and 17B show the results of direct fluorescence polarized coupling assays using N-terminal FITC-derivativeated double i, i+4 stapled peptides on recombinant SARS-CoV-2 5-helix-binding protein and core template sequences (aa1179-1197, SEQ ID NO: 10). Figure 17A shows the different binding activities of the stapled peptides based on double staple locations, reflected by the change in fluorescence polarization (ΔmP) at a 4 μM 5-HB protein concentration. Figure 17B shows the dose-response curves for the fluorescent double-stapled peptides to the 5-HB protein, highlighting in each example that double staple insertion results in enhanced binding activity compared to the core template sequence without staples. Figure 18 shows the results of a direct fluorescence polarized coupling assay using recombinant SARS-CoV-2 5-helix-binding protein and N-terminal FITC-derivativeized double i,i+4 staple peptide in the context of a longer HR2 (SEQ ID NO: 9) and an alternative HR2-type (SEQ ID NO: 110) sequence. The plot demonstrates the comparative binding activity of the double staple peptide to 5-HB of SARS-CoV-2. In both cases, the insertion of the double staple yields a staple peptide with dose-responsive 5-HB binding activity.

[0200] By integrating FPA data across i, i+4 and i, i+7 staple scans, and evaluating double-staple constructs across peptide templates of different lengths and sequences, we found that (1) single-staple peptides with significant binding activity can maintain target affinity in the context of double-staple peptides, even if the second staple may be less effective or ineffective as a single-staple peptide (e.g., compare single i, i+4 staple N, T, and O peptides with i, i+4 double-staple N, T, and O, T peptides), and (2) two peptides that may each be less effective or ineffective as single-staple peptides. (3) It is further evident that by combining staples, peptides with improved binding activity in the context of double-staple peptides can be obtained (for example, compare a single i,i+4 staple O,S peptide with an i,i+4 double-staple O,S peptide); (4) a combination of double staples that yields favorable binding activity in the context of one HR2 template sequence can also yield favorable binding activity in the context of a separate HR2 template sequence (for example, compare the similar favorable binding activities of O,T double-staple peptides in the context of HR2 and EK1 template sequences; Figure 18). Thus, while such binding data can guide repeat peptide design, synthesis and testing of individual constructs are ultimately required to identify and validate the definitive direct binder for SARS-CoV-2 5-HB.

[0201] An alternative approach to measuring the binding activity of the SARS-CoV-2 HR2 staple peptide involved performing a competitive ELISA assay in which serial dilutions of the staple peptide compete with the longer HR2 peptide for binding to the recombinant 5-helix bundle of SARS-CoV-2. In particular, this binding assay measures activity different from direct FPA in that the staple peptide construct must be able to compete with and disrupt the interaction between another HR2 peptide and the 5-HB protein target. Microwells were coated overnight at 4°C with 50 μl of PBS containing neutraavidin (4 μg / ml). The wells were washed twice with PBS containing 0.05% Tween 20 (PBS-T) and blocked at 37°C for 45 minutes with 4% BSA in PBS-T. Next, 50 μl of 250 nM biotinylated PEG was added. 2- SARS-CoV-2 HR2 (SEQ ID NO: 9) was added to PBS-T containing 1% BSA, incubated for 1 hour with shaking, and subsequently washed four times with 300 μl of PBS-T. Next, 1:2 serial dilutions of SARS-CoV-2 peptide starting at 10 μM containing 50 nM recombinant 5-HB in 50 μL of PBS-T containing 1% BSA were added to the plate, shaken at room temperature for 2 hours, and then washed four times with 300 μL of PBS-T. Finally, 50 μL of a 1:5000 dilution of 6×His-tagged-HRP conjugated goat polyclonal was added. After incubation at room temperature for 40 minutes, the wells were washed five times and colored by adding 50 μl of tetramethylbenzidine (TMB) solution. After 20 minutes, the wells containing the TMB solution were stopped by adding 50 μl of H2SO4 (2M), and the absorbance at 450 nm was read using a microplate reader (Molecular Devices). Using Prism software (Graphpad), the concentration of the competing peptide corresponding to the maximum half-volume signal (IC50) was determined by interpolation of the resulting binding curves. Each peptide competitor was tested in triplicate in at least two separate experiments.

[0202] Figures 19A–19C show the results of a competitive ELISA binding assay in which the interaction between the SARS-CoV-2 5-HB protein and the SARS-CoV-2 stapleless HR2 sequence corresponding to SEQ ID NO: 9 was competed by serial dilutions of i, i+4 staple scan libraries of the core template sequence (SEQ ID NO: 10) with N-terminal extension (aa1169–1178) (SEQ ID NO: 103). Figure 19A shows the complete dose-response competitive binding curve, and Figures 19B and 19C highlight the comparative competitive binding activity of each construct at doses of 3 μM and 10 μM, respectively. Figure 20 shows the results of a competitive ELISA binding assay in which the interaction between the SARS-CoV-2 5-HB protein and the SARS-CoV-2 stapleless HR2 sequence corresponding to SEQ ID NO: 9 was competed by double-stapled and stitched peptides of the core template SARS-CoV-2 HR2 sequence corresponding to SEQ ID NO: 10 at a fixed dose (10 μM). The staple-free core template sequence (SEQ ID NO: 10) could not compete with the longer HR2 template sequence (SEQ ID NO: 9) for binding to 5-HB, but selective double-staple peptides (staple combinations O, S, and K, T) and stitch peptides (staple combinations H, L) of the core template sequence could partially disrupt the binding interaction at a dose of 10 μM. Figure 21 shows the results of a competitive ELISA binding assay in which the interaction between the SARS-CoV-2 5-HB protein and the SARS-CoV-2 staple-free HR2 sequence corresponding to SEQ ID NO: 9 was competed with dose-responsive treatment of the longer HR2 sequence corresponding to SEQ ID NO: 9 using double-staple and stitch peptides. The effectiveness in disrupting the 5-HB / HR2 interaction depended on the type of staples and staple arrangement of the double staples and stitch in the core template sequence (SEQ ID NO: 10) in the context of the longer HR2 peptide (SEQ ID NO: 9). Figure 22 shows the results of a competitive ELISA binding assay in which the interaction between the SARS-CoV-2 5-HB protein and the SARS-CoV-2 stapleless HR2 sequence corresponding to SEQ ID NO: 9 was competed for by dose-responsive treatment with double-stapled and stitched peptides of an alternative HR2 sequence corresponding to SEQ ID NO: 110.The effectiveness in disrupting the 5-HB / HR2 interaction depended on the type and arrangement of double staples and stitches of the core template sequence (SEQ ID NO: 258) in the context of a longer HR2-type peptide (SEQ ID NO: 110), with double staples N,S producing the most potent competitive inhibitors in this group.

[0203] Integrating competitive ELISA data across i, i+4 staple scans of various double-staple and stitch constructs within a core template HR2 sequence with N-terminal extension (SEQ ID NO: 103) (SEQ ID NO: 10), as well as the core template sequence (SEQ ID NO: 10), a longer HR2 sequence (SEQ ID NO: 9), and an alternative HR-2 type sequence (SEQ ID NO: 110), (1) competitive binding activity of staple peptides can be enhanced by adding an N-terminal or N-terminal and C-terminal sequence to the staple core template sequence (container of SEQ ID NO: 10, SEQ ID NO: 9, and SEQ ID NO: 110). (1) Compare the N,S double staples in the Kist; (2) In the context of SEQ ID NO: 103, the C-terminal staple position is generally preferred over the N-terminal staple position (Figures 19B and 19C); and (3) it becomes clear that several double staple positions exhibit binding activity across both direct and competitive binding assays, and in the context of alternative HR2 sequences (SEQ ID NO: 9, SEQ ID NO: 110) (see, for example, double staples N,S and O,S in Figures 18, 21, and 22).

[0204] Example 5: Evaluation of the antiviral activity of SARS-CoV-2-S HR2 staple peptide To test the ability of SARS-CoV-2 HR2 staple peptide to inhibit SARS-CoV-2 infection of cultured cells, Vero E6 cells plated in a 384-well format were treated with serial dilutions of the staple peptide (e.g., a starting dose of 10 μM) for 1 hour in three consecutive sets, followed by a 4-hour challenge with SARS-CoV-2, achieving control infection of 10%–20% of cells (a predetermined optimal infectivity to evaluate the dynamic range of the test compound in the assay). The infected cells were then washed, fixed with 4% paraformaldehyde, washed again with PBS, and immunostained with anti-SARS-CoV-2 nucleocapsid monoclonal antibody, followed by anti-mouse Ig secondary antibody (Alexa Fluor 488; Life Technologies), with the cell bodies counterstained with HCS CellMask blue. Cells were imaged across the z-plane using a Nikon Ti Eclipse automated microscope and analyzed with CellProfiler software, and infection efficiency was calculated by dividing the number of infected cells by the total number of cells. Control cytotoxicity assays were performed using the Cell-Titer Glo (Promega) assay and the LDH release (Roche) assay.

[0205] Figure 23 shows the antiviral activity of exemplary double-staple and stitched peptides of the core template sequence of SEQ ID NO: 10 and the double-staple peptide of the longer HR2 sequence corresponding to SEQ ID NO: 9. The peptides were screened at 25 μM for their ability to inhibit infection of Vero E6 cells by live wild-type SARS-CoV-2 virus, and the percentage of infected cells was plotted. In each case, the staple peptide inhibited infection compared to treatment with vehicle controls. Figure 24 shows the hits from peptide screening in Vero E6 cells subjected to SARS-CoV-2 infection, followed by IC for inhibiting SARS-CoV-2 in assays. 50Further dose-response studies were conducted, as exemplified by the double-stapled core template sequence (SEQ ID NO: 52) with staples O and T having a minimum of 6 μM. Figure 25 shows the different antiviral activities of double-stapled and stitched peptides of the core template sequence (SEQ ID NO: 10) as evaluated at high throughput by an antibody-based SARS-CoV-2 detection platform in infected Vero E6 cells. Figure 26 shows that double i, i+7 staples and stitches at the indicated positions outside the core template sequence (SEQ ID NO: 10) in the context of a longer HR2 peptide sequence (SEQ ID NO: 9) did not result in compounds with antiviral activity. Figure 27 shows the different antiviral activities of exemplary double-stapled and stitched peptides of the core template sequence (SEQ ID NO: 10) in the context of a longer HR2 peptide sequence corresponding to SEQ ID NO: 9. Constructs with double i, i+4 staples O, S exhibited the most potent antiviral activity, followed by compounds with O, T; I, R; and N, S staples, while N, T and H, L constructs showed no effect in the assay. Figure 28 shows the different antiviral activities of exemplary double-staple and stitch peptides of alternative core template sequences in the context of its longer HR2-type peptide sequence corresponding to SEQ ID NO: 110. Constructs with double i, i+4 staples N, S exhibited the most potent antiviral activity, followed by peptides containing N, T staples, while other compounds in this group showed no significant effect.

[0206] In the alternative antiviral assay system, SARS-CoV-2 pseudovirus was used instead of wild-type SARS-CoV-2 virus, and ACE2-expressing 293T cells were used instead of Vero E6 cells. A 293T-hsACE2 stable cell line (catalog number C-HA101) with a GFP (catalog number RVP-701G, lot number CG-113A) reporter and pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain) particles were used (Integral Molecular). Neutralization assays were performed according to the manufacturer's protocol. Briefly, a single dose of peptide (5 μM final dose) was incubated with 5 μL of pseudotyped SARS-CoV-2-GFP at 37°C for 1 hour in a 384-well black clear-bottom plate. Subsequently, 30 μL of 1,000 293T-hsACE2 cells were added (in 10% FBS DMEM, phenol red-free medium), and the plates were placed in a humidified incubator for 48 or 72 hours. Hoechst 33342 and DRAQ7 dyes were added, and the plates were imaged at 10x magnification on a Molecular Devices ImageXpress Micro Confocal Laser. GFP(+) cells were counted and plotted using Prism software (Graphpad). Figure 29 shows the antiviral activity of double-stapled and stitch peptides of the core template sequence (SEQ ID NO: 10) compared to a staple-free core template sequence that shows no antiviral activity, as assessed by a SARS-CoV-2 pseudovirus assay that counts the number of infected cells by IXM microscopy based on fluorescence from ACE2-expressing 293T cells infected with GFP-expressing pseudovirus. Figure 30 shows the different antiviral activities of double-stapled and stitch peptides of the core template sequence (SEQ ID NO: 10) in the context of its longer HR2 sequence (SEQ ID NO: 9), as assessed by a SARS-CoV-2 pseudovirus assay that counts the number of infected cells by IXM microscopy based on fluorescence from ACE2-expressing 293T cells infected with GFP-expressing pseudovirus.Figure 31 shows the different antiviral activities of double-staple peptides of a core template sequence (SEQ ID NO: 10) with C-terminal derivatization by GSGSGC (SEQ ID NO: 256)-(PEG4-chol)-carboxamide, with or without N-terminal peptide extension (aa1168-1176). Figure 32 shows the different antiviral activities of double-staple and stitch peptides of an alternative core template sequence in the context of its longer HR2 type sequence (SEQ ID NO: 110).

[0207] By integrating antiviral data for various double-staple and stitch peptides across template sequences of varying lengths and compositions, it was further revealed that (1) incorporating staples or stitches can convert unstaple-free template sequences from little to no activity peptides into active antiviral agents (see, for example, Figures 27, 28, and 29); and (2) the effect of incorporating staples can have different effects on antiviral activity depending on the length of the template sequence and the alternative composition of the sequence template. For example, the N,S double staple produced a more active peptide in the context of SEQ ID NO: 110, while the O,S double staple had a greater benefit in the context of SEQ ID NO: 9 (see Figures 27 and 28); (3) Considering the distinctions between direct FPA, competitive ELISA, live SARS-CoV-2 infectivity assays in Vero E6 cells, and SARS-CoV-2 pseudovirus assays in ACE2-expressing 293T cells, staple constructs with direct or competitive binding activity similarly demonstrated antiviral activity in one or more SARS-CoV-2 infectivity assays, including, for example, HR2 sequences with double staples N,S;O,S; and O,T. As another example, N,T and N,S double staples conferred enhanced 5-HB competitive binding activity in the context of SEQ ID NO: 110 compared to SEQ ID NO: 9, and similarly showed enhanced antiviral activity against wild-type SARS-CoV-2 infectivity assays in Vero E6 cells (compare the double staple N,T and N,S constructs in Figures 21 vs. 22, and Figures 27 and 28); (4) Double staples or stitches outside the core template sequence did not show antiviral effect, in contrast to the beneficial effect of staples within the core template sequence (compare Figure 26 with Figure 27); (5) Staple type, staple location, presence of one or more staples, template sequence length, and template sequence composition may affect the functional activity of staple and stitch peptides in the SARS-CoV-2 HR2 domain.

[0208] Example 6: Determination of whether the SAH-SARS-CoV-2 peptide associates with the plasma membrane and co-localizes with SARS-CoV-2 during infection. FITC-labeled SAH-SARS-CoV-2 peptides will be brought into contact with cultured cells (e.g., Vero cells, Huh770 cells, Calu-371 cells, 293T cells, primary nasal cells, lung epithelial cells, or alveolar cells) to determine whether they associate with the plasma membrane and / or are taken up via the pinosome pathway, which will be tested by measuring the accumulation of FITC-SAH-SARS-CoV-2 on the plasma membrane and / or in intracellular vesicles labeled with cytotracker red. Co-localization of FITC-SAH-SARS-CoV-2 peptides with rhodamine (R18)-labeled SARS-CoV-2 during cell contact and uptake will also be investigated to determine the ability of SAH-SARS-CoV-2 peptides to target SARS-CoV-2 during the infection process.

[0209] Example 7: Investigation of SAH-SARS-CoV-2 inhibition in vivo for COVID-19 infection. To investigate the ability of the SAH-SARS-CoV-2 peptide to inhibit SARS-CoV-2 infection in vivo, anesthetized mice were intranasally administered either a vehicle or the SAH-SARS-CoV-2 peptide (e.g., 250 μM, 25 μL), followed 4–24 hours later by administration of the SARS-CoV-2 virus (e.g., USA-WA1 / 2020; Hong Kong VM20001061) (10 4 Transnasal infection with PFU is performed. Mice are sacrificed 20 hours after infection, frozen sections of nasal epithelium are prepared, immunostained with anti-SARS-CoV-2 nucleocapsid antibody and fluorescent anti-mouse Ig secondary antibody, counterstained with DAPI, and imaged using a fluorescence microscope.

[0210] Example 8: Evaluation of whether the SAH-SARS-CoV-2 peptide both prevents and treats COVID-19 infection in vitro. Vero E6 cells (60,000 cells / well) plated in a 384-well format were (a) exposed to SARS-CoV-2 alone; (b) treated with SAH-SARS-CoV-2 after 4 hours of SARS-CoV-2 exposure; and (c) infected with SARS-CoV-2 after 4 hours of SAH-SARS-CoV-2 exposure. The Vero cells were then imaged 24 hours after infection by anti-SARS-CoV-2 immunostaining and high-content fluorescence microscopy.

[0211] Example 9: Photoreactive SAH-SARS-CoV-2 peptide for protein capture and binding site analysis To identify and confirm SAH-SARS-CoV-2 targets in the context of SARS-CoV-2 cell infection, derivatized staple peptides are used for proteomic analysis. First, a photoreactive SAH-SARS-CoV-2 construct is synthesized in which (1) a non-native amino acid containing a photoreactive benzophenone functional group (Fmoc-Bpa) is substituted at a separate site adjacent to the interaction surface of the HR2 domain, and (2) the N-terminus of the peptide is capped with biotin for robust streptavidin-based target recovery. Next, photoreactive SAH-SARS-CoV-2 (pSAH-SARS-CoV-2) is added to cultured cells exposed to the SARS-CoV-2 virus, and upon UV irradiation, pSAH-SARS-CoV-2 intercalates into the target protein(s). Infected cells are lysed, pelletized, and the isolated supernatant is subjected to SA pulldown to recover the pSAH crosslinked proteins. The complex is eluted by heating in a loading buffer, then trypsinized, and subjected to MS-based identification using reversed-phase nanoflow LC / MS / MS with an online LTQ-Orbitrap mass spectrometer (Thermo Scientific). The MS data is processed using SEQUEST and Mascot software to classify the protein targets.

[0212] A specific hit is defined as a protein that is uniquely observed in pSAH-SARS-CoV-2 treated and irradiated samples, but not in unirradiated controls or pSAH-SARS-CoV-2 mutant treated samples. This methodology allows for the identification of those amino acid residues in target proteins specifically modified by pSAH-SARS-CoV-2, and thus reveals the site(s) of SAH-SARS-CoV-2 peptide interaction.

[0213] Example 10: Structured antigen for COVID-19 vaccination Structurally constrained SARS-CoV-2 HR peptides were conjugated to a protein carrier (e.g., KLH), followed by immunization of rabbits, collection of antiserum, and ELISA-based immunogenicity testing. For a given structurally constrained SARS-CoV-2 HR construct, the unmodified template peptide and three alternatively conjugated staple analogs were compared by neutralization immunogenicity testing. After a preliminary blood collection (approximately 5 mL of serum), two NZW female rabbits (6-8 weeks old) per immunogen received a primary intramuscular (IM) injection (250 μg with Freund's complete, CpG-ODN, or Ribi adjuvant) on day 1, followed by IM boosts (100 μg with the corresponding adjuvant) on days 21, 42, 63, 84, and 105, and production breeding on days 52, 73, 94, and 112. To monitor and compare specific antibody titers, a direct ELISA assay is performed on each production breeze. Briefly, a 96-well microtiter plate is coated overnight at 4°C with individual SARS-CoV-2 HR immunogen (5 μg / mL). The wells are washed twice with PBS containing 0.05% Tween 20 and blocked with 3% BSA at 37°C for 45 minutes. Serial dilutions of rabbit antiserum are then added to the plate in triplicates and incubated at 37°C for 2 hours. After three washes, a 1:500 dilution of alkaline phosphatase-labeled goat anti-rabbit IgG (in PBS / 1% BSA) is added, and the plate is incubated at room temperature for 40 minutes. The wells are washed, exposed to alkaline phosphatase substrate for 30 minutes, and analyzed by a microplate reader at 405 nm.

[0214] In addition to direct N-terminal conjugation of structured SARS-CoV-2 HR peptides (e.g., via an incorporated cysteine ​​thiol) or incorporation of lysine for conjugation to the non-interacting surface of SAH-SARS-CoV-2 peptides, olefinic derivatization of hydrocarbon staples is also performed, resulting in the proposed "neutralizing surface" of the construct being oriented outward, while the non-neutralizing surface embedded in the protein or lipid conjugate is maintained (e.g., KLH14, bovine serum albumin, cholera toxin, micelles). Using catalytic osmium tetroxide, the olefin is first dihydroxylated, followed by cyclization with thionyl chloride or carbonyldiimidazole. Then, the electrophilic cyclic sulfite or carbonate is reacted with sodium azide, which is reduced to an amine using phosphine. Reaction with the bifunctional reagent 3-thiopropionic acid incorporates a thiol, which is then used to attach a support (e.g., maleimide-KLH). As an alternative approach, peptides are presented in the context of lipid membranes that can facilitate neutralizing antibody recognition. For example, peptides are differentially conjugated to 1,3-dipalmitoyl-glycero-2-phosphoethanolamine, which is then combined with dodecylphosphocholine (DPC) to generate immunogenic tethericelles.

[0215] DNA-primed-protein-boosted immunization strategies have been shown to be more effective than protein-only or DNA-only vaccination for obtaining HIV-1 neutralizing antibodies. We will test a similar approach for SARS-CoV-2 using a lead structured COVID-19 HR conjugate that replaces the timed protein boost with a structured peptide boost, following a published immunization protocol.

[0216] Example 11: Determination of whether staple SAH-SARS-CoV-2 peptide inhibits SARS-CoV-2 infection in infected cells in culture. In this test, cells (e.g., Vero cells, Huh770 cells, Calu-371 cells, 293T cells, primary nasal cells, lung epithelial cells, or alveolar cells) are plated in 24-well plates at a rate of 30,000 cells / well. The following day, the cells are treated with the indicated stapled serial dilutions of the SAH-SARS-CoV-2 peptide (e.g., a starting dose of 10 μM) or a volume-equivalent DMSO vehicle, and subsequently infected with SARS-CoV-2 at 0.1 MOI for 2 hours. The infection medium is removed 2 hours after infection and replaced with a medium containing 5% FBS with the indicated serial dilutions of the SAH-SARS-CoV-2 peptide as described above. The cells are then incubated at 37°C and collected after 24 hours to determine viral infectivity (e.g., antibody-based detection or qPCR as described above).

[0217] Example 12: Evaluation of whether the staple SAH-SARS-CoV-2 peptide inhibits SARS-CoV-2-induced syncytium formation. In this study, cells (e.g., Vero cells, Huh770 cells, Calu-371 cells, 293T cells, primary nasal cells, lung epithelial cells, or alveolar cells) are plated and processed as described in Example 11, except that the number of viral syncytia is counted 48 hours after infection. Syncytia are counted in three different wells, at four separate locations per well.

[0218] Example 13: Investigation of whether staple SAH-SARS-CoV-2 peptide prevents viral infection in culture. In this study, cells (e.g., Vero cells, Huh770 cells, Calu-371 cells, 293T cells, primary nasal cells, lung epithelial cells, or alveolar cells) are plated and treated the following day with serial dilutions of the indicated SAH-SARS-CoV-2 peptide (e.g., a starting dose of 10 μM) or a volume-equivalent DMSO vehicle, followed by infection with the SARS-CoV-2 virus within 30 minutes. The supernatant is collected 24 hours after infection and applied to cells plated the previous day in 24-well plates at 60,000 cells / well. A plaque assay is performed using the collected supernatant, and titers are determined 5 days after infection.

[0219] Example 14: Determination of whether the stapled SAH-SARS-CoV-2 peptide inhibits intranasal SARS-CoV-2 infection in a sequence-specific manner. Four groups (n=10 / group) of K18-hACE2 (Jackson Laboratory) mice were anesthetized and intranasally treated with stapled SAH-SARS-CoV-2, stapled SAH-SARS-CoV-2 negative control peptide (e.g., 125 μM in 1.2% DMSO), or a volume-equivalent vehicle. One hour after treatment, three groups of mice were divided into 10 groups. 4 PFU / mice were intranasally inoculated with a single dose of SARS-CoV-2, and a fourth group received a placebo. 24 hours after infection, the mice were sacrificed, their noses were collected and sectioned, immunostained for SARS-CoV-2, counterstained with DAPI, and imaged using a fluorescence microscope.

[0220] Example 15: Evaluation of whether prophylactic intranasal treatment with stapled SAH-SARS-CoV-2 peptide inhibits SARS-CoV-2 lung infection. In this study, four groups (n=10 / group) of K18-hACE2 (Jackson Laboratory) mice were anesthetized and intranasally treated with stapled SAH-SARS-CoV-2, or stapled SAH-SARS-CoV-2 negative control peptide (e.g., 125 μM in 1.2% DMSO), or a volume-equivalent vehicle. After 24 hours, the mice were divided into three groups of 10. 4A single dose of SARS-CoV-2 was intranasally inoculated into pfu / mice. The fourth group was treated with a volume-equivalent vehicle to induce pseudoinfection. For evaluation by autopsy, mice were euthanized 4 days later (peak of viremia), and viral load was quantified by qPCR from supernatant samples of lung homogenate prepared as described using a tissue lysis device (Qiagen). See Bao L et al. Nature. 2020. Epub 2020 / 05 / 08;doi:10.1038 / s41586-020-2312-y. After 1% paraformaldehyde perfusion, left lung lobes were collected from two mice in each group and subsequently cryopreserved by OCT. Tissue sections (5 μm) were treated overnight with anti-SARS-CoV-2 nucleocapsid antibody, followed by treatment with fluorescent anti-Ig secondary antibody for 1 hour. Sections were washed, mounted in a medium containing DAPI (blue), observed with an Olympus fluorescence microscope, and analyzed by ImageJ. To evaluate the ability of the lead staple peptide to treat or mitigate established SARS-CoV-2 infection, 10 K18-hACE2 mice (n=10 / arm; 5 males, 5 females) were given 10 samples on day 1. 4 PFU is administered intranasally with a viral dose, followed by daily treatment with staple peptide or vehicle via the oropharynx, intraperitoneal, intravenous, or subcutaneously for 10 days (days 2-12). An alternative design delays administration to 3-5 days post-inoculation to simulate treatment initiation based on symptoms or a positive test. Mice are continuously monitored to record body weight and clinical signs, scoring disease progression as weight loss greater than 10%, respiratory distress, and / or stunted growth. The doses and routes of the most effective compounds are then investigated in both preventive and treatment studies to determine the minimum dose to protect the mice. The same experimental design is used, except that four treatment groups (n=10; 5 males, 5 females) are administered the original dose, followed by three tapering doses in 4-fold increments.

[0221] Example 16: Investigation of whether administration of staple SAH-SARS-CoV-2 peptide as a nanoparticle preparation increases lung delivery. In this study, three groups (n=10) of K18-hACE2 (Jackson Laboratory) mice were intratracheally treated with Cy5-labeled staple SAH-SARS-CoV-2 administered in 50 μl volumes, either alone (e.g., 100 μM) or in combination with nanoparticles (NPs) formed from nanochitosan polymer (Zhang et al., Nature Medicine, 2005) (1:2.5, peptide:NP). The control group received a volume-equivalent vehicle. Mice were sacrificed 24 hours post-treatment, and lungs were collected after 1% paraformaldehyde perfusion, followed by cryopreservation by OCT, tissue sectioning, and fluorescence detection of the Cy5-labeled staple peptide.

[0222] Example 17: Evaluation of whether intratracheal administration of stapled SAH-SARS-CoV-2 peptide as a nanoparticle preparation 48 hours prior to SARS-CoV-2 inoculation significantly suppresses viral infection in the lungs. In this study, four groups of K18-hACE2 (Jackson Laboratory) mice (n=10 per group) were anesthetized and administered intratracheally with either a volume-equivalent vehicle containing staple nanoparticles (NPs); SAH-SARS-CoV-2 peptide alone (e.g., 250 μM peptide in 1.2% DMSO); SAH-SARS-CoV-2 peptide combined with NPs (1:2.5, peptide:NP); or the volume-equivalent vehicle alone. Forty-eight hours after administration, a single dose of SARS-CoV-2 (1 x 10⁶) was administered intranasally to the mice in all four groups. 4 The mice were inoculated with pfu / mice. The fifth treatment group (n=10) received intratracheal administration of a volume-equivalent vehicle, followed by placebo inoculation 48 hours later. The mice were sacrificed 4 days after infection and evaluated as described in Example 15.

[0223] Other Embodiments Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. In certain embodiments, for example, the following items are provided: (Item 1) A structurally stable polypeptide comprising an amino acid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 94% identical to the sequence described in SEQ ID NO: 10 (IQKEIDRLNEVAKNLNESL), wherein the amino acid sequence is the following (position 1 is the N-terminal isoleucine of SEQ ID NO: 10, and position 19 is the C-terminal leucine of SEQ ID NO: 10): (i) 7th and 11th place; (ii) 10th and 14th place; (iii) 12th and 16th place; (iv) 14th and 18th place (v) 2nd and 9th place; (vi) 4th and 11th place; (vii) 9th and 16th place; (viii) 2nd and 6th place; (ix) 8th and 12th place; (x) 9th and 13th place; (xi) 11th and 15th place; (xii) 14th and 18th place; (xiii) 15th and 19th place; (xiv) 7th and 14th place; (xv) 3rd and 10th place; (xvi) 6th and 13th place; (xvii) 13th and 17th place; (xviii) 3rd and 7th place; (xix) 3rd, 7th, 13th, and 17th place; (xx) 3rd, 7th, 14th, and 18th place; (xxi) 2nd, 6th, 14th, and 18th place; (xxii) 2nd, 6th, 13th, and 17th place; (xxiii) 3rd, 10th, and 17th place; (xiv) 2nd, 9th, and 13th place; (xv) 3rd, 10th, and 14th place; (xvi) 6th, 13th, and 17th; or (xvii) 7th, 14th, and 18th, The amino acid at a position selected from is replaced by an α,α-disubstituted unnatural amino acid having an olefin side chain; and If the amino acid sequence contains further substitutions, those substitutions are (A) or (B): (A) Positions 4, 8, 10, 13, 15, 17 and 18 of SEQ ID NO: 10 are either not substituted with α,α-disubstituted unnatural amino acids having olefin side chains, or they are substituted with conservative amino acid substitutions; Positions 1, 5, 7, and 11, if substituted, are substituted with conservative amino acid substitutions or α,α-disubstituted unnatural amino acids having olefin side chains; and The remaining positions of SEQ ID NO: 10 may be substituted with any amino acid, or with an α,α-disubstituted unnatural amino acid having an olefin side chain; or (B) One or more of positions 1, 3, 5, 6, 8, 10, 12, 13, 15, 17, and 19 of SEQ ID NO: 10 are not substituted, or if substituted, are replaced by conservative amino acid substitutions, and Based on the fact that one or more of positions 2, 4, 7, 9, 11, 14, 16, and 18 of SEQ ID NO: 10 can be replaced by any amino acid or α,α-disubstituted unnatural amino acid having an olefin side chain; The structurally stabilized peptide is 15 to 100 amino acid long, and optionally 19 to 45 amino acid long; and A structurally stabilized polypeptide having one or more of the following properties: (i) binding to the recombinant 5-helix bundle of the SARS-CoV-2 S protein; (ii) disrupting the interaction between the 5-helix bundle and SEQ ID NO: 10; (iii) being an alpha helix; (iv) being protease resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2. (Item 2) The structurally stabilized polypeptide according to item 1, wherein the amino acid sequence is at least 70% identical to the sequence shown in Sequence ID No. 10. (Item 3) The structurally stabilized polypeptide described in item 1, wherein the amino acid sequence is at least 80% identical to the sequence shown in Sequence ID No. 10. (Item 4) A structurally stabilized polypeptide according to any one of items 1 to 3, wherein the aforementioned amino acid sequence is sequence number 50. (Item 5) A structurally stabilized polypeptide according to any one of items 1 to 3, wherein the aforementioned amino acid sequence includes the sequence of SEQ ID NO: 52. (Item 6) A structurally stabilized polypeptide according to any one of items 1 to 3, wherein the aforementioned amino acid sequence includes the sequence of SEQ ID NO: 51. (Item 7) A structurally stabilized polypeptide according to any one of items 1 to 3, wherein the amino acid sequence is (i) at least 75%, at least 80%, at least 85%, at least 90%, or at least 92% identical to any one of the sequences of SEQ ID NOs: 133, 40, 136, 42, 30, 113, 34, 36, 134, 39, 135, 42, 137, 50, 52, 51, 31-33, 37, 41, and 44-49; or (ii) a structurally stabilized polypeptide according to any one of items 1 to 3, comprising any one of the sequences of SEQ ID NOs: 133, 40, 136, 42, 30, 113, 34, 36, 134, 39, 135, 42, 137, 50, 52, 51, 31-33, 37, 41, and 44-49. (Item 8) A structurally stabilized polypeptide according to any one of items 1 to 7, further comprising the amino acid sequence ISGINASVVN (SEQ ID NO: 250) added to the N-terminus of the aforementioned amino acid sequence. (Item 9) A structurally stabilized polypeptide according to any one of items 1 to 7, further comprising the amino acid sequence DISGINASVVN (SEQ ID NO: 251) added to the N-terminus of the aforementioned amino acid sequence. (Item 10) A structurally stabilized polypeptide according to any one of items 1 to 9, further comprising the amino acid sequence IDLQEL (SEQ ID NO: 252) added to the C-terminus of the aforementioned amino acid sequence. (Item 11) A structurally stabilized polypeptide according to any one of items 1 to 9, further comprising the amino acid sequence IDLQELGKYEQYI (SEQ ID NO: 253) added to the C-terminus of the aforementioned amino acid sequence. (Item 12) A structurally stabilized polypeptide according to any one of items 1 to 9, further comprising the amino acid sequence IDLQELGSGSGC (SEQ ID NO: 254) added to the C-terminus of the aforementioned amino acid sequence. (Item 13) A structurally stabilized polypeptide according to any one of items 1 to 9, further comprising the amino acid sequence IDLQELGKYEQYIGSGSGC (SEQ ID NO: 255) added to the C-terminus of the aforementioned amino acid sequence. (Item 14) A structurally stabilized polypeptide as described in any one of items 1 to 13, further comprising polyethylene glycol. (Item 15) A structurally stabilized polypeptide as described in any one of items 1 to 14, further containing cholesterol. (Item 16) A structurally stabilized polypeptide as described in any one of items 1 to 13, further comprising GSGSGC (SEQ ID NO: 256)-(PEG4-chol)-carboxamide. (Item 17) A structurally stable polypeptide comprising an amino acid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 94% identical to the sequence described in SEQ ID NO: 110 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL), wherein the amino acid sequence of SEQ ID NO: 110 is as follows (position 1 is the N-terminal serine and position 36 is the C-terminal leucine): (i) 13th, 20th, and 27th (ii) 14th, 21st, and 28th; (iii) 13th, 17th, 24th, and 28th place; (iv) 14th, 18th, 24th, and 28th; (v) 13th, 17th, 25th, and 29th; or (vi) 14th, 18th, 25th, and 29th, The amino acid at a selected position is replaced by an α,α-disubstituted unnatural amino acid having an olefin side chain; If the aforementioned amino acid sequence has further substitutions (multiple substitutions are possible), they are (A) (A) If one or more of positions 4, 8, 10, 13, 15, 17, and 18 of SEQ ID NO: 110 are not substituted with α,α-disubstituted non-natural amino acids having an olefin side chain, they are either unsubstituted or substituted with a conservative amino acid substitution; If one or more of positions 1, 5, 7, and 11 of sequence number 110 are substituted, the substitutions are by conservative amino acid substitutions; Based on the fact that one or more of the remaining positions in SEQ ID NO: 110 can be substituted with any amino acid; and The peptide has a length of 15 to 100 amino acids; and A structurally stabilized polypeptide having one or more of the following properties: (i) binding to the recombinant 5-helix bundle of the SARS-CoV-2 S protein; (ii) disrupting the interaction between the 5-helix bundle and SEQ ID NO: 258; (iii) being an alpha helix; (iv) being protease resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2. (Item 18) A structurally stabilized polypeptide as described in item 17, wherein the amino acid sequence is at least 70% identical to the sequences shown in SEQ ID NOs. 175-180. (Item 19) A structurally stabilized polypeptide according to item 17, wherein the amino acid sequence is at least 70% identical to the sequence shown in SEQ ID NO: 177 or 179. (Item 20) A structurally stabilized polypeptide as described in item 17, wherein the amino acid sequence is identical to the sequence shown in Sequence ID No. 179. (Item 21) A structurally stabilized polypeptide as described in item 17, wherein the amino acid sequence is identical to the sequence shown in sequence number 177. (Item 22) A structurally stabilized polypeptide according to any one of items 17 to 21, further comprising the amino acid sequence GSGSGC (SEQ ID NO: 256) added to the C-terminus of the aforementioned amino acid sequence. (Item 23) A structurally stabilized polypeptide as described in items 17-21, further comprising polyethylene glycol. (Item 24) A structurally stabilized polypeptide as described in any one of items 17-21, further containing cholesterol. (Item 25) A structurally stabilized polypeptide as described in any one of items 17 to 21, further comprising GSGSGC (SEQ ID NO: 256)-(PEG4-chol)-carboxamide. (Item 26) A peptide comprising the amino acid sequence described in Sequence ID No. 9, wherein at least two amino acids separated by 2, 3, or 6 amino acids are replaced by α,α-disubstituted unnatural amino acids having olefin side chains, the peptide being 45 amino acid length or less and binding to the recombinant 5-helix bundle COVID-19 S protein. (Item 27) A peptide comprising the amino acid sequence described in Sequence ID No. 10, wherein at least two amino acids separated by 2, 3, or 6 amino acids are replaced by α,α-disubstituted non-natural amino acids having olefin side chains, having a maximum amino acid length of 45 amino acids, and binding to the recombinant 5-helix bundle COVID-19 S protein. (Item 28) A structurally stabilized peptide comprising the amino acid sequence described in Sequence ID No. 9, wherein at least two amino acids separated by two, three, or six amino acids are replaced by α,α-disubstituted unnatural amino acids having olefin side chains, having a length of 45 amino acids or less, and possessing one or more of the following properties: (i) binding to the SARS-CoV-2 recombinant 5-helix bundle S protein; (ii) inhibiting the interaction between the 5-helix bundle and the SARS-CoV-2 HR2 peptide (Sequence ID No. 9); (iii) being an alpha helix; (iv) being protease resistant; (v) inhibiting the fusion of SARS-CoV-2 with host cells; and / or (vi) inhibiting cell infection by SARS-CoV-2. (Item 29) A structurally stabilized peptide as described in item 28, wherein the amino acid sequence includes the sequence described in any one of SEQ ID NOs: 11-29, 153, 154, 156, 158, 160, or 162. (Item 30) The peptide described in item 26, with the following amino acid sequence: (a)

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Claims

1. A structurally stabilized peptide, wherein the following is the same as in SEQ ID NO: 10 (position 1 is the N-terminal isoleucine of SEQ ID NO: 10, and position 19 is the C-terminal leucine of SEQ ID NO: 10): (i) 7th and 11th place; (ii) 10th and 14th place; (iii) 12th and 16th place; (iv) 14th and 18th (v) 2nd and 9th place; (vi) 4th and 11th place; (vii) 9th and 16th place; (viiii) 2nd and 6th place; (ix) 8th and 12th place; (x) 9th and 13th place; (xi) 11th and 15th place; (xii) 15th and 19th place; (xiiii) 7th and 14th place; (xiv) 3rd and 10th place; (xv) 6th and 13th place; (xvi) 13th and 17th; or (xvii) 3rd and 7th place, The amino acid sequence is as described in Sequence ID No. 10 (IQKEIDRLNEVAKNLNESL), except that two amino acids at positions selected from are replaced by α,α-disubstituted non-natural amino acids having crosslinked olefin side chains, and the 0, 1, or 2 further amino acid substitutions are not at the positions of the two α,α-disubstituted non-natural amino acids having crosslinked olefin side chains. If the amino acid sequence includes the one or two further substitutions, those substitutions are (A) or (B): (A) A conservative amino acid substitution at any one of positions 1, 4, 5, 7, 8, 10, 11, 13, 15, 17, and 18 of SEQ ID NO: 10, or a substitution with any amino acid at any one of positions 2, 3, 6, 9, 12, 14, 16, and 19 of SEQ ID NO: 10; or (B) Based on any of the conservative amino acid substitutions at one or more of the positions 1, 3, 5, 6, 8, 10, 12, 13, 15, 17, and 19 of SEQ ID NO: 10; The structurally stabilized peptide has a length of 19 to 45 amino acids; and The structurally stabilized peptide possesses the following properties: (i) it binds to the recombinant 5-helix bundle of the SARS-CoV-2 S protein; (ii) it disrupts the interaction between the 5-helix bundle and SEQ ID NO: 10; (iii) it is alpha-helical; (iv) it is protease-resistant; (v) it inhibits the fusion of SARS-CoV-2 with host cells; and / or (vi) it inhibits SARS-CoV-2 infection of cells. A structurally stabilized peptide having one or more of the following:

2. A structurally stabilized peptide according to claim 1, comprising one amino acid sequence of any of the sequences of SEQ ID NOs: 133, 40, 136, 42, 30, 113, 34, 36, 134, 39, 135, 42, 137, 31-33, 37, and 41, excluding 0, 1, or 2 amino acid substitutions, wherein the 1 or 2 amino acid substitutions, if present, are not at the positions of the two α,α-disubstituted unnatural amino acids having olefin side chains.

3. The structurally stabilized peptide according to claim 1, comprising the one or two further amino acid substitutions, wherein the one or two further amino acid substitutions are conservative amino acid substitutions.

4. A structurally stabilized peptide with the following amino acid sequence: (a) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 121】 (Sequence ID 30) [wherein the formula, if the amino acid substitution is present, it is not at positions 2 and 9; the olefin side chains of 8 and X are crosslinked with each other]; (b) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 122】 (Sequence ID 31) [wherein the formula, if present, the amino acid substitution is not at the 3rd and 10th positions; the olefin side chains of 8 and X are crosslinked with each other]; (c) Having 0, 1 or 2 amino acid substitutions, 【Chemical 123】 (Sequence ID 32) [wherein the formula, if present, the amino acid substitution is not at positions 6 and 13; the olefin side chains of 8 and X are crosslinked with each other]; (d) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 124】 (Sequence ID 33) [wherein the formula, if present, the amino acid substitution is not at positions 7 and 14; the olefin side chains of 8 and X are crosslinked with each other]; (e) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 125】 (Sequence ID 34) [wherein the formula, if present, the amino acid substitution is not at positions 9 and 16; the olefin side chains of 8 and X are crosslinked with each other]; (f) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 126】 (Sequence ID 35) [wherein the formula, if present, the amino acid substitution is not at positions 10 and 17; the olefin side chains of 8 and X are crosslinked with each other]; (h) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 127】 (Sequence ID 36) [wherein the formula, if the amino acid substitution is present, it is not at the 2nd and 6th positions; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other; (i) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 128】 (Sequence ID 37) [In the formula, if the amino acid substitution is present, it is not at the 3rd and 7th positions; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other; (j) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 129】 (Sequence ID 38) [wherein the formula, if the amino acid substitution is present, it is not at the 6th and 10th positions; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other; (k) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 130】 (Sequence ID 39) [wherein the formula, if the amino acid substitution is present, it is not at the 9th and 13th positions; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other; (l) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 131】 (SEQ ID NO: 40) [wherein, when the amino acid substitution is present, it is not at positions 10 and 14; X １ and X ２ of the olefin side chains are cross-linked to each other]; (m) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 132】 (Sequence ID 41) [wherein the formula, if the amino acid substitution is present, it is not at positions 13 and 17; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other; (n) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 133】 (Sequence ID 42) [wherein the formula, if the amino acid substitution is present, it is not at positions 14 and 18; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other; (o) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 144】 (Sequence ID 113) [wherein the formula, if present, the amino acid substitution is not at positions 4 and 11; the olefin side chains of 8 and X are crosslinked with each other]; (p) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 145】 (Sequence ID 133) [wherein the formula, if the amino acid substitution is present, it is not at positions 7 and 11; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other; (q) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 146】 (Sequence ID 134) [wherein the formula, if the amino acid substitution is present, it is not at the 8th and 12th positions; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other; (r) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 147】 (Sequence ID 135) [wherein the formula, if the amino acid substitution is present, it is not at positions 11 and 15; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other; (s) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 148】 (Sequence ID 136) [wherein the formula, if the amino acid substitution is present, it is not at the 12th and 16th positions; X １ and X ２ The olefin side chains are crosslinked with each other; or (t) Having 0, 1 or 2 amino acid substitutions, 【Chemistry 149】 (Sequence ID 137) [wherein the formula, if the amino acid substitution is present, it is not at positions 15 and 19; X １ and X ２ The aforementioned olefin side chains are crosslinked with each other. , including, and During the ceremony, 8 = (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine; X, X １ , and X ２ = (S)-α-(4'-pentenyl)alanine; If the amino acid sequence contains one or two substitutions, those substitutions are (A) or (B): (A) 8, X, X, X １ Or X ２ Otherwise, a conservative amino acid substitution in one or two of the positions 1, 4, 5, 7, 8, 10, 11, 13, 15, 17, and 18, numbered according to sequence number 10, or 8, X, X, X １ Or X ２ Otherwise, substitution with any amino acid at one or two of the 2nd, 3rd, 6th, 9th, 12th, 14th, 16th, and 19th positions numbered according to Sequence ID No. 10; or (B) Based on either a conservative amino acid substitution at one or two of the positions 1, 3, 5, 6, 8, 10, 12, 13, 15, 17, and 19 numbered according to Sequence ID No. 10, or a substitution with any amino acid at one or two of the positions 2, 4, 7, 9, 11, 14, 16, and 18 numbered according to Sequence ID No. 10; The structurally stabilized peptide has a length of 19 to 45 amino acids; and The structurally stabilized peptide possesses the following properties: (i) it binds to the recombinant 5-helix bundle of the SARS-CoV-2 S protein; (ii) it disrupts the interaction between the 5-helix bundle and SEQ ID NO: 10; (iii) it is alpha-helical; (iv) it is protease-resistant; (v) it inhibits the fusion of SARS-CoV-2 with host cells; and / or (vi) it inhibits SARS-CoV-2 infection of cells. A structurally stabilized peptide having one or more of the following:

5. 8 = (R)-α-(7'-octenyl)alanine; and X, X １ , and X ２ The structurally stabilized peptide according to claim 4, wherein the peptide is (S)-α-(4'-pentenyl)alanine.

6. The structurally stabilized peptide according to claim 4, wherein the one or two amino acid substitutions are conservative substitutions.

7. A structurally stabilized peptide according to any one of claims 1 to 6, further comprising the amino acid sequence GSGSGC (SEQ ID NO: 256) added to the C-terminus of the aforementioned amino acid sequence.

8. The structurally stabilized peptide according to any one of claims 1 to 7, wherein the peptide is acylated and amidated.

9. A structurally stabilized peptide according to any one of claims 1 to 8, further comprising polyethylene glycol.

10. A structurally stabilized peptide according to any one of claims 1 to 9, further comprising cholesterol.

11. A nanoparticle composition comprising a structurally stabilized peptide according to any one of claims 1 to 10, wherein the nanoparticles are optionally PLGA nanoparticles, and further optionally the lactic acid:glycolic acid ratio of the PLGA nanoparticles is in the range of 2:98 to 100:

0.

12. A pharmaceutical composition comprising a structurally stabilized peptide according to any one of claims 1 to 10, or nanoparticles according to claim 11, and a pharmaceutically acceptable carrier.

13. A composition for treating coronavirus infection in human subjects requiring treatment for coronavirus infection, comprising a structurally stabilized peptide according to any one of claims 1 to 10, or nanoparticles according to claim 11.

14. A composition for preventing coronavirus infection in human subjects requiring prevention of coronavirus infection, comprising a structurally stabilized peptide according to any one of claims 1 to 10, or nanoparticles according to claim 11.

15. The composition according to claim 13 or 14, wherein the coronavirus infection is caused by a beta coronavirus.

16. The composition according to any one of claims 13 or 14, wherein the coronavirus infection is caused by SARS-CoV-2.

17. The composition according to any one of claims 13 to 14, wherein the subject is a human.

18. A method for producing a structurally stabilized peptide, comprising: (a) providing a peptide having the sequence described in any one of SEQ ID NOs: 30-42, 113, or 133-137; and (b) crosslinking the peptide. In the formula, 8 = (R)-α-(7'-octenyl)alanine; and X, X １ , and X ２ The method is that it is (S)-α-(4'-pentenyl)alanine.

19. The method according to claim 18, wherein the crosslinking of the peptide is performed by a ruthenium-catalyzed metathesis reaction.

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