Respiratory syncytial virus surface glycoprotein peptides, conjugates and uses thereof

Abstract

The present disclosure relates to structurally stable (e.g., stapled, e.g., hydrocarbon stapled) respiratory syncytial virus (RSV) peptides and variants thereof, and structurally stable (e.g., stapled, e.g., hydrocarbon stapled) RSV peptides conjugated to polyethylene glycol (PEG) and / or cholesterol (or variants thereof, e.g., mercaptocholesterol), e.g., PEG (n)-cholesterol or PEG (n)-mercaptocholesterol derivatizations, e.g., polyethylene glycol (n)-cholesterol, e.g., polyethylene glycol (n)-cholesterol, e.g., polyethylene glycol (n)-cholesterol, e.g., polyethylene glycol (n)-cholesterol, e.g., polyethylene glycol (n)-cholesterol. And methods of using such structurally stable peptides and conjugates to prevent and treat RSV infection in subjects (e.g., humans).

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 489,096, filed March 8, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application includes a sequence list, which has been electronically submitted in XML file format and is incorporated herein by reference in its entirety. The XML copy was created on March 6, 2024, named 00530-0418WO1_SL.xml, and has a size of 332,258 bytes. Technical Field

[0005] This disclosure relates to structurally stable (e.g., pinned, hydrocarbon-pinned) respiratory syncytial virus (RSV) peptides and structurally stable (e.g., pinned, hydrocarbon-pinned) RSV peptides conjugated with polyethylene glycol (PEG) and / or cholesterol (or variants thereof, such as thiocholesterol), such as PEG(n)-cholesterol or PEG(n)-thiocholesterol derivatization, and methods for preventing and treating RSV infection in subjects (e.g., humans) using such structurally stable peptide conjugates. Background Technology

[0006] Respiratory syncytial virus (RSV) infection causes 64 million respiratory illnesses and 166,000 deaths worldwide each year. Drug-resistant RSV strains have been reported (Adams et al., Clin. Infect. Disease, 51:185-188, 2010; Douglas et al., J. Virol., 49:2560-2466, 2005).

[0007] New and improved strategies are needed for the prevention and / or treatment of RSV infection. Summary of the Invention

[0008] This application relates to compositions and methods of disclosing peptide stabilization technologies (e.g., pinning, such as hydrocarbon pinning) that generalize and reinforce the structure of a bioactive helix. In some instances, peptide pinning is combined with methods for derivatizing cholesterol or cholesterol variants (e.g., thiocholesterol) (e.g., PEG(n)-cholesterol or PEG(n)thiocholesterol) to produce optimized and targeted preventative and therapeutic agents for the prevention and / or treatment of RSV infection. By inserting a “pin” (e.g., a full hydrocarbon pin) into an RSV peptide, the bioactive helical structure is restored, and significant protease resistance is conferred by burying the otherwise unstable amide bond at the core of the helical structure and / or by inhibiting the amide bond in a manner that prevents it from being recognized and proteased by the body’s proteases. Hereinafter, hydrocarbon pinning and PEG(n)-cholesterol or PEG(n)thiocholesterol-derived (hydrocarbon pinning conjugate) peptide inhibitors of RSV are disclosed. These structurally stable peptides and conjugates are used for the prevention and / or treatment of RSV infection.

[0009] This document provides a conjugate comprising (i) a structurally stable peptide and (ii) cholesterol or thiocholesterol; wherein the cholesterol or thiocholesterol is directly or via a linker linked to a C-terminal amino acid of the structurally stable peptide; wherein the structurally stable peptide comprises an internally cross-linked amino acid sequence comprising 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 100), excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO: 100, and wherein the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO: 258); wherein two of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three or six amino acids; wherein the conjugate binds RSV. 5-Helical bundle protein and / or wherein the conjugate inhibits and / or prevents RSV infection of cells; and wherein the length of the conjugate is 25 to 45 amino acids, optionally wherein the length of the conjugate is 30 amino acids. In some examples, the two substitutions with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are at amino acids corresponding to positions 1 and 8 of the sequence shown in SEQ ID NO: 100, at amino acids corresponding to positions 3 and 10 of the sequence shown in SEQ ID NO: 100, or at amino acids corresponding to positions 17 and 24 of the sequence shown in SEQ ID NO: 100.

[0010] This document also provides a conjugate comprising (i) a structurally stable peptide and (ii) cholesterol or thiocholesterol; wherein the cholesterol or thiocholesterol is directly or via a linker linked to a C-terminal amino acid of the structurally stable peptide; wherein the structurally stable peptide comprises an internally cross-linked amino acid sequence comprising the sequence X1X2X3FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:282) [where X1 is any amino acid, optionally S; X2 and X3 are any negatively charged amino acids], excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO:282, and wherein the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO:258); wherein two of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three or six amino acids; wherein the conjugate binds RSV. 5-Helical bundle protein and / or wherein the conjugate inhibits RSV infection of cells and / or prevents RSV infection of cells; and wherein the length of the conjugate is 32 to 45 amino acids, optionally wherein the length of the conjugate is 32 amino acids. In some examples, two substitutions are made with non-natural amino acids having α,α-disubstituted olefinic side chains that are cross-linked with each other at amino acids corresponding to positions 4 and 11 of the sequence shown in SEQ ID NO:282, at amino acids corresponding to positions 6 and 13 of the sequence shown in SEQ ID NO:282, and at amino acids corresponding to positions 20 and 27 of the sequence shown in SEQ ID NO:282.In one example, the structurally stable peptide comprises or consists of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:280), excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO:280 (e.g., amino acids corresponding to positions 4 and 11 of the sequence shown in SEQ ID NO:282; amino acids corresponding to positions 6 and 13 of the sequence shown in SEQ ID NO:282; or amino acids corresponding to positions 20 and 27 of the sequence shown in SEQ ID NO:282), and wherein the internally crosslinked amino acid sequence does not include the sequence NAGKST (SEQ ID NO:258); wherein two of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having crosslinked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having crosslinked olefinic side chains are separated by three or six amino acids; wherein the conjugate binds RSV. 5-Helical bundle protein and / or wherein the conjugate inhibits RSV infection of cells and / or prevents RSV infection of cells; and wherein the length of the conjugate is 32 to 45 amino acids, optionally wherein the length of the conjugate is 30 amino acids.

[0011] This article also provides a conjugate comprising (i) a structurally stable peptide and (ii) cholesterol or thiocholesterol; wherein the cholesterol or thiocholesterol is directly or via a linker linked to a C-terminal amino acid of the structurally stable peptide; wherein the structurally stable peptide comprises an amino acid sequence having an internal crosslinking of the following formula:

[0012]

[0013] Or a pharmaceutically acceptable salt thereof; wherein each of R1 and R2 is H or C1 to C2. 10The conjugate is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocycloalkyl, any of which may be substituted or unsubstituted; wherein x is 3 or 6; wherein each R3 is independently alkylene, alkenylene, or alkynylene, any of which may be substituted or unsubstituted; wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; wherein the internally crosslinked amino acid sequence comprises 25 to 29 consecutive amino acids of the sequence of SEQ ID NO: 100, excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO: 100, and wherein the internally crosslinked amino acid sequence does not include the sequence NAGKST (SEQ ID NO: 258); wherein the conjugate binds to RSV 5-helical bundle protein and / or wherein the conjugate inhibits RSV infection of cells and / or prevents RSV infection of cells; and wherein the length of the conjugate is 25 to 45 amino acids, optionally wherein the length of the conjugate is 30 amino acids. In some instances, R3 is an internal crosslink between amino acids corresponding to positions 1 and 8 of the sequence shown in SEQ ID NO:100, between amino acids corresponding to positions 3 and 10 of the sequence shown in SEQ ID NO:100, or between positions 17 and 24 of the sequence shown in SEQ ID NO:100. In some instances, the internally crosslinked amino acid sequence also includes or consists of the sequence X1X2X3, wherein X1 is any amino acid, optionally S; X2 and X3 are any negatively charged amino acids immediately upstream of the first amino acid in SEQ ID NO:100 (i.e., immediately upstream of the first F in SEQ ID NO:100). In some instances, X1X2X3 = SDE. In one example, the amino acid sequence comprises or consists of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:280), except for two to six amino acid substitutions relative to the sequence of SEQ ID NO:280, and wherein the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO:258); wherein two of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three or six amino acids; wherein the conjugate binds RSV 5-helical bundle protein and / or wherein the conjugate inhibits RSV infection of cells and / or prevents RSV infection of cells; and wherein the length of the conjugate is 32 to 45 amino acids, optionally wherein the length of the conjugate is 30 amino acids.

[0014] In some examples of the aforementioned conjugates, the conjugates contain cholesterol. In some examples, the conjugates contain cholesterol, and the linker contains PEG. In some examples, the conjugates contain PEG(n)-cholesterol directly linked to a C-terminal amino acid of a structurally stable peptide, wherein n is 1-36, optionally wherein n is 4, 5, 6, 7, 8, 12, 16, or 20. In some examples, the conjugates contain Formula II directly linked to a C-terminal amino acid of a structurally stable peptide:

[0015]

[0016] Where n is 1-36, and optionally n is 4, 5, 6, 7, 8, 12, 16 or 20.

[0017] In some examples of the aforementioned conjugates, the conjugates comprise thiocholesterol. In some examples, the conjugates comprise thiocholesterol, and the linker comprises PEG. In some examples, the conjugates comprise PEG(n)-thiocholesterol directly linked to a C-terminal amino acid of a structurally stable peptide, wherein n is 1-36, optionally wherein n is 4, 5, 6, 7, 8, 12, 16, or 20. In some examples, the conjugates comprise Formula III directly linked to a C-terminal amino acid of a structurally stable peptide:

[0018]

[0019] Where n is 1-36, and optionally n is 4, 5, 6, 7, 8, 12, 16 or 20.

[0020] In some examples of the aforementioned conjugates, the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three amino acids, optionally wherein each of the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains is (S)-α-(4'-pentenyl)alanine.

[0021] In some examples of the aforementioned conjugates, the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by six amino acids, optionally wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are (R)-α-(7'-octenyl)alanine and (S)-α-(4'-pentenyl)alanine.

[0022] In some examples of the aforementioned conjugates, the structurally stable peptide comprises the sequence shown in any one of SEQ ID NO: 27, 29, 36, 43, and 46. In some examples of the aforementioned conjugates, the structurally stable peptide comprises the sequence shown in any one of SEQ ID NO: 49, 51, 58, 65, and 68.

[0023] In some examples of the aforementioned conjugates, the conjugates comprise the sequences shown in any of SEQ ID NO: 5-26 and 71-92. In some examples, the conjugates comprise the sequences shown in any of SEQ ID NO: 71, 73, 80, 87, and 90. In some examples, the conjugates comprise the sequences shown in any of SEQ ID NO: 5, 6, 14, 21, and 24. In some examples, the conjugates comprise the sequences shown in SEQ ID NO: 21 or 281.

[0024] In some examples of the aforementioned conjugates, the conjugates contain internally cross-linked peptides with a length of 25 to 34, 26 to 33, 27 to 32, 28 to 31, 29, 30, 31 or 32 amino acids.

[0025] This document also provides conjugates comprising 8DASISQXNEKINQSLAFIRKSDELLHNV* (SEQ ID NO:265) or thereof, wherein 8 is internally cross-linked with X, wherein 8 is (R)-α-(7'-octenyl)alanine, wherein X is (S)-α-(4'-pentenyl)alanine, and wherein * is a conjugate of the formula...

[0026]

[0027] Where n is 1-36, and optionally n is 16 or 20.

[0028] This document also provides conjugates comprising FD8SISQVNXKINQSLAFIRKSDELLHNV* (SEQ ID NO:261) or thereof, wherein 8 is internally cross-linked with X, wherein 8 is (R)-α-(7'-octenyl)alanine, wherein X is (S)-α-(4'-pentenyl)alanine, and wherein * is a conjugate of the formula

[0029]

[0030] Where n is 1-36, and optionally n is 16 or 20.

[0031] This document also provides conjugates comprising or consisting of FDASISQVNEKINQSL8FIRKSDXLLHNV* (SEQ ID NO: 266), wherein 8 is internally cross-linked with X, wherein 8 is (R)-α-(7'-octenyl)alanine, wherein X is (S)-α-(4'-pentenyl)alanine, and wherein * is a conjugate of the formula...

[0032]

[0033] Where n is 1-36, and optionally n is 16 or 20.

[0034] This article also provides a structurally stable peptide comprising: an internally cross-linked amino acid sequence comprising 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:100), excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO:100; wherein two of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three or six amino acids; wherein the structurally stable peptide binds to RSV 5-helical bundle protein and / or wherein the structurally stable peptide inhibits RSV infection of cells and / or prevents RSV infection of cells; and wherein the length of the structurally stable peptide is 25 to 30 amino acids, optionally wherein the length of the structurally stable peptide is 29 amino acids. In some instances, the internally cross-linked amino acid sequence also includes or consists of the sequence X1X2X3, wherein X1 is any amino acid, optionally S; X2 and X3 are any negatively charged amino acids immediately upstream of the first amino acid of SEQ ID NO:100 (i.e., immediately upstream of the first F of SEQ ID NO:100). In some instances, X1X2X3 = SDE. In one example, the amino acid sequence comprises or consists of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:280), except for two to six amino acid substitutions relative to the sequence of SEQ ID NO:100, and wherein the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO:258); wherein two of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three or six amino acids; wherein the conjugate binds RSV 5-helical bundle protein and / or wherein the conjugate inhibits RSV infection of cells and / or prevents RSV infection of cells; and wherein the length of the conjugate is 25 to 45 amino acids, optionally wherein the length of the conjugate is 30 amino acids.

[0035] This article also provides a structurally stable peptide comprising:

[0036] An amino acid sequence with internal cross-links as follows:

[0037]

[0038] Or its pharmaceutically acceptable salt;

[0039] Each of R1 and R2 is H or C1 to C. 10 The alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocycloalkyl group, any of which is substituted or unsubstituted; wherein x is 3 or 6; wherein each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the internally crosslinked amino acid sequence comprises 25 to 29 consecutive amino acids of the sequence of SEQ ID NO: 100, excluding substitutions of two to six amino acids relative to the sequence of SEQ ID NO: 100; wherein the structurally stable peptide binds to RSV 5-helical bundle protein and / or wherein the structurally stable peptide inhibits RSV infection of cells and / or prevents RSV infection of cells; and wherein the length of the structurally stable peptide is 25 to 30 amino acids, optionally wherein the length of the structurally stable peptide is 29 amino acids.

[0040] In some examples of the aforementioned structurally stable peptides, the structurally stable peptides do not contain amino acids corresponding to positions 517-522, 517-521, 517-520, 517-519, or 517-518 of the RSV-F protein (according to the sequence number shown in SEQ ID NO:1). In some examples, the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO:258).

[0041] In some examples of the aforementioned structurally stable peptides, the structurally stable peptide is 29 amino acids in length and comprises 29 consecutive amino acids of the sequence SEQ ID NO:100, excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO:100. In some examples of the aforementioned structurally stable peptides, the structurally stable peptide is 32 amino acids in length and comprises 32 consecutive amino acids of the sequence SEQ ID NO:280, excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO:280.

[0042] In some of the aforementioned examples of structurally stable peptides, the structurally stable peptide comprises the amino acid sequence shown in any of SEQ ID NO:27-70. In some examples, the structurally stable peptide comprises the amino acid sequence shown in any of SEQ ID NO:27, 29, 36, 43, and 46. In some examples, the structurally stable peptide comprises the amino acid sequence shown in any of SEQ ID NO:49, 51, 58, 65, and 68.

[0043] This article also provides a peptide comprising the amino acid sequence of any of SEQ ID NO:27-70, excluding zero to six additional substitutions, wherein the peptide does not contain the sequence NAGKST (SEQ ID NO:258).

[0044] This article also provides a pharmaceutical composition comprising any of the aforementioned conjugates, any of the aforementioned structurally stable peptides, or any of the aforementioned peptides, and a pharmaceutically acceptable carrier.

[0045] This article also provides a method for treating RSV infection in a subject in need, comprising administering to the subject a therapeutically effective amount of any of the aforementioned conjugates, any of the aforementioned structurally stable peptides, or any of the aforementioned peptides. In some instances, the subject is a human being.

[0046] This article also provides a method for preventing RSV infection in a subject of need, comprising administering to the subject a therapeutically effective amount of any of the aforementioned conjugates, any of the aforementioned structurally stable peptides, or any of the aforementioned peptides. In some instances, the subject is a human being.

[0047] This document also provides a method for preparing a structurally stable peptide, the method comprising: (a) providing a peptide having 25 to 29 consecutive amino acids comprising the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 100), except for two to six amino acid substitutions relative to the sequence of SEQ ID NO: 100; wherein two of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having olefinic side chains are separated by three or six amino acids; and (b) crosslinking the peptide to prepare the structurally stable peptide, and optionally purifying the structurally stable peptide. In some examples, the crosslinking is carried out by a ruthenium-catalyzed metathesis reaction. In some examples, the method further comprises derivatizing the resin-bound amine of the structurally stable peptide on a resin with PEG containing a carboxylic acid and / or cholesterol or thiocholesterol. In some examples, the method further comprises formulating the structurally stable peptide into a sterile pharmaceutical composition.

[0048] This article also provides a pharmaceutical composition comprising (a) a means for treating or preventing RSV infection in a subject, and (b) a pharmaceutically acceptable carrier; optionally, wherein the subject is a human. In some instances, the means for treating or preventing RSV infection is a structurally stable RSV peptide or a cholesterol or thiocholesterol conjugate thereof.

[0049] In another aspect, this disclosure is characterized by a peptide comprising or consisting of the amino acid sequence X1X2X3FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:282) or thereof, the amino acid sequence having 0, 1, 2, or 3 amino acid substitutions (optionally, wherein these substitutions include positions 20 and 27 of SEQ ID NO:282), wherein X1 is any amino acid, optionally S, and X2 and X3 are negatively charged amino acids. In one case, the peptide comprises or consists of the amino acid sequence SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:280) or thereof, the amino acid sequence having 0, 1, 2, or 3 amino acid substitutions (optionally, wherein the substitutions include positions 20 and 27 of SEQ ID NO:280).

[0050] In another aspect, this disclosure relates to an internally cross-linked peptide comprising or consisting of the sequence X1X2X3FDASISQVNEKINQSL8FIRKSDXLLHNV (SEQ ID NO:284), wherein X1 is any amino acid, optionally S; X2 and X3 are negatively charged amino acids; “8” = (R)-α-(7'-octenyl)alanine; and “X” = (S)-α-(4'-pentenyl)alanine. In one case, the peptide is an internally cross-linked peptide comprising or consisting of the amino acid sequence SDEFDASISQVNEKINQSL8FIRKSDXLLHNV (SEQ ID NO:285), wherein “8” = (R)-α-(7'-octenyl)alanine; and “X” = (S)-α-(4'-pentenyl)alanine.

[0051] In another aspect, this disclosure is characterized by conjugates comprising the amino acid sequence X1X2X3FDASISQVNEKINQSL8FIRKSDXLLHNV* (SEQ ID NO:286), wherein X1 is any amino acid, optionally S; X2 and X3 are negatively charged amino acids; “8” = (R)-α-(7'-octenyl)alanine; “X” = (S)-α-(4'-pentenyl)alanine; and * = linker-lipid. In one example, * comprises Z-PEG(n)-cholesterol or Z-PEG(n)-mercaptocholesterol, wherein n = 1-36, and wherein Z is a diamino acid (e.g., lysine or ornithine). In one case, the conjugate comprises or consists of the amino acid sequence SDEFDASISQVNEKINQSL8FIRKSDXLLHNV* (SEQ ID NO:281), wherein “8” = (R)-α-(7'-octenyl)alanine; “X” = (S)-α-(4'-pentenyl)alanine; and * = linker-lipid. In one example, * comprises Z-PEG(n)-cholesterol or Z-PEG(n)-mercaptocholesterol, wherein n = 1-36, and wherein Z is a diamino acid (e.g., lysine or ornithine).

[0052] In some cases, the aforementioned peptides, internally cross-linked peptides, or conjugates can be used in methods for treating or preventing RSV infection in a subject of need, the method comprising administering to the subject a therapeutically effective amount of any one of the aforementioned peptides, internally cross-linked peptides, or conjugates. In some instances, the subject is a human being.

[0053] This disclosure also relates to pharmaceutical compositions comprising any one of the aforementioned peptides, internally cross-linked peptides, or conjugates, and a pharmaceutically acceptable carrier. Attached Figure Description

[0054] Figure 1 The mechanism of action of RSV virus-host membrane fusion is shown in the top figure, and the mechanism of action of RSV-F stapled lipopeptide in inhibiting membrane fusion and viral infection is shown in the bottom figure.

[0055] Figure 2 The amino acid sequence of the RSV F protein is shown (SEQ ID NO:1). Heptapeptide repeat domain 1 (HR1) (SEQ ID NO:2) is shown in bold. Heptapeptide repeat domain 2 (HR2) (SEQ ID NO:3) is underlined.

[0056] Figure 3 This is a schematic diagram of the RSV glycoprotein (F) protein, including the sequences of the HR1 (SEQ ID NO:2) and HR2 (SEQ ID NO:3) fusion domains.

[0057] Figure 4 The diagram shows the alignment of the HR1 region (top) and HR2 region (bottom) of various RSV viral strains. The HR1 sequences, from top to bottom, are SEQ ID NO: 2, 2, 93, 94, and 95; the HR2 sequences, from top to bottom, are SEQ ID NO: 3, 3, 96, 97, and 97. For each of HR1 and HR2, the bottom sequences (SEQ ID NO: 98 and 99) represent conserved amino acids.

[0058] Figure 5 A variety of tethered amino acids containing olefinic chains are shown, which can be used to generate hydrocarbon-tethered RSV-F HR2 peptides with tethers spanning positions i, i+3; i, i+4; and i, i+7. The top row, from left to right, shows: (R)-α-(7'-octenyl)alanine, (S)-α-(7'-octenyl)alanine, (R)-α-(4'-pentenyl)alanine, (S)-α-(4'-pentenyl)alanine, (R)-α-(2'-propenyl)alanine, and dipentenylglycine.

[0059] Figure 6 Multiple stud compositions and stud scans in multiple studded peptides are shown to generate a library of multiple studded RSV-F HR2 peptides for conjugation with cholesterol or cholesterol variant moieties (e.g., PEG-mercaptocholesterol or PEG-cholesterol).

[0060] Figure 7 Multiple nail compositions in tandem suturing peptides are shown to generate a library of suturing RSV-F HR2 peptides for conjugation with cholesterol or cholesterol variant moieties (e.g., PEG-mercaptocholesterol or PEG-cholesterol).

[0061] Figure 8 This illustration depicts exemplary methods for designing, synthesizing, and identifying optimal pinned peptide constructs targeting RSV-F fusion devices, including generating Ala scans, pin scans, and libraries with variable N-terminal and C-terminal deletions, additions, and derivatizations for conjugation to cholesterol or cholesterol variant moieties (e.g., PEG-mercaptocholesterol or PEG-cholesterol). Single and double pinned and stitched constructs, including alanine and pinned and stitched scans, are used to identify optimal pinned peptides conjugated to cholesterol or cholesterol variant moieties (e.g., PEG-mercaptocholesterol or PEG-cholesterol moieties with variable PEG chain lengths), and their application in in vitro and in vivo analyses.

[0062] Figure 9 The dose-response curves of a single stabilizing peptide (SEQ ID NO: 4; 8 is (R)-α-(7'-octenyl)alanine, and X is (S)-α-(4'-pentenyl)alanine) are shown when A549 cells infected with GFP-RSV virus are treated.

[0063] Figure 10 The crystal structure of the RSV six-helix bundle (RCSB PDB: 1G2C) is shown. The first and last amino acids of the extended helix are labeled (corresponding to Phe-488 and Val-516 in the sequence of SEQ ID NO:1).

[0064] Figure 11 Synthetic protocols for converting thiocholesterol or cholesterol into carboxylic acids are shown for the easy resin-on-resin derivatization of stapled peptides containing a cholesterol moiety (or a cholesterol variant, such as thiocholesterol). DCM: dichloromethane; TFA: trifluoroacetic acid; eq: equivalent; RT: room temperature; min: minutes; hr: hours; vol: volume; Δ: heat.

[0065] Figure 12 A synthetic scheme for the resin derivatization of a PEG-linked thiocholesterol moiety-stuck peptide sequence (using SEQ ID NO:5 as an example) is shown. DMF: dimethylformamide; HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; DIEA: N,N-diisopropylethylamine. SEQ ID NOs 262-264 are disclosed in order of appearance.

[0066] Figure 13 The dose-response curve for a peptide with the sequence SEQ ID NO:5, having a C-terminal (PEG)4 linker and an additional thiocholesterol (“PEG4-TC”), is shown. The dashed line at 50 represents the vector infection level.

[0067] Figure 14 An exemplary structurally stable RSV-HR2 peptide sequence is shown with C-terminal derivatization, wherein (* = Lys(PEG4-mercaptocholesterol; 8 is (R)-α-(7'-octenyl)alanine), and X is (S)-α-(4'-pentenyl)alanine) (SEQ ID NO:5-26).

[0068] Figure 15 To illustrate the differential antiviral activity of the pinned peptide against RSV-GFP in A549 cells after 48 hours at a 2 μM dose, the pinned peptide has the sequence shown (* = Lys(PEG4-mercaptocholesterol; 8 for (R)-α-(7'-octenyl)alanine), and X for (S)-α-(4'-pentenyl)alanine).

[0069] Figure 16A graph illustrating the dose-response curve of a stapled peptide having the amino acid sequence of SEQ ID NO:51, wherein an additional PEG(n)-thiocholesterol (“TC”) is linked to the stapled peptide via a C-terminal lysine residue, wherein n is 0, 4, 8, 12, 16 or 20 (SEQ ID NO:267-272 from top to bottom).

[0070] Figure 17 A graph illustrating the dose-response curve of a stapled peptide having the amino acid sequence of SEQ ID NO:65, wherein an additional PEG(n)-thiocholesterol is linked to the stapled peptide via a C-terminal lysine residue, wherein n is 0, 4, 8, 12, 16 or 20 (SEQ ID NO:273-278 from top to bottom).

[0071] Figure 18 The solubility of two peptides was recorded: SEQ ID NO:21 (visible clumps) and SEQ ID NO:281 (completely soluble). The buffer conditions were 15 mg / mL in 15% DMSO and PBS (pH 7.4). Detailed Implementation

[0072] This disclosure is particularly based on structurally stable (e.g., pinned, hydrocarbon-pinned) RSV peptides, and it has been found that they can be esterified (e.g., with PEG and / or cholesterol (or a variant of cholesterol, e.g., thiocholesterol), e.g., PEG(n)-cholesterol or PEG(n)-thiocholesterol esterification) to selectively bind RSV and exhibit antiviral activity against RSV. Therefore, this disclosure provides methods (e.g., methods for treating, developing treatments for RSV infection or disease, and for preventing RSV infection or disease, such as converting cholesterol / thiocholesterol to carboxylic acids for resin derivatization) and compositions (e.g., structurally stable RSV peptides and PEG(n)-cholesterol or PEG(n)-thiocholesterol conjugates). Thus, the peptides and compositions disclosed herein can be used for the prevention and / or treatment of RSV infection.

[0073] RSV peptide

[0074] RSV unfolds the discrete heptapeptide repeat domain of its F protein to form RSV-SFB, a structure that allows the virus to penetrate the host cell membrane. A furin-like protease cleaves the RSV-F precursor, resulting in the formation of two subunits stabilized by disulfide bonds (Gonzalez-Reyes et al., Proc. Natl. Acad. Sci., USA, 98:9859-9864, 2003; Sugrue et al., Gen. Virol., 82:1375-1386, 2001). This cleavage also exposes a previously hidden peptide fusion motif at the N-terminus of the F1 subunit, leading to the formation of the FSV-F lollipop structure (Matthews et al., J. Virol., 74:5911-5920, 2000; Smith et al., Protein Eng., 15:365-371, 2002). Once the fusion peptide is inserted into the host cell membrane, the F protein itself refolds to form a trimer hairpin or "6-helix bundle." Trimeric hairpins originate from an undercharacterized conformational change that causes the antiparallel assembly of the C-terminal heptapeptide repeat (HR2) region with the N-terminal heptapeptide repeat (HR1), which is juxtaposed with the fusion peptide (Cianci et al., Proc. Natl. Acad. Sci., USA, 101:15046-15051, 2004). Following pore opening, the HR-induced conformational change achieves a new equilibrium state considered necessary for pore stabilization and expansion (Cianci, ibid., Melikyan, Retrovirology, 5:111, 2008; Melikyan et al., Proc. Natl. Acad. Sci., USA, 102:8728-8733, 2005), and allows penetration of host cells. The compositions and methods disclosed herein can be used to prevent or treat RSV infection by inhibiting this process.

[0075] This article provides the RSV-F HR2 peptide. RSV infection is mediated on the cell surface by the RSV-F protein, which has two heptapeptide repeat domains: HR1 and HR2. The amino acid sequence of an exemplary RSV-F protein sequence (SEQ ID NO:1) is shown in [image / image / description]. Figure 2 The amino acid sequence of an exemplary RSV-F protein HR1 is shown in SEQ ID NO:2. The amino acid sequence of an exemplary RSV-F protein HR2 is shown in SEQ ID NO:3.

[0076] The RSV-F proteins HR1 and HR2 in different RSV strains show high homology. See also Figure 4 To understand the comparison of exemplary amino acid sequences of the HR1 and HR2 sequences of exemplary RSV strains.

[0077] In some instances, the HR2 peptide described herein comprises or is composed of the amino acid sequence of SEQ ID NO:100. In some instances, the HR2 peptide is composed of the amino acid sequence of SEQ ID NO:100. In some instances, the HR2 peptide does not contain the amino acid sequence NAGKST (SEQ ID NO:258). In some instances, the HR2 peptide does not contain the amino acid sequence NAGKS (SEQ ID NO:259). In some instances, the HR2 peptide does not contain the amino acid sequence NAGK (SEQ ID NO:260). In some instances, the HR2 peptide does not contain the amino acid sequence NAG. In some instances, the HR2 peptide does not contain the amino acid sequence NA. In some instances, the HR2 peptide does not contain the amino acid corresponding to positions 517-522, 517-521, 517-520, 517-519, or 517-518 of SEQ ID NO:1 (or the corresponding amino acid of the RSV-F protein of another strain (see, for example)). Figure 4 In some instances, the HR2 peptide is 25 to 34 amino acids in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some instances, the HR2 peptide is 30 amino acids in length. In some instances, the HR2 peptide also comprises or consists of the sequence X1X2X3, wherein X1 is any amino acid, optionally S; X2 and X3 are any negatively charged amino acids immediately upstream of the first amino acid of SEQ ID NO:100 (i.e., immediately upstream of the first F of SEQ ID NO:100). In some instances, X1X2X3 = SDE. In one instance, the amino acid comprises or consists of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:280).

[0078] In some instances, the RSV-F HR2 peptide described herein comprises or is composed of the amino acid sequence of SEQ ID NO:100 or 280, except that it contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., relative to the amino acid sequence of SEQ ID NO:100 or 280), such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved and / or non-conserved amino acid substitutions.

[0079] "Conservative amino acid substitution" refers to the substitution of an amino acid by replacing one amino acid residue 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), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), and amino acids with acidic side chains and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine).

[0080] Those skilled in the art will understand that the comparison of F proteins from different RSV strains can be used to identify conserved residues and residues suitable for substitution (e.g., conserved or non-conserved substitutions). For example, in such a comparison, residues that are unchanged between two or more different RSV strains (see, for example, Figure 4 The residues that differ due to conserved amino acid substitutions in two or more different RSV strains (see, for example, Figure 4) can be unmodified or substituted with conserved amino acid substitutions. In this comparison, the non-conserved residues in two or more different RSV strains (see, for example, Figure 4) can also be unmodified or substituted with conserved amino acid substitutions. Figure 4 The residue can be unmodified or replaced by any amino acid. In some instances, in such comparisons, residues conserved but located on the non-interacting surface of HR2 between two or more different RSV strains can be replaced by any amino acid. For example, considering... Figure 4The comparison in SEQ ID NO:3 allows for the substitution of a conserved amino acid at position 33 and a non-conserved amino acid at position 40. In some instances, the substituted amino acid is selected from L-Ala, D-Ala, Aib, Sar, Ser, substituted alanine, or substituted glycine derivatives. Methods for identifying the interaction surfaces of peptides are known in the art (see, for example, Broglia et al., Proteinsci., 14(10):2668-81, 2005; Hammond et al., J. Pharm. Sci., 98(1):4589-603, 2009; Ng and Yang, J. Phys. Chem. B., 111(50):13886-93, 2007; and Bird et al., PNAS USA, 197:14093, 2010).

[0081] In some instances, the peptides described herein comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 93% of the same amino acid sequence as the sequence shown in SEQ ID NO: 100 or 280. In some instances, the peptides described above possess one or more of the following properties (e.g., 1, 2, 3, 4, or 5): (i) being α-helical; (ii) being protease-resistant; (iii) binding to RSV 5-helical bundle protein; (iv) inhibiting RSV infection of cells; and / or (v) preventing RSV infection of cells. In some instances, the peptides inhibit RSV infection of cells and / or prevent RSV infection of cells in pseudovirus and / or live RSV virus assays. RSV pseudovirus assays are known in the art, see, for example, Haid et al., 2015. J Virol 90:3065–3073, which is incorporated herein by reference in its entirety.

[0082] Methods for determining the percentage identity between amino acid sequences are known in the art. For example, sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The determination of the percentage identity between two amino acid sequences is performed using the BLAST 2.0 procedure. Vacancies-free alignment is used, and the sequence comparison is performed using default parameters (Blossom 62 matrix, vacancy cost of 11, vacancy cost per residue of 1, λ ratio of 0.85). The mathematical algorithm used in the BLAST program is described by Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).

[0083] In some instances, the RSV-F HR2 peptide described herein (e.g., SEQ ID NO: 100, 280) contains at least 1, at least 2, at least 3, at least 4, or at least 5 (e.g., 1, 2, 3, 4, 5, 6) amino acids added to the N-terminus of the peptide. In some instances, the RSV-F HR2 peptide described herein (e.g., SEQ ID NO: 100, 280) contains at least 1, at least 2, at least 3, at least 4, or at least 5 (e.g., 1, 2, 3, 4, 5, 6) amino acids added to the C-terminus of the peptide. In some instances, the RSV-F HR2 peptide described herein (e.g., SEQ ID NO: 100, 280) contains at least 1, at least 2, at least 3, at least 4, or at least 5 (e.g., 1, 2, 3, 4, 5, 6) amino acids deleted at the N-terminus of the peptide. In some instances, the RSV-FHR2 peptide described herein (e.g., SEQ ID NO:100) contains at least one, at least two, at least three, at least four, or at least five (e.g., one, two, three, four, five, or six) amino acids deleted at the C-terminus of the peptide. In some instances, the RSV-FHR2 peptide does not contain the amino acid sequence NAGKST (SEQ ID NO:258).

[0084] In some instances, the peptide comprises an amino acid sequence having 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2 substitutions, insertions, and / or deletions relative to SEQ ID NO:100. In some instances, the peptide comprises 2, 3, 4, 5, or 6 substitutions, insertions, and / or deletions relative to SEQ ID NO:100 or 280. In some instances, the peptide having substitutions, insertions, and / or deletions relative to SEQ ID NO:100 or 280 as described above has one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) is α-helical; (ii) is protease resistant; (iii) binds to RSV 5-helical bundle protein; (iv) inhibits RSV infection of cells; and / or (v) prevents RSV infection of cells. In some instances, peptides inhibit RSV infection of cells in pseudovirus and / or live RSV assays and / or prevent RSV infection of cells in pseudovirus and / or live RSV assays.

[0085] In some instances, the peptide is 25 to 45 amino acids in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). In some instances, the peptide is 28 to 40 amino acids in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40). In some instances, the peptide is 25 to 35 amino acids long (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35). In some instances, the peptide is 29 or 30 amino acids long. In some instances, the peptide is 32 or 33 amino acids long.

[0086] In some instances, the peptides described above possess one or more of the following properties (e.g., 1, 2, 3, 4, or 5): (i) being α-helical; (ii) being protease-resistant; (iii) binding to RSV 5-helical bundle proteins; (iv) inhibiting RSV infection of cells; and / or (v) preventing RSV infection of cells. In some instances, the peptides inhibit RSV infection of cells in pseudovirus and / or live RSV virus assays and / or prevent RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0087] In some instances, each of the RSV-F HR2 peptides described above binds to the RSV 5-helical bundle protein. In some instances, each of the RSV-F HR2 peptides described above binds to the RSV 5-helical bundle protein and prevents or blocks the fusion of the RSV membrane and the host membrane.

[0088] Methods for determining whether a peptide (e.g., the RSV-F HR2 peptide, structurally stable peptide, or structurally stable peptide conjugate described herein) binds to RSV 5-helical bundle protein are known in the art, such as high-resolution clear native electrophoresis (hrCNE). See, for example, Example 3 of WO 2013 / 102211, which is incorporated herein by reference in its entirety.

[0089] Methods for determining whether a peptide (e.g., the RSV-F HR2 peptide, structurally stable peptide, or structurally stable peptide conjugate described herein) prevents or blocks RSV membrane-host membrane fusion are known in the art, such as cytotoxicity and immunofluorescence. In some instances, if less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, or less than 50% of cells are infected with RSV or RSV pseudovirus at a multiplicity of infection of 0.1, 0.5, 1, or 10 in the presence of the peptide, then the peptide prevents or blocks RSV membrane-host membrane fusion. In some instances, if less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, or less than 50% of cells exhibit RSV membrane-host membrane fusion after infection with RSV at a multiplicity of infection of 0.1, 0.5, 1, or 10 in the presence of the peptide, then the peptide prevents or blocks RSV membrane-host membrane fusion.

[0090] Methods for determining whether a peptide (e.g., the RSV-F HR2 peptide, structurally stable peptide, or structurally stable peptide conjugate described herein) inhibits RSV infection of cells are known in the art, such as cytotoxicity and immunofluorescence, and are described in the working examples. In some instances, if less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, or less than 50% of cells in the presence of the peptide are infected with RSV or RSV pseudoviruses at a multiplicity of infection of 0.1, 0.5, 1, or 10, then the peptide (e.g., the RSV-F HR2 peptide, structurally stable peptide, or structurally stable peptide conjugate described herein) inhibits RSV infection of cells. In some instances, if, under the same conditions, the RSV infection level of a cell population in the presence of the peptide is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% lower than the RSV infection level of a cell population in the absence of the peptide, then the peptide (e.g., the RSV-F HR2 peptide, a structurally stable peptide, or a structurally stable peptide conjugate described herein) inhibits cell infection. In some instances, the multiplicity of infection for RSV infection is 0.1, 0.5, 1, or 10.

[0091] In some instances, at least two (e.g., 2, 3, 4, or 5) amino acids (e.g., separated by 3 or 6 amino acids) of the RSV-F HR2 peptide described herein (e.g., SEQ ID NO:100 or its modified form, as described herein) are replaced by α,α-disubstituted non-natural amino acids (e.g., non-natural amino acids substituted with α-methyl and α-alkenyl groups) each having an olefinic side chain, wherein the α,α-disubstituted non-natural amino acids can crosslink with each other to form one or more nails or sutures (see the section "Structurally Stable Peptides" below). The type of substitution performed can be guided, for example, by alignment of the HR2 peptide with two or more RSV-F protein sequences (see, for example, Figure 4 The guidance on modifiable amino acids provided in the “Structurally Stable Peptides” section below also applies to the RSV-F HR2 peptide described herein.

[0092] In some cases, RSV-F HR2 peptides (or structurally stable peptides) are esterified. See the “Structurally Stable Peptide Conjugates” section below. These esterified peptides are interchangeably referred to herein as “structurally stable peptide conjugates” and “conjugates”. In some cases, RSV-F HR2 peptides are modified to include cholesterol, for example via a linker containing polyethylene glycol (PEG). In some cases, RSV-F HR2 peptides are modified to include thiocholesterol, for example via a linker containing PEG. In some cases, RSV-F HR2 peptides (e.g., SEQ ID NO: 100, 280) include the following formula attached to the C-terminus of the peptide:

[0093]

[0094] In some cases, the sulfur atom in Formula II is replaced by an oxygen atom. In some cases, the RSV-F HR2 peptide (e.g., SEQ ID NO: 100, 280) includes the following formula attached to the C-terminus of the peptide:

[0095]

[0096] In some instances, n in Equation II or Equation II is 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 instances, n = 16. In some instances, n = 20.

[0097] structurally stable peptides

[0098] This document also provides structurally stable RSV-F HR2 peptides. In some instances, the structurally stable peptide is a structurally stable (e.g., pinned, hydrocarbon-pinned) form of the RSV-F HR2 peptide described herein (see, for example, the “RSV peptide” section above). In some instances, the structurally stable (e.g., pinned, hydrocarbon-pinned) RSV-F HR2 peptide is derived from the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:100) or SEQ ID NO:280.

[0099] In some instances, the structurally stable peptide comprises an internally cross-linked amino acid sequence comprising 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 100), excluding two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions relative to the sequence of SEQ ID NO: 100; wherein two of the two or more amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by six amino acids. In some instances, the structurally stable peptide has 2 to 6 amino acid substitutions relative to the sequence of SEQ ID NO: 100. In some instances, the structurally stable peptide has 4 amino acid substitutions relative to the sequence of SEQ ID NO: 100. In some instances, the structurally stable peptide has 3 amino acid substitutions relative to the sequence of SEQ ID NO: 100. In some instances, the structurally stable peptide has two amino acid substitutions relative to the sequence of SEQ ID NO:100. In some instances, substitutions with non-natural amino acids having α,α-disubstituted olefinic side chains that are crosslinked with each other are made at positions (i) 1 and 8 (see, for example, SEQ ID NO:27), (ii) 3 and 10 (see, for example, SEQ ID NO:29), (iii) 10 and 17 (see, for example, SEQ ID NO:36), (iv) 17 and 24 (see, for example, SEQ ID NO:43), or (v) 20 and 27 (see, for example, SEQ ID NO:46), relative to the sequence number of SEQ ID NO:100 (where position 1 is the N-terminal phenylalanine of SEQ ID NO:100). In some instances, substitutions with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are made at positions 3 and 10 (see, for example, SEQ ID NO:29), relative to SEQ ID NO:100 (where position 1 is the N-terminal phenylalanine of SEQ ID NO:100). In some instances, substitutions with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are made at positions 17 and 24 (see, for example, SEQ ID NO:43), relative to SEQ ID NO:100 (where position 1 is the N-terminal phenylalanine of SEQ ID NO:100).In some instances, the internally cross-linked amino acid sequence also includes or consists of the sequence X1X2X3, wherein X1 is any amino acid, optionally S; X2 and X3 are any negatively charged amino acids immediately upstream of the first amino acid of SEQ ID NO:100 (i.e., immediately upstream of the first F of SEQ ID NO:100). In some instances, X1X2X3 = SDE. In one example, the amino acid sequence comprises or consists of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:280), except for two to six amino acid substitutions relative to the sequence of SEQ ID NO:100, and wherein the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO:258); wherein two of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three or six amino acids; wherein the conjugate binds RSV 5-helical bundle protein and / or wherein the conjugate inhibits RSV infection of cells and / or prevents RSV infection of cells; and wherein the length of the conjugate is 25 to 45 amino acids, optionally wherein the length of the conjugate is 30 amino acids.

[0100] In some instances, the structurally stable peptide comprises an internally cross-linked amino acid sequence comprising 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 100), excluding two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions relative to the sequence of SEQ ID NO: 100; wherein two of the two or more amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three amino acids. In some instances, the structurally stable peptide has 2 to 6 amino acid substitutions relative to the sequence of SEQ ID NO: 100. In some instances, the structurally stable peptide has 4 amino acid substitutions relative to the sequence of SEQ ID NO: 100. In some instances, the structurally stable peptide has 3 amino acid substitutions relative to the sequence of SEQ ID NO: 100. In some instances, the structurally stable peptide has two amino acid substitutions relative to the sequence of SEQ ID NO:100.

[0101] In some instances, the structurally stable peptide comprises an internally cross-linked amino acid sequence comprising 30 to 32 consecutive amino acids of the sequence shown in SEQ ID NO:280, excluding two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions relative to the sequence of SEQ ID NO:280; wherein two of the two or more amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three amino acids. In some instances, the structurally stable peptide has 2 to 6 amino acid substitutions relative to the sequence of SEQ ID NO:280. In some instances, the structurally stable peptide has 4 amino acid substitutions relative to the sequence of SEQ ID NO:280. In some instances, the structurally stable peptide has 3 amino acid substitutions relative to the sequence of SEQ ID NO:280. In some instances, the structurally stable peptide has two amino acid substitutions relative to the sequence of SEQ ID NO:280.

[0102] In some instances, structurally stable peptides have one or more modifications (e.g., substitution, insertion, addition, or deletion) as described in the “RSV peptides” section above.

[0103] In some instances, structurally stable (e.g., pinned, such as hydrocarbon-pinned) peptides are those shown in Table 1 below. In some instances, this disclosure covers structurally stable peptides of Table 1 that further comprise three N-terminal amino acids X1X2X3, where X1 is any amino acid, optionally S; X2 and X3 are any negatively charged amino acids immediately upstream of the first amino acid of SEQ ID NO:100 (i.e., immediately upstream of the first F of SEQ ID NO:100). In some instances, X1X2X3 = SDE. In some instances, the structurally stable peptide comprises or consists of the amino acid sequence of any of SEQ ID NO:27-70. In some instances, the structurally stable peptide comprises or consists of the amino acid sequence of any of SEQ ID NO:27, 29, 36, 43, and 46. In some instances, the structurally stable peptide comprises or consists of the amino acid sequence of any of SEQ ID NO:49, 51, 58, 65, and 68. In some instances, the structurally stable peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:29. In some instances, the structurally stable peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:51. In some instances, the structurally stable peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:43. In some instances, the structurally stable peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:65. In some instances, the structurally stable peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:281.

[0104]

[0105] In Table 1, relative to SEQ ID NO:27-48, “X1” = α,α-disubstituted non-natural amino acid having an olefinic side chain crosslinked with X2 (e.g., non-natural amino acid substituted with α-methyl and α-alkenyl); “X2” = α,α-disubstituted non-natural amino acid having an olefinic side chain crosslinked with X1 (e.g., α-methyl, α-alkenyl non-natural amino acid); in Table 1, relative to SEQ ID NO:49-70, “8” = (R)-α-(7'-octenyl)alanine; “X” = (S)-α-(4'-pentenyl)alanine.

[0106] Note that the bolded residues in Table 1 identify pinned amino acids. In some instances (e.g., the peptides described in Table 1), structurally stable peptides are monopinned peptides.

[0107] This disclosure covers each peptide listed in Table 1 and structurally stable peptides and their variants (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) amino acid substitutions, insertions, and / or deletions, except at the peg positions (i.e., the bolded residues in Table 1). In some instances, variants have 1 to 10 amino acid substitutions, insertions, and / or deletions, except at the peg positions (i.e., the bolded residues in Table 1). In some instances, variants have 1 to 5 amino acid substitutions, insertions, and / or deletions, except at the peg positions (i.e., the bolded residues in Table 1). In some instances, variants have 1 to 3 amino acid substitutions, insertions, and / or deletions, except at the peg positions (i.e., the bolded residues in Table 1). In some instances, the variants have 1 to 10, 10 to 5, 1 to 3, 2, or 1 amino acid substitution, insertion, and / or deletion relative to the amino acid sequences shown in any of SEQ ID NO:31-35 and 38, except at the peg positions (i.e., the bolded residues in Table 1). In some instances, the variants have 1 to 10, 10 to 5, 1 to 3, 2, or 1 amino acid substitution, insertion, and / or deletion relative to the amino acid sequences shown in any of SEQ ID NO:27, 29, 36, 43, and 46 or SEQ ID NO:49, 51, 58, 65, and 68, except at the peg positions (i.e., the bolded residues in Table 1). In some instances, the structurally stable peptide does not contain the amino acid sequence NAGKST (SEQ ID NO:258). In some instances, the structurally stable peptide does not contain the amino acid sequence NAGKS (SEQ ID NO:259). In some instances, the structurally stable peptide does not contain the amino acid sequence NAGK (SEQ ID NO:260). In some instances, the structurally stable peptide does not contain the amino acid sequence NAG. In some instances, the structurally stable peptide does not contain the amino acid sequence NA. In some instances, the structurally stable peptide does not contain the amino acids corresponding to positions 517-522, 517-521, 517-520, 517-519, or 517-518 of SEQ ID NO:1 (or the corresponding amino acids of the RSV-F protein of another strain (see example)). Figure 4In some instances, the structurally stable peptide is 25 to 34 amino acids in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some instances, the structurally stable peptide is 30 amino acids in length. In some instances, the structurally stable peptide has one or more of the following properties (e.g., 1, 2, 3, 4, or 5): (i) is α-helical; (ii) is protease resistant; (iii) binds to RSV 5-helical bundle protein; (iv) inhibits RSV infection of cells; and / or (v) prevents RSV infection of cells. In some instances, the peptide inhibits RSV infection of cells in pseudovirus and / or live RSV virus assays and / or prevents RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0108] In some instances, the structurally stable peptide comprises an amino acid sequence having 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2 substitutions, insertions, and / or deletions relative to SEQ ID NO:100. In some instances, the structurally stable peptide having substitutions, insertions, and / or deletions relative to SEQ ID NO:100 as described above is (i) α-helical; (ii) protease-resistant; (iii) binds to RSV 5-helical bundle protein; (iv) inhibits RSV infection of cells; and / or (v) prevents RSV infection of cells. In some instances, the peptide inhibits RSV infection of cells in pseudovirus and / or live RSV virus assays and / or prevents RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0109] In some instances, this document discloses peptides containing 0 to 10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions compared to one of the structurally stable peptides in Table 1 (wherein the substitution is not at a peg position in Table 1). In some instances, this document discloses peptides that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 93% identical to one of the structurally stable peptides in Table 1. In some instances, this document discloses peptides that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 93% identical to the amino acid sequences shown in any of SEQ ID NO:27, 29, 36, 43, 46 or any of SEQ ID NO:49, 51, 58, 65, and 68 (wherein the variation is not at a peg position in any of SEQ ID NO:27, 29, 36, 43, and 46 or SEQ ID NO:49, 51, 58, 65, and 68, respectively). It should be understood that the variation in a particular sequence is not at a peg position (i.e., the bolded residues in Table 1). In some instances, this document discloses peptides that are 100% identical to one of the structurally stable peptides in Table 1. In some instances, this document discloses peptides that are 100% identical to the amino acid sequences shown in any of SEQ ID NO:27, 29, 36, 43, and 46 or any of SEQ ID NO:49, 51, 58, 65, and 68. In some instances, the structurally stable peptide does not contain the amino acid sequence NAGKST (SEQ ID NO:258). In some instances, the structurally stable peptide does not contain the amino acid sequence NAGKS (SEQ ID NO:259). In some instances, the structurally stable peptide does not contain the amino acid sequence NAGK (SEQ ID NO:260). In some instances, the structurally stable peptide does not contain the amino acid sequence NAG. In some instances, the structurally stable peptide does not contain the amino acid sequence NA. In some instances, the structurally stable peptide does not contain the amino acids corresponding to positions 517-522, 517-521, 517-520, 517-519, or 517-518 of SEQ ID NO:1 (or the corresponding amino acids of the RSV-F protein of another strain (see, for example)). Figure 4In some instances, the structurally stable peptide is 25 to 34 amino acids in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some instances, the structurally stable peptide is 30 amino acids in length. In some instances, the structurally stable peptide has one or more of the following properties (e.g., 1, 2, 3, 4, or 5): (i) is α-helical; (ii) is protease resistant; (iii) binds to RSV 5-helical bundle protein; (iv) inhibits RSV infection of cells; and / or (v) prevents RSV infection of cells. In some instances, the peptide inhibits RSV infection of cells in pseudovirus and / or live RSV virus assays and / or prevents RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0110] In some instances, any substitution as described herein may be a conservative substitution. In other instances, any substitution as described herein may be a non-conservative substitution.

[0111] In some instances, the structurally stable peptide does not contain the amino acid sequence NAGKST (SEQ ID NO:258). In some instances, the structurally stable peptide does not contain the amino acid sequence NAGKS (SEQ ID NO:259). In some instances, the structurally stable peptide does not contain the amino acid sequence NAGK (SEQ ID NO:260). In some instances, the structurally stable peptide does not contain the amino acid sequence NAG. In some instances, the structurally stable peptide does not contain the amino acid sequence NA. In some instances, the structurally stable peptide does not contain the amino acids corresponding to positions 517-522, 517-521, 517-520, 517-519, or 517-518 of SEQ ID NO:1 (or the corresponding amino acids of the RSV-F protein of another strain (see example)). Figure 4 In some instances, the length of the structurally stable peptide is 25 to 34 amino acids (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some instances, the length of the structurally stable peptide is 30 amino acids.

[0112] In some instances, the non-natural amino acids that can be used as stapled 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'-pentenyl)glycine; and α,α-bis(7'-octenyl)glycine.

[0113] In some instances, the internal stud replaces the side chain of two amino acids, i.e., each stud is between two amino acids separated by, for example, six amino acids. In some instances, the amino acids forming the stud are located at each of the stud positions i and i+7. For example, when the peptide has the sequence ...X1, X2, X3, X4, X5, X6, X7, X8, X9..., crosslinks between X1 and X8 (i and i+7) are a useful form of hydrocarbon studation for the peptide. The use of i and i+4 studs, multiple crosslinks (e.g., two, three, four or more), or tandem sutures are also envisioned. Further descriptions of the preparation and use of hydrocarbon-studded peptides can be found, for example, in U.S. Patent Publications 2012 / 0172285, 2010 / 0286057, and 2005 / 0250680, the contents of which are incorporated herein by reference in their entirety.

[0114] “Peptide pinning” is a term coined by synthetic methodology, in which two olefinic side chains (e.g., crosslinkable side chains) present in a peptide chain are covalently linked (e.g., “pinned together”) using a ring-closed metathesis (RCM) reaction to form a crosslinked ring (see, for example, Blackwell et al., J. Org. Chem., 66:5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). Structural stabilization can be achieved, for example, by pinning peptides (see, for example, Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are incorporated herein by reference in their entirety). In some cases, the pins are hydrocarbon pins.

[0115] In some instances, the nails used in this article are all-hydrocarbon nails.

[0116] In some instances, the pins used herein are lactam pins; UV cycloaddition pins; oxime pins; thioether pins; double-click pins; bis-lactam pins; bis-aryl pins; or any combination of two or more of them. Stable peptides as described herein include studded peptides, as well as peptides containing multiple studs or any other chemical strategy for structural enhancement (see, for example, 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). 7 October 2009; (37): 5588–5590; Lau et al., Chem. Soc. Rev., 2015, 44: 91–102; and Gunnoo et al., Org. Biomol. Chem., 2016, 14: 8002–8013; Each of these references is incorporated herein by reference in its entirety.

[0117] Peptides are "structurally stable" because they retain their native secondary structure. For example, stapled peptides that tend to have an α-helical secondary structure can maintain their native α-helical conformation. This secondary structure increases the peptide's resistance to proteolytic cleavage and heat, and can increase target binding affinity, hydrophobicity, plasma membrane binding, and / or cell permeability. Therefore, the stapled (crosslinked) peptides described herein exhibit improved biological activity and pharmacology compared to their corresponding non-stapled (uncrosslinked) peptides.

[0118] In some instances, the structurally stable peptides described herein comprise an amino acid sequence comprising 25 to 29 consecutive amino acids of the sequence of SEQ ID NO: 100, excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO: 100; wherein the structurally stable peptide comprises an amino acid sequence having internal crosslinks having the following formula:

[0119]

[0120] Or its pharmaceutically acceptable salt;

[0121] Each of R1 and R2 is H or C1 to C10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl or heterocycloalkyl, any of which may be substituted or unsubstituted;

[0122] Where x is 3 or 6;

[0123] Each R3 is independently alkylene, alkenylene, or ynylene, any of which may be substituted or unsubstituted;

[0124] Where z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0125] The structurally stable peptide binds to RSV 5-helical bundle protein and / or the structurally stable peptide inhibits RSV infection of cells and / or prevents RSV infection of cells; and

[0126] The length of the structurally stable peptide is 25 to 30 amino acids, optionally 29 amino acids. In some examples, each [Xaa]... x [Xaa] is identified in Table 2. xOr a variant thereof having one amino acid substitution. In some instances, the amino acid sequence comprises 25 to 30 (e.g., 25, 26, 27, 28, 29, 30) consecutive amino acids of the sequence shown in SEQ ID NO:100, having 2 to 5 (e.g., 2, 3, 4, 5) amino acid substitutions relative to the sequence shown in SEQ ID NO:100. In some instances, the amino acid sequence comprises 25 to 30 (e.g., 25, 26, 27, 28, 29, 30) consecutive amino acids of the sequence shown in SEQ ID NO:100, having 2 to 4 (e.g., 2, 3, 4) amino acid substitutions relative to the sequence shown in SEQ ID NO:100. In some instances, the amino acid sequence comprises 25 to 30 (e.g., 25, 26, 27, 28, 29, 30) consecutive amino acids of the sequence shown in SEQ ID NO:100, having two or three amino acid substitutions relative to the sequence shown in SEQ ID NO:100. In some instances, the amino acid sequence comprises 25 to 30 (e.g., 25, 26, 27, 28, 29, 30) consecutive amino acids of the sequence shown in SEQ ID NO:100, having two amino acid substitutions relative to the sequence shown in SEQ ID NO:100. In some instances, the structurally stable peptide does not contain the amino acid sequence NAGKST (SEQ ID NO:258). In some instances, the structurally stable peptide does not contain the amino acid sequence NAGKS (SEQ ID NO:259). In some instances, the structurally stable peptide does not contain the amino acid sequence NAGK (SEQ ID NO:260). In some instances, the structurally stable peptide does not contain the amino acid sequence NAG. In some instances, the structurally stable peptide does not contain the amino acid sequence NA. In some instances, the structurally stable peptide does not contain the amino acids corresponding to positions 517-522, 517-521, 517-520, 517-519, or 517-518 of SEQ ID NO:1 (or the corresponding amino acids of the RSV-F protein of another strain (see example...) Figure 4 In some instances, the structurally stable peptides described above possess one or more of the following properties (e.g., 1, 2, 3, 4, or 5): (i) being α-helical; (ii) being protease-resistant; (iii) binding to RSV 5-helical bundle proteins; (iv) inhibiting RSV infection of cells; and / or (v) preventing RSV infection of cells. In some instances, the peptides inhibit RSV infection of cells in pseudovirus and / or live RSV virus assays and / or prevent RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0127] In some instances, substitutions to the continuous amino acid sequence of SEQ ID NO:100 in Formula I (except for substitutions introducing a linker group R3) are conserved. In some instances, substitutions to the continuous amino acid sequence of SEQ ID NO:100 in Formula I (except for substitutions introducing a linker group R3) are non-conservative. Methods for determining the type of substitution are as described herein, see, for example, the “RSV peptide” section above. In some instances, the structurally stable peptide is 25 to 45 amino acids in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). In some instances, the length of the structurally stable peptide is 25 to 40 amino acids (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40). In some instances, the length of the structurally stable peptide is 25 to 35 amino acids (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35). In some instances, the length of the structurally stable peptide is 25 to 30 amino acids (e.g., 25, 26, 27, 28, 29, or 30). In some instances, the length of the structurally stable peptide is 30 amino acids. In some instances, the structurally stable peptides described above possess one or more of the following properties (e.g., 1, 2, 3, 4, or 5): (i) being α-helical; (ii) being protease-resistant; (iii) binding to RSV 5-helical bundle proteins; (iv) inhibiting RSV infection of cells; and / or (v) preventing RSV infection of cells. In some instances, the peptides inhibit RSV infection of cells in pseudovirus and / or live RSV virus assays and / or prevent RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0128] In some instances of equation (I),

[0129] Each R1 and R2 is independently H or C1 to C 10 Alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocycloalkyl;

[0130] R3 can be alkyl, alkenyl, or alkynyl; [R4—K—R4] n Each of them is replaced by 0-6 R5s;

[0131] R4 is an alkyl, alkenyl, or alkynyl group;

[0132] R5 can be a halogen, alkyl group, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R6, a fluorescent part, or a radioactive isotope;

[0133] K is O, S, SO, SO2, CO, CO2, CONR6, or

[0134]

[0135] R6 is H, alkyl, or a therapeutic agent;

[0136] n is an integer from 1 to 4;

[0137] x is 3 or 6;

[0138] Each y is an independent integer between 0 and 100;

[0139] z is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

[0140] And each Xaa is an amino acid independently.

[0141] In some instances, [Xaa] of equation (I) w [Xaa] of formula (I) x [Xaa] of formula (I) y Each of these is as described for any of the constructs 1-22 in Table 2. For example, for construct 3 of Table 2, [Xaa] w [Xaa] x and [Xaa] y The structurally stable peptide, [Xaa] w [Xaa] x and [Xaa] y They are: FD, SISQVN (SEQ ID NO:220) and KINQSLAFIRKSDELLHNV (SEQ ID NO:242).

[0142] Table 2. Sequences of [Xaa]w, [Xaa]x, and [Xaa]y for constructs 1-22 of formula (I).

[0143]

[0144]

[0145] In some instances, the structurally stable peptide comprises or is composed of construct 3 of Table 2. In some instances, the structurally stable peptide comprises or is composed of construct 17 of Table 2. In some instances, the structurally stable peptide comprises or is composed of any of constructs 1-22 of Table 2, except for at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid substitutions or deletions (e.g., up to a total of 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions or deletions relative to the sequence of any of constructs 1-22).

[0146] In some instances, the sequences shown in Table 2 above may have at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid substitutions or deletions (e.g., up to a total of 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions or deletions relative to the sequence of SEQ ID NO:100). RSV-F HR2 peptides may include any amino acid sequence described herein.

[0147] In some instances, the structurally stable peptide of formula (I) comprises the sequence of the construct shown in Table 2 above and has one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) is α-helical; (ii) is protease-resistant; (iii) binds to RSV 5-helical bundle protein; (iv) inhibits RSV infection of cells; and / or (v) prevents RSV infection of cells. In some instances, the structurally stable peptide inhibits RSV infection of cells in pseudovirus and / or live RSV virus assays and / or prevents RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0148] The chain of formula (I) may include alkyl, alkenyl, or ynyl moieties (e.g., C5, C8, C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 11 Or C 12 Alkyl, C5, C8 or C 11 Alkenyl or C5, C8, C 11 Or C 12 Alkyne group). The amino acid in the chain can be α-disubstituted (e.g., C1-C3 or methyl).

[0149] In some examples of formula (I), each y is independently an integer from 0 to 15 or from 3 to 15. In some examples of formula (I), R1 and R2 are each independently H or a C1-C6 alkyl group. In some examples of formula (I), R1 and R2 are each independently C1-C3 alkyl groups. In some examples of formula (I), at least one of R1 and R2 is methyl. For example, both R1 and R2 can be methyl. In some examples of formula (I), R3 is C... 11Alkyl group and x is 6. In some examples of formula (I), x is 6 and R3 is C. 11 Alkenyl. In some instances, R3 is a straight-chain alkyl, alkenyl, or alkynyl group. In some instances, R3 is —CH2—CH2—CH2—CH═CH—CH2—CH2—CH2—.

[0150] In another aspect of formula (I), both α,α-disubstituted stereocenters are either in the R configuration or the S configuration (e.g., i, i+4 crosslinking), or one stereocenter is R and the other stereocenter is S (e.g., i, i+7 crosslinking). Therefore, formula (I) is described as follows:

[0151]

[0152] The stereocenters of both C' and C″ disubstituted products can be R-configured, or both can be S-configured. When x in formula (I) is 6, the stereocenter of C' disubstituted products is R-configured, and the stereocenter of C″ disubstituted products is S-configured. The R3 double bond in formula (I) can be E or Z stereochemical configuration.

[0153] In some instances of equation (I), R3 is [R4—K—R4]. n Furthermore, R4 is a straight-chain alkyl, alkenyl, or alkynyl group.

[0154] As used herein, the term "alkyl" alone or in combination with other terms refers to a saturated hydrocarbon group that can be straight-chain or branched. In some instances, an alkyl group contains 1 to 7, 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkyl moiety 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, etc. In some instances, the alkyl group is methyl, ethyl, or propyl. The term "alkylene" refers to the alkyl group that is linked to it.

[0155] As used herein, "alkenyl" alone or in combination with other terms refers to an alkyl group having one or more carbon-carbon double bonds. In some instances, the alkenyl moiety contains 2 to 6 or 2 to 4 carbon atoms. Example alkenyl groups include, but are not limited to, vinyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, etc.

[0156] As used herein, "alkynyl" alone or in combination with other terms refers to an alkyl group having one or more carbon-carbon triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, etc. In some instances, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.

[0157] As used herein, "alkynyl" alone or in combination with other terms refers to an alkyl group having one or more carbon-carbon triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, etc. In some instances, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.

[0158] As used herein, the term "cycloalkylalkyl" alone or in combination with other terms refers to a group of the formula cycloalkyl-alkyl-. In some instances, the alkyl moiety has 1 to 4, 1 to 3, 1 to 2, or 1 carbon atom. In some instances, the alkyl moiety is methylene. In some instances, the cycloalkyl moiety has 3 to 10 ring members or 3 to 7 ring members. In some instances, the cycloalkyl is monocyclic or bicyclic. In some instances, the cycloalkyl moiety is monocyclic. In some instances, the cycloalkyl moiety is C10. 3-7 Monocyclic cycloalkyl.

[0159] As used herein, the term "heteroarylalkyl" alone or in combination with other terms refers to a group of the formula heteroaryl-alkyl-. In some instances, the alkyl moiety has 1 to 4, 1 to 3, 1 to 2, or 1 carbon atom. In some instances, the alkyl moiety is methylene. In some instances, the heteroaryl moiety is a monocyclic or bicyclic group having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some instances, the heteroaryl moiety has 5 to 10 carbon atoms.

[0160] As used herein, the term "substituted" means that a hydrogen atom is replaced by a non-hydrogen group. It should be understood that substitution at a given atom is limited by valence.

[0161] As used herein, “halogen” or “halogen” alone or in combination with other terms includes fluorine, chlorine, bromine, and iodine. In some instances, halogen is F or Cl.

[0162] While this document provides hydrocarbon-based chains, other chains can also be used in the structurally stable RSV-F HR2 peptide described herein. For example, the chain may include one or more of an ether, thioether, ester, amine, amide, or triazole moiety. In some cases, naturally occurring amino acid side chains may be incorporated into the chain. For example, the chain may be coupled with a functional group such as a hydroxyl group in serine, a thiol group in cysteine, a primary amine in lysine, an acid in aspartic acid or glutamic acid, or an amide in asparagine or glutamine. Thus, the chain can be generated using naturally occurring amino acids instead of using a chain made by coupling two non-naturally occurring amino acids. A single non-naturally occurring amino acid can also be used with a naturally occurring amino acid. Crosslinking bonds containing triazoles (e.g., 1,4-triazole or 1,5-triazole) can be used (see, for example, Kawamoto et al., 2012 Journal of Medicinal Chemistry 55:1137; WO 2010 / 060112). In addition, other methods for performing different types of pinning are well known in the art and can be used with the RSV-F HR2 peptide described herein (see, for example, lactam pinning: Shepherd et al., J. Am. Chem. Soc., 127:2974-2983 (2005); UV-cycloaddition pinning: Madden et al., Bioorg. Med. Chem. Lett., 21:1472–1475 (2011); disulfide pinning: Jackson et al., Am. Chem. Soc., 113:9391-9392 (1991); oxime pinning: Haney et al., Chem. Commun., 47:10915–10917 (2011); thioether pinning: Brunel and Dawso). n, Chem. Commun., 552–2554 (2005); Optically switchable pinning: JR Kumita et al., Proc. Natl. Acad. Sci. USA, 97:3803–3808 (2000); Double-click pinning: Lau et al., Chem. Sci., 5:1804–1809 (2014); Dilactam pinning: JCPhelan et al., J. Am. Chem. Soc., 119:455–460 (1997); and bisarylated pinning: AM Spokoyny et al., J. Am. Chem. Soc., 135:5946–5949 (2013)).

[0163] Furthermore, it can be envisioned that the length of the tether can vary. For example, a shorter tether can be used when a relatively high degree of constraint on the second-order α-helix structure is desired, while in some instances, a less constrained second-order α-helix structure is desired, thus requiring a longer tether.

[0164] In addition, while this article provides tandems spanning amino acids i to i+7 to provide tandems primarily on a single facet of the α-helix, tandems can be synthesized to span any combination of many amino acids and can also be combined to assemble multiple tandems.

[0165] In some instances, the hydrocarbon chains described herein (i.e., crosslinks) can be further manipulated. In one instance, the double bonds of the hydrocarbon alkenyl chains (e.g., synthesized using ruthenium-catalyzed ring-closed metathesis (RCM)) can be oxidized (e.g., via epoxidation, amino hydroxylation, or dihydroxylation) to provide one of the following compounds.

[0166]

[0167] One of the epoxide or free hydroxyl moieties can be further functionalized. For example, the epoxide can be treated with a nucleophile that provides additional functional groups that can be used, for example, to link therapeutic agents. Alternatively, this derivatization can be achieved through synthetic operations at the amino or carboxyl terminus of the peptide or via amino acid side chains. Other agents can be attached to the functionalized chain, such as agents that promote peptide entry into cells.

[0168] In some instances, α-disubstituted amino acids are used in peptides to improve the stability of the α-helix secondary structure. However, α-disubstituted amino acids are not essential, and instances using single α-substituents (e.g., in chained amino acids) are also envisioned.

[0169] Structurally stable (e.g., strung) peptides may include drugs, toxins, polyethylene glycol derivatives, secondary peptides, carbohydrates, etc. When a polymer or other agent is linked to a structurally stable (e.g., strung) peptide, it may be desirable for the composition to be substantially homogeneous.

[0170] In some instances, structurally stable (e.g., stapled) peptides can also be modified, for example, to further promote mucosal adhesion, membrane binding, or increase in vivo stability. For instance, acylation or polyethylene glycol modification of structurally stable peptides can improve bioavailability, increase blood circulation, alter pharmacokinetics, modify immunogenicity, and / or reduce the required administration frequency.

[0171] In some instances, the structurally stable (e.g., pinned) peptides disclosed herein possess enhanced ability to bind to or penetrate cell membranes (e.g., relative to unstable peptides). See, for example, International Publication WO 2017 / 147283, which is incorporated herein by reference in its entirety.

[0172] In some instances, the structurally stable peptide is the peptide described in the accompanying drawings or working examples.

[0173] In some instances, the structurally stable peptides described herein (e.g., in Table 1) form part of the structurally stable peptide conjugates described in the “Structurally Stable Peptide Conjugates” section below.

[0174] Structurally stable peptide conjugates

[0175] This document also provides conjugates comprising the structurally stable peptide described herein (see the “Structurally Stable Peptides” section above, e.g., peptides in Table 1 or constructs in Table 2, or variants thereof) and cholesterol (or cholesterol variants, such as thiocholesterol), wherein the cholesterol (or cholesterol variants, such as thiocholesterol) is directly or via a linker (e.g., PEG(n), where n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) linked to the C-terminal amino acid of the structurally stable peptide. The terms “conjugate” and “structurally stable peptide conjugate” are used interchangeably herein. In some instances, the conjugate comprises a structurally stable peptide and cholesterol, wherein the cholesterol is directly or via a linker (e.g., PEG(n), where n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some instances, the conjugate comprises a structurally stable peptide and a thiocholesterol, wherein the thiocholesterol is linked directly or via a linker (e.g., PEG(n), where n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). The conjugate possesses one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) is α-helical; (ii) is protease-resistant; (iii) binds to RSV 5-helical bundle protein; (iv) inhibits RSV infection of cells; and / or (v) prevents RSV infection of cells. In some instances of the conjugate, the structurally stable peptide is conjugated to a thiocholesterol. In some examples of conjugates, the structurally stable peptide is conjugated to thiocholesterol via a linker comprising PEG (e.g., PEG(n), where n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some examples of conjugates, the structurally stable peptide is conjugated to cholesterol. In some examples of conjugates, the structurally stable peptide is conjugated to cholesterol via a linker comprising PEG (e.g., PEG(n), where n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some examples, the structurally stable peptide of the conjugate is the structurally stable form of the peptide described in the "RSV Peptide" section above. In some examples, the structurally stable peptide of the conjugate is the structurally stable peptide described in the "Structurally Stable Peptides" section above. In some examples, the structurally stable peptide of the conjugate is the structurally stable peptide described in the working examples or figures herein.

[0176] In some instances, the structurally stable peptide of the conjugate comprises an internally cross-linked amino acid sequence comprising 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:100), excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO:100, and wherein the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO:258); wherein two of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three or six amino acids; wherein the conjugate binds RSV 5-helical bundle protein and / or wherein the conjugate RSV 5-Helical bundle protein and / or prevention of RSV infection of cells; and wherein the length of the conjugate is 25 to 45 amino acids (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45).

[0177] In some instances, the structurally stable peptide of the conjugate comprises or is composed of the amino acid sequences in Table 1 (e.g., any of SEQ ID NO: 27-70). In some instances, the structurally stable peptide of the conjugate comprises or is composed of the amino acid sequences in any of SEQ ID NO: 27, 29, 36, 43, and 46 or any of SEQ ID NO: 49, 51, 58, 65, and 68. In some instances, the structurally stable peptide of the conjugate comprises or is composed of the amino acid sequence of SEQ ID NO: 29. In some instances, the structurally stable peptide of the conjugate comprises or is composed of the amino acid sequence of SEQ ID NO: 51. In some instances, the structurally stable peptide of the conjugate comprises or is composed of the amino acid sequence of SEQ ID NO: 43. In some instances, the structurally stable peptide of the conjugate comprises or is composed of the amino acid sequence of SEQ ID NO: 65. In some instances, the structurally stable peptide of the conjugate comprises or is composed of any of the constructs in Table 2. In some instances, the structurally stable peptide of the conjugate comprises or is composed of any of or is composed of any of the constructs 1, 3, 10, 17, or 20 in Table 2. In some instances, the structurally stable peptide of the conjugate comprises or is composed of any of the constructs 3 in Table 2. In some instances, the structurally stable peptide of the conjugate comprises or is composed of any of the peptides in Table 1, except for substitutions, insertions, and / or deletions of 1 to 10, 1 to 5, 1 to 3, 2, or 1 amino acid (except at the peg position). In some instances, the structurally stable peptide of the conjugate comprises or is composed of any of the constructs in Table 2, except for substitutions, insertions, and / or deletions of 1 to 10, 1 to 5, 1 to 3, 2, or 1 amino acid (except at the peg position).

[0178] The addition of PEG molecules can improve the pharmacokinetic and pharmacodynamic properties of structurally stable peptides. For example, polyethylene glycolation can reduce renal clearance and yield more stable blood drug concentrations. PEG is a water-soluble polymer and can be represented as a peptide linked to the following formula:

[0179] XO--(CH2CH2O) n --CH2CH2--Y, where n is 2 to 10,000, and X is H or a terminal modification, such as C. 1-4Alkyl group; and Y is an amide, carbamate, or urea bond linked to an amino group (including, but not limited to, the ε-amine or N-terminus of lysine) of a structurally stable peptide. Y may also be a maleimide linked 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 skilled in the art. PEG can be linear or branched. Various forms of PEG, including various functionalized derivatives, are commercially available.

[0180] In some instances, such as those used herein, PEG acts as a linker or spacer between one of the peptides (e.g., the structurally stable peptides in Table 1 or the constructs in Table 2) and the cholesterol or thiocholesterol moiety.

[0181] In some instances, the PEG molecule includes a cholesterol moiety. In some instances, the cholesterol moiety is thiocholesterol. In some instances, the sulfur in the thioether moiety of thiocholesterol is replaced with an oxygen atom to produce an ether moiety in cholesterol derivatization.

[0182] In some instances, the PEG molecule comprises the following formula (Formula II), 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):

[0183]

[0184] In some instances, the PEG molecule comprises the following formula (Formula III), 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):

[0185]

[0186] In some instances, n = 16 for each of Equations II and III. In some instances, n = 17 for each of Equations II and III. In some instances, n = 18 for each of Equations II and III. In some instances, n = 19 for each of Equations II and III. In some instances, n = 20 for each of Equations II and III.

[0187] PEG with degradable bonds in the main chain can be used. For example, PEG can be prepared from ester bonds that are easily hydrolyzed. Degradable PEG bonds are described in WO 99 / 34833, WO 99 / 14259 and US6,348,558, each of which is incorporated herein by reference in its entirety.

[0188] In some instances, macromolecular polymers (e.g., PEG) are linked to structurally stable (e.g., pinned) peptides as described herein via intermediate linkers. In some instances, the linker consists of 1 to 20 amino acids linked by peptide bonds, wherein these amino acids are selected from 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well known to those skilled in the art. In other instances, 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In still other instances, the linker consists primarily of sterically unhindered amino acids, such as glycine and alanine. Non-peptide linkers are also possible. For example, alkyl linkers, such as -NH(CH2), may be used. n C(O)-, where n = 2-20. These alkyl linkers can be further substituted with any non-sterically hindered group, such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. U.S. Patent No. 5,446,090 describes bifunctional PEG linkers and their use in conjugates formed with peptides at the ends of each PEG linker.

[0189] Exemplary structurally stable peptide conjugates are provided in Table 3 below. It should be noted that this disclosure also covers structurally stable peptide conjugates in Table 3 that further comprise three N-terminal amino acids X1X2X3, wherein X1 is any amino acid, optionally S; X2 and X3 are any negatively charged amino acids immediately upstream of the first amino acid of the sequence listed in Table 3. In some instances, X1X2X3 = SDE. In some instances, the structurally stable peptide conjugate comprises or consists of the amino acid sequence shown in any of SEQ ID NO:71-92. In some instances, the structurally stable peptide conjugate comprises or consists of the amino acid sequence shown in any of SEQ ID NO:6-26. In some instances, the structurally stable peptide conjugate comprises or consists of the sequence shown in SEQ ID NO:5 or SEQ ID NO:73. In some instances, the structurally stable peptide conjugate comprises or consists of the sequence shown in SEQ ID NO:21 or SEQ ID NO:87. In some instances, the structurally stable peptide conjugate comprises or consists of the amino acid sequence shown in any of SEQ ID NO:71-92, except for 1 to 10, 1 to 5, 1 to 3, 2, or 1 amino acid substitution, insertion, and / or deletion (except at the peg and conjugation sites). In some instances, the structurally stable peptide conjugate comprises or consists of the amino acid sequence shown in any of SEQ ID NO:6-26, except for 1 to 10, 1 to 5, 1 to 3, 2, or 1 amino acid substitution, insertion, and / or deletion (except at the peg and conjugation sites). In some instances, the structurally stable peptide conjugate has one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) is α-helical; (ii) is protease resistant; (iii) binds to RSV 5-helical bundle protein; (iv) inhibits RSV infection of cells; and / or (v) prevents RSV infection of cells.

[0190]

[0191] In Table 3, relative to SEQ ID NO:71-92, “X1” = an α,α-disubstituted non-natural amino acid having an olefinic side chain crosslinked with X2; “X2” = an α,α-disubstituted non-natural amino acid having an olefinic side chain crosslinked with X1, and * includes Z-PEG(n)-cholesterol or Z-PEG(n)-mercaptocholesterol, where n = 1-36, and where Z is a diamino acid (e.g., lysine or ornithine). In Table 3, relative to SEQ ID NO:5-26, “8” = (R)-α-(7'-octenyl)alanine; “X” = (S)-α-(4'-pentenyl)alanine, and * = Lys(ε-(PEG)4-mercaptocholesterol). In some examples of SEQ ID NO:71-92 or 5-26, * is Lys(ε-(PEG)4-cholesterol) or Lys(ε-(PEG)4-mercaptocholesterol), where n = 1-36. In some instances of SEQ ID NO:71-92, * = one of the following formulas II or III, 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). In some instances of SEQ ID NO:71-92, * = one of the following formulas II or III, where n = 16. In some instances of SEQ ID NO:71-92, * = one of the following formulas II or III, where n = 20. The two formulas marked with “*” include:

[0192]

[0193] In some instances of SEQ ID NO:71-92, * = Equation II, where n = 1-36 (e.g., 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 instances of SEQ ID NO:71-92, * = Equation II, where n = 16. In some instances of SEQ ID NO:71-92, * = Equation II, where n = 20. In some instances of SEQ ID NO:71-92, * = Equation III, where n = 1-36 (e.g., 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 instances of SEQ ID NO:71-92, * = Equation III, where n = 16. In some instances of SEQ ID NO:71-92, * = Equation III, where n = 20. In some instances of SEQ ID NO:5-26, * = one of the following formulas IV or V, 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). In some instances of SEQ ID NO:5-26, * = one of the following formulas IV or V, where n = 16. In some instances of SEQ ID NO:5-26, * = one of the following formulas IV or V, where n = 20. The two formulas marked with “*” include:

[0194]

[0195]

[0196] In some instances of SEQ ID NO:5-26, * = formula IV, where n = 1-36 (e.g., 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 instances of SEQ ID NO:5-26, * = formula IV, where n = 16. In some instances of SEQ ID NO:5-26, * = formula IV, where n = 20. In some instances of SEQ ID NO:5-26, * = formula V, where n = 1-36 (e.g., 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 instances of SEQ ID NO:5-26, * = formula V, where n = 16. In some instances of SEQ ID NO:5-26, * = formula V, where n = 20. It should be understood that the above conjugates can be modified to include additional amino acids at the N-terminus and / or C-terminus (e.g., adding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), and / or have N-terminal and / or C-terminal deletions (e.g., deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). In some instances, the conjugates are derived from SEQ ID NO:100 (e.g., containing the amino acid sequence of SEQ ID NO:100 or its internal cross-linked form or variants thereof, or composed of it). In some instances, the conjugates do not contain the amino acid sequence NAGKST (SEQ ID NO:258). In some instances, the conjugates do not contain the amino acid sequence NAGKS (SEQ ID NO:259). In some instances, the conjugates do not contain the amino acid sequence NAGK (SEQ ID NO:260). In some instances, the conjugates do not contain the amino acid sequence NAG. In some instances, the conjugates do not contain the amino acid sequence NA. In some instances, the conjugate does not contain the amino acids corresponding to positions 517-522, 517-521, 517-520, 517-519, or 517-518 of SEQ ID NO:1 (or the corresponding amino acids of the RSV-F protein of another strain (see, for example)). Figure 4 In some instances, the length of the conjugate is 25 to 34 amino acids (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some instances, the length of the conjugate is 30 amino acids.

[0197] This disclosure covers each structurally stable peptide conjugate and its variants (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) amino acid substitutions, insertions, and / or deletions, except at peg locations (i.e., the bolded residues in Table 3) and esterification locations (i.e., * in Table 3). In some instances, the variants have 1 to 10 amino acid substitutions, insertions, and / or deletions, except at peg locations (i.e., the bolded residues in Table 3) and esterification locations (i.e., * in Table 3). In some instances, the variants have 1 to 5 amino acid substitutions, insertions, and / or deletions, except at peg locations (i.e., the bolded residues in Table 3) and esterification locations (i.e., * in Table 3). In some instances, the variant has one to three amino acid substitutions, insertions, and / or deletions, except at peg locations (i.e., the bolded residues in Table 3) and at esterification locations (i.e., * in Table 3). In some instances, the variant has one to ten, ten to five, one to three, two, or one amino acid substitution, insertion, and / or deletion, except at peg locations (i.e., the bolded residues in Table 3) and at esterification locations (i.e., * in Table 3).

[0198] In some instances, the structurally stable peptide conjugates are 25 to 34 amino acids in length (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some instances, the structurally stable peptide conjugates are 25 to 30 amino acids in length (e.g., 25, 26, 27, 28, 29, or 30). In some instances, the structurally stable peptide conjugates are 30 amino acids in length. In some instances, the above-mentioned structurally stable peptide conjugates possess one or more (1, 2, 3, 4, or 5) of the following properties: (i) being α-helical; (ii) being protease-resistant; (iii) binding to RSV 5-helical bundle protein; (iv) inhibiting RSV infection of cells; and / or (v) preventing RSV infection of cells.

[0199] In some instances, the conjugate has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with one of the monopeptides in Table 3 (wherein the variation in the sequence is neither at the pinning site nor at the esterification site). It should be understood that the variation is neither at the pinning site (i.e., the bolded residues in Table 3) nor at the esterification site (i.e., * in Table 3). In some instances, the conjugate is 100% identical to the conjugates in Table 3. In some instances, the conjugate is 25 to 34 amino acids long (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some instances, the conjugate is 25 to 30 amino acids long (e.g., 25, 26, 27, 28, 29, or 30). In some instances, the conjugate is 30 amino acids long. In some instances, the conjugates described above possess one or more (1, 2, 3, 4, 5) of the following properties: (i) being α-helical; (ii) being protease resistant; (iii) binding to RSV 5-helical bundle protein; (iv) inhibiting RSV infection of cells; and / or (v) preventing RSV infection of cells.

[0200] In some instances, any substitutions described herein are conservative substitutions. In other instances, any substitutions described herein are non-conservative substitutions.

[0201] Pharmaceutical Composition

[0202] One or more of any structurally stable (e.g., pinned) peptides or structurally stable (e.g., pinned) peptide conjugates described herein may be formulated as pharmaceutical compositions or used in pharmaceutical compositions. Pharmaceutical compositions may be used in the methods of treatment or prevention described herein. In some instances, the pharmaceutical composition comprises a structurally stable peptide described herein, along with a pharmaceutically acceptable carrier. In some instances, the pharmaceutical composition comprises a structurally stable peptide conjugate described herein, along with a pharmaceutically acceptable carrier. In some instances, the pharmaceutical composition comprises a structurally stable (e.g., pinned) peptide or a structurally stable (e.g., pinned) peptide conjugate that contains or is composed of the same amino acid sequence as shown in Table 1 or Table 3 or the constructs shown in Table 2. In some instances, the pharmaceutical composition comprises a structurally stable (e.g., pinned) peptide or a structurally stable (e.g., pinned) peptide conjugate containing the same amino acid sequence as shown in Table 1 or Table 3 or the construct shown in Table 2, except that the peptide also contains three N-terminal amino acids X1X2X3, where X1 is any amino acid, optionally S; and X2 and X3 are any negatively charged amino acids. In some instances, the pharmaceutical composition comprises a structurally stable peptide conjugate and a pharmaceutically acceptable carrier, the structurally stable peptide conjugate comprising or consisting of the amino acid sequence of any one of SEQ ID NO:27, 29, 36, 43, and 46 or any one of SEQ ID NO:49, 51, 58, 65, and 68. In some instances, the pharmaceutical composition comprises a structurally stable peptide and a pharmaceutically acceptable carrier, the structurally stable peptide comprising or consisting of the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:51. In some instances, the pharmaceutical composition comprises a structurally stable peptide and a pharmaceutically acceptable carrier, the structurally stable peptide comprising or consisting of the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:65. In some instances, the pharmaceutical composition comprises a structurally stable peptide and a pharmaceutically acceptable carrier, the structurally stable peptide comprising or consisting of the amino acid sequence of SEQ ID NO:281. In some instances, the pharmaceutical composition comprises a structurally stable peptide conjugate and a pharmaceutically acceptable carrier, the structurally stable peptide conjugate comprising or consisting of the amino acid sequence of any one of SEQ ID NO:71, 73, 80, 87, and 90 or any one of SEQ ID NO:5, 6, 14, 21, and 24. In some instances, the pharmaceutical composition comprises a structurally stable peptide conjugate and a pharmaceutically acceptable carrier, the structurally stable peptide conjugate comprising or consisting of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:73.In some instances, the pharmaceutical composition comprises a structurally stable peptide conjugate and a pharmaceutically acceptable carrier, the structurally stable peptide conjugate comprising or consisting of the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:87. In some instances, the pharmaceutical composition comprises a structurally stable (e.g., pinned) peptide or a structurally stable (e.g., pinned) peptide conjugate comprising or consisting of the same amino acid sequence as shown in Tables 1, 2, or 3, except for 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution, insertion, or deletion. It should be understood that the amino acid substitution, insertion, or deletion is not at the pinned position (e.g., not at position 1 or 8 of the amino acid sequence of SEQ ID NO:6). In some instances, the pharmaceutical composition comprises a structurally stable (e.g., pinned) peptide or a structurally stable (e.g., pinned) peptide conjugate containing or consisting of an amino acid sequence identical to or consisting of any of the amino acid sequences in SEQ ID NO: 27, 29, 36, 43, 46, 49, 51, 58, 65, 68, 71, 73, 80, 87, 90, 5, 6, 14, 21, and 24, with 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution, insertion, or deletion. These changes to the amino acid sequence can be made on the non-interacting α-helical sides of these peptides (i.e., amino acids that do not interact with the 5-helical bundles of the RSV-F protein) and / or on the interacting α-helical sides (i.e., amino acids that interact with the 5-helical bundles of the RSV-F protein), as long as they are not at pinned positions (e.g., at positions 1 and 8 of the amino acid sequence in SEQ ID NO: 6). Such compositions can be formulated or adapted to be administered to subjects via any route (e.g., any route approved by the U.S. Food and Drug Administration (FDA)). Exemplary methods are described in the FDA's CDER Data Standards Manual, version 004 (available at fda.give / cder / dsm / DRG / drg00301.htm). For example, the composition may be formulated or adapted for administration by inhalation (e.g., oral and / or nasal inhalation (e.g., via a nebulizer or spray)), injection (e.g., intravenous, intra-arterial, subdermal, intraperitoneal, intramuscular and / or subcutaneous); and / or for oral administration, mucosal administration and / or topical administration (including topical (e.g., nasal) sprays, eye drops and / or solutions).

[0203] In some instances, the pharmaceutical composition is formulated or adapted for administration by nasal spray / drops, nebulization, subcutaneous administration, or intravenous administration. In some instances, the pharmaceutical composition is formulated or adapted for administration by a local mode (e.g., nasal spray, nebulization).

[0204] In some instances, a pharmaceutical composition may comprise an effective amount of one or more structurally stable (e.g., pinned) peptides or structurally stable (e.g., pinned) peptide conjugates. As used herein, the terms “effective amount” and “effective treatment” refer to the amount or concentration of the agent (e.g., a structurally stable (e.g., pinned) peptide or structurally stable (e.g., pinned) peptide conjugate) or the pharmaceutical composition described herein, used over a period of time (including acute or chronic application and periodic or continuous application), which effectively produces the desired effect or physiological outcome (e.g., treatment of infection) upon its application.

[0205] The pharmaceutical compositions disclosed herein may include one or more structurally stable (e.g., stapled) peptides or structurally stable (e.g., stapled) peptide conjugates as described herein, and any pharmaceutically acceptable carrier and / or mediator. In some instances, the pharmaceutical compositions may also contain one or more additional therapeutic agents in amounts that effectively modulate the disease or disease symptoms.

[0206] The term “pharmaceutically acceptable carrier or adjuvant” means a carrier or adjuvant that can be administered together with a compound of the present disclosure (e.g., a structurally stable (e.g., a stapled) peptide or a structurally stable (e.g., a stapled) peptide conjugate) to a patient or a subject from another species provided herein, and which, when administered in a dose sufficient to deliver a therapeutic amount of the compound, does not impair the pharmacological activity of the compound and is non-toxic.

[0207] In some instances, the pharmaceutical compositions disclosed herein comprise one or more of acetate, citrate, and / or maleate. In some instances, the pharmaceutical compositions may comprise water or phosphate-buffered saline (PBS). In some instances, the pharmaceutical compositions may comprise chitosan.

[0208] The pharmaceutical compositions disclosed herein may comprise one or more pharmaceutically acceptable salts. In some instances, pharmaceutically acceptable salts include salts comprising: hydrochloride, sodium salt, sulfate, acetate, phosphate or diphosphate, chloride, potassium salt, maleate, calcium salt, citrate, mesylate, nitrate, tartrate, aluminum salt, gluconate, and any combination thereof.

[0209] The pharmaceutical compositions disclosed herein may contain any conventional, non-toxic, and pharmaceutically acceptable carrier, adjuvant, or mediator. In some instances, the pH of the formulation may be adjusted using pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form. As used herein, parenteral administration includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0210] In some instances, one or more structurally stable (e.g., pinned) peptides or structurally stable (e.g., pinned) peptide conjugates disclosed herein may be further conjugated to, for example, a carrier protein. Such conjugated compositions may be monovalent or multivalent. For example, a conjugated composition may include one structurally stable (e.g., pinned) peptide conjugate disclosed herein conjugated to a carrier protein. Alternatively, as another example, a conjugated composition may include two or more structurally stable (e.g., pinned) peptide conjugates disclosed herein further conjugated to a carrier.

[0211] When two entities are "joined" together, they are connected through direct or indirect covalent or non-covalent interactions. In some instances, the connection is covalent. In others, it is non-covalent. Non-covalent interactions include hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. Indirect covalent interactions occur when two entities are optionally covalently connected through a linker group.

[0212] Carrier proteins may include any protein that increases or enhances stability, half-life, tissue exposure, and / or immunogenicity in subjects. Exemplary carrier proteins are described in the art (see, for example, Fattom et al., Infect. Immun., 58:2309-2312, 1990; Devi et al., Proc. Natl. Acad. Sci. USA 88:7175-7179, 1991; Li et al., Infect. Immun. 57:3823-3827, 1989; Szu et al., Infect. Immun. 59:4555-4561, 1991; Szu et al., J. Exp. Med. 166:1510-1524, 1987; and Szu et al., Infect. Immun. 62:4440-4444, 1994). The polymer carrier can be a natural or synthetic material containing one or more primary and / or secondary amino groups, azide groups, or carboxyl groups. The carrier can be water-soluble.

[0213] Methods for preparing stapled or stitched peptides partially derived from PEG(n)-thiol cholesterol or PEG(n)-cholesterol

[0214] In one aspect, this disclosure is characterized by a method for preparing structurally stable peptides derived from PEG(n)-thiocholesterol or the PEG(n)-cholesterol moiety. The fully resin-on-resin synthesis method involves (a) providing a peptide comprising at least two non-natural amino acids having olefinic side chains (e.g., SEQ ID NO: 27-70), (b) crosslinking the peptide, in some instances by a ruthenium-catalyzed metathesis reaction, and (c) derivatizing the C-terminus on the resin with a variable-length PEG connector linked to the thiocholesterol or cholesterol moiety.

[0215] In some instances, the method includes cleaving a structurally stable peptide from a resin. Cleavage of structurally stable resins is known in the art. In some instances, cleavage occurs prior to derivatizing the C-terminus of the resin with a variable-length PEG linker attached to a thiocholesterol or cholesterol moiety. See, for example, de Vries et al., Science, March 26, 2021; 371(6536):1379-138; and Figueira et al., J. Virol. 91, e01554-16 (2016); each of these references is incorporated herein by reference in its entirety. In other instances, cleavage is performed after the step of derivatizing the C-terminus of the resin with a variable-length PEG linker attached to a thiocholesterol or cholesterol moiety.

[0216] In instances where the cleavage step is performed prior to the derivatization step, the method includes using a compound having one of the following formulas:

[0217]

[0218] Where n is 1-36. In some instances, n is 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. Therefore, compounds having one of the above formulas are also disclosed herein.

[0219] Pinned peptide synthesis: Following our reported method for generating all-hydrocarbon pinned peptides, pinned peptide fusion inhibitors were synthesized using Fmoc-based solid-phase peptide synthesis (Bird et al., Curr. Protocol. Chem., Biol., 3(3):99-117(2011); Bird et al., Methods Enzymol., 446:369-86 (2008). To achieve various pin lengths, α-methyl amino acids and α-alkenyl amino acids were mounted in specific pairings at discrete positions, for example, for the i, i+7 positioning, an S-pentenylalanine residue (S5) and an R-octenylalanine residue (R8) were used. For the pinning reaction, a Grubbs first-generation ruthenium catalyst dissolved in dichloroethane was added to the resin-bound peptide. To ensure maximum conversion, three to five rounds of pinning were performed. After the addition of the PEG(n)-mercaptocholesterol or PEG(n)-cholesterol moiety (see below), the peptide was then cleaved from the resin using trifluoroacetic acid, precipitated using a hexane:ethyl ether (1:1) mixture, air-dried, and purified by LC-MS. All peptides were quantified by amino acid analysis.

[0220] Synthesis of suture peptides: The methods for synthesizing the suture peptides described herein are known in the art. However, the following exemplary methods may be used. Synthetic chemical transformations and protecting group methods (protection and deprotection) for synthesizing the compounds described herein are known in the art and include, 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 V, VCH Publishers (1989); TW Greene and PGM Watts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette (ed.), Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0221] C-terminal derivatization of stapled or sewn peptides was performed using a resin-based synthesis method with PEG(n)-mercaptocholesterol or PEG(n)-cholesterol: To generate carboxy-mercaptocholesterol or carboxy-cholesterol reagents for peptide derivatization via solid-phase synthesis, mercaptocholesterol 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 mercaptocholesterol or sodium hydride or potassium tert-butoxide for cholesterol) were added with stirring. Then, five equivalents of tert-butyl bromoacetate were added, and the reaction mixture was stirred at room temperature for 2 hours, followed by stirring at 40°C for 30 minutes. Two volumes of trifluoroacetic acid (relative to the solvent) were added, and the reaction mixture was stirred at room temperature for 30 minutes. The reaction progress was monitored by TLC (19:1 Hex:EtOAc for mercaptocholesterol, 3:1 Hex:EtOAc for cholesterol) with KMnO4 staining. For example, (thiol)cholesterol migrates with the solvent front, where (thiol) ethers slow the migration by about 20%, and TFA hydrolysis brings the spot to baseline. The reaction mixture was added to 5 volumes of water and 1 volume of DCM. The solvent layer was washed with 0.1 M HCl and brine and dried over sodium sulfate. The solvent was removed by Rotovap to give an orange heavy oil, which was used without further purification. The yield was close to quantitative. Purity was greater than 90% by NMR comparison of olefin protons with a fresh CH2 singlet. For peptide derivatization with thiol or cholesterol, the completed resin-bound peptide sequence was treated with 20% piperidine / DMF, then capped with acetic anhydride to block the N-terminal amine, and then the C-terminal side chain lysine amine was revealed by treatment with 2% hydrazine in DMF (5x, 10 min each time). Amines were acylated with Fmoc-protected PEG(n) amino acids (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)) by treating the olefin three times with a Grubbs(I) catalyst for 2 hours each time. After completion, Fmoc was removed from the C-terminal NH of the PEG reagent, and the amine was acylated with carboxyl-mercaptocholesterol (or carboxyl-cholesterol) for 30 minutes. TFA cleavage yielded a crude product of excellent purity, which was further purified using semi-preparative HPLC.

[0222] The peptide sequences of the present invention can be prepared by chemical synthesis methods well known to those skilled in the art. See, for example, Fields et al., Synthetic Peptides: A User's Guide, Chapter 3, edited by Grant, WHFreeman & Co., New York, NY, 1992, p. 77. Therefore, peptides can be synthesized using automated Merrifield techniques for solid-phase synthesis, using side-chain protected amino acids, on, for example, an Applied Biosystems peptide synthesizer (model 430A or 431), with α-NH2 chemically protected by t-Boc or Fmoc.

[0223] One method for preparing the peptides described herein is using solid-phase peptide synthesis (SPPS). The C-terminal amino acid is linked to a cross-linked polystyrene resin via an acid-labile linker molecule. This resin is insoluble in the solvent used for synthesis, making it relatively simple and rapid to wash away excess reagents and byproducts. The N-terminus is protected with an Fmoc group, which is stable in acid but can be removed by base. Any side-chain functional groups are protected with base-stable, acid-labile groups.

[0224] Longer peptides can be prepared by linking individual synthetic peptides together using natural chemical linkages. The insertion of linking amino acids can be performed as described in the following literature: e.g., Young and Schultz, J Biol Chem. 2010, April 9; 285(15):11039-11044. Alternatively, longer synthetic peptides can be synthesized using well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct the gene encoding the peptide of the present invention, the amino acid sequence is reverse-translated to obtain the nucleic acid sequence encoding that amino acid sequence, preferably using the codons best suited for the organism to which the gene is to be expressed. Next, the synthetic gene is typically prepared by synthesizing oligonucleotides encoding the peptide and any regulatory elements (if desired). The synthetic gene is inserted into a suitable cloning vector and transfected into host cells. The peptide is then expressed under suitable conditions for the selected expression system and host. The peptide is purified and characterized using standard methods.

[0225] Peptides can be prepared in high-throughput, combinatorial manner (e.g., using high-throughput multichannel combinatorial synthesizers available from companies such as Advanced Chemtech or GyrosProtein Technologies). Peptide bonds can be replaced with the following bonds, for example, to increase the physiological stability of the peptide: anti-anti bond (C(O)-NH); reduced amide bond (NH-CH2); thiomethylene bond (S-CH2 or CH2-S); oxomethylene bond (O-CH2 or CH2-O); ethylene bond (CH2-CH2); thioamide bond (C(S)-NH); trans olefin bond (CH=CH); fluorinated trans olefin bond (CF=CH); ketomethylene bond (C(O)-CHR or CHR-C(O), where R is H or CH3); and fluorinated ketomethylene bond (C(O)-CFR or CFR-C(O), where R is H or F or CH3).

[0226] Peptides can be further modified by acetylation, amidation, biotinylation, cinnamicylation, farnesylation, fluoresceinization, formylation, myristylation, palmitoylation, and other esterifications, specifically including thiocholesterol or cholesterol modification, phosphorylation (Ser, Tyr, or Thr), stearylation, succinylation, and sulfonation using the resin-based methods disclosed herein. As described above, peptides can be conjugated to or contain variable-length linker atoms or portions, such as variable-length polyethylene glycol (PEG) moieties; alkyl groups (e.g., C1-C20 straight-chain or branched alkyl groups); fatty acid groups; and combinations thereof. Non-natural amino acids with α,α-disubstituted olefinic side chains of varying 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 instances, the suture peptides contain kinks between i, i+4, and i+8. Such suture peptides can be prepared in the context of SEQ ID NO:100. In some instances, the amino acids forming the nails or sutures are (R)-2-(4'-pentenyl)alanine, 2,2-bis(4-pentenyl)glycine, and (S)-2-(4'-pentenyl)alanine, respectively, at positions i, i+4, and i+8 of the suture. In some instances, for the peptide (the four helically stable turns) in which i is connected to i+7 and i+7 is connected to i+14 of the suture: one R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine), one bispentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine), and one R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine) are used. In some instances, for peptides where i connects to i+7 and i+7 connects to i+14 (the four helical stable turns): an S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine), a bispentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine), and an R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine) are used.In some instances, for peptides where i connects to i+7, i+7 connects to i+14 (the four helically stable turns): an S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine), a bispentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine), and an S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine) are used. In some instances, for peptides where i connects to i+7, i+7 connects to i+14 (the four helically stable turns): an R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine), a bispentenylglycine (e.g., α,α-bis(7'-octenyl)glycine), and an S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine) are used. In some instances, for peptides where i connects to i+7, i+7 connects to i+14 (the four helically stable turns): one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine), one bis(octenyl)glycine (e.g., α,α-bis(7'-octenyl)glycine), and one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine) are used. In some instances, for peptides where i connects to i+7, i+7 connects to i+14 (the four helically stable turns): one S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine), one bis(octenyl)glycine (e.g., α,α-bis(7'-octenyl)glycine), and one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine) are used. In some instances, for peptides where i connects to i+7, i+7 connects to i+14 (the four helical turns are stable): an S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine), a bis(octenyl)glycine (e.g., α,α-bis(7'-octenyl)glycine), and an 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 imparts an R-alkyl-stereoisomer. Additionally, 8-iodooctene is used instead of 5-iodopentene. Inhibitors are synthesized on a solid support using solid-phase peptide synthesis (SPPS) on MBHA resin or Rink Amide AM resin (see, for example, WO 2010 / 148335).

[0227] Fmoc-protected α-amino acids (excluding 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-tetramethylammonium hexafluorophosphate (HCTU) and Rink Amide MBHA are commercially available from, for example, Novabiochem (San Diego, CA). Dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), fluorescein isothiocyanate (FITC), and piperidine are commercially available from, for example, Sigma-Aldrich. The synthesis of olefinic amino acids has been reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0228] Similarly, methods suitable for obtaining (e.g., synthesizing), suturing, and purifying the peptides disclosed herein are also known in the art (see, for example, Bird et al., Methods in Enzymol., 446:369-386 (2008); Bird et al., Current Protocols in Chemical Biology, 2011; Walensky et al., Science, 305:1466-1470 (2004); Schafmeister et al., J. Am. Chem. Soc., 122:5891-5892 (2000); U.S. Patent Application No. 12 / 525,123, filed March 18, 2010; and U.S. Patent No. 7,723,468, granted May 25, 2010, each of which is incorporated herein by reference in its entirety).

[0229] In some instances, the peptides are substantially free of non-stitched or non-pinned peptide contaminants, or are isolated. Methods for purifying peptides include, for example, synthesizing the peptide on a solid-phase support. Following cyclization, a variety of alternative solvents and purification schemes are known in the art for the separation and purification of peptides and pinned peptides, and solvents including, but not limited to, DMSO, DMSO / dichloromethane mixtures, DMSO / NMP mixtures, or mixtures / solutions excluding DMSO can be used. DMSO / dichloromethane or DMSO / NMP mixtures may contain about 30%, 40%, 50%, or 60% DMSO. In one specific example, a 50% / 50% DMSO / NMP solution is used. The solution can be incubated for 1 hour, 6 hours, 12 hours, or 24 hours, and then the resin can be washed, for example, with dichloromethane or NMP. In one example, the resin is washed with NMP. The solution can be shaken and an inert gas can be bubbled through the solution.

[0230] The properties of the C-terminal PEG(n)-thiocholesterol or PEG(n)-cholesterol-derived suture or stapled peptides of this disclosure can be determined, for example, using the methods described below and in the examples.

[0231] Determine the characterization and anti-inflammatory properties of pegged HR2 peptides derived from PEG(n)-thiol cholesterol or PEG(n)-cholesterol. Determination of RSV activity

[0232] Determination of α-helicality: The compound was dissolved in an aqueous solution (e.g., 5 μM potassium phosphate solution at pH 7 or distilled H₂O to a concentration of 25 μM–50 μM). Circular dichroism (CD) spectra were obtained on a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard measurement parameters (e.g., temperature, 20 °C; wavelength, 190 nm–260 nm; step resolution, 0.5 nm; velocity, 20 nm / sec; cumulative, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helical content of each peptide was calculated by dividing the average residue ellipticity by the report value of the model helical decapeptide (Yang et al., Methods Enzymol., 1986).

[0233] Determination of melting temperature (Tm): The cross-linked or unmodified template peptide is dissolved in distilled H2O or other buffer or solvent (e.g., at a final concentration of 50 μM), and Tm is determined by measuring the change in ellipticity over a temperature range (e.g., 4 °C to 95 °C) on a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard parameters (e.g., wavelength, 222 nm; step resolution, 0.5 nm; speed, 20 nm / sec; cumulative, 10; response, 1 sec; bandwidth, 1 nm; heating rate: 1 °C / min; path length, 0.1 cm).

[0234] In vitro protease resistance assay: The amide bonds of the peptide backbone are readily hydrolyzed by proteases, thus facilitating rapid degradation of peptide compounds in vivo. However, peptide helix formation typically buries and / or twists and / or shields the amide backbone, thereby preventing or significantly delaying proteolytic cleavage. The peptiform macrocyclic compounds of the present 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 pinned peptides. For example, the peptiform macrocyclic compound and the corresponding uncrosslinked peptide are incubated with trypsin agarose, and the reaction is quenched at different time points by centrifugation and subsequent HPLC injection to quantify the residual substrate by UV absorption at 280 nm. Briefly, the peptiform macrocyclic compound and the peptiform precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S / E ~ 125) for 0 min, 10 min, 20 min, 90 min, and 180 min. The reaction was quenched by high-speed benchtop centrifugation; the remaining substrate in the supernatant was quantified by HPLC-based detection of the 280 nm peak. The protein hydrolysis reaction exhibited first-order kinetics, and the rate constant k was determined by a curve of ln[S] versus time.

[0235] Peptide-like macrocyclic compounds and / or their corresponding uncrosslinked peptides can be incubated individually with fresh mouse, rat, and / or human serum (e.g., 1 mL–2 mL) at 37 °C for, for example, 0 h, 1 h, 2 h, 4 h, 8 h, and 24 h. Samples with different macrocyclic compound concentrations can be prepared by serial dilution with serum. To determine the level of intact compounds, the following procedure can be used: for example, extract the sample by transferring 100 μL of serum to a 2 mL centrifuge tube, adding 10 μL of 50% formic acid and 500 μL of acetonitrile, and centrifuging at 14,000 RPM for 10 min at 4 ± 2 °C. The supernatant is then transferred to a fresh 2 mL tube and evaporated on a Turbovap at 37 °C under N2 < 10 psi. The sample is reconstituted in 100 μL of 50:50 acetonitrile:water and analyzed by LC-MS / MS. Equivalent or similar procedures for testing in vitro stability are known and can be used to determine the stability of macrocyclic compounds in serum.

[0236] Plasma stability assay: The stability of the peptide was tested in freshly drawn mouse plasma collected in lithium heparin tubes. Triple aliquots of 500 μL plasma doped with 10 μM of the single peptide were incubated. The samples were gently shaken in a 37°C orbital shaker, and 25 μL aliquots were taken at 0, 5, 15, 30, 60, 240, 360, and 480 minutes and added to 100 μL of a mixture containing 10% methanol:10% water:80% acetonitrile to prevent further peptide degradation. During the assay, the samples were kept on ice and then transferred to a MultiScreen Solvinert 0.45 μm low-binding hydrophilic PTFE plate (Millipore). The filtrate was analyzed directly by LC-MS / MS. The peptide was detected as a doubly or tricharged ion using a Sciex 5500 mass spectrometer. The percentage of remaining peptide was determined by the reduction in peak area, and the half-life was calculated by logarithmic transformation.

[0237] In vivo protease resistance assay: A key benefit of peptide stapling is translating in vitro protease resistance into significantly improved in vivo pharmacokinetics. Liquid chromatography / mass spectrometry-based assays were used to detect and quantify stapled peptide levels in plasma. For pharmacokinetic analysis, the peptide was dissolved in sterile aqueous 5% dextran (1 mg / mL) and administered to C57BL / 6 mice (Jackson Laboratory) via tail vein bolus or intraperitoneal injection (e.g., 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg). Blood was collected via retro-orbital puncture at 5, 30, 60, 120, and 240 minutes after administration to five animals at each time point. Plasma was harvested after centrifugation (2,500 x g, 5 min, 4 °C) and stored at -70 °C until assay. Peptide concentrations in plasma were determined by reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry (Aristoteli et al., Journal of Proteome Res., 2007; Walden et al., Analytical and Bioanalytical Chem., 2004). Study samples were determined along with a series of seven calibration standard peptides ranging from 1.0 μg / mL to 50.0 μg / mL in plasma, drug-free plasma measured with and without internal standards, and three quality control samples (e.g., 3.75 μg / mL, 15.0 μg / mL, and 45.0 μg / mL). Standard curves were constructed by plotting the analyte / internal standard peak area ratio against the known drug concentration in each calibration standard. Linear least squares regression was performed with weights proportional to the inverse of the analyte concentration normalized to the number of calibration standards. The slope and y-intercept of the best-fit line were used to calculate the drug concentration in the study samples. The plasma concentration-time curves were analyzed using WinNonlin Professional 5.0 software (Pharsight Corp., Cary, NC) via a standard non-compartmental method to obtain pharmacokinetic parameters such as initial and terminal plasma half-lives, peak plasma levels, total plasma clearance, and apparent volume of distribution.

[0238] In the context of blocking viral fusion and transmission before and after infection, the persistence of the stapled peptides of the present invention in the nasal mucosa after topical application (i.e., nasal drops) and in the respiratory mucosa after intranasal application or nebulization was examined. Mice were exposed to a single treatment at a series of intervals via nasal drops or a nebulizer prior to intranasal infection with RSV, and the relative mucosal stability and prophylactic efficacy of the stapled peptide constructs derived with PEG(n)-thiocholesterol or PEG(n)-cholesterol described herein were measured using the duration of protection against mucosal infection (assessed histologically as described above or by PCR as described below).

[0239] In vitro binding assays: To assess the binding and affinity of peptide-mimicking macrocyclic compounds and precursor peptides to receptor proteins, fluorescence polarization assays (FPA) can be used, for example. FPA techniques use polarized light and fluorescent tracers to measure molecular orientation and mobility. When excited with polarized light, fluorescent tracers attached to molecules or peptides that then bind to proteins with high apparent molecular weights (e.g., FITC) (e.g., FITC-labeled peptides bound to large proteins) emit higher levels of polarized fluorescence due to their slower rotational rate upon protein binding, compared to fluorescent tracers attached alone to smaller molecules or peptides (e.g., FITC-labeled peptides free in solution).

[0240] In vitro displacement assays for characterizing peptide-protein interaction antagonists: To assess the binding and affinity of compounds that antagonize interactions between antagonistic peptides and receptor proteins, fluorescence polarization assays (FPA) are used, for example, utilizing fluoresceinized peptides or peptide-like macrocyclic compounds derived from template peptide sequences. FPA techniques use polarized light and fluorescent tracers to measure molecular orientation and mobility. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules that then bind to proteins with high apparent molecular weights (e.g., FITC-labeled peptides bound to large proteins) emit higher levels of polarized fluorescence due to their slower rotation rates compared to standalone FITC-derived molecules (e.g., FITC-labeled peptides free in solution). Competitive binding FPA experiments will detect compounds that antagonize interactions between fluoresceinized peptides and receptor proteins, such as unlabeled pinned peptides and their conjugates, and will allow for the quantification and comparison of the different efficiencies of compounds in disrupting the interaction.

[0241] Five-Helix Bundle Protein Generation and Fluorescence Polarization Assay: A C-terminal hexa-His-tagged (SEQ ID NO:279) recombinant five-helix bundle (5HB) protein was designed, containing five of six helices that comprise the core of a hairpin RSV-F trimer, linked via a short peptide linker according to the design of gp41 5-HB (Root et al., Science, 291(5505):884-8(2001); Bird et al., J Clin Invest. May 2014; 124(5):2113-24). The plasmid was transformed into Escherichia coli BL21(DE3), cultured in Luria broth, and induced overnight at 37°C with 0.1 M isopropyl β-D-thiogalactoside. Cells were harvested by centrifugation at 5,000g for 20 min, resuspended in buffer A (100 mM NaH2PO4, 20 mM Tris, 8 M urea; pH 7.4), and lysed by stirring overnight at 4°C. The mixture was clarified by centrifugation (35,000g, 30 min) and then bound to a Ni-NTA agarose (Qiagen) column at room temperature. Bound 5-HB was 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 in 10 kDa Amicon centricon (diluted and reconcentrated 7 times) to yield a protein solution of approximately 1 mg / mL. Protein purity was assessed by SDS-PAGE and determined to be >90%. The fluoresceinized derivative (25 nM) of the peptide of the present invention was incubated with a specified concentration of 5-HB protein in room temperature binding buffer (50 mM sodium phosphate, 100 mM NaCl; pH 7.5). Direct binding activity at equilibration (e.g., 10 minutes) was measured by fluorescence polarization using a SpectraMax M5 microplate reader (BMGLabtech). For competitive binding assays, fixed concentrations of FITC-peptide and 5-HB protein reflecting the EC90 of direct binding were then incubated with serial dilutions of acetylated SAH-RSV peptide to generate competitive curves for comparative analysis. Binding assays were performed in triplicate, and Kis was calculated using Prism software (GraphPad) via nonlinear regression analysis of the competitive binding isotherms.

[0242] Determination of binding activity to RSV 5-helical bundles: In some instances, the methods disclosed herein include direct and competitive screening assays. For example, the method may include determining whether a pharmaceutical agent alters (e.g., reduces) the binding of one or more peptides and their conjugates disclosed herein to RSV (e.g., to RSV 5-helical bundles). In some instances, the method includes (i) determining the binding level between one or more peptides and their conjugates disclosed herein and RSV (e.g., to RSV 5-helical bundles) (e.g., in the absence of a pharmaceutical agent); (ii) detecting the binding level between one or more peptides (e.g., one or more peptides in (i)) and RSV (e.g., to RSV 5-helical bundles) in the presence of a pharmaceutical agent, wherein a change (e.g., a reduction) in the binding level between the one or more peptides and RSV (e.g., to RSV 5-helical bundles) indicates that the pharmaceutical agent is a candidate for binding to RSV; and (iii) selecting the candidate pharmaceutical agent. In some instances, step (i) includes contacting one or more peptides with RSV (e.g., with RSV 5-helical bundles) and detecting the binding level between the one or more peptides and RSV (e.g., with RSV 5-helical bundles). In some instances, step (ii) includes contacting one or more peptides and an agent with RSV (e.g., with RSV 5-helical bundles) and detecting the binding level between the one or more peptides and RSV (e.g., with RSV 5-helical bundles). RSV (e.g., RSV 5-helical bundles) may be contacted with one or more peptides and an agent simultaneously or at different times (e.g., one or more peptides may be contacted with RSV (e.g., with RSV 5-helices) before or after the agent). In some embodiments, the candidate agent is administered to a suitable animal model (e.g., an animal model of RSV) to determine whether the agent reduces RSV infection levels in the animals.

[0243] In some instances, one or both of the peptide and the RSV helical bundle may include a label, thereby allowing the detection of the peptide and / or the RSV helical bundle. In some instances, the peptide includes a label. In some instances, the RSV helical bundle includes a label. In some instances, both the peptide and the RSV helical bundle include labels. The label can be any label known in the art, including but not limited to fluorescent labels, radioisotope labels, or enzyme labels. In some instances, the label itself is directly detectable (e.g., radioisotope labeling or fluorescent labeling). In some instances, (e.g., in the case of enzyme labeling), the label is indirectly detectable, for example by catalyzing a chemical change in a substrate compound or composition that is directly detectable.

[0244] Competitive RSV 5-HB binding assay by ELISA: Microwells were coated overnight at 4°C with 50 μl of PBS containing neutral avidin (4 μg / ml). The wells were washed twice with PBS containing 0.05% Tween 20 (PBS-T) and blocked at 37°C for 45 min with 4% BSA in PBS-T. Next, 50 μl of 250 nM biotinylated PEG was added... 2- RSV HR2 (SEQ ID NO: 100) was added to PBS-T containing 1% BSA and incubated with shaking for 1 hour, followed by washing four times with 300 μl of PBS-T. Then, a 1:2 serial dilution of the stabilizing peptide of the present invention (starting from 10 μM, containing 50 nM recombinant 5-HB in 50 μL of PBS-T containing 1% BSA) was added to the plate, and the plate was shaken at room temperature for 2 hours, followed by washing four times with 300 μl of PBS-T. Finally, 50 μL of a 1:5000 dilution of a goat polyclonal antibody conjugated with a 6X His tag-HRP (“HHHHHH”, as disclosed in SEQ ID NO: 279) was added. After incubation at room temperature for 40 minutes, the wells were washed five times and developed with 50 μl of tetramethylbenzidine (TMB) solution. After 20 minutes, the wells containing TMB solution were terminated by adding 50 μl of H2SO4 (2M), and the absorbance was read at 450 nm using a molecular device. The concentration of the competing peptide corresponding to the half-maximum signal (IC50) was determined by interpolating the resulting binding curves using Prism software (Graphpad). Each peptide competitor was tested in triplicate in at least two separate experiments.

[0245] Cell localization assay: To measure the localization of peptides or cross-linked polypeptides on or within cells, intact cells were incubated with fluoresceinized cross-linked polypeptides derived from PEG(n)-mercaptocholesterol or PEG(n)-cholesterol (5 μM) at 37°C for 4 hours in serum-free medium or in medium supplemented with human serum. Cells were washed twice with medium and incubated with trypsin (0.25%) at 37°C for 10 minutes. Cells were washed again and resuspended in PBS. Alternatively, the localization could be measured using a FACSCalibur flow cytometer or Cellomics' KineticScan. RTM HCS microplate reader for analyzing cell fluorescence.

[0246] Antiviral efficacy assay: The efficacy of the peptide-cholesterol conjugate of the present invention in preventing and treating live human respiratory syncytial virus infection with green fluorescent protein (RSV-GFP1) was evaluated in monolayer cell cultures. A549 cells plated in 384-well plates were treated with serial dilutions of the conjugate peptide (e.g., 1 μM–5 μM starting dose, or a fixed dose such as 2 μM) for 30 min in quadruplicates, followed by the addition of GFP-RSV live virus assay (0.75 μL–1.25 μL of virus per well) and incubation for 48–72 h. Infected cells were then washed with PBS. Hoechst 33342 (a cell-penetrating nuclear dye) and DRAQ7 (a cell-non-penetrating nuclear dye) were added, and the plates were imaged at 4x magnification on a Molecular Devices ImageXpress MicroConfocal laser. GFP(+) cells were counted, and the total GFP(+) cells or the percentage of GFP(+) cells was plotted using Prism software (Graphpad). Cytotoxicity was determined by the ratio of DRAQ7(+)Hoechst 33342(+) cells to DRAQ7(-)Hoechst 33342(+) cells.

[0247] How to use

[0248] This disclosure is characterized by methods for treating or preventing RSV infection in a subject (e.g., a human) using any structurally stable (e.g., pinned) peptide or structurally stable peptide conjugate (or pharmaceutical compositions comprising said structurally stable peptide or structurally stable peptide conjugate). In some instances, the treatment reduces, inhibits, or improves the infection suffered by the subject (e.g., a human). In some instances, the subject is an animal. In some instances, the subject is a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey or a human). In some instances, the subject is a domesticated animal (e.g., a dog or cat). In some instances, the subject is a human. In some instances, such terms refer to non-human animals (e.g., non-human animals such as pigs, horses, cows, cats, or dogs). In some instances, such terms refer to pets or farm animals. In some instances, such terms refer to humans.

[0249] The structurally stable (e.g., pinned) peptides or structurally stable peptide conjugates (or pharmaceutical compositions comprising them) described herein may be used to treat subjects (e.g., humans) suffering from RSV infection. In some instances, structurally stable peptides (or pharmaceutical compositions comprising them) are used to treat RSV infection in subjects (e.g., humans). In some instances, the subject is a human.

[0250] The structurally stable (e.g., pinned) peptides or structurally stable peptide conjugates (or pharmaceutical compositions comprising them) described herein may be used to prevent RSV infection in a subject (e.g., a human). In some instances, structurally stable peptides (or pharmaceutical compositions comprising them) are used to prevent RSV infection in a subject (e.g., a human). In some instances, structurally stable peptide conjugates (or pharmaceutical compositions comprising them) are used to prevent RSV infection in a subject (e.g., a human). In some instances, the subject is a human.

[0251] Therefore, this article provides a method for treating RSV infection in a subject (e.g., a human) in need, the method comprising administering to the subject a therapeutically effective amount of the structurally stable peptide described herein (or a pharmaceutical composition comprising the structurally stable peptide).

[0252] This article also provides a method for preventing RSV infection in a subject (e.g., a human) in need, comprising administering to the subject a therapeutically effective amount of the structurally stable peptide described herein (or a pharmaceutical composition comprising the structurally stable peptide).

[0253] Therefore, this article provides a method for treating RSV infection in a subject (e.g., a human) of need, comprising administering to the subject a therapeutically effective amount of the structurally stable peptide conjugate described herein (or a pharmaceutical composition comprising the structurally stable peptide conjugate).

[0254] This article also provides a method for preventing RSV infection in a subject (e.g., a human) in need, comprising administering to the subject a therapeutically effective amount of the structurally stable peptide conjugate described herein (or a pharmaceutical composition comprising the structurally stable peptide conjugate).

[0255] In some instances, the aforementioned methods include administering to a subject (e.g., a human) the peptides or variants thereof described in Table 1 (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 substitutions, insertions, or deletions), the constructs or variants thereof described in Table 2 (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 substitutions, insertions, or deletions), or the conjugates or variants thereof described in Table 3 (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 substitutions, insertions, or deletions). In some instances, the peptides or constructs of Tables 1-3 also contain three N-terminal amino acids X1X2X3, where X1 is any amino acid, optionally S; and X2 and X3 are any negatively charged amino acids.

[0256] In some instances, the method includes administering to a subject (e.g., a human) a structurally stable peptide (or a pharmaceutical composition comprising thereof), the structurally stable peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NO:27, 29, 36, 43, and 46 or any one of SEQ ID NO:49, 51, 58, 65, and 68. In some instances, the method includes administering to a subject (e.g., a human) a structurally stable peptide (or a pharmaceutical composition comprising thereof), the structurally stable peptide comprising or consisting of the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:51. In some instances, the method includes administering to a subject (e.g., a human) a structurally stable peptide (or a pharmaceutical composition comprising thereof), the structurally stable peptide comprising or consisting of the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:65.

[0257] In some instances, the method includes administering to a subject (e.g., a human) a structurally stable peptide conjugate (or a pharmaceutical composition comprising thereof), the structurally stable peptide conjugate comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 71, 73, 80, 87, and 90 or any one of SEQ ID NO: 5, 6, 14, 21, and 24. In some instances, the method includes administering to a subject (e.g., a human) a structurally stable peptide conjugate (or a pharmaceutical composition comprising thereof), the structurally stable peptide conjugate comprising or consisting of the amino acid sequence of SEQ ID NO: 5 or 73. In some instances, the method includes administering to a subject (e.g., a human) a structurally stable peptide conjugate (or a pharmaceutical composition comprising thereof), the structurally stable peptide conjugate comprising or consisting of the amino acid sequence of SEQ ID NO: 21, 281, or 87.

[0258] In some instances, the method includes administering to a subject (e.g., a human) a variant of a structurally stable peptide (or a pharmaceutical composition comprising thereof), the variant comprising or consisting of an amino acid sequence of any one of SEQ ID NO:27, 29, 36, 43 and 46 or any one of SEQ ID NO:49, 51, 58, 65 and 68 (e.g., having 1 to 10, 1 to 5, 1 to 3, 2 or 1 amino acid substitutions, insertions and / or deletions, respectively, relative to any one of SEQ ID NO:27, 29, 36, 43 and 46 or any one of SEQ ID NO:49, 51, 58, 65 and 68, wherein the amino acid substitutions and / or deletions are not in a pinned position, and wherein the structurally stable peptide binds to RSV 5-helical bundle protein and / or inhibits RSV infection of cells and / or prevents RSV infection of cells).

[0259] In some instances, the method includes administering to a subject (e.g., a human) a variant of a structurally stable peptide conjugate (or a pharmaceutical composition comprising thereto), the variant comprising or consisting of the amino acid sequence of any one of SEQ ID NO:71, 73, 80, 87, and 90 or any one of SEQ ID NO:5, 6, 14, 21, and 24 (e.g., having 1 to 10, 1 to 5, 1 to 3, 2, or 1 amino acid substitution, insertion, and / or deletion, respectively, relative to any one of SEQ ID NO:71, 73, 80, 87, and 90 or any one of SEQ ID NO:5, 6, 14, 21, and 24, wherein the amino acid substitution and / or deletion is not at a pinned position, and wherein the structurally stable peptide conjugate binds to RSV 5-helical bundle protein and / or inhibits RSV infection of cells and / or prevents RSV infection of cells).

[0260] In some instances, the method includes administering to a subject (e.g., a human) a structurally stable peptide (or a pharmaceutical composition comprising it) as described in the “Structurally Stable Peptides” section above. In some instances, the method includes administering to a subject (e.g., a human) a structurally stable peptide conjugate (or a pharmaceutical composition comprising it) as described in the “Structurally Stable Peptide Conjugates” section above. In some instances, the method includes administering to a subject (e.g., a human) a structurally stable peptide or structurally stable peptide conjugate (or a pharmaceutical composition comprising it) as described in the accompanying drawings or working examples. In one instance, the method described herein relates to administering a peptide or conjugate comprising the amino acid sequence of SEQ ID NO:281.

[0261] In some instances, the subject (e.g., a person) is infected with RSV. In some instances, the subject (e.g., a person) is at risk of RSV infection (e.g., a subject who has been exposed to an infected subject with RSV). In some instances, the subject (e.g., a person) is suspected of having RSV infection (e.g., a subject who has been exposed to an infected subject with RSV and exhibits one or more symptoms of RSV). Methods for determining whether a subject is infected with RSV are known in the art.

[0262] In some instances, methods of treating or preventing RSV infection further include repeatedly administering to a subject (e.g., a person) a therapeutically effective amount of the structurally stable peptide or structurally stable peptide conjugate (or a pharmaceutical composition comprising the thereof) described herein, required for the treatment or prevention of RSV infection. In some instances, methods of treating or preventing RSV infection further include testing a subject (e.g., a person) to determine if the subject has an RSV infection, and subsequently administering a therapeutically effective amount of the structurally stable peptide or structurally stable peptide conjugate (or a pharmaceutical composition comprising the thereof) described herein. Subjects may be selected for treatment based, for example, determining that the subject is at risk of acquiring RSV infection or has RSV infection.

[0263] The structurally stable peptides or structurally stable peptide conjugates (or pharmaceutical compositions comprising them) described herein can be administered orally, intranasally, intravenously, subcutaneously, intramuscularly, or topically, including through the skin, nose, sinuses, eyes, oropharynx, respiratory tree, and lungs. In some instances, administration is via local respiratory application, including application to the nasal mucosa, sinus mucosa, oropharyngeal mucosa, or respiratory tree (including the lungs). In some instances, local application includes application to the skin or eyes. In some instances, the conjugates described herein improve bioavailability, increase blood circulation, alter pharmacokinetics, reduce immunogenicity, and / or reduce the required frequency of administration.

[0264] The specific dosage and treatment regimen for any particular patient or subject will depend on a number of factors, including the activity of the particular compound used, age, weight, general health status, sex, diet, time of administration, excretion rate, combination of drugs, disease, severity and duration of the condition or symptoms, the patient's or subject's predisposition to the disease, condition or symptoms, and the judgment of the treating physician or veterinarian.

[0265] An effective amount may be administered, applied, or dosed once or multiple times. The therapeutically effective amount (i.e., the effective dose) of a therapeutic compound (e.g., a structurally stable peptide or a structurally stable peptide conjugate) depends on the selected therapeutic compound. The composition may be administered once or multiple times daily to once or multiple times weekly, including every other day. Those skilled in the art will understand that certain factors may affect the dose and duration required for effective treatment of a subject, including but not limited to the risk or severity of disease or condition, prior treatment, the subject's overall health and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the therapeutic compound described herein may comprise a single treatment or a series of treatments. For example, an effective amount may be administered at least once.

[0266] This article also provides the use of the structurally stable peptides or structurally stable peptide conjugates (or pharmaceutical compositions comprising them) described herein in the preparation of a medicament for treating RSV infection in a subject (e.g., a human).

[0267] This article also provides the use of the structurally stable peptides or structurally stable peptide conjugates (or pharmaceutical compositions comprising them) described herein in the preparation of a medicament for the prevention of RSV infection in a subject (e.g., a human).

[0268] Example

[0269] The following embodiments are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. The specific materials mentioned are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can develop equivalent methods or reactants without inventive thinking and without departing from the scope of the invention.

[0270] Example 1: Design and synthesis of RSVHR2 domain-dependent lipopeptides to inhibit viral-host membrane fusion. To block RSV infection .

[0271] In order to design peptides that effectively block the fusion of RSV with host cells ( Figure 1 A series of differentially targeted chemical staples were designed and stapled peptides were partially derivatized at the C-terminus with PEG(n)-thiocholesterol or PEG(n)-cholesterol. The designed peptides were synthesized on a resin via solid-phase synthesis. The differentially targeted chemical staples were positioned within the RSV HR2 domain (i.e., amino acids 488-516 of SEQ ID NO:1) of the surface (F) glycoprotein sequence of human RSV in a manner described in (see [link to relevant documentation]). Figure 2-4 ): Substitution of native olefin residues with α,α-disubstituted non-native olefin residues (e.g., "X" represents (S)-α-(4'-pentenyl)alanine, and "8" represents (R)-α-(7'-octenyl)alanine installed at selected i, i+7 positions, or "X" represents (S)-α-(4'-pentenyl)alanine installed at each of the selected i, i+4 positions) and combinations thereof in the form of double nails or sutures, followed by ruthenium-catalyzed olefin metathesis (see, Figure 5-7Methods for designing, synthesizing, and identifying optimal pinned peptide constructs targeting RSV fusion devices include generating Ala scans (e.g., mutants), pinning scans, and libraries with variable N-terminal and C-terminal deletions, additions, and derivatizations for partial conjugation with PEG-thiocholesterol or PEG-cholesterol (see, Figure 8). The spiked RSVHR2 peptide was constructed by replacing two naturally occurring amino acids at positions i and i+7 (i.e., side-attached with 7 amino acids) with non-natural (R)-2-(((9H–fluorene-9-yl)methoxy)carbonylamino)-2-methyl-dec-9-enoic acid / (R)-α-(7'-octenyl)alanine / (Fmoc-R8) and (S)-2-(((9H–fluorene-9-yl)methoxy)carbonylamino)-2-methyl-hept-6-enoic acid / (S)-α-(4'-pentenyl)alanine / (Fmoc-S5) amino acids to generate a spike spanning two α-helix turns, or by replacing two naturally occurring amino acids at positions i and i+4 with two non-natural S5 amino acids to generate a spike spanning one α-helix turn. The 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 in full by reference).

[0272] Example 2: Identification of the optimally pinned RSVHR2 peptide with C-terminal PEG4-thiocholesterol for pseudotype and active formulations Antiviral activity was achieved in virus assays.

[0273] To enhance the potency of the RSV HR2 helix, thereby improving the drug-like properties stabilized by individual insertion of full-hydrocarbon nails, differentially nailed RSV HR2 constructs were further derivatized at the C-terminus with a PEG(n)-thiocholesterol moiety. For example, the single i, i+7 nailed peptide of SEQ ID NO:4 blocked RSV viral infection with an IC50 > 5 μM. Therefore, iterative optimization activities were initiated by “nailing” the α-helical regions spanning residues Phe 488 and Val 516 across the RSVHR2 domain (see, Figure 10 ).

[0274] Pinning scans were performed to identify residues and binding surfaces critical to the interaction, which determined the design of the optimized construct and negative control mutant (see [link to documentation]). Figure 5 Depending on the experimental application, the N-terminus of the peptide is capped with an acetyl group or a fluorophore (e.g., FITC, rhodamine). This produces an N-terminally acetyl-capped peptide.

[0275] Bi-stapled peptides are generated by attaching two S5-S5, two R8-S5, or other combinations of cross-linked non-natural amino acids; similar principles are used to generate multiple-stapled or stitched peptides (see, Figure 6 ).

[0276] In order to enable peptide derivatization with thiocholesterol on resin, according to Figure 11 The schematic diagram shown illustrates the synthesis of carboxyl-thiol cholesterol. To generate stabilized lipopeptides with the RSV HR2 domain (see...),... Figure 12 In the procedure described in the original text, the completed resin-bound peptide (e.g., SEQ ID NO:5) is capped with an acetyl group (using acetic anhydride), and then deprotected by treating the C-terminal side chain lysine amine with 2% hydrazine. The PEG(n) amino acid (e.g., n = 1-36) protected with Fmoc is then acylated with an olefin crosslinking agent by treatment with a Grubbs(I) catalyst. Fmoc is removed from the C-terminal NH of the PEG(n) amino acid, and the amine is then acylated with a carboxyl-mercaptocholesterol group. After peptide deprotection and cleavage, the final peptide product is purified by reversed-phase high-performance liquid chromatography / mass spectrometry (LC / MS). The same procedure can be performed to derivatize with cholesterol using the cholesterol moiety instead of the mercaptocholesterol moiety.

[0277] This chemical approach involves attaching the PEG4-mercaptocholesterol moiety to the C-terminus of the sequence shown in SEQ ID NO:51, thereby generating a conjugate having the sequence of SEQ ID NO:5. The resulting conjugate of SEQ ID NO:5 exhibited nanomolar antiviral activity (IC50 = 130 nM; see [link to relevant documentation]) in an infectivity assay using live RSV virus. Figure 13 ), where IC50 is 1.3 × 10 -7 M. Therefore, removing six C-terminal residues from the SEQ ID NO:4 sequence and adding the C-terminal PEG-thiocholesterol moiety increased the antiviral activity of the peptide by an order of magnitude (comparative). Figures 9 to 13 ).

[0278] To identify the optimal location for nail insertion, an exemplary i, i+7 nail scan library of RSV HR2 (SEQ ID NO: 1 aa 488-516) derived from PEG(n)-thiocholesterol was used. Figure 11-12The synthetic scheme described in the paper produces (thus generating SEQ ID NO:5-26). The differential antiviral activity of this library was evaluated in an RSV infectivity paper. In the GFP-RSV live virus assay, peptides having the sequences of SEQ ID NO:5, 6, 14, 21 and 24 showed potent activity in a variety of pinned RSV HR2 peptides (see, Fig. 15). In summary, these data indicate that (1) attaching the PEG4-mercaptocholesterol moiety to the C-terminus of structurally stable RSV HR2 peptides using a resin-on-resin method can confer potent antiviral activity in a group of differentially pinned i, i+7 peptides with low or no activity (compare Fig. 9 and 15). Figure 13 ).

[0279] Example 3: Determining the optimal PEG linker length within a pinned RSVHR2 peptide with C-terminal PEG(n)-thiocholesterol To achieve antiviral activity in pseudotype and live virus assays.

[0280] To determine the optimal PEG chain length for linking the stapled RSV HR2 peptide to the thiol cholesterol moiety, a series of PEG(n) analogs were generated according to the above-described synthetic method, where n equals 0, 4, 8, 12, 16, or 20. The antiviral activity of this series of i, i+7 stapled RSV HR2 peptides, bearing SEQ ID NO: 51 and 65 and thiol cholesterol separated by variable-length PEG-linkers, was examined in a GFP-RSV live virus assay (0.75 μL–1.25 μL virus / well; cells: A549; peptide dose 2 μM; readings at 48 hours). See also Figure 16 and Figure 17 For example, the superiority of PEG16 and PEG20 linkers compared to PEG4, PEG8, and PEG12 is unexpected and needs to be experimentally determined in RSV antiviral assays. C-terminal derivatization of PEG16-mercaptocholesterol and PEG20-mercaptocholesterol of SEQ IQ NO:5 yields compounds with IC50s of 49 nM and 60 nM, respectively (see [link to relevant documentation]). Figure 16 Compared to RSV HR2 peptides with only a single corresponding spike and an additional 6 HR2 amino acids at the C-terminus, this reflects an improvement in antiviral activity of more than two orders of magnitude (see, Figure 4 Notably, C-terminal derivatization of the substituted monopeptide PEG16-mercaptocholesterol and PEG20-mercaptocholesterol of SEQ ID NO:21 yields compounds with IC50 values ​​of 18 nM and 8.2 nM, respectively (see, Figure 17Therefore, the combination of pinning scan, differential peptide truncation, and PEG scanning revealed the pinning position, sequence length, and PEG chain length of the pinned lipopeptide that produced SEQ ID NO:21, which exhibited significantly enhanced anti-RSV activity (single-digit nanomolar anti-RSV activity), reflecting a three-order-of-magnitude improvement over the single pinned RSV HR2 peptide of SEQ ID NO:4, and corresponding superiority over previously reported longer dual-pinned anti-RSV peptides lacking PEG-thiocholesterol derivatization (Bird et al., JCI, 2014, 124(5):2113–2124).

[0281] Example 4: Rescuing poor solubility by adding three natural N-terminal residues .

[0282] Poor solubility was observed for the lipopeptide with SEQ ID NO:21, but the insolubility challenge was completely resolved by attaching three native N-terminal residues, “SDE” (which includes two negatively charged amino acids), resulting in complete solubility at 15 mg / mL in 15% DMSO and PBS (pH 7.4). Sequence optimization changed the pI from 8 (which corresponds to poor solubility at neutral pH) to pI 4.7 (complete solubility).

[0283] Other implementation plans

[0284] Although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not 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.

[0285] All publications, patents, patent applications, websites, and accession / database sequences (including polynucleotide and polypeptide sequences) cited herein are incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent, patent application, website, or accession / database sequence were specifically and individually indicated to be incorporated by reference.

Claims

1. A conjugate comprising (i) a structurally stable peptide and (ii) cholesterol or thiocholesterol; The cholesterol or thiocholesterol is directly or via a linker attached to the C-terminal amino acid of the structurally stable peptide; The structurally stable peptide comprises an internally cross-linked amino acid sequence comprising (i) 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:100), or (ii) the sequence SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:280), except for two to six amino acid substitutions relative to the sequence of SEQ ID NO:100 or 281, and wherein the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO:258); The substitution of two amino acids in the two to six amino acid substitutions is carried out with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three or six amino acids. The conjugate binds to RSV 5-helical bundle protein and / or the conjugate inhibits RSV infection of cells and / or prevents RSV infection of cells; and The length of the conjugate is 25 to 45 amino acids, and optionally the length of the conjugate is 30 amino acids.

2. A conjugate comprising (i) a structurally stable peptide and (ii) cholesterol or thiocholesterol; The cholesterol or thiocholesterol is directly or via a linker attached to the C-terminal amino acid of the structurally stable peptide; The structurally stable peptide comprises an internally cross-linked amino acid sequence having the following formula: Or its pharmaceutically acceptable salt; Each of R1 and R2 is H or C1 to C. 10 Alkyl, alkenyl, ynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl or heterocycloalkyl, any one of which may be substituted or unsubstituted; Where x is 3 or 6; Each R3 is independently alkylene, alkenylene, or ynylene, any of which may be substituted or unsubstituted; Where z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; The internally cross-linked amino acid sequence comprises (i) 25 to 29 consecutive amino acids of the sequence of SEQ ID NO: 100, or (ii) the sequence SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 280), except for two to six amino acid substitutions relative to the sequence of SEQ ID NO: 100 or 281, and the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO: 258); The conjugate binds to RSV 5-helical bundle protein and / or the conjugate inhibits RSV infection of cells and / or prevents RSV infection of cells; and The length of the conjugate is 25 to 45 amino acids, and optionally the length of the conjugate is 30 amino acids.

3. The conjugate according to claim 1 or 2, wherein the conjugate comprises the cholesterol, and optionally wherein the linker comprises PEG.

4. The conjugate according to claim 1 or 2, wherein the conjugate comprises the thiocholesterol, and optionally wherein the linker comprises PEG.

5. The conjugate according to claim 1 or 2, wherein the conjugate comprises PEG(n)-cholesterol directly linked to the C-terminal amino acid of the structurally stable peptide, wherein n is 1-36, optionally wherein n is 4, 5, 6, 7, 8, 12, 16 or 20.

6. The conjugate according to claim 1 or 2, wherein the conjugate comprises PEG(n)-thiocholesterol directly linked to the C-terminal amino acid of the structurally stable peptide, wherein n is 1-36, optionally wherein n is 4, 5, 6, 7, 8, 12, 16 or 20.

7. The conjugate according to claim 1 or 2, wherein the conjugate comprises Formula III directly linked to the C-terminal amino acid of the structurally stable peptide: Where n is 1-36, and optionally n is 4, 5, 6, 7, 8, 12, 16 or 20.

8. The conjugate according to claim 1 or 2, wherein the conjugate comprises Formula II directly linked to the C-terminal amino acid of the structurally stable peptide: Where n is 1-36, and optionally n is 4, 5, 6, 7, 8, 12, 16 or 20.

9. The conjugate according to claim 1, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three amino acids, optionally wherein each of the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains is (S)-α-(4'-pentenyl)alanine.

10. The conjugate according to claim 1, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by six amino acids, optionally wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are (R)-α-(7'-octenyl)alanine and (S)-α-(4'-pentenyl)alanine.

11. The conjugate according to claim 1, wherein the two substitutions are made with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains at the amino acid positions corresponding to positions 1 and 8 of the sequence shown in SEQ ID NO: 100, at the amino acid positions corresponding to positions 3 and 10 of the sequence shown in SEQ ID NO: 100, or at the amino acid positions corresponding to positions 17 and 24 of the sequence shown in SEQ ID NO:

100.

12. The conjugate according to claim 2, wherein R3 is an internal crosslink between amino acids corresponding to positions 1 and 8 of the sequence shown in SEQ ID NO: 100, between positions 3 and 10 of the sequence shown in SEQ ID NO: 100, or between positions 17 and 24 of the sequence shown in SEQ ID NO:

100.

13. The conjugate according to any one of claims 1 to 8, wherein the conjugate comprises the sequence shown in any one of SEQ ID NO: 5-26 and 71-92.

14. The conjugate according to any one of claims 1 to 8, wherein the structurally stable peptide comprises the sequence shown in any one of SEQ ID NO: 27, 29, 36, 43 and 46.

15. The conjugate according to any one of claims 1 to 8, wherein the structurally stable peptide comprises the sequence shown in any one of SEQ ID NO: 49, 51, 58, 65, 68 and 281.

16. The conjugate according to claim 1 or 2, wherein the conjugate comprises the sequence shown in any one of SEQ ID NO: 71, 73, 80, 87 and 90.

17. The conjugate according to claim 1 or 2, wherein the conjugate comprises the sequence shown in any one of SEQ ID NO: 5, 6, 14, 21 and 24.

18. The conjugate according to any one of claims 1 to 17, wherein the length of the conjugate is 25 to 34, 26 to 33, 27 to 32, 28 to 31, 29 or 30 amino acids.

19. A conjugate comprising or consisting of 8DASISQXNEKINQSLAFIRKSDELLHNV* (SEQ ID NO:265), wherein 8 is internally cross-linked with X, wherein 8 is (R)-α-(7'-octenyl)alanine, wherein X is (S)-α-(4'-pentenyl)alanine, and wherein * is a conjugate of the formula... Where n is 1-36, and optionally n is 16 or 20.

20. A conjugate comprising or consisting of FD8SISQVNXKINQSLAFIRKSDELLHNV* (SEQ ID NO: 261), wherein 8 is internally cross-linked with X, wherein 8 is (R)-α-(7'-octenyl)alanine, wherein X is (S)-α-(4'-pentenyl)alanine, and wherein * is a conjugate of the formula... Where n is 1-36, and optionally n is 16 or 20.

21. A conjugate comprising or consisting of FDASISQVNEKINQSL8FIRKSDXLLHNV* (SEQ ID NO: 266), wherein 8 is internally cross-linked with X, wherein 8 is (R)-α-(7'-octenyl)alanine, wherein X is (S)-α-(4'-pentenyl)alanine, and wherein * is a conjugate of the formula... Wherein n is 1-36, optionally n is 16 or 20, and optionally the conjugate comprises three N-terminal amino acids X1X2X3 immediately upstream of the first F of SEQ ID NO:266, wherein X1 is any amino acid, optionally S, and X2 and X3 are negatively charged amino acids.

22. A structurally stable peptide, said structurally stable peptide comprising: The amino acid sequence comprising 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:100), excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO:100; The substitution of two amino acids in the two to six amino acid substitutions is carried out with α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having cross-linked olefinic side chains are separated by three or six amino acids. The structurally stable peptide binds to RSV 5-helical bundle protein and / or the structurally stable peptide inhibits RSV infection of cells and / or prevents RSV infection of cells; and The structurally stable peptide is 25 to 32 amino acids in length, optionally 32 amino acids in length, and optionally contains three N-terminal amino acids X1X2X3 immediately upstream of the first F of SEQ ID NO:100, wherein X1 is any amino acid, optionally S, and X2 and X3 are negatively charged amino acids.

23. A structurally stable peptide, said structurally stable peptide comprising: An amino acid sequence with internal cross-links as follows: Or its pharmaceutically acceptable salt; Each of R1 and R2 is H or C1 to C. 10 Alkyl, alkenyl, ynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl or heterocycloalkyl, any one of which may be substituted or unsubstituted; Where x is 3 or 6; Each R3 is independently alkylene, alkenylene, or ynylene, any of which may be substituted or unsubstituted; Where z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and The internally cross-linked amino acid sequence comprises 25 to 29 consecutive amino acids of the sequence of SEQ ID NO:100, excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO:100; The structurally stable peptide binds to RSV 5-helical bundle protein and / or the structurally stable peptide inhibits RSV infection of cells and / or prevents RSV infection of cells; and The structurally stable peptide is 25 to 30 amino acids in length, and optionally, the structurally stable peptide is 29 amino acids in length.

24. The structurally stable peptide according to claim 22 or 23, wherein the structurally stable peptide does not contain amino acids corresponding to positions 517-522, 517-521, 517-520, 517-519 or 517-518 of the RSV-F protein (according to the sequence number shown in SEQ ID NO:1).

25. The structurally stable peptide according to claim 22 or 23, wherein the internally cross-linked amino acid sequence does not include the sequence NAGKST (SEQ ID NO:258).

26. The structurally stable peptide according to claim 22 or 23, wherein the structurally stable peptide is 29 amino acids in length and comprises 29 consecutive amino acids of the sequence of SEQ ID NO:100, excluding two to six amino acid substitutions relative to the sequence of SEQ ID NO:

100.

27. The structurally stable peptide according to any one of claims 22 to 26, wherein the structurally stable peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 27-70.

28. The structurally stable peptide according to any one of claims 22 to 26, wherein the structurally stable peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 27, 29, 36, 43 and 46.

29. The structurally stable peptide according to any one of claims 22 to 26, wherein the structurally stable peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 49, 51, 58, 65, 281, 285 and 68.

30. A peptide comprising the amino acid sequence of any one of SEQ ID NO:27-70, except for zero to six additional substitutions, wherein the peptide does not contain the sequence NAGKST (SEQ ID NO:258).

31. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 21, the structurally stable peptide of any one of claims 22 to 29 or the peptide of claim 30, and a pharmaceutically acceptable carrier.

32. A method for treating RSV infection in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1 to 21, the structurally stable peptide of any one of claims 22 to 29, or the peptide of claim 30.

33. A method for preventing RSV infection in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1 to 21, the structurally stable peptide of any one of claims 22 to 29, or the peptide of claim 30.

34. The method of claim 32 or 33, wherein the subject is a human being.

35. A method for preparing structurally stable peptides, the method comprising: I.(a) Provides a peptide having an amino acid sequence comprising 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:100), except for two to six amino acid substitutions relative to the sequence of SEQ ID NO:100; The two amino acid substitutions of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having olefinic side chains are separated by three or six amino acids. as well as (b) Crosslinking the peptide to prepare the structurally stable peptide, and optionally purifying the structurally stable peptide; or II. (a') Provides a peptide comprising the sequence SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO:280), except for two to six amino acid substitutions relative to the sequence of SEQ ID NO:280; The two amino acid substitutions of the two to six amino acid substitutions are made with α,α-disubstituted non-natural amino acids having olefinic side chains, wherein the α,α-disubstituted non-natural amino acids having olefinic side chains are separated by three or six amino acids. as well as (b') Crosslinking the peptide to prepare the structurally stable peptide, and optionally purifying the structurally stable peptide.

36. The method of claim 35, wherein the crosslinking is carried out by a ruthenium-catalyzed metathesis reaction.

37. The method of claim 35 or 36, further comprising derivatizing the structurally stable peptide onto a resin with a resin-bound amine containing a carboxylic acid PEG and / or cholesterol or thiocholesterol.

38. The method according to any one of claims 35 to 37, the method further comprising formulating the structurally stable peptide into a sterile pharmaceutical composition.

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