CN108395471B - Polypeptide for inhibiting MERS-CoV infection - Google Patents

Polypeptide for inhibiting MERS-CoV infection Download PDF

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CN108395471B
CN108395471B CN201810224816.0A CN201810224816A CN108395471B CN 108395471 B CN108395471 B CN 108395471B CN 201810224816 A CN201810224816 A CN 201810224816A CN 108395471 B CN108395471 B CN 108395471B
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王潮
刘克良
张培宇
孟广鹏
梁国栋
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Abstract

The present invention relates to polypeptide compounds that are resistant to MERS-CoV infection, in particular, to polypeptide compounds having at least 80% identity to the general formula (I): r1‑U‑Leu‑Asp‑Leu‑Thr‑Tyr‑Glu‑Xa‑Leu‑X1‑Leu‑Gln‑X2‑Val‑Val‑Lys‑X3‑Leu‑Asn‑Glu‑X4‑Tyr‑W‑R2(I) Its stereoisomers, mixtures thereof, its pharmaceutically acceptable salts and/or functionally equivalent variants thereof. The invention also relates to a pharmaceutical composition containing such a polypeptide compound, its stereoisomers, mixtures thereof, pharmaceutically acceptable salts thereof and/or functionally equivalent variants thereof, and the use of such a polypeptide compound, its stereoisomers, mixtures thereof, pharmaceutically acceptable salts thereof and/or functionally equivalent variants thereof for the treatment or prevention of a disease associated with MERS-CoV infection, in particular middle east respiratory syndrome.

Description

Polypeptide for inhibiting MERS-CoV infection
Technical Field
The invention belongs to the field of biomedicine, and relates to a polypeptide compound for resisting MERS-CoV infection, in particular to a polypeptide compound with at least 80% of identity with a general formula (I):
R1-U-Leu-Asp-Leu-Thr-Tyr-Glu-Xa-Leu-X1-Leu-Gln-X2-Val-Val-Lys-X3-Leu-Asn-Glu-X4-Tyr-W-R2(I)，
stereoisomers thereof, mixtures thereof, pharmaceutically acceptable salts thereof and/or functionally equivalent variants thereof. The invention also relates to a pharmaceutical composition containing such a polypeptide compound, its stereoisomers, mixtures thereof, pharmaceutically acceptable salts thereof and/or functionally equivalent variants thereof, and the use of such a polypeptide compound, its stereoisomers, mixtures thereof, pharmaceutically acceptable salts thereof and/or functionally equivalent variants thereof for the treatment or prevention of a disease associated with MERS-CoV infection, in particular middle east respiratory syndrome.
Background
In 2012, a new type of coronavirus, known as middle east respiratory syndrome coronavirus (MERS-CoV), was prevalent in the middle east. The virus can cause SARS-like disease, leading to failure of multiple organ functions, with approximately 40% mortality among people. Because MERS-CoV has the ability of being handed-off, the possibility of large-scale prevalence in the population exists, and therefore, intensive research on MERS-CoV is urgently needed as soon as possible to find a drug design target point and develop a corresponding anti-MERS-CoV drug for clinical treatment of patients and prevention of the potential pandemic possibility of MERS-CoV.
MERS-CoV fusion proteins are mainly composed of S1 subunit and S2 subunit, wherein, S1 subunit is responsible for specific recognition binding with CD26 receptor on the surface of host cell, S2 subunit is responsible for mediating the fusion between virus and target cell membrane, S2 subunit can be further divided into Fusion Peptide (FP), N-terminal heptad repeat (NHR, also known as HR1) and C-terminal heptad repeat (CHR, also known as HR2) functional region, S1 subunit is combined with CD26 receptor, S2 subunit is changed in conformation, its FP is inserted into host cell membrane, and HR1 and HR2 regions are induced to interact to form six strands α helix bundle (6HB) structure, thereby mediating the fusion of virus and host cell membrane.
Over the past few decades, chemists and biologists have employed various ingenious approaches to enhance α -helix structures, including non-covalent and covalent based strategies, one of which is polypeptide binding technology.
The general idea of polypeptide binding technology is to covalently bridge the side chains of amino acid residues at positions i → i +3 or i → i +4 or i → i +7 of the polypeptide sequence on the side of the α helix that does not interact with the target protein through orthogonal reactions, thereby locking the α helix conformation of the polypeptide.
Disclosure of Invention
The inventor constructs a high-activity metabolically stable MERS-CoV fusion inhibitor based on a MERS-CoV HR2 α spiral region polypeptide sequence, thereby providing a solution for treating and preventing MERS-CoV.
Thus, in one aspect, the invention relates to polypeptide compounds having at least 80% identity to general formula (I):
R1-U-Leu-Asp-Leu-Thr-Tyr-Glu-Xa-Leu-X1-Leu-Gln-X2-Val-Val-Lys-X3-Leu-Asn-Glu-X4-Tyr-W-R2(I)，
stereoisomers thereof, mixtures thereof, pharmaceutically acceptable salts thereof and/or functionally equivalent variants thereof;
wherein XaIs an amino acid residue capable of hydrophobic interaction with MERS-COV HR2, selected from the following D-or L-form hydrophobic amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), norleucine (n-Leu), and methionine (Met);
X1、X2、X3and X4Each is an independently selected D-or L-form amino acid;
u and W are absent or are each independently selected one or more additional D or L amino acids;
R1selected from: h, a polymer derived from polyethylene glycol, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group and R5-CO-, wherein R5Selected from: h, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclic group, and a substituted or unsubstituted heteroarylalkyl group;
R2selected from: -NR3R4，-OR3and-SR3Wherein R is3And R4Independently selected from: h, a polymer derived from polyethylene glycol, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted aralkyl group; and
R1and R2Is not a D-or L-form amino acid.
In a preferred embodiment, the present invention relates to a polypeptide compound having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to general formula (I) or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof.
In a preferred embodiment, XaAn amino acid selected from the following D-or L-forms: methionine, leucine, isoleucine, valine and norleucine.
In a preferred embodiment, R1Selected from: h, acetyl, lauroyl, myristoyl and palmitoyl.In a preferred embodiment, R1Selected from: h or R5-CO-; wherein R is5Selected from: h or substituted or unsubstituted C1-C24An alkyl group.
In a preferred embodiment, R2Selected from: -NR3R4and-OR3Wherein R is3And R4Independently selected from the group consisting of H, methyl, ethyl, hexyl, dodecyl and hexadecyl. In a preferred embodiment, R2Selected from: -NR3R4OR-OR3Wherein R is3And R4Independently selected from: h or substituted or unsubstituted C1-C24An alkyl group.
Without being bound by theory, it is believed that X in formula (I) is present when the polypeptide compound of the invention is in the α helix conformation1、X2、X3And X4On the side that does not interact with the target protein, but is exposed on the side that is in contact with the solvent. Furthermore, the inventors have surprisingly found that at X1、X2、X3And X4The introduction of one or more α helix stabilizing amino acids can help stabilize the polypeptide compounds of the invention in the α helix conformation, thereby effectively inhibiting MERS-CoV fusion and are suitable for the treatment and prevention of MERS-CoV.
Thus, in some embodiments, X1、X2、X3And X4Is α helix stable amino acid in a preferred embodiment, X is1、X2、X3And X4Are independently α helix stable amino acids in a preferred embodiment, X is1And X2、X1And X3、X2And X3And/or X3And X4Is α helix-stabilized amino acid in a preferred embodiment, X1、X2And X3Is α helix-stabilized amino acid in a preferred embodiment, X1、X3And X4Is α helix-stabilized amino acid in a preferred embodiment, X2、X3And X4Is α helix-stabilized amino acid in a preferred embodiment, X1、X2、X3And X4Is α helix-stabilized amino acids.
In such embodiments, at X1、X2、X3And X4The amino acid residues in (A) which are not the α helix-stabilising amino acid may each independently be selected from any amino acid, preferably from D-or L-amino acids selected from glutamine, alanine and serine.
As used herein, "α helix-stabilizing amino acids" means amino acids capable of increasing the stability of the α helix conformation of MERS-CoV fusion inhibitor peptides α helix-stabilizing amino acids are well known in the art suitable α helix-stabilizing amino acids suitable for the purposes herein include, but are not limited to, alanine, norvaline, norleucine, α -aminobutyric acid, α -aminoisobutyric Acid (AIB), leucine, isoleucine, valine, and the like in some embodiments, the α helix-stabilizing amino acid is any amino acid that is part of the α helix of a naturally occurring protein, e.g., Leu, Phe, Ala, Met, Gly, Ile, Ser, Asn, Glu, Asp, Lys, Arg.
In a preferred embodiment, the α helix-stabilized amino acid is an α -disubstituted amino acid, in a particular embodiment, the α -disubstituted amino acid comprises a first substituent and a second substituent, each bonded to α carbon, wherein the first substituent and the second substituent are each independently selected from the group consisting of C1-C4 alkyl optionally substituted with hydroxy, amide, mercapto, halo, or the first substituent and the second substituent form a ring with the α carbon to which they are attached (e.g., C3-C8 ring). in an exemplary embodiment, the first substituent and the second substituent are each independently selected from the group consisting of methyl, ethyl, propyl, and n-butyl, or the first substituent and the second substituent form a ring octane or cycloheptane (e.g., 1-aminocyclooctane-1-carboxylic acid). in an exemplary embodiment, the first substituent is the same as the second substituentIn exemplary embodiments, the first substituent and the second substituent are each C1 or C2 alkyl, in exemplary embodiments, the first substituent and the second substituent are each methyl, such that the α -disubstituted amino acid is α -aminoisobutyric Acid (AIB), in other exemplary embodiments, the α -disubstituted amino acid is 1-aminocyclopropane-1-carboxylic Acid (ACPC), in other exemplary embodiments, the α -disubstituted amino acid may also be a α -disubstituted amino acid having an olefinic side chain5) (S) -2- (4' -pentenyl) alanine (S)5) (S) -2- (4 '-octenyl) alanine (S8), (R) -2- (4' -octenyl) alanine (R8) and 2-amino-2- (4-pentenyl) -6-heptenoic acid (B)5)。
In a preferred embodiment, the invention relates to a polypeptide compound having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID No.1-37 or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof.
In another aspect, the present inventors also cross-link two or more amino acid residues in the polypeptide compounds of the present invention using polypeptide binding techniques to form so-called bound peptides.
As used herein, the term "binding peptide" refers to a peptide that is stabilized in a desired conformation by intramolecular Cross-links in a preferred embodiment, the binding peptide is a peptide that is stabilized in α helix conformation by intramolecular Cross-links the description of binding peptides can be found, for example, in Verdine GL, Hilinski GJ (2012), "stacked peptides for intracellular targets", Methods in enzymology.503: 3-33; Walensky LD, BirdGH (2014), "Hydrocarbon-stacked peptides: primers, practive, and progress", Journal of medical Chemistry,57(15): 6275-88; Blackwell Helbsren E., Grbscrib H, (1998), "high efficiency Synthesis of catalysis synchronization of simulation, simulation 3281", incorporated herein by means of simulation 3223, and simulation of simulation, simulation of proteins, simulation, and simulation, incorporated herein in its entirety.
The present inventors have surprisingly found that a binding peptide comprising an intramolecular cross-linker at a specific amino acid position therein, constructed based on the MERS-CoV HR2 α helix region polypeptide sequence, is a very highly active MERS-CoV fusion inhibitor, which is effective in the treatment and prevention of MERS-CoV infection and diseases associated with MERS-CoV infection.
In particular, the inventors have found that in the polypeptide sequence of formula (I), at X1、X2、X3And X4Formation of crosslinks between two or more of them can result in very highly active MERS-CoV fusion inhibitors, which can be effective in the treatment and prevention of MERS-CoV infection and diseases associated with MERS-CoV infection.
Thus, in one aspect, the invention relates to polypeptide compounds having at least 80% identity to general formula (I):
R1-U-Leu-Asp-Leu-Thr-Tyr-Glu-Xa-Leu-X1-Leu-Gln-X2-Val-Val-Lys-X3-Leu-Asn-Glu-X4-Tyr-W-R2(I)，
stereoisomers thereof, mixtures thereof, pharmaceutically acceptable salts thereof and/or functionally equivalent variants thereof;
wherein XaIs an amino acid residue capable of hydrophobic interaction with MERS-COV HR2, selected from the following D-or L-form hydrophobic amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), norleucine (n-Leu), and methionine (Met);
X1、X2、X3and X4Each is an independently selected D-or L-form amino acid, and is in X1、X2、X3And X4Form a cross-linked body between two or more of them;
u and W are absent or are each independently selected one or more additional D or L amino acids;
R1selected from: h, polymers derived from polyethylene glycol, acyclic substituted orUnsubstituted aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroarylalkyl group, substituted or unsubstituted aryl group, substituted or unsubstituted aralkyl group and R5-CO-, wherein R5Selected from: h, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heterocyclic group, and a substituted or unsubstituted heteroarylalkyl group;
R2selected from: -NR3R4，-OR3and-SR3Wherein R is3And R4Independently selected from: h, a polymer derived from polyethylene glycol, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted aralkyl group; and
R1and R2Is not a D-or L-form amino acid.
In a preferred embodiment, the present invention relates to a polypeptide compound having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to general formula (I) or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof.
In a preferred embodiment, XaAn amino acid selected from the following D-or L-forms: methionine, leucine, isoleucine, valine and norleucine.
In a preferred embodiment, R1Selected from: h, acetyl, lauroyl, myristoyl and palmitoyl. In a preferred embodiment, R1Selected from: h or R5-CO-; wherein R is5Selected from: h or substituted or unsubstituted C1-C24An alkyl group.
In a preferred embodiment, R2Selected from: -NR3R4and-OR3Wherein R is3And R4Independently selected from the group consisting of H, methyl, ethyl, hexyl, dodecyl and hexadecyl. In a preferred embodiment, R2Selected from: -NR3R4OR-OR3Wherein R is3And R4Independently selected from: h or substituted or unsubstituted C1-C24An alkyl group.
In a preferred embodiment, at X1And X2、X1And X3、X2And X3And/or X3And X4Cross-linked bodies are formed between the two. In a preferred embodiment, at X1、X2And X3Cross-linked bodies are formed between the two. In a preferred embodiment, at X1、X3And X4Cross-linked bodies are formed between the two. In a preferred embodiment, at X2、X3And X4Cross-linked bodies are formed between the two. In a preferred embodiment, at X1、X2、X3And X4Cross-linked bodies are formed between the two.
In a preferred embodiment, the polypeptide compounds of the invention comprise one or more residues at X1And X2、X1And X3、X2And X3And X3And X4Cross-linked body formed between them.
Without being bound by theory, it is believed that X in formula (I) is present when the polypeptide compound of the invention is in the α helix conformation1、X2、X3And X4On the side not interacting with the target protein, but exposed on the side in contact with the solvent, and by passing X1、X2、X3And X4May help stabilize the polypeptide compound of the invention in the α helix conformation1、X2、X3And X4May be selected from any amino acids as long as they are capable of linking to each other through a cross-linker as described herein. It is within the ability of one skilled in the art to select the appropriate natural or unnatural amino acid depending on the particular form of crosslinking desiredWithin the range. In such embodiments, at X1、X2、X3And X4The amino acid residues in (b) that do not form cross-links may each independently be selected from any amino acid, preferably from the following D-or L-form amino acids: glutamine, alanine and serine.
As used herein, the term "crosslinker" or grammatical variants thereof refers to an intramolecular linkage of two peptide domains (i.e., e.g., two loops of a helical peptide). when a peptide has a helical secondary structure, the crosslinker can be a macrocycle that is the core of the outer (not part of the helical peptide structure) or the inherent (not crosslinked) helical peptide structure.
Any cross-linker known in the art may be used as long as it is capable of converting X of formula (I)1、X2、X3And X4Two or more amino acids at a position are linked.
In one embodiment, the crosslinker may be a non-covalent or covalent crosslinker.
Any non-covalent cross-linker known in the art may be used as long as it is capable of converting X of formula (I)1、X2、X3And X4Two or more amino acids at a position are linked. In preferred embodiments, the non-covalent crosslinks may be selected from, but are not limited to, crosslinks that contain hydrogen bonds, electrostatic interactions, or metal chelation (see, e.g., V.Azzarito, K.Long, N.S.Murphy and A.J.Wilson, nat. chem.,2013,5, 161-laid 173; V.Haridas, Eur.J.Org.chem.,2009, 5112-laid 5128; all of which are incorporated herein by reference in their entirety).
Any covalently cross-linked body known in the art may also be used as long as it is capable of converting X of the general formula (I)1、X2、X3And X4Two or more amino acids at a position are linked. In a preferred embodiment of the process according to the invention,the covalent crosslinks may be selected from, but are not limited to, crosslinks containing hydrocarbon linkages, intramolecular amide linkages, disulfide linkages, thioether linkages, or Cu (I) -catalyzed azide-alkyne cycloaddition (CuAAC) reactions (see, e.g., YH Lau et al, "Peptide stabilization techniques based on differential catalysis chemistry," chemistry, 2015,46 (15); White CJ, Yudin AK, "conjugation protocols for Peptide catalysis," Nature chemistry,2011,3(7):509, which are all incorporated herein by reference in their entirety).
In one embodiment, X of formula (I) may also be attached using a linker1、X2、X3And X4Examples of such linkers can include, for example, α, an omega-diaminoalkane linker, a 1, 4-diaminopropane linker, or a 1, 5-diaminopentane linker, a photoswitchable linker, a functionalized double-click linker (based on the CuAAC reaction), and the like ((see, e.g., YH Lau et al, supra; White CJ et al, supra)).
In a preferred embodiment, the cross-linker comprising a hydrocarbon linkage is an all-hydrocarbon cross-linker. In a preferred embodiment, at X1、X2、X3And X4The amino acid residue in which the all-hydrocarbon crosslink is formed is an α -disubstituted amino acid having an olefinic side chain1、X2、X3And X4Non-limiting examples of such α -disubstituted amino acids having olefinic side chains can include, for example, (R) -2- (4' -pentenyl) alanine (R)5) (S) -2- (4' -pentenyl) alanine (S)5) (S) -2- (4 '-octenyl) alanine (S8), (R) -2- (4' -octenyl) alanine (R8) and 2-amino-2- (4-pentenyl) -6-heptenoic acid (B)5). For a detailed description of all-carbon crosslinks, see, e.g., Loren D.Walensky and GregoBird H, bird, "Hydrocarbon-staged Peptides: Principles, Practice, and Progress", J Med chem.2014Aug 14; 57(15) 6275-6288, which is incorporated herein by reference in its entirety.
In a preferred embodiment, the present invention relates to a polypeptide compound having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID No.38-69 or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof.
The present inventors have found that the polypeptide compound of the present invention is capable of effectively inhibiting the fusion of MERS-CoV to cells.
Thus, in preferred embodiments, the polypeptide compounds of the invention are useful for inhibiting MERS-CoV fusion. In a preferred embodiment, the polypeptide compounds of the invention are useful for treating or preventing diseases associated with MERS-CoV infection. In a preferred embodiment, the MERS-CoV infection-associated disease is middle east respiratory syndrome.
Thus, in one aspect, the present invention also relates to a pharmaceutical composition comprising at least one polypeptide compound of the present invention or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof, together with at least one pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The peptide itself may be present in the composition in any of a wide variety of forms. For example, two or more peptides may simply be mixed together, or may be more closely associated by complexation, crystallization, or ionic or covalent bonding.
In one aspect, the present invention relates to MERS-CoV fusion inhibitors comprising at least one polypeptide compound of the invention or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof.
The peptide or pharmaceutical composition thereof or MERS-CoV fusion inhibitor thereof as described herein can be administered in a number of ways depending on whether local or systemic administration is desired and depending on the area to be treated. In some embodiments, the peptide or pharmaceutical composition thereof or MERS-CoV fusion inhibitor thereof can be administered to a patient by: oral or rectal, or transmucosal, or enteral, or intramuscular, or subcutaneous, or intramedullary, or intrathecal, or directly intraventricular, or intravenous, or intravitreal, or intraperitoneal, or intranasal, or intraocular.
Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffers, phosphate buffered saline, buffered sodium chloride, vegetable oils, Minimum Essential Media (MEM), MEM with HEPES, and the like.
In one aspect, the invention relates to the use of a polypeptide compound of the invention, or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof, and/or a functionally equivalent variant thereof, for inhibiting MERS-CoV fusion.
In one aspect, the present invention relates to the use of a polypeptide compound of the present invention or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof for the treatment or prevention of a disease associated with MERS-CoV infection.
In a preferred embodiment, the MERS-CoV infection-associated disease is middle east respiratory syndrome.
In one aspect, the invention relates to the use of a polypeptide compound of the invention or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof in the preparation of a MERS-CoV fusion inhibitor.
In one aspect, the present invention relates to the use of a polypeptide compound of the present invention or a stereoisomer thereof, a mixture thereof, a pharmaceutically acceptable salt thereof and/or a functionally equivalent variant thereof for the manufacture of a medicament for the treatment or prevention of a disease associated with MERS-CoV infection.
In a preferred embodiment, the MERS-CoV infection-associated disease is middle east respiratory syndrome.
In one aspect, the present invention relates to a method of preparing a polypeptide compound of the present invention, comprising: a. synthesizing a polypeptide having at least 80% identity to general formula (I), and b1、X2、X3And X4Two or more of which are connected by cross-linking.In a preferred embodiment, X is attached by crosslinking1And X2、X1And X3、X2And X3And/or X3And X4. In a preferred embodiment, X is attached by crosslinking1、X2And X3. In a preferred embodiment, X is attached by crosslinking1、X3And X4. In a preferred embodiment, X is attached by crosslinking2、X3And X4. In a preferred embodiment, X is attached by crosslinking1、X2、X3And X4。
In an embodiment of the invention, the Synthesis of the polypeptide compounds as described herein, their stereoisomers, mixtures thereof, their pharmaceutically acceptable salts and/or functionally equivalent variants thereof can be performed according to any conventional method known in the art, such as Solid phase peptide Synthesis methods [ Stewart j.m.y Young j.d., "Solid phase peptide Synthesis,2 d edition ] (1984), Pierce Chemical Company, Rockford, Illinois; bodanzsky M.y Bodanzsky A., "The practice of Peptide Synthesis", (1994), Springer Verlag, Berlin; lloyd Williams P.et al, "Chemical applications to the Synthesis of Peptides and Proteins" (1997), CRC, Boca Raton, FL, USA ], Synthesis in solution, enzymatic Synthesis [ Kullmann W. "Proteins as catalysts for enzymatic syntheses of Peptides" (1980), J.biol.chem.,255(17),82348238] or any combination thereof. The compounds can also be obtained by fermentation of genetically modified or unmodified bacterial strains, with the aim of producing the desired sequence, or by controlled hydrolysis of proteins of animal, fungal or preferably plant origin, free of peptide stretches containing at least the desired sequence. For example, a nucleic acid sequence encoding a polypeptide amino acid sequence as described herein, and optionally with appropriate amino acid modifications, can be used to produce a compound of the invention.
Merely by way of example, the process for obtaining the polypeptide compounds of the invention, their stereoisomers and mixtures thereof may comprise the following stages:
-coupling the amino acids protected at the N-terminus and free at the C-terminus with the amino acids free at the N-terminus and protected at the C-terminus or bound to a solid support;
-elimination of the group protecting the N-terminus;
-repeating the coupling procedure and eliminating the group protecting the N-terminus until the desired peptide sequence is obtained;
-elimination of a group protecting the C-terminus or cleavage of the solid support;
-optionally, cross-linking two or more amino acids in the peptide sequence by means of a suitable cross-linker.
Preferably, the C-terminus is bound to a solid support and the process is carried out in a solid phase, thus comprising coupling the N-terminally protected and C-terminally free amino acids with the N-terminally free and C-terminally bound amino acids to a polymer support; elimination of the group protecting the N-terminus; and repeating the procedure as many times as necessary to obtain the desired length of compound, ultimately followed by cleavage of the synthesized compound from the initial polymeric support.
Throughout the synthesis, the functional groups of the amino acid side chains remain conveniently protected with temporary or permanent protecting groups and can be deprotected simultaneously or orthogonally to the process of cleaving the peptide from the polymeric support.
Alternatively, solid phase synthesis can be performed with a convergent strategy: the peptide is coupled to a polymeric carrier or to a peptide or amino acid that is previously bound to a polymeric carrier. Convergent Synthesis strategies are widely known to the person skilled in the art and are described in Lloyd-Williams P.et al, ` Convergent Solid-Phase Peptide Synthesis `, (1993), Tetrahedron,49(48), 11065-11133.
The process of the invention can include additional stages of N-terminal and C-terminal deprotection and/or cleavage of the peptide from the polymeric support in no different order, using standard procedures and conditions known in the art; these end-functional groups can be modified thereafter. Optional modification of the N-terminus and C-terminus can be performed while the peptide of formula (I) is immobilized to the polymeric support or once the peptide has been detached from the polymeric support.
Optionally, R1Can be introduced in such a way that: in the presence of a suitable base and solventThe N-terminus of the compound of the present invention is reacted with R by nucleophilic substitution reaction1-X compound reaction, wherein R1Have the foregoing meaning and X is a leaving group such as and not limited to tosyl group, mesyl group, halogen group, and the like; wherein the fragment having a functional group not involved in the formation of an N-C bond is suitably protected with a temporary or permanent protecting group.
Optionally and/or additionally, R2Residues can be introduced as follows: the compound HR is prepared by reacting the compound HR in the presence of a suitable solvent and a base such as N, N-Diisopropylethylamine (DIEA) or triethylamine or an additive such as 1-hydroxybenzotriazole (HOBt) or 1-hydroxyazabenzotriazol (HOAt) and a dehydrating agent such as carbodiimide, uronium salt, phosphonium salt or amidinium salt2Wherein R is2is-OR3、-NR3R4or-SR3Reaction with complementary fragments corresponds to compounds of formula (I) in which R2is-OH or by pre-forming an acid halide with e.g. thionyl chloride, and thereby obtaining a peptide according to the invention of general formula (I) wherein the fragment has functional groups not involved in the N-C bond formation suitably protected with a temporary or permanent protecting group; or alternatively other R2The residues may be introduced by a process in which the simultaneously incorporated peptide is cleaved from the polymeric support.
Those skilled in the art will readily appreciate that the deprotection/cleavage steps at the C-terminus and N-terminus and their subsequent derivatization can be performed in a different order according to procedures known in the art.
The term "protective group" relates to a group that blocks an organic functional group and is capable of being removed under controlled conditions. Protecting groups, their relative reactivity and the conditions under which they remain inert are known to those skilled in the art.
Examples of representative protecting groups for amino groups are inter alia amides such as amidoacetate, amidobenzoic acid, amidopivalate; carbamates such as benzyloxycarbonyl (Cbz or Z), 2-Chlorobenzyl (CIZ), p-nitrobenzyloxycarbonyl (pNZ), tert-butoxycarbonyl (Boc), 2,2, 2-trichloroethoxycarbonyl (Troc), 2- (trimethylsilyl) ethyloxycarbonyl (Teoc), 9-fluorenylmethyloxycarbonyl (Fmoc) or allyloxycarbonyl (Alloc), trityl (Trt), methoxytrityl (Mtt), 2, 4-dinitrophenyl (Dnp), N-1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethyl (Dde), 1- (4, 4-dimethyl-2, 6-dioxo-cyclohexylidene) -3-methylbutyl (ivDde), 1- (1-adamantyl) -1-methylethoxycarbonyl (Adpoc), preferably Boc or Fmoc.
Examples of protective groups representative of carboxyl groups are esters, such as tert-butyl ester (tBu), allyl ester (All), triphenylmethyl ester (Trt ester), cyclohexyl ester (cHx), benzyl ester (Bzl), o-nitrobenzyl ester, p-methoxybenzyl ester, trimethylsilylethyl ester, 2-phenylisopropyl ester, fluorenylmethyl ester (Fm), 4- (N- [1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -3-methylbutyl ] amino) benzyl ester (Dmab), in particular; preferred protecting groups according to the invention are All, tBu, cHx, Bzl and Trt esters.
The side chains of trifunctional amino acids can be protected during the synthetic process with temporary or permanent protecting groups orthogonal to the N-terminal and C-terminal protecting groups.
The hydroxyl group of the tyrosine side chain can be protected with a 2-bromobenzyloxycarbonyl group (2-BrZ), tBu, All, Bzl or 2, 6-dichlorobenzyl (2,6-diclZ) or the like. The histidine side chain can be protected with a protecting group selected from: tos, Dnp, methyl (Me), Boc, benzyloxymethyl (Bom), Bzl, Fmoc, Mts, Trt and Mtt. The amide groups of the glutamine and asparagine side chains can be protected by the Trt group or the xanthenyl group (Xan) or can be used unprotected. For protecting the carboxyl group of the side chains of aspartic acid and glutamic acid, esters such as tBu ester, All ester, triphenylmethyl ester (Trt ester), cHx ester, Bzl ester, o-nitrobenzyl ester, p-methoxybenzyl ester, trimethylsilylethyl ester, 2-phenylisopropyl ester, Fm ester or Dmab ester and the like can be used. The arginine side chain may be protected by a protecting group selected from: tos, 4-methoxy-2, 3, 6-trimethylbenzenesulfonyl (Mtr), Alloc, nitro, 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and 2,2,5,7, 8-pentamethylchroman-6-sulfonyl (Pmc). The indole group of the tryptophan side chain can be protected by a formyl group (For), Boc, Mts or can be used unprotected. To protect the amino group of the lysine side chain, amides such as acetic acid amide, benzoic acid amide, pivalic acid amide; carbamates such as Cbz or Z, ClZ, pNZ, Boc, Troc, Teoc, Fmoc or Alloc, Trt, Mtt, Dnp, Dde, ivDde, Adpoc, and the like. The methionine side chain can be used in sulfoxide form, in sulfone form or unprotected. The methionyl (sulfoxide) and methionyl (sulfone) side chains are unprotected.
In a preferred embodiment, the protecting group strategy used is the following: the amino group is protected by Boc, the carboxyl group by Bzl, cHx or All, the tyrosine side chain by 2-BrZ or Bzl, the histidine side chain by Tos or Bom, the aspartic and glutamic acid side chains by Bzl, cHx or All, glutamine and asparagine used without side-chain protection, methionine used without side-chain protection, the arginine side chain by Tos, the tryptophan side chain by For or Mts and the lysine side chain by ClZ, Fmoc or Alloc.
In a further preferred embodiment, the protecting group strategy used is the following: the amino group is protected by Fmoc, the carboxyl group by tBu, All or Trt ester, the tyrosine side chain by tBu, the histidine side chain by Trt or Mtt group, the aspartic acid and glutamic acid side chains by tBu or All, glutamine and asparagine by the side chain Trt group, methionine is used without side chain protection, the arginine side chain by Pmc or Pbf, the tryptophan side chain is used with Boc protection or without protection, and the lysine side chain is protected by Boc, Trt or Alloc.
Examples of these and other protective groups, their introduction and removal, can be found in the literature [ Atheron B.and Shepard R.C., "Solid Phase Peptide Synthesis: A reactive approach", (1989), IRL Oxford University Press ]. The term "protecting group" also includes polymeric supports in solid phase synthesis.
Possible solid supports for use in the process of the invention in the case of synthesis occurring wholly or partly in the solid phaseThe body involves a polystyrene support, polyethylene glycol grafted to polystyrene, and the like, such as, but not limited to, p-methylbenzhydrylamine resin (MBHA) [ Matsueda G.R.et al, "A p-methylene benzyl amine resin for improved-phase synthesis of peptide amides", (1981), Peptides,2,4550]2-Chlorotriphenylmethyl resin [ Barlos K.et al, "Darstellung gesch ü tzer PeptideFragmente und Einstatzsuturerter triphenylmethylHarze", (1989), Tetrahedron Lett.,30,39433946; Barlos K.et al, "" Veresterung von Partiell gesch ü tzten Peptidegement Harmit]，
Figure BDA0001601004420000161
Resin (Rapp Polymer GmbH),
Figure BDA0001601004420000162
resins (Matrix Innovation, Inc.), and the like, which may or may not include labile linkers such as 5- (4-aminomethyl-3, 5-dimethoxyphenoxy) Pentanoic Acid (PAL) [ Albericio F. et al, "" preparation and application of the 5- (4- (9-fluoro methyl-phenoxy) aminomethyl-3, 5-dimethyl-phenoxy) valric acid (PAL) handle for the soluble-phase synthesis of C-basic peptide amides units derivatives "" (1990), J.Org.Chem.,55,37303743]2- [ 4-aminomethyl- (2, 4-dimethoxyphenyl)]Phenoxyacetic Acid (AM) [ Rink H., "Solid-phase synthesis of protected peptide fragments using a trialkyl-diphenyl-methyl ester resin", (1987), Tetrahedron Lett.,28,3787-],Wang[Wang S.S.,"p-Alkoxybenzyl Alcohol Resin and p-Alkoxybenzyloxycarbonylhydrazide Resin forSolid Phase Synthesis of Protected Peptide Fragments",(1973),J.Am.Chem.Soc.,95,1328-1333]Etc. which allow for simultaneous deprotection and cleavage of the peptide from the polymeric support.
Definition of
The abbreviations used in the present invention have the following meanings:
ala (Alanine, A) Alanine
Asn (Asparagine, N) Asparagine
Asp (Aspartic acid, D) Aspartic acid
B5(2-amino-2- (4-pentenyl) -6-heptanoic acid) 2-amino-2- (4-pentenyl) -6-heptenoic acid
DCM (Dichloromethane) dichloroethane
DMF (N, N-Dimethyl malonate) dimethylformamide
Fmoc (fluoromethoxy) fluorenylmethyloxycarbonyl
Gln (Glutamine, Q) Glutamine
Glu (Glutamic acid, E) Glutamic acid
Gly (Glycine, G) Glycine
HBTU 2- (1H-1-hydroxybenzotriazole) -1,1,3, 3-tetramethyluronium hexafluorophosphate
His (Histidine, H) Histidine
HOBt (1-Hydroxybenzotriazol anhydrous) 1-hydroxybenzotriazole
HR1 (NHR) N-terminal repeat
HR2(C-terminal peptide repeat, CHR) C terminal repetitive sequence
HPLC (high Performance liquid chromatography) high Performance liquid chromatography
Ile (Isoleucine, I) Isoleucine
Leu (Leucine, L) Leucine
Lys (Lysine, K) Lysine
MERS (middle East respiratory syndrome) middle East respiratory syndrome
MERS-CoV middle east respiratory syndrome coronavirus
Met (Methionine, M) Methionine
nIle (Norleucine, Z) Norleucine
R5((R) -2- (4 '-pentenyl) alanine) (R) -2- (4' -pentenyl) alanine
S5((S) -2- (4 '-pentenyl) alanine) (S) -2- (4' -pentenyl) alanine
S8((S) -2- (4 '-octyl) alanine) (S) -2- (4' -octenyl) alanine
Ser (S) Serine
TFA (trifluoroacetic acid) trifluoroacetic acid
Thr (Threonine, T) Threonine
Tyr (Tyrosine, Y) Tyrosine
Val (Valine, V) Valine
As defined herein, the terms "polypeptide," "protein," "peptide," and "amino acid sequence" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogues, and it may be interrupted by chemical moieties other than amino acids. The term also includes amino acid polymers that have been naturally or artificially modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a label or bioactive component). The term "peptide" includes two or more naturally occurring or synthetic amino acids linked by a covalent bond (e.g., an amide bond).
In the context of the present disclosure, the term "amino acid" is defined as having at least one primary, secondary, tertiary or quaternary amino group and at least one acid group, wherein the acid group may be a carboxylic acid, sulfonic acid or phosphoric acid or mixtures thereof the amino group may be "α", "β", "γ" to "ω" relative to the acid group suitable amino acids include, but are not limited to, the D-and L-isomers of the 20 common naturally occurring amino acids found in peptides (e.g., a, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V (as known by single letter or three letter abbreviations)) as well as naturally occurring and non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways.
The backbone of an "amino acid" may be substituted with one or more groups selected from halogen, hydroxyl, guanidino, heterocyclic groups. Thus the term "amino acid" also includes within its scope glycine, alanine, valine, leucine, isoleucine, norleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamine, lysine, arginine, histidine, taurine, betaine, N-methylalanine and the like. Amino acids in the forms (L) and (D) are included.
The term "amino acid side chain" refers to the moiety attached to the α -carbon of an amino acid.A.i., alanine amino acid side chain is methyl, phenylalanine amino acid side chain is phenylmethyl, cysteine amino acid side chain is thiomethyl, aspartic acid amino acid side chain is carboxymethyl, tyrosine amino acid side chain is 4-hydroxybenzyl, and the like.
As used herein, the term "olefinic" and synonymous variations thereof (e.g., alkene or alkene groups) denotes a monovalent group derived from a straight or branched chain hydrocarbon moiety having at least one carbon-carbon double bond by removal of a single hydrogen atom. The alkenyl moiety contains the indicated number of carbon atoms. E.g. C2-C10Meaning that the group may have 2 to 10 carbon atoms. This means that it has C2，C3，C4，C5，C6，C7，C8，C9Or C10An alkenyl group. The term "lower alkenyl" means C2-C8An alkenyl chain. This means that it has C2，C3，C4，C5，C6，C7Or C8A lower alkenyl group. In thatWithout any numerical designation, "alkenyl" is a chain (straight or branched) having 2 to 20 carbon atoms therein.
The ethylenic group includes, for example, an ethenyl group, a propenyl group, a butenyl group, a 1-methyl-2-buten-1-yl group and the like which may have one or more substituents. Olefinic group substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety. Examples of substituents include, but are not limited to, the following groups: aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, oxo, imino, thioxo, cyano, isocyano, amino, azido, nitro, alkylamino, heteroalkylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, aliphatic thioxo, heteroaliphatic thioxo, alkylthioxo, heteroalkylthioxo, arylthioxo, heteroarylthioxo, each of which may or may not be further substituted.
As used herein, the term "acyclic aliphatic group" encompasses linear or branched alkyl, alkenyl, and alkynyl groups.
The term "alkyl" refers to a linear or branched saturated group having from 1 to 24, preferably from 1 to 16, more preferably from 1 to 14, even more preferably from 1 to 12, still more preferably 1, 2,3, 4, 5 or 6 carbon atoms and bonded to the rest of the molecule by a simple bond, including for example and without limitation, methyl, ethyl, isopropyl, isobutyl, tert-butyl, heptyl, octyl, decyl, dodecyl, lauryl, hexadecyl, octadecyl, pentyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexyl and the like.
The term "alkenyl group" refers to a linear or branched group having from 2 to 24, preferably from 2 to 16, more preferably from 2 to 14, even more preferably from 2 to 12, still more preferably 2,3, 4, 5 or 6 carbon atoms, having one or more carbon-carbon double bonds, preferably having 1, 2 or 3 carbon-carbon double bonds, the bonds being conjugated or unconjugated, which is bonded to the rest of the molecule by a simple bond, including for example and without limitation vinyl (-CH)2＝CH2) Allyl (-CH)2-CH＝CH2) Oleyl radicalLinoleylene group, and the like.
The term "alkynyl group" refers to a linear or branched group having from 2 to 24, preferably from 2 to 16, more preferably from 2 to 14, even more preferably from 2 to 12, still more preferably 2,3, 4, 5 or 6 carbon atoms, having one or more carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds, the bonds being conjugated or unconjugated, which is bonded to the rest of the molecule by a simple bond, including for example and without limitation, ethynyl, 1-propynyl, 2-propynyl, 1-butyl, 2-butyl, 3-butyl, pentyl, such as 1-pentyl, and the like. Alkynyl groups can also contain one or more carbon-carbon double bonds, including for example and without limitation the groups but-1-en-3-ynyl, pent-4-en-1-ynyl, and the like.
The term "alicyclic group" is used herein to encompass, for example and without limitation, cycloalkyl or cycloalkenyl or cycloalkynyl groups.
The term "cycloalkyl" refers to a saturated monocyclic or polycyclic aliphatic group having 3 to 24, preferably 3 to 16, more preferably 3 to 14, even more preferably 3 to 12, still more preferably 3, 4, 5 or 6 carbon atoms and bonded to the rest of the molecule by a simple bond, including for example and without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclohexyl, dimethylcyclohexyl, octahydroindene, decahydronaphthalene, dodecahydrophenalene and the like.
The term "cycloalkenyl" refers to a non-aromatic mono-or polycyclic aliphatic group having 5 to 24, preferably 5 to 16, more preferably 5 to 14, even more preferably 5 to 12, still more preferably 5 or 6 carbon atoms, having one or more carbon-carbon double bonds, preferably 1, 2 or 3 carbon-carbon double bonds, the bonds being conjugated or unconjugated, which is bonded to the rest of the molecule by simple bonds, including for example and without limitation cyclopent-1-en-1-yl groups and the like.
The term "cycloalkynyl" refers to a non-aromatic mono-or polycyclic aliphatic group having from 8 to 24, preferably from 8 to 16, more preferably from 8 to 14, even more preferably from 8 to 12, still more preferably 8 or 9 carbon atoms, having one or more carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds, the bonds being conjugated or unconjugated, which is bonded to the remainder of the molecule by a simple bond, including for example and without limitation, cyclooct-2-yn-1-yl groups and the like. Cycloalkynyl groups can also contain one or more carbon-carbon double bonds, including for example and without limitation, cyclooct-4-en-2-ynyl groups and the like.
The term "aryl group" refers to an aromatic group having 6 to 30, preferably 6 to 18, more preferably 6 to 10, still more preferably 6 or 10 carbon atoms, containing 1, 2,3 or 4 aromatic rings, bonded or fused by a carbon-carbon bond, including for example and without limitation phenyl, naphthyl, diphenyl, indenyl, phenanthryl or anthryl, and the like; or an aralkyl group.
The term "aralkyl" refers to an alkyl group substituted with an aromatic group, having from 7 to 24 carbon atoms and including, for example and without limitation, - (CH)2)1-6-phenyl, - (CH)2)1-6- (1-naphthyl), - (CH)2)1-6- (2-naphthyl), - (CH)2)1-6-CH (phenyl)2And the like.
The term "heterocyclyl group" refers to a 3-to 10-membered hydrocarbonized ring in which one or more of the atoms in the ring, preferably 1, 2 or 3 of the atoms in the ring, is an element other than carbon, such as nitrogen, oxygen or sulfur, and may be saturated or unsaturated. For the purposes of the present invention, a heterocycle can be a monocyclic, bicyclic or tricyclic ring system, which may include fused ring systems; and the nitrogen, carbon or sulfur atom in the residual heterocycle may be optionally oxidized; the nitrogen atoms may be optionally quaternized; and the residual heterocyclic group may be partially or fully saturated or aromatic. The term heterocyclyl most preferably refers to a 5 or 6 membered ring. Examples of saturated heterocyclyl groups are dioxane, piperidine, piperazine, pyrrolidine, morpholine and thiomorpholine. Examples of aromatic heterocyclyl radicals, also referred to as heteroaromatic radicals, are pyridine, pyrrole, furan, thiophene, benzofuran, imidazoline, hydroquinone, quinoline, pyridazine and naphthyridine.
The term "heteroarylalkyl group" refers to an alkyl group substituted with a substituted or unsubstituted aromatic heterocyclyl group, the alkyl group having from 1 to 6 carbon atoms and the aromatic heterocyclyl group having from 2 to 24 carbon atoms and from 1 to 3 atoms other than carbon, and includes, for example and without limitation, - (CH)2)1-6-imidazolyl, - (CH)2)1-6-triazolyl, - (CH)2)1-6-a thienyl group,-(CH2)1-6-furyl, - (CH)2)1-6Pyrrolidinyl and the like.
The term "halogen" or variants such as "halide" or "halo" as used herein refers to fluorine, chlorine, bromine and iodine.
The term "heteroatom" or variants such as "hetero-" as used herein refers to O, N, NH and S.
The term "alkoxy" as used herein refers to a straight or branched chain alkoxy group. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy and the like.
The term "amino" as used herein refers to-NRaRbA radical of the form (I) in which RaAnd RbIndependently selected from the group including, but not limited to, hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl.
It is to be understood that the compounds of the present invention as described herein can each be substituted with any number of substituents or functional moieties. Generally, the term "(substituted)" (whether or not it follows the term "optionally") and substituents contained in the formulae of the present invention all refer to the replacement of a hydrogen radical in a given structure with the radical of the indicated substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at each position. The term "substituted" as used herein is intended to include substitution with all permissible substituents of organic compounds, any of which are described herein.
For example, substituents include, but are not limited to, the following groups that result in the formation of a stabilizing moiety: aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiocarbonyl, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, and halo, and any combination thereof, including but not limited to the following groups: aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, aliphatic thio, heteroaliphatic thio, alkylthio, heteroalkylthio, arylthio, heteroarylthio, acyloxy and the like. The present invention encompasses any and all such combinations to obtain stable substituents/moieties. For purposes of the present invention, a heteroatom such as nitrogen may have a hydrogen substituent and/or any suitable substituent as described herein that satisfies the valence of the heteroatom and results in the formation of a stable moiety.
The compounds may contain one or more asymmetric centers and thus exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included herein. The compounds may also be represented in multiple tautomeric forms, in which case all tautomeric forms of the compounds described herein are expressly included herein (e.g., alkylation of a ring system may result in alkylation of multiple sites, all such reaction products are expressly included herein). All such isomeric forms of such compounds are expressly included herein. All crystal forms of the compounds described herein are expressly included herein.
The term "functionally equivalent variants", as used in the present invention, refers to all those compounds derived from the sequences of the compounds of the invention which are modified by substitution of one or two amino acids, provided that the mentioned compounds maintain at least 20%, at least 50%, at least 80% of the function based on the function of the corresponding non-modified compound. In a particular embodiment, the function of the compounds of the invention and their functionally equivalent variants is determined by quantifying their ability to inhibit neuronal exocytosis, in particular by quantifying the inhibition of SNARE complex formation. The primary function of SNARE fusion complexes is to allow neurotransmitter-filled vesicles to approach and contact presynaptic cellular membranes and to allow calcium-mediated fusion of those vesicles, which results in neurotransmitter release. However, in the case of addition of a compound or functionally equivalent variant that inhibits the formation of the SNARE complex, the concentration of the released neurotransmitter is reduced relative to cells that have not been treated with the inhibitory compound or functionally equivalent variant. In the context of the present invention, modifications of the compounds of the invention to produce functionally equivalent variants are carried out as follows: one or both amino acids of the peptide are replaced with one of the amino acids whose corresponding properties are equivalent. The following amino acids are considered characteristic equivalents: glutamic acid (E) and aspartic acid (D); threonine (T) and serine (S); valine (V), leucine (L), isoleucine (I) and norleucine (n-L); asparagine (N) and glutamine (Q); lysine (K), arginine (R) and histidine (H) and finally the aromatic amino acids phenylalanine (F), tryptophan (W) and tyrosine (Y).
The compounds of the invention or functionally equivalent variants are considered to be conditions which inhibit the formation of the SNARE complex: the formation of SNARE complexes is reduced by at least 15%, 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 100% relative to a cell control to which any inhibitory compound has been added.
The compounds of the present invention can exist as stereoisomers or mixtures of stereoisomers; for example, the amino acids constituting them can have, independently of one another, the configuration L-, D-or racemic. It is thus possible to obtain isomeric mixtures as well as racemic mixtures or mixtures of diastereomers, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and the asymmetric carbons present in the isomeric or isomeric mixtures. Preferred structurally pure isomers of the compounds of the invention, i.e. enantiomers or diastereomers.
For example, in the description of AA1In the case of being able to be-Lys-, AA is to be understood1Selected from-L-Lys-, -D-Lys-, or a mixture of both, racemic or non-racemic. The preparation procedures described in this document allow the person skilled in the art to obtain the respective stereoisomers of the compounds of the invention by selecting amino acids of the correct configuration.
Pharmaceutically acceptable salts of the peptides of the invention are also within the field of the invention. The term "pharmaceutically acceptable salt" means a salt whose use in animals and more particularly in humans is recognized and includes salts used to form base addition salts, whether they are inorganic or organic, inorganic salts such as and not limited to lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum, and the like, or organic salts such as and not limited to ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine, and the like; or acid addition salts, whether they are organic or inorganic, organic such as and not limited to acetate, citrate, lactate, malonate, maleate, tartrate, fumarate, benzoic, aspartate, glutamate, succinate, oleate, trifluoroacetate, oxalate, pamoate or gluconate and the like, or inorganic such as and not limited to chloride, sulfate, borate or carbonate and the like. The nature of the salt is not critical provided that it is cosmetically or pharmaceutically acceptable. Pharmaceutically acceptable Salts of the peptides of the invention can be obtained by conventional methods well known in the art (Berge s.m. et al, "Pharmaceutical Salts", (1977), j.pharm.sci.,66,119, which is incorporated herein by reference in its entirety).
Examples
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The solid phase synthesis carrier Rink amide resin used in the examples is Tianjin Nankai synthesis responsibility Co., Ltd; HBTU, HOBt, DIEA and Fmoc protected natural amino acids or D-form unnatural amino acids were produced by Gill Biochemical Co, Shanghai and Kyokanas technologies, Inc., Beijing Ohio. N-methylpyrrolidone (NMP) is a product of ACROS company; trifluoroacetic acid (TFA) is a product of Beijing Bomeijie science and technology Limited; DMF and DCM are products of Samsung Corp.Korea; the chromatographic pure acetonitrile is a product of Fisher company. Other reagents are domestic analytical pure products unless otherwise specified.
Examples 1 to 37: preparation of Compounds 1-37
Polypeptide synthesis was performed using standard Fmoc solid phase methods. Rink Amide resin is selected, and a peptide chain is extended from a C end to an N end. The condensing agent is HBTU/HOBt/DIEA. The deprotection agent is piperidine/DMF solution. The lysate is trifluoroacetic acid (TFA), and the crude peptide is dissolved in water and then freeze-dried for storage. Separating and purifying by medium pressure liquid chromatography or High Pressure Liquid Chromatography (HPLC), wherein the pure peptide content is more than 90%. And determining the molecular weight of the peptide sequence by matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS).
Peptide sequences were synthesized using a CEM microwave polypeptide synthesizer.
The synthesis conditions were as follows:
protection of amino acids: 0.2M of a solution in DMF,
0.45M HBTU/HOBt in DMF as activator,
activating alkali: a 2M solution of DIEA in NMP,
deprotection agent: 20% v/v piperidine in DMF,
blocking reagent: 20% v/v acetic anhydride in DMF.
Weighing 0.23g (0.1mmol) of Rink Amide resin, placing the Rink Amide resin in a reactor of a CEM microwave polypeptide synthesizer, preparing amino acid, an activating agent, activated alkali, a deprotection reagent and a blocking reagent according to the concentration, and synthesizing by using the CEM microwave full-automatic polypeptide synthesizer. After completion, the peptide resin was washed with DMF for 3 times, shrunk with anhydrous methanol, and vacuum-dried at room temperature to obtain 1.25g of peptide resin.
Lysis buffer (volume percent): trifluoroacetic acid: m-cresol: thioanisole: water 85:5:5: 5.
Cleavage of peptide resin: weighing 1.25g of peptide resin synthesized by a microwave synthesizer, putting the peptide resin into a 250ml eggplant-shaped bottle, carrying out ice bath, and carrying out electromagnetic stirring. The lysate was prepared by adding 10ml of 1g of peptide resin. The TFA needs to be cooled for 30min in an ice bath in advance or stored in a refrigerator for use in advance; adding the prepared lysate into peptide resin under ice bath condition, electromagnetically stirring, allowing the resin to turn orange red, reacting for 30min under ice bath condition, removing the ice bath, continuing stirring at room temperature for reacting for 90min, after the reaction is completed, adding 200ml of cold diethyl ether under vigorous stirring, separating out white precipitate, and continuing stirring for 30 min; the precipitate was filtered off using a G4 sand-core funnel, washed repeatedly with cold ether 3 times and dried. And adding 50ml of double distilled water and 5ml of acetonitrile to fully dissolve the solid, performing suction filtration, and freeze-drying the filtrate to obtain 1.04g of crude peptide.
Purification of the crude peptide: the crude peptide is purified by medium or high pressure chromatography. The chromatographic column is C18 column, and the eluent is acetonitrile, water and small amount. The method comprises the following specific operation steps: 1.00g of crude peptide was weighed, 20ml of water and 5ml of acetonitrile were added to dissolve the solid, and the mixture was centrifuged for 10min (3000 rpm) to obtain a supernatant. The column was equilibrated beforehand with 200ml of a 15% acetonitrile/water/0.1% glacial acetic acid solution. After the sample is loaded, the sample is continuously washed by 200ml of 15 percent acetonitrile/water/0.1 percent glacial acetic acid solution, and the eluent components are detected by a high performance liquid phase. And gradually increasing the acetonitrile content according to the liquid phase detection result until the main peak of the purified polypeptide is eluted. Combining the eluates, rotary evaporating to remove most solvent, lyophilizing pure polypeptide, detecting content by HPLC (high performance liquid chromatography) of more than 90%, and determining molecular weight by MALDI-TOF.
Examples 38 to 57: preparation of Compounds 38-57
Figure BDA0001601004420000261
The polypeptide synthesis method is the same as example 1, except that after the linear polypeptide is completed, solid-phase olefin metathesis reaction is carried out, and side chain olefin groups are covalently linked to synthesize the binding peptide. The method comprises the following specific steps: transferring the polypeptide resin into a light-proof eggplant-shaped bottle, introducing nitrogen for protection for 5min, weighing a first-generation Grubbs catalyst of 25mg, dissolving the Grubbs catalyst in 5ml of dichloroethane, adding the Grubbs catalyst into a reaction system, stirring for 6h under the protection of the nitrogen, and repeating once after the reaction is finished. After the reaction, the reaction mixture was washed with DMF for 3 times, then contracted with anhydrous methanol, and vacuum-dried at room temperature to obtain 1.02g of peptide resin, and the cleavage purification method was the same as in example 1.
Examples 58 to 69: preparation of Compounds 58-69
Figure BDA0001601004420000262
The polypeptide synthesis method is the same as that in example 1, in the subsequent solid-phase olefin metathesis reaction, the feeding amount of the catalyst needs to be increased, and the specific steps are as follows: transferring the polypeptide resin into a light-proof eggplant-shaped bottle, introducing nitrogen for protection for 5min, weighing a first-generation Grubbs catalyst 38mg, dissolving in 8ml dichloroethane, adding the reaction system, stirring for 6h under the protection of nitrogen, and repeating once after the reaction is finished. After the reaction, the mixture was washed with DMF for 3 times, then contracted with anhydrous methanol, and vacuum-dried at room temperature to obtain 1.01g of peptide resin, and the cleavage purification method was the same as in example 1.
Example 70: evaluation of the Activity of Compounds to inhibit MERS-CoV S2 subunit-mediated cell fusion (IC)50)
1) The first day: digesting 293T and preparing cell suspension, adding 2-4 multiplied by 10 to each well in a 6-well plate5And (4) cells.
2) The next day: after culturing for 12-24 h, performing transfection when the density is about 80%, wherein one hole is transfected with pAAV-IRES-EGFP-MERS-S plasmid (293T/MERS/EGFP) for fusion experiment; one well of the transfected pAAV-IRES-EGFP (293T/EGFP) was used as a negative control.
3) And (4) after 8-12 h of transfection, replacing a fresh DMEM cell culture solution.
4) The fourth day: after transfection for 36-48 h, the cells can express stronger fluorescence, and a subsequent fusion inhibition test can be carried out.
5) Digesting Huh-7 cells 6-12 h before fusion to prepare cell suspension, and adding 4-5 multiplied by 10 to each hole4And (4) cells. Culturing at 37 deg.C for use.
6) Prepare the polypeptide with gradient dilution, each hole 55 u L reaction system.
7) 293T/MERS/EGFP cells, and 293T/EGFP cells were digested with EDTA. A single cell suspension was prepared.
8) Add 10 per well to drug plate4And 293T/MERS/EGFP cells. Meanwhile, culture wells without drugs are arranged to serve as positive controls (293T/MERS/EGFP + Huh-7), and 293T/EGFP cell wells are arranged to serve as negative controls (293T/EGFP + Huh-7). The final volume was controlled at 110. mu.L.
9) Incubating at room temperature for 30min to allow the drug to fully act on the cells.
10) The culture medium of Huh-7 cells was removed and 100. mu.L of the mixture of drug and cells was added to the Huh-7 cells.
11) And (3) observing the fusion condition for 24h, adding equal volume of 4% of poly formic acid for fixation when the positive control group (293T/MERS/EGFP + Huh-7) cells have obvious fusion, and stopping the fusion.
12) And (4) carrying out photographing analysis by using a fluorescence microscope or a high content screening system. Calculating the inhibition rate of the polypeptide on cell fusion, and calculating the IC of the polypeptide by using CalcuSyn software50The value is obtained.
The results of the activity test according to the above method are shown in Table 1 below (in which the residues in italics in the sequences numbered 38-69 represent the non-natural amino acid residues after crosslinking).
Table 1: inhibitory Activity (IC) against MERS-CoV S2 protein-mediated cell fusion50)
Figure BDA0001601004420000271
Figure BDA0001601004420000281
Figure BDA0001601004420000291
Figure BDA0001601004420000301
Figure BDA0001601004420000311
Figure BDA0001601004420000321
As can be seen from the activity results in Table 1, all peptide compounds showed inhibition of MERS-CoV cell fusion activity, with compounds 36-38, 46-48, 67 inhibiting MERS-CoV fusion activity to low μ M levels, comparable to the positive control HR2P-M2 (compound 70).
Example 71: circular dichroism determination of the conformation of compounds
A small amount of the polypeptide was dissolved in 50mM PBS (pH 7.2), the supernatant was taken after sufficient dissolution, the concentration of the polypeptide stock solution was calibrated, diluted to a final concentration of 50. mu.M, and the ellipticity was measured by a circular dichrograph, and the percentage of helicity of the sample was calculated using a molar ellipticity peak at 222nm equal to 33000 as 100% α -helices.
Table 2:
Figure BDA0001601004420000322
Figure BDA0001601004420000331
Figure BDA0001601004420000341
Figure BDA0001601004420000351
as can be seen from the results of table 2, the peptide compounds of the present invention all exhibited a certain degree of α helicity.
Example 72: preparation and Activity detection of Compounds 71-78
Compounds 71-78 shown in Table 3 below were prepared in the same manner as described in examples 1-69 above, and tested for inhibition of MERS-CoV S2 subunit-mediated cell fusion activity (IC) according to the method described in example 7050) The results are shown in Table 3.
Table 3: inhibitory Activity (IC) against MERS-CoV S2 protein-mediated cell fusion50)
Compound (I) Sequence of IC50(μM)
71 L-LDLTYEMLSLQS5VVKS5LNESY 3.75±0.75
72 L-LDLTYEMLSLQQVVKS5LNES5Y 0.63±0.12
73 TL-LDLTYEM LSLQS5VVKS5LNESY 4.56±1.03
74 TL-LDLTYEM LSLQQVVKS5LNES5Y 2.68±0.92
75 LDLTYEMLSLQS5VVKS5LNESY-I 5.24±1.32
76 LDLTYEMLSLQQVVKS5LNES5Y-I 2.65±0.51
77 LDLTYEMLSLQS5VVKS5LNESY-ID 0.87±0.14
78 LDLTYEMLSLQQVVKS5LNES5Y-ID 1.02±0.33
As is clear from the results in Table 3, the addition of additional amino acids to the N-terminus or C-terminus of the peptide compounds 21 of examples 1-69 did not affect the inhibitory activity thereof.
Example 73: preparation and Activity detection of Compounds 79-81
Compounds 79 to 81 shown in Table 3 below were prepared in the same manner as described in examples 1 to 69 above, and tested for inhibition of MERS-CoV S2 subunit-mediated cell fusion activity (IC) according to the method described in example 7050) The results are shown in Table 4.
Table 4: inhibitory Activity (IC) against MERS-CoV S2 protein-mediated cell fusion50)
Figure BDA0001601004420000352
Figure BDA0001601004420000361
From the results in Table 4, it is clear that the amino acid substitution of the amino acid at the position not corresponding to X in the formula (I) in the peptide 21 compounds of examples 1 to 69 did not affect the inhibitory activity.
Example 74: preparation and Activity detection of Compounds 82-85
Compounds 82-85 shown in Table 5 below were prepared in the same manner as described in examples 1-69 above, and tested for inhibition of MERS-CoV S2 subunit-mediated cell fusion activity (IC) according to the method described in example 7050) The results are shown in Table 5.
Table 5: inhibitory Activity (IC) against MERS-CoV S2 protein-mediated cell fusion50)
Compound (I) Sequence of IC50(μM)
82 LDLTYEZ LSLQS5VV KS5LNESY 0.641±0.005
83 LDLTYEI LSLQS5VV KS5LNESY 0.248±0.009
84 LDLTYEL LSLQS5VV KS5LNESY 2.233±0.004
85 LDLTYEV LSLQS5VV KS5LNESY 3.219±0.017
From Table 5 and the results in Table 1, it is clear that X in the formula (I)aCan be any hydrophobic amino acid without affecting the inhibitory activity of the polypeptide compound.
Example 75: preparation and Activity detection of Compound 86
The same procedures as described in examples 1 to 69 above were used to prepare the compound 86 shown in Table 6 below, and the compound was tested for its inhibition of MERS-CoV S2 subunit-mediated cell fusion activity (IC) according to the procedure described in example 7050) The results are shown in Table 6.
Table 6: inhibitory Activity (IC) against MERS-CoV S2 protein-mediated cell fusion50)
Compound (I) Sequence of IC50(μM)
86 LDLTYEE LSLQS5VV KS5LNESY >50
From the results in Table 6, it is found that X in the formula (I)aIn the case of non-hydrophobic amino acids, the inhibitory activity of the polypeptide compound is lost. These results demonstrate that X of formula (I) of the present inventionaSelected from hydrophobic amino acids and not from non-hydrophobic amino acids.
Example 76: preparation and Activity detection of Compounds 87-89
Compounds 87-89 shown in Table 6 below were prepared in the same manner as described in examples 1-69 above, and tested for inhibition of MERS-CoV S2 subunit-mediated cell fusion activity (IC) according to the method described in example 7050) The results are shown in Table 7.
Table 7: inhibitory Activity (IC) against MERS-CoV S2 protein-mediated cell fusion50)
Compound (I) Sequence of IC50(μM)
87 LDLTYEM LSLQBVV KBLNESY 2.54±0.38
88 LDLTYEM LBLQBVV KALNESY 6.66±2.15
89 LDLTYEM LSLQQVV KBLNEBY 3.04±1.41
Wherein B is α -aminoisobutyric acid
Example 76: pseudoviral Activity assay
The present inventors also performed a pseudoviral activity test on the polypeptide compounds of the present invention to demonstrate from the viral level that the short peptides are able to inhibit MERS-CoV infection with an effect comparable to the positive control HR2P-M2 (i.e. compound 70) (table 8).
Packaging of MERS-CoV pseudoviruses
1. 293T cells were digested 16h before transfection and plated in 10cm tissue culture dishes (2X 10)6/dish)。
2. Cells were replaced with pre-warmed fresh DMEM medium 2h prior to transfection.
3. Eukaryotic expression plasmid pcDNA3.1-MERS-S encoding MERS-CoV S protein, and HIV-1 genomic plasmid (pNL4-3.luc. RE) encoding luciferase lacking Env were co-transfected with calcium phosphate transfection reagents, each plasmid was transfected with 20. mu.g.
4. After 8-10 h of transfection, 10mL of fresh DMEM culture solution containing 10% FBS and 1% penicillin streptomycin was replaced.
After 5.72 h, transfection supernatants containing MERS-CoV pseudovirus were collected.
Cell debris was removed by centrifugation at 6.4000 rpm for 4min, filtered through a 0.45 μm sterile filter, aliquoted and stored at-80 ℃ until needed.
MERS-CoV pseudovirus titer detection
1. Huh-7 cells were digested 12h in advance, and a single cell suspension was prepared using DMEM and cell concentration was adjusted.
104 cells were added per well in 2.96 well plates. 5% CO at 37 ℃2Culturing for 12h for later use.
3. Pseudoviruses were diluted in 96-well plates using DMEM medium in a 2-fold gradient.
4. The cell culture supernatant of Huh-7 in the 96-well plate was removed, and 100. mu.L of the diluted virus dilution was added.
5. 37℃5％CO2After 12h of culture, virus solution was removed and 200. mu.L of fresh DMEM medium was replaced.
6. After 72 hours of virus addition, the supernatant was aspirated off and the fluorescence value was determined.
7. The virus dilution with the fluorescence value 1000 times larger than that of the blank control is selected for subsequent pseudo virus neutralization experiments.
Firefly enzyme (luciferase) detection
1. The 5 × cell lysate was diluted to 1 × working concentration using ddH 2O. Equilibrating to room temperature.
2. The medium of the cells to be tested in the 96-well plate was carefully aspirated off, the cells were washed once with PBS, the action was gentle to prevent cell shedding, and PBS was removed as much as possible.
3. And adding 20 mu L of 1 multiplied cell lysate into each hole, and then placing the hole on a vortex mixer to shake and mix uniformly for 30-60 min until the cells are completely lysed.
4. Draw 20. mu.L of lysate to a 96-well opaque white microplate.
5. Add 100. mu.L firefly enzyme assay reagent to each well.
6. Detection of fluorescence in multifunctional microplate reader
Polypeptide activity detection
1. Polypeptides were solubilized using DMSO and the polypeptide concentration was determined.
2. Digesting target cells Huh-7 16h before experiment, preparing cell suspension, and adding 10 cells into each hole after adjusting cell concentration4And (4) cells.
Diluting the polypeptide drug polypeptide in a 3.96 pore plate by using a DMEM culture solution containing 10% FBS in a 2-fold gradient manner, wherein each pore is 60 mu L; the polypeptide is added slowly while stirring, so as to prevent the precipitation of the polypeptide due to the addition of a large amount of polypeptide in a short time.
4. Dilutions were made according to the MERS-CoV pseudovirus titer determined previously, and 60. mu.L of the diluted pseudovirus was added to each well of the drug dilution plate.
5. Acting at room temperature for 30min to make the medicine and virus fully act.
6. Then, the culture supernatant of the cells was removed, and 100. mu.L of the mixture of the drug and the virus was added to each well.
Fresh DMEM medium containing 10% FBS was replaced after 12h incubation at 7.37 ℃.
8. Culturing for 72h, and determining firefly enzyme.
9. Calculating and making an inhibition rate curve according to the corresponding relation between the fluorescence value and the concentration of the medicament, and calculating the half effective dose IC of the medicament50。
The results of the activity assay according to the above method are shown in Table 8 below.
Table 8: inhibitory Activity against MERS-CoV pseudoVirus infection
Figure BDA0001601004420000391
While specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
<120> polypeptide inhibiting MERS-CoV infection
<130>IDC180037
<160>89
<170>PatentIn version 3.5
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Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Ala
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>2
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<212>PRT
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Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Gln Val Val Lys Ala
1 5 10 15
Leu Asn Glu Ser Tyr
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<210>3
<211>21
<212>PRT
<213> Artificial sequence
<400>3
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Gln Val Val Lys Ala
1 5 10 15
Leu Asn Glu Ser Tyr
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<210>4
<211>21
<212>PRT
<213> Artificial sequence
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Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Gln Val Val Lys Ala
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Leu Asn Glu Ser Tyr
20
<210>5
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<212>PRT
<213> Artificial sequence
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Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Gln Val Val Lys Ala
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Leu Asn Glu Ser Tyr
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<210>6
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<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
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<223> Xaa is (S) -2- (4' -pentenyl) alanine
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Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
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<223> Xaa is (S) -2- (4' -pentenyl) alanine
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Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
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<213> Artificial sequence
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1 5 10 15
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Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
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Leu Asn Glu Ser Tyr
20
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<213> Artificial sequence
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<221>misc_feature
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<223> Xaa is (R) -2- (4' -pentenyl) alanine
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Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Xaa Val Val Lys Xaa
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Leu Asn Glu Ser Tyr
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<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
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<221>misc_feature
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<223> Xaa is (R) -2- (4' -pentenyl) alanine
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Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Xaa Val Val Lys Xaa
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Leu Asn Glu Ser Tyr
20
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<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
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<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>14
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>15
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<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
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<223> Xaa is (R) -2- (4' -pentenyl) alanine
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<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>15
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>16
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<212>PRT
<213> Artificial sequence
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<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
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<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>16
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Xaa
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20
<210>17
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<212>PRT
<213> Artificial sequence
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<221>misc_feature
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<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>17
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Gln Val Val Lys Xaa
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<212>PRT
<213> Artificial sequence
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<221>misc_feature
<222>(16)..(16)
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<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>18
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>19
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>19
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>20
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>20
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>21
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>21
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>22
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>22
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>23
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>23
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>24
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>24
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>25
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>25
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>26
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>26
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>27
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>27
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>28
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>28
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>29
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>29
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>30
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<400>30
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>31
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>31
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>32
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>32
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>33
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>33
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>34
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>34
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>35
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>35
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>36
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(9)..(9)
<223> Xaa is (S) -2- (4' -octenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>36
Leu Asp Leu Thr Tyr Glu Met Leu Xaa Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>37
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(9)..(9)
<223> Xaa is (R) -2- (4' -octenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<400>37
Leu Asp Leu Thr Tyr Glu Met Leu Xaa Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>38
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>38
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>39
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>39
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 1015
Leu Asn Glu Ser Tyr
20
<210>40
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>40
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>41
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>41
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>42
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>42
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>43
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>43
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>44
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>44
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>45
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>45
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>46
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>46
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>47
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>47
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>48
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>48
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>49
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>49
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Gln Val Val Lys Xaa
15 10 15
Leu Asn Glu Xaa Tyr
20
<210>50
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>50
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>51
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>51
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>52
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>52
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>53
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>53
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>54
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>54
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>55
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>55
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>56
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>56
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>57
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>57
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>58
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>58
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>59
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>59
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>60
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>60
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>61
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>61
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>62
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>62
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>63
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>63
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>64
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>64
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>65
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>65
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>66
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>66
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>67
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16,20)
<223> Xaa is covalently crosslinked
<400>67
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>68
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(9)..(9)
<223> Xaa is (S) -2- (4' -octenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(9,16,20)
<223> Xaa is covalently crosslinked
<400>68
Leu Asp Leu Thr Tyr Glu Met Leu Xaa Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>69
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(9)..(9)
<223> Xaa is (R) -2- (4' -octenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is 2-amino-2- (4-pentenyl) -6-heptenoic acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (R) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(9,16,20)
<223> Xaa is covalently crosslinked
<400>69
Leu Asp Leu Thr Tyr Glu Met Leu Xaa Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>70
<211>36
<212>PRT
<213> Artificial sequence
<400>70
Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp Leu Glu Tyr Glu Met
1 5 10 15
Lys Lys Leu Glu Glu Val Val Lys Lys Leu Glu Glu Ser Tyr Ile Asp
20 25 30
Leu Lys Glu Leu
35
<210>71
<211>22
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(13)..(13)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(17)..(17)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(13,17)
<223> Xaa is covalently crosslinked
<400>71
Leu Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys
1 5 10 15
Xaa Leu Asn Glu Ser Tyr
20
<210>72
<211>22
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(17)..(17)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(21)..(21)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(17,21)
<223> Xaa is covalently crosslinked
<400>72
Leu Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys
1 5 10 15
Xaa Leu Asn Glu Xaa Tyr
20
<210>73
<211>23
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(14)..(14)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(18)..(18)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(14,18)
<223> Xaa is covalently crosslinked
<400>73
Thr Leu Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val
1 5 10 15
Lys Xaa Leu Asn Glu Ser Tyr
20
<210>74
<211>23
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(18)..(18)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(22)..(22)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(18,22)
<223> Xaa is covalently crosslinked
<400>74
Thr Leu Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Gln Val Val
1 5 10 15
Lys Xaa Leu Asn Glu Xaa Tyr
20
<210>75
<211>22
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>75
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr Ile
20
<210>76
<211>22
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>76
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr Ile
20
<210>77
<211>23
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>77
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr Ile Asp
20
<210>78
<211>23
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>78
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr Ile Asp
20
<210>79
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>79
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Glu Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>80
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16,20)
<223> Xaa is covalently crosslinked
<400>80
Leu Asp Leu Thr Tyr Glu Met Leu Ser Lys Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20
<210>81
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>81
Leu Asp Leu Glu Tyr Glu Met Lys Ser Leu Glu Xaa Val Val Lys Xaa
1 5 10 15
Leu Glu Glu Ser Tyr
20
<210>82
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>82
Leu Asp Leu Thr Tyr Glu Glx Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>83
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>83
Leu Asp Leu Thr Tyr Glu Ile Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>84
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>84
Leu Asp Leu Thr Tyr Glu Leu Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>85
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>85
Leu Asp Leu Thr Tyr Glu Val Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>86
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is (S) -2- (4' -pentenyl) alanine
<220>
<221>misc_feature
<222>(12,16)
<223> Xaa is covalently crosslinked
<400>86
Leu Asp Leu Thr Tyr Glu Glu Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>87
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is α -aminoisobutyric acid
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is α -aminoisobutyric acid
<400>87
Leu Asp Leu Thr Tyr Glu Met Leu Ser Leu Gln Xaa Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>88
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(9)..(9)
<223> Xaa is α -aminoisobutyric acid
<220>
<221>misc_feature
<222>(12)..(12)
<223> Xaa is α -aminoisobutyric acid
<400>88
Leu Asp Leu Thr Tyr Glu Met Leu Xaa Leu Gln Xaa Val Val Lys Ala
1 5 10 15
Leu Asn Glu Ser Tyr
20
<210>89
<211>21
<212>PRT
<213> Artificial sequence
<220>
<221>misc_feature
<222>(16)..(16)
<223> Xaa is α -aminoisobutyric acid
<220>
<221>misc_feature
<222>(20)..(20)
<223> Xaa is α -aminoisobutyric acid
<400>89
Leu Asp Leu Thr Tyr GluMet Leu Ser Leu Gln Gln Val Val Lys Xaa
1 5 10 15
Leu Asn Glu Xaa Tyr
20

Claims (7)

1. The polypeptide compound, a mixture thereof or pharmaceutically acceptable salts thereof, wherein the polypeptide compound is shown as any one of SEQ ID NO.38-40, 48-50 and 68.
2. A pharmaceutical composition comprising at least one polypeptide compound according to claim 1, mixtures thereof or pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
3. A MERS-CoV fusion inhibitor comprising at least one polypeptide compound of claim 1, a mixture thereof, or a pharmaceutically acceptable salt thereof.
4. Use of the polypeptide compound of claim 1, mixtures thereof or pharmaceutically acceptable salts thereof in the preparation of MERS-CoV fusion inhibitors.
5. Use of the polypeptide compound of claim 1, a mixture thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating or preventing a disease associated with MERS-CoV infection.
6. The use of claim 5, wherein the MERS-CoV infection-associated disease is middle east respiratory syndrome.
7. A method of making the polypeptide compound of claim 1, comprising:
a. synthesizing the polypeptide of any one of SEQ ID NO.6-8, 16-18, 36, and
b. the α -disubstituted amino acids with olefinic side chains in the polypeptides are linked by all-carbon crosslinks.
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Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109320593A (en) * 2018-11-05 2019-02-12 中国人民解放军军事科学院军事医学研究院 Inhibit the helical polypeptide and application thereof of HIV infection
CA3173973A1 (en) * 2020-03-04 2021-09-10 Dana-Farber Cancer Institute, Inc. Antiviral structurally-stabilized sars-cov-2 peptides and uses thereof
CN111349150B (en) * 2020-03-24 2021-02-09 北京中科微盾生物科技有限责任公司 Polypeptide for inhibiting novel coronavirus and application thereof
US10975126B1 (en) 2020-04-23 2021-04-13 King Faisal University MERS-CoV inhibitor peptides
US11479582B2 (en) 2020-10-16 2022-10-25 King Faisal University Anti-SARS-CoV-2 fusion peptides

Citations (2)

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Publication number Priority date Publication date Assignee Title
CN104072592A (en) * 2013-03-26 2014-10-01 复旦大学 Polypeptide having inhibitory effect on novel coronavirus HCoV-EMC/2012 infection and application thereof
CN107022008A (en) * 2016-01-30 2017-08-08 复旦大学 Suppress polypeptide and its application of human coronary virus's infection wide spectrum

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN104072592A (en) * 2013-03-26 2014-10-01 复旦大学 Polypeptide having inhibitory effect on novel coronavirus HCoV-EMC/2012 infection and application thereof
CN107022008A (en) * 2016-01-30 2017-08-08 复旦大学 Suppress polypeptide and its application of human coronary virus's infection wide spectrum

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
中东呼吸综合征冠状病毒(MERS-CoV)融合抑制剂的研究;刘奇;《中国博士学位论文全文数据库医药卫生科技辑》;20160115(第1期);参见第18页图5，第22页图7，第82页表5，第86页第2段 *

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