Use of a membrane fusion inhibiting polypeptide in combating infection by a paramyxoviridae virus

Abstract

The present disclosure relates to an antiviral use of a membrane fusion inhibiting polypeptide for the treatment of a disease caused by a viral infection of the Paramyxoviridae family.

Description

[0001] This disclosure claims priority to Chinese Patent Application No. 2025101651370, filed on February 14, 2025, and Chinese Patent Application No. 2025113287192, filed on September 17, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This disclosure belongs to the field of biomedical technology and relates to the antiviral use of a membrane fusion inhibitory peptide. Background Technology

[0003] Paramyxoviridae is a family of enveloped, single-stranded, negative-sense RNA viruses with a broad host range, encompassing humans, mammals, and birds. Many of these viruses can cause significant infectious diseases in humans and livestock, making them of great research importance in public health, clinical medicine, and animal husbandry. According to the International Committee on Taxonomy of Viruses (ICTV) classification standards (2024), this family includes multiple subfamilies and genera, exhibiting a wide range of pathogenicity. In addition to causing respiratory infections, they can also lead to systemic diseases such as measles, mumps, and nervous system infections. Some viruses are highly infectious or highly lethal, posing a significant threat to human health, and the development of related prevention and control technologies has important clinical value.

[0004] The following are representative viruses in the Paramyxoviridae family that are closely related to human health, disease prevention and control, and related technology development:

[0005] 1. Respiroviruses: The main members are human parainfluenza viruses (hPIV), a class of negative-sense single-stranded RNA viruses, including types 1-4. PIV-1 and PIV-2 are the main causes of acute laryngitis (laryngotracheobronchitis, typically presenting as a "barking cough") in infants and young children, while PIV-3 is a common cause of severe pneumonia and bronchiolitis in infants and young children. Adult infections often present as mild upper respiratory tract infections. PIV-4 was previously considered less pathogenic, but recent studies show that it can also cause severe acute respiratory infections and poses a higher risk to the elderly.

[0006] Different serotypes exhibit varying epidemiological characteristics and pathogenicity in the population: hPIV-1 is the main pathogen of croup (laryngotracheobronchitis) in children; hPIV-3 is a significant cause of bronchiolitis and pneumonia in infants and young children, with over 60% of children infected with hPIV-3 before the age of 2, and the infection rate approaching 80% by age 4. Epidemiological data shows that approximately 13% of lower respiratory tract infections in children under 5 years old worldwide are caused by hPIV, along with 4%-14% of related hospitalizations and approximately 4% of related deaths.

[0007] Currently, specific prevention and control measures for this type of virus are still relatively limited. This type of virus can be transmitted through droplets, and infection can cause lower respiratory tract infections in infants and young children and immunocompromised individuals. Clinical manifestations include cough, wheezing, and difficulty breathing. Its pathogenic mechanism is related to cell fusion mediated by viral surface fusion proteins and immune damage to the respiratory mucosa, making it one of the respiratory pathogens that require key attention in clinical practice.

[0008] hPIV infection can occur multiple times throughout a person's life, and natural infection does not provide durable immune protection. This characteristic significantly increases the overall burden of the disease, especially in infants and young children whose immune systems are not yet fully developed, often leading to recurrent infections and increased hospitalization rates. Similar to hMPV, there are currently no approved specific antiviral drugs or vaccines for hPIV infection; clinical treatment mainly relies on symptomatic and supportive care (such as antipyretics, fluid replacement, oxygen therapy, and mechanical ventilation when necessary). For some severely ill patients, although broad-spectrum antiviral drugs such as ribavirin have been tried, their efficacy is still unclear and there are risks of side effects, limiting their widespread use.

[0009] 2. Morbillivirus: The core virus is measles virus, which is highly contagious and mainly spreads through droplets. Typical symptoms after infection include high fever and a characteristic rash. It can also invade the lungs, causing measles pneumonia (a complication in 5%-10% of patients and a leading cause of death in measles patients), and may lead to long-term neurological sequelae such as subacute sclerosing panencephalitis. Although measles vaccines are included in routine immunization programs, its prevalence in unimmunized populations still poses a threat to public health. In addition, this genus also includes canine distemper virus (CDV) and peste des petits ruminants virus (PPRV), which are highly pathogenic to dogs, sheep, and other animals, respectively.

[0010] 3. Rubulavirus: The representative virus is mumps virus, which is transmitted through droplets or close contact. Children are the main susceptible population. After infection, the main manifestation is non-suppurative swelling of the parotid gland. Complications such as orchitis, pancreatitis, and meningoencephalitis may also occur. In severe cases, it can lead to reproductive function damage or neurological sequelae. Although mumps vaccine is widely used, there are still cases of epidemics in some areas due to insufficient vaccination coverage.

[0011] 4. Henipavirus: This genus includes Nipah virus and Hendra virus. Both use fruit bats as their natural host and can be transmitted to humans and livestock (such as pigs and horses) through contact with infected animals or their secretions. Human infection can cause acute encephalitis and severe pneumonia. The mortality rate of Nipah virus infection is as high as 40%-75%, and the mortality rate of Hendra virus infection is about 50%. Currently, there are no specific treatments or marketed vaccines. It is a newly emerging and highly infectious disease virus that is a key area for global prevention and control. Its pathogenic mechanism is closely related to the virus's strong invasiveness on the nervous and respiratory systems, making it an important direction for the development of antiviral drugs and early diagnostic technologies.

[0012] The Paramyxoviridae family contains a variety of viruses that have a significant impact on human health and livestock farming. The infectious diseases they cause have urgent prevention and control needs in clinical diagnosis and treatment, public health and industry. The development of related antiviral drugs, vaccines, diagnostic reagents and prevention and control technologies has important practical value and application prospects.

[0013] In summary, hPIV, as an important viral pathogen causing acute lower respiratory tract infections globally, exhibits the following characteristics: (1) It mainly affects infants, the elderly, and people with weakened immune systems, often leading to severe lower respiratory tract infections and even death. (2) It is distributed globally, and its epidemic season overlaps to some extent with other common respiratory viruses. (3) It is difficult to obtain lasting protection after natural infection, and reinfection is likely to occur. (4) Currently, there are no approved specific antiviral drugs or preventive vaccines, and clinical treatment options are limited. (5) With the widespread attention paid to respiratory virus-related diseases, diagnostic, treatment, and preventive measures for hPIV have shown significant research and development and market potential.

[0014] Therefore, developing drugs or vaccines that can prevent and / or treat hPIV not only addresses a significant unmet medical need in current clinical practice, but also has important socio-economic value and broad industrialization prospects. However, there is currently a lack of anti-hPIV drugs or vaccines that are both effective and safe. Summary of the Invention

[0015] To address the lack of effective and safe anti-hPIV preventive and / or therapeutic drugs or vaccines in the prior art, this disclosure provides the antiviral use of a membrane fusion inhibitory peptide.

[0016] Based on the inventors’ prior research, this disclosure uses a polypeptide template such as sequence (1) (SEQ ID NO: 103):

[0017] (1) Z1-WJJLVOOSJJFDOOIJJVNOOIJJSLOOIJJSDOOLJJVNOOLJJTNOOITTI-Z3-Z2

[0018] In the template, Z1 is an amino terminus (NH2-) or its modification, and Z2 is a carboxyl terminus (-COOH) or its modification. Unless otherwise specified, Z1 is an acetyl group (Ac-) and Z2 is an amide group (-NH2), which respectively block the amino terminus and carboxyl terminus of the polypeptide to enhance the stability of the polypeptide.

[0019] J represents an acidic amino acid residue, which may be, but is not limited to, glutamic acid and aspartic acid residues; O represents a basic amino acid residue, which may be, but is not limited to, lysine and arginine residues; a salt bridge is formed between J and O, which plays a general stabilizing role in the secondary structure of the polypeptide; the remaining positions are specific amino acid residues, which occupy the corresponding positions in the viral CHR natural sequence, mainly hydrophobic amino acid residues, forming a hydrophobic surface that contacts and matches with the drug target, producing a specific interaction. 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.

[0020] We further designed specific peptides using a training method for a peptide-receptor binding activity prediction model.

[0021] This disclosure solves the above-mentioned technical problems through the following technical solutions.

[0022] The first aspect of this disclosure provides the use of a modified polypeptide in the preparation of a medicament for the prevention and / or treatment of diseases caused by viral infections of the Paramyxoviridae family;

[0023] The polypeptide comprises one or more amino acid sequences selected from those 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.

[0024] Formula I:

[0025] 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 -X30 -LX 32 -X 33 -X 34 -X 35 -X 36 -X 37 -X 38 (SEQ ID NO: 122);

[0026] Where X1 is D or does not exist; X2 is E or does not exist; X3 is W, F, D, S or does not exist; X4 is D or does not exist; X5 is E, A, K or does not exist; X6 is F, S or does not exist; X7 is D, N, L or does not exist; X8 is K, Q, L or does not exist; X9 is K or does not exist; X 11 For E or N; X 13 For V or E; X 14 For N or K; X 15 For K, R, or E; X 16 For K or R; X 20 For S, I, or L; X 22 For K or R; X 23 For K or R; X 24 For I or H; X 25 For E or N; X 29 For K or R; X 30 For K or R; X 32 E or does not exist; X 33 E, V, or not present; X 34 V, N, S, or not present; X 35 It is N, K, D or does not exist; X 36 K or not present; X 37 For K, A, or not present; X 38 It is L, A, or does not exist;

[0027] Formula II (SEQ ID NO: 123):

[0028] 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 ;

[0029] Where X'1 is F, W, D, L, or Y; X'2 is V or does not exist; X'3 is K or does not exist; X'4 is D, K, or does not exist; X'5 is F, I, or does not exist; X'6 is D or does not exist; X'8 is L or does not exist; X'9 is V or does not exist; X' 10 F or not present; X' 11 For E or D; X' 12 For A or I; X' 13 S or does not exist; X' 14 It is either I or does not exist; X' 23 For E or Q; X' 26 For A or E; X' 27 For F, E, or K; X' 30 L or not present; X' 31 A or not present; X' 32 F or not present; X' 33 It is either I or does not exist; X' 34 R or does not exist; X' 41 H or not present; X' 42 It is N or does not exist; X' 43 V or does not exist; X' 44 It is N or does not exist; X' 45 A or not present; X' 46 G or not present; X' 47 For K, L, or not present; X' 48 S or does not exist; X' 49 If T or not present; X' 50 It is T or does not exist;

[0030] Formula III (SEQ ID NO: 124):

[0031] 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 ;

[0032] Where 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 For K or R; X'' 14 For K or R; X'' 20 For K or R; X'' 21 For K or R; X'' 27 For K or R; X'' 28 For K or R; X'' 30 For E or H; X'' 31 For E or N; X'' 33 =N or does not exist; X'' 34 It is either A or does not exist;

[0033] Formula IV (SEQ ID NO: 125):

[0034] WDEFDASISQ-X''' 11 -NEKINQSLEEIRKSDELLHN-X''' 32 -X''' 33 -X''' 34 -X''' 35 ;

[0035] Where X''' 11 V or does not exist; X''' 32 V, N, or not present; X''' 33 It is N, A, or does not exist; X''' 34 A, L, or not present; X''' 35 It is either L or does not exist.

[0036] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from those shown in SEQ ID NO: 52-102.

[0037] In some embodiments, the modification is a protecting group modification.

[0038] In some preferred embodiments, the protecting group modification is selected from amino protecting group modification, carboxyl protecting group modification, and side chain protecting group modification.

[0039] In some preferred embodiments, the modified polypeptide has an R1-XX-R2R3 structure, wherein XX is the polypeptide as described above, R1 is an amino-terminal protecting group; R2 is a linker arm that is absent or arbitrarily substituted; and R3 is a carboxyl-terminal protecting group.

[0040] In some specific embodiments, the polypeptide comprises one or more amino acid sequences selected from those shown in Formula I-1, Formula II-1, Formula III-1, Formula IV-1 and SEQ ID NO: 106-121:

[0041] Formula I-1 (SEQ ID NO: 126):

[0042] R1-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 -R2-R3;

[0043] Formula II-1 (SEQ ID NO: 127):

[0044] R1-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 -R2-R3;

[0045] Formula III-1 (SEQ ID NO: 128):

[0046] 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;

[0047] Formula IV-1 (SEQ ID NO: 129):

[0048] R1-WDEFDASISQ-X’’’ 11 -NEKINQSLEEIRKSDELLHN-X’’’ 32 -X’’’ 33 -X’’’ 34 -X’’’ 35 -R2-R3. [[ID=SS]]

[0049] SEQ ID NO: 106: R1-ADAFRLEVNDASSKINESIEESLLSLEKLHNVNATA-R2-R3;

[0050] SEQ ID NO: 107: R1-FDAFIQEINVNEDQSLEQSDELLLELHLLHSLLH-R2-R3;

[0051] SEQ ID NO: 108: R1-FAEFNQKINQVNEKIEESLEEIRKSDEELHNVNATT-R2-R3;

[0052] SEQ ID NO: 109: R1-SDEQVNEKINQSLAFIRRIRKLLHN-R2-R3;

[0053] SEQ ID NO: 110: R1-ILELVNKKIEQSLKFIEKSDKLLEN-R2-R3;

[0054] SEQ ID NO: 111: R1-SLEQVNKKINQSLKVNKKSDKLLEN-R2-R3;

[0055] SEQ ID NO: 112: R1-FDEEVNKKIEQSLKINQSLEEIRKS-R2-R3;

[0056] SEQ ID NO: 113: R1-SISQVNEKINEIQSLEEKSDKLLKS-R2-R3;

[0057] SEQ ID NO: 114: R1-VNKKIEEEKQSLKKQSDKIEESDEN-R2-R3;

[0058] SEQ ID NO: 115: R1-FDELVNKKIEKIEEVNKKSLKLLES-R2-R3;

[0059] SEQ ID NO: 116: R1-FEVNRRRIEQSLEKSLESLEEEEHSDKKLHNELH-R2-R3;

[0060] SEQ ID NO: 117: R1-SLEQVNKINKKIDKIEESLKKIEESDKKSLEVNKKL-R2-R3;

[0061] SEQ ID NO: 118: R1-SLEQVNKINEKINKISQSLKKIEESDKKSDEVNAGL-R2-R3;

[0062] SEQ ID NO: 119: R1-SLEQVNKKIEQSLESLKKSDKINQSLEEVNKSDELL-R2-R3;

[0063] SEQ ID NO: 120: R1-SLEQVNEKINQSLAFIRKSDEINQSDEEVNKSDELL-R2-R3;

[0064] SEQ ID NO: 121: R1-SDELVNKKIEFDKKINQSLKKIEESDKKKL-R2-R3.

[0065] In some embodiments, R1 is acetyl.

[0066] In some embodiments, R2 is -R4-R5(R6)-, wherein R4 is a peptide; 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 attached to R5.

[0067] In some specific implementations, R5 is lysine.

[0068] In some specific implementations, R3 is -NH2.

[0069] In some preferred embodiments, the amino acid sequence of R4 is (EAAAK). m Or (GSGSG) m m is a natural number selected from 0 to 5, such as 0, 1, 2, 3, 4 or 5.

[0070] In some preferred embodiments, the lipophilic compound group is selected from one or more of the following: cholesterol, cholesterol monosuccinate, 2-cholesterolacetic 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 valerate, 2-cholesterol isovalerate, 3-cholesterol valerate, 5-cholesterol valerate, 2-cholesterol hexanoic acid, 6-cholesterol hexanoic acid, 2-cholesterol heptanoic acid, 7-cholesterol heptanoic acid, 2-cholesterol octanoic acid, 8-cholesterol octanoic acid, bromoacetate cholesterol ester, cholesterol formyl chloride, palmitic acid, stearic acid, fatty acids containing 3-20 carbon atoms, and fatty diacids containing 3-20 carbon atoms; the lipophilic compound group can interact with the cell membrane or viral envelope to enhance the binding of the peptide to the cell membrane or viral envelope.

[0071] In some specific embodiments, the lipophilic compound is cholesterol succinate monoester.

[0072] In some specific embodiments, the modified polypeptide comprises one or more amino acid sequences selected from those shown in SEQ ID NO:1-51.

[0073] In some embodiments, the modified polypeptide may be in the form of a pharmaceutical salt. The pharmaceutical salts include: acetate, lacturonate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, tartrate, methanesulfonate, borates, methyl bromide, bromide, methyl nitrate, calcium edetate, methyl sulfate, dextrocamphorsulfonic acid, mucilage, carbonate, naphthalenesulfonate, chloride, nitrate, clavate, N-methylglucosamine, citrate, ammonium salt, dihydrochloride, oleate, ethylenediaminetetraacetic acid, oxalate, and ethylenedisulfonate. Phosphate, dihydroxynaphthyl propionate, lauryl propionate, palmitate, ethanesulfonate, pantothenate, fumarate, phosphate / bisphosphonate, glucono-p-phosphate, polygalacturonate, gluconate, salicylate, glutamate, stearate, p-hydroxyacetaminophen, sulfate, hydroxybenzoate, basic acetate, succinate, hydrobromide, tannin, hydrochloride, tartrate, hydroxynaphthyl propionate, 8-chlorotheophylline, iodide, toluenesulfonate, triethyliodine, lactic acid, valerate, etc.

[0074] Depending on the intended use, the pharmaceutical salt may be formed from cations such as sodium, potassium, bismuth, etc., or from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxylate.

[0075] These salts can be prepared using standard methods, such as by reacting a 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, hydrobroms, acetates, and primates can be used as dosage forms; in the presence of an acidic group or an alcohol group, pharmaceutically acceptable esters such as acetates, maleates, and chloromethyl trimethylacetate, as well as esters known in the literature for improving solubility and hydrolysis, can be used as sustained-release and prodrug formulations. In some embodiments, the drug is a solvate or chelate of the polypeptide.

[0076] In some embodiments, the viruses of the Paramyxoviridae family are selected from viruses of the genera Respirovirus, Rubulavirus, Morbillivirus, and Henipavirus.

[0077] In some preferred embodiments, the virus of the respiratory virus genus is human parainfluenza virus (hPIV). The human parainfluenza virus includes subtypes hPIV-1, hPIV-2, hPIV-3, and hPIV-4.

[0078] In some preferred embodiments, the virus of the mumps virus genus is mumpsvirus.

[0079] In some preferred embodiments, the virus of the Measlesvirus genus is measles virus.

[0080] In some preferred embodiments, the virus of the Hennipavirus genus is selected from nipahvirus and Hendravirus.

[0081] A second aspect of this disclosure provides the use of a pharmaceutical composition, pharmaceutical formulation, or cassette in the preparation of a medicament for the prevention and / or treatment of viral infections of the Paramyxoviridae family;

[0082] The pharmaceutical composition or pharmaceutical formulation comprises a modified polypeptide, a nucleic acid molecule encoding the polypeptide portion of the modified polypeptide, and / or a carrier or transformant comprising the nucleic acid molecule; the modified polypeptide or a virus of the Paramyxoviridae family is as described in the first aspect;

[0083] The pharmaceutical preparation is obtained by formulating the modified polypeptide, the nucleic acid molecule, the carrier or transformant into a formulation suitable for administration.

[0084] A third aspect of this disclosure provides a method for preventing and / or treating infection with a virus of the Paramyxoviridae family, comprising administering to a subject in need an effective amount of a modified polypeptide, a pharmaceutical composition containing the modified polypeptide, a pharmaceutical preparation, or a kit thereof; wherein the modified polypeptide, pharmaceutical composition, pharmaceutical preparation, kit, virus of the Paramyxoviridae family, or virus of the Pneumoviridae family are as described above.

[0085] A fourth aspect of this disclosure provides a modified polypeptide, a pharmaceutical composition containing the same, a pharmaceutical preparation, or a kit for the prevention and / or treatment of viral infections of the Paramyxoviridae family; said modified polypeptide, pharmaceutical composition, pharmaceutical preparation, kit, or virus of the Paramyxoviridae family as described above.

[0086] In some embodiments, the pharmaceutical composition or pharmaceutical preparation uses a pharmaceutically acceptable carrier or excipient.

[0087] In some preferred embodiments, the pharmaceutical composition or pharmaceutical preparation is a vaccine, and the vaccine further comprises an immune adjuvant.

[0088] In some implementations, the medicine box also includes instructions for use.

[0089] In this disclosure, the drug may be administered via the gastrointestinal tract or parenteral route.

[0090] In some embodiments, the parenteral administration is selected from injection administration (e.g., subcutaneous or intramuscular injection), mucosal administration, transdermal administration, and nebulized administration (e.g., nasal or pulmonary nebulization).

[0091] In some embodiments, the dosage form of the drug is selected from: oral dosage form, injection dosage form, inhalation dosage form, and lyophilized dosage form.

[0092] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this disclosure.

[0093] All reagents and raw materials used in this disclosure are commercially available.

[0094] The beneficial effects achieved by this disclosure are as follows:

[0095] Experiments have confirmed that the peptide designed in this invention can effectively inhibit human parainfluenza virus (hPIV) infection. This peptide has core advantages such as a clear mechanism of action, high clinical safety, and significant drug resistance barrier, providing a novel solution for the prevention and treatment of human parainfluenza virus diseases. Detailed Implementation

[0096] definition

[0097] To make this disclosure easier to understand, certain terms are first defined. Furthermore, it should be noted that whenever a range of values ​​or parameters is enumerated, the purpose is to indicate that intermediate values ​​and ranges of these referenced values ​​also become part of this disclosure.

[0098] As used in this article, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical objects of the article. By way of example, “an element” refers to one element or more elements, such as multiple elements.

[0099] As used herein, the terms “including” and “comprising” mean “including, but not limited to” and are used interchangeably. In some embodiments, the terms “including” and “comprising” mean “consisting of”.

[0100] The term “or” is used here to mean and / or the term “and / or” and is used interchangeably with it, unless the context clearly indicates otherwise.

[0101] The abbreviations used in this disclosure have the following meanings:

[0102] Ala (Alanine, A)

[0103] Arginine (R)

[0104] Asn(Asparagine, N) asparagine

[0105] Aspartic acid (Aspartic acid, D)

[0106] DCM (Dichloromethane)

[0107] DMF (N,N-Dimethyl malonate)

[0108] Env (Envelope glycoprotein)

[0109] ESI-MS (Electronic spray ion mass spectroscopy)

[0110] Fmoc (Fluorenylmethoxycarbonyl)fluorenemethoxycarbonyl

[0111] Glycine (Glycine)

[0112] Gln (Glutamine, Q)

[0113] Glu (Glutamic acid, E)

[0114] 6-HB (six-helix bundle)

[0115] HBTU 2-(1H-1-hydroxybenzotriazole)-1,1,3,3-tetramethylhexafluorophosphate

[0116] His (Histidine, H) histidine

[0117] HoBt (1-Hydroxyl benzotiazole anhydrous) 1-hydroxybenzotriazole

[0118] NHR (N-terminal heptad repeat) N-terminal heptad repeat sequence

[0119] CHR (C-terminal heptad repeat) is a C-terminal heptad repeat sequence.

[0120] HIV (Human Immunodeficiency Virus)

[0121] HPLC (High Performance Liquid Chromatography)

[0122] Ile (Isoleucine, I) isoleucine

[0123] Leucine (L-leucine)

[0124] Methionine (M) (Met)

[0125] Lysine (K)

[0126] Phe (Phenylalanine, F) phenylalanine

[0127] hPIV (human parainfluenza virus)

[0128] Serine (S)

[0129] TFA (Trifluoroacetic acid)

[0130] Threonine (Threonie, T)

[0131] Tyr (Tyrosine, Y) tyrosine

[0132] Valine (V)

[0133] The single-letter amino acid residues are represented as follows:

[0134] 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.

[0135] As used herein, the term "peptide" typically refers to a short polymer of amino acid monomers linked by peptide bonds. It typically contains fewer than 50 monomer units. However, the term "peptide" does not exclude molecules having more than 50 monomer units. Long peptides, also known as polypeptides, typically have 50–600 monomer units, more specifically 50–300 monomer units. Furthermore, "peptide" is also defined herein to include any peptide-based molecule, including peptide analogs.

[0136] Peptide analogs can typically comprise naturally occurring or non-naturally occurring amino acids for the purposes of this invention. For example, they can comprise amino acids selected from isomers or chiral analogs (D-amino acids or L-amino acids). Additionally, the analogues may comprise one or more amino acids preferably selected from the following: hydroxyproline, β-alanine, 2,3-diaminopropionic acid, α-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, tert-butylalanine, tert-butylglycine, N-methylisoleucine, phenylglycine, alanine cyclohexyl ester, leucine, naphthylalanine, pyridylalanine, 3-benzothiophene alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-tic isoquinoline-3-carboxylic acid [β]-2-thiophene alanine, methionine sulfoxide, arginine, N-acetyllysine, 2,4-diaminobutyric acid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, ε-aminohexanoic acid, δ-aminovaleric acid, 2,3-diaminobutyric acid.

[0137] The peptides, peptide analogs, or derivatives thereof disclosed herein are preferably synthesized using chemical methods known to those skilled in the art. For example, the synthesized peptides are prepared using known solid-phase, liquid-phase, or peptide condensation techniques, or any combination thereof, and may contain native and / or non-native amino acids. Typically, the chemical synthesis method involves sequentially adding one or more amino acids to the resulting peptide chain. Typically, the amino or carboxyl group of the first amino acid is protected with a suitable protecting group. The protected or derived amino acid is then linked to an inert solid support or used in solution by adding the next amino acid in the sequence having a suitably protected complementary (amino or carboxyl) group, under conditions allowing for the formation of an amide bond. The protecting group is then removed from the newly added amino acid residue, and then the next amino acid (suitably protected) is added, and so on. After the desired amino acids have been linked in the correct order, any remaining protecting groups (and any solid support, if solid-phase synthesis techniques are used) are sequentially or simultaneously removed to obtain the final polypeptide. These methods are suitable for synthesizing peptides (such as peptide analogs) or derivatives thereof for the purposes of this invention. Typical protecting groups include tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (BzI); biphenylisopropyloxycarboxyl-carbonyl, tert-amyloxycarbonyl, isoborneolyloxycarbonyl, o-bromobenzeneoxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrobenzenesulfonyl, etc. Typical solid supports are cross-linked polymer supports. These can include divinylbenzene cross-linked styrene polymers, such as divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers, and divinylbenzene-diphenylmethylaminopolystyrene copolymers.

[0138] The peptides, peptide analogs, or derivatives thereof disclosed herein can also be prepared using recombinant protein or peptide preparation. To facilitate the preparation of recombinant peptides or proteins, it is preferable to isolate or synthesize at least one nucleic acid encoding it. Typically, the nucleic acid encoding the recombinant protein or peptide is isolated using known methods, such as, for example, amplification (e.g., using PCR), or isolated from nucleic acids derived from an organism or from a nucleic acid library using one or more restriction enzymes. To express the protein or peptide in a recombinant manner, the nucleic acid encoding the protein / peptide is operatively linked to a promoter or other regulatory sequence capable of regulating expression in a cell-free or cellular system. For example, a nucleic acid containing a sequence encoding a peptide or protein is operatively linked to a suitable promoter and held for a period of time in suitable cells under conditions sufficient for expression to occur. Typical expression vectors for in vitro expression, cell-free expression, or cell-based expression are described and are well known to those skilled in the art. In this context, the cell-free expression system may include E. coli S30 fraction, rabbit reticulum cell lysate, and malt extract, and the cell system may be selected from bacteria (e.g., E. coli), insects, plants, or mammalian cells (e.g., 293, COS, CHO, 1OT cells, 293T cells).

[0139] The pharmaceutical compositions disclosed herein may be solutions with or without a buffer solution or compositions containing a pharmaceutically acceptable carrier. In this disclosure, the pharmaceutical compositions may be administered in a solution. They may be administered in a non-buffered solution, such as in physiological saline or in water. Alternatively, they may be administered in a suitable buffered solution. This buffer solution may include acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and volumetric molar osmotic pressure concentration of the buffer solution of the pharmaceutical composition may be adjusted to suit its administration to a subject.

[0140] In some embodiments, the buffer solution further comprises a reagent for controlling the molar osmotic pressure concentration of the solution, such that the molar osmotic pressure concentration is maintained at a desired value, such as the physiological value in human plasma. Solutes that can be added to the buffer solution to control the molar osmotic pressure concentration include (but are not limited to) proteins, peptides, amino acids, non-metabolitic polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the reagent for controlling the molar osmotic pressure concentration of the solution is a salt. In some embodiments, the reagent for controlling the molar osmotic pressure concentration of the solution is sodium chloride or potassium chloride.

[0141] The pharmaceutical compositions disclosed herein can be administered at a dose sufficient to inhibit viral infection. Typically, suitable doses of the polypeptides of this 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.

[0142] The pharmaceutical composition can be administered once daily, or it can be administered two, three, or more sub-dose at appropriate intervals throughout the day, or even via continuous infusion or delivery using a controlled-release formulation. In this case, the amount of compound contained in each sub-dose must be correspondingly less to achieve the total daily dose. Dosage units can also be compounded for delivery over several days, for example using conventional sustained-release formulations that provide sustained release of the compound over a timeframe of several days. Sustained-release formulations are well known in the art and are particularly useful for delivering reagents at specific sites, thus allowing their use with the reagents of this disclosure. In this embodiment, the dosage unit comprises a corresponding plurality of daily doses.

[0143] In some embodiments, a single dose of the pharmaceutical composition can be administered continuously, with subsequent doses given 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 may include administration every 1-3 days for 4-7 consecutive days; if infection recurs, administration may continue for another 4-7 days, with a treatment cycle of 1-7 times. In some embodiments of this disclosure, a single dose of the pharmaceutical composition of this disclosure is given weekly. In other embodiments of this disclosure, a single dose of the pharmaceutical composition of this disclosure is given monthly.

[0144] Those skilled in the art will understand 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, prior treatment, the subject's overall health and / or age, and other pre-existing conditions. Furthermore, treating a subject with a therapeutically effective dose of the composition may comprise a single treatment or a series of treatments. As described elsewhere herein, the effective dose and in vivo half-life of the various compounds covered by this disclosure can be estimated using conventional methods or based on in vivo testing using suitable animal models.

[0145] Depending on whether local or systemic treatment is desired and depending on the area to be treated, the pharmaceutical compositions of this disclosure can be administered in a variety of ways. Administration can be local (e.g., via a skin patch); pulmonary; such as by inhalation or blowing in a powder or aerosol, including via a nebulizer; intratracheal; intranasal; epidermal; and percutaneous, oral, or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, for example, via an implanted device; or intracranial, such as administration within the brain parenchyma, intrasheath, or ventricle.

[0146] The pharmaceutical compositions disclosed herein (which can be conveniently present in unit dosage forms) can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include steps such as combining the active ingredients with a pharmaceutical carrier or excipient. Generally, these pharmaceutical compositions are prepared by the following steps: uniformly and finely combining the active ingredients with a liquid carrier or a finely dispersed solid carrier, or both, and, if necessary, shaping the product.

[0147] The pharmaceutical formulations provided in this disclosure include the pharmaceutical compositions described herein.

[0148] Some formulations disclosed herein also incorporate a carrier material into the pharmaceutical composition. The carrier material includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. A variety of formulations can be formulated using these materials, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit dosage forms into tablets. 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, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegration. Disintegrants include, for example, dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors include, for example, sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption enhancers include, for example, quaternary ammonium salts, sodium dodecyl sulfate, etc.; lubricants include, for example, talc, silica, corn starch, stearates, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. To formulate unit-dose dosage forms into 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, semi-synthetic glycerides, etc. To formulate unit-dose dosage forms into injectable formulations, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. The above dosage forms can be administered via injection, including subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection; intracavitary administration, such as rectal and vaginal administration; respiratory administration, such as nasal administration; and mucosal administration. Injection is the preferred route of administration.

[0149] As used herein, the term "carrier" can refer to a compound that facilitates the transport and / or complexation of another compound. The carriers of this disclosure are preferably suitable as carriers for polypeptide or nucleic acid molecules, for example, for regulating solubility in physiologically acceptable liquids, transport of polypeptide or nucleic acid molecules, and cellular uptake. Therefore, the carriers of this disclosure can be components suitable for the storage and delivery of polypeptide or nucleic acid molecules. For example, the carrier can be a cationic or polycationic carrier or a compound that can serve as a transfection agent or complexing agent. In this context, particularly preferred carriers are cationic or polycationic compounds, including protamine, nucleolar protein, spermine or spermidine, or other cationic peptides or proteins such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell-penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, membrane-penetrating peptides, VP22-derived or similar peptides, HSVVP22 (Herpes simplex), MAP, KALA or protein transduction domain (PTD), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, one or more MPG-peptides, Pep-1, L-oligomers, calcitonin peptides, and antennal-derived peptides (particularly from Drosophila melanogaster). antennapedia), pAntp, pIsl, FGF, lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptide, SAP, or histone. In this disclosure, the cationic or polycationic carrier is preferably a cationic or polycationic peptide or protein, which preferably contains or is further modified to contain at least one structural moiety capable of forming disulfide bonds, preferably a -SH structural moiety.

[0150] In some embodiments, the pharmaceutical compositions disclosed herein can be formulated as any of a number of possible formulations, such as, but not limited to, any one of the following groups: oral formulations, injections, inhalers, and lyophilized formulations; preferably, inhalers, lyophilized formulations, subcutaneous injections, or intramuscular injections.

[0151] In this disclosure, the pharmaceutical compositions are typically administered orally, parenterally, via inhalation spray, topically, rectally, nasally, orally, via an implanted reservoir. Parenterally administration, as described herein, includes subcutaneous, intravenous, intramuscular, intra-articular, intra-tubercular, intrasynovial, intrasternal, intrasheath, intrahepatic, intralesional, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, and sublingual injection or infusion techniques.

[0152] The sterile injectable form of the pharmaceutical compositions disclosed herein may be an aqueous or oily suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The sterile injectable formulation may also be a sterile injectable solution or suspension in a non-toxic, externally applicable diluent or solvent, such as a solution in 1,3-butanediol. Available excipients and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, non-volatile oils are conventionally used as solvents or suspension media. For this purpose, any mild, non-volatile oil, including synthetic monoglycerides or diglycerides, may be used. Fatty acids, such as oleic acid and its glyceride derivatives, are effective in the preparation of injectables; natural pharmaceutical oils, such as olive oil or castor oil, especially in their polyoxyethyleneized form, are also effective in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants conventionally used in the formulation of pharmaceutical dosage forms (including emulsions and suspensions). Other commonly used surfactants, such as Tweens, Spans, and other emulsifiers or biocompatibility enhancers conventionally used in the preparation of pharmaceutical solids, liquids, or dosage forms thereof, may also be used for the purpose of formulating the pharmaceutical composition.

[0153] In some embodiments, the administration method of the formulation product is selected from any of the following: oral administration, injection administration, mucosal administration, transdermal administration, and nebulization administration; preferably, pulmonary nebulization administration, subcutaneous injection, or intramuscular injection.

[0154] As used in this article, the term "vaccine" generally refers to a preventive or therapeutic substance that provides at least one antigen or antigenic function.

[0155] As used herein, the term "adjuvant" generally refers to an agent that does not itself confer immunity. Therefore, adjuvants may typically not confer immunity themselves, but assist the immune system in various ways to enhance antigen-specific immune responses, for example, by promoting the presentation of antigens to the immune system. Thus, for example, adjuvants may preferably modulate antigen-specific immune responses by modulating, for example, cytokine expression / secretion, improved antigen presentation, the nature of the switching of immune response arm, etc., thereby modulating antigen-specific (adaptive cellular and / or humoral immune responses).

[0156] 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.

[0157] As used herein, "effective amount" is intended to include a dose sufficient to achieve treatment of a disease (e.g., by weakening, improving, or maintaining the existing disease or symptoms of one or more diseases) when administered to a patient for the treatment of a disease caused by a viral infection of the Paramyxoviridae family. This "effective amount" can vary depending on how the agent is administered, the disease and its severity, and the patient's medical history, age, weight, family history, genetic composition, stage of the pathological process mediated by a virus of the Paramyxoviridae family, the type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated. "Effective amount" also includes a dose that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The compounds used in the methods of this disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0158] The following examples are used to illustrate this disclosure, but are not intended to limit the scope of this disclosure. 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.

[0159] Example 1: Peptide Design

[0160] This embodiment designs peptides based on the CHR sequence of the F1 subunit of the viral F protein. First, a series of 36 peptides covering the viral CHR region are designed, and the corresponding peptides are synthesized. Based on the results of molecular interaction and activity detection, the peptides are further designed and optimized. The inventors used sequence (1) (SEQ ID NO: 103):

[0161] Based on Z1-WJJLVOOSJJFDOOIJJVNOOIJJSLOOIJJSDOOLJJVNOOLJJTNOOITTI-Z3-Z2, derivatives were prepared by introducing systematic mutations, including mutating E and K in the salt bridge to the corresponding D and R. Simultaneously, the binding sites were fine-tuned to improve the binding of the peptide to the target. This completed the peptide design in this embodiment, and the results are shown in Tables 1-1 to 1-10. The control peptides are shown in Table 1-11.

[0162] 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, glutamic acid residue and aspartic acid residue; O is a basic amino acid residue, which may be, but is not limited to, lysine residue and arginine residue.

[0163] Z3 is a fatty acid modification group, including a linker arm and a lipophilic group. The lipophilic group binds to the target cells of the drug, increasing the drug's activity. The linker arm connects the polypeptide chain and the lipophilic group and provides a suitable stereochemistry and conformation to enable the polypeptide drug to bind better to the target.

[0164] Table 1-1 Peptide sequence design (Equation I-1)

[0165]

[0166] Table 1-2 Peptide sequence design (Formula II-1)

[0167]

[0168] Table 1-3 Peptide sequence design (Equation III-1)

[0169]

[0170] Table 1-4 Peptide Sequence Design (Formula IV-1)

[0171]

[0172] Table 1-5 Peptide Sequence Design

[0173]

[0174] Chol represents cholesterol succinate monoester, which is represented by the following structure: an ester formed by the carboxyl group of cholesterol succinate monoester linked to the lysine amino group of the polypeptide.

[0175] .

[0176] Table 1-6 Sequence design of peptides (these core sequences correspond to general formula I)

[0177]

[0178] Table 1-7 Sequence design of peptides (these core sequences correspond to general formula II)

[0179]

[0180] Table 1-8 Sequence design of peptides (these core sequences correspond to general formula III)

[0181]

[0182] Table 1-9 Sequence Design of Peptides (These core sequences correspond to general formula IV)

[0183]

[0184] Table 1-10 Peptide Sequence Design (Core Sequence)

[0185]

[0186] Table 1-11 Comparison Examples

[0187]

[0188] The definition of Chol is as described above.

[0189] Example 2: Synthesis of Naked Peptides

[0190] 1. Chemical reagents required in the preparation process

[0191] 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), ethylene dithiol (EDT), anisole sulfide (TA), triisopropylsilane (TIPS), phenol, etc., were all purchased from major chemical reagent suppliers and were not further purified before use.

[0192] Protected amino acid raw materials used in peptide synthesis 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-fluorenyloxycarbonyl, Dde is 1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl, Boc is tert-butoxycarbonyl, tBu is tert-butyl, OtBu is tert-butoxy, Trt is triphenylmethyl, and Pbf is (2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl.

[0193] 2. Synthesis of peptide resins

[0194] Using Rink Amide MBHA resin as the carrier resin, peptide resins were prepared by sequentially coupling the peptides with the corresponding protected amino acids of the peptide amino acid sequence through de-Fmoc protection and coupling reactions.

[0195] 2.1 Integrating the first protected amino acid into the main chain

[0196] 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 DMF solution of the protected amino acid while shaking. Shake and react at room temperature for 5 min to obtain the activated protected amino acid solution for later use.

[0197] Take 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g * 0.3 g), protect it with 25% PIPE / DMF solution (volume ratio) for 20 min (twice), wash and filter to obtain Fmoc-free resin.

[0198] The activated solution of the first protected amino acid was added to the resin that had been de-Fmoc-treated, and the coupling reaction was carried out for 60 min. After filtration and washing, the resin containing the first protected amino acid was obtained.

[0199] 2.2 Integrating other protective amino acids into the main chain

[0200] Using the same method as described above for adding the first protected amino acid to the main chain, other protected amino acids corresponding to the polypeptide are added sequentially to obtain a resin containing main chain amino acids.

[0201] 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.

[0202] Each step of the reaction was controlled by the Kaiser Test. If the condensation of a certain amino acid was incomplete, the condensation was repeated once until the desired target peptide was obtained.

[0203] 3. Preparation of crude product

[0204] Take the above-mentioned peptide resin, add lysis reagent (15 mL / g resin), mix well, and react with shaking at 30℃ for 3 h to lyse the target peptide from the resin and remove the side chain protecting groups. Collect the filtrate of the reaction mixture, wash the resin three times with a small amount of TFA / DCM, combine the filtrates, add anhydrous diethyl ether to precipitate, and centrifuge. Wash the filter cake twice with cold anhydrous diethyl ether, and dry to obtain an off-white powder, which is the crude naked peptide.

[0205] The composition and volume ratio of the cleavage reagent are as follows: trifluoroacetic acid: 1,2-ethylenedithiol: benzyl sulfide: phenol: H2O: triisopropylsilane = 68.5: 10: 10: 5: 3.5: 1.

[0206] 4. Preparation of pure products

[0207] Take the above-mentioned crude naked peptide, add water / acetonitrile and stir to dissolve, centrifuge to remove insoluble matter and set aside for later use.

[0208] Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC): Column type: Agela C18, column dimensions: 10 μm, 100 Å, 50 × 250 mm; mobile phases: mobile phase A (0.05% TFA and 2% acetonitrile aqueous solution) and mobile phase B (90% acetonitrile / aqueous solution); flow rate: 25 mL / min; UV detection wavelength: 220 nm; elution method: gradient elution. The crude product solution was loaded onto the above column, and the corresponding purified fractions were collected. The solvent was removed by direct freeze-drying to obtain the pure trifluoroacetate polypeptide in a fluffy state.

[0209] 5. Characteristics of a pure product

[0210] The trifluoroacetate peptides were redissolved in water and acetonitrile, and a large amount of anion exchange resin (acetate form) was added and stirred for 3 h. After filtration and rinsing the ion exchange resin with a water / acetonitrile mixture, the filtrates were combined and lyophilized to obtain the loosely packed peptide acetate (i.e., the naked peptides in Tables 1-1 to 1-5 and Table 1-11). The chemical structures of the naked peptides were characterized by liquid chromatography-mass spectrometry (LC-MS). The purity, sequence structure, molecular weight, and purity of each naked peptide were determined by analytical high-performance liquid chromatography (Agela C18 column, 4.6 × 250 mm, flow rate 1 mL / min).

[0211] in conclusion:

[0212] HPLC analysis confirmed that the purity of the synthesized naked peptides was greater than 95%, and mass spectrometry analysis confirmed that they had the correct molecular weight.

[0213] Example 3: Synthesis of lipopeptides

[0214] 1. Chemical reagents required in the preparation process

[0215] Chemical reagents used: hydrazine hydrate, cholesterol succinate monoester;

[0216] Protective amino acid raw material used in peptide synthesis: Fmoc-Lys(Dde)-OH.

[0217] 2. Synthesis of the complete sequence of the naked peptide and linker arm.

[0218] The connectors of the connecting arms include: a peptide with the EAAAK sequence (SEQ ID NO: 104) and a peptide with the GGSSG sequence (SEQ ID NO: 105).

[0219] Lipophilic compounds used for modification include: cholesterol succinate monoester.

[0220] 2.1 Main chain synthesis

[0221] (1) Synthesis of peptide resin: Rink Amide MBHA resin was used as the carrier resin. The peptide resin was prepared by sequentially coupling the corresponding protected amino acids of the peptide amino acid sequence through de-Fmoc protection and coupling reaction.

[0222] (2) Integrate the first protected amino acid in the main chain

[0223] 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 DMF solution of the protected amino acid while shaking. Shake and react at room temperature for 5 min to obtain the activated protected amino acid solution for later use.

[0224] Take 0.1 mmol of Rink Amide MBHA resin (0.35 mmol / g * 0.3 g), protect it with 25% PIPE / DMF solution (volume ratio) for 20 min (twice), wash and filter to obtain Fmoc-free resin.

[0225] The activated solution of the first protected amino acid was added to the resin that had been de-Fmoc-treated, and the coupling reaction was carried out for 60 min. After filtration and washing, the resin containing the first protected amino acid was obtained.

[0226] (3) Incorporate other protected amino acids into the main chain

[0227] Using the same method as described above for adding the first protecting amino acid to the main chain, other protecting amino acids corresponding to the peptide were sequentially added to obtain a resin containing the main chain amino acids. 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. Each reaction step was monitored using the Kaiser Test; if the condensation reaction of a certain amino acid was incomplete, the condensation was repeated once until the desired target peptide was obtained.

[0228] 2.2 Sidechain Integration

[0229] (1) Treat the resin with the smallest possible volume of 2% hydrazine hydrate / DMF solution (volume ratio) 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 later use.

[0230] (2) Modification of peptide C-terminal lysine with lipophilic compounds

[0231] C-terminal modification of peptide with lysine-cholesterol monoester: Dissolve 0.3 mmol of cholesterol monoester and 0.3 mmol of HOBt in an appropriate amount of DMF; separately add 0.3 mmol of DIC to the solution containing cholesterol monoester and HOBt, and shake at room temperature for 5 min. Add the prepared solution containing cholesterol monoester, HOBt and DIC to the Dde-free resin obtained in step (1), and couple for 60 min. Filter, wash and dry to obtain peptide resin.

[0232] Other chemical reagents, amino acid raw materials, and operating procedures are the same as in Example 2.

[0233] in conclusion:

[0234] HPLC analysis confirmed that the purity of the synthesized salt-bridged peptides was greater than 98%, and mass spectrometry determined that the molecular weight of the peptides was consistent with the theoretical molecular weight.

[0235] Example 4: Antiviral activity detection

[0236] Human parainfluenza virus (hPIV) is a virus with a typical class I fusion protein—the F protein. The F protein precursor F0 is cleaved into F1 and F2 subunits. During conformational changes, the HRN and HRC regions of the F1 subunit interact to form a six-helix bundle (6-HB), driving the fusion of the viral membrane with the host cell membrane. Using surface plasmon resonance (SPR) technology, hPIV-N51 (139-189) from the HRN region were coupled onto a CM5 chip. As the compound flowed across the chip surface, changes in the SPR signal were monitored in real time to record the dynamic process of binding and dissociation. Furthermore, kinetic analysis was used to obtain the binding rate constant (ka) and dissociation rate constant (kd), thereby calculating the equilibrium dissociation constant (KD = kd / ka).

[0237] 1. Experimental Materials

[0238] CM5 chip, catalog number: 29149603; amino-coupled reagent kit, catalog number: BR100050; HBS-EP+ buffer (10×), sodium acetate (pH 4.0), 10 mM glycine-hydrochloric acid buffer (pH 2.0), all purchased from GE Health.

[0239] 2. Experimental Methods

[0240] CM5 chip coupling: Channels 2 and 4 of the CM5 chip were activated for 7 min with a freshly prepared 1:1 mixture of 50 mM Hydroxysuccinimide (NHS) and 200 mM 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC). hPIV-N51 was then dissolved in sodium acetate at pH 4.0 to a final concentration of 30 μM at a flow rate of 10 μl / min for 600 s. The experimental buffer was HBS-EP+buffer (1×). Finally, the chip was blocked with 1 M ethanolamine. The instrument temperature was set to 25 °C.

[0241] Affinity assay: The buffer used in the experiment was HBS-EP+buffer (1×), and a multi-cycle dynamic mode was selected. Channels 1 and 3 of the chip served as blank reference channels. The test peptide was sequentially flowed through channels 2 and 4 at concentrations of 0 μM, 0.04 μM, 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, 10 μM, and 30 μM, at a flow rate of 30 μl / min. Binding time was 180 s, and dissociation time was 120 s. Finally, 10 mM glycine (pH 2.0) was injected to regenerate the chip. The instrument temperature was set to 25 ℃.

[0242] Data analysis: The data were analyzed using Biacore T200 analysis software (Version 3.2.1), after subtracting the reference channel and zero-concentration background signal, and a 1:1 binding model was selected for analysis.

[0243] The affinity test results of the peptide compounds are shown in Table 2 below:

[0244] Table 2. Experimental data on the affinity of peptides for viruses.

[0245]

[0246]

[0247]

[0248] Where NA: Not detected; M: Molar concentration (mol / L).

[0249] Conclusion: Biological activity is a key quality attribute reflecting the effectiveness of biopharmaceuticals, and its research and validation require special attention. Surface plasmon resonance (SPR) can be used to determine receptor binding activity.

[0250] The designed peptide compound exhibits a KD value between μM and nM with hPIV, indicating moderate binding strength and thus demonstrating clear antiviral activity against hPIV.

[0251] Example 5: Detection of anti-hPIV viral activity - Plaque reduction experiment

[0252] 1. Experimental supplies

[0253] Rhesus monkey kidney cells (LLC-MK2) were obtained from ATCC, catalog number CCL-7.1. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% sodium pyruvate, 1% L-glutamine, 100 U / ml penicillin, and 100 µg / ml streptomycin.

[0254] The HPIV-3 plaque assay medium is DMEM culture medium supplemented with 2% fetal bovine serum, 1% non-essential amino acids, 1% sodium pyruvate, 1% L-glutamine, 100 U / ml penicillin, and 100 µg / ml streptomycin.

[0255] Human parainfluenza virus originates from ATCC.

[0256] 2. Experimental Methods

[0257] This study used a plaque reduction assay to detect the in vitro anti-hPIV virus strain activity of the test sample, with Human Anti-HPIV Monoclonal Antibody, clone PIA174, serving as the experimental control. Eight concentrations of both the test sample and the control antibody were tested in duplicate.

[0258] LLC-MK2 cells were digested with trypsin and diluted to 400,000 cells / mL with cell culture medium containing 2% serum. The diluted cells were then added to 96-well cell culture plates at 100 μL per well (40,000 cells per well). Cells were incubated overnight in a 5% CO2 incubator at 37°C.

[0259] The next day, the serially diluted samples were mixed with an equal volume of virus (approximately 200 PFU per well) and incubated at 37°C and 5% CO2 for 1 hour. The culture medium in the 96-well cell culture plate was then discarded, and 100 μL of the sample-virus mixture was added to the 96-well cell culture plate, incubated at 37°C and 5% CO2 for 2 hours. After 2 hours, the sample-virus mixture was discarded, and 200 μL of experimental culture medium containing the corresponding concentration of the test sample (0.8% CMC) was added. The final concentration of DMSO in the culture medium was 0.5%. The control antibody was directly added to experimental culture medium containing 0.8% CMC. Cell controls (cells, without compound treatment or virus infection) and virus controls (cells infected with virus, without compound treatment) were set up. Cells were cultured in a 5% CO2, 37°C incubator for 1 day.

[0260] Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100. After washing with DPBS, hPIV-specific antibody (1:2000 dilution) was added and incubated at 37°C for 1.5 hours. Secondary antibody (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 approximately 10 minutes. After rinsing thoroughly with running water, the cells were air-dried and the number of spots per well was counted using a microplate imaging counter.

[0261] The raw data was used to calculate the antiviral activity of the samples, and the calculation formula is as follows:

[0262] % Inhibition rate = 100 - (Sample value - Cell control average) / (Virus control average - Cell control average) × 100

[0263] The inhibition rate of the samples was analyzed using a nonlinear fitting method with GraphPad Prism (version 10), and the EC value of the samples was calculated. 50 The fitting formula is: log(inhibitor) vs. response -- Variable slope (four parameters). The results are shown in Tables 3 and 4:

[0264] Table 3 Experimental data on the anti-hPIV3 virus activity of the compounds

[0265]

[0266] Table 4. Experimental data on the antiviral activity of compounds against hPIV1, hPIV2, and hPIV4 viruses.

[0267]

[0268] +++++ represents 1-10nM, ++++ represents 11-50nM, +++ represents 51-100nM, and ++ represents 101-200nM.

[0269] Although specific embodiments of this disclosure have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and such changes are all within the scope of this disclosure. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. The use of a modified polypeptide in the preparation of a drug for treating diseases caused by human parainfluenza virus infection; The polypeptide comprises one or more amino acid sequences selected from those shown in SEQ ID NO: 64-69, SEQ ID NO: 71-73, SEQ ID NO: 76-81, SEQ ID NO: 89, SEQ ID NO: 91-96, and SEQ ID NO: 101-102; wherein: (1) The structure of the polypeptide is R1-XX-R2R3, where XX is an amino acid sequence selected from the sequences shown in SEQ ID NO: 64-69, SEQ ID NO: 71-73, SEQ ID NO: 76-81, SEQ ID NO: 89 and SEQ ID NO: 91-96, R1 is acetyl, R2 is -R4-R5(R6)-, and R3 is -NH2; Wherein, R4 is a peptide segment, and the amino acid sequence of the peptide segment is EAAAK or GGSSG; R5 is lysine; and R6 is a lipophilic compound group attached to R5. The lipophilic compound group is selected from one or more of the following: cholesterol, cholesterol succinate monoester, 2-cholesterolacetic 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 valerate, 2-cholesterol isovaleric acid, 3-cholesterol valerate, 5-cholesterol valerate, 2-cholesterol hexanoic acid, 6-cholesterol hexanoic acid, 2-cholesterol heptanoic acid, 7-cholesterol heptanoic acid, 2-cholesterol octanoic acid, 8-cholesterol octanoic acid, and cholesterol formyl chloride; the lipophilic compound group can interact with the cell membrane or viral envelope to enhance the binding of the peptide to the cell membrane or viral envelope; or, (2) The structure of the polypeptide is R1-XX-R2R3, where XX is an amino acid sequence selected from the sequence shown in SEQ ID NO: 101-102, R1 is acetyl, R2 is -R4-R5-, and R3 is -NH2; wherein, R4 is a peptide segment, the amino acid sequence of which is EAAAK or GGSSG, and R5 is lysine.

2. The application as described in claim 1, characterized in that, The modified polypeptide comprises one or more amino acid sequences selected from those shown in SEQ ID NO: 13-18, 20-22, 25-30, 38, 40-45 and 50-51.

3. The application as described in claim 1 or 2, characterized in that, The drug is a solvate or chelate of the polypeptide.

4. The use of a composition, pharmaceutical preparation, or cassette in the preparation of a medicament for treating human parainfluenza virus infection; The composition comprises a modified polypeptide, a nucleic acid molecule encoding the polypeptide moiety of the modified polypeptide, and / or a carrier or transformant comprising the nucleic acid molecule; the modified polypeptide is as described in any one of claims 1-3; The pharmaceutical preparation is obtained by formulating the modified polypeptide into a formulation suitable for administration; The pillbox contains the modified polypeptide.

5. The application as described in claim 4, wherein the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier or excipient; and / or, the kit further comprises instructions for use.

6. The application as described in any one of claims 1-2 and 4-5, characterized in that, The drug is administered via the gastrointestinal tract or parenteral route; the dosage form of the drug is selected from: oral dosage form, injection, inhalation dosage form, and lyophilized dosage form.

7. The application as described in claim 6, wherein the parenteral administration is selected from injection administration, mucosal administration, transdermal administration, and nebulized administration.

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