Anti-RSV polypeptide membrane fusion inhibitor as well as preparation method and application thereof

Abstract

The invention relates to an anti-RSV polypeptide membrane fusion inhibitor as well as a preparation method and application thereof. The invention discloses a compound, a pharmaceutical salt or a derivative thereof, a pharmaceutical composition and a preparation product containing the compound, and application of the compound and the pharmaceutical salt or the derivative in preparation of medicines for preventing and / or treating diseases caused by RSV or other paramyxoviridae viruses. The polypeptide disclosed by the invention shows the anti-RSV activity and safety which are obviously superior to those of positive control polypeptide. Molecular interaction experiments show that the polypeptide acts on the NHR region of the RSV F protein, and the fusion of the RSV virus and a target cell is prevented by forming an inactive six-helix structure with NHR, or by destroying the RSV NHR structure and inhibiting the process of forming a six-helix bundle by NHR and CHR in the F protein in the key step of RSV infection.

Description

[0001] This disclosure claims the benefit of Chinese Patent Application No. 2025101651370, filed February 14, 2025, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present disclosure belongs to the field of biomedical technology and relates to a polypeptide membrane fusion inhibitor for RSV, its preparation method, and use. Specifically, the disclosure relates to a class of polypeptides, particularly polypeptides that are resistant to human respiratory syncytial virus (RSV) or other related enveloped viruses, and the use of such polypeptides in the preparation of RSV membrane fusion inhibitors and drugs for treating or preventing RSV infection. Background Art

[0003] Human respiratory syncytial virus (RSV) infection causes respiratory diseases, posing a major health threat to infants, the elderly, and immunocompromised people. The vast majority of infants and young children under the age of 2 have been infected with RSV. Every year, 30 million children under the age of 5 worldwide become ill due to RSV infection, approximately 3 million require hospitalization, and approximately 100,000 infants and young children die. RSV infection has become the leading cause of death in infants and young children in developed economies. RSV also infects the elderly. Every year, more than 30 million people over the age of 60 worldwide become ill due to RSV infection, resulting in the death of 500,000 to 600,000 elderly people. In addition, RSV also poses a serious threat to the growing number of immunocompromised people due to cancer, immunotherapy, environmental and other factors. [1] .

[0004] Currently, there are no effective preventive and treatment measures for RSV infection. Ribavirin, the only available treatment for RSV infection, is an older broad-spectrum antiviral drug with poor specificity, inconvenience, and questionable effectiveness. Regarding prevention, the development of newer drugs and vaccines, such as long-acting monoclonal antibodies and mRNA vaccines, is actively advancing. Palivizumab and nirsevimab are monoclonal antibodies currently used to prevent RSV in high-risk infants and young children. They offer some protection, but the protection rate is relatively low. Palivizumab requires monthly injections, is expensive, requires multiple injections during the infection season, and offers only approximately a 50% protection rate. Furthermore, Clesrovimab, developed by Merck, is in the FDA approval process.

[0005] In recent years, RSV vaccine development has made significant progress. Pfizer's PF-06928316 (RSV preF) and GSK's GSK3844766A have received US FDA approval for use in adults over 60 years old, but they are still not approved for use in infants and young children. Currently, four small-molecule RSV fusion inhibitors have entered clinical trials, but all have been discontinued or denied marketing approval for various reasons. This demonstrates the enormous challenges facing the development of RSV therapeutics.

[0006] Although some medicines are available in the field of prevention and treatment of RSV, the protective effect is generally not high or the efficacy is poor or the side effects are many, and it is urgent to develop more effective and safe therapeutic drugs for the respiratory diseases caused by RSV infection. And because RSV is prevalent in healthy people and can infect many times, the RSV virus is difficult to remove in the crowd, and infection is difficult to avoid, especially in infants, the elderly and immunocompromised people. Therefore, the medicine for the treatment of RSV infectious diseases is of great significance. Simultaneously, because RSV spreads and mutates in the crowd for a long time, drug-resistant mutants for existing and future drugs on the market will constantly appear, and it is necessary to continuously launch new drugs to counteract the resistant strains.

[0007] RSV is an enveloped virus that infects cells through virus-cell membrane fusion mediated by its envelope glycoprotein F protein. The F protein belongs to the first type of membrane fusion protein, which forms a non-covalent complex with surface subunits and transmembrane subunits. It is usually embedded in the virus surface in the form of a trimer to form spikes. In the membrane fusion protein complex, three transmembrane subunits make up the root of the spike, forming an unstable high-energy core, which is a direct participant in membrane fusion; three surface subunits are wrapped around the outside of the core and serve as guiding molecules for viral infection. They are responsible for specific cell receptor recognition, determine the cell types that the virus can infect, and stabilize the high-energy structure of the transmembrane subunits. RSV has a similar infection mechanism to HIV-1, influenza, coronavirus, paramyxoviridae, Ebola virus, etc., which are also first-class enveloped viruses. The fusion core structure is a six-helix bundle structure. [2] .

[0008] During viral infection, the surface subunit first binds to specific cell receptors on the cell surface, bringing the virus closer to the target cell. Receptor binding simultaneously causes the dissociation of the surface subunit and transmembrane subunit complex, releasing the unstable transmembrane subunit. The transmembrane subunit first spontaneously releases the N-terminal fusion peptide (FP) to insert into the target cell membrane (spring model). Its C-terminus is anchored in the viral membrane through the transmembrane region, forming a fusion precursor that connects the viral and cell membranes. The fusion precursor, from the viral membrane to the target cell membrane, contains important functional regions such as the transmembrane region, C-terminal heptad repeat (CHR), loop region (Loop), N-terminal heptad repeat (NHR), and FP. The fusion precursor is also an unstable structure. Its NHR and CHR rotate and spontaneously fold in reverse. The three NHRs form the inner core of the trimer, and the three CHRs bind antiparallel to the three grooves formed by the NHR trimer, forming a six-helix bundle structure. The formation of the six-helix bundle draws the viral and cell membranes together, enabling membrane fusion and ultimately allowing the virus to infect the cell. [3] .

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

[0010] In the 1990s, Trimeris, the developer of the first membrane fusion inhibitor, based on its research on peptide-based AIDS membrane fusion inhibitors, investigated peptide RSV membrane fusion inhibitors and discovered that peptides derived from the CHR region of the RSV F protein had anti-RSV activity. These peptides were all derived from the natural sequence of the RSV F protein, and T-118, the most active, served as a representative peptide and became an important reference for subsequent research. [7] .

[0011] However, there is currently a lack of a polypeptide RSV membrane fusion inhibitor with better anti-RSV activity and safety. Summary of the Invention

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

[0013] Based on the existing technology, the inventors proposed a research concept on the interaction of polypeptide fragments that is suitable for the dynamic molecular interaction characteristics of envelope glycoproteins during membrane fusion, which was used for the design and optimization of new membrane fusion inhibitors. They obtained a new RSV fusion inhibitor - salt bridge polypeptide, which has a structure completely different from existing RSV fusion inhibitors but maintains specific interactions.

[0014] This paper uses the study of HIV-1 membrane fusion inhibitors as a model to systematically study the viral membrane fusion mechanism, discover a series of highly active peptides and small molecule membrane fusion inhibitors, and systematically study the mechanism of action and drug design of membrane fusion inhibitors from the perspective of protein folding and protein-protein interaction. It proposes a new design concept and research method for membrane fusion inhibitors that conform to the dynamic Coiled-cholesterol succinate monoester il protein interaction characteristics during membrane fusion. [8-12] In peptide design, the basic hydrophobic and electrostatic interactions are the main control targets according to the folding characteristics of Coiled-cholesterol succinate monoester id protein. [8] , by classifying the physicochemical properties of amino acid residues to simplify the design [9] ; In the study of molecular mechanism of action, we should attach importance to the balance between the binding kinetics and thermodynamics of membrane fusion inhibitors and targets.

[10] , obtained peptides with improved targeting specificity and antiviral activity, and successfully applied the above concept to the design of new membrane fusion inhibitors [11,12] .

[0015] Based on the above reasons, the inventors proposed a research concept on the interaction of polypeptide fragments that is suitable for the dynamic molecular interaction characteristics of envelope glycoproteins during membrane fusion, which is used for the design and optimization of novel membrane fusion inhibitors. This disclosure designs polypeptide sequences based on the common characteristics of enveloped virus fusion mechanisms and the structural characteristics of the RSV viral membrane fusion protein F protein, identifies its target site, and optimizes its sequence at the protein folding level. At the same time, based on the inventors' discovery of the contribution of hydrophobic and electrostatic interactions to the activity of membrane fusion inhibitors, appropriate hydrophobic groups are introduced to further enhance their activity. This completes the present invention.

[0016] In the present invention, a detailed investigation was conducted on the polypeptide sequence from the RSV F protein, and it was found that solubility and the resulting difficulty in synthesis and purification are common in natural RSV polypeptides, becoming an important factor affecting the further modification and drug development of natural RSV polypeptides. The present disclosure creatively adopts a new design concept and research method for membrane fusion inhibitors that are suitable for the dynamic coiled coil protein interaction characteristics during membrane fusion, discovers model polypeptides with anti-RSV activity, mutates, modifies and adjusts the length of the model polypeptides to improve their performance, and discovers a series of polypeptides with significantly higher activity than existing anti-RSV polypeptides, good solubility, easy synthesis, and excellent drugability. The polypeptides discovered in the present disclosure are all composed of natural protein amino acids, are easy to synthesize on a large scale, are convenient for expression in biological vectors, and have excellent metabolic safety. At the same time, modifications can be introduced to further enhance their activity and improve their 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 invention first designs a polypeptide template such as sequence (1) (SEQ ID NO: 103):

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

[0019] In the template, Z1 is the amino terminus (NH2-) or its modification, and Z2 is the carboxyl terminus (-COOH) or its modification. Unless otherwise specified, Z1 is selected from the acetyl group (Ac-) and Z2 is selected from the amide group (-NH2), which respectively block the amino acid and carboxyl termini of the polypeptide to enhance the stability of the polypeptide.

[0020] J is an acidic amino acid residue, which may be, but is not limited to, a glutamic acid residue and an aspartic acid residue; O is a basic amino acid residue, which may be, but is not limited to, a lysine residue and an 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 in the native sequence of RSV CHR [7] , mainly composed of hydrophobic amino acid residues, forming a hydrophobic surface that contacts and matches the drug target, producing a specific interaction. Z3 is a fatty acid modification group, including a linker arm and a lipophilic group, where the lipophilic group binds to the drug's target cells.

[0021] The polypeptide compounds disclosed herein are further designed using a training method for a polypeptide-receptor binding activity prediction model.

[0022] In one aspect, the present disclosure provides a compound, a pharmaceutically acceptable salt thereof, or a derivative thereof, comprising a polypeptide having a sequence selected from 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.

[0023] On the other hand, the present disclosure also provides use of the compound, its pharmaceutically acceptable salt or derivative thereof in the preparation of an anti-RSV polypeptide membrane fusion inhibitor.

[0024] In another aspect, the present disclosure also provides an isolated nucleic acid molecule encoding the compound, a pharmaceutically acceptable salt thereof, or a derivative thereof.

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

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

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

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

[0029] On the other hand, the present disclosure also provides a method for preparing the compound, its pharmaceutically acceptable salt or its derivative, which comprises using a carrier resin, and sequentially coupling with protected amino acids corresponding to the polypeptide amino acid sequence through deprotection and coupling reactions to prepare a salt-bridged naked peptide.

[0030] On the other hand, the present disclosure also provides use of the compound, its pharmaceutically acceptable salt or derivative thereof, the pharmaceutical composition or the formulation product in the preparation of a medicament for preventing and / or treating diseases caused by RSV or other Paramyxoviridae viruses.

[0031] On the other hand, the present disclosure also provides a method for preventing and / or treating diseases caused by RSV or other Paramyxoviridae viruses, which comprises administering an effective amount of the compound, its pharmaceutically acceptable salt or derivative, the pharmaceutical composition or the preparation product to a subject in need.

[0032] On the other hand, the present disclosure also provides the compound, its pharmaceutically acceptable salt or derivative thereof, the pharmaceutical composition or the formulation product for use in preventing and / or treating diseases caused by RSV or other Paramyxoviridae viruses.

[0033] In another aspect, the present disclosure further provides a method for inhibiting RSV, comprising administering the compound, its pharmaceutically acceptable salt or derivative, the pharmaceutical composition or the formulation to a sample.

[0034] In one embodiment, the present disclosure uses RSV-A long strain as the infectious virus, and the anti-RSV activity of the synthesized polypeptide is determined by the Plague Reduction method, and a series of IC 50 A novel anti-RSV peptide with a concentration of less than 20 nanomolars, suitable for clinical drug development.

[0035] In another embodiment, the present disclosure uses SD rats to administer compounds via a nebulizer needle and determine the concentration in lung tissue at different time points to determine lung tissue exposure.

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

[0037] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present disclosure.

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

[0039] The beneficial effects achieved by the present invention are:

[0040] The polypeptide of the present invention shows an anti-RSV activity that is significantly better than that of the positive control polypeptide, and the polypeptide with the highest activity is more than 100 times that of the positive control polypeptide; it shows a safety that is significantly better than that of the positive control polypeptide. Molecular interaction experiments show that the polypeptide acts on the NHR region of the RSV F protein, and inhibits the key step of RSV infection, the process of NHR and CHR forming a six-helix bundle in the F protein, by forming an inactive six-helix structure with NHR, or by destroying the RSV NHR structure, thereby preventing the fusion of RSV virus and target cells. At the same time, the polypeptide of the present invention adopts a unique design strategy, which is completely different from the existing RSV membrane fusion inhibitor sequences and design ideas. The water solubility of the polypeptide is increased by the salt bridge sequence, while maintaining the specific interaction between the polypeptide and the target, ensuring that the polypeptide has higher drugability. The anti-RSV virus activity of the polypeptide sample designed in the present disclosure is very high, and its IC 50 The active IC prepared by the present invention is controlled within 30nM, preferably within 20nM, more preferably within 15nM, and the lowest is 9.9nM. 50 The safety index of peptide samples with a concentration of <30nM was >260, indicating that they can be used as safe and effective alternative anti-RSV drugs. DETAILED DESCRIPTION

[0041] In order to make this disclosure more easily understood, some terms are first defined. In addition, it should be noted that whenever a value or a range of values ​​for a parameter is listed, it is intended to indicate that values ​​and ranges intermediate to these cited values ​​are also part of this disclosure.

[0042] As used herein, the articles "a" and "an" refer to one or to more than one (ie, to at least one) of the grammatical object of the article. By way of example, "an element" refers to one element or more than one element, such as a plurality of elements.

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

[0044] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0045] The abbreviations used in this disclosure have the following meanings:

[0046] Ala (Alanine, A)

[0047] Arg (Arginine, R) Arginine

[0048] Asn (Asparagine, N)

[0049] Asp (Aspartic acid, D)

[0050] DCM (Dichloromethane)

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

[0052] Env (Envelope glycoprotein)

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

[0054] Fmoc (Fluorenylmethoxycarbonyl)

[0055] Gly (Glycine, G) Glycine

[0056] Gln (Glutamine, Q) glutamine

[0057] Glu (Glutamic acid, E) glutamate

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

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

[0060] His (Histidine, H)

[0061] HoBt (1-Hydroxyl benzotiazole anhydrous)

[0062] NHR (N-terminal heptad repeat)

[0063] CHR (C-terminal heptad repeat)

[0064] HIV (Human immunodeficiency virus)

[0065] HPLC (high performance liquid chromatography)

[0066] Ile (Isoleucine, I) Isoleucine

[0067] Leu (Leucine, L)

[0068] Met (Methionine, M)

[0069] Lysine (K)

[0070] Phe (Phenylalanine, F) Phenylalanine

[0071] RSV (Respiratory syncytial Virus)

[0072] Ser (Serine, S) serine

[0073] TFA (Trifluoroacetic acid)

[0074] Thr (Threonine, T) Threonine

[0075] Tyr (Tyrosine, Y) tyrosine

[0076] Val (Valine, V)

[0077] The single-letter amino acid residues are represented 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-Aspartic Acid; 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 one aspect, the present disclosure provides a compound, a pharmaceutically acceptable salt thereof, or a derivative thereof, comprising a polypeptide having a sequence selected from 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 1 (SEQ ID NO: 122):

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

[0082] in,

[0083] X1 is D or does not exist;

[0084] X2 is E or does not exist;

[0085] X3 is W, F, D, S or does not exist;

[0086] X4 is D or does not exist;

[0087] X5 is E, A, K or does not exist;

[0088] X6 is F, S or does not exist;

[0089] X7 is D, N, L or does not exist;

[0090] X8 is K, Q, L or does not exist;

[0091] X9 is K or does not exist;

[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 E or not present;

[0105] X 33 E, V or absent;

[0106] X 34 V, N, S or not present;

[0107] X 35 N, K, D or not present;

[0108] X 36 K or not present;

[0109] X 37 K, A or not present;

[0110] X 38 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] in,

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

[0115] X'2 is V or does not exist;

[0116] X'3 is K or does not exist;

[0117] X'4 is D, K or does not exist;

[0118] X'5 is F, I or does not exist;

[0119] X'6 is D or does not exist;

[0120] X'8 is L or does not exist;

[0121] X'9 is V or does not exist;

[0122] X' 10 F or not present;

[0123] X' 11 is E or D;

[0124] X' 12 A or I;

[0125] X' 13 is S or does not exist;

[0126] X' 14 is 1 or does not exist;

[0127] X' 23 is E or Q;

[0128] X' 26 A or E;

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

[0130] X' 30 is L or does not exist;

[0131] X' 31 A or not present;

[0132] X' 32 F or not present;

[0133] X' 33 is 1 or does not exist;

[0134] X' 34 R or not present;

[0135] X' 41 H or not present;

[0136] X' 42 N or does not exist;

[0137] X' 43 V or not present;

[0138] X' 44 N or does not exist;

[0139] X' 45 A or not present;

[0140] X' 46 is G or does not exist;

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

[0142] X' 48 is S or does not exist;

[0143] X' 49 is T or does not exist;

[0144] X' 50 is T or does not exist;

[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] in,

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

[0149] X''2 is D or does not exist;

[0150] X''3 is E or does not exist;

[0151] X''4 is F or does not exist;

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

[0153] X''7 is 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 N or does not exist;

[0163] X'' 34A or not present;

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

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

[0166] in,

[0167] X''' 11 V or not present;

[0168] X'''' 32 V, N or not present;

[0169] X''' 33 N, A or not present;

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

[0171] X''' 35 L or not present.

[0172] In some embodiments, the compound, its pharmaceutically acceptable salt, or derivative thereof comprises a polypeptide whose sequence is selected from any one or more of SEQ ID NOs: 52 to 102.

[0173] In the present disclosure, the polypeptide defined above is the core sequence contained in the compound, its pharmaceutically acceptable salt or its derivative. The core sequence is the key part for producing RSV inhibition. It can act on the NHR region of the RSV F protein, forming an inactive six-helix structure with the NHR, or by destroying the RSV NHR structure, inhibiting the process of the NHR and CHR forming a six-helix bundle in the F protein, a key step in RSV infection, thereby preventing the fusion of RSV virus with target cells. This inhibitory effect does not depend on modifications at both ends of the polypeptide, modification with a linker or a lipophilic compound, etc.

[0174] In some embodiments, the compound, its pharmaceutically acceptable salt, or derivative thereof comprises a polypeptide having a sequence selected from any one or more of Formula I-1, Formula II-1, Formula III-1, Formula IV-1, and SEQ ID NOs: 106 to 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'[[ID=۸5]] 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; <了

[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] in:

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

[0201] R2 is an absent or arbitrarily substituted linker, preferably -R4-R5(R6)-, wherein R4 is a polypeptide, and its amino acid sequence is preferably (EAAAK) m or (GSGSG) m ; m is a natural number from 0 to 5; specifically, 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-diaminoheptanoic acid, preferably lysine;

[0203] R6 is a lipophilic compound group modified on R5, preferably the lipophilic compound group is selected from the group consisting of cholesterol, cholesterol succinate, 2-cholesterol acetate, 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, cholesterol bromoacetate, cholesterol methyl chloride, palmitic acid, stearic acid, fatty acids containing 3-20 carbon atoms, fatty diacids containing 3-20 carbon atoms, and other groups that can interact with cell membranes or viral membranes to enhance the interaction of the polypeptide with cell membranes or viral membranes; more preferably cholesterol succinate monoester;

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

[0205] In some embodiments, the compound, a pharmaceutically acceptable salt thereof, or a derivative thereof comprises a polypeptide whose sequence is selected from any one or more of SEQ ID NOs: 1 to 51.

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

[0207] In some embodiments, the pharmaceutically acceptable salts include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, methanesulfonate, borate, methyl bromide, bromide, methyl nitrate, calcium edetate, methylsulfate, camphorsulfonic acid, mucate, carbonate, naphthylsulfonate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, ethylenediaminetetraacetate, Sulfonate, pamoate, pamoate, propionate lauryl sulfate, palmitate, ethanesulfonate, pantothenate, fumarate, phosphate / diphosphate, glucoheptonate, polygalacturonate, gluconate, salicylate, glutamate, stearate, p-hydroxyacetamidophenylarsonic acid, sulfate, hydroxybenzoate, subacetate, hepatoacetate, succinate, hydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, 8-chlorotheophylline salt, iodide, toluenesulfonate, triethyl iodide, lactic acid, valerate, etc. Depending on the intended use, pharmaceutically acceptable salts can be formed with cations such as sodium, potassium, and bismuth, or with bases such as ammonia, ethylenediamine, N-methylglutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, diethylamine, piperazine, trishydroxymethylaminomethane, and tetramethylammonium hydroxide. These salts can be prepared using standard methods, for example, by reacting 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 hydrochlorides, hydrobromides, acetates, and pamoates can be used as dosage forms. In the presence of an acidic or alcoholic group, pharmaceutically acceptable esters such as acetates, maleates, and chloromethyl pivalate, 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 the compound, pharmaceutically acceptable salt or derivative thereof as described in the present disclosure in the preparation of an anti-RSV polypeptide membrane fusion inhibitor.

[0209] As described in this article, "anti-RSV polypeptide membrane fusion inhibitors" are specifically designed peptide drugs that inhibit the fusion of the viral envelope with the host cell membrane during RSV infection. By blocking this critical step, the virus cannot enter the host cell, thereby preventing viral replication and spread.

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

[0211] In another aspect, the present disclosure provides a recombinant vector comprising the nucleic acid molecule described in the present disclosure.

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

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

[0214] The pharmaceutical compositions disclosed herein may be solutions with or without a buffer or compositions containing a pharmaceutically acceptable carrier. In the present disclosure, the pharmaceutical compositions may be administered in a solution. They may be administered in an unbuffered solution, such as physiological saline or water. Alternatively, they may be administered in a suitable buffered solution. The buffered solution may include acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffered solution is phosphate-buffered saline (PBS). The pH and osmolality of the buffer of the pharmaceutical composition may be adjusted to make it suitable for administration to a subject.

[0215] In some embodiments, the buffer solution further comprises an agent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, such as the physiological value of human plasma. Solutes that can be added to the buffer solution to control 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. Typically, a suitable dosage of the compound of the present disclosure for use in mammals, particularly humans, can be between 0.1 mg / day and 100 mg / day, for example, between 10 mg / day and 50 mg / day, or, for example, between 20 mg / day and 30 mg / day.

[0217] The pharmaceutical composition can be given once a day, or can be given twice, three times or more sub-doses at appropriate intervals within one day, or can even be given by continuous infusion or sending through a controlled release formulation. In this case, the compound contained in each sub-dose must be correspondingly less, so as to realize a daily total dose. The dosage unit can also be composited and sent in a few days, for example, using a conventional sustained release formulation that provides a lasting compound to release within a few days' timeframe. Sustained release formulations are well known in the art and are particularly useful for delivering reagents at specific sites, and can be used with reagents of the present disclosure thus. In this embodiment, the dosage unit comprises a plurality of corresponding daily doses.

[0218] In other embodiments, a single dose of the pharmaceutical composition can be administered over a long period of time, such that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart. The treatment regimen can be administered once every 1-3 days for 4-7 consecutive days; if the infection recurs, the drug can be administered for an additional 4-7 consecutive days, resulting in a treatment cycle of 1 to 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 skilled in the art will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatment, the subject's overall health and / or age, and other existing conditions. Furthermore, treating a subject with a therapeutically effective dose of a composition may include a single treatment or a series of treatments. As described elsewhere herein, effective dosages and in vivo half-lives of various compounds encompassed by the present disclosure may be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0220] The pharmaceutical compositions of the present disclosure can be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (e.g., via a skin patch); pulmonary; e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer; intratracheal; intranasal; epidermal, as well as transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implant device; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular administration.

[0221] The pharmaceutical compositions of the present disclosure, which may conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the steps of combining the active ingredients with the pharmaceutical carrier or excipient. Generally speaking, the pharmaceutical compositions are prepared by uniformly and finely combining the active ingredients with a liquid carrier or a finely divided solid carrier, or both, and, if desired, 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 disclosed herein also incorporate carrier materials into the pharmaceutical compositions. These carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, and organic acids), poorly soluble carrier materials (such as ethyl cellulose and cholesterol stearate), and enteric-soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose). Water-soluble carrier materials are preferred. These materials can be used to formulate a variety of formulations, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal formulations, buccal tablets, suppositories, and lyophilized powder injections. These formulations can include standard formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit dosage forms into tablets, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, trehalose, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants and adhesives, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; Disintegration agents include dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearin, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium lauryl sulfate; and lubricants include talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or as bilayer and multilayer tablets. To prepare unit dosage forms as pills, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinyl pyrrolidone, gelucine, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dry starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.In addition, to prepare an isotonic injection solution, an appropriate amount of sodium chloride, glucose, or glycerol may be added to the injectable formulation. Conventional cosolvents, buffers, pH adjusters, and the like may also be added. Furthermore, colorants, preservatives, fragrances, flavoring agents, sweeteners, or other materials may be added to the pharmaceutical formulation as needed. The above dosage forms can be administered by injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections, including cavities such as the rectum and vagina, respiratory tracts such as the nasal cavity, and mucosal administration. Injection is preferred among these routes of administration.

[0224] In some embodiments, the pharmaceutical compositions 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 lyophilized; preferably, inhalation, lyophilized, subcutaneous injection, or intramuscular injection.

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

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

[0227] The salt-bridged naked peptide is prepared by using a carrier resin and sequentially coupling with the protected amino acids corresponding to the polypeptide amino acid sequence 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 succinate monoester modification;

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

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

[0232] In another aspect, the present disclosure provides use of the compound, pharmaceutically acceptable salt or derivative thereof, the pharmaceutical composition or formulation described herein in the preparation of a medicament for preventing and / or treating diseases caused by RSV or other Paramyxoviridae viruses.

[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] The Paramyxoviridae family is a family of negative-sense, single-stranded RNA viruses that primarily infect humans and animals. Viruses in this family can cause a variety of respiratory, neurological, 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).

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

[0236] The RSV disclosed herein includes one or more selected from RSV-A subtype, RSV-B subtype and other mutant strains. RSV A subtype strains are, for example, selected from A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22 and A23, etc.; RSV B subtype strains are, for example, selected from B1, B2, B3, B4, B5 and B6, etc.

[0237] In another aspect, the present disclosure provides a method for preventing and / or treating diseases caused by RSV or other Paramyxoviridae viruses, comprising administering to a subject in need thereof an effective amount of 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.

[0238] As used herein, "subject" is intended to include humans or non-human animals, preferably mammals, such as mice. Most preferably, the subject or patient is a human.

[0239] As used herein, "effective amount" is intended to include a dosage that is sufficient to achieve treatment of a disease caused by RSV when administered to a patient for the treatment of the disease (e.g., by weakening, improving, or maintaining the existing disease or one or more disease symptoms). The "effective amount" may vary depending on how the agent is administered, the disease and its severity, and the patient's medical history, age, body weight, family history, genetic makeup, the stage of the pathological process mediated by RSV, the type of previous or concomitant treatment (if any), and other individual characteristics of the patient to be treated. An "effective amount" also includes a dosage that produces a certain desired local or systemic effect under a reasonable benefit / risk ratio applicable to any treatment. The compound used in the method of the present disclosure may be given in an amount that is sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

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

[0241] In another aspect, the present disclosure provides a compound, a pharmaceutically acceptable salt or derivative thereof, a pharmaceutical composition or a formulation product as described herein, for use in preventing and / or treating diseases caused by RSV or other Paramyxoviridae viruses.

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

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

[0244] As used herein, the term "sample" includes similar fluids, cells, or tissues separated 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, lymph, urine, and saliva. Tissue samples can include samples from tissues, organs, or localized areas. For example, a sample can be derived from a specific organ, organ part, or the fluid or cells within these organs. In certain embodiments, a "sample" refers to blood or plasma extracted from the subject.

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

[0246] As used herein, "non-therapeutic purposes" refers to inhibiting RSV replication for research purposes, such as in the laboratory.

[0247] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0248] Example 1: Peptide Design

[0249] Based on the design experience of HIV fusion inhibitors and other viral fusion inhibitors, peptides in the CHR region of the transmembrane subunit of the viral envelope glycoprotein usually show high activity. [3,7]Based on this, the present invention designs polypeptides based on the CHR sequence of the F1 subunit of the F protein corresponding to the RSV virus. According to previous successful experience, a polypeptide with 36 amino acid residues can usually serve as a good starting point 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, examines their activity through molecular interactions and anti-RSV activity experiments, and further designs and optimizes the polypeptides based on the test results. The inventors found that natural sequence RSV CHR polypeptides usually have the disadvantages of poor water solubility and difficulty in synthesis, which poses a great challenge to subsequent drug development. Based on the inventors' experience and previous research results, the present disclosure systematically introduces an EE-KK salt bridge into the non-binding site of the RSV CHR polypeptide sequence to improve its water solubility. The salt bridge can also stabilize the α-helical secondary structure required for its activity. The present disclosure found that the introduction of a salt bridge not only improves water solubility, but also leads to a significant increase in activity, and a shorter polypeptide can be designed.

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

[0251] Based on Z1-WJJLVOOSJJFDOOIJJVNOOIJJSLOOIJJSDOOLJJVNOOLJJTNOOITTI-Z3-Z2, systematic mutations were introduced to prepare its derivatives, including mutating E and K in the salt bridge into corresponding D and R. At the same time, the binding site was fine-tuned to improve the binding of the polypeptide to the target, completing the design of the polypeptide in the embodiment.

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

[0253] Z3 is a fatty acid modification group, including a linker arm and a lipophilic group, wherein the lipophilic group binds to the target cells of the drug to increase the activity of the drug, and the linker arm is used to connect the polypeptide chain and the lipophilic group and provide a suitable three-dimensional space and conformation to enable the polypeptide drug to better bind to the target.

[0254] Table 1-1 Sequence design of polypeptide (Formula I-1)

[0255]

[0256] Table 1-2 Sequence design of polypeptide (Formula II-1)

[0257]

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

[0259]

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

[0261]

[0262] Table 1-5 Peptide sequence design

[0263]

[0264] Chol is cholesterol succinate monoester, which represents the ester formed by the carboxyl group of cholesterol succinate monoester and the amino group of lysine in the polypeptide as shown in the following structure:

[0265] .

[0266] Table 1-6 Sequence design of polypeptides (core sequences corresponding to Formula I)

[0267]

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

[0269]

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

[0271]

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

[0273]

[0274] Table 1-10 Peptide sequence design (core sequence)

[0275]

[0276] Table 1-11 Comparative Examples

[0277]

[0278] Here, the meaning of Chol is as shown above.

[0279] Example 2: Synthesis of naked peptide

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

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

[0282] Protected amino acid raw materials used in the peptide 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, and 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] Rink Amide MBHA resin was used as a carrier resin, and the peptide resin was prepared by coupling with the corresponding protected amino acids in the polypeptide amino acid sequence in sequence through Fmoc removal and coupling reaction.

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

[0286] Dissolve 0.3 mmol of the first protected amino acid and 0.3 mmol of HOBt in an appropriate amount of DMF. Slowly add 0.3 mmol of DIC to the protected amino acid DMF solution under shaking. Shake and react at room temperature for 5 min to obtain the activated protected amino acid solution for later use.

[0287] 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). Wash and filter to obtain the Fmoc-free resin.

[0288] The activated first protected amino acid solution was added to the Fmoc-removed resin, and the coupling reaction was carried out for 60 minutes. The resin containing the first protected amino acid was filtered and washed to obtain the resin.

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

[0290] The same method as described above for inserting the first protected amino acid in the main chain is used to sequentially insert other protected amino acids corresponding to the polypeptide to obtain a resin containing main chain amino acids.

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

[0292] After each step of the above reaction, the reaction is controlled by Kaiser Test. If the condensation reaction of an amino acid is incomplete, the condensation is repeated until the desired target peptide is obtained.

[0293] 3. Preparation of Crude Product

[0294] To the peptide resin, add the cleavage reagent (15 mL / g resin), mix thoroughly, and shake at 30°C for 3 h to cleave the target peptide from the resin and remove the side chain protecting groups. Collect the filtrate from the reaction mixture, wash the resin three times with a small amount of TFA / DCM, combine the filtrates, add anhydrous ether, and precipitate by centrifugation. Wash the filter cake twice with cold anhydrous ether and drain to obtain an off-white powder, which is the crude naked peptide.

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

[0296] 4. Preparation of Pure Product

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

[0298] Purification was performed using reversed-phase HPLC using an Agela C18 column (10 μm, 100Å, 50 × 250 mm). Mobile phases A (0.05% TFA and 2% acetonitrile in water) and B (90% acetonitrile in water) were used at a flow rate of 25 mL / min. UV detection was performed at 220 nm, and the elution method was gradient elution. The crude product solution was loaded onto the aforementioned column, and the corresponding purified fractions were collected and freeze-dried to remove the solvent, yielding the pure trifluoroacetate peptide in a fluffy state.

[0299] 5. Characterization of Pure Products

[0300] The purified trifluoroacetate peptide salt was re-dissolved in water and acetonitrile, and a large amount of anion exchange resin (acetate form) was added and stirred for 3 hours. After filtration and rinsing the ion exchange resin with a water / acetonitrile mixture, the combined filtrates were lyophilized to obtain the fluffy purified peptide acetate salt (i.e., the naked peptides listed in Tables 1-1 to 1-5 and 1-11). The chemical structures of the naked peptides were characterized by liquid chromatography-mass spectrometry (LC-MS / MS). The purity of each naked peptide was determined by analytical HPLC (Agela C18 column, 4.6 × 250 mm, flow rate 1 mL / min). The sequence structure, molecular weight, and purity of each naked peptide were determined.

[0301] in conclusion:

[0302] The purity of the synthesized naked peptides was confirmed to be greater than 95% by HPLC analysis, and the molecular weight was confirmed to be correct by mass spectrometry.

[0303] Example 3: Synthesis of lipopeptides

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

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

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

[0307] 2. Synthesis of the Overall Sequence of Naked Peptide and Linker Sequences

[0308] The linker of the connecting arm includes: a peptide with an EAAAK sequence (SEQ ID NO: 104) and a peptide with a GSGSG sequence (SEQ ID NO: 105);

[0309] Lipophilic compounds for modification include cholesterol succinate.

[0310] 2.1 Synthesis of the main chain

[0311] (1) Synthesis of peptide resin: Rink Amide MBHA resin was used as the carrier resin. Through Fmoc protection removal and coupling reaction, it was sequentially coupled with the protected amino acids corresponding to the polypeptide amino acid sequence to prepare the peptide resin.

[0312] (2) Inserting the first protected amino acid into the main chain

[0313] Dissolve 0.3 mmol of the first protected amino acid and 0.3 mmol of HOBt in an appropriate amount of DMF. Slowly add 0.3 mmol of DIC to the protected amino acid DMF solution under shaking. Shake and react at room temperature for 5 minutes to obtain the activated protected amino acid solution for later 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 minutes (twice). Wash and filter to obtain the Fmoc-free resin.

[0315] The activated first protected amino acid solution was added to the Fmoc-removed resin, and the coupling reaction was carried out for 60 minutes. The resin containing the first protected amino acid was filtered and washed to obtain the resin.

[0316] (3) Adding other protected amino acids to the main chain

[0317] Using the same method as described above for the first protected amino acid in the backbone, the corresponding protected amino acids were sequentially added to the resin, yielding a backbone-containing amino acid resin. Finally, the N-terminus was acetylated and capped with 0.3 mmol AcO + 0.6 mmol DIEA to complete backbone synthesis. Each reaction step was monitored using the Kaiser Test. If the condensation reaction of an amino acid was incomplete, the condensation was repeated until the desired target peptide was obtained.

[0318] 2.2 Sidechain Access

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

[0320] (2) Modification of peptide C-terminal lysine with lipophilic compounds

[0321] Modification of the peptide C-terminal lysine with cholesterol succinate monoester: 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 mixture was shaken at room temperature for 5 minutes. The prepared solution containing cholesterol succinate monoester, HOBt, and DIC was added to the Dde-free resin obtained in step (1), and the coupling reaction was carried out for 60 minutes. The mixture was filtered, washed, and dried to obtain the peptide resin.

[0322] Other chemical reagents, amino acid raw materials and operating steps are the same as those in Example 1.

[0323] in conclusion:

[0324] The HPLC results confirmed that the purity of the synthesized salt-bridge peptides was greater than 98%, and the molecular weight of the peptides was consistent with the theoretical molecular weight as determined by mass spectrometry.

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

[0326] In the examples, the present disclosure uses RSV-A long strain as the infectious virus, and determines the anti-RSV activity of the synthesized polypeptides by CPE detection, thereby identifying a series of novel polypeptides whose activities are significantly superior to those of the control polypeptide fusion inhibitor.

[0327] The compounds in Table 1-1 to Table 1-5, the positive peptides in Table 1-11, and the positive control T-118 were tested for their inhibitory activity against the RSV-A long strain.

[0328] 1. Experimental Materials

[0329] Human laryngeal carcinoma (HEp-2) cells were obtained from the American Type Culture Collection (ATCC) under the catalog number CCL-23. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% nonessential amino acids, 2 mM glutamine, 100 U / mL penicillin, and 100 µg / mL streptomycin. RSV A long strain was obtained from ATCC under the catalog number VR-26.

[0330] 2. Experimental Methods

[0331] This study used a plaque reduction assay to assess the in vitro activity of test samples against the RSV A long strain. Test samples and control compounds were tested at eight concentrations in duplicate. HEp-2 cells were trypsinized and diluted with assay medium to 300,000 cells / mL. 100 μL of the diluted cells were added to a 96-well plate, with 30,000 cells per well. The cells were incubated overnight in a 5% CO2, 37°C incubator. The next day, the test samples were serially diluted in assay medium. These serially diluted samples were mixed with an equal volume of virus (100 PFU per well) and incubated at 37°C, 5% CO2, for 1 hour. The medium in the 96-well plate was then discarded, and 200 μL of the sample-virus mixture was added to the plate and incubated at 37°C, 5% CO2, for 2 hours. After 2 hours, the sample-virus mixture was discarded and 200 μL of maintenance medium containing the corresponding test sample concentrations and 0.8% CMC was added. A cell control (cells without compound treatment or virus infection) and a virus control (cells infected with virus without compound treatment) were set up. The cells were cultured in a 5% CO2, 37°C incubator for 1 day. One day after virus infection, the medium was discarded, and the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% TritonX-100. After washing with DPBS, RSV-specific antibody (RSV antibody, 1:3000 dilution) was added and incubated at 37°C for 1 hour. Secondary antibody (donkey anti-goat IgG, 1:500 dilution) was then added and incubated at 37°C for 1 hour. The secondary antibody was discarded, and TrueBlue solution was added for staining for 10 minutes. After rinsing with running water and air drying, the number of spots per well was counted using a microplate imager. The raw data was used to calculate the antiviral activity of the samples.

[0332] Cytotoxicity and antiviral assays were performed in parallel. HEp-2 cells were seeded at a density of 30,000 cells per well in a microplate and cultured overnight in a 5% CO2, 37°C incubator. The following day, diluted test samples were added. Cell controls (cells without compound treatment) and culture medium controls (culture medium alone, without cells or compound treatment) were set up. The final DMSO concentration in the culture medium was 0.5%. Cells were cultured in a 5% CO2, 37°C incubator for one day. Cell viability was assessed using the CCK8 cell viability assay kit.

[0333] The spot count and cell viability test raw data are used for the antiviral activity and cytotoxicity analysis of the test samples, respectively. The calculation formula is as follows:

[0334] % inhibition rate = 100 - (sample value - average value of cell control) / (average value of virus control - average value of cell control) × 100

[0335] % cell viability = (sample value - average value of medium control) / (average value of cell control - average value of medium control) × 100

[0336] GraphPad Prism (version 10) was used to perform nonlinear fitting analysis on the inhibition rate of the samples and calculate the IC 50 and CC 50 value.

[0337] IC 50 :In the in vitro antiviral activity experiment, IC 50 IC represents the inhibitory effect of the test compound on RSV A long virus infection in Hep-2 cells. Specifically, it means that when the test compound reaches a certain concentration, it can effectively inhibit 50% of RSV A long virus infection in Hep-2 cells. This concentration is the IC 50 .

[0338] CC 50 :In the in vitro antiviral activity experiment, CC 50 It 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 Hep-2 cells to die. 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 anti-RSV activity of the peptide samples was tested using positive peptides 1 to 7 as controls. The cell fusion inhibitory activities of the peptide samples and the positive peptides are shown in Table 2 below:

[0341] Table 2 Cell fusion inhibitory activity

[0342]

[0343]

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

[0345] in conclusion

[0346] 1. The designed salt-bridge naked peptide samples have high anti-RSV virus activity.

[0347] 2. The salt-bridged naked peptide samples prepared in the present disclosure are highly safe, with a safety index SI greater than 1, and can be used as safe and effective alternative anti-RSV drugs.

[0348] Example 5: Efficacy of polypeptides in a study of protection against toxicity in mice

[0349] 1. Experimental Materials

[0350] Female BALB / c mice were obtained from Shanghai Yishang Biotechnology Co., Ltd. Human laryngeal carcinoma (HEp-2) cells were obtained from the American Type Culture Collection (ATCC), catalog number CCL-23. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum and 1% penicillin and streptomycin. The RSV A2 strain was obtained from ATCC, catalog 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 challenged with RSV intranasally on Day 0 with a challenge dose of 1×10 6 pfu (20 μL) per animal. Dosing began 1 hour after challenge on Day 0. Peptide samples, positive controls, and vehicle controls were nebulized once intratracheally for 5 consecutive days (see Table 3 below for dosage and dosing frequency). Lung tissue was collected from all animals 2 hours after the last dose on Day 4. Left lung tissue was quickly frozen in 10-fold volume of virus protection solution and stored below -70°C. Viral titers were determined by plaque assay.

[0353] For analysis, tissue homogenate viral load plaque assay was performed to measure RSV A2 virus in the lungs. Briefly, one day before the sample test, HEp-2 cells were seeded in 12-well plates (plating density of 0.3 × 10 6 cells / mL, 1000 μL of cell suspension per well). On the day of incubation, the supernatant of the tissue sample was collected after homogenization with a tissue homogenizer (kept at low temperature), centrifuged, and the supernatant was collected. The tissue homogenate was diluted with serum-free DMEM and plated in a final volume of 400 μL in a 12-well plate. After a 2-h incubation, the supernatant was discarded, and 1.5 mL of overlay (cell culture medium containing 1% soft agar) was added to each well and incubated in an incubator for approximately 72 h. The overlay was discarded, the cells were fixed, blocked with blocking solution, stained with HRP-RSVG, and color development was stopped with purified water, and air-dried. The developed spots on the plate were counted using a fluorescent (enzyme-linked) immunospot reader, and the viral titer was expressed as plaque-forming units per gram of tissue (PFU / g). Viral titer was calculated as the arithmetic mean ± standard error for all animals in a group.

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

[0355]

[0356] Note: “QD” means once daily.

[0357] One hour after RSV A2 challenge, compounds 13, 14, 17, 19, 20, 21, 26, 27, 28, and 40 were administered via airway aerosol once daily for five consecutive days. Lung tissues of all groups were collected 2 hours after the last administration for viral plaque detection. The results are shown in Table 3.

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

[0359] Example 6: Efficacy of polypeptides in cotton rats' protection against toxicity

[0360] 1. Experimental Materials

[0361] Female cotton rats were obtained from Shanghai Yishang Biotechnology Co., Ltd. The RSV A2 strain was obtained from ATCC, catalog number VR-1540. RT-qPCR primers and probes were obtained from Qingke Biotechnology Co., Ltd., batch number TSP20241227-025-00187. RT-qPCR standards were obtained from Vazyme, batch number 20241008.

[0362] 2. Experimental Methods

[0363] Female cotton rats (6-8 weeks old) were divided into groups (n=5) according to body weight. Mice were manipulated as shown in Table 4 below. All animals were challenged with RSV intranasally on Day 0 at a dose of 1×10 5.5 PFU (50 μL) / animal. Dosing began 1 hour after challenge on Day 0. Peptide samples, positive controls, and vehicle controls were aerosolized once daily for 5 consecutive days. Lung tissue was collected from all animals 2 hours after the last dose on Day 4. Left lung tissue (including part of the trachea) was quickly frozen in 10 volumes of virus protection solution and stored below -70°C. Viral titer was determined by RT-qPCR.

[0364] RT-qPCR steps:

[0365] First, sample homogenization and lung tissue RNA extraction were performed. The supernatant of the tissue sample was collected (kept cold). Approximately 100 μL of the homogenized supernatant was then used to extract total RNA using the VeZol reagent-chloroform-water-magnetic bead method and dissolved in RNase-free water. The RT-qPCR reaction system was prepared as follows: RNA template, probe, primer pair, RT-qPCR buffer, and RNase-free water were used to prepare a one-step RT-qPCR reaction system. Two replicate wells were set up for each sample, with 20 μL of the reaction system per well. A quantitative marker well (full-length RSV A2 N gene plasmid) and positive and negative quality control wells were also set up. The amplification program was then set up, including reverse transcription, initial denaturation, and cyclic amplification. Amplification data were collected using a real-time fluorescence quantitative PCR instrument, and the mean Ct value of the replicate wells was calculated. The Ct value of each sample was incorporated into the quantitative marker well to calculate the copy number. The RSV viral load (copies / gram of lung tissue) was calculated as follows: sample load (copies / 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 compounds in cotton rats

[0368]

[0369] Note: “QD” means once daily.

[0370] One hour after RSV A2 challenge, compounds 13, 14, 17, 19, 20, 21, 26, 27, 28, and 40 were administered via airway aerosol once daily for five consecutive days. Lung tissues of all groups were collected 2 hours after the last administration for virus titer detection. The results are shown in Table 4.

[0371] The results of the efficacy experiments 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 LCutland, Linda Eckert, Daniel Feikin, Tiffany Fitzpatrick, Youyi Fong, BarneyS Graham, Terho Heikkinen, Deborah Higgins, Siddhivinayak Hirve, Keith PKlugman, Leyla Kragten-Tabatabaie, Philippe Lemey, Romina Libster, Yvette Löwensteyn, Asuncion Mejias, Flor M Munoz, Patrick K Munywoki, Lawrence Mwananyanda, Harish Nair, Marta C Nunes, Naveen Thacker, Kody AWaldstein, 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, PeggyGeluykensa, Tom Geversc, Marcia Van Ginderena, Nick Verheyena, Hidong Kimd,Rudy Willebrordse, Jean-François 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), NewEngl. 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, GaëlMcGill, N. Valerio Dorrello,Samuel H. Gellman, Christopher A. Alabi, Rik L.de Swart1, Anne Moscona, Matteo Porotto, Intranasal fusion inhibitorylipopeptide 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-inhibitorylipopeptides maintain high potency against divergent variants of concernincluding 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. PETrEWAY, JR.,Peptides from conserved regions of paramyxovirus fusion (F) proteins arepotent 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 andNegative 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 withsimilar physicochemical properties in coiled-coil interactions, Chem.Commun., 2013,49, 11086.

[0382]

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

[0383]

[11] Chungen Pan, Lifeng Cai, Hong Lu, Lu Lu, and Shibo Jiang, ANovel Chimeric Protein-based HIV-1 Fusion Inhibitor Targeting gp41Glycoprotein with High Potency and Stability, THE JOURNAL OF BIOLOGICALCHEMISTRY VOL. 286, NO. 32, pp. 28425–28434, August 12, 2011.

[0384]

[12] Baohua Zhenga,b, Kun Wangb, Lu Luc, Fei Yud, Maosheng Cheng,Shibo Jiang, Keliang Liua and Lifeng Cai, Hydrophobic mutations in buriedpolar residues enhance HIV-1 gp41 N-terminal heptad repeat–C-terminal heptadrepeat interactions and C-peptides' anti-HIV activity, AIDS 2014, 28:1251–1260.

[0385] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound, a pharmaceutically acceptable salt thereof or a derivative thereof, characterized in that: The compound, its pharmaceutically acceptable salt or derivative thereof comprises a polypeptide having a sequence selected from any one or more of Formula III, Formula IV and SEQ ID NOs: 97 to 102: 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 ; in, X''1 is W or F; X''2 is D or does not exist; X''3 is E or does not exist; X''4 is F or does not exist; X''6 is K or A; X''7 is K or S; X'' 13 is K or R; X'' 14 is K or R; X'' 20 is K or R; X'' 21 is K or R; X'' 27 is K or R; X'' 28 is K or R; X'' 30 is E or H; X'' 31 is E or N; X'' 33 N or does not exist; X'' 34 A or not present; Formula IV: WDEFDASISQ-X''' 11 -NEKINQSLEEIRKSDELLHN-X''' 32 -X''' 33 -X''' 34 -X''' 35 ; in, X''' 11 V or not present; X'''' 32 V, N or not present; X''' 33 N, A or not present; X''' 34 A, L or absent; X''' 35 L or not present.

2. The compound, pharmaceutically acceptable salt or derivative thereof according to claim 1, wherein: The compound, its pharmaceutically acceptable salt or derivative thereof comprises a polypeptide having a sequence selected from any one or more of SEQ ID NOs: 82 to 102.

3. The compound, pharmaceutically acceptable salt or derivative thereof according to claim 1, wherein The compound, its pharmaceutically acceptable salt or derivative thereof comprises a polypeptide having a sequence selected from any one or more of Formula III-1, Formula IV-1 and SEQ ID NOs: 116 to 121: 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; Formula IV-1: R1-WDEFDASISQ-X''' 11 -NEKINQSLEEIRKSDELLHN-X''' 32 -X''' 33 -X''' 34 -X''' 35 -R2-R3; in: R1 is an amino terminal protecting group; R2 is an absent or arbitrarily substituted linker; R3 is a carboxyl terminal protecting group.

4. The compound, pharmaceutically acceptable salt or derivative thereof according to claim 3, characterized in that: The compound, its pharmaceutically acceptable salt or derivative thereof is selected from one or more of the following: (1) R1 is acetyl; (2) R2 is -R4-R5(R6)-, wherein R4 is a polypeptide; R5 is lysine, cysteine, 2,3-diaminopropionic acid, ornithine, 2,4-diaminobutyric acid or 2,7-diaminoheptanoic acid; and R6 is a lipophilic compound group modified on R5; (3) R3 is -NH2.

5. The compound, pharmaceutically acceptable salt or derivative thereof according to claim 4, characterized in that: The amino acid sequence of R4 is (EAAAK) m or (GSGSG) m ; m is a natural number from 0 to 5; and / or The lipophilic compound group is selected from the group consisting of: cholesterol, cholesterol succinate, 2-cholesterol acetate, 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, cholesterol bromoacetate, cholesterol formyl chloride, palmitic acid, stearic acid, fatty acids containing 3-20 carbon atoms, fatty diacids containing 3-20 carbon atoms, and one or more other groups that can interact with cell membranes or viral membranes to enhance the interaction of the polypeptide with cell membranes or viral membranes.

6. The compound, pharmaceutically acceptable salt or derivative thereof according to claim 1, wherein The compound, its pharmaceutically acceptable salt or derivative thereof comprises a polypeptide having a sequence selected from any one or more of SEQ ID NOs: 38 to 51.

7. The compound, pharmaceutically acceptable salt or derivative thereof according to any one of claims 1 to 6, characterized in that: The derivative is a solvate, a chelate or a non-covalent complex.

8. Use of the compound according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, or a derivative thereof in the preparation of an anti-RSV polypeptide membrane fusion inhibitor.

9. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the compound according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, or a derivative thereof.

10. A recombinant vector, characterized in that The recombinant vector contains the nucleic acid molecule according to claim 9.

11. A recombinant cell, characterized in that The recombinant cell contains the nucleic acid molecule according to claim 9 or the recombinant vector according to claim 10.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, or a derivative thereof, the nucleic acid molecule according to claim 9, the recombinant vector according to claim 10, or the recombinant cell according to claim 11; optionally, further comprising a pharmaceutically acceptable carrier or excipient.

13. A preparation product, characterized in that: The preparation product comprises the pharmaceutical composition according to claim 12.

14. The preparation product according to claim 13, wherein The dosage form of the preparation product includes any one of the following groups: oral preparation, injection, inhalation and lyophilized preparation.

15. The preparation product according to claim 14, characterized in that The dosage form of the preparation product is inhalation, lyophilized preparation, subcutaneous injection or intramuscular injection.

16. The preparation product according to claim 13, characterized in that The administration method of the preparation product is selected from any one of the following: oral administration, injection administration, mucosal administration, transdermal administration and atomization administration.

17. The preparation product according to claim 16, characterized in that The administration method of the preparation product is pulmonary atomization, subcutaneous injection or intramuscular injection.

18. A method for preparing the compound according to any one of claims 1 to 6, its pharmaceutically acceptable salt or derivative thereof, characterized in that: The method comprises the following steps: The salt-bridged naked peptide is prepared by using a carrier resin and sequentially coupling with the protected amino acids corresponding to the polypeptide amino acid sequence through deprotection and coupling reactions.

19. The method according to claim 18, 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.

20. The method according to claim 19, wherein The lipophilic compound modification is cholesterol succinate monoester modification.

21. Use of the compound according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, or a derivative thereof, the pharmaceutical composition according to claim 12, or the formulation according to any one of claims 13 to 17 in the preparation of a medicament for preventing and / or treating diseases caused by RSV or other Paramyxoviridae viruses.

22. The use according to claim 21, characterized in that The RSV includes one or more selected from RSV-A subtype, RSV-B subtype and other mutant strains.

23. A method for inhibiting RSV for non-therapeutic purposes, characterized in that: The method comprises administering to the sample the compound according to any one of claims 1 to 6, a pharmaceutically acceptable salt thereof, or a derivative thereof, the pharmaceutical composition according to claim 12, or the preparation product according to any one of claims 13 to 17.

24. The method according to claim 23, wherein The RSV includes one or more selected from RSV-A subtype, RSV-B subtype and other mutant strains.

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