Respiratory syncytial virus surface glycoprotein peptides, conjugates, and their use

Abstract

This disclosure relates to structurally stabilized (e.g., stapled, e.g., hydrocarbon-stapled) respiratory syncytial virus (RSV) peptides and their variants, and structurally stabilized (e.g., stapled, e.g., hydrocarbon-stapled) RSV peptides and their variants conjugated with polyethylene glycol (PEG) and / or cholesterol (or its variants, e.g., thiocholesterol), e.g., PEG(n)-cholesterol or PEG(n)-thiocholesterol derivatization, and to methods for using such structurally stabilized peptides and conjugates in the prevention and treatment of RSV infection in subjects (e.g., humans).

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 489,096, filed on 8 March 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] (Sequence Listing) This application includes a sequence listing, which is submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML copy, created on 6 March 2024, is named 00530-0418WO1_SL.xml and has a size of 332,258 bytes. (Field of Invention) This disclosure relates to structurally stabilized (e.g., stapled, e.g., hydrocarbon-stapled) respiratory syncytial virus (RSV) peptides, and structurally stabilized (e.g., stapled, e.g., hydrocarbon-stapled) RSV peptides conjugated with polyethylene glycol (PEG) and / or cholesterol (or its variants, e.g., thiocholesterol), e.g., the generated PEG(n)-cholesterol or PEG(n)-thiocholesterol derivatization, and to methods for using such structurally stabilized peptide conjugates in the prevention and treatment of RSV infection in subjects (e.g., humans). [Background technology]

[0003] Respiratory syncytial virus (RSV) infection causes 64 million cases of respiratory illness 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).

[0004] There is a need for novel and improved strategies for the prevention and / or treatment of RSV infection.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0006] This application discloses compositions and methods for peptide stabilization techniques (e.g., stapling, e.g., hydrocarbon stapling) that reproduce and enhance the structure of a bioactive helix. In some examples, peptide stapling is combined with methods for derivatization of cholesterol or cholesterol variants (e.g., thiocholesterol) (e.g., PEG(n)-cholesterol or PEG(n)thiocholesterol) to produce optimized and targeted prophylactic and therapeutic agents for the prevention and / or treatment of RSV infection. By inserting "staples" (e.g., total hydrocarbon staples) into the RSV peptide, the bioactive helix structure can be restored, and significant protease resistance can be conferred by embedding the otherwise unstable amide bonds in the core of the helix structure and / or restricting the amide bonds in a manner that prevents their recognition and proteolysis by the body's proteases. Herein, hydrocarbon stapled and PEG(n)-cholesterol or PEG(n)thiocholesterol derivatized (hydrocarbon stapled conjugate) peptide inhibitors of RSV are disclosed. These structurally stabilized peptides and conjugates are used to prevent and / or treat RSV infection.

[0007] This specification provides for a conjugate comprising (i) a structurally stabilized peptide and (ii) cholesterol or thiocholesterol, wherein the cholesterol or thiocholesterol is directly or via a linker to the C-terminus of the structurally stabilized peptide, and the structurally stabilized peptide comprises an internally crosslinked amino acid sequence containing 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (Sequence ID 100), with the exception of 2 to 6 amino acid substitutions compared to the sequence of Sequence ID 100, and the internally crosslinked amino acid sequence comprises the sequence NAGKST (Sequence ID 100). A conjugate is provided which does not contain 258), and in which two of the two to six amino acid substitutions are with α,α-disubstituted unnatural amino acids having crosslinked olefin side chains, and these α,α-disubstituted unnatural amino acids having crosslinked olefin side chains are separated by three or six amino acids, the conjugate binds to the RSV5 helix bundle protein and / or inhibits RSV-mediated cell infection and / or prevents RSV-mediated cell infection, the conjugate is 25 to 45 amino acid long, and optionally, the conjugate is 30 amino acid long. In some examples, the two substitutions with α,α-disubstituted unnatural amino acids having crosslinked olefin side chains are at the amino acids corresponding to positions 1 and 8 of the sequence described in SEQ ID NO: 100, the amino acids corresponding to positions 3 and 10 of the sequence described in SEQ ID NO: 100, or the amino acids corresponding to positions 17 and 24 of the sequence described in SEQ ID NO: 100.

[0008] Furthermore, this specification describes a conjugate comprising (i) a structurally stabilized peptide and (ii) cholesterol or thiocholesterol, wherein the cholesterol or thiocholesterol is directly or via a linker to the C-terminus of the structurally stabilized peptide, and the structurally stabilized peptide is SEQ ID NO Compared to sequence 282, a conjugate is also provided which contains an internally crosslinked amino acid sequence containing the sequence X1X2X3FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 282) [wherein X1 is any amino acid, optionally S, and X2 and X3 are any loaded amino acids], wherein the internally crosslinked amino acid sequence does not contain the sequence NAGKST (SEQ ID NO: 258), two of the 2 to 6 amino acid substitutions are with α,α-disubstituted non-natural amino acids having crosslinked olefin side chains, and these α,α-disubstituted non-natural amino acids having crosslinked olefin side chains are separated by 3 or 6 amino acids, the conjugate binds to the RSV5 helix bundle protein and / or inhibits RSV-mediated cell infection and / or prevents RSV-mediated cell infection, and the conjugate is 32 to 45 amino acid long, optionally 32 amino acid long. In some examples, the two substitutions with α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are located at the amino acids corresponding to positions 4 and 11 of the sequence described in SEQ ID NO: 282, the amino acids corresponding to positions 6 and 13 of the sequence described in SEQ ID NO: 282, or the amino acids corresponding to positions 20 and 27 of the sequence described in SEQ ID NO: 282.For example, a structurally stabilized peptide may contain or consist of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 280), with 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 280 (e.g., amino acids corresponding to positions 4 and 11 of the sequence described in SEQ ID NO: 280, amino acids corresponding to positions 6 and 13 of the sequence described in SEQ ID NO: 282, or amino acids corresponding to positions 20 and 27 of the sequence described in SEQ ID NO: 282), and the internally crosslinked amino acid sequence may not contain the sequence NAGKST (SEQ ID NO: 258), and may contain 2 to 6 amino acid substitutions. Two of the amino acid substitutions are with α,α-disubstituted unnatural amino acids having cross-linked olefin side chains, and these α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are separated by three or six amino acids, the conjugate binds to the RSV5 helix bundle protein and / or inhibits RSV-mediated cell infection and / or prevents RSV-mediated cell infection, the conjugate is 32-45 amino acid long, and optionally 30 amino acid long.

[0009] Furthermore, this specification provides for a conjugate comprising (i) a structurally stabilized peptide and (ii) cholesterol or thiocholesterol, wherein the cholesterol or thiocholesterol is linked directly to the C-terminal amino acid of the structurally stabilized peptide or via a linker, and the structurally stabilized peptide is given the formula:

[0010] [ka] or comprising an internally crosslinked amino acid sequence having a pharmaceutically acceptable salt thereof, wherein each R1 and R2 is H or C1-C 10Conjugates are also provided which are alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which may be substituted or unsubstituted, x is 3 or 6, each R3 is independently alkylene, alkenylene, or alkynylene, any of which may be substituted or unsubstituted, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, the internally crosslinked amino acid sequence contains 25 to 29 consecutive amino acids of the sequence of SEQ ID NO: 100, with 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 100, the internally crosslinked amino acid sequence does not contain the sequence NAGKST (SEQ ID NO: 258), the conjugate binds to the RSV5 helix bundle protein and / or inhibits and / or prevents infection by RSV, the conjugate is 25 to 45 amino acid long, and optionally, the conjugate is 30 amino acid long. In some cases, R3 is an internal crosslink between the amino acids corresponding to positions 1 and 8 of the sequence described in SEQ ID NO: 100, between the amino acids corresponding to positions 3 and 10 of the sequence described in SEQ ID NO: 100, or between positions 17 and 24 of the sequence described in SEQ ID NO: 100. In some cases, the internally crosslinked amino acid sequence further includes or consists of the sequence X1X2X3, where X1 is any amino acid, optionally S, and X2 and X3 are any loaded 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 cases, X1X2X3 = SDE.For example, the amino acid sequence contains or consists of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 280), with 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 280, the internally crosslinked amino acid sequence does not contain the sequence NAGKST (SEQ ID NO: 258), two of the 2 to 6 amino acid substitutions are with α,α-disubstituted unnatural amino acids having crosslinked olefin side chains, the α,α-disubstituted unnatural amino acids having crosslinked olefin side chains are separated by 3 or 6 amino acids, the conjugate binds to the RSV5 helix bundle protein and / or inhibits RSV-mediated cell infection and / or prevents RSV-mediated cell infection, the conjugate is 32 to 45 amino acid long, and optionally 30 amino acid long.

[0011] In some of the aforementioned conjugates, the conjugate contains cholesterol. In some examples, the conjugate contains cholesterol and the linker contains PEG. In some examples, the conjugate contains PEG(n)-cholesterol directly linked to the C-terminal amino acid of a structurally stabilized peptide, where n is 1 to 36, and optionally n is 4, 5, 6, 7, 8, 12, 16, or 20. In some examples, the conjugate contains formula II directly linked to the C-terminal amino acid of a structurally stabilized peptide:

[0012] [ka] Includes, In the formula, n is between 1 and 36, and can be arbitrarily selected as 4, 5, 6, 7, 8, 12, 16, or 20.

[0013] In some of the aforementioned conjugates, the conjugate contains thiocholesterol. In some examples, the conjugate contains thiocholesterol and the linker contains PEG. In some examples, the conjugate contains PEG(n)-thiocholesterol directly linked to the C-terminal amino acid of a structurally stabilized peptide, where n is 1 to 36, and optionally n is 4, 5, 6, 7, 8, 12, 16, or 20. In some examples, the conjugate contains formula III directly linked to the C-terminal amino acid of a structurally stabilized peptide:

[0014] [ka] Includes, In the formula, n is between 1 and 36, and can be arbitrarily selected as 4, 5, 6, 7, 8, 12, 16, or 20.

[0015] In some of the aforementioned conjugate examples, the α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are separated by three amino acids, and optionally, each of the α,α-disubstituted unnatural amino acids having cross-linked olefin side chains is (S)-α-(4'-pentenyl)alanine.

[0016] In some of the aforementioned conjugate examples, the α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are separated by six amino acids, and optionally, the α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are (R)-α-(7'-octenyl)alanine and (S)-α-(4'-pentenyl)alanine.

[0017] In some of the aforementioned conjugates, the structurally stabilized peptide contains the sequence described in one of SEQ ID NOs: 27, 29, 36, 43, and 46. In some of the aforementioned conjugates, the structurally stabilized peptide contains the sequence described in one of SEQ ID NOs: 49, 51, 58, 65, and 68.

[0018] In some of the aforementioned conjugates, the conjugate contains the sequence described in any one of sequence numbers 5-26 and 71-92. In some examples, the conjugate contains the sequence described in any one of sequence numbers 71, 73, 80, 87, and 90. In some examples, the conjugate contains the sequence described in any one of sequence numbers 5, 6, 14, 21, and 24. In some examples, the conjugate contains the sequence described in sequence number 21 or 281.

[0019] In some of the aforementioned conjugates, the conjugate contains an internally crosslinked peptide with an amino acid length of 25-34, 26-33, 27-32, 28-31, 29, 30, 31, or 32 amino acids.

[0020] Furthermore, in this specification, 8DASISQXNEKINQSLAFIRKSDELLHNV * A conjugate containing or consisting of (SEQ ID NO: 265), wherein 8 is internally cross-linked to X, 8 is (R)-α-(7'-octenyl)alanine, and X is (S)-α-(4'-pentenyl)alanine. * However, the formula

[0021] [ka] And, A conjugate is also provided in the formula where n is from 1 to 36, and optionally n is 16 or 20.

[0022] Furthermore, in this specification, FD8SISQVNXKINQSLAFIRKSDELLHNV * A conjugate comprising (SEQ ID NO: 261) or comprising thereof, wherein 8 is internally cross-linked to X, 8 is (R)-α-(7'-octenyl)alanine, and X is (S)-α-(4'-pentenyl)alanine, * However, the formula

[0023] [ka] And, In the formula, a conjugate is provided in which n is from 1 to 36, and n is optionally 16 or 20.

[0024] Furthermore, in this specification, FDASISQVNEKINQSL8FIRKSDXLLHNV * A conjugate containing or consisting of (SEQ ID NO: 266), wherein 8 is internally cross-linked to X, 8 is (R)-α-(7'-octenyl)alanine, and X is (S)-α-(4'-pentenyl)alanine. * However, the formula

[0025] [ka] And, In the formula, a conjugate is provided in which n is from 1 to 36, and n is optionally 16 or 20.

[0026] Furthermore, this specification also provides a structurally stabilized peptide comprising an internally crosslinked amino acid sequence containing 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (Sequence ID 100), with 2 to 6 amino acid substitutions removed compared to the sequence of Sequence ID 100, wherein two of the 2 to 6 amino acid substitutions are α,α-disubstituted unnatural amino acids having crosslinked olefin side chains, and these α,α-disubstituted unnatural amino acids having crosslinked olefin side chains are separated by 3 or 6 amino acids, wherein the structurally stabilized peptide binds to the RSV5 helix bundle protein and / or inhibits and / or prevents infection by RSV, and the structurally stabilized peptide is 25 to 30 amino acid long, and optionally, the structurally stabilized peptide is 29 amino acid long. In some examples, the internally crosslinked amino acid sequence further includes or consists of the sequence X1X2X3, where X1 is any amino acid, optionally S, and X2 and X3 are any loaded amino acids immediately upstream of the first amino acid of SEQ ID NO: 100 (i.e., immediately upstream of the first F in SEQ ID NO: 100). In some cases, X1X2X3 = SDE. For example, the amino acid sequence contains or consists of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 280), with 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 100, the internally crosslinked amino acid sequence does not contain the sequence NAGKST (SEQ ID NO: 258), two of the 2 to 6 amino acid substitutions are with α,α-disubstituted unnatural amino acids having crosslinked olefin side chains, the α,α-disubstituted unnatural amino acids having crosslinked olefin side chains are separated by 3 or 6 amino acids, the conjugate binds to the RSV5 helix bundle protein and / or inhibits RSV-mediated cell infection and / or prevents RSV-mediated cell infection, the conjugate is 25 to 45 amino acid long, and optionally, the conjugate is 30 amino acid long.

[0027] Furthermore, in this specification, formula:

[0028] [ka] or a structurally stabilized peptide comprising an internally crosslinked amino acid sequence having a pharmaceutically acceptable salt thereof, In the formula, each R1 and R2 is H or C1~C 10 A structurally stabilized peptide is also provided, wherein the structurally stabilized peptide is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which may be substituted or unsubstituted, x is 3 or 6, each R3 is independently alkylene, alkenylene, or alkynylene, any of which may be substituted or unsubstituted, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and the internal cross-linking amino sequence contains 25 to 29 consecutive amino acids of the sequence of Sequence ID No. 100, with 2 to 6 amino acid substitutions compared to the sequence of Sequence ID No. 100, and the structurally stabilized peptide binds to the RSV5 helix bundle protein and / or inhibits RSV-mediated cell infection and / or prevents RSV-mediated cell infection, and the structurally stabilized peptide is 25 to 30 amino acid long, and optionally, the structurally stabilized peptide is 29 amino acid long.

[0029] In some of the structurally stabilized peptides mentioned above, the structurally stabilized peptides do not contain the amino acids corresponding to positions 517–522, 517–521, 517–520, 517–519, or 517–518 (numbered according to the sequence described in SEQ ID NO: 1) of the RSV-F protein. In some examples, the internally crosslinked amino acid sequence does not contain the sequence NAGKST (SEQ ID NO: 258).

[0030] In some of the aforementioned structurally stabilized peptides, the structurally stabilized peptide is 29 amino acid long and contains 29 consecutive amino acids of the sequence of SEQ ID NO: 100, with 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 100. In some of the aforementioned structurally stabilized peptides, the structurally stabilized peptide is 32 amino acid long and contains 32 consecutive amino acids of the sequence of SEQ ID NO: 280, with 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 280.

[0031] In some of the aforementioned structurally stabilized peptides, the structurally stabilized peptide contains the amino acid sequence described in one of SEQ ID NOs: 27-70. In some examples, the structurally stabilized peptide contains the amino acid sequence described in one of SEQ ID NOs: 27, 29, 36, 43, and 46. In some examples, the structurally stabilized peptide contains the amino acid sequence described in one of SEQ ID NOs: 49, 51, 58, 65, and 68.

[0032] Furthermore, this specification also provides peptides comprising any one amino acid sequence of SEQ ID NOs. 27 to 70, except for 0 to 6 additional substitutions, wherein the peptide does not contain the sequence NAGKST (SEQ ID NO. 258).

[0033] Furthermore, this specification also provides pharmaceutical compositions comprising any one of the aforementioned conjugates, any one of the aforementioned structurally stabilized peptides, or any one of the aforementioned peptides, and a pharmaceutically acceptable carrier.

[0034] Furthermore, this specification also provides a method for treating RSV infection in a subject requiring treatment of RSV infection, comprising administering to the subject a therapeutically effective amount of any one of the aforementioned conjugates, any one of the aforementioned structurally stabilized peptides, or any one of the aforementioned peptides. In some examples, the subject is a human.

[0035] Furthermore, this specification also provides a method for preventing RSV infection in subjects requiring prevention of RSV infection, comprising administering a therapeutically effective amount of any one of the aforementioned conjugates, any one of the aforementioned structurally stabilized peptides, or any one of the aforementioned peptides to the subject. In some examples, the subject is human.

[0036] Furthermore, this specification also provides a method for producing a structurally stabilized peptide, comprising: (a) providing a peptide having an amino acid sequence containing 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (Sequence ID 100), wherein two of the 2 to 6 amino acid substitutions are α,α-disubstituted unnatural amino acids having olefin side chains, and the α,α-disubstituted unnatural amino acids having olefin side chains are separated by 3 or 6 amino acids; and (b) crosslinking the peptide to produce a structurally stabilized peptide, and optionally purifying the structurally stabilized peptide. In some examples, the crosslinking is performed by a ruthenium-catalyzed metathesis reaction. In some examples, the method further comprises derivatizing the resin-bound amine of the structurally stabilized peptide using PEG and / or cholesterol or thiocholesterol containing a carboxylic acid on a resin. In some examples, the method further comprises formulating the structurally stabilized peptide as a sterile pharmaceutical composition.

[0037] Furthermore, this specification also provides pharmaceutical compositions comprising (a) means for treating or preventing RSV infection in a subject, and (b) a pharmaceutically acceptable carrier, the subject being optionally human. In some examples, the means for treating or preventing RSV infection is a structurally stabilized RSV peptide or its cholesterol or thiocholesterol conjugate.

[0038] In another aspect, the present disclosure features a peptide comprising or consisting of an amino acid sequence X1X2X3FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 282) having 0, 1, 2, or 3 amino acid substitutions (optionally, the substitutions include positions 20 and 27 of SEQ ID NO: 282), where 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 an amino acid sequence SDEFDASISQVNEKINQSLAFIRKSDELLHNV (including or consisting of SEQ ID NO: 280) having 0, 1, 2, or 3 amino acid substitutions (optionally, the substitutions include positions 20 and 27 of SEQ ID NO: 280).

[0039] In another aspect, the present disclosure relates to an internally cross-linked peptide comprising or consisting of the sequence X1X2X3FDASISQVNEKINQSL8FIRKSDXLLHNV (SEQ ID NO: 284), where 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), where "8" = (R)-α-(7'-octenyl)alanine and "X" = (S)-α-(4'-pentenyl)alanine.

[0040] In yet another aspect, the present disclosure features a conjugate comprising the amino acid sequence X1X2X3FDASISQVNEKINQSL8FIRKSDXLLHNV * (SEQ ID NO: 286), where 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, * = linker-lipid. In one example, *It contains Z-PEG(n)-cholesterol or Z-PEG(n)-thiocholesterol, where n=1 to 36, and Z is a diamino acid (e.g., lysine or ornithine). In one case, the conjugate is the amino acid sequence SDEFDASISQVNEKINQSL8FIRKSDXLLHNV * (Sequence ID 281) contains or consists of "8" = (R)-α-(7'-octenyl)alanine and "X" = (S)-α-(4'-pentenyl)alanine, * =Linker-lipid. * This compound contains Z-PEG(n)-cholesterol or Z-PEG(n)-thiocholesterol, where n=1 to 36, and Z is a diamino acid (e.g., lysine or ornithine).

[0041] In some cases, the peptides, internally cross-linked peptides, or conjugates described above are useful in methods for treating or preventing RSV infection in subjects requiring treatment or prevention of RSV infection, the methods comprising administering to the subject a therapeutically effective amount of one of the aforementioned peptides, internally cross-linked peptides, or conjugates. In some examples, the subject is a human.

[0042] The disclosure also features a pharmaceutical composition comprising one of the aforementioned peptides, internally crosslinked peptides, or conjugates, and a pharmaceutically acceptable carrier. [Brief explanation of the drawing]

[0043] [Figure 1] The mechanism of action of RSV virus-host membrane fusion (top) and the mechanism of action of membrane fusion and the inhibition of stapled lipopeptides of RSV-F in viral infection (bottom) are shown. [Figure 2] The amino acid sequence of the RSV F protein (SEQ ID NO: 1) is shown. The 7-amino acid repeat domain 1 (HR1) (SEQ ID NO: 2) is in bold. The 7-amino acid repeat domain 2 (HR2) (SEQ ID NO: 3) is underlined. [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. [Figure 4] Alignments of the HR1 region (top) and HR2 region (bottom) of various RSV virus strains are shown. HR1 sequences, from top to bottom: SEQ ID NOs: 2, 2, 93, 94, and 95, respectively; HR2 sequences, from top to bottom: SEQ ID NOs: 3, 3, 96, 97, and 97, respectively. For each of HR1 and HR2, the sequences below (SEQ ID NOs: 98 and 99, respectively, represent conserved amino acids). [Figure 5] The following are various stapling amino acids containing olefin tethers that can be used to produce hydrocarbon-stapled RSV-F HR2 peptides with staples extending to positions i, i+3; i, i+4; and i, i+7. Top row, left to right: (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 bis-pentenylglycine, respectively. [Figure 6] This paper presents various staple compositions and staple scanning methods for multiple stapled peptides to generate a library of multiple stapled RSV-F HR2 peptides for conjugation to cholesterol or cholesterol variant moieties (e.g., PEG-thiocholesterol or PEG-cholesterol). [Figure 7] This document presents various staple compositions in tandem-stitched peptides for generating a library of stitched RSV-F HR2 peptides for conjugation to cholesterol or cholesterol variant moieties (e.g., PEG-thiocholesterol or PEG-cholesterol). [Figure 8]This figure illustrates an exemplary approach for designing, synthesizing, and identifying optimal stapled peptide constructs for targeting the RSV-F fusion apparatus, including Ala scans, staple scans, and the generation of variable N and C-terminal deletions, additions, and derivatization libraries for conjugation to cholesterol or cholesterol variant moieties (e.g., PEG-thiocholesterol or PEG-cholesterol). Using single and double stapled and stitched constructs, including alanine and staple and stitch scans, optimal stapled peptides are identified for conjugation to cholesterol or cholesterol variant moieties (e.g., PEG-thiocholesterol or PEG-cholesterol moieties with variable PEG chain lengths) and for application in in vitro and in vivo analysis. [Figure 9] The dose-response curve for a single stapled peptide (SEQ ID NO: 4) during treatment of A549 cells infected with GFP-RSV virus is shown, where 8 is (R)-α-(7'-octenyl)alanine and X is (S)-α-(4'-pentenyl)alanine. [Figure 10] The crystal structure of the RSV6 helix bundle (RCSB PDB:1G2C) is shown. The first and last amino acids of the extended helix (corresponding to Phe-488 and Val-516 in the sequence of SEQ ID NO: 1) are shown. [Figure 11] A synthetic scheme for converting thiocholesterol or cholesterol to a carboxylic acid is shown for the easy derivatization on resin of stapled peptides using a cholesterol (or cholesterol variant, e.g., thiocholesterol)-containing moiety. DCM: dichloromethane, TFA: trifluoroacetic acid, eq: equivalent, RT: room temperature, min: minutes, hr: hours, vol: volume, Δ: heating. [Figure 12]The synthetic scheme for the derivatization of a stapled peptide sequence (exemplified by the sequence of SEQ ID NO: 5) using a PEG-linked thiocholesterol moiety on a resin 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. The figure discloses SEQ ID NOs. 262-264 in order of appearance. [Figure 13] The dose-response curve for the peptide having the sequence of Sequence ID No. 5 with a C-terminal (PEG)4 linker and thiocholesterol ("PEG4-TC") attached is shown. The dashed line at 50 represents the vehicle infection level. [Figure 14] The following are exemplary structurally stabilized RSV-HR2 peptide sequences with C-terminal derivatization, where * = Lys (PEG4-thiocholesterol, 8 is (R)-α-(7'-octenyl)alanine) and X is (S)-α-(4'-pentenyl)alanine) (SEQ ID NOs. 5-26). [Figure 15] This graph shows the differential antiviral activity of a stapled peptide with the indicated sequence (*=Lys(PEG4-thiocholesterol, 8 is (R)-α-(7'-octenyl)alanine, and X is (S)-α-(4'-pentenyl)alanine) at a dose of 2 μM against RSV-GFP in A549 cells 48 hours later. [Figure 16] This graph shows the dose-response curves of a stapled peptide having the amino acid sequence of SEQ ID NO: 51, with an added PEG(n)-thiocholesterol ("TC") linked to the stapled peptide via C-terminal lysine, where n is 0, 4, 8, 12, 16, or 20 (SEQ ID NOs: 267-272 from top to bottom, respectively). [Figure 17]This graph shows the dose-response curves of a stapled peptide having the amino acid sequence of SEQ ID NO: 65, which has an added PEG(n)-thiocholesterol linked to the stapled peptide via C-terminal lysine, where n is 0, 4, 8, 12, 16, or 20 (SEQ ID NOs: 273-278 from top to bottom, respectively). [Figure 18] The solubility of two peptides, SEQ ID NO: 21 (visible pellet) and SEQ ID NO: 281 (fully soluble), was recorded. The buffer conditions were 15 mg / mL in 15% DMSO and PBS pH 7.4. [Modes for carrying out the invention]

[0044] This disclosure is based, in particular, on the discovery that structurally stabilized (e.g., stapled, e.g., hydrocarbon-stapled) RSV peptides, and that they can be lipid-conjugated (e.g., using PEG and / or cholesterol (or cholesterol variants, e.g., thiocholesterol), e.g., PEG(n)-cholesterol or PEG(n)-thiocholesterol), to selectively bind to RSV and exhibit antiviral activity against RSV. Accordingly, this disclosure provides methods (e.g., approaches for converting cholesterol / thiocholesterol to carboxylic acids for resin-based derivatization) and compositions (e.g., structurally stabilized RSV peptides and PEG(n)-cholesterol or PEG(n)-thiocholesterol conjugates) for treating, developing, and preventing RSV infection or disease. Accordingly, the peptides and compositions disclosed herein can be used to prevent and / or treat RSV infection.

[0045] RSV peptide RSV arranges the individual seven-amino acid repeat domains of its F protein to form RSV-SFB, a structure that allows the virus to enter the host cell membrane. Furin-like proteases cleave the RSV-F precursor, resulting in the formation of two subunits stabilized by disulfide crosslinking (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 an otherwise hidden peptide fusion motif located at the N-terminus of the F1 subunit, resulting in 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). When the fusion peptide is inserted into the host cell membrane, the F protein refolds itself to form a trimer hairpin or "6-helix bundle." The trimer hairpin originates from an uncharacterized conformational change in which the C-terminal 7-amino acid repeat (HR2) region assembles antiparallel to the N-terminal 7-amino acid repeat (HR1) juxtaposed with the fusion peptide (Cianci et al., Proc. Natl. Acad. Sci., USA, 101:15046-15051, 2004). After the pore opens, the HR-induced conformational change achieves a new equilibrium state that is thought to be essential for stabilizing and expanding the pore (Cianci, supra, Melikyan, Retrovirology, 5:111, 2008, Melikyan et al., Proc. Natl. Acad. Sci., USA, 102:8728-8733, 2005), allowing the protein to enter the host cell. The compositions and methods disclosed herein can be used to prevent or treat RSV infection by inhibiting this process.

[0046] This specification provides the RSV-F HR2 peptide. RSV infection is mediated on the cell surface by the RSV-F protein, which has two 7-amino acid repeat domains, HR1 and HR2. The amino acid sequences of exemplary RSV-F protein sequences are shown in Figure 2. The amino acid sequence of exemplary RSV-F protein HR1 is described in SEQ ID NO: 2. The amino acid sequence of exemplary RSV-F protein HR2 is described in SEQ ID NO: 3.

[0047] The RSV-F proteins HR1 and HR2 exhibit high homology among different RSV strains. See Figure 4 for exemplary amino acid sequence alignments of the HR1 and HR2 sequences in exemplary RSV strains.

[0048] In some cases, the HR2 peptide described herein contains or consists of the amino acid sequence of SEQ ID NO: 100. In some cases, the HR2 peptide consists of the amino acid sequence of SEQ ID NO: 100. In some cases, the HR2 peptide does not contain the amino acid sequence NAGKST (SEQ ID NO: 258). In some cases, the HR2 peptide does not contain the amino acid sequence NAGKS (SEQ ID NO: 259). In some cases, the HR2 peptide does not contain the amino acid sequence NAGK (SEQ ID NO: 260). In some cases, the HR2 peptide does not contain the amino acid sequence NAG. In some cases, the HR2 peptide does not contain the amino acid sequence NA. In some cases, the HR2 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 4)). In some cases, the HR2 peptide is 25–34 amino acid long (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some cases, the HR2 peptide is 30 amino acid long. In certain specific cases, the HR2 peptide further comprises or consists of the sequence X1X2X3, where X1 is any amino acid, optionally S, and X2 and X3 are any loaded amino acids immediately upstream of the first amino acid of SEQ ID NO: 100 (i.e., immediately upstream of the first F in SEQ ID NO: 100). In some cases, X1X2X3 = SDE. In one example, the amino acid sequence comprises or consists of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 280).

[0049] In certain examples, the RSV-F HR2 peptide described herein comprises or consists of the amino acid sequence of SEQ ID NO: 100 or 280, provided that it contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., for the amino acid sequence of SEQ ID NO: 100 or 280), for example, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved and / or non-conserved amino acid substitutions.

[0050] A "conservative amino acid substitution" means that the substitution replaces one amino acid with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in this field. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), and amino acids with acidic side chains and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine).

[0051] Those skilled in the art will understand that alignments of F proteins from different RSV strains are useful for identifying residues suitable for conserved residues and substitutions (e.g., conserved or non-conserved substitutions). For example, residues that do not change between two or more different RSV strains in such alignments (see, e.g., Figure 4) may be either unmodified or substituted with non-natural or conserved amino acid substitutions. Residues that differ between two or more different RSV strains in such alignments due to conserved amino acid substitutions (see, e.g., Figure 4) may be either unmodified or substituted with conserved amino acid substitutions. Non-conserved residues between two or more different RSV strains in such alignments (see, e.g., Figure 4) may be either unmodified or substituted with any amino acid. In some examples, residues that are conserved between two or more different RSV strains in such alignments but located on the non-interaction plane of HR2 may be substituted with any amino acid. For example, considering the alignment in Figure 4, a conserved amino acid substitution is permitted at the amino acid corresponding to position 33 of SEQ ID NO: 3, and a non-conserved amino acid substitution is permitted at position 40 of SEQ ID NO: 3. In certain cases, the substituted amino acid is selected from the group consisting of L-Ala, D-Ala, Aib, Sar, Ser, substituted alanine, or substituted glycine derivatives. Methods for identifying peptide interaction planes are known in the art (see, for example, Brogli et al., Protein sci., 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).

[0052] In some examples, the peptides described herein contain amino acid sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 93% identical to the sequences described in SEQ ID NO: 100 or 280. In some examples, the peptides described herein have one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) being alpha-helix, (ii) being protease-resistant, (iii) binding to the RSV5 helix bundle protein, (iv) inhibiting RSV infection of cells, and / or (v) preventing RSV infection of cells. In some examples, the peptides inhibit RSV infection of cells in a pseudovirus and / or live RSV virus assay, and / or prevent RSV infection of cells in a pseudovirus and / or live RSV virus assay. The RSV pseudovirus assay is 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.

[0053] Methods for determining the percentage of identity between amino acid sequences are known in the art. For example, sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can 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%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Then, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. Determining the percentage of identity between two amino acid sequences is achieved using the BLAST2.0 program. Sequence comparison is performed using gapless alignment with default parameters (Blossom62 matrix, gap cost of 11, gap cost per residue of 1, and lambda ratio of 0.85). The mathematical algorithm used in the BLAST program is described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).

[0054] In some examples, the RSV-F HR2 peptides described herein (e.g., SEQ ID NOs: 100, 280) contain at least one, at least two, at least three, at least four, or at least five (e.g., 1, 2, 3, 4, 5, 6) amino acids added to the N-terminus of the peptide. In some examples, the RSV-F HR2 peptides described herein (e.g., SEQ ID NOs: 100, 280) contain at least one, at least two, at least three, at least four, or at least five (e.g., 1, 2, 3, 4, 5, 6) amino acids added to the C-terminus of the peptide. In some examples, the RSV-F HR2 peptides described herein (e.g., SEQ ID NOs: 100, 280) contain at least one, at least two, at least three, at least four, or at least five (e.g., 1, 2, 3, 4, 5, 6) amino acids deleted at the N-terminus of the peptide. In some cases, the RSV-F HR2 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 amino acids (e.g., 1, 2, 3, 4, 5, 6) deleted at the C-terminus of the peptide. In some cases, the RSV-F HR2 peptide does not contain the amino acid sequence NAGKST (SEQ ID NO: 258).

[0055] In some examples, the peptide contains an amino acid sequence with 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, or two substitutions, insertions, and / or deletions compared to SEQ ID NO: 100. In some examples, the peptide contains 2, 3, 4, 5, or 6 substitutions, insertions, and / or deletions compared to SEQ ID NO: 100 or 280. In some examples, the peptide having the above substitutions, insertions, and / or deletions compared to SEQ ID NO: 100 or 280 has one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) is an alpha-helix, (ii) is protease-resistant, (iii) binds to the RSV5 helix bundle protein, (iv) inhibits RSV-mediated cell infection, and / or (v) prevents RSV-mediated cell infection. In some cases, the peptide inhibits RSV infection of cells and / or prevents RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0056] In some cases, peptides have an amino acid length of 25-45 (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 cases, peptides have an amino acid length of 28-40 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40). In some cases, peptides have an amino acid length of 25-35 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). In some cases, peptides have an amino acid length of 29 or 30. In certain cases, peptides have an amino acid length of 32 or 33.

[0057] In some cases, the peptides described above have one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) being alpha-helix, (ii) being protease-resistant, (iii) binding to the RSV5 helix bundle protein, (iv) inhibiting RSV infection of cells, and / or (v) preventing RSV infection of cells. In some cases, 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.

[0058] In certain cases, each of the RSV-F HR2 peptides mentioned above binds to the RSV5 helix bundle protein. In certain cases, each of the RSV-F HR2 peptides mentioned above binds to the RSV5 helix bundle protein, preventing or blocking fusion between the RSV membrane and the host membrane.

[0059] Methods for determining whether a peptide (e.g., the RSV-F HR2 peptide, structurally stabilized peptides, or structurally stabilized peptide conjugates described herein) binds to an RSV5 helix bundle are known in the art, such as high-resolution clear native electrophoresis (hrCNE). See, for example, Example 3 of International Publication No. 2013 / 102211, which is incorporated herein by reference in its entirety.

[0060] Methods for determining whether a peptide (e.g., the RSV-F HR2 peptide described herein, a structurally stabilized peptide, or a structurally stabilized peptide conjugate) prevents or blocks the fusion of the RSV membrane to the host membrane are known in the art, for example, by cytotoxicity and immunofluorescence. In some examples, the peptide prevents or blocks the fusion of the RSV membrane to the host membrane when 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 infection multiplicity of 0.1, 0.5, 1, or 10 in the presence of the peptide. In some cases, the peptide prevents or blocks RSV membrane-host membrane fusion if, after infection with RSV at infection multiplicity of 0.1, 0.5, 1, or 10 in the presence of the peptide, 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.

[0061] Methods for determining whether a peptide (e.g., the RSV-F HR2 peptide described herein, structurally stabilized peptides, or structurally stabilized peptide conjugates) inhibits RSV infection of cells are known in the art, such as cytotoxicity and immunofluorescence, and are described in the examples. In some examples, the peptide (e.g., the RSV-F HR2 peptide described herein, structurally stabilized peptides, or structurally stabilized peptide conjugates) inhibits RSV infection of cells when 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 with infection multiplicity of 0.1, 0.5, 1, or 10 in the presence of the peptide. In some cases, the peptide (e.g., the RSV-F HR2 peptide, structurally stabilized peptide, or structurally stabilized peptide conjugate described herein) inhibits cell infection if the level of RSV infection in 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 level of RSV infection in a cell population in the absence of the peptide under the same conditions. In some cases, RSV infection has an infection multiplicity of 0.1, 0.5, 1, or 10.

[0062] In some cases, 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, for example, its modified version described herein) are substituted with α,α-disubstituted non-natural amino acids, each having an olefin side chain (e.g., non-natural amino acids substituted with alpha-methyl and alpha-alkenyl groups), and the α,α-disubstituted non-natural amino acids can crosslink with each other to form one or more staples or stitches (see the section “Structurally Stabilized Peptides” below). The type of substitution performed may be guided, for example, by the alignment of HR2 peptides of two or more RSV-F protein sequences (see, for example, Figure 4). The guidance provided in the section “Structurally Stabilized Peptides” below regarding the amino acids that can be altered is equally relevant to the RSV-F HR2 peptide described herein.

[0063] In some cases, the RSV-F HR2 peptide (or structurally stabilized peptide) is lipid-added. See the section “Structurally Stabilized Peptide Conjugates” below. These lipid-added peptides are referred to herein interchangeably with “Structurally Stabilized Peptide Conjugates” and “Conjugates.” In some cases, the RSV-F HR2 peptide is modified to include cholesterol, for example, via a polyethylene glycol (PEG)-containing linker. In some cases, the RSV-F HR2 peptide is modified to include thiocholesterol, for example, via a PEG-containing linker. In some cases, the RSV-F HR2 peptide (e.g., SEQ ID NOs: 100, 280) contains the following formula attached to the C-terminus of the peptide.

[0064] [ka]

[0065] 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 NOs: 100, 280) contains the following formula attached to the C-terminus of the peptide.

[0066] [ka]

[0067] In some cases, Equation II above, or n in Equation II, is n=1 to 36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In some cases, n=16. In some cases, n=20.

[0068] Structurally stabilized peptides Furthermore, structurally stabilized RSV-F HR2 peptides are also provided herein. In some cases, the structurally stabilized peptides are structurally stabilized (e.g., stapled, e.g., hydrocarbon-stapled) versions of the RSV-F HR2 peptides described herein (see, for example, the “RSV Peptides” section above). In some cases, the structurally stabilized (e.g., stapled, e.g., hydrocarbon-stapled) RSV-F HR2 peptides are derived from the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 100) or SEQ ID NO: 280.

[0069] In some examples, the structurally stabilized peptide contains an internally cross-linked amino acid sequence containing 25 to 29 consecutive amino acids of sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 100), with the exception of two or more amino acid substitutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) compared to the sequence of SEQ ID NO: 100, where two of the two or more amino acid substitutions are with α,α-disubstituted unnatural amino acids having cross-linked olefin side chains, and these α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are separated by six amino acids. In some examples, the structurally stabilized peptide has 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 100. In some examples, the structurally stabilized peptide has 4 amino acid substitutions compared to the sequence of SEQ ID NO: 100. In some examples, the structurally stabilized peptide has 3 amino acid substitutions compared to the sequence of SEQ ID NO: 100. In some examples, the structurally stabilized peptide has 2 amino acid substitutions compared to the sequence of SEQ ID NO: 100. In some examples, substitutions with α,α-disubstituted unnatural amino acids having crosslinked olefin side chains are located at positions (i) 1 and 8 (see, e.g., SEQ ID NO: 27), (ii) 3 and 10 (see, e.g., SEQ ID NO: 29), (iii) 10 and 17 (see, e.g., SEQ ID NO: 36), (iv) 17 and 24 (see, e.g., SEQ ID NO: 43), and (v) 20 and 27 (see, e.g., SEQ ID NO: 46) relative to SEQ ID NO: 100 (position 1 is the N-terminal phenylalanine of SEQ ID NO: 100). In some examples, substitutions by α,α-disubstituted unnatural amino acids with cross-linked olefin side chains are located at positions 17 and 24 (see, for example, SEQ ID NO: 43) in SEQ ID NO: 100 (position 1 is the N-terminal phenylalanine of SEQ ID NO: 100).In some examples, the internally crosslinked amino acid sequence further includes or consists of the sequence X1X2X3, where X1 is any amino acid, optionally S, and X2 and X3 are any loaded amino acids immediately upstream of the first amino acid of SEQ ID NO: 100 (i.e., immediately upstream of the first F in SEQ ID NO: 100). In some cases, X1X2X3 = SDE. For example, the amino acid sequence contains or consists of SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 280), with 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 100, the internally crosslinked amino acid sequence does not contain the sequence NAGKST (SEQ ID NO: 258), two of the 2 to 6 amino acid substitutions are with α,α-disubstituted unnatural amino acids having crosslinked olefin side chains, the α,α-disubstituted unnatural amino acids having crosslinked olefin side chains are separated by 3 or 6 amino acids, the conjugate binds to the RSV5 helix bundle protein and / or inhibits RSV-mediated cell infection and / or prevents RSV-mediated cell infection, the conjugate is 25 to 45 amino acid long, and optionally, the conjugate is 30 amino acid long.

[0070] In some examples, the structurally stabilized peptide contains an internally cross-linked amino acid sequence containing 25 to 29 consecutive amino acids of sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 100), with the exception of two or more amino acid substitutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) compared to the sequence of SEQ ID NO: 100, where two of the two or more amino acid substitutions are with α,α-disubstituted unnatural amino acids having cross-linked olefin side chains, and these α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are separated by three amino acids. In some examples, the structurally stabilized peptide has 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 100. In some examples, the structurally stabilized peptide has 4 amino acid substitutions compared to the sequence of SEQ ID NO: 100. In some examples, the structurally stabilized peptide has 3 amino acid substitutions compared to the sequence of SEQ ID NO: 100. In some examples, the structurally stabilized peptide has 2 amino acid substitutions compared to the sequence of SEQ ID NO: 100.

[0071] In some examples, the structurally stabilized peptide contains an internally cross-linked amino acid sequence containing 30 to 32 consecutive amino acids of the sequence described in SEQ ID NO: 280, with the exception of two or more amino acid substitutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) compared to the sequence of SEQ ID NO: 280, where two of the two or more amino acid substitutions are with α,α-disubstituted unnatural amino acids having cross-linked olefin side chains, and these α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are separated by three amino acids. In some examples, the structurally stabilized peptide has 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 280. In some examples, the structurally stabilized peptide has 4 amino acid substitutions compared to the sequence of SEQ ID NO: 280. In some examples, the structurally stabilized peptide has 3 amino acid substitutions compared to the sequence of SEQ ID NO: 280. In some examples, the structurally stabilized peptide has 2 amino acid substitutions compared to the sequence of SEQ ID NO: 280.

[0072] In some cases, structurally stabilized peptides have one or more modifications (e.g., substitution, insertion, addition, or deletion) as described in the "RSV Peptides" section above.

[0073] In some examples, structurally stabilized peptides (e.g., stapled, e.g., hydrocarbon stapled) are the peptides shown in Table 1 below. In certain examples, the disclosure encompasses the structurally stabilized peptides of Table 1 further comprising three N-terminal amino acids X1X2X3, where X1 is any amino acid, optionally S, and X2 and X3 are any loaded amino acids immediately upstream of the first amino acid of SEQ ID NO: 100 (i.e., immediately upstream of the first F in SEQ ID NO: 100). In some cases, X1X2X3=SDE. In some examples, the structurally stabilized peptides contain or consist of any one amino acid sequence of SEQ ID NOs. 27-70. In some examples, the structurally stabilized peptides contain or consist of any one amino acid sequence of SEQ ID NOs. 27, 29, 36, 43, and 46. In some examples, the structurally stabilized peptides contain or consist of the amino acid sequence described in any one of SEQ ID NOs. 49, 51, 58, 65, and 68. In some cases, the structurally stabilized peptide contains or consists of the amino acid sequence described in SEQ ID NO: 29. In some cases, the structurally stabilized peptide contains or consists of the amino acid sequence described in SEQ ID NO: 51. In some cases, the structurally stabilized peptide contains or consists of the amino acid sequence described in SEQ ID NO: 43. In some cases, the structurally stabilized peptide contains or consists of the amino acid sequence described in SEQ ID NO: 65. In some cases, the structurally stabilized peptide contains or consists of the amino acid sequence described in SEQ ID NO: 281.

[0074] [Table 1]

[0075] In Table 1, for Sequence IDs 27-48, "X1" is an α,α-disubstituted unnatural amino acid having an olefin side chain crosslinked to X2 (e.g., an unnatural amino acid substituted with an alpha-methyl group and an alpha-alkenyl group), and "X2" is an α,α-disubstituted unnatural amino acid having an olefin side chain crosslinked to X1 (e.g., an alpha-methyl, alpha-alkenyl unnatural amino acid). In Table 1, for Sequence IDs 49-70, "8" is (R)-α-(7'-octenyl)alanine, and "X" is (S)-α-(4'-pentenyl)alanine.

[0076] Note that the residues in bold in Table 1 identify stapling amino acids. In some examples (e.g., the peptides listed in Table 1), structurally stabilized peptides are single-stapled peptides.

[0077] This disclosure includes all peptides and structurally stabilized peptides listed in Table 1, except those at staple positions (i.e., the bolded residues in Table 1), and their variants (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) with amino acid substitutions, insertions, and / or deletions. In some examples, the variant has 1 to 10 amino acid substitutions, insertions, and / or deletions, excluding those at staple positions (i.e., the bolded residues in Table 1). In some examples, the variant has 1 to 5 amino acid substitutions, insertions, and / or deletions, excluding those at staple positions (i.e., the bolded residues in Table 1). In some examples, the variant has 1 to 3 amino acid substitutions, insertions, and / or deletions, excluding those at staple positions (i.e., the bolded residues in Table 1). In some cases, the variant has 1-10, 10-5, 1-3, 2, or 1 amino acid substitutions, insertions, and / or deletions compared to the amino acid sequence described in any one of SEQ ID NOs. 31-35 and 38, but excluding those at staple locations (i.e., the bolded residues in Table 1). In some cases, the variant has 1-10, 10-5, 1-3, 2, or 1 amino acid substitutions, insertions, and / or deletions compared to the amino acid sequence described in any one of SEQ ID NOs. 27, 29, 36, 43, and 46, or any one of SEQ ID NOs. 49, 51, 58, 65, and 68, but excluding those at staple locations (i.e., the bolded residues in Table 1). In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAGKST (SEQ ID NO. 258). In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAGKS (SEQ ID NO. 259). In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAGK (SEQ ID NO. 260). In some cases, structurally stabilized peptides do not contain the amino acid sequence NAG. In some cases, structurally stabilized peptides do not contain the amino acid sequence NA.In some cases, the structurally stabilized 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, e.g., Figure 4)). In some cases, the structurally stabilized peptide has an amino acid length of 25–34 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some cases, the structurally stabilized peptide has an amino acid length of 30. In some cases, the structurally stabilized peptide has one or more of the following properties (e.g., 1, 2, 3, 4, or 5): (i) it is an alpha-helix, (ii) it is protease-resistant, (iii) it binds to the RSV5 helix bundle protein, (iv) it inhibits RSV-mediated cell infection, and / or (v) it prevents RSV-mediated cell infection. In some cases, the peptide inhibits RSV infection of cells and / or prevents RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0078] In some cases, the structurally stabilized peptides contain amino acid sequences with 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, or two substitutions, insertions, and / or deletions compared to SEQ ID NO: 100. In some cases, the structurally stabilized peptides with substitutions, insertions, and / or deletions compared to SEQ ID NO: 100 as described above are (i) alpha-helices, (ii) protease-resistant, (iii) bind to the RSV5 helix bundle protein, (iv) inhibit RSV-mediated cell infection, and / or (v) prevent RSV-mediated cell infection. In some cases, the peptides inhibit RSV-mediated cell infection in pseudovirus and / or live RSV virus assays and / or prevent RSV-mediated cell infection in pseudovirus and / or live RSV virus assays.

[0079] In some examples, disclosed herein are 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 stabilized peptides in Table 1 (the substitutions are not in the staple positions in Table 1). In some examples, disclosed herein are 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 stabilized peptides in Table 1. In some examples, the peptides disclosed herein 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 sequence described in any one of SEQ ID NOs. 27, 29, 36, 43, and 46, or any one of SEQ ID NOs. 49, 51, 58, 65, and 68 (the mutations are not at the staple positions in any one of SEQ ID NOs. 27, 29, 36, 43, and 46 or SEQ ID NOs. 49, 51, 58, 65, and 68, respectively). It is understood that mutations in a particular sequence are not at the staple positions (i.e., the residues in bold in Table 1). In some examples, the peptides disclosed herein are 100% identical to one of the structurally stabilized peptides in Table 1. In some cases, the peptides disclosed herein are 100% identical to the amino acid sequence described in any one of SEQ ID NOs: 27, 29, 36, 43, and 46, or any one of SEQ ID NOs: 49, 51, 58, 65, and 68. In some cases, the structurally stabilized peptides do not contain the amino acid sequence NAGKST (SEQ ID NO: 258). In some cases, the structurally stabilized peptides do not contain the amino acid sequence NAGKS (SEQ ID NO: 259). In some cases, the structurally stabilized peptides do not contain the amino acid sequence NAGK (SEQ ID NO: 260). In some cases, the structurally stabilized peptides do not contain the amino acid sequence NAG. In some cases, the structurally stabilized peptides do not contain the amino acid sequence NA.In some cases, the structurally stabilized 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, e.g., Figure 4)). In some cases, the structurally stabilized peptide has an amino acid length of 25–34 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some cases, the structurally stabilized peptide has an amino acid length of 30. In some cases, the structurally stabilized peptide has one or more of the following properties (e.g., 1, 2, 3, 4, or 5): (i) it is an alpha-helix, (ii) it is protease-resistant, (iii) it binds to the RSV5 helix bundle protein, (iv) it inhibits RSV-mediated cell infection, and / or (v) it prevents RSV-mediated cell infection. In some cases, the peptide inhibits RSV infection of cells and / or prevents RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0080] In some examples, any substitution described herein may be a conservative substitution. In some examples, any substitution described herein may be a non-conservative substitution.

[0081] In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAGKST (sequence number 258). In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAGKS (sequence number 259). In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAGK (sequence number 260). In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAG. In some cases, the structurally stabilized peptide does not contain the amino acid sequence NA. In some cases, the structurally stabilized peptide does not contain the amino acids corresponding to positions 517-522, 517-521, 517-520, 517-519, or 517-518 of Sequence Number 1 (or the corresponding amino acids of the RSV-F protein of another strain (see, for example, Figure 4)). In some cases, the structurally stabilized peptide has an amino acid length of 25-34 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some examples, the structurally stabilized structure is 30 amino acids long.

[0082] In some cases, unnatural amino acids that can be used as stapling amino acids are (R)-2-(2'-propenyl)alanine, (R)-2-(4'-pentenyl)alanine, (R)-α-(7'-octenyl)alanine, (S)-α-(2'-propenyl)alanine, (S)-α-(4'-pentenyl)alanine, (S)-2-(7'-octenyl)alanine, α,α-bis(4'-pentenyl)glycine, and α,α-bis(7'-octinyl)glycine.

[0083] In some examples, the internal staple replaces the side chains of two amino acids, i.e., each staple lies between two amino acids separated, for example, by six amino acids. In some examples, the amino acids forming the staple are located at staple positions i and i+7, respectively. For example, if a peptide has the sequence ...X1, X2, X3, X4, X5, X6, X7, X8, X9..., then the crosslink between X1 and X8 (i and i+7) is a useful hydrocarbon-stapled form of that peptide. The use of i and i+4 staples, multiple crosslinks (e.g., two, three, four, or more), or tandem stitches is also contemplated. Additional descriptions relating to the preparation and use of hydrocarbon-stapled peptides can be found, for example, in U.S. Patent Application Publications 2012 / 0172285, 2010 / 0286057, and 2005 / 0250680, all of which are incorporated herein by reference in their entirety.

[0084] "Peptide stapling" is a term derived from a synthetic method in which two olefin-containing side chains (e.g., crosslinkable side chains) present in a peptide chain are covalently bonded (e.g., "stapled together") using a ring-closing metathesis (RCM) reaction to form a crosslinking ring (see, e.g., Blackwell et al., J. Org. Chem., 66:5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). Structural stabilization may also be achieved, for example, by stapling the peptide (see, e.g., Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are incorporated herein by reference in their entirety). In some cases, the staples are hydrocarbon staples.

[0085] In some examples, the staples used herein are all-hydrocarbon staples.

[0086] In some examples, the staples used herein are lactam staples, UV-addition cycloadjusted staples, oxime staples, thioether staples, double-click staples, bis-lactam staples, bis-arylated staples, or any two or more combinations thereof. The stabilized peptides described herein include stapled peptides, as well as peptides comprising multiple staples or any other chemical strategies for structural reinforcement (e.g., Balaram P.Cur.Opin.Struct.Biol.1992;2:845, Kemp DS, et al., J.Am.Chem.Soc.1996;118:4240, Orner BP, et al., J.Am.Chem.Soc.2001;123:5382, Chin JW, et al., Int.Ed.2001;40:3806, Chapman RN, et al., J.Am.Chem.Soc.2004;126:12252, Horne WS, et al., Chem.,Int.Ed.2008;47:2853, Madden et al., Chem). See Commun(Camb).2009 Oct 7;(37):5588-5590, Lau et al., Chem.Soc.Rev.,2015,44:91-102, and Gunnoo et al., Org.Biomol.Chem.,2016,14:8002-8013 (each of these is incorporated herein by reference in its entirety).

[0087] Peptides are "structurally stabilized" in the sense that they maintain their native secondary structure. For example, stapling allows peptides that tend to have an α-helix secondary structure to maintain their native α-helix conformation. This secondary structure can increase 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 have improved biological activity and pharmacology compared to the corresponding unstapled (uncrosslinked) peptides.

[0088] In some examples, the structurally stabilized peptides described herein contain an amino acid sequence that includes 25 to 29 consecutive amino acids of the sequence of SEQ ID NO: 100, with the exception of 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 100, and the structurally stabilized peptides have the formula:

[0089] [ka] or comprising an internally crosslinked amino acid sequence having a pharmaceutically acceptable salt thereof, In the formula, each R1 and R2 is H or a C1-C10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and any of these may be substituted or unsubstituted. x is 3 or 6, Each R3 is independently alkylene, alkenylene, or alkynylene, and each of these can be substituted or unsubstituted. z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, A structurally stabilized peptide binds to the RSV5 helix bundle protein, and / or the structurally stabilized peptide inhibits RSV-induced cell infection and / or prevents RSV-induced cell infection. Structurally stabilized peptides are 25-30 amino acid long, and optionally, structurally stabilized peptides are 29 amino acid long. In some examples, each [Xaa] x [Xaa] is identified in Table 2. xor a variant thereof having one amino acid substitution. In some examples, the amino acid sequence contains 25 to 30 (e.g., 25, 26, 27, 28, 29, 30) consecutive amino acids of the sequence described in SEQ ID NO: 100, having 2 to 5 (e.g., 2, 3, 4, 5) amino acid substitutions compared to the sequence described in SEQ ID NO: 100. In some examples, the amino acid sequence contains 25 to 30 (e.g., 25, 26, 27, 28, 29, 30) consecutive amino acids of the sequence described in SEQ ID NO: 100, having 2 to 4 (e.g., 2, 3, 4) amino acid substitutions compared to the sequence described in SEQ ID NO: 100. In some examples, the amino acid sequence contains 25 to 30 (e.g., 25, 26, 27, 28, 29, 30) consecutive amino acids of the sequence described in SEQ ID NO: 100, having 2 or 3 amino acid substitutions compared to the sequence described in SEQ ID NO: 100. In some cases, the amino acid sequence contains 25 to 30 consecutive amino acids (e.g., 25, 26, 27, 28, 29, 30) of the sequence described in SEQ ID NO: 100, which has two amino acid substitutions. In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAGKST (SEQ ID NO: 258). In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAGKS (SEQ ID NO: 259). In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAGK (SEQ ID NO: 260). In some cases, the structurally stabilized peptide does not contain the amino acid sequence NAG. In some cases, the structurally stabilized peptide does not contain the amino acid sequence NA. In some cases, the structurally stabilized 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 4)). In some examples, the structurally stabilized peptides described above possess one or more of the following properties (e.g., 1, 2, 3, 4, or 5):(i) being alpha-helix, (ii) being protease-resistant, (iii) binding to the RSV5 helix bundle protein, (iv) inhibiting RSV-mediated cell infection, and / or (v) preventing RSV-mediated cell infection. In some cases, the peptide inhibits RSV-mediated cell infection and / or prevents RSV-mediated cell infection in pseudovirus and / or live RSV virus assays.

[0090] In certain cases, substitutions to the consecutive amino acid sequence of SEQ ID NO: 100 of formula I (other than substitutions to introduce the linking group R3) are conserved. In certain cases, substitutions to the consecutive amino acid sequence of SEQ ID NO: 100 of formula I (other than substitutions to introduce the linking group R3) are non-conserved. Methods for determining the type of substitution are described herein; see, for example, the section on "RSV peptides" above. In some cases, structurally stabilized peptides have an amino acid length of 25–45 (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 cases, structurally stabilized peptides have an amino acid length of 25–40 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40). In some cases, the structurally stabilized peptides have an amino acid length of 25-35 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). In some cases, the structurally stabilized peptides have an amino acid length of 25-30 (e.g., 25, 26, 27, 28, 29, 30). In some cases, the structurally stabilized peptides have an amino acid length of 30. In some cases, the above structurally stabilized peptides have one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) being an alpha-helix, (ii) being protease-resistant, (iii) binding to the RSV5 helix bundle protein, (iv) inhibiting RSV-mediated cell infection and / or (v) preventing RSV-mediated cell infection. In some cases, the peptide inhibits RSV infection of cells and / or prevents RSV infection of cells in pseudovirus and / or live RSV virus assays.

[0091] In some examples of equation (I), Each R1 and R2 independently corresponds to H or C1-C 10Alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, R3 is alkyl, alkenyl, or alkynyl; [R4-K-R4] n And each of these is replaced by 0 to 6 R5s. R4 is an alkyl, alkenyl, or alkynyl, R5 is a halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO2R6, R6, fluorescent moiety, or radioactive isotope. K is either O, S, SO, SO2, CO, CO2, CONR6, or

[0092] [ka] R6 is H, alkyl, or a therapeutic agent. n is an integer between 1 and 4. x is either 3 or 6, Each y is an independent integer between 0 and 100. z is an integer between 1 and 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), Each Xaa is an amino acid independently.

[0093] In some examples, [Xaa] in equation (I) w [Xaa] of equation (I) x , and [Xaa] of equation (I) y Each of these is as described for one of the structures 1 through 22 in Table 2. For example, [Xaa] for structure 3 in Table 2. w [Xaa] x , and [Xaa] y For structurally stabilized peptides containing [Xaa] w [Xaa] x , and [Xaa] y These are as follows: FD, SISQVN (sequence number 220), and KINQSLAFIRKSDELLHNV (sequence number 242), respectively.

[0094] [Table 2]

[0095] In some examples, the structurally stabilized peptide includes or consists of construct 3 in Table 2. In some examples, the structurally stabilized peptide includes or consists of construct 17 in Table 2. In some examples, the structurally stabilized peptide includes or consists of one of constructs 1 to 22 in Table 2, with the exception of at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid substitutions or deletions (e.g., a total of 2 to 10 substitutions or deletions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) compared to the sequence of any one of constructs 1 to 22).

[0096] In certain examples, the sequences described above in Table 2 may have at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid substitutions or deletions (e.g., a total of 2 to 10 substitutions or deletions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, compared to the sequence of SEQ ID NO: 100)). The RSV-F HR2 peptide may contain any amino acid sequence described herein.

[0097] In some examples, structurally stabilized peptides of formula I contain sequences of the constructs listed in Table 2 above and possess one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) being alpha-helix, (ii) being protease-resistant, (iii) binding to the RSV5 helix bundle protein, (iv) inhibiting RSV-mediated cell infection, and / or (v) preventing RSV-mediated cell infection. In some examples, structurally stabilized peptides inhibit RSV-mediated cell infection in pseudoviral and / or live RSV virus assays, and / or prevent RSV-mediated cell infection in pseudoviral and / or live RSV virus assays.

[0098] The tether of formula (I) is an alkyl, alkenyl, or alkynyl moiety (e.g., C5, C8, C 11 , or C 12 Alkyl, C5, C8, or C 11 Alkenyl, or C5, C8, C 11 , or C 12 It may contain alkynyl amino acids. The tether amino acid may be alpha-disubstituted (e.g., C1-C3 or methyl).

[0099] In some examples of formula (I), each y is independently an integer between 0 and 15 or between 3 and 15. In some examples of formula (I), R1 and R2 are independently H or C1-C6 alkyl. In some examples of formula (I), R1 and R2 are independently C1-C3 alkyl. In some examples of formula (I), at least one of R1 and R2 is methyl. For example, both R1 and R2 may be methyl. In some examples of formula (I), R3 is C 11 It is alkyl, and x is 6. In some examples of formula (I), x is 6, and R3 is C 11 It is an alkenyl. In some examples, R3 is a linear alkyl, alkenyl, or alkynyl. In some examples, R3 is -CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-.

[0100] In another aspect of equation (I), both alpha,alpha disubstitution stereocenters are either in R configuration or S configuration (e.g., i, i+4 bridge), or one stereocenter is R and the other is S (e.g., i, i+7 bridge). Thus, equation (I) can be written as follows:

[0101] [ka] The disubstituted centers of C' and C'' can both be in the R configuration, or they can both be in the S configuration. In equation (I), when x is 6, the C' disubstituted center is in the R configuration and the C'' disubstituted center is in the S configuration. The R3 double bond in equation (I) can have an E or Z stereochemical configuration.

[0102] In some examples of equation (I), R3 is [R4-K-R4] n R4 is a linear alkyl, alkenyl, or alkynyl compound.

[0103] As used herein, the term “alkyl,” used alone or in combination with other terms, refers to a saturated hydrocarbon group that may be linear or branched. In some examples, alkyl groups contain 1-7, 1-6, 1-4, or 1-3 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and n-heptyl. In some examples, the alkyl group is methyl, ethyl, or propyl. The term “alkylene” refers to a linked alkyl group.

[0104] As used herein, "alkenyl," either alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon double bonds. In some examples, the alkenyl moiety contains 2 to 6 or 2 to 4 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, and sec-butenyl.

[0105] As used herein, "alkynyl," either alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propyne-1-yl, propyne-2-yl, and others. In some examples, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.

[0106] As used herein, "alkynyl," either alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propyne-1-yl, propyne-2-yl, and others. In some examples, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.

[0107] As used herein, the term “cycloalkylalkyl,” used alone or in combination with other terms, refers to a group of the formula cycloalkyl-alkyl-. In some examples, the alkyl moiety has 1-4, 1-3, 1-2, or 1 carbon atom. In some examples, the alkyl moiety is methylene. In some examples, the cycloalkyl moiety has 3-10 ring members or 3-7 ring members. In some examples, the cycloalkyl group is monocyclic or bicyclic. In some examples, the cycloalkyl moiety is monocyclic. In some examples, the cycloalkyl moiety is C 3-7 It is a monocyclic cycloalkyl group.

[0108] As used herein, the term “heteroarylalkyl,” used alone or in combination with other terms, refers to a group of the formula heteroaryl-alkyl-. In some examples, the alkyl moiety has 1-4, 1-3, 1-2, or 1 carbon atom. In some examples, the alkyl moiety is methylene. In some examples, 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 examples, the heteroaryl moiety has 5-10 carbon atoms.

[0109] As used herein, the term “substituted” means that a hydrogen atom is replaced by a non-hydrogen group. It should be understood that substitutions in a given atom are limited by their valence.

[0110] As used herein, “halo” or “halogen,” used alone or in combination with other terms, includes fluoro, chloro, bromo, and iodine. In some examples, the halo is F or Cl.

[0111] While hydrocarbon tethers are provided herein, other tethers may also be used in the structurally stabilized RSV-F HR2 peptides described herein. For example, a tether may contain one or more of the following: ethers, thioethers, esters, amines, or amides, or triazole moieties. In some cases, naturally occurring amino acid side chains may be incorporated into the tether. For example, a tether can be coupled with functional groups such as hydroxyl in serine, thiol in cysteine, primary amine in lysine, acid in aspartic acid or glutamic acid, or amide in asparagine or glutamine. Thus, it is possible to prepare tethers using natural amino acids rather than using tethers made by coupling two unnatural amino acids. It is also possible to use a single amino acid that does not exist naturally together with naturally occurring amino acids. Triazole-containing (e.g., 1,4-triazole or 1,5-triazole) crosslinks can be used (see, for example, Kawamoto et al. 2012 Journal of Medicinal Chemistry 55:1137, International Publication No. 2010 / 060112).In addition, other methods for performing different types of stapling are well known in the art and can be used in conjunction with the RSV-F HR2 peptide described herein (e.g., lactam stapling: Shepherd et al., J. Am. Chem. Soc., 127:2974-2983 (2005), UV-cycloaddition stapling: Madden et al., Bioorg. Med. Chem. Lett., 21:1472-1475 (2011), disulfide stapling: Jackson et al., Am. Chem. Soc., 113:9391-9392 (1991), ox oxime stapling: Haney et al., Chem. Commun., 47:10915-10917 (2011), thioether stapling: Brunel and See Dawson, Chem.Commun., 552-2554 (2005), optically switchable stapling: JRKumita et al., Proc.Natl.Acad.Sci.USA, 97:3803-3808 (2000), double-click stapling: Lau et al., Chem.Sci., 5:1804-1809 (2014), bislactam stapling: JCPhelan et al., J.Am.Chem.Soc., 119:455-460 (1997), and bisarylated stapling: AMSpokoyny et al., J.Am.Chem.Soc., 135:5946-5949 (2013).

[0112] It is further assumed that the length of the tether may vary. For example, a shorter tether may be used when it is desirable to provide a relatively high degree of constraint on the secondary alpha-helix structure, but in some examples, it is desirable to provide less constraint on the secondary alpha-helix structure, and therefore a longer tether may be desired.

[0113] Furthermore, while tethers extending from amino acids i to i+7 are provided herein primarily to provide tethers on a single face of an alpha helix, tethers may be synthesized to extend to any combination of the number of amino acids and may be used in combination to position multiple tethers.

[0114] In some cases, the hydrocarbon tethers (i.e., crosslinks) described herein can be further manipulated. In one example, the double bond of a hydrocarbon alkenyl tether (e.g., one synthesized using ruthenium-catalyzed ring-closing metathesis (RCM)) can be oxidized (e.g., via epoxidation, aminohydroxylation, or dihydroxylation) to provide one of the following compounds.

[0115] [ka]

[0116] Either the epoxide moiety or the free hydroxyl moiety can be further functionalized. For example, the epoxide may be treated with a nucleophile, which provides additional functional groups that can be used, for example, to bind therapeutic agents. Such derivatization can alternatively be achieved by synthetic operations of the amino or carboxyl terminus of the peptide, or via amino acid side chains. Other agents, such as agents that promote the entry of peptides into cells, can be bound to the functionalized tether.

[0117] In some cases, alpha-disubstituted amino acids are used in peptides to improve the stability of the alpha-helix secondary structure. However, there are also cases where alpha-disubstituted amino acids are not necessary, and mono-alpha substitutions (e.g., in tethered amino acids) are used instead.

[0118] Structurally stabilized (e.g., stapled) peptides may include drugs, toxins, polyethylene glycol derivatives, secondary peptides, carbohydrates, etc. When polymers or other agents are linked to structurally stabilized (e.g., stapled) peptides, it may be desirable for the composition to be substantially homogeneous.

[0119] Structurally stabilized (e.g., stapled) peptides can also be modified in some cases to further enhance mucosal adhesion, membrane binding, or increase in vivo stability. For example, acylation or PEGylation of structurally stabilized peptides can increase bioavailability, increase blood circulation, alter pharmacokinetics, change immunogenicity, and / or reduce the required administration frequency.

[0120] In some cases, structurally stabilized (e.g., stapled) peptides disclosed herein have enhanced ability to bind to or enter cell membranes (e.g., compared to unstabilized peptides). See, for example, International Publication 2017 / 147283, which is incorporated herein in its entirety by reference.

[0121] In some examples, the structurally stabilized peptide is the peptide described in the drawings or examples.

[0122] In some cases, the structurally stabilized peptides described herein (e.g., in Table 1) form part of the structurally stabilized peptide conjugates described in the following section, “Structurally Stabilized Peptide Conjugates.”

[0123] Structurally stabilized peptide conjugates Furthermore, this specification provides a conjugate comprising a structurally stabilized peptide as described herein (see the section “Structurally Stabilized Peptides” above, e.g., the peptides in Table 1 or the constructs in Table 2, or their variants) and cholesterol (or a cholesterol variant, e.g., thiocholesterol), wherein the cholesterol (or cholesterol variant, e.g., thiocholesterol) is linked to the C-terminal amino acid of the structurally stabilized peptide 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 terms “conjugate” and “structurally stabilized peptide conjugate” are interchangeable herein. In some examples, the conjugate contains a structurally stabilized peptide and cholesterol, with the cholesterol 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). In some examples, the conjugate contains a structurally stabilized peptide and thiocholesterol, with the thiocholesterol 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 has one or more of the following properties (e.g., 1, 2, 3, 4, 5). (i) is an alpha-helix, (ii) is protease-resistant, (iii) binds to the RSV5 helix bundle protein, (iv) inhibits RSV-mediated cell infection, and / or (v) prevents RSV-mediated cell infection. In some examples of conjugates, structurally stabilized peptides are conjugated to thiocholesterol. In some examples of conjugates, structurally stabilized peptides are conjugated to thiocholesterol via a linker containing 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, structurally stabilized peptides are conjugated to cholesterol. In some examples of conjugates, structurally stabilized peptides are conjugated to cholesterol via a linker containing 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 stabilized peptide in the conjugate is a structurally stabilized version of the peptide described in the “RSV Peptides” section above. In some examples, the structurally stabilized peptide in the conjugate is a structurally stabilized peptide described in the “Structurally Stabilized Peptides” section above. In some examples, the structurally stabilized peptide in the conjugate is a structurally stabilized peptide described in the examples or drawings herein.

[0124] In some examples, the structurally stabilized peptide of the conjugate contains an internally cross-linked amino acid sequence containing 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 100), with the exception of 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 100. The internally cross-linked amino acid sequence does not contain the sequence NAGKST (SEQ ID NO: 258), and two of the 2 to 6 amino acid substitutions are with α,α-disubstituted unnatural amino acids having olefin side chains that are cross-linked with each other. α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are separated by 3 or 6 amino acids, and the conjugate binds to the RSV5 helix bundle protein and / or prevents cell infection by the RSV5 helix bundle protein and / or RSV, and the conjugate has an amino acid length of 25-45 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45).

[0125] In some examples, the structurally stabilized peptide of the conjugate contains or consists of the amino acid sequences in Table 1 (e.g., any one of SEQ ID NOs. 27-70). In some examples, the structurally stabilized peptide of the conjugate contains or consists of the amino acid sequences of any one of SEQ ID NOs. 27, 29, 36, 43, and 46, or any one of SEQ ID NOs. 49, 51, 58, 65, and 68. In some examples, the structurally stabilized peptide of the conjugate contains or consists of the amino acid sequence of SEQ ID NO. 29. In some examples, the structurally stabilized peptide of the conjugate contains or consists of the amino acid sequence of SEQ ID NO. 51. In some examples, the structurally stabilized peptide of the conjugate contains or consists of the amino acid sequence of SEQ ID NO. 43. In some examples, the structurally stabilized peptide of the conjugate contains or consists of the amino acid sequence of SEQ ID NO. 65. In some examples, the structurally stabilized peptide of the conjugate contains or consists of any one of the constructs in Table 2. In some examples, the structurally stabilized peptide of the conjugate contains or consists of one of the constructs 1, 3, 10, 17, or 20 in Table 2. In some examples, the structurally stabilized peptide of the conjugate contains or consists of construct 3 in Table 2. In some examples, the structurally stabilized peptide of the conjugate contains or consists of construct 17 in Table 2. In some examples, the structurally stabilized peptide of the conjugate contains or consists of one of the amino acid sequences of the peptides in Table 1, except for 1-10, 1-5, 1-3, 2, or one amino acid substitution, insertion, and / or deletion (except those at the staple position). In some examples, the structurally stabilized peptide of the conjugate contains or consists of one of the constructs in Table 2, except for 1-10, 1-5, 1-3, 2, or one amino acid substitution, insertion, and / or deletion (except those at the staple position).

[0126] The addition of PEG molecules can improve the pharmacokinetic and pharmacodynamic properties of structurally stabilized peptides. For example, PEGylation can reduce renal clearance and lead to more stable plasma concentrations. PEG is a water-soluble polymer, and when linked to a peptide, its formula is: XO--(CH2CH2O) n It can be expressed as --CH2CH2--Y, where n is between 2 and 10,000, and X is H or a terminal modification, for example, C. 1-4 The molecule is alkyl, and Y is an amide, carbamate, or urea linkage to a structurally stabilized peptide amine group (including, but not limited to, the epsilonamine or N-terminus of lysine). Y may also be a maleimide linkage to a thiol group (including, but not limited to, the thiol group of cysteine). Other methods for 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.

[0127] As used herein, PEG functions in some examples as a linker or spacer between one of the peptides (e.g., the structurally stabilized peptides in Table 1 or the constructs in Table 2) and the cholesterol or thiocholesterol moiety.

[0128] In some cases, the PEG molecule contains a cholesterol moiety. In some cases, the cholesterol moiety is thiocholesterol. In some cases, the sulfur in the thioether moiety in thiocholesterol is replaced by an oxygen atom to produce the ether moiety in cholesterol derivatization.

[0129] In some examples, the PEG molecule contains the following formula (Formula II), where n=1 to 36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36).

[0130] [ka]

[0131] In some examples, the PEG molecule contains the following formula (Formula III), where n=1 to 36 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36).

[0132] [ka]

[0133] In some examples for each of equations II and III, n=16. In some examples for each of equations II and III, n=17. In some examples for each of equations II and III, n=18. In some examples for each of equations II and III, n=19. In some examples for each of equations II and III, n=20.

[0134] PEG having a degradable bond in its backbone can be used. For example, PEG can be prepared using an ester bond that is subject to hydrolysis. Degradable PEG bonds are described in International Publication Nos. 99 / 34833, 99 / 14259, and U.S. Patent No. 6,348,558, each of which is incorporated herein by reference in whole.

[0135] In certain examples, a high molecular weight polymer (e.g., PEG) is linked to a structurally stabilized (e.g., stapled) peptide as described herein via an intermediate linker. In certain cases, the linker consists of 1 to 20 amino acids linked by peptide bonds, selected from 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those skilled in the art. In other examples, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other examples, the linker consists mostly of sterically unhindered amino acids such as glycine and alanine. Non-peptide linkers are also possible, e.g., -NH(CH2) n Alkyl linkers such as C(O)- can be used, where n=2 to 20. These alkyl linkers may be further substituted with any non-sterically hindered group such as lower alkyl (e.g., C1 to C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, or phenyl. U.S. Patent No. 5,446,090 describes the use of a bifunctional PEG linker and its use in forming a conjugate having a peptide at each of the PEG linker ends.

[0136] Exemplary structurally stabilized peptide conjugates are provided in Table 3 below. This disclosure also encompasses the structurally stabilized peptide conjugates in Table 3 further comprising three N-terminal amino acids X1X2X3, where X1 is any amino acid, optionally S, and X2 and X3 are any loaded amino acids immediately upstream of the first amino acid in the sequences listed in Table 3. In some cases, X1X2X3 = SDE. In some examples, the structurally stabilized peptide conjugates contain or consist of the amino acid sequence described in any one of SEQ ID NOs. 71-92. In some examples, the structurally stabilized peptide conjugates contain or consist of the amino acid sequence described in any one of SEQ ID NOs. 6-26. In some examples, the structurally stabilized peptide conjugates contain or consist of the sequence described in SEQ ID NOs. 5 or SEQ ID NOs. 731. In some examples, the structurally stabilized peptide conjugates contain or consist of the sequence described in SEQ ID NOs. 21 or SEQ ID NOs. 871. In some cases, a structurally stabilized peptide conjugate contains or consists of the amino acid sequence described in any one of SEQ ID NOs: 71-92, except for 1-10, 1-5, 1-3, 2, or 1 amino acid substitution, insertion, and / or deletion (excluding the staple and conjugation locations). In some cases, a structurally stabilized peptide conjugate contains or consists of the amino acid sequence described in any one of SEQ ID NOs: 6-26, except for 1-10, 1-5, 1-3, 2, or 1 amino acid substitution, insertion, and / or deletion (excluding the staple and conjugation locations). In some cases, a structurally stabilized peptide conjugate has one or more of the following properties (e.g., 1, 2, 3, 4, 5): (i) is an alpha-helix, (ii) is protease-resistant, (iii) binds to the RSV5 helix bundle protein, (iv) inhibits RSV-mediated cell infection, and / or (v) prevents RSV-mediated cell infection.

[0137] [Table 3]

[0138] In Table 3, for sequence numbers 71-92, "X1" = α,α-disubstituted unnatural amino acid having an olefin side chain crosslinked to X2, and "X2" = α,α-disubstituted unnatural amino acid having an olefin side chain crosslinked to X1. * It contains Z-PEG(n)-cholesterol or Z-PEG(n)-thiocholesterol, where n=1 to 36, and Z is a diamino acid (e.g., lysine or ornithine). In Table 3, for SEQ ID NOs. 5 to 26, "8" = (R)-α-(7'-octenyl)alanine and "X" = (S)-α-(4'-pentenyl)alanine. * =Lys(epsilon-(PEG)4-thiocholesterol). In some examples of sequence numbers 71-92 or 5-26, * This is Lys(epsilon-(PEG)4-cholesterol) or Lys(epsilon-(PEG)4-thiocholesterol), and n=1 to 36. In some examples of sequence numbers 71 to 92, * = One of the following equations 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 examples of sequence numbers 71~92, * = One of the following equations II or III, where n=16. In some examples of sequence numbers 71-92, * = One of the following equations II or III, where n=20. * The two equations shown by include the following:

[0139] [ka]

[0140] In some examples of sequence numbers 71-92, *= Equation II, where n = 1 to 36 (for example, n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In some examples of sequence numbers 71 to 92, * = Equation II, where n=16. In some examples of sequence numbers 71-92, * = Equation II, where n=20. In some examples of sequence numbers 71-92, * = Equation III, where n = 1 to 36 (for example, n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In some examples of sequence numbers 71 to 92, * = Equation III, where n=16. In some examples of sequence numbers 71-92, * = Equation III, where n=20. In some examples of sequence numbers 5-26, * = One of the following equations 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 examples of sequence numbers 5~26, * = One of the following equations IV or V, where n=16. In some examples of sequence numbers 5-26, * = One of the following equations IV or V, where n=20. * The two equations shown by include the following:

[0141] [ka]

[0142] In some examples of sequence numbers 5-26, *= Equation IV, where n=1~36 (for example, n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In some examples of sequence numbers 5~26, * = Equation IV, where n=16. In some examples of sequence numbers 5-26, * = Equation IV, where n=20. In some examples of sequence numbers 5-26, * = Equation V, where n = 1 to 36 (for example, n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36). In some examples of sequence numbers 5 to 26, * = Equation V, where n=16. In some examples of sequence numbers 5-26, *= Equation V, where n=20. It should be understood that the above conjugate can be modified to include additional amino acids at the N and / or C-terminus (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids added) and / or to have N and / or C-terminal deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids deleted). In some examples, the conjugate is derived from SEQ ID NO: 100 (e.g., includes or consists of the amino acid sequence of SEQ ID NO: 100 or its internally crosslinked version or variant). In some examples, the conjugate does not include the amino acid sequence NAGKST (SEQ ID NO: 258). In some examples, the conjugate does not include the amino acid sequence NAGKS (SEQ ID NO: 259). In some examples, the conjugate does not include the amino acid sequence NAGK (SEQ ID NO: 260). In some examples, the conjugate does not include the amino acid sequence NAG. In some examples, the conjugate does not include the amino acid sequence NA. In some cases, 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 from another strain (see, for example, Figure 4)). In some cases, the conjugate is 25–34 amino acid long (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some cases, the conjugate is 30 amino acid long.

[0143] This disclosure relates to each structurally stabilized peptide conjugate listed in Table 3, as well as 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 for those at the staple, i.e., the positions of the residues in bold in Table 3, and lipidization (i.e., the residues in Table 3) *This includes (excluding those in the position of ). In some examples, the variant has 1 to 10 amino acid substitutions, insertions, and / or deletions, but excluding those in the staple (i.e., the bolded residues in Table 3) and lipidization (i.e., the residues in Table 3). * Excluding those at the position of ). In some examples, the variant has 1 to 5 amino acid substitutions, insertions, and / or deletions, but excluding those at the staple position (i.e., the bolded residues in Table 3) and lipidization (i.e., Table 3 * Excluding those at the position of ). In some examples, the variant has 1 to 3 amino acid substitutions, insertions, and / or deletions, but excluding those at the staple (i.e., the bolded residues in Table 3) and lipidization (i.e., Table 3 * Excluding those at the positions of ). In some examples, variants have 1-10, 10-5, 1-3, 2, or 1 amino acid substitutions, insertions, and / or deletions, but excluding those at the staple (i.e., the bolded residues in Table 3) and lipidization (i.e., Table 3 * Excluding those in the position indicated by ).

[0144] In some cases, the structurally stabilized peptide conjugate has an amino acid length of 25–34, e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. In some cases, the structurally stabilized peptide conjugate has an amino acid length of 25–30 (e.g., 25, 26, 27, 28, 29, or 30). In some cases, the structurally stabilized peptide conjugate has an amino acid length of 30. In some cases, the above structurally stabilized peptide conjugate has one or more (1, 2, 3, 4, or 5) of the following properties: (i) it is an alpha-helix, (ii) it is protease-resistant, (iii) it binds to the RSV5 helix bundle protein, (iv) it inhibits RSV-mediated cell infection and / or (v) it prevents RSV-mediated cell infection.

[0145] In some cases, the conjugate has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to one of the single stapled peptides in Table 3 (the mutations in the sequence are not at the stapled or lipid-forming sites). The mutations are not at the stapled sites (i.e., the bolded residues in Table 3) but also at the lipid-forming sites (i.e., the residues in Table 3). * It is understood that they are not even in the same category. In some cases, the conjugate is 100% identical to the conjugate in Table 3. In some cases, the conjugate has an amino acid length of 25-34 (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34). In some cases, the conjugate has an amino acid length of 25-30 (e.g., 25, 26, 27, 28, 29, or 30). In some cases, the conjugate has an amino acid length of 30. In some cases, the above conjugate has one or more (1, 2, 3, 4, or 5) of the following properties: (i) it is an alpha-helix, (ii) it is protease-resistant, (iii) it binds to the RSV5 helix bundle protein, (iv) it inhibits RSV-mediated cell infection and / or (v) it prevents RSV-mediated cell infection.

[0146] In some examples, any substitution described herein is a conservative substitution. In some examples, any substitution described herein is a non-conservative substitution.

[0147] Pharmaceutical composition One or more structurally stabilized (e.g., stapled) peptides or structurally stabilized (e.g., stapled) peptide conjugates described herein can be formulated as a pharmaceutical composition or for use in a pharmaceutical composition. The pharmaceutical composition may be used in a method of treatment or prevention described herein. In some examples, the pharmaceutical composition comprises a structurally stabilized peptide described herein and a pharmaceutically acceptable carrier. In some examples, the pharmaceutical composition comprises a structurally stabilized peptide conjugate described herein and a pharmaceutically acceptable carrier. In certain examples, the pharmaceutical composition comprises a structurally stabilized (e.g., stapled) peptide or structurally stabilized (e.g., stapled) peptide conjugate comprising, or consisting of, an amino acid sequence identical to the amino acid sequence described in Table 1 or Table 3 or the construct described in Table 2. In certain examples, the pharmaceutical composition includes a structurally stabilized (e.g., stapled) peptide or a structurally stabilized (e.g., stapled) peptide conjugate containing an amino acid sequence identical to the amino acid sequences listed in Table 1 or Table 3 or the constructs listed in Table 2, except that the peptide further comprises three N-terminal amino acids X1X2X3, where X1 is any amino acid, optionally S, and X2 and X3 are any load amino acids. In certain examples, the pharmaceutical composition includes a structurally stabilized peptide conjugate containing or comprising one of the amino acid sequences of SEQ ID NOs. 27, 29, 36, 43, and 46, or one of the amino acid sequences of 49, 51, 58, 65, and 68, and a pharmaceutically acceptable carrier. In certain examples, the pharmaceutical composition includes a structurally stabilized peptide containing or comprising the amino acid sequence of SEQ ID NOs. 29 or SEQ ID NOs. 51, and a pharmaceutically acceptable carrier. In certain examples, the pharmaceutical composition comprises a structurally stabilized peptide containing or comprising the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 65, and a pharmaceutically acceptable carrier.In certain cases, the pharmaceutical composition comprises a structurally stabilized peptide containing or comprising the amino acid sequence of SEQ ID NO: 281, and a pharmaceutically acceptable carrier. In certain cases, the pharmaceutical composition comprises a structurally stabilized peptide conjugate containing or comprising one of the amino acid sequences of SEQ ID NOs: 71, 73, 80, 87, and 90, or one of the amino acid sequences of SEQ ID NOs: 5, 6, 14, 21, and 24, and a pharmaceutically acceptable carrier. In certain cases, the pharmaceutical composition comprises a structurally stabilized peptide conjugate containing or comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 73, and a pharmaceutically acceptable carrier. In certain cases, the pharmaceutical composition comprises a structurally stabilized peptide conjugate containing or comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 87, and a pharmaceutically acceptable carrier. In certain examples, a pharmaceutical composition includes an amino acid sequence identical to the amino acid sequences listed in Table 1, Table 2, or Table 3, except for 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or one amino acid substitution, insertion, or deletion, or includes a structurally stabilized (e.g., stapled) peptide or a structurally stabilized (e.g., stapled) peptide conjugate thereof. It will be understood that the amino acid substitution, insertion, or deletion is not at a stapled position (e.g., not at position 1 or 8 of the amino acid sequence of SEQ ID NO: 6). In certain examples, the pharmaceutical composition includes an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 27, 29, 36, 43, 46, 49, 51, 58, 65, 68, 71, 73, 80, 87, 90, 5, 6, 14, 21, and 24, except for 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or one amino acid substitution, insertion, or deletion, or a structurally stabilized (e.g., stapled) peptide or a structurally stabilized (e.g., stapled) peptide conjugate comprising an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 27, 29, 36, 43, 46, 49, 51, 58, 65, 68, 71, 73, 80, 87, 90, 5, 6, 14, 21, and 24, or a structurally stabilized (e.g., stapled) peptide conjugate comprising such an amino acid sequence.These changes to the amino acid sequence can be made on the non-interacting alpha-helix plane of these peptides (i.e., for amino acids that do not interact with the 5-helix bundle of the RSV-F protein) and / or on the interacting alpha-helix plane (i.e., for amino acids that interact with the 5-helix bundle of the RSV-F protein), as long as they are not stapling positions (e.g., positions 1 and 8 of the amino acid sequence of SEQ ID NO: 6). Such compositions can be formulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA's CDER Data Standards Manual, version 004 (available at fda.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, subcutaneous, intraperitoneal, intramuscular, and / or subcutaneous), and / or for oral administration, transmucosal administration, and / or topical administration (including topical (e.g., nasal) sprays, eye drops, and / or solutions).

[0148] In some cases, pharmaceutical compositions are formulated or adapted for administration by nasal spray / drops, aerosols, subcutaneous administration, or intravenous administration. In some cases, pharmaceutical compositions are formulated or adapted for administration by topical means (e.g., nasal spray, aerosol).

[0149] In some examples, a pharmaceutical composition may comprise an effective amount of one or more structurally stabilized (e.g., stapled) peptides or structurally stabilized (e.g., stapled) peptide conjugates. As used herein, “effective amount” and “effective to treat” refer to the amount or concentration of the described agent (e.g., structurally stabilized (e.g., stapled) peptides or structurally stabilized (e.g., stapled) peptide conjugates or the pharmaceutical composition described herein) used over a period of time (including acute or chronic administration and periodic or continuous administration) in which it is effective in relation to its administration for producing the intended effect or physiological outcome (e.g., treatment of an infection).

[0150] The pharmaceutical compositions of this disclosure may comprise one or more structurally stabilized (e.g., stapled) peptides or structurally stabilized (e.g., stapled) peptide conjugates as described herein, and any pharmaceutically acceptable carrier and / or vehicle. In some examples, the pharmaceutical compositions may further comprise one or more additional therapeutic agents in amounts effective to achieve the modulation of a disease or disease symptom.

[0151] The term "pharmaceutically acceptable carrier or adjuvant" means a carrier or adjuvant that, when administered in a dose sufficient to deliver a therapeutic amount of the compound of this disclosure (e.g., a structurally stabilized (e.g., stapled) peptide or a structurally stabilized (e.g., stapled) peptide conjugate), can be administered to a patient or subject from another species provided herein and, when administered in a dose sufficient to deliver a therapeutic amount of the compound, does not destroy its pharmacological activity and is non-toxic.

[0152] In some examples, the pharmaceutical compositions of this disclosure comprise one or more of acetates, citrates, and / or maleates. In some examples, the pharmaceutical compositions may comprise water or phosphate-buffered saline (PBS). In some examples, the pharmaceutical compositions may comprise chitosan.

[0153] The pharmaceutical compositions disclosed herein may comprise one or more pharmaceutically acceptable salts. In some examples, 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.

[0154] The pharmaceutical compositions of this disclosure may contain any conventional non-toxic, pharmaceutically acceptable carrier, adjuvant, or vehicle. In some cases, the pH of the formulation may be adjusted with a pharmaceutically acceptable acid, base, or buffer to enhance the stability of the formulated compound or its delivery form. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-bursal, intrasternal, intrathecal, intrafocal, and intracranial injection or infusion techniques.

[0155] In some examples, one or more structurally stabilized (e.g., stapled) peptides or structurally stabilized (e.g., stapled) peptide conjugates disclosed herein may be further conjugated to a carrier protein, for example. Such conjugate compositions may be monovalent or polyvalent. For example, a conjugate composition may comprise one structurally stabilized (e.g., stapled) peptide conjugate disclosed herein, conjugated to a carrier protein. Alternatively, another example may include a conjugate composition comprising two or more structurally stabilized (e.g., stapled) peptide conjugates disclosed herein, further conjugated to a carrier.

[0156] When two entities are "conjugated" to one another, they are linked by direct or indirect covalent or non-covalent interactions. In certain examples, the association is covalent. In other examples, the association is non-covalent. Non-covalent interactions include hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic interactions, and electrostatic interactions. Indirect covalent interactions occur when two entities are covalently connected via a linker group of their choice.

[0157] The carrier protein may include any protein that increases or enhances the stability, half-life, tissue exposure, and / or immunogenicity in the target. Exemplary carrier proteins have been 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 may be a natural or synthetic material containing one or more primary and / or secondary amino groups, azide groups, or carboxyl groups. The carrier may be water-soluble.

[0158] A method for preparing stapled or stitched peptides derivatized using PEG(n)-thiocholesterol or a PEG(n)-cholesterol moiety. In one embodiment, the present disclosure features a method for producing structurally stable peptides derivatized using PEG(n)-thiocholesterol or a PEG(n)-cholesterol moiety. The complete resin synthesis method includes (a) providing a peptide (e.g., SEQ ID NOs. 27-70) comprising at least two non-natural amino acids having olefin side chains; (b) crosslinking the peptide by a ruthenium-catalyzed metathesis reaction in some examples; and (c) derivatizing the C-terminus on the resin using a variable-length PEG linker attached to the thiocholesterol or cholesterol moiety.

[0159] In some embodiments, the method involves cleaving a structurally stabilized peptide from a resin. Cleaving a structurally stabilized resin is known in the art. In some embodiments, the cleavage occurs before derivatization of the C-terminus on the resin using a variable-length PEG linker attached to a thiocholesterol or cholesterol moiety. See, for example, de Vries et al., Science, 2021 Mar 26;371(6536):1379-138, and Figueira et al., J.Virol.91,e01554-16(2016), each of which is incorporated herein by reference in whole. In other embodiments, the cleavage occurs after the step of derivatization of the C-terminus on the resin using a variable-length PEG linker attached to a thiocholesterol or cholesterol moiety.

[0160] If the cleavage step is performed before the derivatization step, the method includes the use of a compound having one of the following formulas:

[0161] [ka] In the formula, n is 1 to 36. In some examples, n is 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. Compounds having one of the above formulas are also disclosed herein.

[0162] Staple-stapled peptide synthesis: Staple-stapled peptide fusion inhibitors were synthesized using Fmoc-based solid-phase peptide synthesis according to our reported method for producing total hydrocarbon staple-stapled peptides (Bird et al., Curr..Protocol.Chem, Biol., 3(3):99-117(2011; Bird et al., Methods) Enzymol., 446:369-86 (2008). To achieve various staple lengths, α-methyl and α-alkenyl amino acids were positioned in specific pairings at individual positions, such as i, i+7, using one S-pentenylalanine residue (S5) and one R-octenylalanine residue (R8). For the stapling reaction, Grubbs first-generation ruthenium catalyst dissolved in dichloroethane was added to the resin-bound peptide. To ensure maximum conversion, 3–5 rounds of stapling were performed. After adding the PEG(n)-thiocholesterol or PEG(n)-cholesterol moiety (see below), the peptide was then cleaved from the resin using trifluoroacetic acid, precipitated using a hexane:ether (1:1) mixture, air-dried, and purified by LC-MS. All peptides were quantified by amino acid analysis.

[0163] Stitched Peptide Synthesis: Methods for synthesizing stitched peptides as described herein are known in the art. However, the following exemplary methods may be used. The synthetic chemical transformations and protecting group methods (protection and deprotection) useful for synthesizing the compounds described herein are known in the art, for example, Bird et al., ACS Chem Biol. (2020) 15(6):1340-1348, Hilinski et al., J Am Chem Soc. (2014) 136(35):12314-22, R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989), TW Greene and PGMWuts, Protective Groups in Organic Synthesis, 3d. Ed., John Wiley and Sons (1999), L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994), and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and This includes Sons (1995) and subsequent editions thereof.

[0164] C-terminal derivatization of stapled or stitched peptides using PEG(n)-thiocholesterol or PEG(n)-cholesterol with a resin-based synthesis approach: To produce carboxythiocholesterol or carboxycholesterol reagents for peptide derivatization by solid-phase synthesis, thiocholesterol was dissolved in 0.1 M dichloromethane (DCM) or cholesterol in tetrahydrofuran (THF) and added to a round-bottom flask. Three equivalents of base (diisopropylethylamine for thiocholesterol, or sodium hydride or potassium t-butoxide for cholesterol) were added with stirring. Next, five equivalents of t-butyl bromoacetic acid were added, and the reaction mixture was stirred at room temperature for 2 hours, followed by 30 minutes at 40°C. Twice the volume of trifluoroacetic acid (relative to the solvent) was added, and the reaction mixture was stirred at room temperature for 30 minutes. The progress of the reaction was monitored by TLC (19:1 Hex:RINKAN for thiocholesterol, 3:1 Hex:RINKAN for cholesterol) using KMnO4 staining. For example, (thio)cholesterol migrated with the solvent tip containing (thio)ether, slowing the migration by approximately 20%, while TFA hydrolysis caused the spot to move to the baseline. The reaction mixture was added to five times the volume of water and one time the volume of DCM. The solvent layer was washed with 0.1 M HCl brine and dried over sodium sulfate. Removal of the solvent by Rotovap yielded orange heavy oil, which was used without further purification. The yield was almost quantitative. Purity was determined to be greater than 90% by olefin proton vs. new CH2 singlet NMR. For peptide derivatization using thiocholesterol or cholesterol, the completed resin-bound peptide sequence was treated with 20% piperidine / DMF, followed by capping with acetic anhydride to block the N-terminal amine, and then the C-terminal side-chain lysineamine was revealed by five treatments of 2% hydrazine in DMF for 10 minutes each.The amine was acylated with an Fmoc-protected PEG(n) amino acid (e.g., n=1-36 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36)), and the olefin was crosslinked at that point by treating it 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 carboxy-thiocholesterol (or carboxy-cholesterol) for 30 minutes. Crude products of excellent purity were obtained by TFA cleavage, which were further purified using half-portion HPLC.

[0165] The peptide sequences of the present invention can be prepared by chemical synthesis methods well known to those skilled in the art. For example, see Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, WH Freeman & Co., New York, NY, 1992, p. 77. Thus, the peptides can be synthesized, for example, on an Applied Biosystems Peptide Synthesizer Model 430A or 431 using side-chain protected amino acids and automated Merrifield technology for solid-phase synthesis using α-NH2 protected by either t-Boc or Fmoc chemistry.

[0166] One method for preparing the peptides described herein is to use solid-phase peptide synthesis (SPPS). The C-terminal amino acid is bonded to a cross-linked polystyrene resin via an acid-unstable bond with a linker molecule. This resin is insoluble in the solvent used in the synthesis, making it relatively easy and quick to wash away excess reagents and by-products. The N-terminus is protected with an Fmoc group that is stable in acid but removable with a base. All side-chain functional groups are protected with base-stable, acid-unstable groups.

[0167] Longer peptides can be prepared by linking individual synthetic peptides using native chemical ligation. Insertion of linked amino acids can be performed, for example, as described in Young and Schultz, J Biol Chem. 2010 Apr 9;285(15):11039-11044. Alternatively, longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct the gene encoding the peptide of the present invention, the amino acid sequence is back-translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably using codons optimal for the organism in which the gene will be expressed. The synthetic gene is then prepared, typically by synthesizing oligonucleotides encoding the peptide and, if necessary, any regulatory elements. The synthetic gene is inserted into a suitable cloning vector and transfected into host cells. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0168] Peptides can be prepared in a high-throughput combinatorial manner using, for example, high-throughput multi-channel combinatorial synthesizers available from Advanced Chemtech or Gyros Protein Technologies. Peptide bonds can be substituted, for example, retro-inverso bonds (C(O)-NH), reducing amide bonds (NH-CH2), thiomethylene bonds (S-CH2 or CH2-S), oxomethylene bonds (O-CH2 or CH2-O), ethylene bonds (CH2-CH2), thioamide bonds (C(S)-NH), trans-olefin bonds (CH=CH), fluoro-substituted trans-olefin bonds (CF=CH), ketomethylene bonds (C(O)-CHR or CHR-C(O)) where R is H or CH3, and fluoro-ketomethylene bonds (C(O)-CFR or CFR-C(O)) where R is H or F or CH3, in order to increase the physiological stability of the peptide.

[0169] Peptides may be further modified by acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceination, formylation, myristoylation, palmitoylation, and other lipidization, phosphorylation (Ser, Tyr, or Thr), stearoylation, succinylation, and sulfurylation, particularly modification of thiocholesterol or cholesterol using the resin-based methods disclosed herein. As described above, peptides may be conjugated to or contain linker atoms or moieties of varying lengths, e.g., polyethylene glycol (PEG) moieties of varying lengths, alkyl groups (e.g., C1-C20 linear or branched alkyl groups), fatty acid radicals, and combinations thereof. α,α-disubstituted unnatural amino acids containing olefin side chains of various lengths can be synthesized by known methods (Williams et al. J.Am.Chem.Soc., 113:9276, 1991; Schafmeister et al., J.Am.Chem Soc., 122:5891, 2000; and Bird et al., Methods Enzymol., 446:369, 2008; Bird et al., Current Protocols in Chemical Biology, 2011). In some examples, the stitched peptides include linkages between i, i+4 and i+4 and i+8. Such stitched peptides can be prepared in association with SEQ ID NO: 100. In some examples, the amino acids that form the staple or stitch are (R)-2-(4'-pentenyl)alanine, 2,2-bis(4-pentenyl)glycine, and (S)-2-(4'-pentenyl)alanine at positions i, i+4, and i+8 of the stitch, respectively. In some examples of peptides where an i-linked, i+7 and i+7 linked stitch is used (four turns of the helix are stabilized), one R-octenylalanine (e.g., (R-α-(7'-octenyl)alanine)), one bis-pentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine) and one R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine) are used.In some examples of peptides where an i+7 stitch linked to i+7 and an i+7 stitch linked to i+14 are used (which stabilizes four turns of the helix), one S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine), one bis-pentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine), and one R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine) are used. In some examples of peptides where an i-linked i at i+7 and an i+7-linked i+14 stitch is used (which stabilizes four turns of the helix), one S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine), one bis-pentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine), and one S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine) are used. In some examples of peptides where i-linked to i+7 and i+7-linked to i+14 stitches are used (which stabilizes four turns of the helix), one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7'-octenyl)glycine), and one S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine) are used. In some examples of peptides where an i-linked to i+7 and an i+7-linked to i+14 stitch is used (which stabilizes four turns of the helix), one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7'-octenyl)glycine), and one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine) are used. In some examples of peptides where i-linked to i+7 and i+7-linked to i+14 stitches are used (which stabilizes four turns of the helix), one S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7'-octenyl)glycine), and one R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine) are used.In some examples of peptides using i+7 linked i and i+14 linked i+7 stitches (which stabilize four turns of the helix), one S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7'-octenyl)glycine), and one S-pentenylalanine (e.g., (S)-α-(4'-pentenyl)alanine) are used. R-octenylalanine is synthesized using the same route, except that the starting chiral auxiliary gives the R-alkyl stereoisomer. Also, 8-iodooctene is used instead of 5-iodopentene. Inhibitors are synthesized on solid supports using solid-phase peptide synthesis (SPPS) on MBHA resin or Rink Amide AM resin (see, for example, International Publication 2010 / 148335).

[0170] 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-tetramethylaminium hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially available, for example, from Novabiochem (San Diego, CA). Dimethylformamide (DMF), N-methyl-2-pyrrolidinone (NMP), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), fluorescein isothiocyanate (FITC), and piperidine are commercially available, for example, from Sigma-Aldrich. The synthesis of olefinic amino acids has been reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0171] Again, preferred methods for obtaining (e.g., synthesizing), stitching, and purifying the peptides disclosed herein are also known in the art (see, for example, Bird et al., Methods in Enzymol., 446:369-386 (2008), Bird et al., Current Protocols in Chemical Biology, 2011, Walensky et al., Science, 305:1466-1470 (2004), Schafmeister et al., J.Am.Chem.Soc., 122:5891-5892 (2000), U.S. Patent Application Publication No. 12 / 525,123 filed March 18, 2010, and U.S. Patent No. 7,723,468 issued May 25, 2010, each of which is incorporated herein by reference in whole).

[0172] In some examples, the peptides are substantially free of or isolated from unstitched or unstapled peptide contaminants. Methods for purifying the peptides include, for example, synthesizing the peptides on a solid support. After cyclization, several alternative solvents and purification schemes for the isolation and purification of the peptides and stapled peptides are known in the art and may use solvents including, but not limited to, DMSO, DMSO / dichloromethane mixtures, DMSO / NMP mixtures, or mixtures / solutions that do not contain DMSO. DMSO / dichloromethane or DMSO / NMP mixtures may contain about 30%, 40%, 50%, or 60% DMSO. In certain examples, a 50% / 50% DMSO / NMP solution is used. The solution may be incubated for 1, 6, 12, or 24 hours, after which the resin may be washed with, for example, dichloromethane or NMP. In one example, the resin is washed with NMP. Shaking may be performed, or an inert gas may be bubbling into the solution.

[0173] The properties of stitched or stapled peptides derivatized with C-terminal PEG(n)-thiocholesterol or PEG(n)-cholesterol of the present disclosure can be assayed, for example, using the methods described below and in the examples.

[0174] Assay for determining the characteristics and anti-RSV activity of staple HR2 peptides derivatized using PEG(n)-thiocholesterol or PEG(n)-cholesterol moiety Assay for determining α-helicity: Dissolve the compound in an aqueous solution (e.g., 5 μM potassium phosphate solution at pH 7, or distilled H2O, up to a concentration of 25–50 μM). Circular dichroism (CD) spectra are obtained using a spectropolarimeter (e.g., Jasco J-710, Aviv) with standard measurement parameters (e.g., temperature, 20°C; wavelength, 190–260 nm; step resolution, 0.5 nm; velocity, 20 nm / sec; accumulation, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helix content of each peptide is calculated by dividing the average residue ellipticity by the value reported for the model helix decapeptide (Yang et al., Methods Enzymol., 1986).

[0175] Assay for determining the melting temperature (Tm): The crosslinked or unmodified template peptide is dissolved in distilled H2O or another buffer or solvent (e.g., to a final concentration of 50 μM). Tm is determined by measuring the change in ellipticity over a temperature range (e.g., 4–95°C) using a spectropolarimeter (e.g., Jasco J-710, Aviv) with standard parameters (e.g., wavelength 222 nm; step resolution 0.5 nm; velocity 20 nm / sec; accumulation 10; response 1 sec; bandwidth 1 nm; temperature rise rate: 1°C / min; path length 0.1 cm).

[0176] In vitro protease resistance assay: The amide bonds in the peptide backbone are susceptible to hydrolysis by proteases, thereby making peptide compounds vulnerable to rapid degradation in vivo. However, peptide helix formation typically fills and / or twists and / or shields the amide backbone, thus preventing or substantially delaying proteolytic cleavage. The peptide-mimicking macrocyclic molecules of the present invention can be subjected to in vitro enzymatic proteolysis (e.g., trypsin, chymotrypsin, pepsin) to evaluate any changes in degradation rate compared to the corresponding uncrosslinked or alternatively stapled polypeptides. For example, the peptide-mimicking macrocyclic molecules and the corresponding uncrosslinked polypeptides are incubated with trypsin agarose, and the reaction is quenched at various time points by centrifugation and subsequent HPLC injection, with residual substrate quantified by UV absorption at 280 nm. Briefly, a peptide-mimicking macrocyclic molecule and a peptide-mimicking precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S / E approximately 125) for 0, 10, 20, 90, and 180 minutes. The reaction is quenched by high-speed benchtop centrifugation, and the residual substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction exhibits first-order kinetics, and the rate constant k is determined from the ln[S] versus time plot.

[0177] Peptide-mimicking macrocyclic molecules and / or corresponding non-crosslinked polypeptides can be incubated with fresh mouse, rat, and / or human serum (e.g., 1-2 mL) at 37°C for, for, e.g., 0, 1, 2, 4, 8, and 24 hours, respectively. Samples of different macrocyclic molecule concentrations can be prepared by serial dilution in serum. To determine the level of undamaged compounds, the following procedure can be used: The sample is extracted, for example, by transferring 100 μL of serum to a 2 ml centrifuge tube, followed by the addition of 10 μL of 50% formic acid and 500 μL of acetonitrile, and centrifugation at 14,000 RPM for 10 minutes at 4+ / -2°C. The supernatant is then transferred to a new 2 ml tube and evaporated on a Turbovap under N2, less than 10 psi, at 37°C. The sample is reconstituted with 100 μL of 50:50 acetonitrile:water and submitted for LC-MS / MS analysis. Equivalent or similar procedures for testing ex vivo stability are known and can be used to determine the stability of macrocyclic molecules in serum.

[0178] Plasma Stability Assay: The stability of stapled peptides can be tested in freshly collected mouse plasma gathered in lithium heparin tubes. Three incubations are set up using 500 μl of plasma supplemented with 10 μM individual peptides. The samples are gently shaken in an orbital shaker at 37°C, and 25 μl aliquots are taken at 0, 5, 15, 30, 60, 240, 360, and 480 minutes, and added to 100 μl of a mixture containing 10% methanol, 10% water, and 80% acetonitrile to stop further degradation of the peptides. The samples are placed on ice during the assay and then transferred to a MultiScreen Solvinert 0.45 μm low-binding hydrophilic PTFE plate (Millipore). The filtrate is analyzed directly by LC-MS / MS. Peptides are detected as bi- or tri-charged ions using a Sciex 5500 mass spectrometer. The percentage of residual peptide is determined by the decrease in chromatographic peak area, and the half-life is calculated by logarithmically transforming the result.

[0179] In vivo protease resistance assay: A key advantage of peptide stapling is the conversion of in vitro protease resistance to significantly improved in vivo pharmacokinetics. Analytical assays based on liquid chromatography / mass spectrometry are used to detect and quantify the levels of stapled peptides in plasma. For pharmacokinetic analysis, the peptides are dissolved in sterile aqueous 5% dextrose (1 mg / mL) and administered to C57BL / 6 mice (Jackson Laboratory) by bolus tail vein or intraperitoneal injection (e.g., 5, 10, 25, 50 mg / kg). Blood is collected by retroorbital puncture 5, 30, 60, 120, and 240 minutes after administration to 5 animals at each time point. Plasma is collected after centrifugation (2,500 × g, 5 min, 4°C) and stored at -70°C until assay. Peptide concentrations in plasma are determined by reversed-phase high-performance liquid chromatography using electrospray ionization mass spectrometry detection (Aristoteli et al., Journal of Proteome Res., 2007; Walden et al., Analytical and Bioanalytical Chem., 2004). Study samples are assayed with a set of seven calibration standards of peptides in plasma at concentrations ranging from 1.0 to 50.0 μg / mL, drug-free plasma assayed with and without the addition of an internal standard, and three quality control samples (e.g., 3.75, 15.0, and 45.0 μg / mL). Calibration curves are constructed by plotting the analyte / internal standard chromatographic peak area ratio against known drug concentrations in each calibration standard. Linear least-squares regression is performed using weighting proportional to the reciprocal of the analyte concentration normalized to the number of calibration standards. The drug concentration in the study samples is calculated using the slope and y-intercept values ​​of the best-fit curve. Plasma concentration-time curves are analyzed using WinNonlin Professional 5.0 software (Pharsight Corp., Cary, NC) with a standard non-compartmental method to obtain pharmacokinetic parameters such as early and late plasma half-lives, peak plasma levels, total plasma clearance, and apparent volume of distribution.

[0180] The persistence of the stapled peptides of the present invention in the nasal mucosa after topical administration (i.e., nasal spray) and in the respiratory mucosa after intranasal application or spray is tested under conditions of pre- and post-infection blockage of viral fusion and dissemination. Mice are exposed to a single treatment with a nasal spray or sprayer at a series of intervals preceding intranasal infection with RSV, and the relative mucosal stability and prophylactic efficacy of the stapled peptide constructs derivatized with PEG(n)-thiocholesterol or PEG(n)-cholesterol as described herein are measured using the duration of protection from mucosal infection (assessed histologically as described above or by PCR as described below).

[0181] In vitro binding assays: To evaluate the binding and affinity of peptide-mimicking macrocyclic molecules and peptide-mimicking precursors to receptor proteins, for example, fluorescence polarization assays (FPAs) can be used. FPA techniques use polarization and fluorescence tracers to measure molecular orientation and mobility. When excited by polarization, fluorescence tracers (e.g., FITCs) bound to molecules or peptides and then to proteins with high apparent molecular weights (e.g., FITC-labeled peptides bound to large proteins) emit higher levels of polarization fluorescence compared to fluorescence tracers bound to smaller molecules or peptides alone (e.g., FITC-labeled peptides free in solution) because of their slower rotational speed upon protein binding.

[0182] In vitro substitution assays to characterize peptide-protein interaction antagonists: To evaluate the binding and affinity of compounds that antagonize the interaction between peptides and receptor proteins, fluorescence polarization assays (FPAs) are used, for example, utilizing fluorescent peptides derived from template peptide sequences or macrocyclic peptide-mimicking molecules. FPA techniques use polarization and fluorescence tracers to measure the orientation and mobility of molecules. When excited by polarization, fluorescence tracers (e.g., FITC) bound to molecules that subsequently bind to proteins with high apparent molecular weights (e.g., FITC-labeled peptides bound to large proteins) emit higher levels of polarization fluorescence due to their slower rotational speed compared to FITC-derivative molecules alone (e.g., FITC-labeled peptides free in solution). Compounds such as unlabeled stapled peptides and their conjugates that antagonize the interaction between fluorescently labeled peptides and receptor proteins can be detected in competitive binding FPA experiments, and the differential potency of the compounds in disrupting the interaction can be quantified and compared.

[0183] 5-Helix Bundle Protein Production and Fluorescence Polarization Assay: Design a C-terminal Hexa-His tagged (SEQ ID NO: 279) recombinant 5-helix bundle (5HB) protein containing 5 of 6 helices, including a hairpin RSV-F trimer core, linked by 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. 2014 May;124(5):2113-24). Transform Escherichia coli BL21(DE3) with the plasmid, culture in Luria broth, and induce overnight at 37°C with 0.1 M isopropyl β-D-thiogalactoside. Cells were collected by centrifugation at 5,000 g for 20 minutes, resuspended in buffer A (100 mM NaH2PO4, 20 mM Tris, 8 M urea, pH 7.4), and dissolved by stirring overnight at 4°C. The mixture was clarified by centrifugation (35,000 g for 30 minutes) and then conjugated to a nickel-nitrilotriacetate (Ni-NTA) agarose (Qiagen) column at room temperature. The conjugated 5-HB was washed with buffer A (pH 6.3), eluted with buffer A (pH 4.5), regenerated by dilution (1:2) with PBS (50 mM sodium phosphate, 100 mM NaCl, pH 7.5), and concentrated with 10 kDa Amicon centricon (7 dilutions and reconcentration) to obtain a protein solution of approximately 1 mg / mL. The purity of the protein is evaluated by SDS-PAGE and determined to be greater than 90%. The fluorescent derivative of the peptide of the present invention (25 nM) is incubated with the 5-HB protein at the indicated concentration in a binding buffer (50 mM sodium phosphate, 100 mM NaCl, pH 7.5) at room temperature. Direct binding activity at equilibrium (e.g., 10 minutes) is measured by fluorescence polarization using a SpectraMax M5 microplate reader (BMG Labtech).Next, for the competitive binding assay, fixed concentrations of FITC-peptide and 5-HB protein, reflecting the EC90 of direct binding, were incubated with a series of dilutions of acetylated SAH-RSV peptide to create competitive curves for comparative analysis. The binding assay was performed in triplicate, and Ki was calculated by nonlinear regression analysis of competitive binding isotherms using Prism software (GraphPad).

[0184] Assays for screening binding activity to RSV5 helix bundles: In some examples, the methods disclosed herein include direct and competitive screening assays. For example, a method may involve determining whether a drug alters (e.g., reduces) the binding of one or more of the peptides disclosed herein and their conjugates to RSV (e.g., RSV5 helix bundles). In some examples, a method may include (i) determining the level of binding (e.g., to RSV5 helix bundles) between one or more of the peptides disclosed herein and their conjugates and RSV (e.g., to RSV5 helix bundles) (e.g., in the absence of a drug), (ii) detecting the level of binding (e.g., reduction) between one or more peptides (e.g., one or more of the peptides in (i)) and RSV (e.g., to RSV5 helix bundles) in the presence of a drug, wherein the alteration (e.g., reduction) of the level of binding (e.g., reduction) between one or more peptides and RSV (e.g., to RSV5 helix bundles) indicates that the drug is a candidate drug that binds to RSV, and (iii) selecting candidate drugs. In some examples, step (i) includes contacting one or more peptides with RSV (e.g., to an RSV5 helix bundle) and detecting the level of binding between one or more peptides and RSV (e.g., to an RSV5 helix bundle). In some examples, step (ii) includes contacting one or more peptides and a drug with RSV (e.g., to an RSV5 helix bundle) and detecting the level of binding between one or more peptides and RSV (e.g., to an RSV5 helix bundle). RSV (e.g., to an RSV5 helix bundle) can be contacted with one or more peptides and a drug simultaneously or at different times (e.g., one or more peptides can be contacted with RSV (e.g., to an RSV5 helix bundle) before or after the drug). In some embodiments, candidate drugs are administered to a suitable animal model (e.g., an animal model of RSV) to determine whether the drug reduces the level of RSV infection in the animal.

[0185] In some examples, one or both of the peptide and / or RSV helix bundle may contain a label that enables detection of the peptide and / or RSV helix bundle. In some examples, the peptide contains the label. In some examples, the RSV helix bundle contains the label. In some examples, both the peptide and the RSV helix bundle contain the label. The label may be any label known in the art, including but not limited to fluorescent labels, radioisotope labels, or enzyme labels. In some examples, the label is directly detectable by itself (e.g., radioisotope labels or fluorescent labels). In some examples, (e.g., in the case of enzyme labels) the label is indirectly detectable by catalyzing a chemical change in, for example, a chemical substrate compound or composition, and the chemical substrate compound or composition is directly detectable.

[0186] Competitive RSV5-HB binding assay by ELISA: Microwells are coated overnight at 4°C with 50 μl of PBS containing nutraavidin (4 μg / ml). Wells are washed twice with PBS (PBS-T) containing 0.05% Tween® 20, and then blocked with 4% BSA in PBS-T at 37°C for 45 minutes. Next, 50 μl of 250 nM biotinylated PEG is added. 2-RSV HR2 (SEQ ID NO: 100) is added to PBS-T containing 1% BSA, incubated for 1 hour with shaking, and then washed four times with 300 μl of PBS-T. Next, a 1:2 series dilution of the stapled peptide of the present invention, starting at 10 μM and containing 50 nM recombinant 5-HB in 50 μl of PBS-T containing 1% BSA, is added to the plate, shaken at room temperature for 2 hours, and then washed four times with 300 μl of PBS-T. Finally, a 1:5000 dilution of goat polyclonal to conjugated 6×His-tagged-HRP (disclosed as SEQ ID NO: 279 "HHHHHH") is added. After incubation at room temperature for 40 minutes, the wells are washed five times and colored by adding 50 μl of tetramethylbenzidine (TMB) solution. After 20 minutes, the well containing the TMB solution is stopped by adding 50 μl of H2SO4 (2M), and the absorbance at 450 nm is read using a microplate reader (Molecular Devices). The concentration of the competing peptide corresponding to the maximum half-volume signal (IC50) is determined by interpolating the resulting binding curve using Prism software (Graphpad). Each peptide competitor is tested in triplicate in at least two separate experiments.

[0187] Cell localization assay: To measure the localization of peptides or cross-linked polypeptides on or inside cells, undamaged cells are incubated with fluorescent cross-linked polypeptides derivatized with PEG(n)-thiocholesterol or PEG(n)-cholesterol (5 μM) in serum-free medium or in medium supplemented with human serum at 37°C for 4 hours, washed twice with medium, and incubated with trypsin (0.25%) at 37°C for 10 minutes. The cells are washed again and resuspended in PBS. Cell fluorescence is analyzed using, for example, a FACSCalibur flow cytometer or a Cellomics KineticScan® HCS Reader.

[0188] Antiviral efficacy assay: The efficiency of the peptide-cholesterol conjugate of the present invention in preventing and treating infection with live human respiratory syncytial virus (RSV-GFP1) possessing green fluorescent protein is evaluated in monolayer cell culture. A549 cells plated in a 384-well format are treated for 30 minutes with a series of dilutions of stapled peptides performed in a quadruple-row (e.g., starting dose of 1–5 μM or fixed dose, e.g., 2 μM), followed by the addition of GFP-RSV live virus assay (0.75–1.25 μL of virus per well) and incubation for 48–72 hours. The infected cells are then washed with PBS. Hoechst33342 (cell-permeable nuclear dye) and DRAQ7 (cell-impermeable nuclear dye) are added, and the plate is imaged at 4x magnification with a Molecular Devices ImageXpress Micro Confocal Laser. GFP(+) cells are counted, and the total GFP(+) cells or the percentage of GFP(+) cells is plotted using Prism software (Graphpad). Cytotoxicity is determined by the ratio of DRAQ7(+)Hoechst33342(+) cells to DRAQ7(-)Hoechst33342(+) cells.

[0189] How to use This disclosure features methods of using any of the structurally stabilized (e.g., stapled) peptides or structurally stabilized peptide conjugates (or pharmaceutical compositions comprising such structurally stabilized peptides or structurally stabilized peptide conjugates) described herein to treat or prevent RSV infection in subjects requiring such treatment (e.g., humans). In some examples, the treatment alleviates, inhibits, or improves the infection that the subject (e.g., humans) is suffering from. In some examples, the subject is an animal. In some examples, the subject is a mammal such as a non-primate (e.g., a cattle, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey or human). In some examples, the subject is a domesticated animal (e.g., a dog or cat). In some examples, the subject is a human. In certain examples, such terms refer to non-human animals (e.g., non-human animals such as a pig, horse, cattle, cat or dog, etc.). In some examples, such terms refer to pets or livestock. In some examples, such terms refer to humans.

[0190] The structurally stabilized (e.g., stapled) peptides or structurally stabilized peptide conjugates (or pharmaceutical compositions containing them) described herein are useful for treating subjects (e.g., humans) having RSV infection. In some examples, the structurally stabilized peptides (or pharmaceutical compositions containing them) are used to treat RSV infection in subjects (e.g., humans). In some examples, the structurally stabilized peptide conjugates (or pharmaceutical compositions containing them) are used to treat RSV infection in subjects (e.g., humans). In some examples, the subjects are humans.

[0191] The structurally stabilized (e.g., stapled) peptides or structurally stabilized peptide conjugates (or pharmaceutical compositions containing them) described herein are useful for preventing a subject (e.g., a human) from having RSV infection. In some examples, the structurally stabilized peptide (or pharmaceutical composition containing it) is used to prevent RSV infection in a subject (e.g., a human). In some examples, the subject is a human.

[0192] Accordingly, this specification provides a method for treating RSV infection in a subject (e.g., a human) requiring treatment of RSV infection, comprising administering to the subject a therapeutically effective amount of a structurally stabilized peptide (or a pharmaceutical composition comprising a structurally stabilized peptide) described herein.

[0193] Furthermore, this specification also provides a method for preventing RSV infection in subjects (e.g., humans) who require prevention of RSV infection, the method comprising administering to the subjects a therapeutically effective amount of a structurally stabilized peptide (or a pharmaceutical composition containing a structurally stabilized peptide) described herein.

[0194] Accordingly, the Specified provides a method for treating RSV infection in a subject (e.g., a human) requiring treatment of RSV infection, comprising administering to the subject a therapeutically effective amount of a structurally stabilized peptide conjugate (or a pharmaceutical composition comprising a structurally stabilized peptide conjugate) as described herein.

[0195] Furthermore, this specification also provides a method for preventing RSV infection in subjects (e.g., humans) who require prevention of RSV infection, the method comprising administering to the subjects a therapeutically effective amount of a structurally stabilized peptide conjugate (or a pharmaceutical composition comprising a structurally stabilized peptide conjugate) as described herein.

[0196] In certain cases, the aforementioned method involves administering to a subject (e.g., a human) a peptide or its variant (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions, insertions, or deletions) listed in Table 1, a construct or its variant (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions, insertions, or deletions) listed in Table 2, or a conjugate or its variant (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions, insertions, or deletions) listed in Table 3. In some cases, the peptides or constructs in Tables 1-3 further contain three N-terminal amino acids X1, X2, and X3, where X1 is any amino acid, optionally S, and X2 and X3 are any load amino acids.

[0197] In certain cases, the method involves administering to a subject (e.g., a human) a structurally stabilized peptide (or a pharmaceutical composition containing the same) containing or comprising one of the amino acid sequences of SEQ ID NOs: 27, 29, 36, 43, and 46, or one of the amino acid sequences of SEQ ID NOs: 49, 51, 58, 65, and 68. In certain cases, the method involves administering to a subject (e.g., a human) a structurally stabilized peptide (or a pharmaceutical composition containing the same) containing or comprising the amino acid sequence of SEQ ID NOs: 29 or 51. In certain cases, the method involves administering to a subject (e.g., a human) a structurally stabilized peptide (or a pharmaceutical composition containing the same) containing or comprising the amino acid sequence of SEQ ID NOs: 43 or 65.

[0198] In certain cases, the method involves administering to a subject (e.g., a human) a structurally stabilized peptide conjugate (or a pharmaceutical composition containing the same) containing or comprising one of the amino acid sequences of SEQ ID NOs. 71, 73, 80, 87, and 90, or one of the amino acid sequences of SEQ ID NOs. 5, 6, 14, 21, and 24. In certain cases, the method involves administering to a subject (e.g., a human) a structurally stabilized peptide conjugate (or a pharmaceutical composition containing the same) containing or comprising the amino acid sequence of SEQ ID NOs. 73. In certain cases, the method involves administering to a subject (e.g., a human) a structurally stabilized peptide conjugate (or a pharmaceutical composition containing the same) containing or comprising the amino acid sequence of SEQ ID NOs. 21, 281, or 87.

[0199] In certain cases, the method involves administering to a subject (e.g., a human) a variant of a structurally stabilized peptide comprising or consisting of any one of SEQ ID NOs: 27, 29, 36, 43, and 46, or any one of SEQ ID NOs: 49, 51, 58, 65, and 68 (for example, each having 1 to 10, 1 to 5, 1 to 3, 2, or 1 amino acid substitutions, insertions, and / or deletions compared to any one of SEQ ID NOs: 27, 29, 36, 43, and 46, or any one of SEQ ID NOs: 49, 51, 58, 65, and 68, wherein the amino acid substitutions and / or deletions are not in staple positions, and the structurally stabilized peptide binds to the RSV5 helix bundle protein and / or inhibits and / or prevents RSV-mediated cell infection) (or a pharmaceutical composition containing the same).

[0200] In certain cases, the method involves administering to a subject (e.g., a human) a variant of a structurally stabilized peptide conjugate comprising or consisting of one of the amino acid sequences of SEQ ID NOs. 71, 73, 80, 87, and 90, or one of the amino acid sequences of SEQ ID NOs. 5, 6, 14, 21, and 24 (for example, each having 1 to 10, 1 to 5, 1 to 3, 2, or 1 amino acid substitutions, insertions, and / or deletions compared to the amino acid sequence described in one of SEQ ID NOs. 71, 73, 80, 87, and 90, or one of SEQ ID NOs. 5, 6, 14, 21, and 24, wherein the amino acid substitutions and / or deletions are not in staple positions, and the structurally stabilized peptide binds to the RSV5 helix bundle protein and / or inhibits and / or prevents RSV-mediated cell infection) (or a pharmaceutical composition comprising the same).

[0201] In certain cases, the method involves administering a structurally stabilized peptide (or a pharmaceutical composition containing the same) as described in the "Structurally Stabilized Peptides" section above to a subject (e.g., a human). In certain cases, the method involves administering a structurally stabilized peptide conjugate (or a pharmaceutical composition containing the same) as described in the "Structurally Stabilized Peptide Conjugates" section above to a subject (e.g., a human). In some cases, the method involves administering a structurally stabilized peptide or structurally stabilized peptide conjugate (or a pharmaceutical composition containing the same) as described in the Figures or Examples to a subject (e.g., a human). In one example, the method described herein involves administering a peptide or conjugate containing the amino acid sequence of SEQ ID NO: 281.

[0202] In some cases, the subject (e.g., a human) is infected with RSV. In some cases, the subject (e.g., a human) is at risk of being infected with RSV (e.g., has been exposed to an RSV-infected subject). In some cases, the subject (e.g., a human) is suspected of being infected with RSV (e.g., has been exposed to an RSV-infected subject and exhibits one or more symptoms of RSV). Methods for determining whether a subject is infected with RSV are publicly known in the art.

[0203] In some examples, a method for treating or preventing RSV infection further comprises repeatedly administering a therapeutically effective amount of a structurally stabilized peptide or structurally stabilized peptide conjugate (or a pharmaceutical composition containing thereof) described herein to a subject (e.g., a human) as required for the treatment or prevention of RSV infection. In some examples, a method for treating or preventing RSV infection further comprises testing a subject (e.g., a human) to determine whether the subject has RSV infection, and then administering a therapeutically effective amount of a structurally stabilized peptide or structurally stabilized peptide conjugate (or a pharmaceutical composition containing thereof) described herein. Subjects may be selected for treatment, for example, on the basis that the subject is at risk of acquiring RSV infection or is determined to have RSV infection.

[0204] The structurally stabilized peptides or structurally stabilized peptide conjugates (or pharmaceutical compositions comprising them) described herein may be administered orally, intranasally, intravenously, subcutaneously, intramuscularly, or topically, including administration to the skin, nose, sinuses, eyes, oropharynx, respiratory tree, and lungs. In some cases, administration is by topical respiratory application, including application to the nasal mucosa, sinus mucosa, oropharyngeal mucosa, or respiratory tree, including the lungs. In some cases, topical application includes application to the skin or eyes. In some cases, the conjugates described herein increase bioavailability, increase blood circulation, alter pharmacokinetics, decrease immunogenicity, and / or reduce the required frequency of administration.

[0205] The specific dosage and treatment regimen for any particular patient or subject will depend on a variety of factors, including the activity of the specific compound being employed, age, weight, general health status, sex, diet, administration time, excretion rate, drug combinations, severity and course of the disease, condition, or symptom, the patient's or subject's predisposition to the disease, condition, or symptom, and the judgment of the treating physician or veterinarian.

[0206] An effective dose may be administered in one or more doses, applications, or prescriptions. The effective therapeutic dose (i.e., effective dosage) of a therapeutic compound (e.g., a structurally stabilized peptide or a structurally stabilized peptide conjugate) depends on the therapeutic compound selected. The composition may be administered at a frequency of once or more times per day, including once every other day, or at a frequency of once or more times per week. Those skilled in the art will understand that certain factors, including but not limited to the risk or severity of the disease or disorder, previous treatments, the subject's general health status and / or age, and other pre-existing diseases, may influence the dosage and timing required to effectively treat the subject. Furthermore, treatment of a subject with an effective dose of a therapeutic compound as described herein may consist of a single treatment or a series of treatments. For example, an effective dose may be administered at least once.

[0207] Furthermore, this specification also provides the use of structurally stabilized peptides or structurally stabilized peptide conjugates (or pharmaceutical compositions containing them) described herein in the manufacture of pharmaceuticals for treating RSV infection in subjects (e.g., humans).

[0208] Furthermore, this specification also provides the use of structurally stabilized peptides or structurally stabilized peptide conjugates (or pharmaceutical compositions containing them) described herein in the manufacture of pharmaceuticals for preventing RSV infection in subjects (e.g., humans). [Examples]

[0209] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, it is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without exercising their inventive capacity and without departing from the scope of the invention.

[0210] Example 1: Design and synthesis of a stapled lipopeptide of the RSV HR2 domain to block RSV infection by inhibiting viral fusion with the host membrane. To design peptides that strongly block RSV fusion to host cells (Figure 1), a series of stapled peptides were designed, each possessing differentially localized chemical staples and derivatized at the C-terminus using PEG(n)-thiocholesterol or PEG(n)-cholesterol moieties. The designed peptides were synthesized on resin by solid-phase synthesis. The differentially localized chemical staples are located within the RSV HR2 domain of the human RSV surface (F) glycoprotein sequence (i.e., amino acids 488-516 of SEQ ID NO: 1) (see Figures 2-4), which substitute native residues with α,α-disubstituted non-native olefin residues in the form of double staples or stitches (e.g., "X" for (S)-α-(4'-pentenyl)alanine located at selected i, i+7 positions and "8" for (R)-α-(7'-octenyl)alanine, or "X" for (S)-α-(4'-pentenyl)alanine located at each of selected i, i+4 positions) and combinations thereof, followed by ruthenium-catalyzed olefin metathesis (see Figures 5-7). Approaches for designing, synthesizing, and identifying optimal stapled peptide constructs for targeting RSV fusion apparatus include Ala scanning (e.g., mutants), staple scanning, and generation of variable N and C-terminal deletions, additions, and derivatization libraries for conjugation to PEG-thiocholesterol or PEG-cholesterol moieties (see Figure 8). Stapled RSV HR2 peptides are constructed by substituting two naturally occurring amino acids with the unnatural (R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-deca-9-enoic acid / (R)-α-(7'-octenyl)alanine / (Fmoc-R8) and (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-hepta-6-enoic acid / (S)-α-(4'-pentenyl)alanine / (Fmoc-S5) amino acids at positions i and i+7 (i.e., adjacent to seven amino acids) to create a staple spanning two α-helix rotations, or by substituting with two unnatural S5 amino acids at positions i and i+4 to create a staple spanning one α-helix rotation.The asymmetric synthesis of α,α-disubstituted amino acids is 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 herein by reference in its entirety).

[0211] Example 2: Identification of optimally stapled RSV HR2 peptides with C-terminal PEG4-thiocolesterol to achieve antiviral activity in pseudotype and live virus assays To enhance the potency of the RSV HR2 helix stabilized by the insertion of only hydrocarbon staples, and thus its drug-like properties, the differentially stapled RSV HR2 constructs were further derivatized at the C-terminus with PEG(n)-thiocolesterol moieties. For example, the singly i,i+7 stapled peptide of SEQ ID NO: 4 blocked infection by RSV virus with an IC50 greater than 5 μM. Therefore, an iterative optimization campaign was initiated by "staple scanning" the alpha-helical region of the RSV HR2 domain spanning residues Phe488 and Val516 (see Figure 10).

[0212] Staple scanning was performed to identify the residues important for interaction and the binding surface, respectively, to determine the design of optimized constructs and negative control mutants (see Figure 5). The peptide N-terminus is capped with an acetyl or fluorophore (e.g., FITC, rhodamine) depending on the experimental application. N-terminal acetyl-capped peptides were generated.

[0213] Doubly stapled peptides were generated by installing two -S5-S5, two R8-S5, or other combinations of crosslinked unnatural amino acids, and multiply stapled peptides or stitched peptides were generated using similar principles (see Figure 6).

[0214] To enable peptide derivatization using thiol - cholesterol on resin, carboxy - thiol - cholesterol was synthesized according to the schematic diagram presented in FIG. 11. To generate stapled lipopeptides of the RSV HR2 domain (see the scheme in FIG. 12), the completed resin - bound peptide (e.g., SEQ ID NO: 5) was capped with an acetyl group (by using acetic anhydride), and subsequently, the C - terminal side - chain lysine amine was deprotected by treatment with 2% hydrazine. The amine was acylated with Fmoc - protected PEG(n) amino acid (e.g., n = 1 - 36), and at that point, the olefin was cross - linked by treatment with Grubbs (I) catalyst. The Fmoc was removed from the C - terminal NH of the PEG(n) amino acid, and the amine was acylated with carboxy - thiol - cholesterol. After peptide deprotection, cleavage, and purification by reverse - phase high - performance liquid chromatography / mass spectrometry (LC / MS), the final peptide product was obtained. The same procedure for derivatization with cholesterol may be carried out using a cholesterol moiety instead of the thiol - cholesterol moiety.

[0215] Applying this chemical scheme, a PEG4 - thiol - cholesterol moiety was added to the C - terminus of the sequence described 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 FIG. 13) in an infectivity assay using live RSV virus, and the IC50 was 1.3×10 -7 M. Thus, removal of the six C - terminal residues of the sequence of SEQ ID NO: 4 and addition of a C - terminal PEG - thiol - cholesterol moiety improved the antiviral activity of the peptide by one order of magnitude (compare FIG. 9 with FIG. 13).

[0216] To identify the optimal site for staple insertion, exemplary i,i+7 staple-scanning libraries of RSV HR2 (sequence number 1, aa488-516) derivatized with PEG(n)-thiocholesterol were generated using the synthetic scheme shown in Figures 11-12 (thus generating sequence numbers 5-26). The differential antiviral activity of this library was evaluated in an RSV infectivity essay. Peptides with sequences 5, 6, 14, 21, and 24 exhibited potent activity among various stapled RSV HR2 peptides in a GFP-RSV live virus assay (see Figure 15). In summary, these data indicate that (1) adding a PEG4-thiocholesterol moiety to the C-terminus of a structurally stabilized RSV HR2 peptide using a resin-based method can confer potent antiviral activity among a panel of differentially stapled i,i+7 peptides that otherwise exhibit lower or no activity (compare Figures 9 and 13).

[0217] Example 3: Determination of the optimal PEG linker length in a stapled RSV HR2 peptide with C-terminal PEG(n)-thiocholesterol to achieve antiviral activity in pseudotyped and live virus assays. To determine the optimal PEG chain length for linking the stapled RSV HR2 peptide to the thiocholesterol moiety, a series of PEG(n) analogs were generated according to the synthetic method described above, where n is equal to 0, 4, 8, 12, 16, or 20. The antiviral activity of this series of i, i+7 stapled RSV HR2 peptides, including SEQ ID NOs. 51 and 65, spaced by PEG linkers of various lengths, and thiocholesterol, was tested in a GFP-RSV live virus assay (0.75–1.25 μL of virus per well, cells: A549, 2 μM peptide dose, readout at 48 hours). See Figures 16 and 17. The superiority of PEG16 and PEG20 linkers, particularly compared to PEG4, PEG8, and PEG12, was unexpected and required experimental determination in the RSV antiviral assay. C-terminal derivatization of PEG16-thiochol and PEG20-thiochol of SEQ ID NO: 5 produced compounds with IC50s of 49 and 60 nM, respectively (see Figure 16), reflecting an improvement of more than two orders of magnitude in antiviral activity compared to the corresponding RSV HR2 peptide, which has only a single staple and six additional HR2 amino acids at the C-terminus (see Figure 4). In particular, C-terminal derivatization of the alternative single-staple-bound peptide of SEQ ID NO: 21 produced compounds with IC50s of 18 and 8.2 nM (see Figure 17). Therefore, the combination of staple scanning, differential peptide truncation, and PEG scanning revealed the staple position, sequence length, and PEG chain length that produce the staple lipopeptide of SEQ ID NO: 21 with a remarkably high increase in anti-RSV activity (single-order nanomolar anti-RSV activity), a three-order-order improvement compared to the single-stapled RSV HR2 peptide of SEQ ID NO: 4, and reflects a corresponding superiority over previously reported longer, double-stapled anti-RSV peptides lacking PEG-thiocholesterol derivatization (Bird et al. JCI, 2014, 124(5):2113-2124).

[0218] Example 4: Rescue of poor solubility by adding three natural N-terminal residues. Poor solubility was observed in the staple lipopeptide with sequence number 21. However, by incorporating three native N-terminal residues "SDE" containing two loaded amino acids, the insolubility issue was completely resolved, resulting in complete solubility at 15 mg / mL in 15% DMSO and PBS pH 7.4. Sequence optimization shifted the pI from 8, corresponding to poor solubility at neutral pH, to 4.7, which is associated with complete solubilization.

[0219] Other Embodiments The present invention is described in detail, but the foregoing description is intended to be illustrative and not to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the following claims.

[0220] All publications, patents, patent applications, internet sites, and database sequences, including both polynucleotide and polypeptide sequences, cited herein are incorporated herein in whole for all purposes to the same extent that each individual publication, patent, patent application, internet site, or database sequence is specifically and individually incorporated for reference.

Claims

1. (i) a structurally stabilized peptide and (ii) cholesterol or thiocholesterol, a conjugate comprising The cholesterol or thiocholesterol is linked directly or via a linker to the C-terminal amino acid of the structurally stabilized peptide. The structurally stabilized peptide, compared to the sequence of SEQ ID NO: 100 or 281, has 2 to 6 amino acid substitutions, and includes (i) 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 100), or (ii) an internally crosslinked amino acid sequence including the sequence SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 280), wherein the internally crosslinked amino acid sequence does not include the sequence NAGKST (SEQ ID NO: 258). Two of the two to six amino acid substitutions are α,α-disubstituted unnatural amino acids having cross-linked olefin side chains, and these α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are separated by three or six amino acids. The conjugate binds to the RSV5 helix bundle protein and / or the conjugate inhibits RSV-induced cell infection and / or prevents RSV-induced cell infection. The aforementioned conjugate has a length of 25 to 45 amino acids, and optionally, the aforementioned conjugate has a length of 30 amino acids.

2. (i) a structurally stabilized peptide and (ii) cholesterol or thiocholesterol, a conjugate comprising The cholesterol or thiocholesterol is linked directly or via a linker to the C-terminal amino acid of the structurally stabilized peptide. The structurally stabilized peptide is given by formula: 【Chemistry 19】 or comprising an internally crosslinked amino acid sequence having a pharmaceutically acceptable salt thereof, In the formula, each R １ and R ２ However, H or C １ ~C １０ Alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and any of these may be substituted or unsubstituted. x is 3 or 6, Each R ３ However, independently, they are alkylene, alkenylene, or alkynylene, and each of these can be substituted or unsubstituted. z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The internally crosslinked amino acid sequence, compared to the sequence of SEQ ID NO: 100 or 281, has 2 to 6 amino acid substitutions, and includes (i) 25 to 29 consecutive amino acids of the sequence of SEQ ID NO: 100, or (ii) the sequence SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 280), and the internally crosslinked amino acid sequence does not include the sequence NAGKST (SEQ ID NO: 258). The conjugate binds to the RSV5 helix bundle protein and / or the conjugate inhibits RSV-induced cell infection and / or prevents RSV-induced cell infection. The aforementioned conjugate has a length of 25 to 45 amino acids, and optionally, the aforementioned conjugate has a length of 30 amino acids.

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

4. The conjugate according to claim 1 or 2, comprising the thiocholesterol, wherein optionally 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 stabilized peptide, where n is 1 to 36, and optionally 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 stabilized peptide, where n is 1 to 36, and optionally n is 4, 5, 6, 7, 8, 12, 16, or 20.

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

8. The conjugate comprises formula II directly linked to the C-terminal amino acid of the structurally stabilized peptide, 【Chemistry 21】 The conjugate according to claim 1 or 2, wherein n is 1 to 36, and n is optionally 4, 5, 6, 7, 8, 12, 16, or 20.

9. The conjugate according to claim 1, wherein the α,α-disubstituted nonnatural amino acids having mutually crosslinked olefin side chains are separated by three amino acids, and optionally each of the α,α-disubstituted nonnatural amino acids having mutually crosslinked olefin side chains is (S)-α-(4'-pentenyl)alanine.

10. The conjugate according to claim 1, wherein the α,α-disubstituted unnatural amino acids having mutually crosslinked olefin side chains are separated by six amino acids, and optionally the α,α-disubstituted unnatural amino acids having mutually crosslinked olefin side chains are (R)-α-(7'-octenyl)alanine and (S)-α-(4'-pentenyl)alanine.

11. The conjugate according to claim 1, wherein two substitutions with α,α-disubstituted unnatural amino acids having mutually crosslinked olefin side chains are in amino acids corresponding to positions 1 and 8 of the sequence described in SEQ ID NO: 100, amino acids corresponding to positions 3 and 10 of the sequence described in SEQ ID NO: 100, or amino acids corresponding to positions 17 and 24 of the sequence described in SEQ ID NO:

100.

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

100.

13. The conjugate according to any one of claims 1 to 8, wherein the conjugate includes the sequence described in any one of sequence numbers 5 to 26 and 71 to 92.

14. The conjugate according to any one of claims 1 to 8, wherein the structurally stabilized peptide comprises the sequence described in any one of SEQ ID NOs: 27, 29, 36, 43, and 46.

15. The conjugate according to any one of claims 1 to 8, wherein the structurally stabilized peptide comprises the sequence described in any one of SEQ ID NOs: 49, 51, 58, 65, 68, and 281.

16. The conjugate according to claim 1 or 2, wherein the conjugate includes the sequence described in any one of sequence numbers 71, 73, 80, 87, and 90.

17. The conjugate according to claim 1 or 2, wherein the conjugate includes the sequence described in any one of sequence numbers 5, 6, 14, 21, and 24.

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

19. 8DASISQXNEKINQSLAFIRKSDELLHNV ＊ A conjugate comprising (SEQ ID NO: 265) or comprising thereof, wherein 8 is internally cross-linked to X, 8 is (R)-α-(7'-octenyl)alanine, and X is (S)-α-(4'-pentenyl)alanine. ＊ However, the formula 【Chemistry 22】 And, A conjugate in which n is between 1 and 36, and can be either 16 or 20.

20. FD8SISQVNXKINQSLAFIRKSDENHV ＊ A conjugate comprising or consisting of ＊ (SEQ ID NO: 261), wherein 8 is internally crosslinked to X, 8 is (R)-α-(7'-octenyl)alanine, X is (S)-α-(4'-pentenyl)alanine, ＊ is of the formula 【Chemistry 23】 And, A conjugate in which n is between 1 and 36, and can be either 16 or 20.

21. FDASISQVNEKINQSL8FIRKSDXLHNV ＊ A conjugate comprising (SEQ ID NO: 266) or comprising thereof, wherein 8 is internally cross-linked to X, 8 is (R)-α-(7'-octenyl)alanine, and X is (S)-α-(4'-pentenyl)alanine. ＊ However, the formula 【Chemistry 24】 And, A conjugate in which n is 1 to 36, and optionally n is 16 or 20, and optionally the conjugate contains three N-terminal amino acids X1X2X3 immediately upstream of the first F in Sequence ID No. 266, where X1 is any amino acid, optionally S, and X2 and X3 are load amino acids.

22. A structurally stabilized peptide comprising an internally crosslinked amino acid sequence containing 25 to 29 consecutive amino acids, compared to the sequence of Sequence ID No. 100, with the exception of 2 to 6 amino acid substitutions, wherein the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (Sequence ID No. 100) has two to six amino acid substitutions removed, Two of the two to six amino acid substitutions are α,α-disubstituted unnatural amino acids having cross-linked olefin side chains, and these α,α-disubstituted unnatural amino acids having cross-linked olefin side chains are separated by three or six amino acids. The structurally stabilized peptide binds to the RSV5 helix bundle protein and / or inhibits RSV infection of cells and / or prevents RSV infection of cells. The structurally stabilized peptide has a length of 25 to 32 amino acids, and optionally the structurally stabilized peptide has a length of 32 amino acids, and optionally the structurally stabilized peptide is Sequence ID No. A structurally stabilized peptide containing three N-terminal amino acids X1, X2, and X3 immediately upstream of the first F of 100, where X1 is any amino acid, optionally S, and X2 and X3 are load amino acids.

23. A structurally stabilized peptide, formula: 【Chemistry 25】 or comprising an internally crosslinked amino acid sequence having a pharmaceutically acceptable salt thereof, In the formula, each R １ and R ２ However, H or C １ ~C １０ Alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, and any of these may be substituted or unsubstituted. x is 3 or 6, Each R ３ However, independently, they are alkylene, alkenylene, or alkynylene, and each of these can be substituted or unsubstituted. z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The internally crosslinked amino acid sequence, compared to the sequence of SEQ ID NO: 100, has 2 to 6 amino acid substitutions removed and contains 25 to 29 consecutive amino acids of the sequence of SEQ ID NO:

100. The structurally stabilized peptide binds to the RSV5 helix bundle protein and / or inhibits RSV infection of cells and / or prevents RSV infection of cells. The structurally stabilized peptide is 25 to 30 amino acid lengths, and optionally, the structurally stabilized peptide is 29 amino acid lengths.

24. A structurally stabilized peptide according to claim 22 or 23, which does not contain amino acids corresponding to positions 517-522, 517-521, 517-520, 517-519, or 517-518 (numbered according to the sequence described in SEQ ID NO: 1) of the RSV-F protein.

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

26. The structurally stabilized peptide according to claim 22 or 23, wherein the structurally stabilized peptide has a length of 29 amino acids and contains 29 consecutive amino acids of the sequence of Sequence ID No. 100, with the exception of 2 to 6 amino acid substitutions compared to the sequence of Sequence ID No.

100.

27. The structurally stabilized peptide according to any one of claims 22 to 26, wherein the structurally stabilized peptide comprises the amino acid sequence described in any one of SEQ ID NOs: 27 to 70.

28. The structurally stabilized peptide according to any one of claims 22 to 26, wherein the structurally stabilized peptide comprises the amino acid sequence described in any one of SEQ ID NOs: 27, 29, 36, 43, and 46.

29. The structurally stabilized peptide according to any one of claims 22 to 26, wherein the structurally stabilized peptide comprises the amino acid sequence described in any one of Sequence ID Nos. 49, 51, 58, 65, 281, 285, and 68.

30. A peptide comprising one amino acid from sequence numbers 27 to 70, with the exception of 0 to 6 additional substitutions, wherein the peptide does not contain sequence NAGKST (sequence number 258).

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

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

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

34. The method according to claim 32 or 33, wherein the subject is a human.

35. A method for producing structurally stabilized peptides, I. (a) To provide a peptide having an amino acid sequence that includes 25 to 29 consecutive amino acids of the sequence FDASISQVNEKINQSLAFIRKSDELLHNV (Sequence ID 100), with 2 to 6 amino acid substitutions removed compared to the sequence of Sequence ID 100, To provide a product in which two of the two to six amino acid substitutions are α,α-disubstituted unnatural amino acids having olefin side chains, and the α,α-disubstituted unnatural amino acids having olefin side chains are separated by three or six amino acids, and (b) Crosslink the peptide to produce the structurally stabilized peptide, and optionally purify the structurally stabilized peptide, or II. (a') To provide a peptide comprising the sequence SDEFDASISQVNEKINQSLAFIRKSDELLHNV (SEQ ID NO: 280) obtained by removing 2 to 6 amino acid substitutions compared to the sequence of SEQ ID NO: 280, To provide a product in which two of the two to six amino acid substitutions are α,α-disubstituted unnatural amino acids having olefin side chains, and the α,α-disubstituted unnatural amino acids having olefin side chains are separated by three or six amino acids, and (b') A method comprising crosslinking the peptide to produce the structurally stabilized peptide, and optionally purifying the structurally stabilized peptide.

36. The method according to claim 35, wherein the crosslinking is performed by a ruthenium-catalyzed metathesis reaction.

37. The method according to claim 35 or 36, further comprising derivatizing the resin-bound amine of the structurally stabilized peptide using PEG and / or cholesterol or thiocholesterol containing a carboxylic acid on the resin.

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

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