Antiviral structurally stapled SARS-CoV-2 peptide-cholesterol conjugates and uses thereof

Abstract

Disclosed herein are cross-linked peptides, alone or conjugated to PEG(n)-cholesterol (or thiocholesterol) moieties, useful for interfering with, inhibiting or preventing coronavirus infection (e.g., infection by SARS-CoV-2). Methods of treating and / or preventing coronavirus infection (e.g., COVID-19) are also disclosed. There are currently no antiviral therapeutics to prevent, treat, or completely treat infection from novel coronavirus (nCoV) pandemics, such as COVID-19 caused by Wuhan nCoV (also known as 2019-nCoV or SARS-CoV-2).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 241,722, filed September 8, 2021, which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file with filename 00530-0414WO1_SL_ST26.xml. This XML file, created on September 6, 2022, is 74,442 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0003] Technical Field The present disclosure relates to structurally stabilized SARS-CoV-2 antiviral peptides, alone or conjugated with PEG(n)-cholesterol or PEG(n)-thiocholesterol derivatization generated to further optimize activity, methods for making such stapled peptide-cholesterol conjugates and methods for using same in the prevention and treatment of coronavirus infections. [Background technology]

[0004] background There are currently no antiviral therapeutics to prevent, treat, or completely cure infections from 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 has caused 603,711,760 cases of respiratory illness and 6,484,136 deaths worldwide as of September 2022. Subsequent variants pose ongoing public health challenges, including hampering the effectiveness of vaccines, antibodies, and molecular therapeutics.

[0005] SARS-CoV-2 contains a surface protein that undergoes a conformational change upon engagement with the host cell, resulting in the formation of a six-helix bundle that integrates the host and viral membranes. Although peptide-based inhibition of the viral fusion process is mechanistically feasible and clinically effective (e.g., fuzeon (i.e., enfurvirtide), approved by the FDA in 2003), the biophysical and pharmacological propensity of peptides, including loss of bioactive forms in vivo and rapid proteolysis (e.g., 100 mg self-injected twice daily), limits 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. Summary of the Invention [Means for solving the problem]

[0006] overview This application relates to compositions and methods that disclose peptide stabilization techniques (e.g., stapling) that recreate and enhance bioactive helical structures combined with PEG(n)-cholesterol or thiocholesterol derivatization methods to generate optimized and targeted prophylactic and therapeutic agents for the prevention and / or treatment of coronavirus (e.g., betacoronavirus, e.g., SARS-CoV-2) infections. By inserting "staples" (e.g., all-carbohydrate staples) into native peptides, bioactive helical structures can be restored and superior protease resistance can be imparted by burying otherwise labile amide bonds in 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. Disclosed herein are carbohydrate-stapled and PEG(n)-cholesterol or thiocholesterol derivatized peptide inhibitors of coronavirus (e.g., betacoronavirus, e.g., SARS-CoV-2). These structurally stabilized peptide inhibitors are used to prevent and / or treat coronavirus (e.g., betacoronavirus, e.g., SARS-CoV-2) infections, such as COVID-19.

[0007] The present disclosure features novel SARS-CoV-2 HR2 stapled peptides. In some examples, the HR2 stapled peptides are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to the amino acid sequence of SEQ ID NO:6. In some examples, the HR2 stapled peptides differ from SEQ ID NO:10 at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions. In certain cases, SEQ ID NO:6 is modified to include a staple at any one of staple constructs D, G, K, or N (see FIG. 12). In some examples, these HR2 stapled peptides bind to recombinant 5-HB of SARS-CoV-2. In one example, the 5-HB of SARS-CoV-2 comprises or consists of the sequence of SEQ ID NO:78. In certain examples, substitutions to SEQ ID NO:6 are made to one or more of the residues on the non-interacting face of the helix (see FIG. 9) or to one or more of the non-binding residues in FIG. 24A. In some examples, the HR2 stapled peptide is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to the amino acid sequence of SEQ ID NO:10, 13, 17, or 20. In some examples, the HR2 stapled peptide differs from SEQ ID NO:10, 13, 17, or 20 at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions, except that the two non-natural amino acids that form the intermolecular staple in SEQ ID NO:10, 13, 17, or 20 are not substituted. In some examples, these HR2 stapled peptides bind to recombinant 5-HB of SARS-CoV-2. In one example, the 5-HB of SARS-CoV-2 comprises or consists of the sequence of SEQ ID NO: 78. In some cases, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions that are substituted are in a non-interacting face or at the interface between the interacting and non-interacting faces of the alpha helix (see FIG. 9). In certain examples, the substitutions are made to one or more of the non-binding residues of FIG. 24A.In certain examples, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids may be deleted at the N- and / or C-terminus of SEQ ID NO: 10, 13, 17 or 20. In some examples, the stapled peptides are 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 or 50 amino acids in length. In some cases, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) N-terminal amino acids upstream of the first stapled amino acid of SEQ ID NO: 10 (i.e., one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) DISGINASVVNIQ (SEQ ID NO: 35) may be deleted. In some cases, one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) C-terminal amino acids downstream of the second stapled amino acid of SEQ ID NO: 10 (i.e., one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) VAKNLNESLIDLQELG (SEQ ID NO: 76) may be deleted. In some cases, the stapled peptide is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 4. and has two unnatural amino acids that form an intermolecular staple found in SEQ ID NO: 10, 13, 17, or 20. In some cases, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions in SEQ ID NO: 4 that are substituted are on the interacting face of the alpha helix. In some cases, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions in SEQ ID NO: 4 that are substituted are on the non-interacting face of the alpha helix or at the interface between the interacting and non-interacting faces (see FIG. 9), or are one or more of the non-binding residues in FIG. 24A. In some examples, these HR2 stapled peptides bind to recombinant 5-HB of SARS-CoV-2. In one example, the 5-HB of SARS-CoV-2 comprises or consists of the sequence of SEQ ID NO: 78.

[0008] In some cases, the amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), where 8 is a (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine group and X is a (S)-α-(4'-pentenyl)alanine or (S)-α-(7'-octenyl)alanine group. In some cases, the structurally stabilized polypeptide comprises an amino acid sequence that is at least 94% identical to the sequence set forth in SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), where 8 is a (R)-α-(7'-octenyl)alanine group and X is a (S)-α-(4'-pentenyl)alanine group. In some cases, the structurally stabilized polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), in which 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group, or a variant thereof with one amino acid substitution. In some cases, the amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13 (DISGINASVVNIQKEI8RLNEVAXNLNESLIDLQELGK), in which 8 is an (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine or (S)-α-(7'-octenyl)alanine group. In some cases, the amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17 (DISGINASVVNIQKEIDRLN8VAKNLNXSLIDLQELGK), where 8 is an (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine or (S)-α-(7'-octenyl)alanine group. In some cases, 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group.In some cases, the amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, where 8 is an (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine or (S)-α-(7'-octenyl)alanine group. In some cases, 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group. In some cases, the amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO:20, where 8 is an (R)-α-(7'-octenyl)alanine or a (R)-α-(4'-pentenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine or a (S)-α-(7'-octenyl)alanine group. In some cases, 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group.

[0009] In some cases, each of the stapled peptides has either GK or K at the C-terminus of the stapled peptide. Each of these stapled peptides can inhibit and / or prevent infection of cells (e.g., lung epithelial cells) by SARS-CoV-2 or variants thereof. In some cases, the stapled peptides inhibit infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays. In some cases, the stapled peptides prevent infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays. In some cases, the stapled peptides are conjugated with cholesterol or thiocholesterol. Conjugation can be via a linker, such as polyethylene glycol. In some cases, the linker is PEG(n), where n=1-36. In some cases, n=3-10, 12, 16, 20, 24, or 36. In some cases, n=4 or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some cases, the linker is a peptide linker. In some cases, the linker has the same or about the same length as (PEG)4 or (PEG)8. In some cases, the linker / cholesterol has the structure shown in FIG. 12 as "*". These stapled peptides and stapled peptide conjugates can be used to treat or prevent SARS-CoV-2 infection in a subject in need thereof. In some cases, the subject is a human subject.

[0010] The disclosure also features linkers that include PEG(n)-cholesterol or PEG(n)-thiocholesterol, where n=3-36. In some cases, n=3-10, 12, 16, 20, 24, or 36. In some cases, n=4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0011] The disclosure also features stapled HR2 SARS-CoV-2 peptides conjugated to cholesterol or thiocholesterol via a linker (e.g., PEG). In some cases, there are 3-36 repeats of PEG. The stapled HR2 peptides can be 18-60 amino acids in length (e.g., 19-50, or 38-50 amino acids in length). In some cases, the peptides include a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:3. In some cases, the peptides include a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:4. In some cases, the 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid positions that are substituted are in the interacting face of the alpha helix. In some cases, the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions that are substituted are in the non-interacting face or at the interface between the interacting and non-interacting faces of the alpha helix (see FIG. 9). In some cases, the staple is inserted at the same position as in SEQ ID NO: 10. In some cases, the staple is inserted at the same position as in SEQ ID NO: 13. In some cases, the staple is inserted at the same position as in SEQ ID NO: 17. In some cases, the staple is inserted at the same position as in SEQ ID NO: 20. In some cases, the stapled peptide inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay. In some cases, the stapled peptide prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay. In some cases, the stapled peptide binds to a polypeptide that includes or consists of the amino acid sequence of SEQ ID NO: 78.

[0012] In a first aspect, the disclosure provides 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 set forth in SEQ ID NO:6 (DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK). In some examples, the amino acids at positions selected from (i) 14 and 21, (ii) 17 and 24, (iii) 20 and 27; (iv) 21 and 28; or (v) 24 and 31 of SEQ ID NO:6 (wherein position 1 of SEQ ID NO:6 is an N-terminal aspartic acid and position 38 is a C-terminal lysine) are replaced with α,α-disubstituted non-natural amino acids having olefinic side chains. In one example, positions 14 and 21 are replaced with α,α-disubstituted non-natural amino acids having olefinic side chains. In one example, positions 17 and 24 are replaced with α,α-disubstituted unnatural amino acids having olefinic side chains. In one example, positions 21 and 28 are replaced with α,α-disubstituted unnatural amino acids having olefinic side chains. In one example, positions 24 and 31 are replaced with α,α-disubstituted unnatural amino acids having olefinic side chains. In some examples, the structurally stabilized peptide is 36-60 amino acids in length, optionally 38-50 amino acids in length (e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids in length. In some examples, the structurally stabilized peptide is 38 amino acids in length. In some examples, the structurally stabilized peptide inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay, and / or the structurally stabilized peptide prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay. In some examples, the structurally stabilized peptide binds to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:78.

[0013] In some examples, the amino acid sequence of the structurally stabilized peptide is at least 70% identical (e.g., at least 70%, 75%, 80%, 85%, 90% or 94% identical) to the sequence set forth in SEQ ID NO: 6. In some examples, the amino acid sequence of the structurally stabilized peptide is at least 80% identical (e.g., at least 80%, 85%, 90% or 94% identical) to the sequence set forth in SEQ ID NO: 6.

[0014] In some examples, the amino acid sequence of the structurally stabilized peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK). In some examples, the amino acid sequence of the structurally stabilized peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13 (DISGINASVVNIQKEI8RLNEVAXNLNESLIDLQELGK). In some examples, the amino acid sequence of the structurally stabilized peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. In some examples, the amino acid sequence of the structurally stabilized peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20.

[0015] In some examples, the structurally stabilized polypeptide is 38-45 (38, 39, 40, 41, 42, 43, 44, or 45) amino acids in length. In some examples, the structurally stabilized polypeptide is 38-40 (38, 39, or 40) amino acids in length.

[0016] In some examples, positions 37 and / or 38 of SEQ ID NO:6 are not substituted.

[0017] In a second aspect, the present disclosure relates to a structurally stabilized peptide comprising an amino acid sequence as set forth in SEQ ID NO: 10, 13, 17 or 20, wherein at least 1 to 18 of the amino acids at positions 3, 4, 6, 9, 11, 13, 15, 18, 20, 22, 25, 27, 29, 32, 34, 35, 37 or 38 of SEQ ID NO: 10 are substituted by any other natural or non-natural amino acid, and the non-natural amino acids at positions 14 and 21, 17 and 24, 21 and 28, or 24 and 31, respectively, of SEQ ID NO: 6 are not substituted, wherein the structurally stabilized peptide is 45 amino acids or less in length, and inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay and / or prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay. In some examples, the structurally stabilized peptide inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay, and / or the structurally stabilized peptide prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay. In some examples, the structurally stabilized peptide binds to a polypeptide comprising or consisting of the sequence of SEQ ID NO:78.

[0018] In a third aspect, the disclosure features a conjugate having any one of the aforementioned structurally stabilized polypeptides (e.g., having any one of SEQ ID NOs: 7-21). In some examples, the conjugate also includes a polyethylene glycol (PEG) and / or a cholesterol moiety (e.g., cholesterol; thiocholesterol). In some examples, the PEG and / or cholesterol are linked to the C-terminus of the structurally stabilized polypeptide. In some examples, the conjugate includes PEG and cholesterol. In some examples, the conjugate comprises PEG(n)-cholesterol, where n is 1-36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36), optionally, n is 4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, the conjugate comprises PEG and thiocholesterol. In some examples, the conjugate comprises PEG(n)-thiocholesterol, where n is 1-36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36), optionally, n is 4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0019] Also featured herein is a conjugate comprising a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or any one of SEQ ID NOs: 13, 17, or 20, and a PEG(4)-cholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide. In some examples, the PEG(4)-cholesterol moiety has the formula: [ka] Includes.

[0020] In some examples, the PEG(4)-cholesterol moiety has the formula: [ka] has.

[0021] Additionally, conjugates are featured herein that include a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or any one of SEQ ID NOs: 13, 17, or 20, and a PEG(4)-thiocholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide. In some examples, the PEG(4)-thiocholesterol moiety has the formula: [ka] Includes.

[0022] In some examples, the PEG(4)-thiocholesterol moiety has the formula: [ka] has.

[0023] Further featured herein is a conjugate comprising a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or any one of SEQ ID NOs: 13, 17, or 20, and a PEG(8)-cholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide. In some examples, the PEG(8)-cholesterol moiety has the formula: [ka] Includes.

[0024] In some examples, the PEG(8)-cholesterol moiety has the formula: [ka] has.

[0025] Also featured herein is a conjugate comprising a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or any one of SEQ ID NOs: 13, 17, or 20, and a PEG(8)-thiocholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide. In some examples, the PEG(8)-thiocholesterol moiety has the formula: [ka] Includes.

[0026] In some examples, the PEG(8)-thiocholesterol moiety has the formula: [ka] has.

[0027] Additionally, conjugates are featured herein that include a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or any one of SEQ ID NOs: 13, 17, or 20, and a PEG(n)-cholesterol moiety (wherein (n)=4, 5, 6, 7, or 8) linked to the C-terminal lysine of the structurally stabilized polypeptide. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, the PEG(n)-cholesterol moiety has the formula: [ka] (wherein n=4, 5, 6, 7, or 8) In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0028] In some examples, the PEG(n)-cholesterol moiety has the formula: [ka] (wherein n=4, 5, 6, 7, or 8) In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0029] Finally, also featured herein is a conjugate comprising a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or any one of SEQ ID NOs: 13, 17, or 20, and a PEG(n)-thiocholesterol moiety (wherein (n)=4, 5, 6, 7, or 8) linked to the C-terminal lysine of the structurally stabilized polypeptide. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, the PEG(n)-thiocholesterol moiety has the formula: [ka] (wherein n=4, 5, 6, 7, or 8) In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0030] In some examples, the PEG(n)-thiocholesterol moiety has the formula: [ka] (wherein n=4, 5, 6, 7, or 8) In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0031] Also featured herein is a conjugate that includes a structurally stabilized polypeptide having an amino acid sequence that is at least 94% identical to SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), or to any one of SEQ ID NOs: 13, 17, or 20, where 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group, and a PEG(4)-cholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide.

[0032] In some examples, the conjugate comprises a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), or any one of SEQ ID NOs: 13, 17, or 20, wherein 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group; and a PEG(4)-cholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide.

[0033] In some examples, the PEG(4)-cholesterol moiety has the formula: [ka] In some examples, the PEG(4)-cholesterol moiety has the formula: [ka] has.

[0034] Also featured herein is a conjugate that includes a structurally stabilized polypeptide having an amino acid sequence that is at least 94% identical to SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or to any one of SEQ ID NOs: 13, 17 or 20, where 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group, and a PEG(4)-thiocholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide.

[0035] In some examples, the conjugate comprises a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), or any one of SEQ ID NOs: 13, 17, or 20, wherein 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group; and a PEG(4)-thiocholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide.

[0036] In some examples, the PEG(4)-thiocholesterol moiety has the formula: [ka] In some examples, the PEG(4)-thiocholesterol moiety has the formula: [ka] has.

[0037] Also featured herein is a conjugate that includes a structurally stabilized polypeptide having an amino acid sequence that is at least 94% identical to SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), or to any one of SEQ ID NOs: 13, 17, or 20, where 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group, and a PEG(8)-cholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide.

[0038] In some examples, the conjugate comprises a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or any one of SEQ ID NOs: 13, 17, or 20, where 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group; and a PEG(8)-cholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide. In some examples, the PEG(8)-cholesterol moiety has the formula: [ka] In some examples, the PEG(8)-cholesterol moiety has the formula: [ka] has.

[0039] Also featured herein is a conjugate that includes a structurally stabilized polypeptide having an amino acid sequence that is at least 94% identical to SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or to any one of SEQ ID NOs: 13, 17 or 20, where 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group, and a PEG(8)-thiocholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide. In some examples, the conjugate comprises a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), or any one of SEQ ID NOs: 13, 17, or 20, wherein 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group; and a PEG(8)-thiocholesterol moiety linked to the C-terminal lysine of the structurally stabilized polypeptide.

[0040] In some examples, the PEG(8)-thiocholesterol moiety has the formula: [ka] Includes.

[0041] In some examples, the PEG(8)-thiocholesterol moiety has the formula: [ka] has.

[0042] Also featured herein is a conjugate comprising a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or any one of SEQ ID NOs: 13, 17, or 20, where 8 is an (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine group, and X is an (S)-α-(4'-pentenyl)alanine or (S)-α-(7'-octenyl)alanine group, and a PEG(n)-cholesterol moiety (where (n)=4, 5, 6, 7, or 8) linked to the C-terminal lysine of the structurally stabilized polypeptide. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, the PEG(n)-cholesterol moiety has the formula: [ka] (wherein n=4, 5, 6, 7, or 8) In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0043] Also featured herein is a conjugate comprising a structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) or any one of SEQ ID NOs: 13, 17, or 20, where 8 is an (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine group, and X is an (S)-α-(4'-pentenyl)alanine or (S)-α-(7'-octenyl)alanine group, and a PEG(n)-thiocholesterol moiety (where (n)=4, 5, 6, 7, or 8) linked to the C-terminal lysine of the structurally stabilized polypeptide. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, the PEG(n)-thiocholesterol moiety has the formula: [ka] (wherein n=4, 5, 6, 7, or 8) In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0044] In some examples, 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group.

[0045] Next, the formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein each R1 and R2 is H or a C1-C10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted; each R3 is independently an alkane, alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each [Xaa] is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; w is DISGINASVVNIQ (SEQ ID NO: 35), and each [Xaa] x is EIDRLN (SEQ ID NO: 36), and each [Xaa] y is VAKNLNESLIDLQELGK (SEQ ID NO: 37), wherein the structurally stabilized peptide inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay and / or prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay.

[0046] In some examples, R1 is an alkyl. In some examples, R1 is a methyl group. In some examples, R3 is an alkyl. In some examples, R3 is a methyl group. In some examples, R2 is an alkenyl. In some examples, R2 is an alkyl. In some examples, R2 is a methyl group. In some examples, R3 is an alkenyl. In some examples, R2 is an alkyl. In some examples, R2 is a methyl group. In some examples, R3 is an alkenyl. In some examples, pharma- ceutically acceptable salts include hydrochloride, sodium, sulfate, acetate, phosphate or diphosphate, chloride, potassium, maleate, calcium, citrate, mesylate, nitrate, tartrate, aluminum, gluconate, and any combination thereof.

[0047] In some examples, the structurally stabilized peptides or pharma- ceutically acceptable salts described herein are up to 50 (e.g., 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) amino acids in length, optionally up to 45 (e.g., 45, 46, 47, 48, 49, or 50) amino acids in length. In some examples, the structurally stabilized peptides or pharma- ceutically acceptable salts thereof are 38 amino acids in length.

[0048] A structurally stabilized peptide comprising formula I-1, wherein formula I-1 is [ka] or a pharma- ceutically acceptable salt thereof; R3 is alkenylene; [Xaa] w is DISGINASVVNIQ (SEQ ID NO:35); [Xaa] x is EIDRLN (SEQ ID NO:36); and [Xaa] y is VAKNLNESLIDLQELGK (SEQ ID NO:37); If necessary, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y wherein the carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2; Conformational stabilized peptides are also featured herein.

[0049] In some examples, the structurally stabilized peptide comprises the formula I-1a. In some examples, the structurally stabilized peptide comprises the formula I-1a. [ka] or a pharma- ceutically acceptable salt thereof (wherein, as appropriate, [Xaa] wThe amino group of the N-terminal aspartic acid in 1~4 [Xaa] is optionally replaced by an alkyl group. y is replaced with -C(O)NH2, where the variables are as indicated for formula IA. The compounds include those of formula I-1a, which are defined by:

[0050] A structurally stabilized peptide comprising formula IA, [ka] or a pharma- ceutically acceptable salt thereof; R3 is alkenylene; [Xaa] w is DISGINASVVNIQ (SEQ ID NO:35); [Xaa] x is EIDRLN (SEQ ID NO:36); and [Xaa] y is VAKNLNESLIDLQELGK (SEQ ID NO:37); If necessary, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y wherein the carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2; Conformational stabilized peptides are also featured herein.

[0051] In some instances, the structurally stabilized peptide comprises formula IA.

[0052] In some instances, the structurally stabilized peptide comprises: [ka] or a pharma- ceutically acceptable salt thereof (wherein, as appropriate, [Xaa] wThe amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y is replaced with -C(O)NH2, where the variables are as indicated for formula IA. The compounds include those of formula Ia, which are defined by:

[0053] In some examples, the structurally stabilized peptide comprises Formula Ia.

[0054] In some instances, the structurally stabilized peptide comprises: [ka] [ka] or a pharma- ceutically acceptable salt thereof, in which, optionally, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y The carboxylic acid group of the C-terminal lysine in is replaced with -C(O)NH2, where the variables are as indicated for formula IA.

[0055] In some instances, the structurally stabilized peptide comprises: [ka] or a pharma- ceutically acceptable salt thereof, in which, as appropriate, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y The carboxylic acid group of the C-terminal lysine in is replaced with -C(O)NH2, where the variables are as indicated for formula IA.

[0056] In some examples, R3 is C 7~15 In some embodiments, R3 is C 9~13 In some embodiments, R3 is C 11 In some examples, R3 is -(CH2) 3~7 -CH=CH-(CH2) 3~7 In some examples, R3 is -(CH2) 5~7 -CH=CH-(CH2) 3~4 In some examples, R3 is -(CH2)6-CH=CH-(CH2)3-. In some examples, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 In some cases, [Xaa] is replaced by w In some cases, the amino group of the N-terminal aspartic acid in [Xaa] is replaced by -N(H)C(O)CH3. y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0057] Another feature of the present disclosure is a conjugate comprising a structurally stabilized peptide having formula (I) or a pharma- ceutically acceptable salt thereof and PEG and / or cholesterol. In some examples, the conjugate comprises PEG and cholesterol. In some examples, the cholesterol is thiocholesterol. In some examples, the conjugate comprises a conjugate comprising PEG(n)-cholesterol, optionally where n is 1-36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36), optionally where n is 4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, the conjugate comprises a conjugate comprising PEG(n)-thiocholesterol, where n is 1-36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36), optionally where n is 4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0058] A structurally stabilized peptide conjugate comprising formula II, [ka] or a pharma- ceutically acceptable salt thereof, R3 is alkenylene; R4 is, ** -C(O)-(C 2~6 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~6 alkylene)-R6, where **is [Xaa] y is the point of attachment to the amino group in the side chain of the C-terminal lysine in R5 is hydrogen or C 1~4 is alkyl; R6 is one of the following: [ka] and optionally each of which is replaced by t occurrences of R7; R7, independently for each occurrence, is 1~3 Alkyl, hydroxyl or C 1~3 represents alkoxyl; [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] x is EIDRLN (SEQ ID NO:36); [Xaa] y is VAKNLNESLIDLQELGK (SEQ ID NO:37); m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and t is 0, 1, 2, or 3; If necessary, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y The carboxylate group of the C-terminal lysine in is replaced by -C(O)NH2) Also featured herein are conformationally stabilized peptide conjugates defined by:

[0059] In some embodiments, the structurally stabilized peptide comprises Formula II. In some embodiments, the structurally stabilized peptide comprises Formula II: [ka] or a pharma- ceutically acceptable salt thereof (wherein, as appropriate, [Xaa] wThe amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y wherein the carboxylic acid group of the C-terminal lysine is replaced with -C(O)NH2, and the variables are as indicated for formula II. In some embodiments, the structurally stabilized peptide consists of formula IIa. In some embodiments, the structurally stabilized peptide comprises formula IIa, [ka] or a pharma- ceutically acceptable salt thereof, in which, optionally, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y The carboxylic acid group of the C-terminal lysine in is replaced with -C(O)NH2, where the variables are as indicated for Formula II.

[0060] In some embodiments, the structurally stabilized peptide comprises: [ka] or a pharma- ceutically acceptable salt thereof, in which, as appropriate, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y The carboxylic acid group of the C-terminal lysine in is replaced with -C(O)NH2, where the variables are as indicated for Formula II.

[0061] In some embodiments, R3 is C 7~15 In some embodiments, R is C 9~13 In some embodiments, R is C 11In some embodiments, R3 is -(CH2) 3~7 -CH=CH-(CH2) 3~7 In some embodiments, R3 is -(CH2) 5~7 -CH=CH-(CH2) 3~4 In some embodiments, R3 is -(CH2)6-CH=CH-(CH2)3-.

[0062] A structurally stabilized peptide conjugate comprising formula III, [ka] or a pharma- ceutically acceptable salt thereof, R4 is, ** -C(O)-(C 2~6 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~6 alkylene)-R6, where ** is [Xaa] y is the point of attachment to the amino group in the side chain of the C-terminal lysine in R5 is hydrogen or C 1~4 is alkyl; R6 is one of the following: [ka] and optionally each of which is replaced by t occurrences of R7; R7, independently for each occurrence, is 1~3 Alkyl, hydroxyl or C 1~3 represents alkoxyl; [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] x is EIDRLN (SEQ ID NO:36); [Xaa] y is VAKNLNESLIDLQELGK (SEQ ID NO:37); m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; p is 2, 3, 4, 5, 6, 7, or 8; z is 2, 3, 4, 5, or 6; and t is 0, 1, 2, or 3; If necessary, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y The carboxylate group of the C-terminal lysine in is replaced by -C(O)NH2) Also featured herein are structurally stabilized peptide conjugates defined by:

[0063] In some instances, the structurally stabilized peptide comprises Formula III.

[0064] In some instances, the structurally stabilized peptide comprises: [ka] or a pharma- ceutically acceptable salt thereof (wherein, as appropriate, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 alkyl), and, optionally, [Xaa] y wherein the carboxylic acid group of the C-terminal lysine is replaced with -C(O)NH2, and the variables are as indicated for formula III.

[0065] In some examples, R4 is ** -C(O)-(C 2~3 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~2 alkylene)-R6, where ** is [Xaa] yIn some embodiments, R4 is the point of attachment to the amino group in the side chain of the C-terminal lysine in ** -C(O)-(CH2CH2)-[O-CH2CH2] m -N(R5)C(O)-(CH2)-R6, where ** is [Xaa] y In some cases, [Xaa] is the point of attachment to the amino group in the side chain of the C-terminal lysine in w The amino group of the N-terminal aspartic acid in 1~4 In some cases, [Xaa] is replaced by w In some cases, the amino group of the N-terminal aspartic acid in [Xaa] is replaced by -N(H)C(O)CH3. y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0066] Formula IV: [ka] or a pharma- ceutically acceptable salt thereof, R4 is -C(O)-(C 2~6 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~6 alkylene)-R6; R5 is hydrogen or C 1~4 is alkyl; R 6 is one of the following: [ka] and optionally each of which is replaced by t occurrences of R7; R7, independently for each occurrence, is 1~3 Alkyl, hydroxyl or C 1~3 represents alkoxyl; R8 is -C(O)-(C 1~4 alkyl); [Xaa] wis DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] x is EIDRLN (SEQ ID NO:36); [Xaa] z is VAKNLNESLIDLQELG (SEQ ID NO:77); m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; p is 2, 3, 4, 5, 6, 7, or 8; z is 2, 3, 4, 5, or 6; and t is 0, 1, 2, or 3) Also featured herein is a conformationally stabilized peptide conjugate represented by:

[0067] In some examples, the structurally stabilized peptide is represented by formula IVa, or a pharma- ceutically acceptable salt thereof, wherein formula IVa is [ka] where the variables are as indicated for Formula IV. It is defined by:

[0068] In some examples, the structurally stabilized peptide is represented by formula IVb, or a pharma- ceutically acceptable salt thereof, wherein formula IVb is: [ka] where the variables are as indicated for Formula IV. It is defined by:

[0069] In some examples, the structurally stabilized peptide is represented by formula IVc, or a pharma- ceutically acceptable salt thereof, wherein formula IVc is: [ka] where the variables are as indicated for Formula IV. It is defined by:

[0070] A structurally stabilized peptide conjugate represented by formula V or a pharma- ceutically acceptable salt thereof, wherein formula V is [ka] (In the formula, R4 is -C(O)-(C 2~6 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~6 alkylene)-R6; R5 is hydrogen or C 1~4 is alkyl; R 6 is one of the following: [ka] and optionally each of which is replaced by t occurrences of R7; R7, independently for each occurrence, is 1~3 Alkyl, hydroxyl or C 1~3 represents alkoxyl; R8 is -C(O)-(C 1~4 alkyl); [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] x is EIDRLN (SEQ ID NO:36); [Xaa] z is VAKNLNESLIDLQELG (SEQ ID NO:77); m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; t is 0, 1, 2, or 3) Also featured herein are structurally stabilized peptide conjugates defined by:

[0071] In some examples, R8 is -C(O)CH3. In some examples, R4 is -C(O)-(C 2~3 Alkylene)-[O-CH2CH2]m -N(R5)C(O)-(C 1~2 In some examples, R4 is -C(O)-(CH2CH2)-[O-CH2CH2] m -N(R5)C(O)-(CH2)-R6. In some examples, R5 is hydrogen.

[0072] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0073] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0074] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0075] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0076] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0077] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0078] In some examples, t is 0. In some examples, m is 4. In some examples, m is 8.

[0079] A structurally stabilized peptide conjugate represented by formula VI, or a pharma- ceutically acceptable salt thereof, wherein formula VI is [ka] (In the formula, R4 is -C(O)-(CH2CH2)-[O-CH2CH2]8-N(H)C(O)-(CH2)-R6; R6 is, [ka] and; [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] x is EIDRLN (SEQ ID NO:36); and [Xaa] z is VAKNLNESLIDLQELG (SEQ ID NO: 77) Also featured herein are structurally stabilized peptide conjugates defined by:

[0080] Also featured herein is a pharmaceutical compound comprising any one of the above-described structurally stabilized peptides, pharma- ceutically acceptable salts, or conjugates thereof, and a pharma- ceutically acceptable carrier.

[0081] Further provided herein are methods of making and using any one of the structurally stabilized peptides, pharma- ceutically acceptable salts, or conjugates thereof. In some examples, provided herein are methods of treating a coronavirus infection in a human subject in need of such treatment, comprising administering to the human subject a therapeutically effective amount of any one of the structurally stabilized peptides described above (e.g., including SEQ ID NOs: 7-21, e.g., including SEQ ID NO: 10 or any one of SEQ ID NOs: 13, 17, or 20), a pharma- ceutically acceptable salt, or conjugate thereof. Also provided herein are methods of preventing a coronavirus infection in a human subject in need of such treatment, comprising administering to the human subject a therapeutically effective amount of any one of the structurally stabilized peptides described above (e.g., including SEQ ID NOs: 7-21, e.g., including SEQ ID NO: 10 or any one of SEQ ID NOs: 13, 17, or 20), a pharma- ceutically acceptable salt, or conjugate thereof.

[0082] Further featured herein is a method of treating a coronavirus infection in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of any one of the structurally stabilized peptides described above (e.g., including SEQ ID NO: 7-21, e.g., including SEQ ID NO: 10 or any one of SEQ ID NO: 13, 17 or 20), a pharma- ceutically acceptable salt, or a conjugate thereof. Also disclosed herein is a method of preventing a coronavirus infection in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of any one of the structurally stabilized peptides described above (e.g., including SEQ ID NO: 7-21, e.g., including SEQ ID NO: 10 or any one of SEQ ID NO: 13, 17 or 20), a pharma- ceutically acceptable salt, or a conjugate thereof. In some examples, the subject is selected from a cow, a pig, a horse, a cat, a dog, a rat, a mouse, or a bat.

[0083] In some instances, the coronavirus infection is caused by a betacoronavirus. In some instances, the coronavirus infection is caused by an alphacoronavirus.

[0084] In some examples, the coronavirus infection is due to infection with SARS-CoV-2. In some examples, the coronavirus infection is due to infection with a variant of SARS-CoV-2. In some examples, the variant is selected from Wuhan-Hu-1, B.1.427 / B.1.429, B.1.617.2, D614G B.1, or Brazilian variant P.1. In some examples, the variant is an Omicron variant. In some examples, the variant is selected from B.1.351, cluster 5, lineage B.1.1.207, lineage B.1.1.7, variants of concern 202102 / 02, lineage B.1.1.317, lineage B.1.1.318, lineage B.1.351, lineage B.1.429, lineage B.1.525, lineage P.1 (also known as lineage B.1.1.28), lineage B.1.1.529, lineage BA.1, lineage BA.1.1, lineage BA.2, lineage BA.3, lineage BA.4 lineage BA.5, D614G, E484K, N501Y, S477G / N, or P681H. In some cases, the variant includes one of Wuhan-Hu-1, B.1.427 / B.1.429, B.1.617.2, D614G B.1, Brazilian variant P.1, B.1.1.7, B.1.351, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, B.1.621.1, B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4 or BA.5.

[0085] The methods disclosed herein also include a method of making one of the structurally stabilized peptides. In some examples, the method of making the structurally stabilized peptide includes (a) providing a peptide having a sequence as shown in SEQ ID NO: 6 or a variant thereof (e.g., at least 1-18 of the amino acids at positions 3, 4, 6, 9, 11, 13, 15, 18, 20, 22, 25, 27, 29, 32, 34, 35, 37 or 38 in SEQ ID NO: 6 are substituted), and (b) crosslinking the peptide, and optionally purifying the structurally stabilized peptide. In some examples, the crosslinking of the peptide is by a ruthenium-catalyzed metathesis reaction. In some examples, the method further includes formulating the structurally stabilized peptide as a sterile pharmaceutical composition.

[0086] Also provided herein is a method for synthesizing one of the conjugates provided herein.In some examples, the method for synthesizing one of the conjugates provided herein comprises: (a) providing a structurally stabilized polypeptide; and (b) derivatizing the resin-bound amine of the structurally stabilized polypeptide with PEG and / or cholesterol containing carboxylic acid on the resin.In some examples, the method for synthesizing one of the conjugates provided herein comprises: (a) providing a structurally stabilized polypeptide; and (b) derivatizing the resin-bound amine of the structurally stabilized polypeptide with PEG and / or thiocholesterol containing carboxylic acid on the resin.

[0087] Also provided herein is a method for synthesizing a heptad repeat domain 2 (HR2) polypeptide. In some examples, the method includes the steps of (a) providing a HR2 polypeptide; and (b) derivatizing the resin-bound amine of the HR2 polypeptide with a carboxylic acid-containing PEG and / or cholesterol on the resin. In some examples, the method includes the steps of (a) providing a HR2 polypeptide; and (b) derivatizing the resin-bound amine of the HR2 polypeptide with a carboxylic acid-containing PEG and / or thiocholesterol on the resin.

[0088] In some examples, the derivatizing step includes dissolving thiocholesterol in dichloromethane (DCM) or cholesterol in tetrahydrofuran (THF) to generate a solution; and incorporating carboxythiocholesterol or carboxycholesterol by solid-phase synthesis by sequentially adding a base, t-butyl ester of bromoacetic acid, and trifluoroacetic acid to the solution. In some examples, the derivatizing step further includes treating the resin-bound structurally stabilized polypeptide with piperidine in a solution containing dimethylformamide (DMF); capping the N-terminus of the structurally stabilized polypeptide with acetic anhydride; deprotecting the C-terminus of the structurally stabilized polypeptide with hydrazine in DMF; acylating the structurally stabilized polypeptide with an Fmoc-protected PEG(n) amino acid; crosslinking the structurally stabilized polypeptide; and isolating the structurally stabilized polypeptide from the resin.

[0089] In some examples, n=1-36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In some examples, n=4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, the C-terminal lysine of SEQ ID NO:6 is substituted with a resin-bound amine, and optionally, the C-terminal lysine of SEQ ID NO:6 is further substituted with a resin-bound carboxylic acid or thiol. In some examples, the cholesterol is a thiocholesterol. In some examples, the conjugate comprises PEG(n)-cholesterol, where n is 1-36, and optionally n is 4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, the conjugate comprises PEG(n)-thiocholesterol, where n is 1-36, and optionally n is 4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, crosslinking the peptides is by a ruthenium-catalyzed metathesis reaction.

[0090] 1. A method for synthesizing a stabilized peptide, comprising subjecting a peptide comprising formula VII to ring-closing metathesis conditions to obtain a stabilized peptide, wherein formula VII is [ka] or a pharma- ceutically acceptable salt thereof, [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] x is EIDRLN (SEQ ID NO:36); [Xaa] yis VAKNLNESLIDLQELGK (SEQ ID NO:37); R9 is -OH or [ka] and; p is 2, 3, 4, 5, 6, 7, or 8; and z is 2, 3, 4, 5, or 6; If necessary, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 (Alkyl) is substituted) Also featured herein is a method defined by:

[0091] In some examples, the ring-closing metathesis conditions include exposing said peptide comprising Formula IV to a Grubbs ring-closing metathesis ruthenium catalyst.

[0092] In some examples, the stabilizing peptide is [ka] or a pharma- ceutically acceptable salt thereof, [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] x is EIDRLN (SEQ ID NO:36); [Xaa] y is VAKNLNESLIDLQELGK (SEQ ID NO:37); R9 is -OH or [ka] and; p is 2, 3, 4, 5, 6, 7, or 8; and z is 2, 3, 4, 5, or 6; If necessary, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4(Alkyl) is substituted) The compounds of formula III are defined as follows:

[0093] In some examples, p is 5. In some examples, z is 3. In some examples, the method further comprises derivatizing the C-terminus of the stabilized peptide with a moiety comprising a polyethylene glycol moiety and a cholesterol or thiocholesterol moiety. In some examples, the method comprises derivatizing the C-terminus of the stabilized peptide to provide ** -C(O)-(C 2~6 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~6 The method further comprises the step of incorporating an alkylene)-R6 group, wherein ** is [Xaa] y R5 is a hydrogen or C 1~4 alkyl; R6 is one of the following: [ka] and optionally each of which is replaced by t occurrences of R7; R7, independently for each occurrence, is 1~3 Alkyl, hydroxyl or C 1~3 represents alkoxyl; m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; t is 0, 1, 2, or 3.

[0094] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0095] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0096] In some examples, t is 0. In some examples, m is 4. In some examples, m is 8. In some examples, R9 is [ka] In some examples, the method further comprises cleaving off the solid support.

[0097] Nanoparticle compositions are also featured herein. In some examples, the nanoparticle compositions include any of the structurally stabilized peptides provided herein (e.g., SEQ ID NOs: 10, 13, 17, 20), pharma- ceutically acceptable salts, or conjugates thereof. In some examples, the nanoparticle compositions are PLGA nanoparticles. In some examples, the nanoparticle compositions include a lactic acid:glycolic acid ratio of PLGA nanoparticles ranging from 2:98 to 100:0. In some examples, the nanoparticle compositions also include chitosan, dextrin, or both.

[0098] formula: [ka] (wherein n is 1 to 36). Also featured herein are peptide linkers comprising PEG(n)-thiocholesterol having the formula:

[0099] Next, the formula: [ka] (wherein n is 1 to 36). Featured herein are PEG(n)-cholesterol-containing peptide linkers comprising:

[0100] In some examples, n of the peptide linker is 4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some examples, the peptide linker comprises a lysine attached to a PEG.

[0101] One of the following formulas: [ka] (wherein n is 1 to 36). Also featured herein are compounds having the formula: In some examples, n is 4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0102] In some examples, for any of the structurally stabilized peptides or conjugates described herein, 8 = (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine; and X = (S)-α-(4'-pentenyl)alanine or (S)-α-(7'-octenyl)alanine. In some examples, 8 = (R)-α-(7'-octenyl)alanine; and X = (S)-α-(4'-pentenyl)alanine.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in carrying out or testing this disclosure, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, shall control. The materials, methods, and examples are only illustrative and are not intended to be limiting.

[0104] Other features and advantages of the disclosure will become apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0105] [Figure 1] FIG. 1 depicts the mechanism of action of SARS-CoV-2 S fusion inhibitor peptides. [Diagram 2] FIG. 2 provides the amino acid sequence of the S protein of SARS-CoV-2 (SEQ ID NO:1). [Diagram 3] FIG. 3 is a schematic diagram of the SARS-CoV-2 spike (S) protein, including the sequence composition of the fusion domains of heptad repeat domain 1 (HR1) (SEQ ID NO: 2) and heptad repeat domain 2 (HR2) (SEQ ID NO: 3). [Figure 4] Figure 4 shows an alignment of the HR1 and HR2 regions of the SARS-CoV-2 and SARS-CoV-1 ("SARS, C.2004") viruses with sequence homology highlighted, with significant sequence identity between the HR2 regions of SARS-CoV-2 and SARS-CoV-1. [Diagram 5] Figure 5 shows various unnatural amino acids containing olefin tethers that can be used to generate hydrocarbon stapled SARS-CoV-2 S peptides with staples spanning positions i, i+3; i, i+4; and i, i+7. Single staple scanning is used to generate a library of single stapled SARS-CoV-2-19 HR2 peptides for conjugation with PEG-thiocholesterol or PEG-cholesterol moieties. [Figure 6] FIG. 6 shows various staple compositions in multiply-stapled peptides and staple scanning to generate a library of multiply-stapled SARS-CoV-2 HR2 peptides for conjugation with PEG-thiocholesterol or PEG-cholesterol moieties. [Figure 7]FIG. 7 shows various staple compositions in tandem stitched peptides to generate a library of stitched SARS-CoV-2 HR2 peptides for conjugation with PEG-thiocholesterol or PEG-cholesterol moieties. [Figure 8] 8 is an illustration of an exemplary approach for designing, synthesizing and identifying optimal stapled peptide constructs targeting the SARS-CoV-2 fusion machinery, including Ala scan, staple scan, and generation of variable N-terminal and C-terminal deletion, addition and derivatization libraries for conjugation with PEG-thiocholesterol or PEG-cholesterol moieties. Single and double stapled and stitched constructs, including alanine and staple and stitch scans, are used to identify optimal stapled peptides for conjugation with PEG-thiocholesterol or PEG-cholesterol moieties and application in in vitro and in vivo analyses. [Figure 9] Figure 9 is a helical wheel drawing of a portion of the SARS-CoV-2 HR2 domain structured as an amphipathic alpha helix (SEQ ID NO: 4), showing a primarily hydrophobic binding interface with adjacent charged or polar residues around the binding interface and on the non-interacting faces. Arrows indicate the hydrophobic moments. [Figure 10] Figure 10 shows a synthetic scheme for converting thiocholesterol or cholesterol to a carboxylic acid for facile on-resin derivatization of stapled peptides with cholesterol-containing moieties. DCM: dichloromethane; TFA: trifluoroacetic acid. [Figure 11A] 11A-11B show a synthetic scheme of the steps for on-resin derivatization of a stapled peptide sequence (SEQ ID NO: 74) with a PEG-linked thiocholesterol moiety (FIG. 11A), and a synthetic scheme of the steps for on-resin derivatization of a stapled peptide sequence (SEQ ID NO: 74) with a PEG-linked cholesterol moiety (FIG. 11B). [Figure 11B] 11A-11B show a synthetic scheme of the steps for on-resin derivatization of a stapled peptide sequence (SEQ ID NO: 74) with a PEG-linked thiocholesterol moiety (FIG. 11A), and a synthetic scheme of the steps for on-resin derivatization of a stapled peptide sequence (SEQ ID NO: 74) with a PEG-linked cholesterol moiety (FIG. 11B). [Figure 12] Figure 12 shows exemplary non-stapled SARS-CoV-2 HR2 peptide sequences (SEQ ID NOs: 5 and 6) and structurally stabilized SARS-CoV-2 HR2 peptide sequences (SEQ ID NOs: 7-21) generated by single i,i+7 staple scanning of the core template sequence (amino acids 1178-1199) with N- and C-terminal sequence extensions (e.g., amino acids 1168-1205) and C-terminal derivatization with PEG-thiocholesterol or PEG-cholesterol moieties of various PEG linker lengths, where X = (R)-α-(7'-octenyl)alanine; and X = (S)-α-(4'-pentenyl)alanine. [Figure 13] Figure 13 shows that the non-stapled HR2 sequence of SEQ ID NO:5 does not exhibit antiviral activity against the Wuhan-Hu-1 fluorescent pseudovirus corresponding to GenBank QHD43416.1 as measured by IXM microscopy, whereas C-terminal derivatization of SEQ ID NO:5 with a PEG4-thiocholesterol moiety to generate the peptide of SEQ ID NO:6 results in dose-responsive antiviral activity (pseudovirus: Wuhan-Hu-1; cells: 293T-ACE2; peptide: 2-fold serial dilutions starting at 5 μM; readout: 72 hours). [Figure 14]Figure 14 shows that the stapled HR2 peptide with on-resin added PEG4-thiocholesterol moieties (staple D, SEQ ID NO: 10) exhibits consistent and potent antiviral activity in pseudovirus assays whether cells are treated with the peptide (1 μM) before or after virus inoculation across a range of SARS-CoV-2 pseudovirus variants, whereas the corresponding non-stapled peptide (SEQ ID NO: 6) is ineffective when applied after virus inoculation and exhibits lower antiviral activity compared to the stapled sequence even when applied before virus inoculation with a range of SARS-CoV-2 pseudovirus variants (pseudoviruses: D614G B.1, Wuhan-Hu-1, B.1.526, B.1.427 / B.1.429, B.1.1.7; cells: 293T-ACE2; readout at 72 hours). [Figure 15] Figure 15 shows the differential antiviral activity of non-stapled and stapled HR2 peptides with PEG4-thiocholesterol moieties added on the resin, with the peptide of SEQ ID NO: 10 (staple D) exhibiting the most potent dose-responsive antiviral activity, followed by the non-stapled peptide of SEQ ID NO: 6. The stapled peptide of SEQ ID NO: 17 (staple K) was the least active in this pseudovirus assay (pseudovirus: B.1.526; cells: 293T-ACE2; 2-fold serial dilutions starting at 1 μM; readout: 72 hours). [Figure 16]Figure 16 shows the differential antiviral activity of a series of i,i+7 stapled HR2 peptides with PEG4-thiocholesterol moieties added on the resin. In a SARS-CoV-2 pseudovirus assay (pseudovirus: D614G B.1; cells: 293T-ACE2; peptide doses of 100, 300, 1000 nM; readout at 48 hours), peptides with SEQ ID NOs: 11, 14 and 15 (staples E, H, I, respectively) show little to no activity, peptides with SEQ ID NOs: 13 and 16 (staples G and J, respectively) display moderate activity, whereas peptide with SEQ ID NO: 10 (staple D) stands out as having uniquely strong activity among the various stapled HR2 peptides. [Figure 17] Figure 17 shows the differential antiviral activity of a series of i,i+7 stapled HR2 peptides with on-resin added PEG4-thiocholesterol moieties. In this SARS-CoV-2 live virus assay (live virus: USA-WA1 / 2020; cells: VeroB6; peptide dose range 4-1000 nM (i.e., each group of bars represents a dilution with a half-fold decrease in peptide concentration from bottom to top, i.e., 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.90625 nM), SEQ ID NOs: 11, 14 and 15. The peptides (staples E, H, I, respectively) showed little to no activity (as measured by the concentration of virus in μM on the x-axis), peptides of SEQ ID NO:13 and 16 (staples G and J) exhibited moderate activity, whereas peptide of SEQ ID NO:10 (staple D) stands out as having uniquely potent activity among the various stapled HR2 peptides, consistent with the pseudovirus assay results shown in FIG. 15. [Figure 18A]Figures 18A-18B show the differential antiviral activity of i,i+7 staple scans of the indicated HR2 peptide sequences (SEQ ID NOs: 7-21) with C-terminal PEG4-thiocholesterol moieties against SARS-CoV-2 (virus: live beta strain; cells: VeroB6; peptide dose 4 μM), with the most active peptide sequences pointed out by asterisks (Figure 18A). As summarized by a helical wheel drawing of the helical portion of the HR2 sequences, a distinct subset of staple positions results in potent antiviral activity (SEQ ID NOs: 10, 13, 17, 20) (Figure 18B). [Figure 18B] Figures 18A-18B show the differential antiviral activity of i,i+7 staple scans of the indicated HR2 peptide sequences (SEQ ID NOs: 7-21) with C-terminal PEG4-thiocholesterol moieties against SARS-CoV-2 (virus: live beta strain; cells: VeroB6; peptide dose 4 μM), with the most active peptide sequences pointed out by asterisks (Figure 18A). As summarized by a helical wheel drawing of the helical portion of the HR2 sequences, a distinct subset of staple positions results in potent antiviral activity (SEQ ID NOs: 10, 13, 17, 20) (Figure 18B). [Figure 19] FIG. 19 shows the antiviral activity of i,i+7 stapled HR2 peptide of SEQ ID NO: 10 with a PEG4-thiocholesterol moiety against GFP-expressing SARS-CoV-2 omicron variant B.1.1.529.1 (BA1) pseudovirus (cells; 293T-ACE2 cells; peptide 2-fold serial dilutions from 2000 nM; 48 hour readout). [Figure 20] Figure 20 shows the antiviral activity of i,i+7 stapled HR2 peptide of SEQ ID NO: 10 with PEG4-thiocholesterol moieties against live SARS-CoV-2 beta and delta strains (cells: VeroB6; peptide dose range 15-4000 nM (i.e., each group of bars represents a peptide dilution with decreasing concentration by a factor of two from bottom to top, i.e., 4000 nM, 2000 nM, 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM). [Figure 21] FIG. 21 shows the antiviral activity of the i,i+7 stapled HR2 peptide of SEQ ID NO: 13 with a PEG4-thiocholesterol moiety measured against GFP-expressing SARS-CoV pseudoviruses, including SARS-CoV-2 Wuhan-hu-1, SARS-CoV-2 omicron BA1.1.529.1 (BA1), SARS-CoV-2 omicron BA1.1.529.2 (BA2), and SARS-CoV-1 (Urbani) (cells; 293T-ACE2 cells; peptide 2-fold serial dilutions from 10 μM; 48 hour readout). [Figure 22] FIG. 22 shows the antiviral activity of the i,i+7 stapled HR2 peptide of SEQ ID NO: 20 with a PEG4-thiocholesterol moiety measured against GFP-expressing SARS-CoV pseudoviruses, including SARS-CoV-2 Wuhan-hu-1, SARS-CoV-2 omicron BA1.1.529.1 (BA1), SARS-CoV-2 omicron BA1.1.529.2 (BA2), and SARS-CoV-1 (Urbani) (cells; 293T-ACE2 cells; peptide 2-fold serial dilutions from 10 μM; 48 hour readout). [Figure 23] FIG. 23 shows the antiviral activity of i,i+7 stapled HR2 peptides of SEQ ID NO: 13 and SEQ ID NO: 20 bearing PEG4-thiocholesterol moieties measured against live SARS-CoV-2 beta strain virus (cells: VeroB6; 4 μM dosing). [Figure 24A]Figures 24A-24B show sequence maps of the i,i+7 stapled HR2 peptide of SEQ ID NO:10 (staple D spanning positions K1181 and E1188) highlighting amino acid positions that are alternatively in contact with the HR1 core or are non-binding (solvent exposed) (Figure 24A). Mutagenesis studies revealed a set of exemplary positions that are relatively unaffected by substitution of native residues with alanine when evaluated for SEQ ID NO:10 with a C-terminal PEG4 thiocholesterol moiety against GFP-expressing SARS-CoV-1 (Urbani) in a pseudovirus assay (cells; 293T-ACE2 cells; 4-fold serial dilution of peptide from 1.25 μM; 48 hour readout) (Figure 24B). [Figure 24B] Figures 24A-24B show sequence maps of the i,i+7 stapled HR2 peptide of SEQ ID NO:10 (staple D spanning positions K1181 and E1188) highlighting amino acid positions that are alternatively in contact with the HR1 core or are non-binding (solvent exposed) (Figure 24A). Mutagenesis studies revealed a set of exemplary positions that are relatively unaffected by substitution of native residues with alanine when evaluated for SEQ ID NO:10 with a C-terminal PEG4 thiocholesterol moiety against GFP-expressing SARS-CoV-1 (Urbani) in a pseudovirus assay (cells; 293T-ACE2 cells; 4-fold serial dilution of peptide from 1.25 μM; 48 hour readout) (Figure 24B). [Fig. 25A-B]Figures 25A-25E show the differential antiviral activity of a series of i,i+7 stapled HR2 peptides with PEG-thiocholesterol moieties of variable PEG length (n=3-8). In this pseudovirus assay, the peptide of SEQ ID NO:10 with a PEG8 linker moiety (staple D) exhibited the most potent dose-responsive activity (as measured by the number of green cells or percent GFP positive, shown on the x-axis) across a series of five SARS-CoV-2 variants, with PEG3 having relatively low activity among the constructs of variable PEG linker length (pseudoviruses / variants: Wuhan-Hu-1 (Figure 25A), B.1.427 / B.1.429 (Figure 25B), B.1.617.2 (Figure 25C), D614G (Figure 25D), and B.1.617.2 (Figure 25E). B.1 (Figure 25D), Brazilian variant P.1 (Figure 25E); Cells: 293T-ACE2; 2-fold serial dilutions starting at 1 μM (i.e., from bottom to top for each cluster of bars: 1 μM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM); Readout: 48 h). [Fig. 25C-D] Figures 25A-25E show the differential antiviral activity of a series of i,i+7 stapled HR2 peptides with PEG-thiocholesterol moieties of variable PEG length (n=3-8). In this pseudovirus assay, the peptide of SEQ ID NO:10 with a PEG8 linker moiety (staple D) exhibited the most potent dose-responsive activity (as measured by the number of green cells or percent GFP positive, shown on the x-axis) across a series of five SARS-CoV-2 variants, with PEG3 having relatively low activity among the constructs of variable PEG linker length (pseudoviruses / variants: Wuhan-Hu-1 (Figure 25A), B.1.427 / B.1.429 (Figure 25B), B.1.617.2 (Figure 25C), D614G (Figure 25D), and B.1.617.2 (Figure 25E). B.1 (Figure 25D), Brazilian variant P.1 (Figure 25E); Cells: 293T-ACE2; 2-fold serial dilutions starting at 1 μM (i.e., from bottom to top for each cluster of bars: 1 μM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM); Readout: 48 h). [Figure 25E]Figures 25A-25E show the differential antiviral activity of a series of i,i+7 stapled HR2 peptides with PEG-thiocholesterol moieties of variable PEG length (n=3-8). In this pseudovirus assay, the peptide of SEQ ID NO:10 with a PEG8 linker moiety (staple D) exhibited the most potent dose-responsive activity (as measured by the number of green cells or percent GFP positive, shown on the x-axis) across a series of five SARS-CoV-2 variants, with PEG3 having relatively low activity among the constructs of variable PEG linker length (pseudoviruses / variants: Wuhan-Hu-1 (Figure 25A), B.1.427 / B.1.429 (Figure 25B), B.1.617.2 (Figure 25C), D614G (Figure 25D), and B.1.617.2 (Figure 25E). B.1 (Figure 25D), Brazilian variant P.1 (Figure 25E); Cells: 293T-ACE2; 2-fold serial dilutions starting at 1 μM (i.e., from bottom to top for each cluster of bars: 1 μM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM); Readout: 48 h). [Figure 26] Figure 26 shows the differential antiviral activity of a series of i,i+7 stapled HR2 peptides (staple D, SEQ ID NO: 10) with PEG-thiocholesterol moieties of variable PEG length (n=3-8). In this SARS-CoV-2 live virus assay, the peptide of SEQ ID NO: 10 with a PEG8 linker moiety (staple D) exhibits the most potent dose-responsive activity, with PEG3 having relatively low activity among the constructs of variable PEG linker length (live virus: South African B.1.351: cells: VeroB6: peptide dose range 4-1000 nM; i.e., 2-fold serial dilutions, from bottom to top for each cluster of bars: 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.90625 nM). [Figure 27]FIG. 27 shows the differential antiviral activity of a series of i,i+7 stapled HR2 peptides of SEQ ID NO: 10 with PEG-thiocholesterol moieties of variable PEG length (n=0, 3-20) measured against GFP-expressing SARS-CoV-2 omicron variant B.1.1.529.1 (BA1) (cells: 293T-ACE2; 3-fold serial dilutions starting at 3.3 μM from bottom to top for each cluster of bars: 3.3 μM, 1.1 μM, 367 nM, 122 nM, 41 nM, 13 nM, 4.5 nM, 1.5 nM; readout: 48 hours). [Figure 28] FIG. 28 shows the differential antiviral activity of a series of i,i+7 stapled HR2 peptides of SEQ ID NO: 10 with PEG-thiocholesterol moieties of variable PEG length (n=0, 3-20) measured against GFP-expressing SARS-CoV-1 (Urbani) (cells: 293T-ACE2; 2-fold serial dilutions starting at 2 μM from bottom to top for each cluster of bars: 2 μM, 1 μM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.2 nM, 15.6 nM; readout: 48 hours). [Figure 29] Figure 29 shows the differential antiviral activity of a series of i,i+7 stapled HR2 peptides of SEQ ID NO: 10 bearing PEG-thiocholesterol moieties of variable PEG length (n=0, 3-20) measured against live SARS-CoV-2 beta strain virus (cells: VeroB6: peptide dose range 7.8-1000 nM; i.e., 2-fold serial dilutions, from bottom to top for each cluster of bars: 4000 nM, 2000 nM, 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.2 nM, 15.6 nM, 7.8 nM). [Diagram 30]FIG. 30 shows that a series of i,i+7 stapled HR2 peptides (staple D, SEQ ID NO: 10) with PEG-thiocholesterol moieties of variable PEG length (n=3-8) exhibit no nonspecific antiviral activity against a vesicular stomatitis virus (VSV) pseudovirus with a murine leukemia virus (MLV) core, whereas the corresponding non-stapled peptide (SEQ ID NO: 6) exhibits some nonspecific antiviral activity (pseudovirus: VSV; cells: 293T-ACE2; peptide, 2.5 μM; readout: 48 h). [Diagram 31] FIG. 31 shows that the non-stapled peptide derivatized with PEG4-thiocholesterol on resin (SEQ ID NO:6) exhibits somewhat improved antiviral activity in this SARS-CoV-2 live virus assay (live virus: South African B.1.351; cells: VeroB6; peptide dose range 4-1000 nM at 2-fold dilutions) compared to the non-stapled peptide derivatized in solution with GSGSGC-PEG4-cholesterol moieties (shown in SEQ ID NO:71). [Diagram 32] FIG. 32 shows that when applied to 293T-ACE2 cells in a pseudovirus assay, the non-stapled HR2 peptide (SEQ ID NO: 6) and the stapled HR2 peptide (SEQ ID NO: 10) derivatized with PEG4-thiocholesterol on the resin do not exhibit non-specific cytotoxicity, whereas the corresponding non-stapled HR2 peptide derivatized in solution with GSGSGC-PEG4-cholesterol moieties kills cells within the dosing range (pseudovirus: D614G B.1; cells: 293T-ACE2; 2-fold serial dilutions starting at 2500 nM (from bottom to top: 2500 nM, 1250 nM, 630 nM, 315 nM, 158 nM, 78 nM); readout: 48 hours). [Diagram 33]Figure 33 shows direct fluorescence polarization binding curves of SEQ ID NO: 10, C-terminally derivatized with PEG8-Chol and N-terminally derivatized with FITC-β-Ala instead of acetyl, combined with serial dilutions of recombinant five-helix bundle (5HB) lacking the third HR2 group. The addition of FITC-HR2 peptide completes the fusogenic six-helix bundle. (Peptide: 5 nM; serial dilutions of 5-HB protein from 1000 nM). [Diagram 34] FIG. 34 shows the differential antiviral activity of SEQ ID NO:10 derivatized at the C-terminus with PEG8-Chol against a panel of GFP-expressing SARS-CoV-2 variant pseudoviruses (293T-ACE2 cells; peptide serial dilutions from 1000 nM; 48 hour readout). [Diagram 35] FIG. 35 shows the differential antiviral activity of SEQ ID NO:10 derivatized at the C-terminus with PEG8-Chol against GFP-expressing SARS-CoV-2 omicron variant pseudoviruses (293T-ACE2 cells; peptide serial dilutions from 250 nM; 48 hour readout). [Diagram 36] FIG. 36 shows the differential antiviral activity of SEQ ID NO:10 derivatized at the C-terminus with PEG8-Chol against live SARS-CoV-2 beta and delta viruses (cells: Vero; peptide serial dilutions starting at 100 nM; peptide serial dilutions starting at 100 nM; 48 hour readout). [Figure 37] FIG. 37 shows the antiviral activity of SEQ ID NO:10 derivatized at the C-terminus with PEG8-Chol against GFP-expressing alphacoronavirus NL63 pseudovirus (293T-ACE2 cells; 3-fold serial dilutions of peptide from 10 μM; 48 hour readout). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0106] Detailed Description The present disclosure is based, inter alia, on the discovery that stabilized (e.g., stapled) peptides can be designed to selectively bind to one or more coronaviruses (e.g., betacoronaviruses, e.g., SARS-CoV-2). Thus, the present disclosure provides novel methods (e.g., approaches to convert cholesterol / thiocholesterol to carboxylic acid for on-resin derivatization) and compositions (e.g., peptides, stabilized peptides, peptide combinations; stabilized peptide combinations; peptide and stabilized peptide combinations; and cholesterol conjugates thereof) for treating, advancing the treatment of, and preventing infections with one or more coronaviruses (e.g., betacoronaviruses, e.g., SARS-CoV-2). Thus, the peptides and compositions disclosed herein can be used to prevent and / or treat COVID-19.

[0107] Coronavirus Peptides The amino acid sequences of exemplary coronavirus surface glycoproteins are provided in Figure 2. (See also GenBank Accession No.: QHD43416.1). An exemplary amino acid sequence of heptad repeat domain 1 (HR1) in SARS-CoV-2 S is shown in Figure 3 as SEQ ID NO:2. An exemplary amino acid sequence of heptad repeat domain 2 (HR2) in SARS-CoV-2 S is also shown in Figure 3 as SEQ ID NO:3.

[0108] Other exemplary amino acid sequences of HR2 in SARS-CoV-2 S are provided as SEQ ID NOs: 5 and 6 in Table 1.

[0109] In certain examples, the SARS-CoV-2 HR1 or HR2 peptides described herein (e.g., SEQ ID NOs: 5 and 6) may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) amino acid substitutions (relative to the amino acid sequence set forth in either one of SEQ ID NOs: 5 and 6), such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) conservative and / or non-conservative amino acid substitutions. In addition, in some examples, at least two (e.g., 2, 3, 4, 5, or 6) amino acids of SEQ ID NOs: 5 and 6 may be substituted with an α,α-disubstituted non-natural amino acid having an olefinic side chain. The type of substitutions made can be guided, for example, by an alignment of the HR2-like regions of the two SARS sequences, SARS-CoV-1 and SARS-CoV-2 (Figure 4). The guidance provided in the structurally stabilized peptides section below regarding amino acids that may be changed is similarly relevant to the peptides described herein. Residues that are not changed between SARS-CoV-1 and SARS-CoV-2 in the above alignment are either unmodified or substituted with a non-natural amino acid or a conservative amino acid. Residues in the alignment that are known to be replaced by conservative substitutions in the HR2-like regions of SARS-CoV-1 and SARS-CoV-2 (e.g., isoleucine in SARS-CoV-1 and SARS-CoV-2 replaced by leucine or methionine) are either not replaced or replaced by a conservative amino acid substitution. Residues that are not conserved between the HR2-like regions of SARS-CoV-1 and SARS-CoV-2 may be replaced with any amino acid. In some examples, residues that are conserved between the HR2-like regions of SARS-CoV-1 and SARS-CoV-2 but are located on the non-interacting face of the HR2 helix can be replaced with any amino acid (see, e.g., Figures 9, 24A and 24B). In some examples, the mutations in SEQ ID NO:6 are not introduced at positions 14 and 21 of SEQ ID NO:6. In some examples, the mutations in SEQ ID NO:6 are not introduced at positions 17 and 24 of SEQ ID NO:6.In some examples, the mutations in SEQ ID NO:6 are not introduced at positions 20 and 27 of SEQ ID NO:6. In some examples, the mutations in SEQ ID NO:6 are not introduced at positions 21 and 28 of SEQ ID NO:6. In some examples, the mutations in SEQ ID NO:6 are not introduced at positions 24 and 31 of SEQ ID NO:6.

[0110] A "conservative amino acid substitution" means that the amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) and acidic side chains and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine).

[0111] In some examples, the SARS-CoV-2 HR2 peptides described herein (e.g., SEQ ID NO: 5 or 6) may also contain 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 NO: 5 or 6) 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. In some examples, the SARS-CoV-2 HR2 peptides described herein (e.g., SEQ ID NO: 5 or 6) 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. In some examples, the SARS-CoV-2 HR2 peptides described herein (e.g., SEQ ID NO: 5 or 6) may also contain at least one, at least two, at least three, at least four, or at least five amino acids deleted at the C-terminus of the peptide.

[0112] In some cases, the peptide is lipidated. In some cases, the peptide is modified to include polyethylene glycol and / or cholesterol. In some cases, the peptide (e.g., SEQ ID NO: 5 or 6) has a nucleotide sequence of the following formula attached to the C-terminus of the peptide: [ka] In some instances, the above formula is attached to the C-terminus of the peptide by a modifiable carbon atom. In some instances, the peptide (e.g., SEQ ID NO: 5 or 6) comprises the following formula attached to the C-terminus of the peptide: [ka] Includes.

[0113] In some instances, the peptide (e.g., SEQ ID NO: 5 or 6) may comprise a nucleotide sequence of the following formula attached to the C-terminus of the peptide: [ka] In some instances, the above formula is attached to the C-terminus of the peptide by a modifiable carbon atom. In some instances, the peptide (e.g., SEQ ID NO: 5 or 6) comprises the following formula attached to the C-terminus of the peptide: [ka] Includes.

[0114] In some examples, n in the above formula is n=1 to 36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In some examples, n=4. In some examples, n=8. In some examples, the sulfur atom in the formula is replaced with an oxygen atom.

[0115] In some examples, the peptides described herein comprise an amino acid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 94% identical to the sequence set forth in SEQ ID NO: 6 (DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK). In some examples, the peptides (i) are alpha-helical; (ii) are protease resistant; (iii) inhibit fusion of SARS-CoV-2 with a host cell; and / or (iv) inhibit infection of cells by SARS-CoV-2. In some examples, the peptides inhibit infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays, and / or the structurally stabilized peptides prevent infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays.

[0116] In some examples, the peptide comprises an amino acid sequence having 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, or 2 substitutions, insertions, and / or deletions compared to SEQ ID NO: 6. In some examples, the peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 substitutions, insertions, and / or deletions compared to SEQ ID NO: 6. In some examples, the peptides having substitutions, insertions, and / or deletions compared to SEQ ID NO: 6 (i) are alpha helical; (ii) are protease resistant; (iii) inhibit fusion of SARS-CoV-2 with a host cell; and / or (iv) inhibit infection of a cell by SARS-CoV-2. In some examples, the peptide inhibits infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays, and / or the structurally stabilized peptide prevents infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays.

[0117] In some examples, the peptides are 36-50 (e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) amino acids in length. In examples where the peptides are modified to include polyethylene glycol and / or cholesterol, the peptides are 19-50 (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, 46, 47, 48, 49, or 50) amino acids in length. In some examples, the peptides range from 19-100, 38-100, 19-60, 38-60, 19-50, 38-50, 19-45, 38-45, 19-40 and 38-40 amino acids in length.

[0118] In some examples, the peptides (i) are alpha helical; (ii) are protease resistant; (iii) inhibit fusion of SARS-CoV-2 with a host cell; and / or (iv) inhibit infection of a cell by SARS-CoV-2. In some examples, the peptides inhibit infection of a cell by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays, and / or the structurally stabilized peptides prevent infection of a cell by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays.

[0119] Structurally stabilized peptides Disclosed herein are stapled SARS-CoV-2 peptides based on portions of the HR2 region. In some examples, the stapled SARS-CoV-2 peptides are based on portions of the SARS-CoV-2 HR2 region. (1168~1205) (DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK (SEQ ID NO: 6)). In some instances, the stapled SARS-CoV-2 peptide derived from SEQ ID NO:9 is 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-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; or SAH-SARS-CoV-2-O (e.g., SEQ ID NO: 7-21). [Table 1-1] [Table 1-2] [Table 1-3]

[0120] In Table 1, "8" = (R)-α-(7'-octenyl)alanine; "X" = (S)-α-(4'-pentenyl)alanine; and * = PEG-thiocholesterol or PEG-cholesterol moiety. In some cases, * = one of the two formulas shown below (wherein n=1 to 36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36)). In some examples, n=4. In some examples, n=8. * The two formulas shown by [ka] Includes.

[0121] In some cases, * The two formulas shown by [ka] Includes.

[0122] It should be understood that the above peptides can be modified to include additional amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids added) at the N- and / or C-terminus and / or have deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids deleted) at the N- and / or C-terminus. In some examples, the stapled SARS-CoV-2 peptide is derived from SEQ ID NO:4.

[0123] Please note that the bold and underlined sequences used herein (e.g., in Table 1) identify the amino acids that are stapled at the N-terminus and C-terminus, as well as the intervening sequences between the staples for each of the disclosed peptides. In some examples (e.g., SEQ ID NOs: 7-21), the structurally stabilized peptides are single-stapled peptides.

[0124] In some examples, SEQ ID NO:6 includes one or more variants. For example, when 25 or 29 positions of SEQ ID NO:6 are substituted, they are substituted with α,α-disubstituted non-natural amino acids having olefinic side chains, or are substituted with any amino acid. In some examples, when one or more of 1, 2, 5, 7, 8, 10, 12, 16, 17, 19, 23, 24, 26, 28, 30, 31, 33, and 36 positions of SEQ ID NO:6 are substituted, they are substituted with conservative amino acid substitutions. In some examples, when one or more of 3, 4, 6, 9, 11, 13, 15, 18, 20, 22, 27, 32, 34, 35, 37, or 38 positions of SEQ ID NO:6 are substituted, they are substituted with any amino acid.

[0125] The present disclosure encompasses each and every peptide and structurally stabilized peptide listed in Table 1, as well as variants thereof. In some examples, the structurally stabilized peptides are 19-50 (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, 46, 47, 48, 49, or 50) amino acids in length. In some examples, the structurally stabilized peptides are 19-60 (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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60) amino acids in length. In some examples, the structurally stabilized peptides range in length from 19-100, 38-100, 19-60, 38-60, 19-50, 38-50, 19-45, 38-45, 19-40 and 38-40 amino acids. In some examples, the structurally stabilized peptides have one or more (1, 2, 3, 4, 5, 6) of the properties listed below: (i) are alpha helical; (ii) are protease resistant; (iii) inhibit fusion of SARS-CoV-2 with a host cell; and / or (iv) inhibit infection of cells by SARS-CoV-2. In some examples, the structurally stabilized peptides inhibit infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays, and / or the structurally stabilized peptides prevent infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays.

[0126] In some examples, the structurally stabilized peptide comprises an amino acid sequence having 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, or 2 substitutions, insertions, and / or deletions compared to SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:17, or SEQ ID NO:20. In some examples, the structurally stabilized peptide having substitutions, insertions, and / or deletions compared to SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:17, or SEQ ID NO:20 (i) is alpha helical; (ii) is protease resistant; (iii) inhibits fusion of SARS-CoV-2 with a host cell; and / or (iv) inhibits infection of a cell by SARS-CoV-2. In some examples, the structurally stabilized peptide inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay, and / or the structurally stabilized peptide prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay. In some examples, the structurally stabilized peptide binds to a polypeptide comprising or consisting of the sequence of SEQ ID NO:78.

[0127] In some examples, disclosed herein are peptides that include 0-10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions compared to one of the single stapled peptides in Table 1 (e.g., SEQ ID NOs: 7-21). In some examples, disclosed herein are peptides that are at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 94%, at least 95%) identical to one of the single stapled peptides in Table 1 (e.g., SEQ ID NOs: 7-21). In some examples, the structurally stabilized peptides are 19-50 (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, 46, 47, 48, 49 or 50) amino acids in length. In some examples, the structurally stabilized peptides are 19-60 (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, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60) amino acids in length. In some examples, the structurally stabilized peptides range in length from 19-100, 38-100, 19-60, 38-60, 19-50, 38-50, 19-45, 38-45, 19-40, and 38-40 amino acids. In some examples, the structurally stabilized peptides have one or more (1, 2, 3, 4, 5, 6) of the properties listed below: (i) are alpha helical; (ii) are protease resistant; (iii) inhibit fusion of SARS-CoV-2 with a host cell; and / or (iv) inhibit infection of cells by SARS-CoV-2. In some examples, the structurally stabilized peptides inhibit infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays, and / or the structurally stabilized peptides prevent infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays.In some examples, the structurally stabilized peptide binds to a polypeptide comprising or consisting of the sequence of SEQ ID NO:78.

[0128] In some examples, the stapled peptide is a peptide comprising or consisting of either of the amino acid sequences of SEQ ID NO:5 or 6, provided that at least two (e.g., 2, 3, 4, 5, 6) amino acids of SEQ ID NO:5 or 6 are replaced with non-natural amino acids capable of forming staples. In some examples, the non-natural amino acids are α,α-disubstituted non-natural amino acids having olefinic side chains. In some examples, the stapled peptide is a peptide comprising or consisting of either of the amino acid sequences of SEQ ID NO:5 or 6, provided that at least two (e.g., 2, 3, 4, 5, 6) amino acids of SEQ ID NO:5 or 6 are replaced with non-natural amino acids capable of forming staples. In some examples, the non-natural amino acids are α,α-disubstituted non-natural amino acids having olefinic side chains. In some examples, the structurally stabilized peptide is 19-50 (e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50) amino acids in length. In some examples, the structurally stabilized peptides are 19-60 (e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60) amino acids in length. In some examples, the structurally stabilized peptides range in length from 19-100, 38-100, 19-60, 38-60, 19-50, 38-50, 19-45, 38-45, 19-40 and 38-40 amino acids. In some examples, the structurally stabilized peptides have one or more (1, 2, 3, 4, 5, 6) of the properties listed below: (i) are alpha helical; (ii) are protease resistant; (iii) inhibit fusion of SARS-CoV-2 with a host cell; and / or (iv) inhibit infection of cells by SARS-CoV-2.In some examples, the structurally stabilized peptide inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay, and / or the structurally stabilized peptide prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay. In some examples, the structurally stabilized peptide binds to a polypeptide comprising or consisting of the sequence of SEQ ID NO:78.

[0129] In some examples, peptides are disclosed herein that include 0-10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions compared to one of the unmodified peptides in Table 1 (e.g., SEQ ID NO: 5 or 6). 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 unmodified peptides in Table 1 (e.g., SEQ ID NO: 5 or 6). In some examples, the substitutions described herein are conservative substitutions. In some examples, the structurally stabilized peptides are 19-50 (e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) amino acids in length. In some examples, the structurally stabilized peptides are 19-60 (e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60) amino acids in length. In some examples, the structurally stabilized peptides range in length from 19-100, 38-100, 19-60, 38-60, 19-50, 38-50, 19-45, 38-45, 19-40 and 38-40 amino acids. In some examples, the structurally stabilized peptide has one or more (1, 2, 3, 4, 5, 6) of the properties listed below: (i) is alpha-helical; (ii) is protease resistant; (iii) inhibits fusion of SARS-CoV-2 with a host cell; and / or (iv) inhibits infection of cells by SARS-CoV-2. In some examples, the structurally stabilized peptide inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay, and / or the structurally stabilized peptide prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay. In some examples, the structurally stabilized peptide binds to a polypeptide comprising or consisting of the sequence of SEQ ID NO:78.

[0130] In some examples, any of the substitutions described herein can be conservative substitutions. In some examples, any of the substitutions described herein are non-conservative substitutions.

[0131] In some examples, unnatural amino acids that may be used as stapling amino acids are (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'-propenyl)glycine; and α,α-bis(7'-octenyl)glycine.

[0132] In some embodiments, the internal staples replace the side chains of two amino acids, i.e., each staple is between two amino acids that are separated by, for example, six amino acids. In some embodiments, the amino acids forming the staples are at each of the i and i+7 positions of the staple. For example, if a peptide has the sequence ...X1, X2, X3, X4, X5, X6, X7, X8, X9..., the bridge between X1 and X8 (i and i+7) is a useful carbohydrate stapled form of the peptide. The use of i and i+4 staples, multiple bridges (e.g., 2, 3, 4, or more), or tandem stitches are also contemplated. Additional description regarding the production and use of carbohydrate-stapled peptides can be found, for example, in U.S. Patent Application Publication Nos. 2012 / 0172285, 2010 / 0286057 and 2005 / 0250680, the contents of all of which are incorporated by reference herein in their entireties.

[0133] "Peptide stapling" is a term derived from the synthesis methodology that uses ring-closing metathesis (RCM) reaction to covalently bond (e.g., "stapled together") two olefin-containing side chains (e.g., crosslinkable side chains) present in a peptide chain to form a crosslinked ring (e.g., see Blackwell et al., J. Org. Chem., 66:5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). For example, the structural stabilization can be achieved by stapling the peptide (e.g., see Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are incorporated herein by reference in their entirety). In some cases, the staple is a carbohydrate staple.

[0134] In some examples, the staples used herein are lactam staples; UV cycloaddition staples; oxime staples; thioether staples; double click staples; bislactam staples; bisarylation staples; or a combination of any two or more of these. The stabilized peptides described herein include stapled peptides as well as peptides containing multiple staples, or any other chemical strategy for structural reinforcement (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 al., Chem Commun (Camb). 2009 Oct. 2009). 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 which is incorporated by reference in its entirety.

[0135] A peptide is "structurally stabilized" in that it maintains its native secondary structure. For example, stapling allows peptides that tend to have α-helical secondary structures to maintain their native α-helical conformation. This secondary structure can increase the peptide's resistance to proteolytic cleavage and heat, as well as increase target binding affinity, hydrophobicity, plasma membrane binding, and / or cell permeability. Thus, the stapled (crosslinked) peptides described herein have improved biological activity and pharmacology compared to the corresponding non-stapled (non-crosslinked) peptides.

[0136] In certain instances, modifications to introduce structural stabilization (e.g., internal crosslinks, e.g., stapling) into the SARS-CoV-2 HR2 peptides described herein may be located on a SARS-CoV-2 HR2 helical face that does not interact with the recombinant five-helix bundle of SARS-CoV-2 or the corresponding native fusion machinery. Alternatively, modifications to introduce stabilization (e.g., internal crosslinks, e.g., stapling) into the SARS-CoV-2 HR2 peptides described herein may be located on a SARS-CoV-2 HR2 helical face that interacts with the five-helix bundle of SARS-CoV-2. In some instances, the SARS-CoV-2 HR2 peptides described herein are stabilized by introducing staples (e.g., carbohydrate staples) at the interface between the interacting and non-interacting helical faces of the SARS-CoV-2 HR2 protein. In some cases, the SARS-CoV-2 HR2 peptides described herein are stabilized by introducing a staple (e.g., a carbohydrate staple) or staples at the interface between the hydrophobic interacting and non-interacting surfaces of the SARS-CoV-2 HR2 protein.

[0137] In some examples, modifications to introduce structural stabilization (e.g., internal crosslinks, e.g., stapling) into the SARS-CoV-2 HR2 peptides described herein include modifications to the following residues: (i) 11 and 18 of SEQ ID NO:6; (ii) 12 and 19 of SEQ ID NO:6; (iii) 13 and 20 of SEQ ID NO:6; (iv) 14 and 21 of SEQ ID NO:6; (v) 15 and 22 of SEQ ID NO:6; (vi) 16 and 23 of SEQ ID NO:6; (vii) 17 and 24 of SEQ ID NO:6; (viii) 18, and 25 of SEQ ID NO:6; (ix) 19 and 26 of SEQ ID NO:6; (x) 20 and 27 of SEQ ID NO:6; (xi) 21 and 28 of SEQ ID NO:6; (xii) 22 and 29 of SEQ ID NO:6; (xiii) 23 and 30 of SEQ ID NO:6; (xiv) 24 and 31 of SEQ ID NO:6; or (xv) 25 and 32 of SEQ ID NO:6 located at the amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to

[0138] In certain examples, the SARS-CoV-2 HR2 peptides described herein (e.g., SEQ ID NO: 5 or 6) may also contain one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions (compared to the amino acid sequence set forth in any one of SEQ ID NO: 5 or 6), such as one or more (e.g., 1, 2, 3, 4, or 5) conservative and / or non-conservative amino acid substitutions. In some examples, the SARS-CoV-2 HR2 peptides described herein (e.g., SEQ ID NO: 5 or 6) may also contain 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 NO: 5 or 6) 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.

[0139] In some examples, the N-terminal aspartic acid in any one of the peptides disclosed herein is replaced with -N(H)C(O)-(C1-4 alkyl). In some examples, the carboxylic acid group of the C-terminal lysine in any one of the peptides disclosed herein is replaced with C(O)NH2. In some examples, the N-terminal aspartic acid in SEQ ID NO:6, SEQ ID NO:10, or SEQ ID NO:20 is replaced with -N(H)C(O)-(C1-4 alkyl). In some examples, the carboxylic acid group of the C-terminal lysine in SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:17, or SEQ ID NO:20 is replaced with C(O)NH2.

[0140] In some examples, the C-terminal lysine for any of the peptides disclosed herein is replaced with ornithine (e.g., L-ornithine), L-2,3-diaminopropionic acid, L-2,7-diaminoheptanoic acid, diaminobutyric acid (e.g., L-2,4-diaminobutyric acid, an amino acid with an alpha carbon amine, or a diamine.

[0141] In one embodiment, the conformationally stabilized SARS-CoV-2 HR2 peptide has the formula (I): [ka] or a pharma- ceutically acceptable salt thereof, Each R1 and R2 is independently H, or C1-C 10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; R3 is alkyl, alkenyl, alkynyl; [R4-K-R4] n each of which is substituted with 0-6 R5; R4 is alkyl, alkenyl, or alkynyl; R5 is halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R6, a fluorescent moiety, or a radioisotope; K is O, S, SO, SO2, CO, CO2, CONR6, or [ka] and; R6 is H, alkyl, or a therapeutic agent; n is an integer from 1 to 4; x is an integer from 2 to 10; each y is independently an integer from 0 to 100; z is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and each Xaa is independently an amino acid. including; A conformationally stabilized peptide, wherein the peptide (i) inhibits fusion of SARS-CoV-2 with a host cell; and / or (ii) inhibits infection of a cell by SARS-CoV-2. In some examples, the structurally stabilized peptide inhibits infection of a cell by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay, and / or the structurally stabilized peptide prevents infection of a cell by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay. In some examples, the structurally stabilized peptide binds to a polypeptide comprising or consisting of the sequence of SEQ ID NO:78.

[0142] In another embodiment, the conformationally stabilized SARS-CoV-2 HR2 peptide comprises formula (IA): [ka] or a pharma- ceutically acceptable salt thereof. In some instances, the conformationally stabilized peptide is of formula (IA):

[0143] In some examples, R3 of formula (IA) is alkenylene. In some examples, [Xaa] wThe amino group of the N-terminal aspartic acid in is replaced with -N(H)C(O)-(C1-4 alkyl), and, if necessary, [Xaa] y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0144] In another aspect, the conformationally stabilized peptide comprises [ka] or a pharma- ceutically acceptable salt thereof. In one embodiment, the structurally stabilized peptide comprises formula (Ia). In some examples, [Xaa] of formula (Ia) w The amino group of the N-terminal aspartic acid in 1~4 [Xaa] is optionally replaced by an alkyl group. y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0145] In some instances, the structurally stabilized peptide comprises: [ka] or a pharma- ceutically acceptable salt thereof.

[0146] In some cases, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 [Xaa] is optionally replaced by an alkyl group. y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0147] In some examples, R3 for any one of formulas (I)-(Id) (i.e., I, Ia, Ib, Ic, Id) is C 7~15 In some embodiments, R3 is C 9~13 In some embodiments, R3 is C 11In some examples, R3 is -(CH2) 3~7 -CH=CH-(CH2) 3~7 In some examples, R3 is -(CH2) 5~7 -CH=CH-(CH2) 3~4 In some examples, R3 is -(CH2)6-CH=CH-(CH2)3-.

[0148] In some cases, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 In some cases, [Xaa] is replaced by w In some cases, the amino group of the N-terminal aspartic acid in [Xaa] is replaced by -N(H)C(O)CH3. y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0149] In another embodiment, there is provided a structurally stabilized peptide conjugate comprising or consisting of formula II, [ka] Disclosed herein are structurally stabilized peptide conjugates defined by:

[0150] In some examples, R3 of formula II is alkenylene. In some examples, R4 is ** -C(O)-(C 2~6 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~6 alkylene)-R6, where ** is [Xaa] y In some embodiments, R5 is a hydrogen or a C 1~4 It is an alkyl.

[0151] In some examples, R6 is one of the following: [ka] and optionally each of these is replaced by t occurrences of R7.

[0152] In some examples, R7, independently for each occurrence, is C 1~3 Alkyl, hydroxyl or C 1~3 In some examples, m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, t is 0, 1, 2, or 3.

[0153] In some cases, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 [Xaa] is optionally replaced by an alkyl group. y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0154] In some instances, the structurally stabilized peptide comprises: [ka] or a pharma- ceutically acceptable salt thereof (wherein, as appropriate, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 In some embodiments, the compound of formula IIa is defined as follows: y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0155] In some instances, the structurally stabilized peptide comprises: [ka] [ka] or a pharma- ceutically acceptable salt thereof. w The amino group of the N-terminal aspartic acid in 1~4 In some cases, [Xaa] is replaced by y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0156] In some examples, R3 of formula (II), (IIa), (IIb), (IIc), or (IId) is C 7~15 In some embodiments, R3 is C 9~13 In some embodiments, R3 is C 11 In some examples, R3 is -(CH2) 3~7 -CH=CH-(CH2) 3~7 In some examples, R3 is -(CH2) 5~7 -CH=CH-(CH2) 3~4 In some examples, R3 is -(CH2)6-CH=CH-(CH2)3-.

[0157] In some examples, a structurally stabilized peptide conjugate comprising or consisting of formula III, [ka] or a pharma- ceutically acceptable salt thereof. ** -C(O)-(C 2~6 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~6 alkylene)-R6, where ** is [Xaa] y In some embodiments, R5 is a hydrogen or a C 1~4 In some examples, R6 is one of the following: [ka] and optionally each of these is replaced by t occurrences of R7.

[0158] In some examples, R7, independently for each occurrence, is C 1~3 Alkyl, hydroxyl or C 1~3 In some examples, m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, p is 2, 3, 4, 5, 6, 7, or 8. In some examples, z is 2, 3, 4, 5, or 6. In some examples, t is 0, 1, 2, or 3. In some examples, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 In some cases, [Xaa] is replaced by y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0159] In some instances, the structurally stabilized peptide comprises: [ka] or a pharma- ceutically acceptable salt thereof.

[0160] In some cases, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 [Xaa] is optionally replaced by an alkyl group. y In some examples, R4 is ** -C(O)-(C 2~3 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~2 alkylene)-R6, where ** is [Xaa] yIn some embodiments, R4 is the point of attachment to the amino group in the side chain of the C-terminal lysine in ** -C(O)-(CH2CH2)-[O-CH2CH2] m -N(R5)C(O)-(CH2)-R6, where ** is [Xaa] y In some cases, [Xaa] is the point of attachment to the amino group in the side chain of the C-terminal lysine in w The amino group of the N-terminal aspartic acid in 1~4 In some cases, [Xaa] is replaced by w In some cases, the amino group of the N-terminal aspartic acid in [Xaa] is replaced by -N(H)C(O)CH3. y The carboxylic acid group of the C-terminal lysine in is replaced by -C(O)NH2.

[0161] In another embodiment, the compound of formula (IV): [ka] or a pharma- ceutically acceptable salt thereof.

[0162] In some examples, R4 is -C(O)-(C 2~6 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~6 In some examples, R5 is hydrogen or C 1~4 In some cases, R 6 is one of the following: [ka] and optionally each of these is replaced by t occurrences of R7.

[0163] In some examples, R7, independently for each occurrence, is C 1~3 Alkyl, hydroxyl or C 1~3In some examples, R8 represents -C(O)-(C 1~4 In some examples, m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, p is 2, 3, 4, 5, 6, 7, or 8. In some examples, z is 2, 3, 4, 5, or 6. In some examples, t is 0, 1, 2, or 3.

[0164] In some examples, the structurally stabilized peptide is represented by formula IVa, or a pharma- ceutically acceptable salt thereof, wherein formula IVa is [ka] It is defined by:

[0165] In some examples, the structurally stabilized peptide is represented by formula IVb, or a pharma- ceutically acceptable salt thereof, wherein formula IVb is: [ka] It is defined by:

[0166] In some examples, the structurally stabilized peptide is represented by formula IVc, or a pharma- ceutically acceptable salt thereof, wherein formula IVc is: [ka] It is defined by:

[0167] In some examples, there is provided a structurally stabilized peptide conjugate represented by formula V or a pharma- ceutically acceptable salt thereof, wherein formula V is [ka] Disclosed herein is a structurally stabilized peptide conjugate defined by:

[0168] In some examples, R4 is -C(O)-(C 2~6 Alkylene)-[O-CH2CH2]m -N(R5)C(O)-(C 1~6 In some examples, R5 is hydrogen or C 1~4 In some cases, R 6 is one of the following: [ka] and optionally each of these is replaced by t occurrences of R7.

[0169] In some examples, R7, independently for each occurrence, is C 1~3 Alkyl, hydroxyl or C 1~3 In some examples, R8 represents -C(O)-(C 1~4 In some cases, [Xaa] is w is DISGINASVVNIQ (SEQ ID NO: 35), [Xaa] x is EIDRLN (SEQ ID NO: 36), and [Xaa] z is VAKNLNESLIDLQELG (SEQ ID NO: 77). In some examples, m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, t is 0, 1, 2, or 3.

[0170] In some examples, R8 is -C(O)CH3. In some examples, R4 is -C(O)-(C 2~3 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~2 In some examples, R4 is -C(O)-(CH2CH2)-[O-CH2CH2] m -N(R5)C(O)-(CH2)-R6. In some examples, R5 is hydrogen. In some examples, R6 is replaced by t occurrences of R7. [ka] It is.

[0171] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0172] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0173] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0174] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0175] In some instances, R6 is replaced by t occurrences of R7. [ka] It is.

[0176] In some examples, t is 0. In some examples, m is 4. In some examples, m is 8.

[0177] In another embodiment, there is provided a structurally stabilized peptide conjugate represented by formula VI, or a pharma- ceutically acceptable salt thereof, wherein formula VI is [ka] Disclosed herein is a structurally stabilized peptide conjugate defined by:

[0178] In some examples, R4 is -C(O)-(CH2CH2)-[O-CH2CH2]8-N(H)C(O)-(CH2)-R6. In some examples, R6 is [ka] It is.

[0179] In some cases, [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35), [Xaa] x is EIDRLN (SEQ ID NO: 36), and [Xaa] z is VAKNLNESLIDLQELG (sequence number 77).

[0180] In some embodiments, [Xaa] of formula (I) w [Xaa] of formula (I) x and [Xaa] of formula (I), (Ia), (II) and (IIa). y Each of the [Xaa] in construct 1 of Table 2 is as described for any one of constructs 1 to 15. For example, [Xaa] in construct 1 of Table 2 is w , [Xaa] x , and [Xaa] y For stabilizing peptides containing, [Xaa] w , [Xaa] x , and [Xaa] y are DISGINASVV (SEQ ID NO: 26), IQKEID (SEQ ID NO: 27), and LNEVAKNLNESLIDLQELGK (SEQ ID NO: 28), respectively. w , [Xaa] x , and [Xaa] y For stabilizing peptides containing, [Xaa] w , [Xaa] x , and [Xaa] y are DISGINASVVN (SEQ ID NO: 29), QKEIDR (SEQ ID NO: 30), and NEVAKNLNESLIDLQELGK (SEQ ID NO: 31), respectively. [Table 2-1] [Table 2-2]

[0181] In certain instances, the sequences shown in Table 2 above may have at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid substitution or deletion. The SARS-CoV-2 HR2 peptide may include any amino acid sequence described herein.

[0182] In some examples, formula (I) comprising the sequence shown in Table 2 above may have one or more of the properties listed below: (i) binds to recombinant SARS-CoV-2 five-helix bundle S protein and / or the corresponding native fusion machinery; (ii) is alpha-helical; (iii) is protease resistant; (iv) inhibits fusion of SARS-CoV-2 with a host cell; and / or (v) inhibits infection of cells by SARS-CoV-2. In some examples, the compound binds to a polypeptide comprising or consisting of the sequence of SEQ ID NO:78.

[0183] The tether of formula (I) may be 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 The tethered amino acids may be alpha disubstituted (e.g., C1-C3 or methyl).

[0184] 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 each independently H or C1-C6 alkyl. In some examples of Formula (I), R1 and R2 are each independently C1-C3 alkyl. In some examples of Formula (I), at least one of R1 and R2 is methyl. For example, R1 and R2 can both 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 In some examples of formula (I), R3 is an alkenyl (e.g., C8 alkenyl) and x is 3. In some examples of formula (I), x is 6 and R3 is a C 11 In some examples, R3 is -CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-.

[0185] In one aspect, the conformationally stabilized COVID-19 HR2 peptide comprises formula (I), or a pharma- ceutically 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 is substituted or unsubstituted; each R3 is independently an alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and (a) Each [Xaa] w is DISGINASVV (SEQ ID NO: 26), and each [Xaa] x is IQKEID (SEQ ID NO: 27), and each [Xaa] y is LNEVAKNLNESLIDLQELGK (SEQ ID NO:28); (b) Each [Xaa] w is DISGINASVVN (SEQ ID NO: 29), and each [Xaa] x is QKEIDR (SEQ ID NO: 30), and each [Xaa] y is NEVAKNLNESLIDLQELGK (SEQ ID NO:31); (c) Each [Xaa] w is DISGINASVVNI (SEQ ID NO: 32), and each [Xaa] x is KEIDRL (SEQ ID NO: 33), and each [Xaa] y is EVAKNLNESLIDLQELGK (SEQ ID NO:34); (d) Each [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35), and each [Xaa] x is EIDRLN (SEQ ID NO: 36), and each [Xaa] y is VAKNLNESLIDLQELGK (SEQ ID NO:37); (e) Each [Xaa] w is DISGINASVVNIQK (SEQ ID NO: 38), and each [Xaa] x is IDRLNE (SEQ ID NO: 39), and each [Xaa] y is AKNLNESLIDLQELGK (SEQ ID NO: 40); (f) Each [Xaa] w is DISGINASVVNIQKE (SEQ ID NO: 41), and each [Xaa] x is DRLNEV (SEQ ID NO: 42), each [Xaa] y is KNLNESLIDLQELGK (SEQ ID NO: 43); (g) Each [Xaa] w is DISGINASVVNIQKEI (SEQ ID NO: 44), and each [Xaa] x is RLNEVA (SEQ ID NO: 45), and each [Xaa] y is NLNESLIDLQELGK (SEQ ID NO: 46); (h) Each [Xaa] w is DISGINASVVNIQKEID (SEQ ID NO: 47), and each [Xaa] xis LNEVAK (SEQ ID NO: 48), and each [Xaa] y is LNESLIDLQELGK (SEQ ID NO:49); (i) Each [Xaa] w is DISGINASVVNIQKEIDR (SEQ ID NO: 50), and each [Xaa] x is NEVAKN (SEQ ID NO: 51), and each [Xaa] y is NESLIDLQELGK (SEQ ID NO:52); (j) Each [Xaa] w is DISGINASVVNIQKEIDRL (SEQ ID NO: 53), and each [Xaa] x is EVAKNL (SEQ ID NO: 54), and each [Xaa] y is ESLID LQELGK (SEQ ID NO:55); (k) Each [Xaa] w is DISGINASVVNIQKEIDRLN (SEQ ID NO: 56), and each [Xaa] x is VAKNLN (SEQ ID NO: 57), and each [Xaa] y is SLIDLQELGK (SEQ ID NO:58); (l) Each [Xaa] w is DISGINASVVNIQKEIDRLNE (SEQ ID NO: 59), and each [Xaa] x is AKNLNE (SEQ ID NO: 60), and each [Xaa] y is LIDLQELGK (SEQ ID NO:61); (m) Each [Xaa] w is DISGINASVVNIQKEIDRLNEV (SEQ ID NO: 62), and each [Xaa] x is KNLNES (SEQ ID NO: 63), and each [Xaa] y is IDLQELGK (SEQ ID NO:64); (n) each [Xaa] w is DISGINASVVNIQKEIDRLNEVA (SEQ ID NO: 65), and each [Xaa] x is NLNESL (SEQ ID NO: 66), and each [Xaa] y is DLQELGK (SEQ ID NO:67); (o) Each [Xaa]w is DISGINASVVNIQKEIDRLNEVAK (SEQ ID NO: 68), and each [Xaa] x is LNESLI (SEQ ID NO: 69), and each [Xaa] y is LQELGK (SEQ ID NO: 70); The conformationally stabilized SARS-CoV-2 HR2 peptide binds to recombinant SARS-CoV-2 five-helix bundle S protein and / or the corresponding native fusion machinery. In some examples, R1 is an alkyl. In some examples, R1 is a methyl group. In some examples, R3 is an alkyl. In some examples, R3 is a methyl group. In some examples, R2 is an alkenyl. In some examples, z is 1.

[0186] In another embodiment of formula (I), the two alpha, alpha disubstituted stereocenters are both in the R or S configuration (e.g., an i,i+4 bridge), or one stereocenter is R and the other is S (e.g., an i,i+7 bridge). Thus, formula (I) in this case is [ka] It is depicted as follows.

[0187] The C' and C" disubstituted stereocenters may both be in the R configuration or they may both be in the S configuration. When x in formula (I) is 6, the C' disubstituted stereocenter is in the R configuration and the C" disubstituted stereocenter is in the S configuration. The R3 double bond in formula (I) may be in the E or Z stereochemical configuration.

[0188] In some examples of formula (I), R3 is [R4-K-R4] n and R4 is a straight chain alkyl, alkenyl, or alkynyl.

[0189] As used herein, the term "alkyl," used alone or in combination with other terms, refers to a saturated hydrocarbon group, which may be linear or branched. In some embodiments, the alkyl group contains 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, n-heptyl, and the like. In some embodiments, the alkyl group is methyl, ethyl, or propyl. The term "alkylene" refers to a linking alkyl group.

[0190] As used herein, "alkenyl," 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 2-6 or 2-4 carbon atoms. Exemplary alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like. The term "alkenylene" refers to a linking alkenyl group (e.g., -CH=CH- or -CH2CH2CH=CHCH2CH2- group).

[0191] As used herein, "alkynyl," used alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Exemplary alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2-6 or 2-4 carbon atoms.

[0192] As used herein, "alkynyl," used alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Exemplary alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2-6 or 2-4 carbon atoms.

[0193] As used herein, the term "cycloalkylalkyl," employed alone or in combination with other terms, refers to a group of formula cycloalkyl-alkyl-. In some embodiments, the alkyl portion has 1 to 4, 1 to 3, 1 to 2, or 1 carbon atom. In some embodiments, the alkyl portion is methylene. In some embodiments, the cycloalkyl portion 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 portion is monocyclic. In some embodiments, the cycloalkyl portion is C 3~7 It is a monocyclic cycloalkyl group.

[0194] As used herein, the term "heteroarylalkyl," used alone or in combination with other terms, refers to a group of formula heteroaryl-alkyl-. In some embodiments, the alkyl portion has 1-4, 1-3, 1-2, or 1 carbon atoms. In some embodiments, the alkyl portion is methylene. In some embodiments, the heteroaryl portion 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 portion has 5-10 carbon atoms.

[0195] As used herein, the term "substituted" means that a hydrogen atom is replaced with a non-hydrogen group. It should be understood that substitution at a given atom is limited by valence.

[0196] As used herein, "halo" or "halogen," employed alone or in combination with other terms, includes fluoro, chloro, bromo, and iodo. In some embodiments, halo is F or Cl.

[0197] In some embodiments, the disclosure features a structurally stabilized (e.g., stapled) peptide comprising the amino acid sequence of any one of SEQ ID NOs: 5 or 6 (or a modified version thereof), in which the side chains of two amino acids separated by six amino acids have been replaced with internal staples.

[0198] The stapled peptides can be 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 amino acids in length. In certain embodiments, the stapled peptides are 19-45 amino acids in length (i.e., 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). In certain embodiments, the stapled peptides are 36-45 amino acids in length (i.e., 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45). In certain embodiments, the stapled peptides are 38-45 amino acids in length (i.e., 38, 39, 40, 41, 42, 43, 44, or 45). In certain embodiments, the stapled peptides are 36-42 amino acids in length (i.e., 36, 37, 38, 39, 40, 41, 42) amino acids. In certain embodiments, the stapled peptides are 38-42 amino acids in length (i.e., 38, 39, 40, 41, 42) amino acids. In certain embodiments, the stapled peptides are 36 amino acids in length. In another particular embodiment, the stapled peptides are 38 amino acids in length. Exemplary COVID-19 HR2 stapled peptides are shown in Tables 1 and 2 and described by Formula (I). In one embodiment, the COVID-19 HR2 stapled peptide comprises or consists of a stapled version of the amino acid sequence of any one of SEQ ID NOs: 7-21 (e.g., the product of a ring-closing metathesis reaction performed on a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 7-21, respectively). In one embodiment, the SARS-CoV-2 HR2 stapled peptide comprises or consists of a stapled version of the amino acid sequence of SEQ ID NO: 5 (e.g., the product of a ring-closing metathesis reaction performed on a peptide comprising the amino acid sequence of SEQ ID NO: 5).In one embodiment, the SARS-CoV-2 HR2 stapled peptide comprises or consists of a stapled version of the amino acid sequence of SEQ ID NO:6 (e.g., the product of a ring-closing metathesis reaction performed on a peptide comprising the amino acid sequence of SEQ ID NO:6).

[0199] In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 11 and 18 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 12 and 19 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 13 and 20 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 14 and 21 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 15 and 22 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 16 and 23 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 17 and 24 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 18 and 25 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 19 and 26 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 20 and 27 of SEQ ID NO:6. In certain embodiments, the two amino acids, each separated by six amino acids, are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 21 and 28 of SEQ ID NO:6.In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 22 and 29 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 23 and 30 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 24 and 31 of SEQ ID NO:6. In certain embodiments, the two amino acids separated by six amino acids are at amino acid positions in the SARS-CoV-2 HR2 peptide corresponding to positions 25 and 32 of SEQ ID NO:6.

[0200] Although a hydrocarbon tether is provided herein, other tethers can be used in the structurally stabilized SARS-CoV-2 HR2 peptides described herein. For example, the tether can include one or more of an ether, a thioether, an ester, an amine, or an amide, or a triazole moiety. In some cases, naturally occurring amino acid side chains can be incorporated into the tether. For example, the tether can be coupled to a functional group, such as a hydroxyl on a serine, a thiol on a cysteine, a primary amine on a lysine, an acid on an aspartate or glutamate, or an amide on an asparagine or glutamine. Thus, rather than using a tether made by coupling two non-naturally occurring amino acids, it is possible to create a tether using a naturally occurring amino acid. It is also possible to use a single non-naturally occurring amino acid with a naturally occurring amino acid. Triazole-containing (e.g., 1,4 triazole or 1,5 triazole) bridges can be used (see, e.g., Kawamoto et al. 2012 Journal of Medicinal Chemistry 55:1137; WO2010 / 060112).Additionally, other methods of performing different types of stapling are known in the art and can be used with the SARS-CoV-2 HR2 peptides 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: JR Kumita 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: JC Phelan et al.,, J. Am. Chem. Soc., 119:455-460 (1997); and Bis-arylation stapling: AM Spokoyny et al., J. Am. Chem. Soc., 135:5946-5949 (2013)).

[0201] It is further envisioned that the length of the tether can be varied: for example, if it is desired to impose a relatively high degree of constraint on the secondary alpha-helical structure, a shorter tether length can be used, whereas in some instances it may be desirable to impose less constraint on the secondary alpha-helical structure, and therefore a longer tether may be desirable.

[0202] Additionally, although tethers spanning amino acids i through i+7 are provided herein to provide tethers that reside primarily on a single face of the alpha helix, tethers can be synthesized spanning any combination of amino acid numbers and can also be used in combination to incorporate multiple tethers.

[0203] In some examples, the hydrocarbon tethers (i.e., bridges) described herein can be further manipulated. In one example, the double bond of a hydrocarbon alkenyl tether (e.g., as synthesized using ruthenium-catalyzed ring-closing metathesis (RCM)) can be oxidized (e.g., by epoxidation, aminohydroxylation, or dihydroxylation) to give one of the following compounds: [ka]

[0204] Either the epoxide moiety or one of the free hydroxyl moieties can be further functionalized. For example, the epoxide can be treated with a nucleophile, which gives an additional functional group that can be used, for example, to attach a therapeutic agent. Alternatively, such derivatization can be achieved by synthetic manipulation of the amino or carboxy terminus of the peptide, or via an amino acid side chain. Other agents, for example, agents that facilitate the entry of the peptide into cells, can be attached to the functionalized tether.

[0205] In some instances, alpha disubstituted amino acids are used in peptides to improve the stability of alpha helical secondary structures, however, alpha disubstituted amino acids are not required and instances in which mono alpha substitutes are used (e.g., in tethering amino acids) are also envisioned.

[0206] The structurally stabilized (e.g., stapled) peptides may include drugs, toxins, derivatives of polyethylene glycol, second peptides, carbohydrates, etc. When a polymer or other agent is linked to the structurally stabilized (e.g., stapled) peptide, it may be desirable for the composition to be substantially homogeneous.

[0207] The addition of polyethylene glycol (PEG) molecules can improve the pharmacokinetic and pharmacodynamic properties of peptides. For example, PEGylation can reduce renal clearance and result in more stable plasma concentrations. PEG is a water-soluble polymer and has the formula: XO--(CH2CH2O) n They may be represented in peptide-linked form as --CH2CH2--Y, where n is 2 to 10,000 and X is H or a terminal modification, e.g., C 1~4 is alkyl; Y is an amide, carbamate or urea linkage to an amine group of the peptide (including but not limited to the epsilon amine of lysine, or the N-terminus). Y may also be a maleimide linkage to a thiol group (including but not limited to the thiol group of cysteine). Other methods for directly or indirectly linking PEG to peptides are known to those of skill in the art. PEG may be linear or branched. Various forms of PEG, including various functionalized derivatives, are commercially available.

[0208] PEG, as used herein, in some examples, functions as a linker or spacer between one of the peptides (e.g., a stapled peptide; e.g., SEQ ID NOs: 7-21) and the cholesterol or thiocholesterol moiety. In some examples, the PEG molecule comprises the following formula, where n=1-36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36): [ka]

[0209] In some examples, the PEG molecule comprises the following formula, where n=1-36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36): [ka]

[0210] In some examples, the PEG molecule comprises the following formula, where n=1-36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36): [ka]

[0211] In some examples, the PEG molecule comprises the following formula, where n=1-36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36): [ka]

[0212] In some examples for each of the above formulas, n=4. In some examples for each of the above formulas, n=5. In some examples for each of the above formulas, n=6. In some examples for each of the above formulas, n=7. In some examples for each of the above formulas, n=8.

[0213] In some cases, the PEG molecule comprises a cholesterol moiety. In some cases, the cholesterol moiety is a thiocholesterol. In some cases, the sulfur of the thioether moiety of the thiocholesterol is replaced with an oxygen atom to generate an ether moiety in cholesterol derivatization.

[0214] PEG with degradable linkage in the backbone can be used.For example, PEG with ester linkage that undergoes hydrolysis can be prepared.Conjugates with degradable PEG linkage are described in WO99 / 34833; WO99 / 14259 and U.S. Patent No. 6,348,558.

[0215] In certain embodiments, a macromolecular polymer (e.g., PEG) is attached to a structurally stabilized (e.g., stapled) peptide described herein via an intermediate linker. In certain embodiments, the linker is composed of 1-20 amino acids linked by peptide bonds, which are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those of skill in the art. In other embodiments, the 1-20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is composed of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Non-peptide linkers are also possible, such as -NH(CH2) n Alkyl linkers such as C(O)- (wherein n=2-20) can be used. These alkyl linkers can be further substituted with any non-sterically hindering 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 bifunctional PEG linkers and their use in forming conjugates having a peptide at each of the PEG linker termini.

[0216] Structurally stabilized (e.g., stapled) peptides can also be modified, for example, in some embodiments to further promote mucoadhesion, membrane binding, or to increase in vivo stability. For example, acylation or PEGylation of a structurally stabilized peptide can increase bioavailability, enhance blood circulation, alter pharmacokinetics, alter immunogenicity, and / or reduce the required dosing frequency.

[0217] In some embodiments, the structurally stabilized (e.g., stapled) peptides disclosed herein have improved ability to bind to or penetrate cell membranes (e.g., compared to non-stabilized peptides). See, e.g., International Publication No. WO2017 / 147283, the entire contents of which are incorporated herein by reference.

[0218] Treatment The present disclosure features methods of using any of the structurally stabilized (e.g., stapled) peptide-cholesterol conjugates described herein (or pharmaceutical compositions including said structurally stabilized peptide-cholesterol conjugates) for the prevention and / or treatment of a coronavirus (e.g., a betacoronavirus, e.g., SARS-CoV-2) infection or coronavirus disease (e.g., COVID-19). The term "treat" or "treating" as used herein refers to alleviating, inhibiting, or ameliorating a disease or infection suffered by a subject (e.g., a human) or other species (e.g., pets; livestock; farm animals). In some examples, the subject is an animal. In some embodiments, the subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys or humans). In some examples, the subject is a domesticated animal (e.g., a dog or cat). In some examples, the subject is a bat, or other species that transmits coronaviruses. In some examples, the subject is a human. In certain embodiments, the term refers to a non-human animal (e.g., a non-human animal such as a pig, horse, cow, cat, or dog). In some embodiments, the term refers to a pet or farm animal. In some embodiments, the term refers to a human.

[0219] The structurally stabilized (e.g., stapled) peptide-cholesterol conjugates (or compositions comprising peptides) described herein may be useful for treating subjects (e.g., human subjects or the above-mentioned species) with coronavirus (e.g., betacoronavirus) infection. The structurally stabilized (e.g., stapled) peptide-cholesterol conjugates (or compositions comprising peptide-cholesterol conjugates) described herein may also be useful for treating human subjects suffering from coronavirus disease or another species provided herein. The structurally stabilized (e.g., stapled) peptide-cholesterol conjugates (or compositions comprising peptide-cholesterol conjugates) described herein may also be useful for treating subjects suffering from coronavirus disease, and the subjects may be mammals, e.g., non-primates (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or primates (e.g., monkeys or humans).

[0220] In certain embodiments, the coronavirus infection is an infection with one of 229E (alphacoronavirus); NL63 (alphacoronavirus); OC43 (betacoronavirus); HKU1 (betacoronavirus); Middle East Respiratory Syndrome (MERS); SARS-CoV; or SARS-CoV-2. In certain embodiments, the coronavirus infection is an infection with a SARS-CoV-2 variant selected from one of D614G B.1 (RVP-702), Wuhan-Hu-1 QHD43416.1 (RVP-701), New York variant B.1.526 (RVP-726) iota, California variant B.1.526 (RVP-713), or UK variant B.1.1.7 with E484K (RVP-717). In certain embodiments, the coronavirus disease is due to COVID-19 infection.

[0221] In certain embodiments, the coronavirus infection is an infection with one of Wuhan-Hu-1, B.1.427 / B.1.429, B.1.617.2, D614G B.1, Brazilian variant P.1, B.1.1.7, B.1.351, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, B.1.621.1, B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4 or BA.5. In certain embodiments, the coronavirus infection is an infection with one of B.1.351, cluster 5, lineage B.1.1.207, lineage B.1.1.7, variants of concern 202102 / 02, lineage B.1.1.317, lineage B.1.1.318, lineage B.1.351, lineage B.1.429, lineage B.1.525, lineage P.1 (also known as lineage B.1.1.28), lineage B.1.1.529, lineage BA.1, lineage BA.1.1, lineage BA.2, lineage BA.3, lineage BA.4 lineage BA.5, D614G, E484K, N501Y, S477G / N, or P681H.

[0222] The structurally stabilized (e.g., stapled) peptide-cholesterol conjugates (or compositions comprising peptide-cholesterol conjugates) described herein may be useful for preventing coronavirus (e.g., betacoronavirus) infection in a human subject or a subject from another species provided herein. The peptide-cholesterol conjugates (or compositions comprising peptide-cholesterol conjugates) described herein may also be useful for preventing coronavirus disease in a subject (e.g., a human subject) or a subject from another species provided herein. In certain embodiments, the coronavirus infection is an infection with one of 229E (alphacoronavirus); NL63 (alphacoronavirus); OC43 (betacoronavirus); HKU1 (betacoronavirus); Middle East Respiratory Syndrome (MERS); SARS-CoV; or SARS-COVID-19. In certain embodiments, the coronavirus disease is due to COVID-19 infection.

[0223] In certain embodiments, a human subject in need thereof or a subject from another species provided herein is administered a peptide as set forth in Tables 1 or 2, or a variant thereof. In certain embodiments, a human subject in need thereof or a subject from another species provided herein is administered a stapled SARS-CoV-2 HR2 peptide-cholesterol conjugate comprising or consisting of SEQ ID NO:6, or a modified version thereof. In certain embodiments, a human subject in need thereof is administered a stapled SARS-CoV-2 HR2 peptide-cholesterol conjugate comprising or consisting of SEQ ID NO:5, or a modified version thereof.

[0224] In certain embodiments, a human subject in need thereof or a subject from another species provided herein is administered any one of peptide-cholesterol conjugates having SEQ ID NOs: 7-21 or variants thereof (as described herein) as set forth in Table 1. Possible variations of these peptide-cholesterol conjugates are described in the structurally stabilized peptides section. Variants of these sequences have at least one (e.g., one, two, three, four, five) of these properties: (i) are alpha-helical; (ii) are protease resistant; (iii) inhibit fusion of SARS-CoV-2 with a host cell; and / or (iv) inhibit infection of cells by SARS-CoV-2. In some examples, structurally stabilized peptides having the variants inhibit infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays, and / or structurally stabilized peptides prevent infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays. In certain embodiments, treatment is performed to prevent transmission between human subjects. In some instances, treatment controls the spread of infection in a population of human subjects.

[0225] In certain embodiments, a human subject in need thereof or a subject from another species provided herein is administered a peptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95% identity to any one of the peptide-cholesterol conjugates having SEQ ID NOs: 7-21. In certain embodiments, a human subject in need thereof or a subject from another species provided herein is administered any one of the peptide-cholesterol conjugates having SEQ ID NOs: 7-21 but having 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, insertions and / or deletions. In certain embodiments, a human subject in need thereof or a subject from another species provided herein is administered any one of a peptide-cholesterol conjugate having an amino acid sequence comprising any one of SEQ ID NOs: 10, 13, 17, or 20, but having 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, insertions, and / or deletions.

[0226] In some embodiments, a human subject or a subject from another species provided herein is infected with a coronavirus (e.g., a betacoronavirus). In some embodiments, a human subject or a subject from another species provided herein is at risk of being infected with a coronavirus (e.g., a betacoronavirus). In some embodiments, a human subject or a subject from another species provided herein is at risk of developing a coronavirus disease (e.g., a betacoronavirus). In some examples, a human subject or a subject from another species provided herein is at risk of being infected with a coronavirus or developing a coronavirus disease if he or she or a subject from another species provided herein lives in an area (e.g., a city, state, country) that is exposed to an active coronavirus outbreak (e.g., an area where at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or more people have been diagnosed with a coronavirus). In some examples, a human subject or a subject from another species provided herein is at risk of being infected with a coronavirus or developing a coronavirus disease if he or she or a subject from another species provided herein lives in a region near (e.g., a region near (e.g., adjacent to) a second region (e.g., a city, state, country) where at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or more people have been diagnosed with a coronavirus) (e.g., a neighboring city, state, country) where he or she or a subject from another species provided herein is exposed to an active coronavirus outbreak. In certain embodiments, the coronavirus disease is due to SARS-CoV-2 infection. In certain embodiments, the subject or a subject from another species provided herein is at risk of having or developing COVID-19.

[0227] In general, the method includes the steps of selecting a subject or a subject from another species provided herein, and administering to the subject or a subject from another species provided herein an effective amount of one or more of the structurally stabilized (e.g., stapled) peptides herein, e.g., in or as a pharmaceutical composition, and optionally repeating the administration for the prevention or treatment of coronavirus infection or disease, which may be administered orally, intranasally, intravenously, intradermally, subcutaneously, intramuscularly, or locally, including to the skin, nose, sinuses, eyes, oropharynx, respiratory tree, and lungs. In some examples, administration is by local respiratory application, including application to the nasal mucosa, to the sinus mucosa, to the oropharynx mucosa, or to the respiratory tree, including the lungs. In some examples, topical application includes application to the skin or eyes. The subject may be selected for treatment, for example, based on determining that the subject is at risk of acquiring or has a coronavirus (e.g., a betacoronavirus, e.g., SARS-CoV-2) infection. The peptide-cholesterol conjugates of the present disclosure can be used to determine whether a subject is infected with coronavirus. In some examples, the peptide-cholesterol conjugates described herein increase bioavailability, promote blood circulation, modify pharmacokinetics, reduce immunogenicity, and / or reduce the required frequency of administration.

[0228] The specific dosage and treatment regimen for any particular patient or subject will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, frequency of administration, excretion rate, drug combination, the severity and course of the disease, condition or symptom, the patient's or subject's predisposition to the disease, condition or symptom, and the judgment of the treating physician or veterinarian.

[0229] An effective amount can be administered in one or more administrations, applications or dosages. The therapeutically effective amount (i.e., effective dosage) of the therapeutic compound depends on the therapeutic compound selected. The composition can be administered from one or more times a day to one or more times a week, including once every other day. Those skilled in the art will understand that certain factors, including but not limited to the risk of acquiring a disease or disorder or the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of the therapeutic compound described herein can include a single treatment or a series of treatments. For example, an effective amount can be administered at least once.

[0230] Pharmaceutical Compositions Any one or more of the structurally stabilized (e.g., stapled) peptide-cholesterol conjugates described herein can be formulated for use as or in a pharmaceutical composition. The pharmaceutical compositions can be used in the treatment or prevention methods described herein (see above). In certain embodiments, the pharmaceutical compositions comprise structurally stabilized (e.g., stapled) peptide-cholesterol conjugates that comprise or consist of an amino acid sequence that is identical to the amino acid sequence shown in Table 1 except for 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 amino acid substitution, insertion, or deletion. These changes to the amino acid sequence can be made to the non-interacting alpha-helical faces of these peptides (i.e., to amino acids that do not interact with the coronavirus six-helix bundle fusion machinery) and / or to the interacting alpha-helical faces (i.e., to amino acids that interact with the coronavirus six-helix bundle fusion machinery). Such compositions can be formulated for or adapted for administration to a subject by any route, for example, any route approved by the U.S. Food and Drug Administration (FDA). Exemplary methods are described in FDA's CDER Data Standards Manual, version number 004, available at fda.give / cder / dsm / DRG / drg00301.htm. For example, the composition can be formulated or adapted for administration by inhalation (e.g., oral and / or nasal inhalation (e.g., by nebulizer or spray)), injection (e.g., intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly, and / or subcutaneously); and / or oral, transmucosal, and / or topical administration (including topical (e.g., nasal) sprays, eye drops, and / or solutions).

[0231] In some examples, the pharmaceutical composition may include an effective amount of one or more structurally stabilized (e.g., stapled) peptide-cholesterol conjugates. The terms "effective amount" and "effective to treat" as used herein refer to the amount or concentration of one or more structurally stabilized (e.g., stapled) peptide-cholesterol conjugates or pharmaceutical compositions described herein that are utilized for a period of time (including acute or chronic administration and intermittent or continuous administration) that is effective within the context of its administration (e.g., treatment of an infectious disease) to produce an intended effect or physiological outcome.

[0232] The pharmaceutical compositions of the present invention may comprise one or more structurally stabilized (e.g., stapled) peptide-cholesterol conjugates described herein and any pharma- ceutically acceptable carrier and / or vehicle. In some cases, the pharmaceutical product may further comprise one or more additional therapeutic agents in an amount effective to achieve modulation of a disease or disease symptoms.

[0233] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered together with a compound of the invention to a patient or a subject from another species provided herein, which does not destroy the pharmacological activity of the compound and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the compound.

[0234] In some examples, the pharmaceutical composition of the present disclosure comprises one or more of acetate, citrate and / or maleate. In some examples, the pharmaceutical composition may comprise water or phosphate buffered saline (PBS). In some examples, the pharmaceutical composition may comprise chitosan.

[0235] The pharmaceutical compositions disclosed herein may include one or more pharma- ceutically acceptable salts.In some examples, pharma-ceutically acceptable salts include the salts that include hydrochloride, sodium, sulfate, acetate, phosphate or diphosphate, chloride, potassium, maleate, calcium, citrate, mesylate, nitrate, tartrate, aluminum, gluconate, and any combination thereof.

[0236] The pharmaceutical composition of the present invention can contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle.In some cases, the pH of the formulation can be adjusted with pharmaceutically acceptable acid, base or buffer to enhance the stability of the compound formulated or its delivery form.The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0237] In some examples, one or more structurally stabilized (e.g., stapled) peptide-cholesterol conjugates disclosed herein can be further conjugated, for example, with a carrier protein. Such conjugated compositions can be monovalent or multivalent. For example, a conjugated composition can include one structurally stabilized (e.g., stapled) peptide-cholesterol conjugate disclosed herein conjugated with a carrier protein. Alternatively, a conjugated composition can include two or more structurally stabilized (e.g., stapled) peptide-cholesterol conjugates disclosed herein further conjugated with a carrier.

[0238] 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 two entities are covalently connected, optionally via a linker group.

[0239] Carrier proteins can include any protein that increases or enhances stability, half-life, tissue exposure, and / or immunogenicity in a subject. Exemplary carrier proteins have been described in the art (see, e.g., 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 polymeric carrier may be a natural or synthetic material containing one or more primary and / or secondary amino groups, azide groups, or carboxyl groups. The carrier may be water-soluble.

[0240] Methods for Making Stapled or Stitched Peptides Derivatized with PEG(n)-Thiocholesterol or PEG(n)-Cholesterol Moieties In one aspect, the disclosure features a method of making a structurally stabilized peptide derivatized with a PEG(n)-thiocholesterol or PEG(n)-cholesterol moiety. The entirely on-resin synthesis method includes (a) providing a peptide (e.g., SEQ ID NOs: 7-21) that includes at least two unnatural amino acids with olefinic side chains, (b) crosslinking the peptide, in some instances by a ruthenium-catalyzed metathesis reaction, and (c) on-resin derivatizing the C-terminus with a PEG linker of variable length connected to a thiocholesterol or cholesterol moiety.

[0241] In some examples, the method includes cleaving the structurally stabilized peptide from the resin. Cleavage of structurally stabilized resins is known in the art. In some examples, the cleaving step is performed before the step of derivatizing the C-terminus on the resin with a PEG linker of variable length connected to a thiocholesterol or cholesterol moiety. See, for example, de Vries et al., Science, 2021 Mar 26;371(6536):1379-138, and Figueira et al., J. Virol. 91, e01554-16 (2016), each of which is incorporated by reference in its entirety. In other aspects, the cleaving step is performed after the step of derivatizing the C-terminus on the resin with a PEG linker of variable length connected to a thiocholesterol or cholesterol moiety.

[0242] In examples where the cleaving step occurs before the derivatizing step, the method includes the use of a compound having one of the following formulas: [ka] where n is 1 to 36. In some examples, n is 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. Thus, compounds having any of the above formulas are also disclosed herein.

[0243] In some aspects, a method for synthesizing a stabilized peptide (i.e., any of the peptides disclosed herein; e.g., SEQ ID NO: 10), comprising subjecting a peptide comprising formula VII to ring-closing metathesis conditions to obtain a stabilized peptide, wherein formula VII is [ka] or a pharma- ceutically acceptable salt thereof. w is DISGINASVVNIQ (SEQ ID NO: 35); x is EIDRLN (SEQ ID NO: 36); [Xaa] y In some examples, [Xaa] is VAKNLNESLIDLQELGK (SEQ ID NO: 37). w , [Xaa] x , and [Xaa] y are disclosed in Table 2. In some examples, R9 is -OH or [ka] In some examples, p is 2, 3, 4, 5, 6, 7, or 8. In some examples, z is 2, 3, 4, 5, or 6. In some examples, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 In some examples, the ring-closing metathesis conditions include exposing the peptide comprising Formula IV to a Grubbs ring-closing metathesis ruthenium catalyst.

[0244] In some examples, the stabilizing peptide is [ka] or a pharma- ceutically acceptable salt thereof, [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] x is EIDRLN (SEQ ID NO:36); [Xaa] y is VAKNLNESLIDLQELGK (SEQ ID NO:37); R9 is -OH or [ka] and; p is 2, 3, 4, 5, 6, 7, or 8; and z is 2, 3, 4, 5 or 6; optionally, [Xaa] w The amino group of the N-terminal aspartic acid in 1~4 (Alkyl) is substituted) In some examples, p is 4. In some examples, p is 5. In some examples, p is 6. In some examples, z is 3. In some examples, the method includes derivatizing the C-terminus of the stabilized peptide with a moiety that includes a polyethylene glycol moiety and a cholesterol or thiocholesterol moiety. In some examples, the method includes derivatizing the C-terminus of the stabilized peptide to provide ** -C(O)-(C 2~6 Alkylene)-[O-CH2CH2] m -N(R5)C(O)-(C 1~6 The method further comprises the step of incorporating an alkylene)-R6 group, wherein ** is [Xaa] y In some embodiments, R5 is a hydrogen or a C 1~4 In some examples, R6 is one of the following: [ka] and m is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and In some examples, t is 0, 1, 2, or 3. In some examples, m is 4. In some examples, m is 8. In some examples, R9 is [ka] In some instances, the peptide is truncated.

[0245] In some examples, the above method further comprises formulating the stabilized peptide or salt thereof into a sterile pharmaceutical composition.

[0246] Stapled peptide synthesis: Our reported method for generating all-hydrocarbon stapled peptides (Bird et al., Curr. Protocol. Chem. Biol., 3(3):99-117 (2011; Bird et al., Methods Enzymol., Stapled peptide fusion inhibitors were synthesized using Fmoc-based solid-phase peptide synthesis according to 446:369-86 (2008). To obtain various staple lengths, α-methyl,α-alkenyl amino acids were incorporated in specific pairings at distinct positions, e.g., at i,i+7 positions positioning the use of one S-pentenylalanine residue (S5) and one R-octenylalanine residue (R8). For the stapling reaction, Grubbs first generation ruthenium catalyst dissolved in dichloroethane was added to the resin-bound peptide. Three to five rounds of stapling were performed to ensure maximum conversion. Then, after addition of PEG(n)-thiocholesterol or PEG(n)-cholesterol moieties (see below), the peptides were cleaved off the resin using trifluoroacetic acid, precipitated using a hexane:ether (1:1) mixture, air-dried, and purified by LC-MS. All peptides were quantified by amino acid analysis.

[0247] Stitched Peptide Synthesis: Methods for synthesizing the stitched peptides described herein are known in the art. Nonetheless, the following exemplary method can be used. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful for the synthesis of the compounds described herein are known in the art and may be found in, for example, Bird et al., ACS Chem Biol. (2020) 15(6):1340-1348; Hilinski et al., J Am Chem Soc. (2014) 136(35):12314-22; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3d. 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 (1999). (1995), and their subsequent editions.

[0248] C-Terminal Derivatization of Stapled or Stitched Peptides with PEG(n)-Thiocholesterol or PEG(n)-Cholesterol Using an On-Resin Synthesis Approach: To generate carboxythiocholesterol or carboxycholesterol reagents for peptide derivatization by solid-phase synthesis, thiocholesterol was dissolved in dichloromethane (DCM) or cholesterol was dissolved in tetrahydrofuran (THF) at 0.1 M and added to a round-bottom flask. Three equivalents of base (diisopropylethylamine for thiocholesterol or sodium hydride or potassium t-butoxide for cholesterol) were added with stirring. Five equivalents of t-butyl ester of bromoacetic acid were then added and the reaction was stirred for 2 hours at room temperature followed by 30 minutes at 40°C. Two volumes (relative to solvent) of trifluoroacetic acid were added and the reaction was stirred for 30 minutes at room temperature. Reaction progress was monitored by TLC (19:1 Hex:EtOAc for thiocholesterol and 3:1 Hex:EtOAc for cholesterol) with KMnO4 staining. For example, (thio)cholesterol migrated with the solvent front, (thio)ethers slowed migration by about 20%, and TFA hydrolysis brought the spot to baseline. The reaction mixture was added to 5 volumes of water and 1 volume of DCM was added. The solvent layer was washed with 0.1 M HCl, brine, and dried over sodium sulfate. Removal of the solvent by Rotovap gave a heavy orange oil that was used without further purification. The yield was nearly quantitative. Purity was determined to be greater than 90% by NMR of the olefinic protons relative to the new CH2 singlet. For peptide derivatization with thiocholesterol or cholesterol, the completed resin-bound peptide sequence was treated with 20% piperidine / DMF, followed by capping with acetic anhydride to block the N-terminal amine, and then the C-terminal side chain lysine amine was revealed by five treatments with 2% hydrazine in DMF for 10 min each.The amine was acylated with Fmoc-protected PEG(n) amino acid (e.g., n=1-36 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36)), at which point the olefin was crosslinked by treatment with Grubbs(I) catalyst three times for 2 h each. Upon completion, Fmoc was removed from the C-terminal NH of the PEG reagent and the amine was acylated with carboxy-thiocholesterol (or carboxy-cholesterol) for 30 min. TFA cleavage afforded a crude product of excellent purity, which was further purified using semi-preparative HPLC.

[0249] The peptide sequences of the present invention can be produced 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, peptides can be synthesized using automated Merrifield solid phase synthesis techniques with α-NH2 protected by either t-Boc or Fmoc chemistry using side chain protected amino acids, for example, on an Applied Biosystems Peptide Synthesizer Model 430A or 431.

[0250] One mode of making the peptides described herein is by using solid phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin by an acid-labile bond using a linker molecule. This resin is insoluble in the solvents used in the synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with an Fmoc group, which is stable in acid but removable by base. Any side chain functional groups are protected with base-stable and acid-labile groups.

[0251] Longer peptides could be made by joining individual synthetic peptides using native chemical ligation. Insertion of linking amino acids could be done, for example, as described in Young and Schultz, J Biol Chem. 2010 Apr 9; 285(15): 11039-11044. Alternatively, longer synthetic peptides could be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding the peptide of the invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably a nucleic acid sequence that is codon-optimized for the organism in which the gene will be expressed. A synthetic gene is then made, typically by synthesizing oligonucleotides encoding the peptide and any regulatory elements, if necessary. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions that are appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0252] Peptides can be made in a high-throughput, combinatorial format, for example, using a high-throughput multi-channel combinatorial synthesizer, such as available from Advanced Chemtech or Gyros Protein Technologies. Peptide bonds can be replaced with retro-inverso bonds (C(O)-NH); reduced 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); fluoro-substituted trans-olefin bonds (CF=CH); ketomethylene bonds (C(O)-CHR) or CHR-C(O) (where R is H or CH3); and fluoro-ketomethylene bonds (C(O)-CFR or CFR-C(O) (where R is H or F or CH3), for example, to increase the physiological stability of the peptide.

[0253] The peptides can be further modified by acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceination, formylation, myristoylation, palmitoylation, and other lipidations, including thiocholesterol or cholesterol modifications using the on-resin method disclosed herein, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation, and sulfurylation, among others. As mentioned above, the peptides can be conjugated to or contain linker atoms or moieties of variable length, such as polyethylene glycol (PEG) moieties of variable length; alkyl groups (e.g., C1-C20 straight or branched alkyl groups); fatty acid radicals; and combinations thereof. Alpha,alpha-disubstituted unnatural amino acids containing olefinic 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, the stitched peptides include linkages between i, i+4, and between i+4 and i+8. Such stitched peptides can be made with respect to SEQ ID NO:4 or SEQ ID NO:6. In some examples, the amino acids that form the staple or stitch are (R)-2-(4'-pentenyl)alanine, 2,2-bis(4-pentenyl)glycine, and (S)-2-(4'-pentenyl)alanine at positions i, i+4, and i+8, respectively, of the stitch.In some examples for peptides (four turns of a stabilized helix) where stitches are used in which i is linked to i+7 and i+7 is linked to i+14: one R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine), 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 (four turns of a stabilized helix) where stitches are used in which i is linked to i+7 and i+7 is linked to i+14: one S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine), 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 (four turns of a stabilized helix) where stitches are used in which i is linked to i+7 and i+7 is linked to i+14: 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 (four turns of a stabilized helix) where stitches are used in which i is linked to i+7 and i+7 is linked to i+14: 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 (four turns of a stabilized helix) where stitches are used where i is linked to i+7 and i+7 is linked to i+14: 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 (four turns of a stabilized helix) where stitches are used in which i is linked to i+7 and i+7 is linked to i+14: 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 (four turns of stabilized helix) where stitches are used where i is linked to i+7 and i+7 is linked to i+14: 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 results in the R-alkyl stereoisomer. Also, 8-iodooctene is used instead of 5-iodopentene. Inhibitors are synthesized on solid support using solid phase peptide synthesis (SPPS) on MBHA resin or Rink Amide AM resin (see, e.g., WO2010 / 148335).

[0254] Fmoc-protected α-amino acids (other than the 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-tetramethyluronium hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially available, for example, from Novabiochem (San Diego, Calif.). 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-based amino acid synthesis has been reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0255] Again, suitable methods for obtaining (e.g., synthesizing), stitching, and purifying the peptides disclosed herein are also known in the art (see, e.g., 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 which is hereby incorporated by reference in their entirety).

[0256] In some examples, the peptide is substantially free of non-stitched or non-stapled peptide contaminants or the peptide is isolated. Methods for purifying peptides include, for example, synthesizing the peptide on a solid support. Several alternative solvents and purification schemes for isolation and purification of peptides and stapled peptides after cyclization are known in the art and may use solvents including, but not limited to, DMSO, DMSO / dichloromethane mixtures, DMSO / NMP mixtures, or mixtures / solutions that do not contain DMSO. The DMSO / dichloromethane or DMSO / NMP mixtures 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, for example, with dichloromethane or NMP. In one example, the resin is washed with NMP. Shaking and bubbling of an inert gas through the solution may be performed.

[0257] The properties of stitched or stapled peptides derivatized with C-terminal PEG(n)-thiocholesterol or PEG(n)-cholesterol of the disclosure can be assayed, for example, using the methods described below and in the Examples.

[0258] Assays to determine the characteristics and anti-SARS-CoV-2 activity of stapled HR2 peptides derivatized with PEG(n)-thiocholesterol or PEG(n)-cholesterol moieties Assay for determining α-helicity: Compounds are dissolved in aqueous solution (e.g., 5 μM potassium phosphate solution at pH 7, or distilled H2O, to a concentration of 25-50 μM). Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard measurement parameters (e.g., temperature, 20 °C; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 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 value reported for a model helical decapeptide (Yang et al., Methods Enzymol., 1986).

[0259] Assay for determining melting temperature (Tm): Dissolve crosslinked or unmodified template peptide in distilled HO or other buffer or solvent (e.g., at a final concentration of 50 µM) and determine Tm by measuring the change in ellipticity over a temperature range (e.g., 4-95 °C) with a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard parameters (e.g., wavelength 222 nm; step resolution, 0.5 nm; rate, 20 nm / sec; accumulation, 10; response, 1 sec; bandwidth, 1 nm; temperature ramp rate: 1 °C / min; path length, 0.1 cm).

[0260] In vitro protease resistance assay: Amide bonds in peptide backbones are susceptible to hydrolysis by proteases, thereby making peptide compounds vulnerable to rapid degradation in vivo. However, peptide helix formation typically buries and / or twists and / or shields the amide backbone, which may prevent or substantially delay proteolytic cleavage. The peptidomimetic macrocycles of the invention can be subjected to in vitro enzymatic proteolysis (e.g., trypsin, chymotrypsin, pepsin) to assess any changes in degradation rate compared to the corresponding uncrosslinked or alternatively stapled polypeptide. For example, the peptidomimetic macrocycles and the corresponding uncrosslinked polypeptides are incubated with trypsin agarose, the reaction is quenched at various time points by centrifugation followed by HPLC injection, and the remaining substrate is quantified by UV absorption at 280 nm. Briefly, peptidomimetic macrocycles and peptidomimetic precursors (5 mcg) are incubated with trypsin agarose (Pierce) (S / E approx. 125) for 0, 10, 20, 90 and 180 min. Reactions are quenched by high speed tabletop centrifugation; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction exhibits first-order kinetics and the rate constant, k, is determined from a ln[S] plot versus time.

[0261] The peptidomimetic macrocycles and / or corresponding uncrosslinked polypeptides can each be incubated with fresh mouse, rat and / or human serum (e.g., 1-2 mL) at 37°C for, e.g., 0, 1, 2, 4, 8 and 24 hours. Samples with different macrocycle concentrations can be prepared by serial dilution with serum. To determine the levels of intact compounds, the following procedure can be used: for example, 100 μL of serum is transferred to a 2 ml centrifuge tube, followed by extraction of the sample by adding 10 μL of 50% formic acid and 500 μL of acetonitrile and centrifugation at 14,000 RPM for 10 minutes at 4+ / -2°C. The supernatant is then transferred to a fresh 2 ml tube and evaporated by Turbovap under N2<10 psi at 37°C. The sample is reconstituted with 100 μL of 50:50 acetonitrile:water and subjected to LC-MS / MS analysis. Equivalent or similar procedures for testing ex vivo stability are known and may be used to determine the stability of the macrocycle in serum.

[0262] Plasma stability assay: The stability of stapled peptides can be tested in freshly drawn mouse plasma collected in lithium heparin tubes. Triplicate incubations with 500 μl of plasma spiked with 10 μM of each peptide are set up. Samples are gently shaken at 37° C. in an orbital shaker and 25 μl aliquots are removed at 0, 5, 15, 30, 60, 240, 360 and 480 min and added to a 100 μl mixture containing 10% methanol: 10% water: 80% acetonitrile to stop further degradation of the peptide. Samples are kept on ice for the duration of the assay and then transferred to a MultiScreen Solvinert 0.45 μm low-binding hydrophobic PTFE plate (Millipore). The filtrate is directly analyzed by LC-MS / MS. Peptides are detected as doubly or triply charged ions using a Sciex 5500 mass spectrometer. The percentage of remaining peptide is determined by the reduction in chromatographic peak area and log transformed to calculate half-life.

[0263] In vivo protease resistance assay: A key advantage of peptide stapling is that in vitro protease resistance translates into significantly improved pharmacokinetics in vivo. Stapled peptide levels are detected and quantified in plasma using a liquid chromatography / mass spectrometry-based analytical assay. For pharmacokinetic analysis, peptides are dissolved in sterile 5% aqueous dextrose (1 mg / mL) and administered to C57BL / 6 mice (Jackson Laboratory) via bolus tail vein or intraperitoneal injection (e.g., 5, 10, 25, 50 mg / kg). Blood is collected by retro-orbital puncture at 5, 30, 60, 120 and 240 min after dosing in five animals at each time point. Plasma is collected after centrifugation (2,500 × g, 5 min, 4 °C) and stored at -70 °C until assayed. Peptide concentrations in plasma are determined by reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry detection (Aristoteli et al., Journal of Proteome Res., 2007; Walden et al., Analytical and Bioanalytical Chem., 2004). Study samples are assayed along with a series of seven calibration standards of peptides in plasma at concentrations ranging from 1.0 to 50.0 μg / mL; drug-free plasma assayed with and without added internal standards; and three quality control 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 ratios versus the known drug concentrations in each calibration standard. Linear least-squares regressions are performed, weighted proportionally to the inverse of the analyte concentration normalized to the number of calibration standards. The slope and y-intercept values ​​of the best-fit line are used to calculate the drug concentration in the study samples. Plasma concentration-time curves are analyzed by standard noncompartmental methods using WinNonlin Professional 5.0 software (Pharsight Corp., Cary, NC) to obtain pharmacokinetic parameters such as early and terminal plasma half-lives, peak plasma levels, total plasma clearance, and apparent volume of distribution.

[0264] The persistence of the stapled peptides of the invention in the nasal mucosa after topical administration (i.e., nasal drops) and in the respiratory mucosa after intranasal application or spray is examined in the context of pre- and post-infection blockade of viral fusion and dissemination. Mice are exposed to a single treatment with nasal drops or nebulizer at a series of intervals preceding intranasal infection with SARS-CoV-2, and the duration of protection from mucosal infection (assessed histologically as above or by PCR as below) is used to measure the relative mucosal stability and prophylactic efficacy of the stapled peptide constructs derivatized with PEG(n)-thiocholesterol or PEG(n)-cholesterol described herein.

[0265] In vitro binding assays: To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins, for example, fluorescence polarization assays (FPA) can be used. FPA techniques use polarized light and fluorescent tracers to measure molecular orientation and mobility. When excited by polarized light, fluorescent tracers (e.g., FITC) bound to molecules or peptides and then to proteins of high apparent molecular weight (e.g., FITC-labeled peptides bound to large proteins) emit higher levels of polarized fluorescence due to their slower turnover rate upon protein binding compared to fluorescent tracers bound only to smaller molecules or peptides (e.g., FITC-labeled peptides free in solution).

[0266] In vitro displacement assays to characterize antagonists of peptide-protein interactions: Fluorescence polarization assays (FPAs) using, for example, fluoresceinated peptides or peptidomimetic macrocycles derived from a template peptide sequence are used to assess the binding and affinity of compounds that antagonize the interaction of peptides with acceptor proteins. FPA techniques use polarized light and fluorescent tracers to measure the orientation and mobility of molecules. When excited by polarized light, fluorescent tracers (e.g., FITC) that are bound to molecules that are then bound to proteins of high apparent molecular weight (e.g., FITC-labeled peptides bound to large proteins) emit higher levels of polarized fluorescence due to their slower rotation rates compared to FITC-derivatized molecules alone (e.g., FITC-labeled peptides free in solution). Compounds that antagonize the interaction of fluoresceinated peptides with acceptor proteins, for example, unlabeled stapled peptides and their conjugates, will be detected in competitive binding FPA experiments, and the differential potency of compounds in disrupting the interaction can be quantified and compared.

[0267] Five-helix bundle protein production and fluorescence polarization assay: A C-terminally 6His-tagged recombinant five-helix bundle (5HB) protein was designed that contains five of the six helices comprising the hairpin SARS-CoV-2 S trimer core, connected by short peptide linkers according to the design of gp41 5-HB (Root et al. Science, 291(5505):884-8 (2001); Bird et al., J Clin Invest. 2014 May;124(5):2113-24). The plasmid was transformed into Escherichia coli BL21(DE3), grown in Luria broth, and induced with 0.1 M isopropyl β-D-thiogalactoside overnight at 37°C. Cells are harvested by centrifugation at 5,000 g for 20 min, resuspended in Buffer A (100 mM NaH2PO4, 20 mM Tris, 8 M urea; pH 7.4) and lysed by overnight stirring at 4°C. The mixture is clarified by centrifugation (30 min, 35,000 g) and then bound to a nickel nitrilotriacetate (Ni-NTA) agarose (Qiagen) column at room temperature. Bound 5-HB is washed with Buffer A (pH 6.3), eluted with Buffer A (pH 4.5), renatured by dilution (1:2) with PBS (50 mM sodium phosphate, 100 mM NaCl; pH 7.5), and concentrated on a 10 kDa Amicon centricon (diluted and reconcentrated 7 times) to obtain an approximately 1 mg / ml protein solution. Protein purity is assessed by SDS-PAGE and determined to be >90%. Fluoresceinated derivatives of the peptides of the present invention (25 nM) are incubated with 5-HB protein at the indicated concentrations in binding buffer (50 mM sodium phosphate, 100 mM NaCl; pH 7.5) at room temperature. Direct binding activity at equilibrium (e.g., 10 min) is measured by fluorescence polarization using a SpectraMax M5 microplate reader (BMG Labtech). Then, for competitive binding assays, a fixed concentration of FITC-peptide reflecting the EC90 of direct binding and 5-HB protein are incubated with serial dilutions of acetylated SAH-SARS-CoV-2 peptide to generate a competitive curve for comparative analysis.Binding assays are performed in triplicate and Ki is calculated by nonlinear regression analysis of competitive binding isotherms using Prism software (GraphPad).

[0268] Assays for screening for binding activity to the SARS-CoV-2 5-helical bundle: In some examples, the methods disclosed herein include direct and competitive screening assays. For example, the methods may include determining whether an agent alters (e.g., reduces) the binding of one or more of the peptides disclosed herein and their conjugates to SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helical bundle). In some examples, the method includes the steps of: (i) determining the binding level of one or more of the peptides disclosed herein and conjugates thereof to SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle) (e.g., in the absence of an agent); and (ii) detecting the binding level of one or more peptides (e.g., one or more peptides of (i)) to SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle) in the presence of an agent, wherein a change (e.g., a decrease) in the binding level of the one or more peptides to SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle) indicates that the agent is a candidate agent that binds to SARS-CoV-2; and (iii) selecting the candidate 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 a level of binding between the one or more peptides with 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 agent with SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle) and detecting a level of binding between the one or more peptides with SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle).SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle) can be contacted with one or more peptides and the agent at the same time or at different times (e.g., one or more peptides can be contacted with SARS-CoV-2 (e.g., to the SARS-CoV-2 5-helix bundle) before or after the agent). In some embodiments, the candidate agent is administered to a suitable animal model (e.g., an animal model of COVID-19) to determine whether the agent reduces the level of COVID-19 infection in the animal.

[0269] In some examples, one or both of the peptide and the SARS-CoV-2 helical bundle may include a label that allows for detection of the peptide and / or the SARS-CoV-2 helical bundle. In some examples, the peptide includes a label. In some examples, the SARS-CoV-2 helical bundle includes a label. In some examples, both the peptide and the SARS-CoV-2 helical bundle include a label. The label may be any label known in the art, including but not limited to a fluorescent label, a radioisotope label, or an enzymatic label. In some examples, the label is itself directly detectable (e.g., a radioisotope label or a fluorescent label). In some examples (e.g., in the case of an enzymatic label), the label is indirectly detectable, for example, by catalyzing a chemical alteration of a chemical substrate compound or composition that is directly detectable.

[0270] Competitive SARS-CoV-2 5-HB binding assay by ELISA: Microwells are coated with 50 μl of PBS containing neutravidin (4 μg / ml) overnight at 4° C. The wells are washed twice with PBS containing 0.05% Tween® 20 (PBS-T) and blocked with 4% BSA in PBS-T for 45 min at 37° C. Then, 50 μl of 250 nM biotinylated PEG2-SARS-CoV-2 HR2 (SEQ ID NO: 6) is added in PBS-T containing 1% BSA and incubated with shaking for 1 h, followed by washing four times with 300 μl of PBS-T. Then, 1:2 serial dilutions of the stapled peptides of the invention starting at 10 μM, containing 50 nM recombinant 5-HB, in 50 μL PBS-T containing 1% BSA, are added to the plate and shaken for 2 hours at room temperature, followed by washing 4 times with 300 μl PBS-T. Finally, 50 μL of a 1:5000 dilution of a goat polyclonal antibody conjugated with 6× His tag-HRP is added. After 40 minutes of incubation at RT, the wells are washed 5 times and developed 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 on a microplate reader (Molecular Devices). The concentration of competitor peptide corresponding to half-maximal signal (IC50) is determined by interpolation of the resulting binding curves using Prism software (Graphpad). Each peptide competitor is tested in triplicate in at least two separate experiments.

[0271] Cellular localization assay: To measure the localization of peptides or crosslinked polypeptides on or within cells, intact cells are incubated with PEG(n)-thiocholesterol or PEG(n)-cholesterol derivatized fluoresceinated crosslinked polypeptides (5 μM) for 4 hours in serum-free medium or in medium supplemented with human serum at 37° C., washed twice with medium, and incubated with trypsin (0.25%) for 10 minutes at 37° C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or a Cellomics KineticScan® HCS Reader.

[0272] Antiviral Efficacy Assay: The efficiency of the peptide-cholesterol conjugates of the present invention in preventing and treating live SARS-CoV-2 virus infection is evaluated in monolayer cell cultures. A virus detection platform was developed for SARS-CoV-2 based on previous screening against 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 are treated with serial dilutions of stapled peptides (e.g., starting doses of 1–5 μM) in triplicate for 1 h, followed by a 4 h challenge with SARS-CoV-2 (e.g., USA-WA1 / 2020 [wild type], South African B.1.351 [beta strain]) or Indian B.1.617.2 [delta strain] to achieve a control infection of 10–25% of cells (predetermined optimal infectivity to evaluate the dynamic range of test compounds in the assay). Infected cells are then washed, fixed with 4% paraformaldehyde, washed again with PBS, and immunostained with an anti-SARS-CoV-2 nucleocapsid monoclonal antibody followed by an anti-Ig secondary antibody (Alexa Fluor 488; Life Technologies) and counterstaining the cell bodies with HCS CellMask blue. Cells are imaged across the z-plane on a Nikon Ti Eclipse automated microscope and analyzed with CellProfiler software, and infection efficiency is calculated by dividing infected cells by total cells. Control cytotoxicity assays are performed using Cell-Titer Glo (Promega) and LDH release (Roche) assays.

[0273] In an alternative approach, qPCR-based virus detection is used 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; Hongkon VM20001061). Cultured cells are treated with serial dilutions of the stapled peptide-cholesterol conjugate of the present invention for 1 hour, followed by challenge with SARS-CoV-2 virus. Samples of culture supernatant are taken, the virus is lysed in the presence of RNAse inhibitors, 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. CDC-validated BHQ-quenched dye pair primers are purchased from IDT, and genome equivalents are calculated from Ct values.

[0274] In yet another approach, the antiviral activity of the stapled peptide-cholesterol conjugates of the invention is evaluated using pseudotyped viruses. We used a 293T-hsACE2 stable cell line (Cat# C-HA101) and pseudotyped SARS-CoV-2 viruses [RVP-701G (Wuhan-Hu-1) RVP-702G (D614G B.1, 20A) RVP-706G (UK variant B.1.1.7, 20I / 501Y.V1) RVP-724G (South African, variant Δ3 B.1.351, 20H / 501Y.V2) RVP-763G (Indian variant, B.1.617.2) RVP-768G (BA.1) RVP-770G (BA.2) RVP-801G (SARS-CoV-1 Urbani) RVP-1002G (VSV with MLV core) receptor (Integral Molecular). Neutralization assays are performed according to the manufacturer's protocol. Pseudotyped VSV virus with MLV core and GFP reporter is used in these assays as a virus specificity control. Briefly, 2-fold serial dilutions of peptides (starting dose 500 or 1000 nM) are incubated with 5 μL of pseudotyped SARS-CoV-2-GFP for 1 hour at 37°C in 384-well black clear bottom plates, followed by addition of 30 μL of 1,000 293T-hsACE2 cells in 10% FBS DMEM, phenol red-free medium, and placing in a humidified incubator for 48 or 72 hours. Hoechst 33342 (cell-permeable nuclear stain) and DRAQ7 (cell-impermeable nuclear stain) are added, and the plate is imaged with a Molecular Devices ImageXpress Micro Confocal Laser at 10x magnification. GFP(+) cells are counted and the total GFP(+) cells or percent GFP(+) cells are plotted using Prism software (Graphpad). Cytotoxicity is determined by the ratio of DRAQ7(+) Hoechst 33342(+) cells to DRAQ7(-) Hoechst 33342(+) cells.

[0275] To evaluate the ability of the lead stapled peptide-cholesterol conjugate to prevent SARS-CoV-2 infection, K18-hACE2 (Jackson Laboratory) mice (n=10 per arm; 5 males, 5 females) were treated intravenously or via the oropharyngeal route with the stapled peptide-cholesterol conjugate or vehicle, and then 4–24 h later, 10 4 A virus dose of PFU is inoculated intranasally. Mice are euthanized 4 days later (peak of viremia) for evaluation by necropsy and by viral load quantified by qPCR from lung homogenate supernatant samples prepared as described using a tissuelyzer (Qiagen). See Bao L et al. Nature. 2020. Epub 2020 / 05 / 08; doi: 10.1038 / s41586-020-2312-y. To evaluate the ability of lead stapled peptides to treat or mitigate established SARS-CoV-2 infection, K18-hACE2 mice (n=10 per arm; 5 males, 5 females) were inoculated with 10 4 A viral dosage of PFU is inoculated intranasally on day 1, followed by oropharyngeal or intraperitoneal treatment with stapled peptide or vehicle daily for 10 days (days 2–12). In an alternative plan, dosing is delayed until 3–5 days after inoculation to simulate symptom- or positive test-driven treatment initiation. Mice are continuously monitored to record weight and clinical signs, and disease progression is scored as >10% weight loss, labored breathing, and / or growth failure. Doses for the most effective compounds and routes are then refined in both prevention and treatment studies to determine the minimal dose to protect mice. The same experimental plan is used, except that four treatment groups (n=10; 5 males, 5 females) receive the original dose, followed by three doses tapered by a factor of 4. Alternative animal models of SARS-CoV-2 infection (e.g., hamsters, ferrets) are also used.

[0276] Clinical trials: Clinical trials can be conducted to determine the suitability of the PEG(n)-thiocholesterol or PEG(n)-cholesterol derivatized stapled peptides 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, where the treatment group is administered the peptide of the present invention, while the control group is administered a placebo or a known antiviral drug. The treatment safety and efficacy of the peptide-cholesterol conjugates of the present invention can thus be evaluated by comparing 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 crosslinked polypeptide or a placebo. After administration of the treatment, the patients are followed up. In both instances, SARS-CoV-2 exposed patient populations treated with the stapled peptide-cholesterol conjugates of the invention will avoid developing infection, or patient populations suffering from SARS-CoV-2 infection will show resolution or reduction in symptoms or their severity compared to placebo treated patient control populations.

[0277] List of embodiments: This disclosure provides the following list of exemplary embodiments.

[0278] Embodiment 1. 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 set forth in SEQ ID NO: 6 (DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK), (i) positions 14 and 21; (ii) positions 17 and 24; (iii) positions 20 and 27; (iv) positions 21 and 28; or (v) 24th and 31st place (position 1 of SEQ ID NO:6 is an N-terminal aspartic acid and position 38 is a C-terminal lysine) is replaced with an α,α-disubstituted unnatural amino acid having an olefinic side chain; the structurally stabilized peptide is 38-60 amino acids in length, optionally 38-50 amino acids in length; A conformationally stabilized polypeptide, wherein the conformationally stabilized peptide inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay, and / or the conformationally stabilized peptide prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay.

[0279] Embodiment 2. The structurally stabilized polypeptide of embodiment 1, wherein the amino acid sequence is at least 70% identical to the sequence set forth in SEQ ID NO:6.

[0280] Embodiment 3. The structurally stabilized polypeptide of embodiment 1, wherein the amino acid sequence is at least 80% identical to the sequence set forth in SEQ ID NO:6.

[0281] Embodiment 4. The structurally stabilized polypeptide of any one of embodiments 1 to 3, wherein the amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK).

[0282] Embodiment 5. The structurally stabilized polypeptide of any one of embodiments 1 to 3, wherein the amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13 (DISGINASVVNIQKEI8RLNEVAXNLNESLIDLQELGK).

[0283] Embodiment 6. The structurally stabilized polypeptide of any one of embodiments 1 to 3, wherein the amino acid sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17 (DISGINASVVNIQKEIDRLN8VAKNLNXSLIDLQELGK).

[0284] Embodiment 7. The structurally stabilized polypeptide of any one of embodiments 1 to 6, which is 38 to 45 amino acids in length.

[0285] Embodiment 8. The structurally stabilized polypeptide of any one of embodiments 1 to 7, which is 38 to 40 amino acids in length.

[0286] Embodiment 9. The structurally stabilized polypeptide of any one of embodiments 1 to 8, wherein positions 37 and / or 38 of SEQ ID NO:6 are not substituted.

[0287] Embodiment 10. A structurally stabilized peptide comprising at least 19 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 10, wherein at least 1-18 of the amino acids at positions 3, 4, 6, 9, 11, 13, 15, 18, 20, 22, 25, 27, 29, 32, 34, 35, 37 or 38 of SEQ ID NO: 10 are substituted with any other natural or non-natural amino acid, and the non-natural amino acids at positions 14 and 21 of SEQ ID NO: 6 are not substituted, and the peptide has a length of 19-45 amino acids, and is suitable for use in pseudoviruses and and / or inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay and / or prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay, and optionally a structurally stabilized peptide in which 19 contiguous amino acids correspond to positions 1179 to 1197 of HR2, except that positions 1181 and 1188 are replaced by unnatural amino acids having olefinic side chains.

[0288] Embodiment 11. A structurally stabilized peptide of embodiment 10 that inhibits infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay and / or prevents infection of cells by SARS-CoV-2 in a pseudovirus and / or live SARS-CoV-2 virus assay.

[0289] Embodiment 12. A conjugate comprising the structurally stabilized polypeptide of any one of embodiments 1 to 11 and polyethylene glycol (PEG) and / or cholesterol, wherein the PEG and / or cholesterol are linked to the C-terminus of the structurally stabilized polypeptide.

[0290] Embodiment 13 The conjugate of embodiment 12, comprising PEG and cholesterol.

[0291] Embodiment 14 The conjugate of embodiment 12 or 13, wherein the cholesterol is thiocholesterol.

[0292] Embodiment 15. The conjugate of embodiment 12 or 13, comprising PEG(n)-cholesterol, where n is 1 to 36, and optionally n is 4, 5, 6, 7, or 8.

[0293] Embodiment 16. The conjugate of any one of embodiments 12-14, comprising a PEG(n)-thiocholesterol, where n is 2-36, and optionally n is 4, 5, 6, 7, or 8. In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0294] Embodiment 17. A structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), a PEG(4)-cholesterol moiety linked to the C-terminal lysine of a conformationally stabilized polypeptide; A conjugate comprising:

[0295] Embodiment 18. The PEG(4)-cholesterol moiety has the formula: [ka] 18. The conjugate of embodiment 17, comprising:

[0296] Embodiment 19. A structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), a PEG(4)-thiocholesterol moiety linked to the C-terminal lysine of a conformationally stabilized polypeptide; A conjugate comprising:

[0297] Embodiment 20. The PEG(4)-thiocholesterol moiety has the formula: [ka] 20. The conjugate of embodiment 19, comprising:

[0298] Embodiment 21. A structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), a PEG(8)-cholesterol moiety linked to the C-terminal lysine of a conformationally stabilized polypeptide; A conjugate comprising:

[0299] Embodiment 22. The PEG(8)-cholesterol moiety has the formula: [ka] 22. The conjugate of embodiment 21, comprising:

[0300] Embodiment 23. A structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), a PEG(8)-thiocholesterol moiety linked to the C-terminal lysine of a conformationally stabilized polypeptide; A conjugate comprising:

[0301] Embodiment 24. The PEG(8)-thiocholesterol moiety has the formula: [ka] 24. The conjugate of embodiment 23, comprising:

[0302] Embodiment 25. A structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), a PEG(n)-cholesterol moiety (where (n)=4, 5, 6, 7, or 8) linked to the C-terminal lysine of a structurally stabilized polypeptide; In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0303] Embodiment 26. The PEG(n)-cholesterol moiety has the formula: [ka] (wherein n=4, 5, 6, 7, or 8) In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0304] Embodiment 27. A structurally stabilized polypeptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), a PEG(n)-thiocholesterol moiety (where (n)=4, 5, 6, 7, or 8) linked to the C-terminal lysine of a structurally stabilized polypeptide; In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0305] Embodiment 28. The PEG(n)-thiocholesterol moiety has the formula: [ka] (wherein n=4, 5, 6, 7, or 8) In some cases, n=4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

[0306] Embodiment 29. The formula: [ka] or a pharma- ceutically acceptable salt thereof, each R1 and R2 is H or a C1-C10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted; each R3 is independently an alkane, alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Each [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 34), and each [Xaa] x is EIDRLN (SEQ ID NO: 35), and each [Xaa] y is VAKNLNESLIDLQELGK (SEQ ID NO: 36) And, the structurally stabilized peptide inhibits infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays and / or prevents infection of cells by SARS-CoV-2 in pseudovirus and / or live SARS-CoV-2 virus assays; A conformationally stabilized peptide or a pharma- ceutically acceptable salt thereof.

[0307] Embodiment 30. The structurally stabilized peptide of embodiment 29, or a pharma- ceutically acceptable salt thereof, wherein R1 is alkyl.

[0308] Embodiment 31. The structurally stabilized peptide of embodiment 29, or a pharma- ceutically acceptable salt thereof, wherein R1 is a methyl group.

[0309] Embodiment 32. The structurally stabilized peptide of embodiment 29, or a pharma- ceutically acceptable salt thereof, wherein R3 is alkyl.

[0310] Embodiment 33. The structurally stabilized peptide of embodiment 29, or a pharma- ceutically acceptable salt thereof, wherein R3 is a methyl group.

[0311] Embodiment 34. The structurally stabilized peptide of embodiment 29, or a pharma- ceutically acceptable salt thereof, wherein R2 is alkenyl.

[0312] Embodiment 35. The structurally stabilized peptide or pharma- ceutically acceptable salt thereof of any one of embodiments 29-34, wherein the pharma- ceutically acceptable salt comprises hydrochloride, sodium, sulfate, acetate, phosphate or diphosphate, chloride, potassium, maleate, calcium, citrate, mesylate, nitrate, tartrate, aluminum, gluconate, and any combination thereof.

[0313] Embodiment 36. The structurally stabilized peptide of any one of embodiments 29 to 35, or a pharma- ceutically acceptable salt thereof, which is at most 50 amino acids in length, optionally at most 45 amino acids in length.

[0314] Embodiment 37. The structurally stabilized peptide of any one of embodiments 29 to 36, or a pharma- ceutically acceptable salt thereof, which is 38 amino acids in length.

[0315] Embodiment 38. A conjugate comprising the structurally stabilized peptide of any one of embodiments 29 to 37 or a pharma- ceutically acceptable salt thereof and PEG and / or cholesterol.

[0316] Embodiment 39 The conjugate of embodiment 38, comprising PEG and cholesterol.

[0317] Embodiment 40 The conjugate of embodiment 38 or 39, wherein the cholesterol is thiocholesterol.

[0318] Embodiment 41. The conjugate of embodiment 38 or 39, comprising PEG(n)-cholesterol, optionally where n is 1 to 36, optionally where n is 4, 5, 6, 7, or 8.

[0319] Embodiment 42. The conjugate of any one of embodiments 38 to 40, comprising PEG(n)-thiocholesterol, where n is 1 to 36, and optionally n is 4, 5, 6, 7, or 8.

[0320] Embodiment 43. A pharmaceutical compound comprising a structurally stabilized peptide of any one of embodiments 1 to 42, a pharma- ceutically acceptable salt, or a conjugate thereof, and a pharma- ceutically acceptable carrier.

[0321] Embodiment 44. A method of treating a coronavirus infection in a human subject in need thereof, comprising administering to the human subject a therapeutically effective amount of a structurally stabilized peptide, a pharma- ceutically acceptable salt thereof, or a conjugate thereof of any one of embodiments 1 to 43.

[0322] Embodiment 45. A method for preventing a coronavirus infection in a human subject in need thereof, comprising administering to the human subject a therapeutically effective amount of a structurally stabilized peptide, a pharma- ceutically acceptable salt thereof, or a conjugate thereof of any one of embodiments 1 to 43.

[0323] Embodiment 46. A method of treating a coronavirus infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a structurally stabilized peptide, pharma- ceutically acceptable salt, or conjugate thereof of any one of embodiments 1 to 43, wherein the subject is selected from a cow, a pig, a horse, a cat, a dog, a rat, a mouse, or a bat.

[0324] Embodiment 47. A method for preventing a coronavirus infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a structurally stabilized peptide, a pharmaceutically acceptable salt thereof, or a conjugate of any one of embodiments 1 to 43, wherein the subject is selected from a cow, a pig, a horse, a cat, a dog, a rat, a mouse, or a bat.

[0325] Embodiment 48 The method of embodiment 46 or 47, wherein the coronavirus infection is due to a betacoronavirus.

[0326] Embodiment 49. The method of any one of embodiments 46-48, wherein the coronavirus infection is due to infection with SARS-CoV-2.

[0327] Embodiment 50. The method of any one of embodiments 46-49, wherein the coronavirus infection is due to infection with a variant of SARS-CoV-2.

[0328] Embodiment 51. The method of embodiment 50, wherein the variant is selected from Wuhan-Hu-1, B.1.427 / B.1.429, B.1.617.2, D614G B.1, or the Brazilian variant P.1.

[0329] Embodiment 52. A method for making a structurally stabilized peptide, comprising: (a) providing a peptide having a sequence as set forth in SEQ ID NO:6 or a variant thereof; and (b) cross-linking the peptide, and, optionally, purifying the structurally stabilized peptide.

[0330] Embodiment 53 The method of embodiment 52, wherein the step of crosslinking the peptides is by a ruthenium-catalyzed metathesis reaction.

[0331] Embodiment 54 The method of embodiment 52 or 53, further comprising formulating the structurally stabilized peptide as a sterile pharmaceutical composition.

[0332] Embodiment 55. A method for synthesizing a conjugate comprising a structurally stabilized polypeptide of any one of embodiments 1-11 or 29-37, comprising the steps of: (a) providing a structurally stabilized polypeptide; and (b) derivatizing a resin-bound amine of the structurally stabilized polypeptide on the resin with a carboxylic acid-containing PEG and / or cholesterol.

[0333] Embodiment 56. A method for synthesizing a conjugate comprising a heptad repeat domain 2 (HR2) polypeptide, comprising the steps of: (a) providing a HR2 polypeptide; and (b) derivatizing the resin-bound amine of the HR2 polypeptide on the resin with a carboxylic acid-containing PEG and / or cholesterol.

[0334] Embodiment 57. A method for synthesizing a conjugate comprising a structurally stabilized polypeptide of any one of embodiments 1-11 or 29-37, comprising the steps of: (a) providing a structurally stabilized polypeptide; and (b) derivatizing a resin-bound amine of the structurally stabilized polypeptide on the resin with a carboxylic acid-containing PEG and / or thiocholesterol.

[0335] Embodiment 58. A method of synthesizing a conjugate comprising an HR2 polypeptide, comprising the steps of: (a) providing an HR2 polypeptide; and (b) derivatizing the resin-bound amine of the HR2 polypeptide on the resin with a carboxylic acid-containing PEG and / or thiocholesterol.

[0336] Embodiment 59. The derivatizing step comprises: Producing a solution by dissolving thiocholesterol in dichloromethane (DCM) or cholesterol in tetrahydrofuran (THF); and adding a base, t-butyl ester of bromoacetic acid, and trifluoroacetic acid to the solution in sequence; or incorporating carboxythiocholesterol or carboxycholesterol by solid phase synthesis by Alternatively, the combined steps can be carried out sequentially after work-up or column chromatography. The method of any one of embodiments 55 to 58.

[0337] Embodiment 60. The derivatizing step comprises: treating the resin-bound conformationally stabilized polypeptide with piperidine in a solution containing dimethylformamide (DMF); capping the N-terminus of the structurally stabilized polypeptide with acetic anhydride; deprotecting the C-terminus of the structurally stabilized polypeptide with hydrazine in DMF; acylation of the structurally stabilized polypeptide with an Fmoc-protected PEG(n) amino acid; crosslinking the conformationally stabilized polypeptide; and Isolating the conformationally stabilized polypeptide from the resin 60. The method of any one of embodiments 55-59, further comprising:

[0338] Embodiment 61. The method of embodiment 60, wherein n=1 to 36.

[0339] Embodiment 62. The method of embodiment 60 or 61, wherein n=4, 5, 6, 7, or 8.

[0340] Embodiment 63. The method of any one of embodiments 55-62, wherein the C-terminal lysine of SEQ ID NO:6 is substituted with a resin-bound amine, and optionally, the C-terminal lysine of SEQ ID NO:6 is further substituted with a resin-bound carboxylic acid or thiol.

[0341] Embodiment 64 The method of any one of embodiments 55 to 63, wherein the cholesterol is thiocholesterol.

[0342] Embodiment 65 The method of any one of embodiments 55 to 63, wherein the conjugate comprises PEG(n)-cholesterol, where n is 1 to 36, and optionally n is 4, 5, 6, 7, or 8.

[0343] Embodiment 66 The method of any one of embodiments 55 to 64, wherein the conjugate comprises PEG(n)-thiocholesterol, where n is 1 to 36, and optionally n is 4, 5, 6, 7, or 8.

[0344] Embodiment 67. The method of any one of embodiments 55 to 66, wherein crosslinking the peptide is by a ruthenium-catalyzed metathesis reaction.

[0345] Embodiment 68. A nanoparticle composition comprising a structurally stabilized peptide of any one of embodiments 1 to 43, a pharma- ceutically acceptable salt, or a conjugate thereof, optionally in the form of a PLGA nanoparticle, and optionally further comprising a lactic acid:glycolic acid ratio of the PLGA nanoparticles in the range of 2:98 to 100:0.

[0346] Embodiment 69. The nanoparticle composition of embodiment 68, further comprising chitosan, dextrin, or both.

[0347] Embodiment 70. Formula [ka] (wherein n is 1 to 36). A linker comprising PEG(n)-thiocholesterol.

[0348] Embodiment 71. Formula [ka] (wherein n is 1 to 36). A linker comprising PEG(n)-cholesterol.

[0349] The linker of embodiment 70 or 71, wherein n is 4, 5, 6, 7, or 8.

[0350] Embodiment 73 The linker of any one of embodiments 70 to 72, further comprising a lysine attached to the PEG.

[0351] 74. Each of the embodiments 8 = (R)-α-(7'-octenyl)alanine or (R)-α-(4'-pentenyl)alanine; and X = (S)-α-(4'-pentenyl)alanine or (S)-α-(7'-octenyl)alanine The structurally stabilized peptide of any one of embodiments 1 to 11 or the conjugate of any one of embodiments 12 to 28, 75. 8 = (R)-α-(7'-octenyl)alanine; and X = (S)-α-(4'-pentenyl)alanine The structurally stabilized peptide of any one of embodiments 1 to 11 or the conjugate of any one of embodiments 12 to 28, EXAMPLES

[0352] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. When specific materials are mentioned, they are merely for illustrative purposes and are not intended to limit the invention. Those skilled in the art may devise equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0353] Example 1 Design and synthesis of stapled SARS-CoV-2 peptides derivatized with C-terminal PEG(n)-thiocholesterol or PEG(n)-cholesterol moieties To design peptides capable of blocking coronavirus-host cell fusion (FIG. 1), a series of stapled peptides with differentially localized chemical staples, derivatized at the C-terminus with PEG(n)-thiocholesterol or PEG(n)-thiocholesterol moieties, were designed and then synthesized on resin by solid phase synthesis. Differentially localized chemical staples were positioned within the SARS-CoV-2 HR2 domain (i.e., amino acids 1168-1205) of the sequence of the surface (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (see Figures 2-4), preferably within the alpha-helical region (i.e., amino acids 1179-1197; see Figure 9), by replacing native residues with α,α-disubstituted non-natural olefin residues in the form of double staples or stitches (e.g., S-pentenylalanine incorporated at "X" and R-octenylalanine incorporated at "8" at selected i, i+7 positions, or S-pentenylalanine incorporated at "X" at each of selected i, i+4 positions) and combinations thereof, followed by ruthenium-catalyzed olefin metathesis (see Figures 5-7). Our approach to designing, synthesizing and identifying optimal stapled peptide constructs targeting the SARS-CoV-2 fusion machinery includes Ala-scanning (e.g., mutants), staple-scanning, and generation of variable N- and C-terminal deletion, addition and derivatization libraries for conjugation with PEG-thiocholesterol or PEG-cholesterol moieties (see FIG. 8). Some preferred designs incorporate staples into the non-interacting amphipathic face of the core SARS-CoV-2 HR2 helix (e.g., SEQ ID NO: 4) or at the interface of the hydrophobic interacting face and the amphipathic face of the helix (see FIG. 9).

[0354] Stapled SARS-CoV-2 HR2 constructs with C-terminal derivatization with PEG(n)-thiocholesterol or PEG(n)-cholesterol moieties were designed by replacing two naturally occurring amino acids at positions i,i+7 (i.e., seven adjacent amino acids) with the unnatural (R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-dec-9-enoic acid (R8) and S-2-(4'-pentenyl)alanine (S5) amino acids to generate a staple that spans two α-helical turns, or by replacing two naturally occurring amino acids at positions i,i+4 with two S5 unnatural amino acids to generate a staple that spans one α-helical turn. Asymmetric synthesis of 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, each of which is incorporated by reference in its entirety).

[0355] "Staple scanning" was performed to identify residues and binding surfaces crucial for the interactions that dictated the design of the optimized constructs and negative control mutants, respectively. Peptide N-termini were capped with acetyl or fluorophores (e.g., FITC, rhodamine) depending on the experimental application.

[0356] Doubly stapled peptides were generated by incorporating two S5-S5, two R8-S5, or other combinations of bridging unnatural amino acids. Multiply stapled or stitched peptides are generated using similar principles.

[0357] To allow for on-resin peptide derivatization with thiocholesterol or cholesterol, carboxy-thiocholesterol or carboxy-cholesterol were synthesized according to the procedure described above (see Methods and Figure 10). The completed resin-bound peptide was capped with an acetyl group (by use of acetic anhydride) followed by deprotection of the C-terminal side chain lysine amine by treatment with 2% hydrazine. The amine was acylated with an Fmoc-protected PEG(n) amino acid (e.g., n=1-36, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36), at which point the olefin was crosslinked by treatment with Grubbs(I) catalyst. Fmoc was removed from the C-terminal NH of the PEG(n) amino acid, and the amine was acylated with carboxy-thiocholesterol or carboxy-cholesterol. The final peptide products were obtained after peptide deprotection and cleavage, and purification by reversed-phase high performance liquid chromatography / mass spectrometry (LC / MS). See full synthetic scheme in Figures 11A-11B. Exemplary i,i+7 stapled SARS-CoV-2 HR2 peptides derivatized with PEG(n)-thiocholesterol and PEG(n)-cholesterol, generated using the synthetic scheme, are listed in Figure 12 (SEQ ID NOs: 7-21).

[0358] Example 2 Identification of optimally stapled SARS-CoV-2 HR2 peptides with C-terminal PEG4-thiocholesterol to achieve antiviral activity in pseudotype and live virus assays We demonstrated for the first time that applying our chemical scheme to add a PEG4-thiocholesterol moiety to the C-terminus of SEQ ID NO:5 (non-stapled peptide with a C-terminal amide) to generate SEQ ID NO:6 (non-stapled peptide with a C-terminal PEG4-thiocholesterol) converted the inactive HR2 peptide into an active construct that exhibited dose-responsive antiviral activity in a pseudovirus assay (see Methods) using Wuhan-Hu-1 pseudovirus, 293T-ACE2 cells, and two-fold serial dilutions of the peptide starting at 500 nM (see FIG. 13). We then demonstrated that the stapled HR2 peptide (staple D, SEQ ID NO: 10) with PEG4-thiocholesterol moieties added on the resin exhibited consistent and potent antiviral activity in pseudovirus assays whether cells were treated with the peptide (1 μM) before or after virus inoculation across a range of SARS-CoV-2 pseudovirus variants, including D614G B.1, Wuhan-Hu-1, B.1.526, B.1.427, and B.1.1.7 (see FIG. 14). Meanwhile, the corresponding non-stapled peptide with PEG4-thiocholesterol moieties (SEQ ID NO: 6) was ineffective when applied after virus inoculation and exhibited lower antiviral activity compared to the stapled sequence even when applied before virus inoculation with the same range of SARS-CoV-2 pseudovirus variants (see FIG. 14).

[0359] We compared a non-stapled construct (SEQ ID NO: 6) with two stapled constructs (SEQ ID NO: 10 and 17) bearing i,i+7 staples on the non-interacting hydrophobic face of the HR2 helix in a pseudovirus assay (pseudovirus: B.1.526; cells: 293T-ACE2; serial dilutions starting at 1 mM). The stapled HR2 peptide of SEQ ID NO: 10 (staple D) exhibited the most potent, dose-responsive antiviral activity, followed by the non-stapled peptide of SEQ ID NO: 6; notably, the stapled peptide of SEQ ID NO: 17 (staple K) was the least active in the pseudovirus assay, even though its staples are also located on the non-interacting face of the HR2 helix (see FIG. 15). These data clearly demonstrate that identifying optimal staple locations is unpredictable and this (these) locations must be determined empirically.

[0360] To extend this analysis, we next evaluated the differential antiviral activity of a series of i,i+7 stapled HR2 peptides with a C-terminal PEG4-thiocholesterol moiety added on the resin. In a SARS-CoV-2 pseudovirus assay (pseudovirus: D614G B.1; cells: 293T-ACE2; peptide doses of 100, 300, 1000 nM), peptides with SEQ ID NOs: 11, 14, and 15 (staples E, H, I, respectively) showed little to no activity, peptides with SEQ ID NOs: 13 and 16 (staples G and J, respectively) exhibited moderate activity, whereas peptide with SEQ ID NO: 10 (staple D) stood out as having uniquely strong activity among the various stapled HR2 peptides (see FIG. 16). We then sought to demonstrate this hierarchy of peptide activity in a live SARS-CoV-2 assay. We tested the differential antiviral activity of the same series of i,i+7 stapled HR2 peptides bearing a C-terminal PEG4-thiocholesterol moiety. In this SARS-CoV-2 live virus assay (live virus: USA-WA1 / 2020; cells: VeroB6; peptide dose range 8-1000 nM), peptides SEQ ID NOs: 11, 14, 15 (staples E, H, I) showed little to no activity, peptides SEQ ID NOs: 13, 16 (staples G, J) displayed moderate activity, whereas peptide SEQ ID NO: 10 (staple D) stood out as having uniquely potent activity among the various stapled HR2 peptides, consistent with the pseudovirus assay results shown in Figure 16 (see Figure 17). By performing a full i,i+7 staple scan of the alpha helical portion of the HR2 sequence and then testing the resulting constructs in a live virus assay using a SARS-CoV-2 beta strain, we again found that SEQ ID NO:10 (staple D) exhibited one of the most potent antiviral activities, along with three additional staple positions (SEQ ID NOs:13, 17 and 20, with staple positions G, K and N, respectively) (Figure 18A).A select few constructs (SEQ ID NOs: 10, 13, 17, 20) that yielded strong antiviral activity were distributed and colocalized in localized regions on the HR2 alpha helix surface, as demonstrated by helical wheel drawing (Figure 18B). Among the SARS-CoV-2 variants tested in the pseudovirus assay, the stapled lipopeptide corresponding to SEQ ID NO: 10 (staple D) with a C-terminal PEG4 thiocholesterol moiety was the most active against the omicron variant (Figure 19) and also exhibited strong antiviral activity against SARS-CoV-2 live virus strains, including beta and delta (Figure 20). Additional constructs corresponding to SEQ ID NOs: 13 and 20 with a C-terminal PEG4 thiocholesterol moiety also exhibited consistent antiviral activity against diverse SARS-CoV-2 variants and SARS-CoV-1 pseudoviruses as well as live SARS-CoV-2 beta strain viruses (Figures 21-23). Mutagenesis within the HR2 sequence, which alternatively contacts the HR1 core or is solvent exposed (Figure 24A), is tolerated, as evidenced by retention of antiviral potency in a SARS-CoV-1 pseudovirus assay, as demonstrated for an exemplary alanine substitution in SEQ ID NO: 10 bearing a C-terminal PEG4 thiocholesterol moiety (Figure 24B).

[0361] Taken together, these data demonstrate that (1) the addition of a PEG4-thiocholesterol moiety to the C-terminus of the HR2 peptide using on-resin methodology can confer potent, dose-responsive antiviral activity; (2) stapling can not only enhance peptide activity relative to non-stapled analogs, but can also achieve antiviral activity whether administered prior to or after virus inoculation in culture; and (3) the supporting pseudovirus and live virus assays enabled the identification of a select few uniquely potent stapled HR2 peptides (SEQ ID NOs: 10, 13, 17, and 20) from a full panel of lesser or no i,i+7 stapled differentially peptides, highlighting that identification of optimal staple positions for achieving potent antiviral activity is unpredictable and must be experimentally determined in SARS-CoV-2 antiviral assays.

[0362] Example 3 Determination of the optimal PEG linker length within a stapled SARS-CoV-2 HR2 peptide with C-terminal PEG(n)-thiocholesterol to achieve antiviral activity in pseudotype and live virus assays To determine the optimal PEG chain length for linking stapled HR2 peptides to thiocholesterol or cholesterol moieties, a series of PEG(n) analogs were generated according to the synthesis method described above, with n being 3, 4, 5, 6, 7, and 8. The antiviral activity of this series of i,i+7 stapled HR2 peptides with SEQ ID NO:10 (staple D) and thiocholesterol spaced by variable length PEG linkers was examined in a pseudovirus assay across a series of five SARS-CoV-2 variants (pseudoviruses: Wuhan-Hu-1, B.1.427 / B.1.429, B.1.617.2, D614G B.1, Brazilian variant P.1; cells: 293T-ACE2: serial dilutions starting at 1 μM). The peptide of SEQ ID NO:10 (staple D) with a PEG8 linker exhibited the most potent dose-responsive activity in this pseudovirus assay (see Figures 25A-25E).

[0363] Next, we sought to demonstrate a hierarchy of peptide activity based on PEG(n) linker length in a live SARS-CoV-2 assay. We tested the differential antiviral activity of the same series of i,i+7 stapled HR2 peptides of SEQ ID NO: 10 with PEG(n) linkers of n=3, 4, 5, 6, 7 or 8 PEG moieties and an added C-terminal thiocholesterol (Staple D) assembled on resin in a SARS-CoV-2 live virus assay (live virus: South African B.1.351; cells: VeroB6; peptide 2-fold serial dilution dose range 1000-4 nM). With regard to the pseudovirus assay, constructs with PEG8 linker moieties presented the most potent dose-responsive activity in this SARS-CoV-2 live virus assay (see Figure 26).

[0364] Next, we generated a series of i,i+7 stapled HR2 peptides of SEQ ID NO: 10 (staple D) with longer PEG(n) linkers corresponding to n=10, 12, 14, 16 and 20. Across SARS-CoV-2 Omicron (FIG. 27) and SARS-CoV-1 (FIG. 28) pseudovirus assays, as well as live SARS-CoV-2 beta strain (FIG. 29) virus assays, the longer linker lengths were as effective as the PEG8 linker length, and in some cases even more effective. In summary, we found that the length of the PEG linker between the stapled HR2 peptide and the thiocholesterol derivatization can be optimized to maximize antiviral activity, with PEG8 and longer PEG lengths having the highest activity as evidenced across pseudovirus and live virus assays, and PEG0 and PEG3 showing the least potent activity compared to the rest of the series of constructs. The superiority of PEG8 and longer linkers, and the inferiority of PEG3 compared to PEG4-8 and longer species, was unexpected and required experimental determination in SARS-CoV-2 antiviral assays.

[0365] Example 4 Specificity of the Antiviral Mechanism of Action of Stapled SARS-CoV-2 HR2 Peptides with C-Terminal PEG(n)-Thiocholesterol To investigate the specificity of the peptides of the invention against the SARS-CoV-2 virus, a series of i,i+7 stapled SARS-CoV-2 HR2 peptides (staple D, SEQ ID NO: 10) with PEG(n)-thiocholesterol moieties of variable PEG length (n=3-8) were tested in a pseudoviral assay of VSV with an MLV core. The corresponding non-stapled peptides exhibited some non-specific antiviral activity (2.5 μM dosing, 48 hours), but none of the stapled HR2 constructs with PEG(n)-thiocholesterol with n=3, 4, 5, 6, 7 or 8 PEG linkers exhibited any anti-VSV pseudoviral activity (see FIG. 30).

[0366] Next, we investigated whether the non-stapled or stapled SARS-CoV-2 HR2 peptides of the invention with PEG(n)-thiocholesterol moieties synthetically produced on resin would exhibit any cytotoxicity within the antiviral dose range. Comparing the peptide of SEQ ID NO:6 of the invention to an alternatively synthesized non-stapled HR2 construct with a GSGSGC linker and PEG4-cholesterol added to the C-terminus in solution (DeVries et al, Science, 2021), we see that the non-stapled HR2 peptide of SEQ ID NO:6 produced according to our on-resin method above exhibited relatively enhanced antiviral activity in a live virus assay (live virus: South African B.1.351; cells: VeroB6: peptide dose range 4-1000 nM) (see Figure 31). Additionally, the non-stapled HR2 peptide of SEQ ID NO:6 and the corresponding i,i+7 stapled HR2 peptide of SEQ ID NO:10 (staple D, PEG4-thiocholesterol) showed no cytotoxic activity over the antiviral dose range as measured by DRAQ7 and Hoechst 33342 staining, whereas the non-stapled HR2 peptide with a GSGSGC linker and PEG4-cholesterol attached to the C-terminus (DeVries et al, Science, 2021) killed cells within the dosing range (pseudovirus: D614G B.1; cells: 293T-ACE2; 2-fold serial dilutions starting at 1 μM; readout: 48 hours) (see Figure 32).

[0367] Example 5 SARS-CoV-2 5-HB Binding Activity and Broad SARS-CoV-2 Antiviral Activity across Variants of Stapled SARS-CoV-2 HR2 Peptides with C-Terminal PEG8-Chol Direct fluorescence polarization binding assays revealed low nanomolar binding affinity of a stapled lipopeptide of the composition corresponding to SEQ ID NO: 10, derivatized at the C-terminus with PEG8-Chol and at the N-terminus with FITC-β-Ala instead of acetyl, to a recombinant five-helix bundle of SARS-CoV-2 (5-HB), where the fusogenic six-helix bundle is completed by the addition of the FITC-stapled lipopeptide (peptide: 5 nM; sequence [ka] 5-HB protein of 1000 nM (serial dilution) (Figure 33). Stapled lipopeptide of SEQ ID NO: 10 derivatized at the C-terminus with PEG8-Chol exhibited potent antiviral activity against (a) a wide variety of SARS-CoV-2 variant pseudoviruses (293-ACE2 cells, GFP-expressing pseudovirus, peptide (serial dilution) from 1000 nM, 48 hour readout) (Figure 34); (b) omicron variants (293T-ACE2 cells, GFP-expressing pseudovirus, peptide (serial dilution) from 250 nM, 48 hour readout) (Figure 35); (c) SARS-CoV-2 beta and delta live viruses (Vero cells, peptide (serial dilution) from 100 nM, 48 hour readout) (Figure 36); and (d) common human coronaviruses, e.g., alphacoronavirus NL63 (Figure 37).

[0368] Example 6 Preparation of stapled SARS-CoV-2 peptides derivatized with a C-terminal PEG(8)-cholesterol moiety The following compound was prepared based on the procedure described in Example 1 above: A structurally stabilized peptide conjugate represented by the formula: [ka] (In the formula, R4 is -C(O)-(CH2CH2)-[O-CH2CH2]8-N(H)C(O)-(CH2)-R6; R6 is, [ka] and; [Xaa] w is DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] x is EIDRLN (SEQ ID NO:36); and [Xaa] z is VAKNLNESLIDLQELG (SEQ ID NO: 77).

[0369] Other embodiments Although the present invention has been described in conjunction with its detailed description, the above description is intended to be illustrative of the invention and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A structurally stabilized peptide comprising an amino acid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 94% identical to the sequence set forth in SEQ ID NO: 6 (DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK), (i) positions 14 and 21; (ii) positions 17 and 24; (iii) positions 21 and 28, or (iv) positions 24 and 31 wherein position 1 of SEQ ID NO:6 is an N-terminal aspartic acid and position 38 is a C-terminal lysine, and an amino acid at a position selected from the group consisting of: the structurally stabilized peptide is 38 to 60 amino acids in length, optionally 38 to 50 amino acids in length; The structurally stabilized peptide inhibits infection of a cell by SARS-CoV-2 in a SARS-CoV-2 pseudovirus and / or a live SARS-CoV-2 virus assay, and / or the structurally stabilized peptide inhibits infection of a cell by SARS-CoV-2 in the SARS-CoV-2 pseudovirus and / or a live SARS-CoV-2 virus assay, and optionally the amino acid sequence comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 10, 13, 17, and 20.

2. formula: 【Chemistry 132】 or a pharmaceutically acceptable salt thereof, wherein Each R １ and R ２ is H or C １ ~C １０ alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted; Each R ３ is independently an alkane, alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; (a) each [Xaa] ｗ is DISGINASVVNIQ (SEQ ID NO: 35), and each [Xaa] ｘ is EIDRLN (SEQ ID NO: 36), and each [Xaa] ｙ is VAKNLNESLIDLQELGK (SEQ ID NO: 37); (b) each [Xaa] w is DISGINASVVNIQKEI (SEQ ID NO:44), each [Xaa] x is RLNEVA (SEQ ID NO:45), and each [Xaa] y is NLNESLIDLQELGK (SEQ ID NO:46); (c) each [Xaa] w is DISGINASVVNIQKEIDRLN (SEQ ID NO: 56), each [Xaa] x is VAKNLN (SEQ ID NO: 57), and each [Xaa] y is SLIDLQELGK (SEQ ID NO: 58); or (d) each [Xaa] w is DISGINASVVNIQKEIDRLNEVA (SEQ ID NO: 65), each [Xaa] x is NLNESL (SEQ ID NO: 66), and each [Xaa] y is DLQELGK (SEQ ID NO: 67); the structurally stabilized peptide inhibits infection of cells by SARS-CoV-2 in a SARS-CoV-2 pseudovirus and / or live SARS-CoV-2 virus assay and / or prevents infection of cells by SARS-CoV-2 in the SARS-CoV-2 pseudovirus and / or live SARS-CoV-2 virus assay; A structurally stabilized peptide or a pharmaceutically acceptable salt thereof.

3. 3. The structurally stabilized peptide of claim 2, comprising formula IA, or a pharmaceutically acceptable salt thereof, wherein formula IA is: 【Chemistry 133】 or a pharmaceutically acceptable salt thereof; R ３ is -(CH 2 ) 6 -CH═CH-(CH 2 ) 3; (a) [Xaa] ｗ is DISGINASVVNIQ (SEQ ID NO: 35); ｘ is EIDRLN (SEQ ID NO: 36); [Xaa] ｙ is VAKNLNESLIDLQELGK (SEQ ID NO: 37); (b) [Xaa] w is DISGINASVVNIQKEI (SEQ ID NO:44), [Xaa] x is RLNEVA (SEQ ID NO:45), and [Xaa] y is NLNESLIDLQELGK (SEQ ID NO:46); (c) [Xaa] w is DISGINASVVNIQKEIDRLN (SEQ ID NO: 56), [Xaa] x is VAKNLN (SEQ ID NO: 57), and [Xaa] y is SLIDLQELGK (SEQ ID NO: 58); or (d) [Xaa] w is DISGINASVVNIQKEIDRLNEVA (SEQ ID NO: 65), [Xaa] x is NLNESL (SEQ ID NO: 66), and [Xaa] y is DLQELGK (SEQ ID NO: 67); [Xaa] ｗ The amino group of the N-terminal aspartic acid in [Xaa] is replaced with —N(H)C(O)CH 3. ｙ The carboxylic acid group of the C-terminal lysine in ２ has been replaced by A structurally stabilized peptide or a pharmaceutically acceptable salt thereof.

4. 4. The structurally stabilized peptide of claim 3, comprising the formula IA, or a pharmaceutically acceptable salt thereof.

5. 【Chemical 170】 4. The structurally stabilized peptide of claim 3, comprising or consisting of: or a pharmaceutically acceptable salt thereof.

6. The structurally stabilized polypeptide of claim 1, comprising the amino acid sequence set forth in SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK), wherein 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group, or a variant thereof having one amino acid substitution in SEQ ID NO:

10. (a) 38 to 45 amino acids in length; or (b) is 38-40 amino acids in length; 2. The structurally stabilized polypeptide of claim 1.

8. A structurally stabilized polypeptide as described in claim 1, wherein positions 37 and / or 38 of SEQ ID NO: 6 are not substituted.

9. A structurally stabilized peptide conjugate represented by Formula VI, or a pharmaceutically acceptable salt thereof, wherein Formula VI is 【158】 is defined by: R ４ is -C(O)-(CH ２ CH ２ )-[O-CH ２ CH ２ ] ８ -N(H)C(O)-(CH ２ )-R ６ and R ６ teeth, 【Chemistry 159】 and [Xaa] ｗ is DISGINASVVNIQ (SEQ ID NO: 35); [Xaa] ｘ is EIDRLN (SEQ ID NO: 36); and [Xaa] ｚ is VAKNLNESLIDLQELG (SEQ ID NO: 77); Structurally stabilized peptide conjugates.

10. A conjugate comprising: (i) a structurally stabilized polypeptide according to any one of claims 1 and 6 to 8 and polyethylene glycol (PEG) and / or cholesterol, wherein the PEG and / or cholesterol are linked to the C-terminus of the structurally stabilized polypeptide; or (ii) a structurally stabilized peptide according to any one of claims 2 to 6, or a pharmaceutically acceptable salt thereof, and PEG and / or cholesterol; Optionally, the conjugate (i) or (ii) comprises PEG and cholesterol. (a) the cholesterol is thiocholesterol; or (b) the conjugate comprises PEG(n)-cholesterol, where n is 1-36, and optionally n is 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36; The conjugate of claim 10.

12. The conjugate of claim 1, (i) PEG(n)-thiocholesterol, wherein n is 2 to 36, and optionally n is 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, according to the conjugate of claim 10; or (ii) PEG(n)-thiocholesterol, wherein n is 1 to 36, and optionally n is 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, in accordance with the conjugate of claim 10; The conjugate of claim 10.

13. A conjugate comprising: a structurally stabilized peptide of SEQ ID NO: 10 (DISGINASVVNIQ8EIDRLNXVAKNLNESLIDLQELGK) in which 8 is an (R)-α-(7'-octenyl)alanine group and X is an (S)-α-(4'-pentenyl)alanine group; a PEG(8)-cholesterol moiety linked to the C-terminal lysine of the structurally stabilized peptide; Including, Optionally, the PEG(8)-cholesterol moiety has the formula: 【126】 , a conjugate comprising:

14. A pharmaceutical compound comprising: (i) a structurally stabilized peptide according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, or a conjugate according to claim 13; or (ii) a structurally stabilized peptide conjugate according to claim 9, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

15. 13. A composition comprising the structurally stabilized peptide of claim 1, or a pharmaceutically acceptable salt thereof, the conjugate of claim 13, or the structurally stabilized peptide conjugate of claim 9, or a pharmaceutically acceptable salt thereof, for use in treating or preventing a coronavirus infection in a human subject in need thereof, wherein optionally the composition is administered intranasally to the human subject.

16. The coronavirus infection is (a) Betacoronavirus; (b) SARS-CoV-2; and / or (c) SARS-CoV-2 variants and optionally the variant is selected from Wuhan-Hu-1, B.1.427 / B.1.429, B.1.617.2, D614G B.1, Brazilian variant P.1, B.1.1.7, B.1.351, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, B.1.621.1, B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4 and BA.5, and further optionally the variant is selected from B.1.351, cluster 5, lineage B.1.1.207, lineage B.1.1.208, lineage B.1.1.209, lineage B.1.1.351, cluster 5 ...351, lineage B.1.1.351, cluster 5, lineage B.1.1.209, lineage B.1.1.351, cluster 5, lineage B.1.1.351, lineage B.1.1.351, cluster 5, lineage B.1.1.351, lineage B.1.1.351, cluster 5, lineage B.1.1.351, lineage 16. The composition of claim 15, wherein the variant is selected from: B.1.1.7, Variant of Concern 202102 / 02, lineage B.1.1.317, lineage B.1.1.318, lineage B.1.351, lineage B.1.429, lineage B.1.525, lineage P.1 (also known as lineage B.1.1.28), lineage B.1.1.529, lineage BA.1, lineage BA.1.1, lineage BA.2, lineage BA.3, lineage BA.4 lineage BA.5, D614G, E484K, N501Y, S477G / N, and P681H.

17. 14. A nanoparticle composition comprising the structurally stabilized peptide of any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, the structurally stabilized peptide conjugate of claim 9, or a pharmaceutically acceptable salt thereof, or the conjugate of claim 13, optionally wherein the nanoparticle composition is a PLGA nanoparticle, optionally further comprising a lactic acid:glycolic acid ratio of the PLGA nanoparticles in the range of 2:98 to 100:0, and optionally wherein the nanoparticle composition further comprises chitosan, dextrin, or both.

18. Formula (i) 【168】 PEG(n)-thiocholesterol comprising: (ii) Formula 【169】 wherein n is 1-36, optionally n is 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, and / or further comprises a lysine attached to said PEG.

19. A composition comprising a structurally stabilized peptide according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, a conjugate according to claim 13, or a structurally stabilized peptide conjugate according to claim 9, or a pharmaceutically acceptable salt thereof, for use in therapy.

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