WO2021164576A1 - Médicament contre une infection à coronavirus et son utilisation - Google Patents

Médicament contre une infection à coronavirus et son utilisation Download PDF

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WO2021164576A1
WO2021164576A1 PCT/CN2021/075497 CN2021075497W WO2021164576A1 WO 2021164576 A1 WO2021164576 A1 WO 2021164576A1 CN 2021075497 W CN2021075497 W CN 2021075497W WO 2021164576 A1 WO2021164576 A1 WO 2021164576A1
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Prior art keywords
polypeptide
seq
amino acid
substituted
functionally equivalent
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PCT/CN2021/075497
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English (en)
Chinese (zh)
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薛彤彤
赵栋
肖亮
戚建英
喻海旻
龙虎
刘登念
沈利
赵忠琼
林丽洋
刘晨
王晶翼
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四川科伦博泰生物医药股份有限公司
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Priority to CN202180010229.1A priority Critical patent/CN115003686A/zh
Publication of WO2021164576A1 publication Critical patent/WO2021164576A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of disease treatment. Specifically, the present invention relates to drugs and their derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants for anti-coronavirus infections, and nucleic acids encoding polypeptides. Molecules, methods of preparing them, and pharmaceutical compositions (e.g., pharmaceutical preparations) containing them. The present invention further relates to the use of the polypeptide and its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants in the prevention and/or treatment of coronavirus infection-related diseases, as well as in the preparation of prevention and / Or use in medicines for the treatment of diseases related to coronavirus infection.
  • coronavirus SARS-CoV-2 belongs to the ⁇ genus of coronaviruses. It has an envelope and the particles are round or elliptical, often pleomorphic, with a diameter of 60-140nm. Its genetic characteristics are obviously different from SARS-CoV and MERS-CoV. Current research shows that the homology with bat SARS-like coronavirus (bat-SL-CoVZC45) is more than 85%.
  • the clinical manifestations of the infection caused by the new coronavirus include: the incubation period is 1-14 days, generally 3-7 days, with fever, Fatigue and dry cough are the main manifestations.
  • a small number of patients have symptoms such as nasal congestion, runny nose, sore throat, and diarrhea; mild patients only have low fever, mild fatigue, etc., without pneumonia; severe patients often have difficulty breathing and/or one week after onset Hypoxemia, in severe cases, rapidly progresses to acute respiratory distress syndrome, septic shock, difficult to correct metabolic acidosis and coagulation dysfunction.
  • the fatality rate of COVID-19 caused by the new coronavirus SARS-CoV-2 is about 3.06%, and the basic infection number Ro reaches 3.77 (Yang Yang et al., BioRxiv, 2020.2.11). COVID-19 poses a serious threat to human survival and health, and it also has a serious impact on social and economic development.
  • the clinical treatment is mainly based on symptomatic and supportive treatment. It is urgent to develop corresponding preventive and therapeutic drugs to control the further spread of the disease and save the lives of critically ill patients.
  • Coronavirus is an enveloped virus of single-stranded RNA. It is divided into four categories: ⁇ , ⁇ , ⁇ , and ⁇ . The ⁇ category can be divided into four subgroups, A-D. So far, six virus molecules (except SARS-CoV-2) have been found to cause different types of diseases. The main clinical manifestations are upper respiratory and gastrointestinal infections. SARS and MERS virus infections can cause severe organ damage such as pneumonia, renal failure, and death. According to statistics, the mortality rate of patients caused by SARS and MERS is 9% and 36% respectively.
  • Coronavirus particles wrap the fatty membrane.
  • S protein spike glycoprotein
  • Spike protein which is the receptor binding site, cytolysis and main antigen site
  • small envelope glycoprotein E protein, Envelope protein, is a small protein that binds to the envelope
  • M protein, Membrane proten is responsible for the transmembrane transport of nutrients, the release of new virus teeth, and the formation of the virus envelope.
  • the envelope of ⁇ group A coronaviruses also has a shorter spike protein, namely hemagglutinin (HE protein, Heamaglutinesterase).
  • HE protein hemagglutinin
  • HE protein Heamaglutinesterase
  • the infection steps of coronavirus include the four processes of adsorption and invasion, gene synthesis, and packaging and release of mature viruses.
  • the key step of virus adsorption and invasion is the specificity of virus receptors.
  • S protein specifically recognizes cell surface receptors and forms a complex, which is a key factor in determining virus invasion.
  • the S protein can be cleaved by host cell enzymes into S1 and S2 subunits, where S1 is responsible for receptor recognition, and S2 initiates the fusion of the virus with the host's membrane.
  • the S1 subunit has a host receptor binding domain (RBD), and related receptors include CD13 (APN), sialic acid (SA), ACE2, CD26 (DPP4), etc.; the S2 subunit has HR1 (heptad repeat 1) and HR2 In the region, HR1 exposes three highly hydrophobic groove structures through the formation of homotrimers, which can bind to HR2 to form a six-helix bundle core structure, which assists the fusion of the virus with the cell membrane, thereby allowing the virus to enter the target cell.
  • RBD host receptor binding domain
  • related receptors include CD13 (APN), sialic acid (SA), ACE2, CD26 (DPP4), etc.
  • HR1 heptad repeat 1
  • HR1 exposes three highly hydrophobic groove structures through the formation of homotrimers, which can bind to HR2 to form a six-helix bundle core structure, which assists the fusion of the virus with the cell membrane, thereby allowing the virus to enter the target cell.
  • SARS-CoV-2 like SARS virus, enters cells through S protein binding to the ACE2 protein on the surface of human cells (Tianlei Ying, bioRxiv, 2020; Vincent Munster, bioRxiv, 2020).
  • McLellan et al. found (Daniel Wrapp et al., BioRxiv, 2020.2.15) that the affinity of SARS-CoV-2 and human ACE2 is 10-20 times that of SARS virus, which further explains that SARS-CoV-2 has a stronger effect than SARS virus. causess of infectious ability.
  • the inventors have developed polypeptides and their derivatives, stereoisomers, and pharmacologically effective anti-coronaviruses (such as SARS-CoV-2) to infect animal cells (especially to block coronavirus infections). Accepted salt or functionally equivalent variant.
  • the polypeptide and its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants competitively block the fusion of SARS-CoV-2 and other coronaviruses into cells to reduce or prevent viral infections Risks, reduce morbidity, and prevent further spread of the virus.
  • the process of SARS-CoV-2 virus fusion into cells involves the binding of the S1 subunit of the S protein on the surface of the virus to the human specific receptor ACE2, and then the S2 subunit initiates the fusion of the virus and the host cell membrane.
  • the S2 subunit initiates the fusion of the virus and the host cell membrane.
  • HR1 and HR2 there are two key regions HR1 and HR2 in the S2 region of the S protein.
  • HR1 will first form a trimer and then combine with three HR2 to form a stable complex, which is a necessary structure for the virus to fuse into the cell.
  • the present invention designs specific binding molecules for different targets in the fusion process, and blocks the fusion process of viruses and human cells.
  • One aspect of the present invention provides a class of polypeptides and derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants thereof, the polypeptides and derivatives, stereoisomers, pharmaceutically acceptable
  • the salt or functionally equivalent variant targets the HR1 region in the S2 subunit of the SARS-CoV-2 virus.
  • Such polypeptides and their derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants can block the combination of HR1 and HR2 in the S2 subunit of the virus to form a hexamer, thereby blocking the virus and the human body Cell fusion inhibits virus infection in the human body.
  • polypeptide molecules and derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants thereof have the amino acid composition of the molecular structure as shown in the general formula (I):
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 and X 7 are each independently selected from one of D-type or L-type amino acids, and the D-type or L-type amino acid is selected from glutamine Acid (E), aspartic acid (D), lysine (K), asparagine (N), arginine (R), glutamine (Q), histidine (H) and phenylalanine Amino acid (F);
  • Z 1 and Z 2 are not present; or Z 1 and Z 2 are each independently selected from one or more D-type or L-type amino acids;
  • R 1 is an N-terminal modification group, which does not exist or is an amino protecting group connected to the primary amine functional group of the N-terminal amino acid;
  • R 2 is a C-terminal modification group, which is absent or is a carboxyl protecting group connected to the carboxyl functional group of the C-terminal amino acid.
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 and X 7 are each independently selected from one of the following D-type or L-type amino acids, the D-type or L-type
  • the type amino acid is selected from glutamic acid (E), aspartic acid (D), lysine (K), asparagine (N) and arginine (R).
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 and X 7 are each independently selected from one of the following D-type or L-type amino acids, the D-type or The L-type amino acid is selected from glutamic acid (E), aspartic acid (D), lysine (K) and arginine (R).
  • X 1 is glutamic acid (E) or lysine (K)
  • X 2 is aspartic acid (D) or lysine (K)
  • X 3 is arginine (R ) Or lysine (K)
  • X 4 is selected from asparagine (N) and glutamic acid (E)
  • X 5 is glutamic acid (E) or aspartic acid (D)
  • X 6 is asparagus Amide (N) or glutamic acid (E)
  • X 7 is glutamic acid (E) or lysine (K).
  • X 1 is glutamic acid (E) or lysine (K)
  • X 2 is aspartic acid (D) or lysine (K)
  • X 3 is arginine (R )
  • X 4 is selected from asparagine (N) and glutamic acid (E)
  • X 5 is glutamic acid (E)
  • X 6 is asparagine (N) and glutamic acid (E)
  • X 7 is Glutamic acid (E) or lysine (K).
  • X 1 is glutamic acid (E)
  • X 2 is aspartic acid (D)
  • X 3 is arginine (R)
  • X 4 is selected from glutamic acid (E)
  • X 5 is glutamic acid (E)
  • X 6 is asparagine (N)
  • X 7 is glutamic acid (E).
  • X 1 is glutamic acid (E)
  • X 2 is aspartic acid (D)
  • X 3 is arginine (R)
  • X 4 is selected from glutamic acid (E)
  • X 5 is glutamic acid (E)
  • X 6 is asparagine (N)
  • X 7 is glutamic acid (E).
  • X 1 is lysine (K)
  • X 2 is aspartic acid (D)
  • X 3 is arginine (R)
  • X 4 is selected from glutamic acid (E)
  • X 5 is glutamic acid (E)
  • X 6 is asparagine (N)
  • X 7 is glutamic acid (E).
  • X 1 is glutamic acid (E)
  • X 2 is aspartic acid (D)
  • X 3 is arginine (R)
  • X 4 is selected from glutamic acid (E)
  • X 5 is glutamic acid (E)
  • X 6 is asparagine (N)
  • X 7 is glutamic acid (E).
  • X 1 is glutamic acid (E)
  • X 2 is lysine (K)
  • X 3 is arginine (R)
  • X 4 is selected from glutamic acid (E)
  • X 5 Is glutamic acid (E)
  • X 6 is glutamic acid (E)
  • X 7 is lysine (K).
  • X 2 is aspartic acid (D)
  • X 3 is arginine (R)
  • X 4 is selected from asparagine (N) and glutamic acid (E); and/or X 5 is glutamic acid (E).
  • X 1 is selected from lysine (K) and glutamic acid (E)
  • X 2 is aspartic acid (D)
  • X 3 is arginine (R)
  • X 4 is selected from Asparagine (N) and glutamic acid (E)
  • X 5 is glutamic acid (E)
  • X 6 is asparagine (N); and/or X 7 is glutamic acid (E).
  • polypeptide molecule of general formula (I) has an amino acid sequence as shown in SEQ ID NO: 1, 2 or 3.
  • the present invention provides a functionally equivalent variant of the polypeptide molecule shown in SEQ ID NO:1 with an amino acid sequence, and the variant has one or more of the polypeptide molecules shown in SEQ ID NO:1.
  • Amino acid substitutions, deletions or additions such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions or additions
  • the polypeptide shown in 1 has at least 75%, at least 77%, at least 80%, at least 83%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; preferably, the functionally equivalent variant has substantially the same or higher antiviral infection activity as the polypeptide shown in SEQ ID NO:1.
  • the present invention provides a functionally equivalent variant of the polypeptide molecule shown in SEQ ID NO: 2 with an amino acid sequence, which has one or more of the polypeptide shown in SEQ ID NO: 2 Amino acid substitutions, deletions or additions (such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions or additions), or with SEQ ID NO:
  • the polypeptide shown in 2 has at least 75%, at least 77%, at least 80%, at least 83%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; preferably, the functionally equivalent variant has substantially the same or higher antiviral infection activity as the polypeptide shown in SEQ ID NO: 2.
  • the present invention provides functionally equivalent variants of the polypeptide molecule shown in SEQ ID NO: 3, the variant having one or more of the polypeptides shown in SEQ ID NO: 3 Amino acid substitutions, deletions or additions (such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions or additions), or with SEQ ID NO:
  • the polypeptide shown in 3 has at least 75%, at least 77%, at least 80%, at least 83%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; preferably, the functionally equivalent variant has substantially the same or higher antiviral infection activity as the polypeptide shown in SEQ ID NO: 3.
  • polypeptide molecules and derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants thereof whose molecular structure has an amino acid composition such as general formula (II) or general formula (III) shows:
  • Z 1 and Z 2 are not present; or Z 1 and Z 2 are each independently selected from one or more D-type or L-type amino acids;
  • R 1 is an N-terminal modification group, which does not exist or is an amino protecting group connected to the primary amine functional group of the N-terminal amino acid;
  • R 2 is a C-terminal modification group, which is absent or is a carboxyl protecting group connected to the carboxyl functional group of the C-terminal amino acid.
  • polypeptide molecule of general formula (II) has an amino acid sequence as shown in SEQ ID NO:4.
  • the present invention provides functionally equivalent variants of the polypeptide molecule shown in SEQ ID NO: 4, the variant having one or more of the polypeptides shown in SEQ ID NO: 4 Amino acid substitutions, deletions or additions (such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions or additions), or with SEQ ID NO:
  • the polypeptide shown in 4 has at least 75%, at least 77%, at least 80%, at least 83%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; preferably, the functionally equivalent variant has substantially the same or higher antiviral infection activity as the polypeptide shown in SEQ ID NO: 4.
  • polypeptide of general formula (III) is shown in SEQ ID NO: 5.
  • the present invention provides functionally equivalent variants of the polypeptide molecule shown in SEQ ID NO: 5, the variant having one or more of the polypeptides shown in SEQ ID NO: 5 Amino acid substitutions, deletions or additions (such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions or additions), or with SEQ ID NO:
  • the polypeptide shown in 5 has at least 75%, at least 77%, at least 80%, at least 83%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity; preferably, the functionally equivalent variant has substantially the same or higher antiviral infection activity as the polypeptide shown in SEQ ID NO: 5.
  • the N-terminal amino acid of the polypeptide molecule represented by the general formula (I), the general formula (II) or the general formula (III) may optionally include an amino protecting group
  • the C-terminal amino acid may optionally include Carboxy protecting group. Therefore, the primary amine functional group of the N-terminal amino acid of the polypeptide molecule represented by the general formula (I), the general formula (II) or the general formula (III) can be free (that is, R 1 is not present) or can be protected by an amino group.
  • the carboxyl functional group of the C-terminal amino acid can be free (that is, R 2 is not present) or substituted with a carboxy protecting group.
  • the protecting group that can be used to protect the amino group is well known to those skilled in the art, and examples thereof include, but are not limited to, tert-oxybutylcarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), Trifluoroacetyl, allyloxycarbonyl, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl (Dde) or 3-nitro-2-pyridine sulfenylidene (Npys) and so on.
  • Boc tert-oxybutylcarbonyl
  • Fmoc fluorenylmethoxycarbonyl
  • Trifluoroacetyl Trifluoroacetyl
  • allyloxycarbonyl 1-(4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl (Dde) or 3-nitro-2-pyridine sulfenylidene
  • Protecting groups that can be used to protect carboxyl groups are well known to those skilled in the art, and examples thereof include, but are not limited to, methyl esters, ethyl esters, benzyl esters, amide groups, hydrazide groups, and the like.
  • R 1 in general formula (I), general formula (II) or general formula (III) is absent or is selected from -NH 2 , -COOH, hydrogen, -OH, derivatization of polyethylene glycol Polymers, substituted or unsubstituted acyclic aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted heteroaryl groups Alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl and R 5 -CO-, wherein R 5 is selected from hydrogen, substituted or unsubstituted acyclic aliphatic group Groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, substituted or unsubstituted heterocyclic groups, and substituted or unsub
  • R 2 is not present in general formula (I), general formula (II) or general formula (III) or is selected from NH 2 , -COOH, hydrogen, OH, -NR 3 R 4 , -OR 3 and -SR 3 , wherein R 3 and R 4 are independently selected from hydrogen, polyethylene glycol derivatives, substituted or unsubstituted acyclic aliphatic groups, substituted or unsubstituted lipids Cyclic, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl; and R 2 is not a D or L amino acid.
  • the "substituted" group in the general formula (I), general formula (II) or general formula (III) is selected from aliphatic, alkyl, alkenyl, alkynyl, hetero Aliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiocarbonyl, cyano, isocyano, amino, azide, nitro, hydroxyl, thiol and halogenated And any combination of them.
  • R 1 in general formula (I), general formula (II) or general formula (III) is absent or is selected from NH 2 , -COOH, hydrogen, OH, polyethylene glycol derivative polymer , Substituted or unsubstituted C 1-12 chain aliphatic group, substituted or unsubstituted C 3-12 alicyclic group, substituted or unsubstituted 3-14 membered heterocyclic group, substituted or Unsubstituted 5-14 membered heteroarylalkyl, substituted or unsubstituted C 6-14 aryl, substituted or unsubstituted C 6-14 aralkyl, and R 5 -CO-, wherein R 5 is selected from hydrogen, substituted or unsubstituted C 1-12 chain aliphatic group, substituted or unsubstituted C 3-12 alicyclic group, substituted or unsubstituted C 6-14 aryl group , Substituted or unsubstituted C 6
  • R 1 in general formula (I), general formula (II) or general formula (III) is absent or is selected from NH 2 , -COOH, hydrogen, OH, polyethylene glycol derivative polymer , Substituted or unsubstituted C 1-12 chain aliphatic group, substituted or unsubstituted C 3-12 alicyclic group, substituted or unsubstituted 3-14 membered heterocyclic group, substituted or Unsubstituted 5-14 membered heteroarylalkyl, substituted or unsubstituted C 6-14 aryl, substituted or unsubstituted C 6-14 aralkyl, and R 5 -CO-, wherein R 5 is selected from hydrogen, substituted or unsubstituted C 1-12 chain aliphatic group, substituted or unsubstituted C 3-12 alicyclic group, substituted or unsubstituted C 6-14 aryl group , Substituted or unsubstituted C 6
  • R 1 in Formula (I), Formula (II), or Formula (III) is R 5 -CO-, wherein R 5 is selected from cholesterol and palmitate.
  • R 1 in Formula (I), Formula (II), or Formula (III) is hydrogen.
  • R 2 is not present in general formula (I), general formula (II) or general formula (III) or is selected from NH 2 , -COOH, hydrogen, OH, -NR 3 R 4 , -OR 3 and -SR 3 , wherein R 3 and R 4 are independently selected from hydrogen, polyethylene glycol derivatives, substituted or unsubstituted C 1-12 chain aliphatic groups, substituted or unsubstituted C 3-12 alicyclic group, substituted or unsubstituted 3-14 membered heterocyclic group, substituted or unsubstituted 5-14 membered heteroarylalkyl, substituted or unsubstituted C 6- 14 aryl, substituted or unsubstituted C 6-14 aralkyl, cholesterol, and palmitate; and R 2 is not a D or L amino acid.
  • R 2 is not present in general formula (I), general formula (II) or general formula (III) or is selected from NH 2 , -COOH, hydrogen, OH, -NR 3 R 4 , -OR 3 and -SR 3 , wherein R 3 and R 4 are independently selected from hydrogen, polyethylene glycol derivatives, substituted or unsubstituted C 1-12 chain aliphatic groups, substituted or unsubstituted C 3-12 alicyclic group, substituted or unsubstituted 3-14 membered heterocyclic group, substituted or unsubstituted 5-14 membered heteroarylalkyl, substituted or unsubstituted C 6- 14 aryl and substituted or unsubstituted C 6-14 aralkyl; and R 2 is not a D or L amino acid.
  • R 2 in general formula (I), general formula (II) or general formula (III) is selected from -NR 3 R 4 , -OR 3 and -SR 3 , wherein R 3 and R 4 are independent Ground is selected from cholesterol, or palmitate.
  • R 2 in Formula (I), Formula (II), or Formula (III) is selected from —NH 2 and —OH.
  • Z 1 and Z 2 are not present in general formula (I), general formula (II) or general formula (III).
  • Z 1 and Z 2 are not present in Formula (I), Formula (II), or Formula (III) , R 1 is hydrogen, and R 2 is selected from -NH 2 and -OH.
  • polypeptide molecules, derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants thereof provided by the present invention include:
  • polypeptide shown in (1) has at least 75%, at least 77%, at least 80%, at least 83%, at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, A polypeptide having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • polypeptide molecule of the present invention has the following amino acid sequence:
  • the substitution of one or several amino acids is a conservative substitution.
  • Another aspect of the present invention provides a class of polypeptides and derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants thereof, the polypeptides and derivatives, stereoisomers, pharmaceutically acceptable Salts or functionally equivalent variants of SARS-CoV-2 virus S protein conserved region part of the polypeptide as the target.
  • the present invention designs polypeptides and their derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants for the relevant conserved regions of the SARS-CoV-2 virus S protein.
  • the polypeptides and their derivatives, stereo Isomers, pharmaceutically acceptable salts or functionally equivalent variants can play a role in blocking viral infections.
  • Another aspect of the present invention provides an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the polypeptide of the present invention and a functionally equivalent variant thereof.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of the invention and functionally equivalent variants thereof operably linked to a heterologous promoter.
  • the isolated nucleic acid molecule of the present invention encodes an amino acid sequence as shown in any one of SEQ ID NO: 1-5.
  • the present invention provides a vector (e.g., cloning vector or expression vector) comprising the isolated nucleic acid molecule of the present invention.
  • a vector e.g., cloning vector or expression vector
  • the vectors of the present invention are, for example, plasmids, cosmids, bacteriophages and the like.
  • the vector can express the polypeptide of the present invention and its functionally equivalent variants in a suitable host cell.
  • the invention provides a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the present invention is a mammalian cell, such as CHO (e.g. CHO-K1, CHO-S, CHO DG44).
  • the present invention provides methods for preparing the polypeptides of the present invention. As prepared according to various methods known in the art.
  • the obtained polypeptide of the present invention is expressed in vitro by means of genetic engineering.
  • the DNA molecule encoding the polypeptide is inserted into the expression vector, and then transfected into host cells (such as E.coli cells, yeast cells, COS cells, CHO cells, etc.), and the host cells are cultured under conditions that allow the expression of the polypeptide.
  • the polypeptide is recovered from the host cell culture of.
  • the vector contains the necessary regulatory elements for expressing the DNA molecule, for example, including a promoter sequence, a transcription initiation sequence, and an optional enhancer sequence. Further, the recovery may also include purification of the polypeptide by means such as immunoaffinity purification.
  • the host cell of the present invention may be a suitable prokaryotic or eukaryotic expression system.
  • E. coli cells of the prokaryotic expression system eukaryotic expression systems such as yeast cells, insect cells, plant cells and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).
  • the polypeptides of the invention can also be chemically synthesized.
  • a solid phase method can be used to synthesize peptides (Stewart, JM and Young, JD, "Solid Phase Peptide Synthesis, 2nd Edition", (1984), Pierce Chemical Company, Rockford, Ill (1984) inois; Bodanzsky My Bodanzsky A .,"The practice of Peptide Synthesis",(1994),Springer Verlag,Berlin;LloydWilliams P.etal.,”Chemical Approaches to the Synthesis of Peptides and Proteins",(1997),CRC,Boca Raton,FL,USA) , Synthesis in solution, enzymatic synthesis (Kullmann W. "Proteases as catalysts for enzymic synthesis of opioid peptides", (1980), J. Biol. Chem., 255(17), 8234-8238) or any combination thereof.
  • the method of polypeptide synthesis may include successively adding one or more amino acids or appropriately protected amino acids to the growing peptide chain.
  • the amino or carboxyl group of the first amino acid is usually protected by a suitable protecting group.
  • the protected or derivatized amino acid is then attached to an inert solid support or by adding the next amino acid to the sequence with a properly protected complementary group (amino or carboxyl) and used in solution under conditions suitable for the formation of amide bonds . Then remove the protecting group from this newly added amino acid residue and add the next amino acid (with appropriate protection), and so on.
  • any remaining protecting groups are successively or simultaneously removed to obtain the final crude polypeptide, which is subjected to further post-processing to obtain the final polypeptide.
  • the synthesis of the polypeptide of the present invention is completed on a solid-phase synthesizer or by manual solid-phase synthesis.
  • the synthesis of the polypeptide of the present invention includes the following steps:
  • the reagent for removing Fmoc protecting group in step (1) consists of piperidine and DMF in a volume ratio of 1:1-10; preferably, the reagent for removing Fmoc protecting group The reagent consists of piperidine and DMF in a volume ratio of 1:4.
  • step (1) solid-phase synthesis is performed on Wang resin or 2-chlorotrityl resin; preferably, the Wang resin substitution degree is 0.4-1.0 mmol/g; The degree of substitution of 2-chlorotrityl resin is 0.4-1.1mmol/g.
  • the coupling agent system described in step (2) includes a condensing agent and a reaction solvent
  • the condensing agent is selected from the group consisting of HBTU/DIEA, HATU/DIEA, HBTU/HOBt/DIEA, HCTU/ One or more of NMM, HATU/HOAt/DIEA, TBTU/DIEA, HOBt/DIC, HOAt/DIC, Cl-HOBt/DIC, PyBOP/HOBt/DIEA, PyAOP/HOBt/DIEA and Oxyma/DIC, so
  • the reaction solvent is selected from one or more of DMF, DCM, NMP and DMSO; preferably, the condensing agent is HOBt/DIC, and the reaction solvent is DMF.
  • the cleavage reagent used in the cleavage of step (3) is composed of two or two of TFA, TIS, H 2 O, EDT, anisole, phenol and p-cresol.
  • the reagent is composed of a mixed solution of TFA, TIS, H 2 O and
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polypeptide of the present invention, which comprises the polypeptide of the present invention or derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants, and pharmaceutically acceptable The carrier and/or excipient.
  • the pharmaceutical composition may also contain additional pharmaceutically active agents.
  • the pharmaceutical composition comprises an additional pharmaceutically active agent, which may be selected from antiviral drugs, immunotherapeutic drugs and/or antibiotics.
  • the pharmaceutically active agent is provided as a separate component or as a different component of the same composition or product. Therefore, the polypeptide or its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.
  • the additional pharmaceutically active agent is selected from the group consisting of ACE inhibitors, ARB, ribavirin, Aluvia, Remdesivir, Acyclovir, En Enfuvirtide, zidovudine, ritonavir, Arbido, lopinavir, Methylprednisolone or any combination thereof.
  • the additional pharmacologically active agent is an antiviral drug, which may be selected from Klitsch (Aluvia lopinavir and ritonavir tablets), Remdesivir (Remdesivir), and Os Tamivir (oseltamivir), zanamivir (zanamivir) and/or peramivir (peramivir).
  • Klitsch Aluvia lopinavir and ritonavir tablets
  • Remdesivir Remdesivir
  • Os Tamivir oseltamivir
  • zanamivir zanamivir
  • peramivir peramivir
  • the antiviral drug is abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, a Arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, complera, darunavir, Delavirdine, didanosine, behenol, dulutvir, edouridine, efavirenz, emtricitabine, enfuvirtide, entecavir (entecavir), famciclovir, Fomivirsen, fosamprenavir, foscarnet, foscarnet, ganciclovir, ibacitabine, imunovir, iodoside, imiquimod (imiquimod), indinavir (indinavir), inosine, type III interferon, type II interferon, type I interferon, lamivudine, lopinavir
  • the pharmaceutical composition comprises at least one polypeptide or a derivative, stereoisomer, pharmaceutically acceptable salt or functional equivalent selected from SEQ ID NOs: 1-5. Variants.
  • the pharmaceutical composition comprises at least two polypeptides selected from SEQ ID NOs: 1-5 or derivatives, stereoisomers, pharmaceutically acceptable salts or functional equivalents thereof Variants.
  • the pharmaceutical composition comprises at least three polypeptides selected from SEQ ID NOs: 1-5 or derivatives, stereoisomers, pharmaceutically acceptable salts or functional equivalents thereof Variants.
  • the pharmaceutical composition comprises at least four polypeptides selected from SEQ ID NOs: 1-5 or derivatives, stereoisomers, pharmaceutically acceptable salts or functional equivalents thereof Variants.
  • the pharmaceutical composition comprises all five polypeptides selected from SEQ ID NOs: 1-5 or derivatives, stereoisomers, pharmaceutically acceptable salts or functional equivalents thereof Variants.
  • the pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections). Liquid, sterile powder for injection and concentrated solution for injection), inhalation, aerosol, etc.
  • the preferred dosage form depends on the intended mode of administration and preventive/therapeutic use.
  • a liquid preparation can be prepared by the following method: incorporating the necessary dose of the polypeptide of the present invention in an appropriate solvent, and optionally, at the same time incorporating other desired ingredients (including but not limited to, pH regulator, surface Active agents, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization.
  • desired ingredients including but not limited to, pH regulator, surface Active agents, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof
  • the lyophilized preparation can be prepared by the following method: incorporating the necessary dose of the polypeptide of the present invention or its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants of the present invention in an appropriate solvent, And optionally, while incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), Subsequent filter sterilization, freeze-drying or direct freezing.
  • desired ingredients including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof.
  • the polypeptide of the present invention or its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants, pharmaceutical compositions are administered by inhalation, such as by inhalation Sprayer or sprayer.
  • inhalation Sprayer or sprayer In a preferred embodiment, the polypeptide of the present invention or its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants, pharmaceutical compositions include, but are not limited to, dry powder inhalants, aerosols It can be administered by inhalation, liquid preparation for nebulizer, or other preparations that can be administered by inhalation.
  • the formulation of the present invention is a liquid formulation (for example, as a liquid formulation for nebulizer, or a liquid formulation for other modes of administration), which contains polypeptide active ingredients, an osmotic pressure regulator, and pH adjustment. And water for injection.
  • the liquid preparation contains 0.05-5 mg/mL of the polypeptide active ingredient, 1-9 mg/mL of an osmotic pressure regulator, a pH regulator and water for injection.
  • the liquid preparation contains 1 mg/mL of the polypeptide active ingredient, 9 mg/mL of an osmotic pressure regulator, a pH regulator and water for injection.
  • the active ingredient of the polypeptide is ACV01 (SEQ ID NO: 1) polypeptide or ACV03 (SEQ ID NO: 2) polypeptide
  • the osmotic pressure regulator is sodium chloride
  • the pH regulator is hydrochloric acid, Acetic acid or sodium hydroxide
  • pH is 7.0-9.0, preferably 7.5-8.5, more preferably 8.0-8.5.
  • the present invention provides an optimized liquid preparation, preferably an inhalation preparation, which contains: 1 mg/mL ACV01 polypeptide, 9 mg/mL sodium chloride, adjusted to pH 8.0-8.5 with hydrochloric acid or sodium hydroxide, Add water for injection to the specified volume.
  • the present invention provides an optimized liquid preparation, preferably an inhalation preparation, which contains 1 mg/mL ACV03 polypeptide, 9 mg/mL sodium chloride, and adjusts the pH to 8.0-8.5 with acetic acid or sodium hydroxide, Add water for injection to the specified volume.
  • an inhalation preparation which contains 1 mg/mL ACV03 polypeptide, 9 mg/mL sodium chloride, and adjusts the pH to 8.0-8.5 with acetic acid or sodium hydroxide, Add water for injection to the specified volume.
  • the formulation of the present invention is a liquid formulation (for example, as a liquid formulation for nebulizer, or a liquid formulation for other modes of administration), which contains polypeptide active ingredients, an osmotic pressure regulator, and a co-solvent. , PH regulator and water for injection.
  • the liquid preparation contains 0.05-5 mg/ml of the polypeptide active ingredient, 1-9 mg/ml of an osmotic pressure regulator, 0.05-100 mg/ml of a co-solvent, a pH regulator, and Water for Injection.
  • the active ingredient of the polypeptide is ACV01 (SEQ ID NO: 1) polypeptide or ACV03 (SEQ ID NO: 2) polypeptide
  • the osmotic pressure regulator is sodium chloride
  • the pH regulator is hydrochloric acid, Acetic acid or sodium hydroxide
  • the co-solvent is arginine, glycine, aspartic acid, glutamine, polysorbate 20 or polysorbate 80
  • the pH is 7.0-9.0, preferably 7.5-8.5, more Preferably 8.0-8.5.
  • the present invention provides an optimized liquid preparation, preferably an inhalation preparation, which contains: 1 mg/mL ACV01 polypeptide or ACV03 polypeptide, 9 mg/mL sodium chloride, 1 mg/mL arginine, and hydrogen Adjust the pH to 8.0-8.5 with sodium oxide or acetic acid or hydrochloric acid solution, and add water for injection to the specified volume.
  • an inhalation preparation which contains: 1 mg/mL ACV01 polypeptide or ACV03 polypeptide, 9 mg/mL sodium chloride, 1 mg/mL arginine, and hydrogen Adjust the pH to 8.0-8.5 with sodium oxide or acetic acid or hydrochloric acid solution, and add water for injection to the specified volume.
  • the present invention provides an optimized liquid preparation, preferably an inhalation preparation, which contains: 1 mg/mL ACV01 polypeptide or ACV03 polypeptide, 9 mg/mL sodium chloride, 5 mg/mL arginine, and hydrogen Adjust the pH to 8.0-8.5 with sodium oxide or acetic acid or hydrochloric acid solution, and add water for injection to the specified volume.
  • an inhalation preparation which contains: 1 mg/mL ACV01 polypeptide or ACV03 polypeptide, 9 mg/mL sodium chloride, 5 mg/mL arginine, and hydrogen Adjust the pH to 8.0-8.5 with sodium oxide or acetic acid or hydrochloric acid solution, and add water for injection to the specified volume.
  • the present invention provides an optimized liquid preparation, preferably an inhalation preparation, which contains: 1 mg/mL ACV01 polypeptide or ACV03 polypeptide, 9 mg/mL sodium chloride, 3 mg/mL polysorbate 20 or poly Sorbate 80, adjust the pH to 8.0-8.5 with sodium hydroxide or acetic acid or hydrochloric acid solution, and add water for injection to the specified volume.
  • an inhalation preparation which contains: 1 mg/mL ACV01 polypeptide or ACV03 polypeptide, 9 mg/mL sodium chloride, 3 mg/mL polysorbate 20 or poly Sorbate 80, adjust the pH to 8.0-8.5 with sodium hydroxide or acetic acid or hydrochloric acid solution, and add water for injection to the specified volume.
  • the present invention provides an optimized liquid preparation, preferably an inhalation preparation, which contains: 1 mg/mL ACV01 polypeptide or ACV03 polypeptide, 9 mg/mL sodium chloride, 0.05 mg/mL polysorbate 20 or Polysorbate 80, adjust the pH to 8.0-8.5 with sodium hydroxide or acetic acid or hydrochloric acid solution, and add water for injection to the specified volume.
  • an inhalation preparation which contains: 1 mg/mL ACV01 polypeptide or ACV03 polypeptide, 9 mg/mL sodium chloride, 0.05 mg/mL polysorbate 20 or Polysorbate 80, adjust the pH to 8.0-8.5 with sodium hydroxide or acetic acid or hydrochloric acid solution, and add water for injection to the specified volume.
  • the formulation of the present invention may be a lyophilized powder, which is prepared by adding 1-50 mg/mL of protective agent such as mannitol, sorbitol, sucrose or lactose on the basis of the aforementioned liquid formulation.
  • protective agent such as mannitol, sorbitol, sucrose or lactose
  • the freeze-dried powder is prepared into a solution according to the specified volume when in use.
  • the polypeptide active ingredient or polypeptide includes the polypeptide or derivatives, stereoisomers, pharmaceutically acceptable salts or the like provided by the present invention. Functionally equivalent variants.
  • polypeptide or derivatives, stereoisomers, pharmaceutically acceptable salts, or functionally equivalent variants of the polypeptides of the present invention may be present in the pharmaceutical composition in a unit dosage form for ease of administration.
  • the preventive or therapeutically effective amount of the polypeptide or its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants of the present invention may vary according to the following factors: the severity of the disease to be prevented or treated
  • the dosage regimen can be adjusted to obtain the best objective response (for example, therapeutic or preventive response).
  • the best objective response for example, therapeutic or preventive response
  • it can be administered as a single dose, it can be administered multiple times over a period of time, or the dose can be reduced or increased proportionally to the urgency of the treatment situation.
  • Another aspect of the present invention provides a method or use, which comprises combining the polypeptide of the present invention or its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants, pharmaceutical compositions It is used to prevent and/or inhibit the fusion of coronavirus and cells.
  • Another aspect of the present invention provides a method for preventing and/or inhibiting the fusion of coronavirus with cells, the method comprising administering an effective amount of the polypeptide of the present invention or its derivatives, stereoisomers, Pharmaceutically acceptable salts or functionally equivalent variants, pharmaceutical compositions.
  • the method or use is for preventing the fusion of coronavirus and cells.
  • the method or use is for inhibiting the fusion of coronavirus and cells.
  • the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2.
  • the coronavirus is SARS-CoV-2.
  • the preventive and/or therapeutic method or use includes combining the polypeptide or derivatives, stereoisomers, or pharmaceutically acceptable Salts or functionally equivalent variants and pharmaceutical compositions are used to prevent and/or treat coronavirus infections or diseases related to them in subjects (such as humans).
  • Another aspect of the present invention provides a method for preventing and/or treating a coronavirus infection or a disease related thereto in a subject, the method comprising administering to a subject in need thereof an effective amount of the polypeptide of the present invention or Its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants, pharmaceutical compositions.
  • the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2; in a more preferred embodiment, the coronavirus is SARS-CoV-2.
  • the disease associated with coronavirus infection is COVID-19 or Middle East Respiratory Syndrome (MERS) or severe acute respiratory syndrome (SARS); in a more preferred embodiment, the coronavirus The disease associated with viral infection is COVID-19.
  • MERS Middle East Respiratory Syndrome
  • SARS severe acute respiratory syndrome
  • Another aspect of the present invention provides the use of the polypeptide of the present invention or its derivatives, stereoisomers, pharmaceutically acceptable salts or functionally equivalent variants, and pharmaceutical compositions to prepare medicines, which are used for prevention and / Or inhibit the fusion of coronavirus and cells, or be used to prevent and/or treat coronavirus infection or diseases related to it in a subject (such as a human).
  • the drug is used to inhibit the fusion of the coronavirus SARS-CoV, MERS-CoV or SARS-CoV-2 with cells, or to prevent the coronavirus SARS-CoV in a subject (e.g., human), MERS-CoV or SARS-CoV-2 infection-related diseases; in a more preferred embodiment, the coronavirus is SARS-CoV-2.
  • the disease associated with coronavirus infection is COVID-19 or Middle East Respiratory Syndrome (MERS) or severe acute respiratory syndrome (SARS); in a more preferred embodiment, the coronavirus The disease associated with viral infection is COVID-19.
  • the pharmaceutical compositions, or preparations of the present invention are used for preparing
  • the drugs are used to prevent and/or treat the deterioration and recurrence of diseases or conditions associated with SARS-CoV-2 infection in a subject (for example, a human).
  • the disease is COVID-19.
  • the present invention provides a method of preventing and/or treating a disease or condition associated with coronavirus infection in a subject. In another aspect, the present invention provides a method of delaying a disease or condition associated with coronavirus infection in a subject. In another aspect, the present invention provides a method of reducing or inhibiting the recurrence of a disease or condition associated with coronavirus infection in a subject.
  • the above-mentioned method includes administering an effective amount of the polypeptide of the present invention, or a derivative thereof, or a stereoisomer, a derivative thereof, a pharmaceutically acceptable salt thereof, and/or a function thereof to a subject in need thereof Equivalent variants, pharmaceutical compositions, formulations.
  • the disease or condition associated with coronavirus infection is COVID-19 or Middle East Respiratory Syndrome (MERS) or Severe Acute Respiratory Syndrome (SARS); in a more preferred embodiment, the disease or condition related to coronavirus infection is COVID-19.
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • the subject is a mammal. In certain more preferred embodiments, the subject is a human.
  • a container such as a plastic or glass vial, such as a hollow needle or syringe barrel with a cap
  • a container such as a plastic or glass vial, such as a hollow needle or syringe barrel with a cap
  • the present invention also provides an injection device or aerosol inhalation device, which comprises any polypeptide or derivative, stereoisomer, pharmaceutically acceptable salt, or functionally equivalent variant, composition, or formulation of the present invention.
  • ACV04 SEQ ID NO.3 DISGI NASVV NIQEE IKRLE EVAKE LNKSL IDLQE L
  • ACV05 SEQ ID NO.4 QYGSF CTQLN RALTG IAVEQ
  • ACV06 SEQ ID NO.5 YQDVN CTEVP VAIHA DQLTP
  • ACV02 SEQ ID NO.6 SLDQI NVTFL DLEYE MKKLE EAIKK LEESY IDLKE L
  • polypeptide As defined herein, the terms “polypeptide”, “protein”, “peptide” and “amino acid sequence” are used interchangeably herein and refer to a polymer of amino acid residues of any length.
  • the polymer can be linear or branched, it can contain modified amino acids or amino acid analogs, and can be interrupted by non-amino acid chemical moieties.
  • the term also includes the polymerization of amino acids that have been naturally or artificially modified (for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a label or biologically active component). Things.
  • the term “peptide” includes two or more naturally occurring or synthetic amino acids connected by covalent bonds (e.g., amide bonds).
  • the term "derivative" refers to a polypeptide that has the same biological activity as the polypeptide after replacing one or several groups of an amino acid in the amino acid sequence of the polypeptide with another group. For example, one or more amino acid residues of the parent polypeptide are introduced into one or more substituents selected from amides, carbohydrates, alkyl groups, acyl groups, esters, and pegylation.
  • stereoisomer means an isomer formed by having one or more stereoisomeric centers, and each isomeric center may exist in the form of R or S configuration or a combination thereof.
  • polypeptides described herein may have one or more double bonds, and each double bond may exist in the E (trans) or Z (cis) configuration or a combination thereof.
  • a specific stereoisomer, regioisomer, diastereomer, enantiomer or epimer shall be understood to include all possible stereoisomers, structural differences Conformers, diastereomers, enantiomers or epimers and mixtures thereof.
  • the polypeptides described herein include all stereoisomers, structural isomers, diastereomers, enantiomers, or epimers that differ in configuration and their corresponding mixtures.
  • the preferred structurally pure isomers of the polypeptides of the invention that is, enantiomers or diastereomers.
  • Techniques for transforming specific stereoisomers or keeping specific stereoisomers intact, as well as techniques for resolving mixtures of stereoisomers are well known in the art, and those skilled in the art can select suitable methods for specific situations.
  • the term "pharmaceutically acceptable salt” means a salt whose use in animals and more particularly in humans is recognized, and includes salts used to form base addition salts, whether they are Whether it is an inorganic salt or an organic salt, inorganic salts such as but not limited to lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, or organic salts such as but not limited to ethylamine, diethylamine, ethylenediamine, Ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine, etc.; or acid addition salts, whether they are organic or inorganic salts, organic salts such as but not limited to acetate, citrate, Lactate, malonate, maleate, tartrate, fumarate, benzoic acid, aspartate, glutamate, succinate, oleate, trifluoroacetate, Oxalate, pamoate or gluconate, etc., or inorganic salts such as
  • the term "functionally equivalent variant” refers to a polypeptide derived from the polypeptide sequence of the present invention modified by amino acid substitution, deletion or addition, or a derived polypeptide having certain sequence identity with the polypeptide of the present invention The condition is that the mentioned derivative polypeptide maintains at least 20%, at least 50%, at least 80%, or at least 100% of the function based on the corresponding non-modified polypeptide.
  • the functionally equivalent variants also include polypeptides with increased functional activity compared to non-modified polypeptides. In this article, when it is specifically pointed out that "have substantially the same or higher ... activity", the substantially same activity means that the activity of the functionally equivalent variant is at least 50% of the activity of the original polypeptide.
  • the function of the polypeptide of the present invention and its functionally equivalent variants can be determined by quantifying its ability to inhibit virus infection of cells.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 fruit fly cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • the molecules are the same at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput.
  • conservative substitution means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties, including The ability to form covalent bonds or hydrogen bonds, etc.) is replaced by residues. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamate), uncharged polar side chains (e.g., glycine , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • alanine, valine, leucine, isoleucine Acid, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999) ; And Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which is incorporated herein by reference).
  • amino acids are usually represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • polyethylene glycol derivative polymer refers to a polymer formed with polyethylene glycol as a basic structural unit, for example, amine, maleic acid formed with polyethylene glycol as a structural unit Imines, azides, NHS esters, mercaptans, esters, etc.
  • acyclic aliphatic group includes linear or branched alkyl, alkenyl, and alkynyl groups.
  • alkyl refers to a linear or branched saturated group having 1 to 24, preferably 1 to 16, more preferably 1 to 14, even more preferably 1 to 12, still more preferably 1, 2, 3, 4 , 5 or 6 carbon atoms and bonded to the rest of the molecule by simple bonds, including for example and not limited to methyl, ethyl, isopropyl, isobutyl, tert-butyl, heptyl, octyl, decyl, ten Dialkyl, lauryl, hexadecyl, octadecyl, pentyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexyl, etc.
  • alkenyl group refers to a linear or branched group having 2 to 24, preferably 2 to 16, more preferably 2 to 14, even more preferably 2 to 12, still more preferably 2, 3, 4, 5 or 6 carbon atoms, with one or more carbon-carbon double bonds, preferably with 1, 2 or 3 carbon-carbon double bonds, the bonds are conjugated or unconjugated, which are bonded to the rest of the molecule through simple bonds
  • alkynyl group refers to a linear or branched group having 2 to 24, preferably 2 to 16, more preferably 2 to 14, even more preferably 2 to 12, still more preferably 2, 3, 4, 5 or 6 carbon atoms, with one or more carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds, the bonds are conjugated or unconjugated, which are bonded to the rest of the molecule by simple bonds , Including, for example and not limited to, ethynyl group, 1-propynyl, 2-propynyl, 1-butyl, 2-butyl, 3-butyl, pentyl, 1-pentyl and the like.
  • Alkynyl groups can also contain one or more carbon-carbon double bonds, including for example and not limited to the groups but-1-en-3-ynyl, pent-4-en-1-ynyl and the like.
  • alicyclic is used in the present invention to encompass, for example, and not limited to, cycloalkyl or cycloalkenyl or cycloalkynyl groups.
  • cycloalkyl refers to a saturated monocyclic or polycyclic aliphatic group having 3 to 24, preferably 3 to 16, more preferably 3 to 14, even more preferably 3 to 12, still more preferably 3,4 , 5 or 6 carbon atoms and bonded to the rest of the molecule by a simple bond, including for example and not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclohexyl, dimethyl ring Hexyl, octahydroindene, decahydronaphthalene, dodecahydrophenalene, etc.
  • cycloalkenyl refers to a non-aromatic monocyclic or polycyclic aliphatic group having 5 to 24, preferably 5 to 16, more preferably 5 to 14, even more preferably 5 to 12, still more preferably 5 or 6 carbon atoms, with one or more carbon-carbon double bonds, preferably 1, 2 or 3 carbon-carbon double bonds, the bonds are conjugated or unconjugated, which are bonded to the rest of the molecule through simple bonds, including For example and not limited to cyclopent-1-en-1-yl groups and the like.
  • cycloalkynyl refers to a non-aromatic monocyclic or polycyclic aliphatic group having 8 to 24, preferably 8 to 16, more preferably 8 to 14, even more preferably 8 to 12, still more preferably 8 or 9 carbon atoms, with one or more carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds, the bonds are conjugated or unconjugated, which are bonded to the rest of the molecule through simple bonds, including For example and not limited to cyclooct-2-yn-1-yl groups and the like. Cycloalkynyl groups can also contain one or more carbon-carbon double bonds, including, for example and not limited to, cyclooct-4-en-2-ynyl groups and the like.
  • aryl refers to an aromatic group having 6 to 30, preferably 6 to 18, more preferably 6 to 10, still more preferably 6 or 10 carbon atoms, which contains 1, 2, 3 or 4 aromatic groups.
  • Group rings, bonded or fused through carbon-carbon bonds include, but are not limited to, phenyl, naphthyl, diphenyl, indenyl, phenanthryl, or anthracenyl, etc.; or aralkyl groups.
  • aralkyl refers to an alkyl group substituted with an aromatic group, having 7 to 24 carbon atoms and including but not limited to -(CH 2 ) 1-6 -phenyl, -(CH 2 ) 1-6 -(1-naphthyl), -(CH 2 ) 1-6 -(2-naphthyl), -(CH 2 ) 1-6 -CH(phenyl) 2 and the like.
  • heterocyclyl refers to a 3-10 membered hydrocarbonated ring, in which one or more of the atoms in the ring, preferably 1, 2 or 3 of the atoms in the ring are elements other than carbon such as nitrogen, oxygen or Sulfur and can be saturated or unsaturated.
  • the heterocyclic ring may be a monocyclic, bicyclic or tricyclic ring system, which may include a fused ring system; and the nitrogen, carbon or sulfur atoms in the residue heterocyclic ring may be optionally oxidized; the nitrogen atom may be optionally quaternized ; And the residue heterocyclyl may be partially or fully saturated or aromatic.
  • heterocyclyl most preferably refers to a 5- or 6-membered ring.
  • saturated heterocyclic groups are dioxane, piperidine, piperazine, pyrrolidine, morpholine and thiomorpholine.
  • aromatic heterocyclic groups also called heteroaromatic groups, are pyridine, pyrrole, furan, thiophene, benzofuran, imidazoline, hydroquinone, quinoline, pyridazine and naphthyridine.
  • heteroarylalkyl refers to an alkyl group substituted with a substituted or unsubstituted aromatic heterocyclic group, the alkyl group has 1 to 6 carbon atoms and the aromatic heterocyclic group has 2 to 24 carbon atoms and 1 to 3 non-carbon atoms, and include, for example and not limited to -(CH 2 ) 1-6 -imidazolyl, -(CH 2 ) 1-6 -triazolyl, -(CH 2 ) 1-6 -thienyl, -(CH 2 ) 1-6 -furyl, -(CH 2 ) 1-6 -pyrrolidinyl and the like.
  • halogen or halo refers to fluorine, chlorine, bromine and iodine.
  • substituents include, but are not limited to, the following groups that result in the formation of stable moieties: aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclyl, aryl, heteroaryl, acyl, oxo , Imino, thiocarbonyl, cyano, isocyano, amino, azido, nitro, hydroxy, thiol and halo and any combination thereof, including but not limited to the following groups: aliphatic amino, hetero Aliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic, alkoxy, heteroalkoxy Group, aryloxy group, heteroaryloxy group, aliphapha
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituents as described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.
  • the term "pharmaceutical composition” refers to a drug treatment obtained by mixing or combining more than one active ingredient, which includes fixed and non-fixed combinations of active ingredients.
  • the term “fixed combination” refers to the simultaneous administration of at least one compound described herein and at least one additional pharmaceutically active agent to a patient in the form of a single entity or a single dosage form.
  • the term “non-fixed combination” refers to the simultaneous administration, co-administration, or sequential administration of at least one compound described herein and at least one additional pharmaceutically active agent to a patient in the form of separate entities, wherein such Two or more compounds are administered that provide effective levels in the patient's body. These also apply to cocktail therapy, such as the administration of three or more active ingredients.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is Known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, Diluent, agent to maintain osmotic pressure, agent to delay absorption, preservative.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include but are not limited to sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugar, NaCl and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol) and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, paraben, chlorobutanol, phenol, sorbic acid and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredients in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , Lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate).
  • prevention refers to the use of blocking, reducing, inhibiting, preventing and/or delaying a disease or condition or symptom (for example, respiratory disease and symptom, infection or autoimmune disease) in A method implemented by the occurrence of a subject in the body, and a method for reducing the incidence of infectious diseases in the subject.
  • treatment refers to a method performed in order to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, those used to relieve symptoms, narrow the scope of the disease, stabilize (ie, no longer worsen) the state of the disease, and delay or slow down the disease after the subject is sick.
  • treatment can also refer to prolonging the survival period compared to the expected survival period (if not receiving treatment).
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject e.g., human
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing diseases e.g., respiratory diseases and symptoms, infections or autoimmune diseases
  • an effective amount for preventing diseases refers to an amount sufficient to prevent, prevent, or delay diseases (e.g., respiratory diseases and symptoms, infections or autoimmune diseases).
  • the amount that occurs; the effective amount for treating the disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who have already suffered from the disease. It is completely within the abilities of those skilled in the art to determine such an effective amount.
  • the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general conditions such as age, weight and sex, the way the drug is administered, and other treatments that are administered at the same time and many more.
  • the determination of the appropriate dose is performed by the clinician, for example, using parameters or factors known or suspected to affect treatment in the art.
  • the dosage starts with an amount slightly less than the optimal dosage, and it is subsequently increased in smaller increments until the desired or optimal effect is achieved relative to any negative side effects.
  • Important diagnostic measures include, for example, a measure of the symptoms of inflammation or the level of inflammatory cytokines produced.
  • the typical non-limiting range of the therapeutically or preventively effective amount of the polypeptide of the present invention is 0.01-1000 mg/kg, such as 0.1-500 mg/kg, 1-250 mg/kg, or 1-50 mg/kg.
  • the specific dosing regimen should be adjusted over time according to the needs of the patient and the professional evaluation of the doctor; the dosage range given here is for illustrative purposes only, and is not limited The use or scope of the pharmaceutical composition or immunogenic composition of the present invention.
  • the polypeptide, its derivative, its stereoisomer, its mixture, its pharmaceutically acceptable salt and/or its functionally equivalent variant in the pharmaceutical composition of the present invention produce at least one of the following organisms in a subject Physical activity: binds to ACE2, inhibits the binding of S1 and ACE2; binds to S2, inhibits the formation of 6-HB; binds to HR1, inhibits the formation of 6-HB; binds to the conserved regions of S protein, inhibits SARS-CoV-2 and Fusion of cells.
  • polypeptide, its derivatives, or its stereoisomers, mixtures, pharmaceutically acceptable salts and/or functionally equivalent variants in the pharmaceutical composition of the present invention can effectively inhibit SARS-CoV-2 and
  • the fusion of cells has great clinical value for the prevention and treatment of diseases related to SARS-CoV-2 infection.
  • Figure 1 Flow cytometric detection of HEK293T-ACE2 cells. Flow cytometric detection showed that the fluorescence expression was obvious, and the positive rate was 100%. HEK293T cells expressing ACE2 were successfully constructed.
  • Figure 2 ACV02 blocking virus infection ACE2 high-expressing cell activity test.
  • Figure 3 ACV03 blocking virus infection ACE2 high-expressing cell viability test.
  • Figure 4 ACV06 blocking virus infection ACE2 high-expressing cell viability test.
  • Figure 5 ACV01 and ACV03 blocking virus infection ACE2 high-expressing cell activity test.
  • Figure 6 The results of plaque count detection of the in vitro inhibitory activity of ACV03 on SARS-CoV-2.
  • each cleavage peptide resin weighs 470 g and 475 g.
  • the method of each cleavage is as follows: add peptide resin to the peptide cleavage reactor, add 4L of cleavage reagent composed of TFA and water with a volume ratio of 95:5, which has been cooled to about 0°C, and react with stirring for 2 hours at room temperature. . After the reaction is completed, the reaction solution is filtered to 40L of methyl tert-butyl ether which has been cooled to about -10°C in advance, and white solids are produced.
  • step (2) The crude product obtained in step (2) was weighed and purified by 0.1% TFA, NH 4 OAc was exchanged for salt to obtain a peptide solution, and the finished product (weight 42 g, purity 99.8%) was obtained by freeze-drying.
  • the ketone method detects and judges the end of the reaction, and obtains H-Gln(Trt)-Wang resin.
  • step (2) Weigh the crude product obtained in step (2) and purify it with 0.1% TFA and KH 2 PO 4 , exchange salt with NH 4 OAc to obtain a refined peptide solution, and freeze-dry the refined peptide to obtain a finished product (weight 240.5 mg, purity 88.1%).
  • step (2) The crude product obtained in step (2) was weighed and purified by 0.1% TFA, NH 4 OAc was replaced with salt to obtain a refined peptide solution, and the finished product (weight 174.4 mg, purity 87.9%) was obtained by freeze-drying.
  • the solid phase synthesis method was used to synthesize ACV02 (the amino acid sequence is shown in SEQ ID NO. 6, see S. Xia, L. Yan, W. Xu, AS Agrawal, A. Algaissi, C.-TKTseng,Q.Wang,L.Du,W.Tan,IAWilson,S.Jiang,B.Yang,L.Lu,A pan-coronavirus fusion inhibitor targeting the HR1domain of humancoronavirusspike.Sci.Adv. 5.
  • ACV04 amino acid sequence is shown in SEQ ID NO. 3).
  • S-Virus pseudoviral particles When S-Virus pseudoviral particles are incubated with cells overexpressing ACE2, after S protein binds to the receptor ACE2, it undergoes a series of allosteric and fusion with the cell membrane, transduces the reporter gene to the target cell, and expresses it in the target cell Luciferase.
  • the polypeptide of the present invention inhibits virus from infecting target cells by blocking S protein allosteric/membrane fusion, so this experiment can be used to detect whether the polypeptide inhibits virus from infecting target cells, so as to speculate whether to block the virus S protein allosteric and protein ACE2 Membrane fusion mediated by binding to S protein.
  • a plasmid expressing human ACE2 (containing APC fluorescent marker gene) is constructed using conventional genetic engineering methods in the field, transfected into HEK293T cells, and subjected to pressure screening to obtain HEK293T-ACE2 expressing ACE2 and carrying APC fluorescent signal.
  • flow cytometry verification as shown in Figure 1, the fluorescence expression is obvious, and the positive rate is 100%, which proves that HEK293T-ACE2 cells expressing ACE2 were successfully constructed.
  • plasmid containing the GFP gene
  • S protein of SARS-Cov-2 co-transfect the plasmid with the lentiviral backbone plasmid and packaging plasmid into HEK293T cells, and package to form lentiviral particles
  • Harvest the virus supernatant and obtain the S pseudovirus particle S-Virus that is chimeric with the S protein of SARS-Cov-2 on the virus envelope.
  • ACV02, ACV03, and ACV06 The experimental results of ACV02, ACV03, and ACV06 are shown in Figure 2, Figure 3 and Figure 4.
  • ACV02, ACV03 and ACV06 can effectively block the pseudovirus particles from infecting overexpressing ACE2 cells, and as the concentration increases, the inhibition rate is all gradually increases, the highest inhibition rate ACV02 about 50%, ACV03 ACV06 highest inhibition rate and 80%, ACV03 blocking activity IC 50 of 0.37 ⁇ M.
  • HEK293T-ACE2 expressing ACE2 (contains APC fluorescent marker gene, constructed in Experimental Example 1) and S pseudovirus particles S-Virus (2*E4 per cell, 150ul) /Well, constructed in experimental example 1) mix; culture in a 37°C 5% CO 2 incubator for 4 hours, flow cytometry to detect APC and GFP fluorescence and cell fusion, microplate reader reads the signal value, and plots based on the data .
  • Experiment selection age 4 year-old cynomolgus monkeys purchased from Hainan Jingang Biotechnology Co., Ltd.
  • male and female were given 0.25 mg/kg of the polypeptide compound of the present invention (ACV01 and ACV03) intravenously at a concentration of 0.1 mg/ml
  • the administration volume is 2.5ml/kg
  • the administration time is 2min/mouse.
  • TK blood sampling was performed on the first day of administration.
  • the blood sampling time points were before administration and 5min, 30min, 1h, 2h, 4h, 8h, 24h after administration.
  • blood samples were collected for hematology and serum biochemical indicators.
  • the polypeptide compounds ACV01 and ACV03 of the present invention are administered to cynomolgus monkeys by a single intravenous administration, and the safety is good, the non-toxic reaction dose (NOAEL) is 0.25 mg/kg, and there is no difference between male and female.
  • NOAEL non-toxic reaction dose
  • ACV03 polypeptide is dissolved to 1mg/mL (250uM) in DMEM medium, then diluted to 200uM, and sterilized with 0.22uM filter;
  • ACV03 polypeptide is diluted with DMEM (200uM, 100uM, 50uM, 25uM) to 7 concentration points, 3 replicate holes;
  • SARS-CoV-2 virus stock solution is diluted 1:4 with DMEM to prepare virus working solution;
  • step 5 Remove the cell culture solution in step 1, take 200uL of each dilution of the drug, add them to the cell culture plate in turn, and incubate in a CO 2 incubator at 37°C for 2 hours;
  • step 6 mix 190uL+10uL virus working solution of each concentration gradient drug; positive control: 190uL DMEM+10uL virus working solution; negative control: 200uL DMEM, incubate at room temperature for 2 hours;
  • step 7 Remove the polypeptide dilution in step 5, add the virus polypeptide mixture and control in step 6 to the cell culture plate in sequence, and incubate in a CO 2 incubator at 37°C for 2 hours;
  • Table 1 Fluorescence quantitative PCR and plaque count results of virus titer
  • Formulation example 1 ACV01 inhalation formulation 1
  • Component Dosage Active ingredient (ACV01 hydrochloride) 2mg
  • the drug solution is coarsely filtered through a 0.45 ⁇ m filter membrane, and then sterilized and filtered through a 0.22 ⁇ m filter membrane to a temporary storage tank; and then divided into a 2ml/bottle inhalation container. It can then be optionally packaged and stored.
  • the inhalant prepared by the above method can be atomized by a medicinal atomizer, and the user inhales the atomized active ingredient to receive administration.
  • Preparation Example 1 Refer to the preparation method of Preparation Example 1, and prepare a preparation with the following composition: 1 mg/ml ACV01 polypeptide hydrochloride, 9 mg/ml sodium chloride, 1 mg/ml arginine, and sodium hydroxide or hydrochloric acid solution to adjust the pH To 8.0 ⁇ 8.5, add water for injection to the specified volume.
  • a preparation consisting of 1 mg/ml ACV01 polypeptide hydrochloride, 9 mg/ml sodium chloride, and 5 mg/ml arginine was prepared. Adjust the pH to 8.0-8.5 with sodium hydroxide or hydrochloric acid solution, and add water for injection to the specified volume.
  • preparation method of preparation example 1 prepare a preparation with the following composition: 1 mg/ml ACV01 polypeptide hydrochloride, 9 mg/ml sodium chloride, 0.05 mg/ml polysorbate 80 or 20; sodium hydroxide or Adjust the pH of the hydrochloric acid solution to 8.0-8.5, and add water for injection to the specified volume.
  • a preparation consisting of ACV01 polypeptide at 1 mg/ml, sodium chloride at 9 mg/ml, and polysorbate 80 or 20 at 3 mg/ml was prepared. Adjust the pH to 8.0-8.5 with sodium hydroxide or hydrochloric acid solution, and add water for injection to the specified volume.
  • Formulation example 6 ACV03 inhalation formulation 1
  • Component Dosage Active ingredient (ACV03 acetate) 2mg Sodium chloride 18mg Acetic acid/sodium hydroxide Right amount, adjust pH Water for Injection To 2ml
  • the drug solution is coarsely filtered through a 0.45 ⁇ m filter membrane, and then sterilized and filtered through a 0.22 ⁇ m filter membrane to be temporarily stored; and then divided into 2ml/bottle inhalation containers. It can then be optionally packaged and stored.
  • the inhalant prepared by the above method can be atomized by a medicinal atomizer, and the user inhales the atomized active ingredient to receive administration.
  • ACV03 polypeptide acetate 9mg/mL sodium chloride, 1mg/mL arginine, adjust the pH to 8.0-8.5 with sodium hydroxide or acetic acid solution, and add water for injection to the specified volume.
  • ACV03 polypeptide acetate 9mg/mL sodium chloride, 5mg/mL arginine, adjust the pH to 8.0-8.5 with sodium hydroxide or acetic acid solution, and add water for injection to the specified volume.
  • preparation method of preparation example 6 prepare a preparation with the following composition: 1 mg/mL ACV03 polypeptide acetate, 9 mg/mL sodium chloride, 0.05 mg/mL polysorbate 20 or polysorbate 80, and use hydroxide Adjust the pH to 8.0-8.5 with sodium or acetic acid solution, and add water for injection to the specified volume.
  • preparation method of preparation example 6 prepare a preparation with the following composition: 1 mg/mL ACV03 polypeptide acetate, 9 mg/mL sodium chloride, 3 mg/mL polysorbate 20 or polysorbate 80, with sodium hydroxide Or adjust the pH to 8.0-8.5 with acetic acid solution, and add water for injection to the specified volume.
  • ACV01 polypeptide solution with water for injection, and add sodium chloride separately.
  • concentrations of ACV01 and sodium chloride are shown in Table 4 respectively. Adjust the pH to 7 with sodium hydroxide solution and dilute hydrochloric acid solution (if necessary), and respectively Filter to obtain formulations 5-6.
  • a solution of ACV03 polypeptide salt with a concentration of 1 mg/ml was prepared with water for injection, and sodium chloride was added to obtain the solution.
  • the solution was divided into 2 groups, and the pH of the two groups was adjusted to 6.0 and 8.0 with sodium hydroxide solution and acetic acid solution (if necessary), and filtered respectively to obtain inhalation preparations 7 and 8.
  • formulations of formulations 7 and 8 were stored for 4 days under refrigerated conditions, the content of the active ingredient ACV03 was measured, and compared with the content of the active ingredient at day 0, the peak area change rate of the content of the active ingredient was calculated.

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Abstract

L'invention concerne un polypeptide contre une infection à coronavirus et son utilisation. L'invention concerne en particulier un polypeptide contre une infection à coronavirus et un dérivé, un stéréoisomère, un sel pharmaceutiquement acceptable ou un variant fonctionnellement équivalent de celui-ci, une molécule d'acide nucléique codant pour le polypeptide, son procédé de préparation et une composition pharmaceutique (par exemple une préparation pharmaceutique) le comprenant. L'invention concerne en outre une utilisation du polypeptide et du dérivé, du stéréoisomère, du sel pharmaceutiquement acceptable ou du variant fonctionnellement équivalent de celui-ci dans la prévention et/ou le traitement de maladies associées à une infection à coronavirus, et une utilisation dans la préparation d'un médicament pour la prévention et/ou le traitement de maladies associées à une infection à coronavirus.
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WO2023069728A1 (fr) * 2021-10-22 2023-04-27 Wisconsin Alumni Research Foundation Peptides qui inhibent l'infection par le sars-cov-2, le virus qui provoque la maladie covid-19
CN113908254B (zh) * 2021-10-19 2024-05-28 山西锦波生物医药股份有限公司 一种干粉吸入剂及其制备方法和用途

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113908254B (zh) * 2021-10-19 2024-05-28 山西锦波生物医药股份有限公司 一种干粉吸入剂及其制备方法和用途
WO2023069728A1 (fr) * 2021-10-22 2023-04-27 Wisconsin Alumni Research Foundation Peptides qui inhibent l'infection par le sars-cov-2, le virus qui provoque la maladie covid-19

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