A polypeptide membrane fusion inhibitor against rsv and methods of making and using the same

Abstract

The present application relates to a polypeptide membrane fusion inhibitor against RSV and its preparation method and use. The present application discloses a compound, a pharmaceutical salt thereof or a derivative thereof, a pharmaceutical composition containing the same, and a preparation product, and the use thereof in the preparation of a medicine for preventing and / or treating diseases caused by RSV or other paramyxoviridae viruses. The polypeptide of the present application shows significantly better anti-RSV activity and safety than the positive control polypeptide. Molecular interaction experiments show that the polypeptide acts on the NHR region of RSV F protein, inhibits the process of NHR forming a six-helix bundle with CHR in F protein, which is a key step of RSV infection, by forming an inactive six-helix structure with NHR or by destroying the structure of RSV NHR, thereby preventing the fusion of RSV virus with target cells.

Description

[0001] The present disclosure claims priority to Chinese patent application 2025101651370 with a filing date of 2025 / 2 / 14. The present disclosure incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present disclosure belongs to the field of biomedical technology, and relates to a polypeptide membrane fusion inhibitor against RSV, and a preparation method and use thereof. In particular, it relates to a class of polypeptides, especially a class of polypeptides against human respiratory syncytial virus (RSV) or other related enveloped viruses, and the use of the polypeptides in the preparation of RSV membrane fusion inhibitors and in the preparation of drugs for treating or preventing RSV infection. BACKGROUND

[0003] Human respiratory syncytial virus (RSV) infection causes respiratory diseases, which poses a major health threat to infants, the elderly, and immunocompromised populations. Most infants under the age of 2 have been infected with RSV, and every year 300 million children under the age of 5 worldwide fall ill due to RSV infection, about 3 million of whom need to be hospitalized, resulting in about 100,000 infant deaths. RSV infection has become the leading cause of infant mortality in developed economies. RSV also infects the elderly, and every year more than 300 million people over the age of 60 worldwide fall ill due to RSV infection, resulting in 500,000 to 600,000 deaths. In addition, RSV also poses a serious threat to an increasingly large population of immunocompromised individuals due to cancer, immunotherapy, environmental and other factors [1] .

[0004] Currently, there is a lack of effective prevention and treatment measures for RSV infection, and the only drug available for the treatment of RSV infection, ribavirin, is an old broad-spectrum antiviral drug, which has the disadvantages of poor specificity and inconvenience of use, and the effect is questionable. In terms of prevention, the development of new drugs and vaccines such as long-acting monoclonal antibodies and mRNA vaccines is actively advancing. Palivizumab and Nirsevimab are currently used for RSV prevention in high-risk infants, and can provide some degree of protection, but the protection rate is relatively low. Palivizumab needs to be injected once a month, is expensive, needs to be injected multiple times in an infection season, and only has a protection rate of about 50%. In addition, Clesrovimab developed by Merck is in the process of applying for marketing at FDA.

[0005] In recent years, RSV vaccine research has made great progress. Pfizer's PF-06928316 (RSV preF) and GSK's GSK3844766A have been approved by the US FDA for people over 60 years old, but have not been approved for infants and young children. Currently, there are four small molecule RSV fusion inhibitors in the clinical stage, but they have been terminated in clinical trials or failed in marketing approval for various reasons. This shows that the development of RSV treatment drugs faces great challenges.

[0006] Although there are some drugs in the prevention and treatment of RSV, the protective effect is generally not high or the curative effect is poor or the side effects are more, and it is urgent to develop more effective and safe treatment drugs for respiratory diseases caused by RSV infection. And because RSV is prevalent in healthy populations and can be infected multiple times, RSV virus is difficult to eliminate in the population, and infection is difficult to avoid, especially in infants, the elderly and immunocompromised populations. Therefore, drugs for treating RSV infectious diseases have great significance. At the same time, due to the long-term spread and variation of RSV in the population, drug-resistant mutant strains will continue to appear against existing and future marketed drugs, and new drugs need to be continuously introduced to combat resistant strains.

[0007] RSV belongs to enveloped viruses, which infect cells through virus-cell membrane fusion mediated by its envelope glycoprotein F protein. F protein belongs to the first class of membrane fusion proteins, which forms a non-covalent complex with surface and transmembrane subunits, and is usually embedded on the surface of the virus in the form of a trimer to form a spike. In the membrane fusion protein complex, 3 transmembrane subunits form the root of the spike, forming an unstable high-energy core as a direct participant in membrane fusion; 3 surface subunits are wrapped on the outside of the core, responsible for specific cell receptor recognition as a guiding molecule for viral infection, determining the type of cells that can be infected by the virus, while stabilizing the high-energy structure of the transmembrane subunit. RSV has a similar infection mechanism as HIV-1, influenza, coronavirus, paramyxoviridae, Ebola virus, etc. belonging to the first class of enveloped viruses, and the fusion core structure is a six-helix bundle structure [2] .

[0008] During the process of viral infection, the surface subunit first binds to specific cell receptors on the cell surface to make the virus close to the target cell. The receptor binding causes the dissociation of the surface subunit and the transmembrane subunit complex, releasing the unstable transmembrane subunit. The transmembrane subunit first releases the N-terminal fusion peptide (FP) into the target cell membrane spontaneously (spring model), and its C-terminal is anchored in the viral membrane through the transmembrane region, forming a fusion precursor connecting the viral membrane and the cell membrane. The fusion precursor contains important functional regions such as the transmembrane region, the C-terminal heptad repeat (CHR), the loop region (Loop), the N-terminal heptad repeat (NHR), and the FP from the viral membrane to the target cell membrane. The fusion precursor is also an unstable structure, and its NHR and CHR rotate to spontaneously reverse fold, in which three NHR form a trimer core, and three CHR are combined in the three grooves formed by the NHR trimer to form a six-helix bundle structure. The formation of the six-helix bundle pulls the viral membrane and the cell membrane together, allowing membrane fusion to occur and allowing the virus to ultimately infect the cell [3] .

[0009] During the process of membrane fusion, NHR and CHR form a six-helix bundle through specific molecular recognition, and they can be targets for each other. Polypeptides and their derivatives from NHR and CHR can be used as exogenous drugs to form a hybrid six-helix bundle structure with the corresponding target from the viral membrane fusion protein through specific polypeptide sequence binding, preventing the formation of the endogenous six-helix bundle structure required for viral-cell membrane fusion, thereby preventing viral-cell membrane fusion and achieving the purpose of blocking and inhibiting viral infection. This strategy has been successful in HIV-1 and new crown drug development [4-6] .

[0010] In the 1990s, Trimeris, the developer of the first membrane fusion inhibitor, investigated polypeptide RSV membrane fusion inhibitors based on research on polypeptide AIDS membrane fusion inhibitors and found that polypeptides from the CHR region of RSV F protein had anti-RSV activity. These polypeptides are all natural sequences from RSV F protein, and T-118, which has the highest activity, is used as a representative polypeptide for subsequent research [7] .

[0011] However, there is still a lack of a polypeptide RSV membrane fusion inhibitor with higher anti-RSV activity and safety. SUMMARY

[0012] To solve the technical problem that the anti-RSV activity and safety of the polypeptide RSV membrane fusion inhibitor in the prior art are not high enough, the present disclosure provides an anti-RSV polypeptide membrane fusion inhibitor and a preparation method and use thereof.

[0013] On the basis of the prior art, the present inventors propose a polypeptide fragment interaction research concept suitable for the dynamic molecular interaction characteristics of envelope glycoproteins in the membrane fusion process, for the design and optimization of novel membrane fusion inhibitors, and obtain a novel RSV fusion inhibitor-salt bridge polypeptide which is completely different from the existing RSV fusion inhibitors but maintains specific interactions.

[0014] The present disclosure takes HIV-1 membrane fusion inhibitor research as a model to systematically study the viral membrane fusion mechanism, finds a series of highly active polypeptide and small molecule membrane fusion inhibitors, and systematically studies the mechanism of action and application of drug design of membrane fusion inhibitors from the perspective of protein folding and protein-protein interaction, and proposes a new design concept and research method of membrane fusion inhibitors in accordance with the dynamic Coiled-cholesterol succinic monoester il protein interaction characteristics in the membrane fusion process [8-12] . In polypeptide design, basic hydrophobic and electrostatic interactions are taken as the main control target according to the characteristics of Coiled-cholesterol succinic monoester id protein folding [8] , the classification of the physicochemical properties of amino acid residues is used to simplify the design [9] ; in the study of molecular mechanism of action, the balance of binding kinetics and binding thermodynamics of membrane fusion inhibitors and target points is emphasized

[10] , polypeptides with improved target specificity and antiviral activity are obtained, and the above-mentioned concept is successfully applied to the design of novel membrane fusion inhibitors [11,12] .

[0015] For the above reasons, the present inventors propose a polypeptide fragment interaction research concept suitable for the dynamic molecular interaction characteristics of envelope glycoproteins in the membrane fusion process, for the design and optimization of novel membrane fusion inhibitors. The present disclosure designs polypeptide sequences according to the common characteristics of the fusion mechanism of enveloped viruses and the structural characteristics of the RSV viral membrane fusion protein F protein, determines the target point thereof, optimizes the sequence thereof from the perspective of protein folding, and further improves the activity thereof according to the contribution of hydrophobic and electrostatic interactions to the activity of membrane fusion inhibitors found by the inventors. Thus, the present invention is completed.

[0016] In the present application, the polypeptide sequences from RSV F protein are investigated in detail, and it is found that solubility and the difficulty of synthesis and purification caused thereby are common in natural RSV polypeptides, which is an important reason affecting the further modification and drug development of natural RSV polypeptides. The present disclosure creatively adopts a new design concept and research method suitable for the characteristics of dynamic coiled coil protein interaction in the membrane fusion process, finds a model polypeptide with anti-RSV activity, and adjusts the mutation, modification and length of the model polypeptide to improve its performance, finds a series of polypeptides with activity significantly higher than the existing anti-RSV polypeptides, good solubility, easy synthesis and excellent drug properties. The polypeptides found in the present disclosure are all composed of natural protein amino acids, easy to be synthesized on a large scale, convenient for biological carrier expression, with excellent metabolic safety, and at the same time, modifications can be introduced to further improve its activity and performance, and are expected to be developed into a new generation of anti-RSV drugs and other drugs with similar mechanisms of action.

[0017] The present application first designs a polypeptide template as sequence (1) (SEQ ID NO: 103):

[0018] (1) Z1-WJJLVOOSJJFDOOIJJVNOOIJJSLOOIJJSDOOLJJVNOOLJJTNOOITTI-Z3-Z2

[0019] In the template, Z1 is the amino terminal (NH2-) or its modification, and Z2 is the carboxyl terminal (-COOH) or its modification. If not specified, Z1 is acetyl (Ac-), and Z2 is amide (-NH2), which respectively blocks the amino acid and carboxyl terminal of the polypeptide to enhance the stability of the polypeptide.

[0020] J is an acidic amino acid residue, which can be but is not limited to glutamic acid residue and aspartic acid residue; O is a basic amino acid residue, which can be but is not limited to lysine residue and arginine residue; a salt bridge is formed between J and O, which generally stabilizes the secondary structure of the polypeptide; the remaining positions are specific amino acid residues, which occupy the corresponding positions of the RSV CHR natural sequence [7] The hydrophobic amino acid residues are mainly used to form a hydrophobic surface, which contacts and matches with the drug target, and specific interaction is generated. Z3 is a fatty acid modification group, including a connecting arm and a lipophilic group, wherein the lipophilic group binds to the target cells of the drug.

[0021] Further, a training method of a polypeptide and receptor binding activity prediction model is used to design the polypeptide compounds of the present disclosure.

[0022] On one hand, this disclosure provides a compound, its pharmaceutical salt, or a derivative thereof, said compound, its pharmaceutical salt, or a derivative thereof comprising a polypeptide selected from any one or more of the sequences shown in Formula I, Formula II, Formula III, Formula IV, SEQ ID NO: 57-59, SEQ ID NO: 82-88, and SEQ ID NO: 97-102.

[0023] On the other hand, this disclosure also provides the use of the said compound, its pharmaceutical salt or derivative thereof in the preparation of anti-RSV peptide membrane fusion inhibitors.

[0024] On the other hand, this disclosure also provides an isolated nucleic acid molecule that encodes the compound, its pharmaceutical salt, or a derivative thereof.

[0025] On the other hand, this disclosure also provides a recombinant vector containing the said nucleic acid molecule.

[0026] On the other hand, this disclosure also provides a recombinant cell containing the said nucleic acid molecule or recombinant vector.

[0027] On the other hand, this disclosure also provides a pharmaceutical composition comprising the compound, its pharmaceutical salt or a derivative thereof, the nucleic acid molecule, the recombinant vector or the recombinant cell.

[0028] On the other hand, this disclosure also provides a formulation product comprising the pharmaceutical composition.

[0029] On the other hand, this disclosure also provides a method for preparing the compound, its pharmaceutical salt or derivative thereof, comprising using a carrier resin to sequentially couple with the corresponding protected amino acids of the polypeptide amino acid sequence through deprotection and coupling reactions to obtain a salt-bridged naked peptide.

[0030] On the other hand, this disclosure also provides the use of the said compound, its pharmaceutical salt or derivative thereof, the said pharmaceutical composition or the said formulation product in the preparation of a medicament for the prevention and / or treatment of diseases caused by RSV or other paramyxoviridae viruses.

[0031] On the other hand, this disclosure also provides a method for preventing and / or treating diseases caused by RSV or other paramyxoviridae viruses, comprising administering an effective amount of the compound, its pharmaceutical salt or derivatives thereof, the pharmaceutical composition or the formulation product to a subject in need.

[0032] On the other hand, this disclosure also provides the compounds, their pharmaceutical salts or derivatives thereof, the pharmaceutical compositions or the formulation products for the prevention and / or treatment of diseases caused by RSV or other paramyxoviridae viruses.

[0033] In another aspect, the present disclosure also provides a method for inhibiting RSV, comprising administering the compound, the pharmaceutical salt or the derivative thereof, the pharmaceutical composition or the preparation product to a sample.

[0034] In one embodiment, the present disclosure uses RSV-A long strain as the infecting virus, determines the anti-RSV activity of the synthesized polypeptide by Plague Reduction method, and determines the IC 50 A novel anti-RSV polypeptide with less than 20 nanomoles, suitable for clinical drug development.

[0035] In another embodiment, the present disclosure uses SD rats, determines the concentration in lung tissue at different time points by atomizing needle administration of the compound, and determines the lung tissue exposure.

[0036] In another embodiment, the present disclosure uses mice and cotton rats as RSV A2 challenge models to determine the efficacy.

[0037] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present disclosure.

[0038] The reagents and raw materials used in the present disclosure are commercially available.

[0039] The present disclosure has the following beneficial effects:

[0040] The polypeptide of the present disclosure shows significantly better anti-RSV activity than the positive control polypeptide, and the polypeptide with the highest activity is more than 100 times the activity of the positive control polypeptide; and shows significantly better safety than the positive control polypeptide. Molecular interaction experiments show that the polypeptide acts on the NHR region of RSV F protein, forms an inactive six-helix structure with NHR, or destroys the NHR structure of RSV, thereby inhibiting the process of NHR and CHR forming a six-helix bundle in F protein, which is a key step of RSV infection, thereby preventing the fusion of RSV virus and target cells. At the same time, the polypeptide of the present disclosure uses a unique design strategy, which is completely different from the existing RSV membrane fusion inhibitor sequence and design idea. The salt bridge sequence increases the water solubility of the polypeptide, while maintaining the specific interaction of the polypeptide with the target, ensuring that the polypeptide has higher drugability. The anti-RSV virus activity of the polypeptide sample designed by the present disclosure is very high, and the IC 50 Controlled within 30 nM, preferably within 20 nM, more preferably within 15 nM, and the lowest is 9.9 nM. The active IC 50 The polypeptide sample with IC DETAILED DESCRIPTION

[0041] To enable the disclosure to be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or a range of values of a parameter are recited it is intended that the intervening technically- equivalent values are also encompassed unless the context clearly dictates otherwise.

[0042] The articles "a" and "an" as used herein mean one or more than one (i.e., at least one) of the grammatical article's object. By way of example, "an element" means one element or more than one element, e.g., multiple elements.

[0043] The term "including" as used herein is intended to mean "including, but not limited to" and is used interchangeably with the phrase "including but not limited to".

[0044] The term "or" as used herein is intended to mean "and / or" unless the context clearly indicates otherwise.

[0045] Abbreviations used in the disclosure have the following meanings:

[0046] Ala (Alanine, A) Propionic acid

[0047] Arg (Arginine, R) Arginine

[0048] Asn (Asparagine, N) Asparagine

[0049] Asp (Aspartic acid, D) Aspartic acid

[0050] DCM (Dichloromethane) Dichloromethane

[0051] DMF (N,N-Dimethyl malonate) Dimethylformamide

[0052] Env (Envelope glycoprotein) Envelope glycoprotein

[0053] ESI-MS (Electronic spray ion mass spectroscopy) Electronic spray ion mass spectroscopy

[0054] Fmoc (Fluorenylmethoxycarbonyl) Fluorenylmethoxycarbonyl

[0055] Gly (Glycine, G) Glycine

[0056] Gln (Glutamine, Q) Glutamine

[0057] Glu (Glutamic acid, E) Glutamic acid

[0058] 6-HB (six-helix bundle)

[0059] HBTU 2-(1H-1-hydroxybenzotriazole)-1,1,3,3-tetramethylhexafluorophosphate

[0060] His (Histidine, H) histidine

[0061] HoBt (1-Hydroxyl benzotiazole anhydrous) 1-hydroxybenzotriazole

[0062] NHR (N-terminal heptad repeat) N-terminal heptad repeat sequence

[0063] CHR (C-terminal heptad repeat) is a C-terminal heptad repeat sequence.

[0064] HIV (Human Immunodeficiency Virus)

[0065] HPLC (high performance liquid chromatography)

[0066] Ile (Isoleucine, I) isoleucine

[0067] Leucine (L-leucine)

[0068] Methionine (M) (Met)

[0069] Lysine (K)

[0070] Phe (Phenylalanine, F) phenylalanine

[0071] RSV (Respiratory Syncytial Virus)

[0072] Serine (S)

[0073] TFA (Trifluoroacetic acid)

[0074] Threonine (Threonie, T)

[0075] Tyr (Tyrosine, Y) tyrosine

[0076] Val (Valine, V) Valine

[0077] Single letter amino acid residue designations are as follows:

[0078] A: L-alanine; C: L-cysteine; D: L-aspartic acid; E: L-glutamic acid; F: L-phenylalanine; G: L-glycine; H: L-histidine; I: L-isoleucine; K: L-lysine; L: L-leucine; M: L-methionine; N: L-asparagine; P: L-proline; Q: L-glutamine; R: L-arginine; S: L-serine; T: L-threonine; V: L-valine; W: L-tryptophan; Y: L-tyrosine.

[0079] In an aspect, the present disclosure provides a compound, a pharmaceutically acceptable salt thereof, or a derivative thereof, comprising a polypeptide selected from a sequence represented by any one or more of Formula I, Formula II, Formula III, Formula IV, SEQ ID NOs: 57~59, SEQ ID NOs: 82~88, and SEQ ID NOs: 97~102:

[0080] Formula I (SEQ ID NO: 122):

[0081] X1-X2-X3-X4-X5-X6-X7-X8-X9-I-X 11 -E-X 13 -X 14 -X 15 -X 16 -IEE-X 20 -L-X 22 -X 23 -X 24 -X 25 -ESD-X 29 -X 30 -L-X 32 -X 33 -X 34 -X 35 -X 36 -X 37 -X 38 ;

[0082] wherein,

[0083] X1is D or absent;

[0084] X2is E or absent;

[0085] X3is W, F, D, S, or absent;

[0086] X4is D or absent;

[0087] X5is E, A, K or absent;

[0088] X6is F, S or absent;

[0089] X7is D, N, L or absent;

[0090] X8is K, Q, L or absent;

[0091] X9is K or absent;

[0092] X 11 is E or N;

[0093] X 13 is V or E;

[0094] X 14 is N or K;

[0095] X 15 is K, R or E;

[0096] X 16 is K or R;

[0097] X 20 is S, I or L;

[0098] X 22 is K or R;

[0099] X 23 is K or R;

[0100] X 24 is I or H;

[0101] X 25 is E or N;

[0102] X 29 is K or R;

[0103] X 30 is K or R;

[0104] X 32 is E or absent;

[0105] X 33 is E, V or absent;

[0106] X 34 is V, N, S or absent;

[0107] X 35 is N, K, D or absent;

[0108] X 36 is K or absent;

[0109] X 37 is K, A, or absent;

[0110] X 38 is L, A, or absent;

[0111] Formula II (SEQ ID NO: 123):

[0112] X'1-X'2-X'3-X'4-X'5-X'6-E-X'8-X'9-X' 10 -X' 11 -X' 12 -X' 13 -X' 14 -SQVNEKIN-X' 23 -SL-X' 26 -X' 27 -IR-X' 30 -X' 31 -X' 32 -X' 33 -X' 34 -KSDELL-X' 41 -X' 42 -X' 43 -X' 44 -X' 45 -X' 46 -X' 47 -X' 48 -X' 49 -X' 50 ;

[0113] wherein,

[0114] X'1is F, W, D, L, or Y;

[0115] X'2is V or absent;

[0116] X'3is K or absent;

[0117] X'4is D, K, or absent;

[0118] X'5is F, I, or absent;

[0119] X'6is D or absent;

[0120] X'8is L or absent;

[0121] X'9is V or absent;

[0122] X' 10 is F or absent;

[0123] X' 11 is E or D;

[0124] X' is A or I; 12 is A or I;

[0125] X' is S or absent; 13 is S or absent;

[0126] X' is I or absent; 14 is I or absent;

[0127] X' is E or Q; 23 is E or Q;

[0128] X' is A or E; 26 is A or E;

[0129] X' is F, E, or K; 27 is F, E, or K;

[0130] X' is L or absent; 30 is L or absent;

[0131] X' is A or absent; 31 is A or absent;

[0132] X' is F or absent; 32 is F or absent;

[0133] X' is I or absent; 33 is I or absent;

[0134] X' is R or absent; 34 is R or absent;

[0135] X' is H or absent; 41 is H or absent;

[0136] X' is N or absent; 42 is N or absent;

[0137] X' is V or absent; 43 is V or absent;

[0138] X' is N or absent; 44 is N or absent;

[0139] X' is A or absent; 45 is A or absent;

[0140] X' is G or absent; 46 is G or absent;

[0141] X' is K, L, or absent; 47 is K, L, or absent;

[0142] X' is S or absent; 48 is S or absent;

[0143] X' is T or absent; 49 is T or absent;

[0144] X' is T or absent; 50 is T or absent;

[0145] Formula III (SEQ ID NO: 124):

[0146] X''1-X''2-X''3-X''4-D-X''6-X''7-IEEVN-X'' 13 -X'' 14 -IEESL-X'' 20 -X'' 21 -IEESD-X'' 27 -X'' 28 -L-X'' 30 -X'' 31 -V-X'' 33 -X'' 34 ;

[0147] wherein,

[0148] X''1is W or F;

[0149] X''2is D or absent;

[0150] X''3is E or absent;

[0151] X''4is F or absent;

[0152] X''6is K or A;

[0153] X''7is K or S;

[0154] X'' 13 is K or R;

[0155] X'' 14 is K or R;

[0156] X'' 20 is K or R;

[0157] X'' 21 is K or R;

[0158] X'' 27 is K or R;

[0159] X'' 28 is K or R;

[0160] X'' 30 is E or H;

[0161] X'' 31 is E or N;

[0162] X'' 33 is N or absent;

[0163] X'' 34A or absent;

[0164] Formula IV (SEQ ID NO: 125):

[0165] WDEFDASISQ-X' 11 -NEKINQSLEEIRKSDELLHN-X' 32 -X' 33 -X' 34 -X' 35 ;

[0166] wherein,

[0167] X' 11 V or absent;

[0168] X'' 32 V, N or absent;

[0169] X' 33 N, A or absent;

[0170] X' 34 A, L or absent;

[0171] X' 35 L or absent.

[0172] In some embodiments, the compound, a pharmaceutically acceptable salt thereof, or a derivative thereof comprises a polypeptide having a sequence selected from any one or more of the sequences set forth in SEQ ID NOs: 52-102.

[0173] In the present disclosure, the polypeptide defined above is the core sequence comprised by the compound, a pharmaceutically acceptable salt thereof, or a derivative thereof, which is the key part for generating the RSV inhibitory effect, and is capable of acting on the RSV F protein NHR region, forming an inactive six-helix structure with the NHR, or destroying the RSV NHR structure, thereby inhibiting the process of the formation of the six-helix bundle of the NHR and the CHR in the F protein, which is a key step of RSV infection, so as to prevent the fusion of the RSV virus and the target cell. The inhibitory effect does not depend on the modification, the connecting arm, or the modification of the lipophilic compound at both ends of the polypeptide.

[0174] In some embodiments, the compound, a pharmaceutically acceptable salt thereof, or a derivative thereof comprises a polypeptide having a sequence selected from any one or more of the sequences set forth in Formula I-1, Formula II-1, Formula III-1, Formula IV-1, and SEQ ID NOs: 106-121:

[0175] Formula I-1 (SEQ ID NO: 126):

[0176] R1-X1-X2-X3-X4-X5-X6-X7-X8-X9-I-X 11 -E-X 13 -X 14 -X 15 -X 16 -IEE-X 20 -L-X 22 -X 23 -X 24 -X 25 -ESD-X 29 -X 30 -L-X 32 -X 33 -X 34 -X 35 -X 36 -X 37 -X 38 -R2-R3;

[0177] Formula II-1 (SEQ ID NO: 127):

[0178] R1-X'1-X'2-X'3-X'4-X'5-X'6-E-X'8-X'9-I-X' 11 -X' 12 -X' 13 -X' 14 -SQVNEKIN-X' 23 -SL-X' 26 -X' 27 -IR-X' 30 -X' 31 -X' 32 -X' 33 -X' 34 -KSDELL-X' 41 -X' 42 -X' 43 -X' 44 -X' 45 -X' 46 -X' 47 -X' 48 -X' 49 -X' 50 -R2-R3;

[0179] Formula III-1 (SEQ ID NO: 128):

[0180] R1-X''1-X''2-X''3-X''4-D-X''6-X''7-IEEVN-X'' 13 -X'' 14 -IEESL-X'' 20-X'' 21 -IEESD-X'' 27 -X'' 28 -L-X'' 30 -X'' 31 -V-X'' 33 -X'' 34 -R2-R3;

[0181] Formula IV-1 (SEQ ID NO: 129):

[0182] R1-WDEFDASISQ-X''' 11 -NEKINQSLEEIRKSDELLHN-X''' 32 -X''' 33 -X''' 34 -X''' 35 -R2-R3;

[0183] SEQ ID NO: 106: R1-ADAFRLEVNDASSKINESIEESLLSLEKLHNVNATA-R2-R3;

[0184] SEQ ID NO: 107: R1-FDAFIQEINVNEDQSLEQSDELLLELHLLHSLLH-R2-R3;

[0185] SEQ ID NO: 108: R1-FAEFNQKINQVNEKIEESLEEIRKSDEELHNVNATT-R2-R3;

[0186] SEQ ID NO: 109: R1-SDEQVNEKINQSLAFIRRIRKLLHN-R2-R3;

[0187] SEQ ID NO: 110: R1-ILELVNKKIEQSLKFIEKSDKLLEN-R2-R3;

[0188] SEQ ID NO: 111: R1-SLEQVNKKINQSLKVNKKSDKLLEN-R2-R3;

[0189] SEQ ID NO: 112: R1-FDEEVNKKIEQSLKINQSLEEIRKS-R2-R3;

[0190] SEQ ID NO: 113: R1-SISQVNEKINEIQSLEEKSDKLLKS-R2-R3;

[0191] SEQ ID NO: 114: R1-VNKKIEEEKQSLKKQSDKIEESDEN-R2-R3;

[0192] SEQ ID NO: 115: R1-FDELVNKKIEKIEEVNKKSLKLLES-R2-R3;

[0193] SEQ ID NO: 116: R1-FEVNRRRIEQSLEKSLESLEEEEHSDKKLHNELH-R2-R3;

[0194] SEQ ID NO: 117: R1-SLEQVNKINKKIDKIEESLKKIEESDKKSLEVNKKL-R2-R3;

[0195] SEQ ID NO: 118: R1-SLEQVNKINEKINKISQSLKKIEESDKKSDEVNAGL-R2-R3;

[0196] SEQ ID NO: 119: R1-SLEQVNKKIEQSLESLKKSDKINQSLEEVNKSDELL-R2-R3;

[0197] SEQ ID NO: 120: R1-SLEQVNEKINQSLAFIRKSDEINQSDEEVNKSDELL-R2-R3;

[0198] SEQ ID NO: 121: R1-SDELVNKKIEFDKKINQSLKKIEESDKKKL-R2-R3;

[0199] wherein:

[0200] R1 is an amino-terminal protecting group, preferably acetyl;

[0201] R2 is absent or a linking arm which is optionally substituted, preferably -R4-R5(R6)-, wherein R4 is a polypeptide, preferably having an amino acid sequence of (EAAAK) m or (GSGSG) m ; m is a natural number from 0 to 5; in particular, m can be any natural number from 0, 1, 2, 3, 4 and 5;

[0202] R5 is lysine, cysteine, 2,3 diaminopropionic acid, ornithine, 2,4 diaminobutyric acid or 2,7 diaminheptanoic acid, preferably lysine;

[0203] R6 is a lipophilic compound group that is modified to R5, preferably the lipophilic compound group is selected from one or more of: cholesterol, cholesterol succinate monoester, 2-cholesterol acetic acid, 2-cholesterol propionic acid, 3-cholesterol propionic acid, 2-cholesterol butyric acid, 2-cholesterol isobutyric acid, 3-cholesterol butyric acid, 3-cholesterol isobutyric acid, 4-cholesterol butyric acid, 2-cholesterol valeric acid, 2-cholesterol isovaleric acid, 3-cholesterol valeric acid, 5-cholesterol valeric acid, 2-cholesterol caproic acid, 6-cholesterol caproic acid, 2-cholesterol heptanoic acid, 7-cholesterol heptanoic acid, 2-cholesterol octanoic acid, 8-cholesterol octanoic acid, cholesteryl bromoacetate, cholesteryl formyl chloride, palmitic acid, stearic acid, a fatty acid comprising 3-20 carbon atoms, a fatty diacid comprising 3-20 carbon atoms, and other groups that can interact with a cell membrane or viral membrane to enhance the interaction of the polypeptide with the cell membrane or viral membrane; more preferably cholesterol succinate monoester;

[0204] R3 is a carboxyl terminal protecting group, preferably -NH2.

[0205] In some embodiments, the compound, pharmaceutically acceptable salt thereof, or derivative thereof comprises a sequence selected from any one or more of the polypeptides set forth in SEQ ID NOs: 1-51.

[0206] In some embodiments, the derivative is a solvate, chelate, or non-covalent complex.

[0207] In some embodiments, the pharmaceutically acceptable salts include: acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, methylsulfate, borate, bromomethane, bromide, nitromethyle, calcium edetate, methylsulfate, dextrocamphorate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium, dihydrochloride, oleate, edetate, oxalate, edisylate, embonate, glutarate, formate, hydroxynaphthoate, fumarate, glutamate, stearate, glycollylarsanilate, pantothenate, fumarate, phosphate / diphosphate, glucoheptonate, polygalacturonate, gluconate, salicylate, glutamate, stearate, p-hydroxyethyIaminoarsanilic, sulfate, hydroxybenzoate, subacetate, hydrabamine, succinate, hydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, 8-chlorotheophyllinate, iodide, tosylate, triethyl iodide, lactate, valerate, and the like. Depending on the application, the pharmaceutically acceptable salts can be formed with cations such as sodium, potassium, bismuth, and the like, or with bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, diethylamine, piperazine, tris-hydroxymethylaminomethane, and hydroxytetramethylammonium, and the like. These salts can be prepared using standard procedures, for example, by reaction of the free acid with an organic or inorganic base. In the presence of a basic group such as an amino group, acidic salts such as hydrochloride, hydrobromide, acetate, pamoate, and the like can be used as dosage forms; in the presence of an acidic group or an alcoholic group, pharmaceutically acceptable esters such as acetate, maleate, chloromethyltrimethylacetate, and the like, as well as esters known in the literature for improving solubility and hydrolysis, can be used as sustained release and prodrug formulations.

[0208] In another aspect, the present disclosure provides use of a compound as described herein, a pharmaceutically acceptable salt thereof, or a derivative thereof, in the manufacture of a polypeptide membrane fusion inhibitor against RSV.

[0209] As described herein, a "polypeptide membrane fusion inhibitor against RSV" is a specially designed polypeptide drug that is capable of inhibiting the fusion of the viral envelope with the host cell membrane during the RSV infection process. By preventing this critical step, the virus cannot enter the host cell, thereby preventing the replication and spread of the virus.

[0210] In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding a compound as described herein, a pharmaceutically acceptable salt thereof, or a derivative thereof.

[0211] In another aspect, the present disclosure provides a recombinant vector containing a nucleic acid molecule as described herein.

[0212] In another aspect, the present disclosure provides a recombinant cell containing the nucleic acid molecule as described in the present disclosure or the recombinant vector as described in the present disclosure.

[0213] In another aspect, the present disclosure provides a pharmaceutical composition comprising the compound as described in the present disclosure, a pharmaceutically acceptable salt thereof or a derivative thereof, the nucleic acid molecule described in the present disclosure, the recombinant vector described in the present disclosure or the recombinant cell described in the present disclosure; optionally, further comprising a pharmaceutically acceptable carrier or excipient.

[0214] The pharmaceutical composition of the present disclosure can be a solution with or without a buffer or a composition containing a pharmaceutically acceptable carrier. In the present disclosure, the pharmaceutical composition can be administered in a solution. It can be administered in a non-buffered solution, for example, in physiological saline or in water. Alternatively, it can also be administered in a suitable buffered solution. The buffered solution can include acetate, citrate, prolamine, carbonate or phosphate, or any combination thereof. In a preferred embodiment, the buffered solution is phosphate buffered saline (PBS). The pH and the osmolality of the buffer of the pharmaceutical composition can be adjusted such that it is suitable for administration to a subject.

[0215] In some embodiments, the buffered solution further comprises an agent for controlling the osmolality of the solution such that the osmolality is maintained at a desired value, for example, at the physiological value of human plasma. Solutes that can be added to the buffered solution to control the osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolizable polymers, vitamins, ions, sugars, metabolites, organic acids, lipids or salts. In some embodiments, the agent for controlling the osmolality of the solution is a salt. In certain embodiments, the agent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0216] The pharmaceutical composition of the present disclosure can be administered at a dose sufficient to inhibit RSV. Generally, a suitable dose of the compound of the present disclosure for a mammal, particularly a human, can be between 0.1 mg / day and 100 mg / day, for example, between 10 mg / day and 50 mg / day, for example, between 20 mg / day and 30 mg / day.

[0217] The pharmaceutical composition can be administered once a day, or the pharmaceutical composition can be administered in two, three or more sub-doses at appropriate intervals throughout the day, or even continuously with an infusion or delivery by a controlled release formulation. In this case, the compound contained in each sub-dose must correspondingly be less so as to achieve the total daily dose. Dose units can also be compounded for delivery over several days, for example using conventional sustained release formulations that provide a sustained release of the compound over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering agents at a particular site, and can be used with the agents of the present disclosure. In this embodiment, the dose unit contains a corresponding plurality of daily doses.

[0218] In other embodiments, a single dose of the pharmaceutical composition can be sustained for a long duration, such that subsequent doses are administered at intervals of no more than 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks. Treatment regimens can be one administration per 1-3 days, for 4-7 consecutive days; if the infection recurs, the 4-7 consecutive days of administration can be repeated, for a treatment cycle of 1-7 administrations. In some embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once per week. In other embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once per month.

[0219] Those of skill in the art will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatments, the general health and / or age of the subject, and other existing diseases. In addition, treatment of a subject with a therapeutically effective dose of the composition can include a single treatment or a series of treatments. Effective doses and the in vivo half-lives of the various compounds encompassed by the present disclosure can be estimated

[0220] The pharmaceutical compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., through a skin patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizers; intratracheal; intranasal; epidermal and transdermal, oral or parenteral. Parenteral administration includes subcutaneous, intravenous, intramuscular, intra- peritoneal or intramuscular injection or infusion; subdermal, e.g., via implantation devices; or intracranial, e.g., intracerebral or intrathecal injection. The compounds of the present disclosure can be administered in a form

[0221] The pharmaceutical compositions of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier or excipient. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0222] In another aspect, the present disclosure provides a formulation product comprising the pharmaceutical composition of the present disclosure.

[0223] Certain formulations of the present disclosure also incorporate a carrier material into the pharmaceutical composition. The carrier materials include, but are not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate and carboxymethyl cellulose, etc.). Preferred among these are water-soluble carrier materials. Using these materials, a variety of formulations can be prepared, including, but not limited to, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, lyophilized powder injections, etc. These can be ordinary formulations, sustained-release formulations, controlled-release formulations, and various microparticle drug delivery systems. In order to prepare a unitary drug delivery dosage form into a tablet, a variety of carriers known in the art can be widely used. Examples of the carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, trehalose, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerol, polyethylene glycol, ethanol, propyl alcohol, starch paste, dextrin, sugar syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants such as dry starch, alginate, agar powder, alginic acid, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glycerol tri-stearate, cocoa butter, hydrogenated oil, etc.; absorption accelerators such as quaternary ammonium salt, sodium dodecylsulfate, etc.; lubricants such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further prepared into coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to prepare a unitary drug delivery dosage form into a pill, a variety of carriers known in the art can be widely used. Examples of the carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or dough, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare a unitary drug delivery dosage form into a suppository, a variety of carriers known in the art can be widely used. Examples of the carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare a unitary drug delivery dosage form into an injection preparation such as a solution, an emulsion, a lyophilized powder injection, and a suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc.In addition, for the preparation of isotonic injection solutions, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation, and in addition, a conventional solubilizing agent, a buffer, a pH adjusting agent, etc. can be added. In addition, if necessary, a coloring agent, a preservative, a flavoring agent, a corrigent, a sweetener or other materials can be added to the pharmaceutical preparation. The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc., cavity administration, such as rectal and vaginal administration; respiratory tract administration, such as nasal cavity; mucosal administration. The above administration routes are preferably injection administration.

[0224] In some embodiments, the pharmaceutical composition of the present disclosure can be formulated into any of a number of possible formulations, such as, but not limited to, any of the following groups: oral, injection, inhalation and lyophilization; preferably inhalation, lyophilization, subcutaneous injection or intramuscular injection.

[0225] In some embodiments, the administration mode of the preparation product is selected from any of the following: oral administration, injection administration, mucosal administration, transdermal administration and atomization administration; preferably pulmonary atomization administration, subcutaneous injection or intramuscular injection administration.

[0226] In another aspect, the present disclosure provides a method for preparing a compound, a pharmaceutically acceptable salt thereof or a derivative thereof as described in the present disclosure, the method comprising the following steps:

[0227] The salt-bridged naked peptide is prepared by sequentially coupling with protected amino acids corresponding to the polypeptide amino acid sequence using a carrier resin through deprotection and coupling reactions.

[0228] In some embodiments, the method further comprises one or more of the following steps:

[0229] (1) lipophilic compound modification; preferably cholesterol succinic monoester modification;

[0230] (2) N-terminal acetylation capping; and,

[0231] (3) C-terminal amidation capping.

[0232] In another aspect, the present disclosure provides the use of a compound, a pharmaceutically acceptable salt thereof or a derivative thereof as described in the present disclosure, a pharmaceutical composition as described in the present disclosure or a preparation product as described in the present disclosure in the preparation of a medicament for preventing and / or treating a disease caused by RSV or other paramyxoviridae virus.

[0233] As used herein, "disease caused by RSV" is intended to include any disease associated with respiratory syncytial virus infection. Exemplary diseases caused by RSV include upper respiratory tract infection, lower respiratory tract infection (e.g. bronchiolitis or pneumonia), viral otitis media, asthma or wheezing-related diseases, chronic lung disease or other possible complications.

[0234] Paramyxoviridae is a family of RNA viruses, which are negative-sense single-stranded RNA viruses, that primarily infect humans and animals. Viruses in this family can cause a variety of respiratory, nervous system, and systemic diseases. As used herein, “other Paramyxoviridae viruses” include, but are not limited to, Measles virus (MeV), Mumps virus (MuV), and Newcastle disease virus (NDV), among others.

[0235] In some embodiments, the RSV comprises one or more selected from the group consisting of RSV-A subtypes, RSV-B subtypes, and other mutant strains.

[0236] The RSV described in the present disclosure comprises one or more selected from the group consisting of RSV-A subtypes, RSV-B subtypes, and other mutant strains. RSV A subtype strains are, for example, selected from the group consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, and A23, among others; and RSV B subtype strains are, for example, selected from the group consisting of B1, B2, B3, B4, B5, and B6, among others.

[0237] In another aspect, the present disclosure provides a method of preventing and / or treating a disease caused by RSV or other Paramyxoviridae viruses, the method comprising administering to a subject in need thereof an effective amount of a compound as described in the present disclosure, a pharmaceutically acceptable salt thereof, or a derivative thereof, a pharmaceutical composition as described in the present disclosure, or a formulation product as described in the present disclosure.

[0238] As used herein, “subject” is intended to include a human or non-human animal, preferably a mammal, such as a mouse. Most preferably, the subject or patient is a human.

[0239] As used herein, “effective amount” is intended to include the dosage that is sufficient to effect treatment (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of the disease) of a disease caused by RSV when administered to a patient for such treatment. The “effective amount” can vary depending on how the agent is administered, the disease and its severity, and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by RSV, type of previous or concomitant treatments, if any, and other individual characteristics of the patient to be treated. An “effective amount” also includes a dosage that produces some desired, local or systemic, effect at a reasonable benefit / risk ratio applicable to any treatment. The compounds used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0240] In some embodiments, the RSV comprises one or more selected from the group consisting of RSV-A subtypes, RSV-B subtypes, and other mutant strains.

[0241] In another aspect, the present disclosure provides a compound as described herein, a pharmaceutically acceptable salt thereof, or a derivative thereof, a pharmaceutical composition as described herein, or a formulation product as described herein, for use in the prevention and / or treatment of a disease caused by RSV or other paramyxoviridae virus.

[0242] In some embodiments, the RSV comprises one or more selected from the group consisting of RSV-A subtypes, RSV-B subtypes, and other mutant strains.

[0243] In another aspect, the present disclosure provides a method of inhibiting RSV, the method comprising administering to a sample a compound as described herein, a pharmaceutically acceptable salt thereof, or a derivative thereof, a pharmaceutical composition as described herein, or a formulation product as described herein.

[0244] As used herein, the term "sample" includes similar fluids, cells, or tissues isolated from a subject, as well as a collection of fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum, serosal fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, and saliva, among others. A tissue sample can include a sample from a tissue, organ, or local region. For example, a sample can be derived from a particular organ, organ portion, or fluid or cells within these organs. In certain embodiments, a "sample" refers to blood or plasma drawn from the subject.

[0245] In some embodiments, the method is for non-therapeutic purposes; and / or, the RSV comprises one or more selected from the group consisting of RSV-A subtypes, RSV-B subtypes, and other mutant strains.

[0246] "Non-therapeutic purposes" as described herein refers to inhibiting RSV replication for scientific research purposes in, for example, a laboratory.

[0247] The following examples are intended to illustrate the present application but not to limit the scope of the present application. If not specifically mentioned, the technical means used in the examples are the conventional means well known to those skilled in the art, and the raw materials used are commercially available.

[0248] Example 1: Polypeptide design

[0249] Based on the experience of designing HIV fusion inhibitors and other viral fusion inhibitors, polypeptides of the CHR region of the transmembrane subunit of viral envelope glycoprotein generally show high activity [3,7]Based on this, the present disclosure designs polypeptides according to the CHR sequence of the F1 subunit of the F protein of the RSV virus. Polypeptides of 36 amino acid residues are usually a good starting point according to previous successful experience, and can contain up to 11 binding sites [3] The present disclosure first designs a series of 36 peptides covering the RSV CHR region, synthesizes the corresponding polypeptides, and investigates their activity through molecular interaction and anti-RSV activity experiments, and further designs and optimizes the polypeptides according to the test results. The inventors found that the natural sequence RSV CHR polypeptide usually has the disadvantages of poor water solubility and difficulty in synthesis, which brings great challenges to subsequent drug development. According to the experience and previous research results of the inventors, the present disclosure introduces EE-KK salt bridge into the non-binding site of the RSV CHR polypeptide sequence to improve its water solubility, and the salt bridge can also stabilize the α-helix secondary structure required for its activity. The present disclosure found that the introduction of the salt bridge not only improves the water solubility, but also can significantly improve the activity, and a shorter polypeptide can be designed.

[0250] Based on the above considerations, in the present disclosure, on the basis of sequence (1) (SEQ ID NO: 103):

[0251] Z1-WJJLVOOSJJFDOOIJJVNOOIJJSLOOIJJSDOOLJJVNOOLJJTNOOITTI-Z3-Z2, a derivative is prepared by introducing systematic mutations, including mutating E and K in the salt bridge to corresponding D and R, and fine-tuning the binding site to improve the binding of the polypeptide to the target, and the design of the polypeptides in the examples is completed.

[0252] Wherein, Z1 is an amino terminal (NH2-) or a modification thereof; Z2 is a carboxyl terminal (-COOH) or a modification thereof; J is an acidic amino acid residue, which can be but is not limited to glutamic acid residue and aspartic acid residue; O is a basic amino acid residue, which can be but is not limited to lysine residue and arginine residue.

[0253] Z3 is a fatty acid modification group, including a connecting arm and a lipophilic group, wherein the lipophilic group binds to the target cell of the drug to increase the activity of the drug, and the connecting arm is used to connect the polypeptide chain and the lipophilic group and provide appropriate steric space and conformation for the polypeptide drug to better bind to the target.

[0254] Table 1-1 Sequence design of polypeptides (formula I-1)

[0255]

[0256] Table 1-2 Sequence design of polypeptides (formula II-1)

[0257]

[0258] Table 1-3 Sequence design of polypeptides (Formula III-1)

[0259]

[0260] Table 1-4 Sequence design of polypeptides (Formula IV-1)

[0261]

[0262] Table 1-5 Sequence design of polypeptides

[0263]

[0264] wherein Chol is cholesteryl succinate monoester, which represents an ester formed by the carboxyl group in cholesteryl succinate monoacid and the amino group of lysine in the polypeptide

[0265] .

[0266] Table 1-6 Sequence design of polypeptides (the core sequence corresponds to Formula I)

[0267]

[0268] Table 1-7 Sequence design of polypeptides (the core sequence corresponds to Formula II)

[0269]

[0270] Table 1-8 Sequence design of polypeptides (the core sequence corresponds to Formula III)

[0271]

[0272] Table 1-9 Sequence design of polypeptides (the core sequence corresponds to Formula IV)

[0273]

[0274] Table 1-10 Sequence design of polypeptides (core sequence)

[0275]

[0276] Table 1-11 Control examples

[0277]

[0278] wherein Chol has the same meaning as described above.

[0279] Example 2: Synthesis of naked peptides

[0280] 1. Chemical reagents required in the preparation process

[0281] The chemical reagents used, such as various Fmoc amino acids, N,N'-diisopropyl carbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), N,N-dimethylformamide (DMF), piperidine (PIPE), indantrione, acetic anhydride (Ac2O), N,N-diisopropyl ethylamine (DIEA), trifluoroacetic acid (TFA), ethanedithiol (EDT), benzyl sulfide (TA), triisopropylsilane (TIPS), phenol, etc. were purchased from major chemical reagent suppliers and were not further purified before use.

[0282] The protected amino acid raw materials used in the polypeptide synthesis process include Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH. The abbreviations have well-known definitions: Fmoc is 9-fluorenylmethyloxycarbonyl, Dde is 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, Boc is tert-butyloxycarbonyl, tBu is tert-butyl, OtBu is tert-butoxy, Trt is trityl, and Pbf is (2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl.

[0283] 2. Synthesis of peptide resin

[0284] Using Rink Amide MBHA resin as the carrier resin, the peptide resin was prepared by deprotection and coupling reaction with the corresponding protected amino acids of the polypeptide amino acid sequence in sequence.

[0285] 2.1 Accessing the first protected amino acid of the main chain

[0286] Take 0.3 mmol of the first protected amino acid and 0.3 mmol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.3 mmol of DIC, and slowly add it to the protected amino acid DMF solution under shaking, and shake at room temperature for 5 min to obtain an activated protected amino acid solution, which is ready for use.

[0287] Take 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g*0.3 g), deprotect with 25% PIPE / DMF solution (volume ratio) for 20 min (twice), and wash and filter to obtain Fmoc-free resin.

[0288] Add the activated first protected amino acid solution to the Fmoc-free resin, couple for 60 min, filter and wash to obtain the resin containing the first protected amino acid.

[0289] 2.2 Access other protected amino acids of the main chain

[0290] Access other protected amino acids corresponding to the polypeptide in sequence by the same method as accessing the first protected amino acid of the main chain, to obtain the resin containing the amino acids of the main chain.

[0291] Finally, acetylate the N-terminus with 0.3 mmol Ac2O+0.6 mmol DIEA to complete the synthesis of the main chain.

[0292] After each of the above reactions, control the reaction by Kaiser Test, and if the condensation reaction of a certain amino acid is not complete, repeat the condensation once until the desired target peptide segment is obtained.

[0293] 3. Preparation of crude product

[0294] Take the above peptide resin, add cleavage reagent (cleavage reagent 15 mL / g resin), mix well, and shake at 30°C for 3 h to cleave the target polypeptide from the resin and remove the side chain protecting group. Collect the filtrate of the reaction mixture, wash the resin with a small amount of TFA / DCM for 3 times, combine the filtrate, add anhydrous ether for precipitation, and centrifuge. Wash the filter cake with cold anhydrous ether for precipitation twice, and dry to obtain a white powder, which is the crude naked peptide.

[0295] The composition and volume ratio of the cleavage reagent are as follows: trifluoroacetic acid: 1, 2-ethanedithiol: benzyl thioether: phenol: H2O: triisopropylsilane = 68.5: 10: 10: 5: 3.5: 1.

[0296] 4. Preparation of pure product

[0297] Take the above crude naked peptide, add water / acetonitrile to stir and dissolve, centrifuge to remove insoluble matter, and reserve.

[0298] Purification by reverse phase high performance liquid chromatography: column type: Agela C18, column size: 10 μm, 100 Å, 50 x 250 mm, mobile phase: mobile phase A (0.05% TFA and 2% acetonitrile in water) and mobile phase B (90% acetonitrile in water), flow rate of mobile phase: 25 mL / min, UV detection wavelength: 220 nm, elution mode: gradient elution. The crude product solution was loaded onto the above-mentioned column, and the purified components were collected and directly freeze-dried to remove the solvent, thereby obtaining the fluffy trifluoroacetate salt peptide product.

[0299] 5. Characterization of the product

[0300] The trifluoroacetate salt peptide product was redissolved with water and acetonitrile, and a large amount of anion exchange resin (acetate form) was added and stirred for 3 h. After filtration and washing of the ion exchange resin with a water / acetonitrile mixture, the filtrate was freeze-dried to obtain the fluffy peptide acetate product (i.e., the naked peptide in Tables 1-1 to 1-5 and Table 1-11). The chemical structure of the naked peptide was characterized by liquid chromatography-mass spectrometry, and the purity of each naked peptide was detected by an analytical high performance liquid chromatograph (column type: Agela C18, column size: 4.6 x 250 mm, flow rate: 1 mL / min), and the sequence structure, molecular weight, and purity of each naked peptide were determined.

[0301] Conclusion:

[0302] The naked peptides synthesized were confirmed by analytical HPLC to have a purity of greater than 95%, and mass spectrometry confirmed that they had the correct molecular weight.

[0303] Example 3: Synthesis of lipopeptides

[0304] 1. Chemical reagents required in the preparation process

[0305] Chemical reagents used: hydrazine hydrate, cholesterol succinate monoester;

[0306] Protective amino acid raw material used in the synthesis of the peptide: Fmoc-Lys(Dde)-OH.

[0307] 2. Synthesis of the overall sequence of the naked peptide and the linker sequence

[0308] Among them, the linker of the linker arm includes: the peptide of EAAAK sequence (SEQ ID NO: 104), the peptide of GSGSG sequence (SEQ ID NO: 105);

[0309] The lipophilic compound for modification includes: cholesterol succinate monoester.

[0310] 2.1 Synthesis of the main chain

[0311] (1) Synthesis of peptide resin: using Rink Amide MBHA resin as carrier resin, through deprotection of Fmoc and coupling reaction, the peptide resin was prepared by coupling with the corresponding protected amino acids of polypeptide amino acid sequence in turn.

[0312] (2) Accessing the first protected amino acid of the main chain

[0313] Take 0.3 mmol of the first protected amino acid and 0.3 mmol of HOBt, and dissolve them with an appropriate amount of DMF; take another 0.3 mmol of DIC, and slowly add it to the protected amino acid DMF solution under shaking, and shake at room temperature for 5 min to obtain an activated protected amino acid solution, which is ready for use.

[0314] Take 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g*0.3 g), and deprotect it with 25% PIPE / DMF solution (volume ratio) for 20 min (twice), and then wash and filter to obtain the Fmoc-deprotected resin.

[0315] Add the activated first protected amino acid solution to the Fmoc-deprotected resin, and couple for 60 min, then filter and wash to obtain the resin containing the first protected amino acid.

[0316] (3) Accessing other protected amino acids of the main chain

[0317] Using the same method as described above for accessing the first protected amino acid of the main chain, the corresponding other protected amino acids of the polypeptide are sequentially accessed to obtain the resin containing the main chain amino acids. Finally, the N-terminus is acetylated with 0.3 mmol of Ac2O+0.6 mmol of DIEA to complete the synthesis of the main chain. After each step, Kaiser Test is used to detect and control the reaction. If the condensation reaction of a certain amino acid is not complete, repeat the condensation once until the desired peptide segment is obtained.

[0318] 2.2 Accessing the side chain

[0319] (1) Treat the resin with as small a volume as possible of 2% hydrazine hydrate / DMF solution (volume ratio) to remove the Dde protecting group of the C-terminal lysine side chain (10 min, twice), and then filter and wash to obtain the Dde-deprotected resin for later use.

[0320] (2) Modification of the C-terminal lysine lipophilic compound of the polypeptide

[0321] C-terminal lysine cholesterol succinate monoester modification: 0.3 mmol of cholesterol succinate monoester and 0.3 mmol of HOBt were dissolved in an appropriate amount of DMF; 0.3 mmol of DIC was slowly added to the solution containing cholesterol succinate monoester and HOBt, and the reaction was oscillated at room temperature for 5 min. The prepared solution containing cholesterol succinate monoester, HOBt and DIC was added to the Dde-removed resin obtained in step (1), and the coupling reaction was performed for 60 min. After filtration, washing and drying, the peptide resin was obtained.

[0322] Other chemical reagents, amino acid raw materials and operation steps were consistent with those in Example 1.

[0323] Conclusion:

[0324] The analysis of HPLC results confirmed that the purity of the synthesized salt bridge polypeptides was greater than 98%, and the mass spectrum determined that the molecular weight of the polypeptides was consistent with the theoretical molecular weight.

[0325] Example 4: Anti-RSV activity detection of polypeptides

[0326] In the examples, the disclosure uses RSV-A long strain as the infection virus, and determines the anti-RSV activity of the synthesized polypeptides by CPE detection, and determines a series of new polypeptides with significantly better activity than the control polypeptide fusion inhibitor.

[0327] The inhibitory activity of the compounds in Tables 1-1 to 1-5, the positive polypeptide in Table 1-11 and the positive control T-118 on RSV-A long strain were detected.

[0328] 1. Experimental materials

[0329] Human laryngeal cancer cells (HEp-2) cells were from the American Type Culture Collection (ATCC), with the item number CCL-23. The cells were cultured in DMEM medium added with 10% fetal bovine serum, 1% sodium pyruvate, 1% non-essential amino acids, 2 mM glutamine, 100 U / mL penicillin and 100 µg / mL streptomycin. RSV A long strain was from ATCC, with the item number VR-26.

[0330] 2. Experimental method

[0331] The plaque reduction assay was used to test the activity of the test samples against RSV A long strain in vitro. The test samples and control compounds were tested at 8 concentrations in duplicate. HEp-2 cells were trypsinized and diluted to 300,000 cells per ml with assay medium. The diluted cells were added to the 96-well cell assay plates at 100 μL per well, 30,000 cells per well. The cells were incubated overnight in a 37°C incubator with 5% CO2. The next day, the test samples were diluted with assay medium in a 2-fold dilution series. The diluted samples were mixed with an equal volume of virus (100 PFU per well) and incubated at 37°C for 1 h in a 5% CO2 incubator. Then the culture medium was removed from the 96-well cell assay plates and the sample-virus mixture (200 μL) was added to the 96-well cell assay plates and incubated at 37°C for 2 h in a 5% CO2 incubator. After 2 h, the sample-virus mixture was removed and 200 μL of maintenance medium containing 0.8% CMC with the corresponding concentration of test sample was added. Cell controls (cells without compound treatment or virus infection) and virus controls (cells infected with virus without compound treatment) were set up. The cells were incubated for 1 day in a 37°C incubator with 5% CO2. After 1 day of virus infection, the culture medium was removed and the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After washing the plates with DPBS, RSV-specific antibody (RSV antibody, 1:3000 dilution) was added and incubated at 37°C for 1 h. Then secondary antibody (donkey anti-goat IgG, 1:500 dilution) was added and incubated at 37°C for 1 h. The secondary antibody was removed and TrueBlue solution was added and stained for 10 min; after washing with running water, the plates were air-dried and the number of plaques per well was counted using a microplate imaging reader. The raw data were used to calculate the antiviral activity of the test samples.

[0332] Cytotoxicity experiments were tested in parallel with the antiviral experiments. HEp-2 cells were seeded into microplates at a density of 30,000 cells per well and incubated overnight in a 37°C incubator with 5% CO2. The next day, the diluted test samples were added. Cell controls (cells without compound treatment) and medium controls (medium only without cells or compound treatment) were set up. The final concentration of DMSO in the medium was 0.5%. The cells were incubated for 1 day in a 37°C incubator with 5% CO2. Cell viability was detected using the cell viability detection kit CCK8.

[0333] The raw data from the plaque count and cell viability tests were used to analyze the antiviral activity and cytotoxicity of the test samples, respectively. The calculation formulae are as follows:

[0334] % Inhibition = 100 - (sample value - cell control average value) / (virus control average value - cell control average value) x 100

[0335] Cell survival rate = (sample value - medium control average value) / (cell control average value - medium control average value) x 100

[0336] The inhibition rate of the sample was analyzed by non-linear fitting using GraphPad Prism (version 10), and the IC 50 and CC 50 values of the sample were calculated.

[0337] IC 50 : In the in vitro antiviral activity experiment, IC 50 represents the index of the inhibitory ability of the test compound on RSV A long strain infection of Hep-2 cells, specifically, when the test compound reaches a certain concentration, it can effectively inhibit 50% of the infection of RSV A long strain on Hep-2 cells, and this concentration is the IC 50 .

[0338] CC 50 : In the in vitro antiviral activity experiment, CC 50 is used to measure the toxicity of the test compound to Hep-2 cells. Specifically, when the test compound is at a certain concentration, it can cause 50% of the Hep-2 cells to die, and this concentration is defined as CC 50 . The fitting formula is: log (inhibitor) vs. response -- Variable slope (four parameters).

[0339] Safety index SI: SI = IC 50 / CC 50

[0340] The positive polypeptides 1 to 7 were used as controls to detect the anti-RSV virus activity of the polypeptide sample. The cell fusion inhibition activity of the polypeptide sample and the positive polypeptide is shown in Table 2:

[0341] Table 2 Cell fusion inhibition activity

[0342]

[0343]

[0344] wherein the safety index (SI) is CC 50 / IC 50 ; NA: cannot be calculated, not provided.

[0345] Conclusion

[0346] 1. The anti-RSV virus activity of the designed salt bridge naked peptide sample is very high.

[0347] 2. The salt bridge denatured peptide sample prepared by the method of the present disclosure has high safety, and the safety index SI is all >1, and can be used as a safe and effective anti-RSV virus alternative drug.

[0348] Example 5: Pharmacodynamics of polypeptides in the study of protection of mice from infection

[0349] 1. Experimental materials

[0350] Female BALB / c mice were from Shanghai Yishang Biotechnology Co., Ltd. Human laryngeal cancer cells (HEp-2) were from the American Type Culture Collection (ATCC), with the item number CCL-23. The cells were cultured in DMEM medium added with 10% (v / v) fetal bovine serum and 1% penicillin and streptomycin. RSV A2 strain was from ATCC, with the item number VR-1540.

[0351] 2. Experimental methods

[0352] Eight-week-old female BALB / c mice were grouped according to body weight (n = 6). All animals were subjected to RSV nasal infection on Day 0, with an infection dose of 1 × 10 6 pfu (20 μL) per mouse. From 1 h after infection on Day 0, polypeptide samples, positive controls and solvent controls were administered by tracheal atomization once, for 5 consecutive days (dose and frequency of administration are shown in Table 3 below). At 2 h after the last administration on Day 4, lung tissues were collected from all animals, and the left lung tissues were quickly frozen in 10 times the volume of virus protection solution and stored at -70°C. Virus titers were determined by the plaque method.

[0353] For analysis, lung RSV A2 virus was determined by the plaque method of tissue homogenate virus load. Briefly, the day before sample testing, HEp-2 cells were inoculated in 12-well plates according to the standard operating procedures for HEp-2 cell culture, with a plating density of 0.3 × 10 6 cells / mL, and 1000 μL of cell suspension per well. On the day of incubation, the supernatant of the tissue homogenate sample was taken (maintained in a low-temperature environment), and after centrifugation, the supernatant was taken. The tissue homogenate sample was diluted with serum-free DMEM, and inoculated in 12-well plates at a final volume of 400 μL. After 2 h of incubation, the homogenate supernatant was discarded, and 1.5 mL of a cover layer (containing 1% soft agar in cell culture medium) was added to each well, and incubated in an incubator for about 72 h. The cover layer was discarded, the cells were fixed, blocked with blocking solution, stained with HRP-RSVG, and stopped with water, and air-dried. The color spots on the well plate were counted using a fluorescence (enzyme-linked) immunospot analyzer, and the virus titer was expressed as plaque-forming units per gram of tissue (PFU / g). The virus titer was calculated as the arithmetic mean ± standard error of all animals in a group.

[0354] Table 3 RSV A2 plaque detection results and statistics of each group

[0355]

[0356] Note: "QD" means once a day.

[0357] Compound 13, 14, 17, 19, 20, 21, 26, 27, 28, 40 were administered by airway nebulization once a day for 5 consecutive days at 1h after RSV A2 challenge in mice; lung tissues of all groups were collected at 2h after the last administration for virus plaque detection, and the results are shown in Table 3.

[0358] The results of the pharmacodynamic experiment of the compounds in mice showed that RSV compounds 13, 14, 17, 19, 20, 21, 26, 27, 28, 40 could significantly reduce the viral titer of lung tissues.

[0359] Example 6: Pharmacodynamics of polypeptides in cotton rat challenge protection study

[0360] 1. Experimental materials

[0361] Female cotton rats were from Shanghai Yishang Biotechnology Co., Ltd. RSV A2 strain was from ATCC, with item number VR-1540, RT-qPCR primers + probe primers were from Genview Biotechnology Co., Ltd., with batch number TSP20241227-025-00187, and RT-qPCR standard was from Vazyme, with batch number 20241008.

[0362] 2. Experimental methods

[0363] Female cotton rats (6-8 weeks old) were grouped according to body weight (n=5). The mice were operated according to the table 4 shown below. All animals were challenged with RSV by nose drop at Day 0, with a challenge dose of 1x10 5.5 pfu (50 μL) per mouse. From 1h after challenge at Day 0, polypeptide samples, positive controls and vehicle controls were administered by airway nebulization once a day for 5 consecutive days. All animals were collected for lung tissues at 2h after the last administration at Day 4. The left lung tissues (containing part of the trachea) were quickly frozen in 10 times the volume of virus protection solution and stored below -70℃, and the viral titer was detected by RT-qPCR.

[0364] Steps of RT-qPCR:

[0365] First, homogenization of the samples and RNA extraction from lung tissue were performed. The supernatant from the homogenized tissue samples was collected (while maintaining a low temperature). Then, approximately 100 μL of the homogenate supernatant was used to extract total RNA from the tissue using the VeZol reagent-chloroform-aqueous phase-magnetic bead method, dissolved in RNase-free water. The RT-qPCR reaction system was prepared as follows: RNA template, probe, primer pairs, RT-qPCR buffer, and RNase-free water were used to prepare a one-step RT-qPCR reaction system. Two replicates were set for each sample, with 20 μL of the one-step RT-qPCR reaction system per well. Quantitative standard curve wells (RSV A2 N gene full-length plasmid), positive control wells, and negative control wells were also included. The amplification program was then set according to reverse transcription, pre-denaturation, and cyclic amplification. Amplification data were collected using a real-time quantitative PCR instrument, and the mean CT value of each replicate was calculated. The CT value of each sample was input into the quantitative standard curve to calculate the copy number. The formula for calculating RSV viral load (copy number / gram of lung tissue) is: Sample load (copy number / gram of lung tissue) = copy number / relative tissue input (g).

[0366] The experimental results are shown in Table 4:

[0367] Table 4. Results of efficacy tests of the compounds in cotton rats.

[0368]

[0369] Note: "QD" means once a day.

[0370] One hour after RSV A2 challenge in cotton rats, compounds 13, 14, 17, 19, 20, 21, 26, 27, 28, and 40 were administered via inhalation once daily for five consecutive days. Two hours after the last administration, lung tissue from all groups was collected for viral titer detection, and the results are shown in Table 4.

[0371] The efficacy results of the compounds in cotton rats showed that RSV compounds 13, 14, 17, 19, 20, 21, 26, 27, 28, and 40 could significantly reduce the viral titer in lung tissue.

[0372] References:

[0373] [1] Natalie I Mazur, Jonne Terstappen, Ranju Baral, Azucena Bardaji, Philippe Beutels, Ursula J Buchholz, Cheryl Cohen, James E Crowe Jr, Clare L Cutland, Linda Eckert, Daniel Feikin, Tiffany Fitzpatrick, Youyi Fong, Barney S Graham, Terho Heikkinen, Deborah Higgins, Siddhivinayak Hirve, Keith P Klugman, Leyla Kragten-Tabatabaie, Philippe Lemey, Romina Libster, Yvette Lovensteyn, Asuncion Mejias, Flor M Munoz, Patrick K Munywoki, Lawrence Mwananyanda, Harish Nair, Marta C Nunes, Octavio Ramilo, Peter Richmond, Tracy J Ruckwardt, Charles Sande, Padmini Srikantiah, Naveen Thacker, Kody A Waldstein, Dan Weinberger, Joanne Wildenbeest, Dexter Wiseman, Heather J Zar, Maria Zambon, Louis Bont. Respiratory syncytial virus prevention within reach: the vaccine and monoclonal antibody landscape, Lancet Infect Dis (2022), https: / / doi.org / 10.1016 / S1473-3099(22)00291-2.

[0374] [2] Dirk Roymansa, Hendrik L. De Bondta, Eric Arnoultb, Peggy Geluykensa, Tom Geversc, Marcia Van Ginderena, Nick Verheyena, Hidong Kimd, Rudy Willebrordse, Jean-Francois Bonfantib, Wouter Bruinzeele, Maxwell D. Cummingsa, Herman van Vlijmena, and Koen Andries, Binding of a potent small-molecule inhibitor of six-helix bundle formation requires interactions with both heptad-repeats of the RSV fusion protein, PNAS, January 5, 2010, vol. 107, no. 1, 308-313.

[0375] [3] Lifeng Cai and Shibo Jiang, Development of Peptide and Small-Molecule HIV-1 Fusion Inhibitors that Target gp41, ChemMedChem 2010, 5, 1813-1824.

[0376] [4] A. Lazzarin, B. Clotet, D. Cooper, J. Reynes, K. Arasteh, M. Nelson, C. Katlama, H. Stellbrink, J. Delfraissy, J. Lange, L. Huson, R. DeMasi, C. Wat, J. Delehanty, C. Drobnes, M. Salgo, (TORO 2 Study Group), New Engl. J. Med. 2003, 348, 2186-2195.

[0377] [5] Rory D. de Vries, Katharina S. Schmitz, Francesca T. Bovier, Camilla Predella, Jonathan Khao, Danny Noack, Bart L. Haagmans, Sander Herfst, Kyle N. Stearns, Jennifer Drew-Bear, Sudipta Biswas, Barry Rockx, Gaël McGill, N. Valerio Dorrello, Samuel H. Gellman, Christopher A. Alabi, Rik L. de Swart1, Anne Moscona, Matteo Porotto, Intranasal fusion inhibitory lipopeptide prevents direct-contact SARS-COV-2 transmission in ferrets, Science 371, 1379-1382 (2021).

[0378] [6] Yuanmei Zhu, Xiaojing Dong, Nian Liu, Tong Wu, Huihui Chong, Xiaobo Lei, Lili Ren, Jianwei Wang & Yuxian He, SARS-COV-2 fusion-inhibitory lipopeptides maintain high potency against divergent variants of concern including Omicron, Emerging Microbes & Infections, 2022, VOL. 11, 1819-1827. https: / / doi.org / 10.1080 / 22221751.2022.2098060

[0379] [7] D. M. LAMBERT*, S. BARNEY, A. L. LAMBERT, K. GUTHRIE, R. MEDINAS, D. E. DAVIS, T. BUCY, J. ERICKSON, G. MERUTKA, AND S. R. PETTIGREW, JR., Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion, Proc. Natl. Acad. Sci. USA Vol. 93, pp.2186-2191, March 1996.

[0380] [8] Lifeng Cai and Miriam Gochin, The Role of Amphiphilicity and Negative Charge in Glycoprotein 41 Interactions in the Hydrophobic Pocket, J. Med. Chem. 2009, 52, 4338-4344 DOI: 10.1021 / jm900190q.

[0381] [9] Guiying Zhang, Kun Wang, Baohua Zheng, Maosheng Cheng, Yanni Li, Keliang Liu and Lifeng Cai, Exchangeability of amino acid residues with similar physicochemical properties in coiled-coil interactions, Chem. Commun., 2013, 49, 11086.

[0382]

[10] Lifeng Cai, Chungen Pan, Liang Xu, Yuan Shui, Keliang Liu, and Shibo Jiang, Interactions between different generation HIV-1 fusion inhibitors and the putative mechanism underlying the synergistic anti-HIV-1 effect resulting from their combination, FASEB J. 26, 1018 - 1026 (2012). www.fasebj.org.

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[11] Chungen Pan, Lifeng Cai, Hong Lu, Lu Lu, and Shibo Jiang, A Novel Chimeric Protein-based HIV-1 Fusion Inhibitor Targeting gp41 Glycoprotein with High Potency and Stability, THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 32, pp. 28425 - 28434, August 12, 2011.

[0384]

[12] Baohua Zheng a,b, Kun Wang b, Lu Lu c, Fei Yu d, Maosheng Cheng, Shibo Jiang, Keliang Liu a and Lifeng Cai, Hydrophobic mutations in buried polar residues enhance HIV-1 gp41 N-terminal heptad repeat-C-terminal heptad repeat interactions and C-peptides' anti-HIV activity, AIDS 2014, 28:1251 - 1260.

[0385] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to those details can be made within the scope of the application as disclosed in the above teachings. The foregoing detailed description has set forth various embodiments of the application via specific examples. It is to be understood, however, that the application is not limited to these embodiments, but embraces all changes and alterations to the application that fall within the scope of the appended claims and their equivalents.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound or a pharmaceutically acceptable salt thereof comprises a polypeptide of a sequence selected from the group consisting of Formula III: Formula III: X''1-X''2-X''3-X''4-D-X''6-X''7-IEEVN-X'' 13 -X'' 14 -IEESL-X'' 20 -X'' 21 -IEESD-X'' 27 -X'' 28 -L-X'' 30 -X'' 31 -V-X'' 33 -X'' 34 ; wherein, X''1 is W or F; X''2 is D or absent; X''3 is E or absent; X''4 is F or absent; X''6 is K or A; X''7 is K or S; X 13 K or R; X 14 R is K or R; X 20 K or R; X 21 K or R; X 27 K or R; X 28 K or R; X 30 E or H; X 31 E or N; X'' 33 is N or absent; X'' 34 is A or absent; the sequence of Formula III is selected from any one of SEQ ID NO: 91~95; The compound is a polypeptide of a sequence of Formula III-1: Formula III-1: R1-X''1-X''2-X''3-X''4-D-X''6-X''7-IEEVN-X'' 13 -X'' 14 -IEESL-X'' 20 -X'' 21 -IEESD-X'' 27 -X'' 28 -L-X'' 30 -X'' 31 -V-X'' 33 -X'' 34 -R2-R3; wherein, R1 is an amino terminal protection group, and R1 is acetyl; R2 is -R4-R5(R6)-, wherein R4 is EAAAK or GSGSG; R5 is lysine; R6 is a lipophilic compound group modified to R5, and the lipophilic compound group is selected from the group consisting of one or more of cholesteryl succinate monoester, 2-cholesterol acetic acid, 2-cholesterol propionic acid, 3-cholesterol propionic acid, 2-cholesterol butyric acid, 2-cholesterol isobutyric acid, 3-cholesterol butyric acid, 3-cholesterol isobutyric acid, 4-cholesterol butyric acid, 2-cholesterol valeric acid, 2-cholesterol isovaleric acid, 3-cholesterol valeric acid, 5-cholesterol valeric acid, 2-cholesterol hexanoic acid, 6-cholesterol hexanoic acid, 2-cholesterol heptanoic acid, 7-cholesterol heptanoic acid, 2-cholesterol octanoic acid, 8-cholesterol octanoic acid, cholesteryl bromoacetic acid, cholesteryl formyl chloride; R3 is a carboxyl terminal protection group, and R3 is -NH2.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein The compound is a polypeptide of a sequence selected from any one of SEQ ID NO: 40~44.

3. Use of the compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the manufacture of a polypeptide membrane fusion inhibitor against RSV; the RSV is RSV-A subtype.

4. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2.

5. The pharmaceutical composition of claim 4, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

6. A formulation product, characterized by, The preparation product comprises the pharmaceutical composition according to claim 4 or 5.

7. The formulation product according to claim 6, characterized in that, The dosage form of the preparation product comprises any one of the following group: oral dosage, injection dosage, inhalation dosage and lyophilized dosage.

8. The formulation product according to claim 7, characterized in that, The dosage form of the preparation product is inhalation dosage, lyophilized dosage, subcutaneous injection dosage or intramuscular injection dosage.

9. The formulation product according to claim 8, characterized in that, The administration mode of the preparation product is selected from any one of the following group: oral administration, injection administration, mucosal administration, transdermal administration and atomization administration.

10. The formulation product according to claim 9, characterized in that, The administration mode of the preparation product is pulmonary atomization administration, subcutaneous injection or intramuscular injection administration.

11. A process for preparing a compound as claimed in claim 1 or 2, or a pharmaceutically acceptable salt thereof, characterized in that, The method comprises the following steps: The salt-bridge naked peptide is prepared by sequentially coupling with the protected amino acids corresponding to the polypeptide amino acid sequence through deprotection and coupling reaction using a carrier resin.

12. The method of claim 11, wherein, The method further comprises one or more of the following steps: (1) lipophilic compound modification; (2) N-terminal acetylation capping; and, (3) C-terminal amidation capping.

13. The method of claim 12, wherein, The lipophilic compound modification is cholesteryl succinate monoester modification.

14. Use of the compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, the pharmaceutical composition according to claim 4 or 5, or the preparation product according to any one of claims 6~10 in the manufacture of a medicament for preventing and / or treating a disease caused by RSV; the RSV is RSV-A subtype.

15. A method of inhibiting RSV in vitro for non-therapeutic purposes, characterized in that, The method comprises administering to the sample a compound as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition as described in claim 4 or 5, or a preparation product as described in any one of claims 6 to 10; and the RSV is RSV-A subtype.

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