WO2023155318A1 - 一种优化病毒膜融合抑制剂的方法及广谱抗冠状病毒脂肽和应用 - Google Patents

一种优化病毒膜融合抑制剂的方法及广谱抗冠状病毒脂肽和应用 Download PDF

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WO2023155318A1
WO2023155318A1 PCT/CN2022/094001 CN2022094001W WO2023155318A1 WO 2023155318 A1 WO2023155318 A1 WO 2023155318A1 CN 2022094001 W CN2022094001 W CN 2022094001W WO 2023155318 A1 WO2023155318 A1 WO 2023155318A1
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lipopeptide
eaaak
membrane fusion
formula
group
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PCT/CN2022/094001
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English (en)
French (fr)
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何玉先
朱园美
种辉辉
刘念
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悦康药业集团股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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 disclosure relates to a method for optimizing viral membrane fusion inhibitors, broad-spectrum anti-coronavirus lipopeptides and applications.
  • Membrane fusion is an extremely important biological phenomenon. Physiological processes such as the formation of fertilized eggs and intracellular vesicle transport are all realized through membrane fusion. Many viruses that seriously endanger human health also infect host cells through membrane fusion, such as AIDS virus (HIV), influenza virus, hepatitis virus, Ebola virus, Zika virus, SARS coronavirus, Middle East respiratory syndrome (MERS) ) coronavirus, and the new type of coronavirus (SARS-CoV-2) that is currently ravaging humans, etc. Viral membrane fusion is mediated by fusion proteins located on the surface of virus particles, such as the HIV envelope protein gp41 subunit and the coronavirus spike S protein S2 subunit.
  • HIV HIV envelope protein gp41 subunit and the coronavirus spike S protein S2 subunit.
  • Fusion proteins generally contain important functional regions such as fusion peptide (FP), heptad repeat domain 1 (HR1), heptad repeat domain 2 (HR2) and transmembrane domain (TM) in sequence.
  • FP fusion peptide
  • HR1 heptad repeat domain 1
  • HR2 heptad repeat domain 2
  • TM transmembrane domain
  • peptides derived from the HR1 and HR2 regions of many viruses can be used as viral membrane fusion inhibitors, and their mechanism of action is to competitively bind to the fusion protein in the pre-fusion state, thereby blocking the formation of the 6-HB structure.
  • the HIV treatment drug T20 enfuvirtide
  • the HIV treatment drug T20 is the only viral membrane fusion inhibitor approved by the US FDA for clinical use, but the research and development of antiviral drugs targeting this target has been receiving much attention.
  • lipopeptides modified based on lipid compounds such as fatty acids and cholesterol, etc.
  • Coronavirus is a single-stranded positive-sense RNA virus with an envelope, which is divided into four genera: ⁇ , ⁇ , ⁇ and ⁇ .
  • CoVs currently known to infect humans include HCoV-229E and HCoV-NL63 of the genus ⁇ and HCoV-OC43, CoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2 of the genus ⁇ .
  • HCoV-229E, HCoV-NL63, HCoV-OC43, and CoV-HKU1 are common epidemic pathogens that usually cause only common cold symptoms, accounting for about 10% to 30% of upper respiratory tract infections in adults, but are harmful to children, the elderly, and immunocompromised patients.
  • SARS-CoV SARS-CoV
  • MERS-CoV MERS-CoV
  • SARS-CoV-2 are highly pathogenic pathogens that cause severe lung disease with high mortality.
  • SARS-CoV-2 shares 79.5% and 96% sequence identity with SARS-CoV and bat coronavirus SL-CoV-RaTG13, respectively, and uses the same cellular receptor (ACE2).
  • ACE2 cellular receptor
  • VOCs variant strains
  • Alpha, Beta, Gamma, Delta, and Omicron variant strains
  • other strains often lead to a decline in the effectiveness of vaccines and drugs or even loss of activity. Therefore, it is necessary to develop efficient and broad-spectrum coronavirus inhibitors.
  • the disclosed scientific research team has been committed to the research and development of viral membrane fusion inhibitor drugs, and designed a lipopeptide-based broad-spectrum coronavirus membrane fusion inhibitor, which has strong inhibitory activity against SARS-CoV-2 and its mutant strains ( References 2-5).
  • a linker between the polypeptide sequence and the lipid group (such as fatty acid and cholesterol, etc.) to act as a linking arm.
  • the expected binding sites for lipid groups are viral or cell membranes, while peptides are enriched in the target region, so the binding sites for lipid groups and peptides are different. Since the polypeptide tends to form a stable secondary structure and has strong structural rigidity, a flexible linker is usually used to connect the polypeptide and the lipid group, so that the polypeptide and the lipid group can respectively form a suitable conformation , bound to their respective binding sites.
  • Common flexible linkers have a combination of glycine (G) and serine (S), such as (GGGGS)n or (GSGSG)n, etc., by changing the size of n, the distance between the domains can be enlarged and reduced; another common
  • the flexible linker is a small molecule polyethylene glycol (PEG) n, wherein n is mostly between 2 and 24.
  • the lipopeptides of the coronavirus membrane fusion inhibitors reported in the literature also use flexible linkers, such as IPB02V1-IPB02V5 are all PEG 8 (reference 2), and IPB24-IPB27 are PEG 4 , PEG 5 , PEG 6 and PEG 8 ( Reference 4), EKL1C is GSG (Reference 6), EK1C4 is tandem GSGSG and PEG 4 (Reference 7), [SARS HRC -PEG 4 ] 2 -chol is PEG 4 (Reference 8), etc.
  • flexible linkers such as IPB02V1-IPB02V5 are all PEG 8 (reference 2), and IPB24-IPB27 are PEG 4 , PEG 5 , PEG 6 and PEG 8 ( Reference 4), EKL1C is GSG (Reference 6), EK1C4 is tandem GSGSG and PEG 4 (Reference 7), [SARS HRC -PEG 4 ] 2 -chol is PEG 4 (Reference 8),
  • the common rigid linker (rigid linker) for the preparation of fusion proteins has the (EAAAK) n sequence capable of forming an ⁇ -helix, which has internal hydrogen bonds and a closely linked peptide chain backbone, which is rigid and stable; another type of rigid linker has Pro- rich sequence (XP)n, where X can specify any amino acid, preferably alanine, lysine or glutamic acid; (XP)n sequence has no helical structure, but where proline can increase the backbone stiffness and effectively separate the structure area.
  • EAAAK Pro- rich sequence
  • X Pro- rich sequence
  • X can specify any amino acid, preferably alanine, lysine or glutamic acid
  • (XP)n sequence has no helical structure, but where proline can increase the backbone stiffness and effectively separate the structure area.
  • the lipopeptide virus membrane fusion inhibitor prepared so far has no precedent of using a rigid linker.
  • This disclosure creatively uses the rigid linker EAAAK sequence to prepare a broad-spectrum coronavirus membrane fusion inhibitor lipopeptide, endowing the polypeptide with a significant helical structure, and significantly Improved antiviral activity and stability of the inhibitor.
  • the present disclosure uniquely provides a method for optimizing a virus membrane fusion inhibitor, a broad-spectrum anti-coronavirus lipopeptide and its application.
  • the present disclosure finds for the first time that compounds of formula I, IPB29 and IPB30, with a rigid linker EAAAK, especially IPB29, have at least 70-fold increased activity against the new coronavirus compared to compounds without this linker, compared to compounds with a flexible linker, such as PEG Alternatively, compounds with GSGSG had an approximately 8-fold increase in inhibitory activity.
  • the reason for the increase in the activity of the lipopeptide IPB29 is that the rigid linker EAAAK significantly increases the helical content of the lipopeptide and improves the stability at the same time.
  • this lipopeptide IPB29 can inhibit many different types of new coronaviruses, including but not limited to SARS-CoV-2 and its various mutants, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43 and HCoV -NL63 and other coronaviruses.
  • the present disclosure provides a compound or a pharmaceutically acceptable salt or derivative thereof
  • the compound is a compound shown in formula (I) or a compound shown in formula (II);
  • X 1 is an amino terminal protecting group
  • X 2 is a polypeptide, and the amino acid sequence is (EAAAK)n or A[(EAAAK)n]A; n is a natural number below 5, representing the number of repetitions of the EAAAK sequence;
  • X 3 is lysine or cysteine or 2,3-diaminopropionic acid (Dap) or ornithine (Orn) or 2,4-diaminobutyric acid (Dab) or 2 ,7-diaminoheptanoic acid (Dah);
  • X4 is a lipophilic compound group modified at X3 ;
  • X 4 is a lipophilic compound group modified on K in X 2 ;
  • X 5 is a carboxyl terminal protecting group.
  • X 1 is any group in acetyl (Ac), amino (NH 2 ), maleyl, succinyl, tert-butoxycarbonyl or benzyloxy or other hydrophobic groups or macromolecule carrier groups .
  • X 5 is any group in amino group (NH 2 ), carboxyl group, hydroxyl group, amide group or tert-butoxycarbonyl group or other hydrophobic groups or macromolecular carrier groups.
  • the lipophilic compound is cholesterol monosuccinate, 2-cholesterol acetate, 2-cholesterol propionate, 3-cholesterol propionate, 2-cholesterol butyrate, 2-cholesterol isobutyrate, 3-cholesterol butyrate acid, 3-cholesterol isobutyrate, 4-cholesterol butyrate, 2-cholesterol valerate, 2-cholesterol isovalerate, 3-cholesterol valerate, 5-cholesterol pentanoate, 2-cholesterol hexanoate, 6-cholesterol hexanoate Acid, 2-cholesterol heptanoic acid, 7-cholesterol heptanoic acid, 2-cholesteryl octanoic acid, 8-cholesteryl octanoic acid, cholesteryl bromoacetate, fatty acids with 8 to 20 carbon atoms (such as octadecanoic acid), dihydro (nerve ) Lipid compounds such as sphingosine and vitamin E.
  • the lipophilic compound is stearyl chloride.
  • the compound is lipopeptide IPB29.
  • Lipopeptide IPB29 is a compound represented by formula (I), X 1 is Ac, X 2 is EAAAK, X 3 is lysine, the lipophilic compound is cholesterol succinic acid monoester, and X 5 is NH 2 .
  • the compound is lipopeptide IPB30.
  • the lipopeptide IPB30 is a compound represented by formula (I), X 1 is Ac, X 2 is EAAAK, X 3 is lysine, the lipophilic compound is stearyl chloride, and X 5 is NH 2 .
  • EAAAK is the form in which n is equal to 1 in (EAAAK)n.
  • the present disclosure also protects a multimer as follows (a1) or (a2) or (a3):
  • the present disclosure also protects the application of any of the above-mentioned compounds or their pharmaceutically acceptable salts or derivatives, which are as follows (b1) or (b2) or (b3) or (b4):
  • the present disclosure also protects a product comprising any of the above-mentioned compounds or pharmaceutically acceptable salts or derivatives thereof; the function of the product is as follows (c1) or (c2):
  • linker polypeptides in the preparation of products that enhance the antiviral activity and/or stability of viral membrane fusion inhibitors; the amino acid sequence of the linker polypeptides is (EAAAK)n or A[(EAAAK)n]A ; n is a natural number below 5.
  • the disclosure also protects a method for enhancing the antiviral activity and/or stability of a viral membrane fusion inhibitor, comprising the steps of: linking a linker polypeptide to a viral membrane fusion inhibitor; the amino acid sequence of the linker polypeptide is (EAAAK)n or A[(EAAAK)n]A; n is a natural number below 5.
  • the present disclosure also protects a method for preparing a modified viral membrane fusion inhibitor, comprising the following steps (d1) or (d2):
  • (d1) linker polypeptide with amino acid sequence (EAAAK)n or A[(EAAAK)n]A is used as connecting arm to connect virus membrane fusion inhibitor before transformation and X 3 (X 4 ) group to obtain lipopeptide;
  • X 3 is lysine or cysteine or 2,3-diaminopropionic acid (Dap) or ornithine (Orn) or 2,4 -diaminobutyric acid (Dab) or 2,7-diaminoheptanoic acid (Dah);
  • X 4 is a lipophilic compound group;
  • n is a natural number below 5;
  • linker polypeptide with amino acid sequence (EAAAK)n or A[(EAAAK)n]A is used as a connecting arm to connect the virus membrane fusion inhibitor and X4 group before transformation to obtain lipopeptide;
  • X4 is lipophilic Compound group, modified on K in the linker polypeptide;
  • n is a natural number below 5;
  • the lipopeptide is the transformed viral membrane fusion inhibitor.
  • the present disclosure also protects a modified viral membrane fusion inhibitor, which is (e1) or (e2):
  • (e1) a lipopeptide obtained by using a linker polypeptide with an amino acid sequence of (EAAAK)n or A[(EAAAK)n]A as a tether to connect the virus membrane fusion inhibitor before transformation and the X 3 (X 4 ) group;
  • X 4 is modified on X 3 ;
  • X 3 is lysine or cysteine or 2,3-diaminopropionic acid (Dap) or ornithine (Orn) or 2,4 -diaminobutyric acid (Dab) or 2,7-diaminoheptanoic acid (Dah);
  • X 4 is a lipophilic compound group;
  • n is a natural number below 5;
  • a linker polypeptide with an amino acid sequence of (EAAAK)n or A[(EAAAK)n]A is used as a connecting arm to connect the virus membrane fusion inhibitor before transformation and the lipopeptide obtained by the X4 group;
  • X4 is lipophilic Compound group, modified on K in the linker polypeptide;
  • n is a natural number below 5;
  • the lipopeptide is the transformed viral membrane fusion inhibitor.
  • the present disclosure also protects a pharmaceutical compound, which is any of the above-mentioned compounds or pharmaceutically acceptable salts or derivatives thereof, or any of the above-mentioned multimers.
  • the use of the pharmaceutical compound is as follows (f1) or (f2) or (f3) or (f4) or (f5) or (f6):
  • the present disclosure also protects a method for treating or/and preventing coronavirus-infected animals, comprising administering the pharmaceutical compound to recipient animals to inhibit coronavirus-infected animals.
  • Any of the above X 2 is a rigid joint.
  • linker polypeptides described above is a rigid linker polypeptide.
  • Any of the above X 2 is a rigid linker with an ⁇ -helical structure.
  • linker polypeptides described above is a rigid linker polypeptide with an ⁇ -helical structure.
  • n can be 1 or 2 or 3 or 4 or 5.
  • Any of the above X2 functions as a connecting arm, which can significantly increase the ⁇ -helical structure, thereby improving the stability and antiviral activity of the compound or its pharmaceutically acceptable salt or derivative thereof.
  • linker polypeptides described above acts as a connecting arm, which can significantly increase the ⁇ -helical structure, thereby improving the stability and antiviral activity of the viral membrane fusion inhibitor.
  • the lipid compound is linked through the amino group of its side chain.
  • lipid compound When X3 is cysteine, the lipid compound is linked through the sulfhydryl group of its side chain.
  • the preferred lipophilic compound is cholesterol succinate monoester attached to the side chain of lysine by amidation reaction.
  • the preferred lipophilic compound is cholesteryl bromoacetate.
  • amino acids have well-known meanings in the art, for example, S is serine, V is valine, N is asparagine, I is isoleucine, Q is glutamine , K is lysine, E is glutamic acid, D is aspartic acid, R is arginine, L is leucine, A is alanine, G is glycine, Y is tyrosine, C is Cysteine etc.
  • the amino acid may be an L-form amino acid.
  • One or more (such as 2-5, 2-4 or 2-3) amino acids in the polypeptide can also be replaced by amino acids with a D-type conformation, artificially modified amino acids, rare amino acids that exist in nature, etc., to Improve the bioavailability, stability and/or antiviral activity of the polypeptide.
  • D-type amino acids refer to amino acids corresponding to L-type amino acids constituting proteins.
  • Artificially modified amino acids refer to the common L-type amino acids that make up proteins after modification such as methylation and phosphorylation.
  • Rare amino acids that exist in nature include uncommon amino acids that make up proteins and amino acids that do not make up proteins, such as 5-hydroxylysine, methylhistidine, ⁇ -aminobutyric acid, homoserine, etc.
  • the present disclosure provides a new method for designing a lipopeptide which is a virus membrane fusion inhibitor.
  • the lipopeptide inhibitor prepared by this method or strategy has significantly increased helical structure characteristics and significantly increased antiviral activity.
  • the pharmaceutically acceptable salt, solvate, chelate or non-covalent complex formed by the compound, the drug prodrug based on the compound, or any mixture of the above forms are also the content of the present disclosure.
  • the present disclosure provides methods for preventing and treating diseases caused by coronaviruses comprising the compounds of the present disclosure.
  • the present disclosure also provides a pharmaceutical composition comprising the compound according to the present disclosure for the preparation of prevention and treatment of diseases caused by coronavirus.
  • the pharmaceutical composition is used in the prevention and treatment of diseases caused by coronaviruses.
  • coronaviruses described in the present disclosure include, but are not limited to, various coronaviruses embodied in the examples.
  • SARS-CoV-2 SARS-CoV-2 wild strain and its various mutant strains, the complete genome sequence of the wild strain can be found in GenBank: MN908947.3, and the mutant strains can be alpha, beta, gamma Strains such as horse, delta and Omicron), SARS-CoV, MERS-CoV, and other human coronaviruses (such as HCoV-229E, HCoV-OC43 and HCoV-NL63, etc.).
  • the compounds include different optical isomers, racemates and/or mixtures thereof.
  • a single enantiomer or diastereoisomer such as an optically active isomer, can be obtained by asymmetric synthesis or racemate resolution.
  • the resolution of the racemate can be achieved by different methods, such as conventional recrystallization with resolution aids, or chromatography.
  • the compounds include different cis and/or trans isomers with double bonds.
  • the derivative of the compound may be a solvate of the compound, a complex of the compound, a chelate of the compound or a non-covalent complex of the compound.
  • a derivative of a compound may also be a prodrug based on the compound (eg, an ester or amide derivative of the compound).
  • any form of mixture of the above compounds may be contained.
  • the modified virus membrane fusion inhibitor prepared by the disclosed method has significantly increased helical structure characteristics, thereby significantly increasing antiviral activity and/or stability.
  • the chemical structural formula is used to define the compound exactly.
  • the compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optically active Enantiomers or diastereomers.
  • any chemical structure within the scope of the description herein, whether it contains the above-mentioned similar structures in part or in the whole structure, includes all possible enantiomers and diastereoisomers of this compound, including Any single stereoisomer (such as a single geometric isomer, a single enantiomer or a single diastereoisomer) and any mixture of these isomers are contemplated.
  • the mixture of these racemic isomers and stereoisomers can also be further resolved into enantiomers or stereoisomers of their constituents by those skilled in the art using chiral separation techniques or chiral molecular synthesis methods .
  • the disclosed drug can be directly administered to the patient, or mixed with a suitable carrier or excipient and then administered to the patient, so as to achieve the purpose of treating and/or preventing coronavirus infection.
  • the carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), insoluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials Materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Of these, water-soluble carrier materials are preferred.
  • These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Buccal tablets, suppositories, freeze-dried powder injections, etc. It can be common preparations, sustained-release preparations, controlled-release preparations and various microparticle drug delivery systems. Various carriers known in the art can be widely used for tableting unit dosage forms.
  • carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid Aluminum, etc.; wetting agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia mucilage, gelatin paste, sodium carboxymethylcellulose , shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, Sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors,
  • Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.
  • Various carriers known in the art can be widely used for pelletizing the unit dosage form.
  • carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin , ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecylsulfonate, methylcellulose, ethylcellulose, etc.
  • Various carriers known in the art can be widely used for formulating the unit dosage form into a suppository.
  • carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like.
  • all diluents commonly used in this field can be used, for example, water, ethanol, polyethylene glycol, 1, 3-Propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like.
  • an appropriate amount of sodium chloride, glucose or glycerin can be added to the preparation for injection, and in addition, conventional solubilizers, buffers, pH regulators, etc. can also be added.
  • colorants, preservatives, fragrances, correctives, sweeteners or other materials can also be added to the pharmaceutical preparations, if necessary.
  • the above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal administration; respiratory tract administration, such as nasal cavity; mucosal administration.
  • the above route of administration is preferably injection, atomized inhalation, nasal spray or nasal drop administration.
  • the dosage of the drug of the present disclosure depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight and individual response of the patient or animal, the specific active ingredient used, the route of administration and the times etc.
  • the above dose can be administered in a single dose or divided into several, eg two, three or four doses.
  • the disclosed drug can be directly used for the treatment and prevention of coronavirus infection alone, and can also be used in combination with one or more other antiviral drugs to achieve the purpose of improving the overall therapeutic effect.
  • antiviral drugs include, but are not limited to, neutralizing antibodies, protease inhibitors, RNA-dependent RNA polymerase (RdRp) inhibitors, virus entry inhibitors, and the like.
  • the above-mentioned neutralizing antibody can be Ambavirumab (BRII-196), Romisvirumab (BRII-198), Casirivimab (Casirivimab), Idevimab (Imdevimab), Solovir One or more monoclonal antibodies (Sotrovimab), bamlanivimab (Bamlanivimab), etc.;
  • the protease inhibitors can be paxlovid, darunavir, lopinavir /Ritonavir (Lopinavir/Ritonavir), etc.
  • the RdRp inhibitor can be Monupiravir (Molnupiravir), Favipiravir (Favipiravir), Remdesivir (Remdesivir), One or more of Sofosbuvir and the like;
  • the virus entry inhibitor can be one or more of Arbidol, hydroxychloroquine and the like.
  • the specific therapeutically effective dosage level will depend on a number of factors, including the disorder being treated and the severity of the disorder; the activity of the particular active ingredient employed; the particular composition employed. ; patient's age, weight, general health, sex and diet; time of administration, route of administration and excretion rate of the specific active ingredient employed; duration of treatment; Drugs; and similar factors well known in the medical arts. For example, it is practice in the art to start dosages of the active ingredient at levels lower than that required to obtain the desired therapeutic effect and to gradually increase the dosage until the desired effect is obtained.
  • the inventors of the present disclosure have found for the first time that compounds of formula (I) IPB29 and IPB30, especially IPB29, with a rigid linker EAAAK, have at least 70-fold increased activity against the new coronavirus compared to compounds with a flexible linker. (eg, PEG or GSGSG) had an approximately 8-fold increase in inhibitory activity.
  • lipopeptide IPB29 the reason for the increased activity of lipopeptide IPB29 is that the rigid linker EAAAK significantly increased the helical content of this lipopeptide while improving its stability. Moreover, this lipopeptide IPB29 can inhibit many different types of new coronaviruses.
  • the disclosed compound is stable in nature and is a highly efficient and broad-spectrum novel coronavirus membrane fusion inhibitor, which is used to prepare a pharmaceutical composition for preventing and treating diseases caused by coronavirus, and the pharmaceutical composition is used for preventing and treating coronavirus Diseases caused by viruses.
  • Figure 1 shows the ⁇ -helix content (left panel) and thermal stability (right panel) of lipopeptides as viral membrane fusion inhibitors.
  • Figure 2 shows the ⁇ -helix content (left panel) and thermal stability (right panel) of the complex of lipopeptide and target sequence polypeptide as a virus membrane fusion inhibitor.
  • FIG 3 shows the activity of lipopeptides in inhibiting SARS-CoV-2 infection of 293T/ACE2 cells (left panel) or Huh-7 cells (right panel).
  • Figure 4 shows the inhibitory activity of lipopeptides on 293T/ACE2 cells infected by various SARS-CoV-2 mutant strains.
  • Figure 5 shows the inhibitory activity of lipopeptides on Huh-7 cells infected by various SARS-CoV-2 mutant strains.
  • Figure 6 shows the activity of lipopeptides to inhibit other coronaviruses.
  • Figure 7 is the in vitro cytotoxicity test of lipopeptides.
  • Figure 8 is the stability analysis of lipopeptides IBP24 and IPB29.
  • Figure 9 shows the inhibitory effect of novel lipopeptides on cell membrane fusion mediated by the S protein of SARS-CoV-2.
  • the target cells in the left picture are 293T/ACE2 cells, and the target cells in the right picture are Huh-7 cells.
  • the disclosed scientific research team has been committed to the research and development of viral membrane fusion inhibitor drugs, and designed a lipopeptide-based broad-spectrum coronavirus membrane fusion inhibitor, which has strong inhibitory activity against SARS-CoV-2 and its mutant strains ( References 2-5).
  • a linker between the polypeptide sequence and the lipid group (such as fatty acid and cholesterol, etc.) to act as a linking arm.
  • the expected binding sites for lipid groups are viral or cell membranes, while peptides are enriched in the target region, so the binding sites for lipid groups and peptides are different. Since the polypeptide tends to form a stable secondary structure and has strong structural rigidity, a flexible linker is usually used to connect the polypeptide and the lipid group, so that the polypeptide and the lipid group can respectively form a suitable conformation , bound to their respective binding sites.
  • Common flexible linkers have a combination of glycine (G) and serine (S), such as (GGGGS)n or (GSGSG)n, etc., by changing the size of n, the distance between the domains can be enlarged and reduced; another common
  • the flexible linker is a small molecule polyethylene glycol (PEG) n, where n is mostly between 2 and 24.
  • the lipopeptides of coronavirus membrane fusion inhibitors reported in the literature all use flexible linkers, such as IBP02V1 ⁇ IBP02V5 are all PEG8 (reference 2), IPB24 ⁇ IPB27 are PEG 4 , PEG 5 , PEG 6 and PEG 8 respectively (reference Document 4), EKL1C is GSG (Reference 6), EK1C4 is GSGSG and PEG 4 in series (Reference 7), [SARS HRC -PEG 4 ] 2 -chol is PEG 4 (Reference 8), etc.
  • the common rigid linker (rigid linker) for the preparation of fusion proteins has the (EAAAK) n sequence capable of forming an ⁇ -helix, which has internal hydrogen bonds and a closely linked peptide chain backbone, which is rigid and stable; another type of rigid linker has Pro- rich sequence (XP)n, where X can specify any amino acid, preferably alanine, lysine or glutamic acid, (XP)n sequence has no helical structure, but where proline can increase the backbone stiffness and effectively separate the structure area.
  • EAAAK Pro- rich sequence
  • Embodiment 1 the preparation of lipopeptide
  • lipopeptides were prepared respectively: lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB20, lipopeptide IPB24, and lipopeptide IPB28.
  • lipopeptide IPB29 and lipopeptide IPB30 have EAAAK (SEQ ID NO: 4) rigid linker.
  • the amino acid parts of lipopeptide IPB29 and lipopeptide IPB30 are shown in SEQ ID NO:1.
  • SEQ ID NO: 1 SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAKK.
  • Lipopeptide IPB20 (which does not have a linker as a connecting arm), lipopeptide IPB24 (which has a flexible linker PEG 4 as a connecting arm, derived from Fmoc-NH-PEG 4 -CH 2 CH 2 COOH as a synthetic raw material), lipopeptide IPB28 (with a flexible linker GSGSG (SEQ ID NO: 5) as a tether) was used as a control.
  • lipopeptide IPB29 and lipopeptide IPB30 satisfy the following general formula:
  • Lipopeptide IPB29 Lipopeptide IPB29, X 1 is the amino terminal protecting group Ac, X 2 is the EAAAK rigid linker, X 3 is the lysine residue, X 4 is the cholesterol succinic acid monoester group modified on X 3 , X 5 is Carboxy-terminal protecting group NH 2 .
  • Lipopeptide IPB30 Lipopeptide IPB30, X 1 is the amino-terminal protecting group Ac, X 2 is the EAAAK rigid linker, X 3 is the lysine residue, X 4 is the stearoyl chloride group modified on X 3 , X 5 is the carboxyl terminal Protecting group NH 2 .
  • the sequence structures of the five lipopeptides are shown in Table 1.
  • Ac represents an acetyl group
  • NH 2 represents an amino group
  • EAAAK represents a short peptide with an amino acid sequence of EAAAK.
  • the protected amino acid raw materials used in the peptide synthesis process include Fmoc-Lys(Dde)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc -Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu) -OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH.
  • Fmoc is 9-fluorenylmethoxycarbonyl
  • Dde is 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl
  • Boc is tert-butoxy Carbonyl
  • tBu is tert-butyl
  • OtBu is tert-butoxy
  • Trt is trityl
  • Pbf is (2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran- 5-yl)sulfonyl.
  • Rink Amide MBHA resin as the carrier resin, through de-Fmoc protection and coupling reaction, sequentially coupled with the corresponding protected amino acids of the polypeptide amino acid sequence to prepare the peptide resin.
  • the activated first protected amino acid solution was added to the Fmoc-depleted resin, the coupling reaction was carried out for 60 minutes, and the resin was filtered and washed to obtain the resin containing the first protected amino acid Fmoc-Lys(Dde).
  • cleavage reagent cleavage reagent 15mL/g resin
  • cleavage reagent 15mL/g resin cleavage reagent 15mL/g resin
  • the filtrate of the reaction mixture was collected, and the resin was washed three times with a small amount of TFA/DCM.
  • anhydrous ether was added to precipitate, and centrifuged.
  • the filter cake was washed twice with cold anhydrous diethyl ether and dried to obtain a off-white powder which was the crude lipopeptide.
  • the chemical structures of the lipopeptides in Table 1 were characterized by MALDI-TOF mass spectrometry, and their purity was given by analytical high-performance liquid chromatography (Agela C18-4.6 ⁇ 250mm, flow rate 1mL per minute). The results showed that the purity of the synthesized lipopeptides was greater than 95%.
  • the secondary structure ( ⁇ -helix) and thermal stability of the test lipopeptide and the interaction between the test lipopeptide and the target sequence mimic polypeptide were determined by circular dichroism (CD) technology, and the specific method refers to the published by the inventor. Papers (References 4 and 5).
  • the target sequence mimics the polypeptide N52, which is derived from the HR1 sequence of the S2 subunit of the SARS-CoV-2 spike protein.
  • the sequence structure of N52 is as follows:
  • lipopeptide to be tested lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB20, lipopeptide IPB24 or lipopeptide IPB28 prepared in Example 1.
  • Test complex a mixture of test lipopeptide and N52.
  • test lipopeptide or the test complex
  • 10 ⁇ M refers to the test lipopeptide and N52
  • concentrations are all 10 ⁇ M
  • step 2 2. Move the solution obtained in step 1 to the corresponding cuvette, and use a Jasco spectropolarimeter (model J-815) to scan the change of the molar ellipticity [ ⁇ ] ⁇ of the solution in the wavelength range of 195-270nm, typical ⁇ -helix
  • the structure can have the largest negative peaks at 208nm and 222nm.
  • the peak value is -33000degree.cm2.dmol-1 as the standard of 100% ⁇ -helix content.
  • the molar ellipticity at is calculated as a percentage of ⁇ -helical content.
  • step 3 Add the solution obtained in step 1 into the thermal stability detection cuvette, and adjust the CD temperature control module to scan the change of solution [ ⁇ ] 222 with temperature at a speed of 2°C per minute at 20-98°C. The melting curve was smoothed, and the midpoint temperature (Tm) of the thermal dissociation transition was calculated by Origin software to reflect the degree of thermal stability of the helix.
  • the CD results of the tested lipopeptides are shown in Figure 1.
  • the ⁇ -helix content of IPB20 without flexible or rigid linker was 51%; the ⁇ -helix content of IPB24 and IPB28 introduced with flexible linker was 19% and 20%, respectively, showing that the introduction of flexible linker improved the overall rotation freedom of lipopeptides degree, which increases the overall molecular entropy of the lipopeptide, which is unfavorable for stabilizing the secondary structure of the peptide;
  • the ⁇ -helix content of IPB29 and IPB30 introduced into the rigid linker is 74% and 62%, respectively, indicating that the introduction of the EAAAK sequence significantly increases the helix of the lipopeptide
  • the structure may be due to the conjugation of the E-K salt bridge in the EAAAK linker to the salt bridge in the ⁇ -helix secondary structure of the polypeptide, which stabilizes its secondary structure, and A is more inclined to form an ⁇ -helix structure than G.
  • the relatively high content of its own ⁇ -helix can effectively reduce the binding entropy between the polypeptide part of the lipopeptide and the target, increase the binding constant, and improve the activity; the relatively stable secondary structure is also conducive to the protease hydrolysis of the polypeptide antibody, improving the stability in vivo , is also beneficial for antiviral activity.
  • Each lipopeptide can interact with the target sequence mimic polypeptide to form a complex, which has a typical ⁇ -helix structure, in which the ⁇ -helix content of the IPB20-N52 complex, IPB24-N52 complex and IPB28-N52 complex is 66%, 48% and 37%, and the ⁇ -helical content of the IPB29-N52 complex and IPB30-N52 complex were 68% and 53%, respectively (left panel of Figure 2).
  • the Tm values of IPB20-N52 complex, IPB24-N52 complex and IPB28-N52 complex were 90 °C, 90 °C and 89 °C, respectively, and the Tm values of IPB29-N52 complex and IPB30-N52 complex were 86 °C and 78°C (right panel of Figure 2). This shows that the lipopeptide introduced into the EAAAK linker can maintain specific and stable binding to the target while increasing the ability to bind to cell membranes and viral membranes.
  • Embodiment 3 the inhibitory effect of lipopeptide to new coronavirus SARS-CoV-2 and its mutant
  • 293T cells are products of the American Type Culture Collection (ATCC, catalog number CRL-3216).
  • Huh-7 cells are products of the National Experimental Cell Resource Sharing Service Platform.
  • 293T/ACE2 cells are described in reference 2.
  • Test cells 293T/ACE2 cells or Huh-7 cells.
  • lipopeptide to be tested lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB20, lipopeptide IPB24 or lipopeptide IPB28 prepared in Example 1.
  • a plasmid expressing the S protein of SARS-CoV-2 (defined as pCoV2-S), which is described in "a plasma expressing the S protein of SARS” in the "Single-cycle infection assay” of the Materials and Methods section of reference 5 -CoV-2".
  • the HIV backbone plasmid pNL4-3.luc.RE was provided by the AIDS Reagent and Reference Items Program of the National Institutes of Health (Cat. No. 3418).
  • SARS-CoV-2 pseudovirus SARS-CoV-2 pseudovirus (SARS-CoV-2PV, hereinafter referred to as SARS-CoV-2WT)" in reference 2.
  • each Wells contained 50 ⁇ L of lipopeptide solution. Set up 9 dilutions, and each dilution has 3 replicate wells. Set up control wells to which DMEM medium (50 ⁇ L per well) was added.
  • step (1) After completing step (1), add 50 ⁇ L (500 TCID 50 of virus) of the pseudovirus prepared in step 1 to each well, and then incubate at room temperature for 30 minutes.
  • step (3) Adjust the pre-cultured test cells to a cell suspension with a concentration of 10 ⁇ 10 4 cells/mL, add DEAE-dextran to a concentration of 15 ⁇ g/mL, and then add it to the 96 wells that completed step (2) plate (100 ⁇ L/well), and cultured in a 37° C., 5% CO 2 cell incubator for 48 hours.
  • step (3) discard the supernatant, then add cell lysate at 30 ⁇ L/well, lyse at room temperature for 15 minutes, then add luciferase substrate (Promega), and use a microplate chemiluminescence detector The relative fluorescence unit (RLU) was measured, and the inhibition rate curve and half inhibitory concentration (IC 50 ) of the drug were calculated.
  • RLU relative fluorescence unit
  • the results are shown in Figure 3.
  • the IC 50 values of IPB20, IBP24, IBP28, IBP29 and IBP30 for inhibiting SARS-CoV-2 infection in 293T/ACE2 cells were 63.85nM, 5.51nM, 6.91nM, 0.57nM and 4.19nM;
  • IPB20, IBP24, IBP28, IBP29 and IBP30 The IC 50 values for inhibiting SARS-CoV-2 infection of Huh-7 cells were 39nM, 2.43nM, 2.88nM, 0.53nM and 2.77nM, respectively.
  • IPB29 and IPB20 The difference between IPB29 and IPB20 is that IPB29 contains a rigid linker EAAAK, and IPB20 has no rigid or flexible linker; compared with IPB20, the antiviral activity of IPB29 in 293T/ACE2 cells increased by about 112 times, and the antiviral activity of IPB29 in Huh-7 cells The activity was increased about 74-fold.
  • IPB29 and IPB24 The difference between IPB29 and IPB24 is that IPB29 contains a rigid linker EAAAK, and IPB24 contains a flexible linker PEG 4 ; compared with IPB24, the antiviral activity of IPB29 in 293T/ACE2 cells increased by about 10 times, and the antiviral activity of IPB29 in Huh-7 cells The activity was increased about 5-fold.
  • IPB29 contains a rigid linker EAAAK
  • IPB28 contains a flexible linker GSGSG
  • the antiviral activity of IPB29 in 293T/ACE2 cells increased by about 12 times
  • the antiviral activity of IPB29 in Huh-7 cells improved by about 5 times.
  • the experimental results showed that the EAAAK linker significantly enhanced the inhibitory activity of lipopeptides as inhibitors, such as the activity of IPB29 and IPB30 against SARS-CoV-2.
  • lipopeptide IPB29 lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB24 or lipopeptide IPB28 prepared in Example 1.
  • step 1 of step 1 For the method, see step 1 of step 1. The only difference is that the plasmid expressing the S protein of SARS-CoV-2 is replaced by a plasmid expressing the S protein of a mutant (single point mutation or representative epidemic strain) of SARS-CoV-2.
  • the prepared pseudovirus of the D614G mutant of SARS-CoV-2 is the "D614G PV" in reference 2.
  • Step 2 of Step 1 The method is the same as Step 2 of Step 1.
  • the IC 50 values of IPB24, IPB28, IPB29 and IPB30 against Omicron strains were 4.51nM, 4.51nM, 0.47nM and 1.78nM, respectively, and the IC 50 values of IPB24, IPB28 and IPB30 were about that of IPB29 10x, 10x and 4x.
  • the results when the tested cells are Huh-7 cells are shown in Figure 5.
  • the IC 50 values of IPB24, IPB28, IPB29 and IPB30 for inhibiting Delta strain infection were 3.46nM, 4.34nM, 0.56nM and 2.17nM, respectively, and the IC 50 values of IPB24, IPB28 and IPB30 were about IPB29 6 times, 8 times and 4 times.
  • the IC 50 values of IPB24, IPB28, IPB29 and IPB30 for inhibiting the infection of Omicron strains were 2.56nM, 2.46nM, 0.46nM and 1.46nM, respectively, and the IC 50 values of IPB24, IPB28 and IPB30 were about IPB29 6 times, 5 times and 3 times.
  • novel lipopeptides such as IPB29 and IPB30
  • viral membrane fusion inhibitors had good inhibitory activity against various mutants (mutant strains) of SARS-CoV-2.
  • Embodiment 4 the inhibitory effect of lipopeptide to other coronaviruses
  • lipopeptide IPB29 lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB24 or lipopeptide IPB28 prepared in Example 1.
  • Test cells 293T/ACE2 cells or Huh-7 cells.
  • coronavirus pseudoviruses prepared various other coronavirus pseudoviruses respectively, and other coronaviruses are specifically as follows: bat-derived coronavirus (bat RaTG13), pangolin-derived coronavirus (PCoV-GD or PCoV-GX), SARS-CoV, MERS-CoV, HCoV - NL63 and HCoV-229E.
  • Step 1 of Example 3 For the method, refer to 1 of Step 1 of Example 3. The only difference is that the plasmid expressing the S protein of SARS-CoV-2 is replaced by a plasmid expressing the S protein of other coronaviruses.
  • the prepared SARS-CoV pseudovirus is "SARS-CoV PV" in reference 2.
  • the prepared MERS-CoV pseudovirus is "MERS-CoV PV" in reference 2.
  • the prepared HCoV-NL63 pseudovirus is "HCoV-NL63PV" in reference 2.
  • the prepared HCoV-229E pseudovirus is "HCoV-229E PV" in reference 2.
  • the method is the same as step 2 of embodiment 3.
  • the results are shown in Figure 6. The results showed that the novel lipopeptide could effectively inhibit the infection of the above seven viruses.
  • the IC 50 values of IPB24, IPB28 and IPB30 for inhibiting the infection of bat RaTG13 strain, PCoV-GD strain and PCoV-GX strain were about 4 times, 6 times and 5 times higher than that of IPB29, respectively.
  • the IC50 values of IPB24, IPB28 and IPB30 for inhibiting the infection of SARS-CoV strains, MERS-CoV strains, HCoV-NL63 strains and HCoV-229E strains were about 11 times higher than those of IPB29. , 7x, 9x, and 7x.
  • lipopeptides IPB29 and IPB30 have strong inhibitory effects on other coronaviruses, especially IPB29; compared with other lipopeptides, IPB29 has a strong inhibitory effect on SARS-CoV, PCoV-GD and PCoV which are closely related to SARS-CoV-2. -GX still showed the strongest inhibitory activity.
  • lipopeptide IPB29 lipopeptide IPB29, lipopeptide IPB30, lipopeptide IPB24 or lipopeptide IPB28 prepared in Example 1.
  • Test cells 293T/ACE2 cells or Huh-7 cells.
  • the in vitro cytotoxicity detection of the tested lipopeptide was carried out using CCK-8 cell proliferation/toxicity detection kit (manufacturer: Abbkine, article number: KTC011001). Specific steps: (1) Dilute the lipopeptide to be tested in a 3-fold gradient in a 96-well cell culture plate, and finally, each well contains 100 ⁇ L lipopeptide solution; set 9 dilutions, and each dilution has 3 replicate wells .
  • step (2) Set up a control well with DMEM medium (100 ⁇ L per well); (2) Add 10 ⁇ 104 cells/mL of the test cell suspension to the 96-well cell culture plate that completed step (1), 100 ⁇ L/well , cultivated at 37°C and 5% CO 2 for 48 hours; (3) After completing step (2), add 20 ⁇ L of CCK-8 solution to each well, continue to incubate the culture plate in the incubator for 2 hours, and then use a microplate reader to Absorbance at 450 nm (OD450) was measured. GraphPad Prism software was used to create inhibition rate curves and calculate the half cytotoxic drug concentration (CC 50 ).
  • the results are shown in Figure 7.
  • the CC 50 values of IBP24, IBP28, IBP29 and IBP30 for 293T/ACE2 cells were 14.36 ⁇ M, 12.28 ⁇ M, 23.94 ⁇ M and 45.46 ⁇ M, and the CC 50 values for Huh-7 cells were 15.02 ⁇ M and 15.97 ⁇ M, 22.75 ⁇ M and 44.43 ⁇ M.
  • IBP29 and IBP30 have relatively low cytotoxicity, especially IPB30 modified with stearyl chloride group.
  • IC 50 data is the data obtained in Example 3
  • the four polypeptides all have extremely high selectivity therapeutic index (TI).
  • TI selectivity therapeutic index
  • the TI values of IPB24, IPB28, IPB29 and IPB30 were as high as about 3184, 2723, 50936 and 25539, respectively, and the TI values of IPB29 were about 16 of IPB24, IPB28 and IPB30, respectively. times, 19 times and 2 times.
  • the TI values of IPB24, IPB28, IPB29 and IPB30 were as high as about 5867, 6492, 49457 and 30432, respectively, and the TI values of IPB29 were about 8 times that of IPB24, IPB28 and IPB30. times, 8 times and 1.6 times. It can be seen that IBP29 and IBP30 have higher therapeutic index than IBP24 and IBP28, so they are more druggable.
  • Embodiment 6 the stability research of lipopeptide inhibitor
  • the inventors compared and analyzed the stability of representative lipopeptides IPB24 and IPB29 from multiple perspectives, including digestion with protease, digestion with liver microsomes, incubation with human serum, and long-term storage at 37°C.
  • Lipopeptides to be tested lipopeptide IPB29 and lipopeptide IPB24 prepared in Example 1.
  • the method for detecting the antiviral activity of the lipopeptide is the same as Step 1 of Example 3 (test cells: 293T/ACE2 cells).
  • Proteinase K, trypsin and ⁇ -chymotrypsin were all purchased from Sigma-Aldrich with product numbers P2308, T4799 and C4129, respectively.
  • the lipopeptide and the protease to be tested were mixed at final concentrations of 2 mg/mL and 0.1 mg/mL respectively, incubated at 37°C for 0, 30, 60, 120 or 180 minutes, and then the antiviral activity of the lipopeptide was detected.
  • Phase I Metabolic Stability Kit Human liver microsome (mixed) reagent was purchased from Beijing Huizhi Taikang Pharmaceutical Technology Co., Ltd., the catalog number is 0111A1.03. The experimental method was carried out according to the instructions provided by the manufacturer. First, mix 10 ⁇ L of solution A, 2 ⁇ L of solution B and 28 ⁇ L of 0.1M PBS buffer in the kit evenly, pre-incubate at 37°C for 5 minutes, then dispense 40 ⁇ L/tube, warm in a 37°C water bath, and prepare the pre-incubation solution for later use.
  • the tested lipopeptides containing 20% human serum and a final concentration of 150 ⁇ M were mixed, incubated at 37° C. for 0, 5, 30, 60, 120 or 180 minutes, and then the antiviral activity of the lipopeptides was detected.
  • the tested lipopeptide aqueous solution with a concentration of 300 ⁇ M was placed at 37° C. for different times, and then the change of its antiviral activity was detected.
  • IPB24 and IPB29 are more sensitive to the treatment of 20% human serum, especially the antiviral activity of IPB24 decreased by about 24 times after incubation for 5 minutes; after incubation for 30, 60, 120 and 180 minutes, the antiviral activity of IPB24 decreased respectively About 32, 36, 37 and 42 times.
  • IPB29 showed significantly improved resistance to human serum, and its antiviral activity decreased by about 4, 10, 12, 13 and 13 times at the above-mentioned time points from 5 to 180 minutes, respectively.
  • the antiviral activity of IPB24 decreased gradually with time after being stored at 37°C for 3, 7, 14, 21 and 28 days, especially after 28 days, it decreased by more than 5 times. In comparison, the antiviral activity of IPB29 did not change or was much smaller under the same storage conditions. The longer-term temperature stability of lipopeptides is for further study.
  • IPB29 showed obvious stability advantage over IPB24, and the stability was greatly enhanced, which further supported the better druggability of IPB29 helical lipopeptide carrying EAAAK sequence.
  • Example 7 Inhibitory effect of novel lipopeptide on cell-cell membrane fusion mediated by S protein of SARS-CoV-2
  • Lipopeptides to be tested lipopeptide IPB29, lipopeptide IPB30, and lipopeptide IPB20 prepared in Example 1.
  • Test cells 293T/ACE2 cells, Huh-7 cells.
  • the present disclosure conducted a cell-cell fusion inhibition experiment based on the DSP system. For specific methods, see the Cell-cell fusion assay section in references 4 and 5. Proceed as follows:
  • polypeptide was diluted in a 3-fold gradient in a 96-well plate, and 3 duplicate wells and 9 dilution gradients were set. Add the diluted polypeptide to the effector cells and incubate for 1 hour at 37°C, 5% CO2 in a cell incubator.
  • Pan-coronavirus fusion inhibitors possess potent inhibitory activity against HIV-1, HIV-2, and simian immunodeficiency virus. Emerg Microbes Infect 10: 810-821.

Abstract

提供了一种广谱抗新冠病毒脂肽、其制备方法、包含该脂肽的病毒膜融合抑制剂以及该在脂肽在制备预防和治疗冠状病毒所致疾病的药物组合物中的用途。该脂肽的结构如式（I）或式（II）所示，其中X1位氨基保护基团，X2为多肽，氨基酸序列为(EAAAK)n或A[(EAAAK)n]A，n为5以下的自然数，表示EAAAK序列的重复次数，X3为赖氨酸或半胱氨酸或2,3-二氨基丙酸或鸟氨酸或2,4-二氨基丁酸或2,7-二氨基庚酸，X4为亲脂性化合物基团，X5为羧基端保护基团。

Description

一种优化病毒膜融合抑制剂的方法及广谱抗冠状病毒脂肽和应用 技术领域
本公开涉及一种优化病毒膜融合抑制剂的方法及广谱抗冠状病毒脂肽和应用。
背景技术
膜融合是一极其重要的生物学现象，比如受精卵的形成和细胞内囊泡运输等生理过程都是通过膜融合实现的。众多严重危害人类健康的病毒也是通过膜融合感染宿主细胞，如艾滋病病毒(HIV)、流感病毒、肝炎病毒、埃博拉病毒、寨卡病毒、非典(SARS)冠状病毒、中东呼吸综合征(MERS)冠状病毒、以及当前正在肆虐人类的新型冠状病毒(SARS-CoV-2)等等。病毒膜融合是由位于病毒颗粒表面的融合蛋白所介导，如HIV的包膜蛋白gp41亚基和冠状病毒刺突S蛋白的S2亚基。融合蛋白在序列结构上通常依次含有融合肽(FP)、七肽重复域1(HR1)、七肽重复域2(HR2)和跨膜区(TM)等重要功能区。在病毒膜融合过程中，融合蛋白通常发生剧烈的构象变化，首先是FP暴露出来并插入到靶细胞膜，紧接着HR1形成三聚体螺旋，而HR2则反向折叠于HR1三聚体所形成的沟槽中，导致一个典型的六螺旋束结构(6-HB)，从而拉近病毒膜和细胞膜发生融合反应，使病毒的基因物质通过融合孔进入到靶细胞内。研究发现，来源于众多病毒HR1和HR2区域的多肽可以作为病毒膜融合抑制剂，其作用机制就是竞争性地与处于融合前状态的融合蛋白结合，从而阻断6-HB结构的形成。目前，HIV治疗药物T20(恩夫韦肽)是唯一一个美国FDA批准用于临床的病毒膜融合抑制剂，但针对该靶点的抗病毒药物研发一直备受重视。为改善多肽的半衰期和抗病毒活性，基于脂类化合物(如脂肪酸和胆固醇等)修饰的脂肽(lipopeptide)是近年来病毒膜融合抑制剂药物研发的重点方向(参考文献1)。
冠状病毒(CoV)是具有囊膜的单股正链RNA病毒，分为α、β、γ和δ四个属。目前已知感染人类的CoV包括α属的HCoV-229E和HCoV-NL63和β属的HCoV-OC43、CoV-HKU1、SARS-CoV、MERS-CoV和SARS-CoV-2。HCoV-229E、HCoV-NL63、HCoV-OC43和CoV-HKU1是常见的流行病原体，通常只引起普通感冒症状，约占成人上呼吸道感染的10％至30％，但对儿童、老年人和免疫功能低下的患者仍可以造成严重甚至致命的疾病。SARS-CoV、MERS-CoV和SARS-CoV-2则属于高致病病原体，导致严重的肺部疾病，病死率高。SARS-CoV-2与SARS-CoV和蝙蝠冠状病毒SL-CoV-RaTG13分别具有79.5％和96％的序列同源性并使用相同的细胞受体(ACE2)，SARS-CoV-2比SARS-CoV具有更强的传播能力。截至2022年1月底，全球累计报告确诊新冠(COVID-19)病例近3.6亿，其中超过560万例患者死亡(www.who.int)。SARS-CoV-2在流行中不断产生特别令人关注的变异株(VOC)，如阿尔法(Alpha)、贝塔(Beta)、伽马(Gamma)、德尔塔(Delta)和奥密克戎(Omicron)等毒株，往往导致疫苗和药物的效 果下降甚至失去活性。因此，需要研发高效且广谱的冠状病毒抑制剂。
也就是说，本领域存在研发高效且广谱的冠状病毒抑制剂的需求，这种抑制剂能够抑制多种不同类型，包含不同突变的冠状病毒。
发明内容
本公开科研团队一直致力于病毒膜融合抑制剂药物的研究与开发，设计出基于脂肽的广谱冠状病毒膜融合抑制剂，对SARS-CoV-2及其突变株具有较强的抑制活性(参考文献2-5)。
在设计基于脂肽的病毒膜融合抑制剂中，通常需要在多肽序列和脂类基团(如脂肪酸和胆固醇等)之间加入一个接头(linker)起到连接臂的作用。脂类基团的预期结合位点为病毒或细胞膜，而多肽在靶点区富集，因此脂类基团和多肽的结合位点不同。由于多肽倾向于形成稳定的二级结构，具有较强的结构刚性，因此多肽和脂类基团之间通常选用柔性接头(flexible linker)连接，使多肽和脂类基团能够分别形成合适的构象，结合到各自的结合位点。同时，在充分发挥各自的作用的同时，避免因空间位阻等原因而相互影响。常见柔性接头有甘氨酸(G)和丝氨酸(S)的组合，如(GGGGS)n或(GSGSG)n等，通过改变n的大小可以将结构域之间的距离放大和减小；另一常见的柔性接头为小分子聚乙二醇(PEG)n，其中n多在2至24之间。目前文献报道的冠状病毒膜融合抑制剂脂肽也都是采用柔性接头，如IPB02V1～IPB02V5均为PEG 8(参考文献2)，IPB24～IPB27分别为PEG 4、PEG 5、PEG 6和PEG 8(参考文献4)，EKL1C为GSG(参考文献6)，EK1C4为串联的GSGSG和PEG 4(参考文献7)，[SARS HRC-PEG 4] 2-chol为PEG 4(参考文献8)等。制备融合蛋白常见的刚性接头(rigid linker)有能够形成α-螺旋的(EAAAK)n序列，其具有内部氢键和密切联系肽链骨干，刚性且稳定；另一种类型的刚性接头具有Pro-rich序列(XP)n，其中X可指定任何氨基酸，优选丙氨酸，赖氨酸或谷氨酸；(XP)n序列没有螺旋结构，但其中脯氨酸可以增加骨架硬度，并且有效分离结构域。
目前制备的脂肽类病毒膜融合抑制剂尚无采用刚性接头的先例，本公开创造性地使用刚性接头EAAAK序列制备了广谱冠状病毒膜融合抑制剂脂肽，赋予多肽显著的螺旋结构，并显著提高了抑制剂的抗病毒活性和稳定性。
本公开独创性地提供了一种优化病毒膜融合抑制剂的方法及广谱抗冠状病毒脂肽和应用。
本公开首次发现：具有刚性接头EAAAK的具有式I的化合物IPB29和IPB30，尤其是IPB29，相对于没有此接头的化合物，抑制新冠病毒的活性提高了至少70倍，相对于具有柔性接头，例如PEG或者GSGSG的化合物抑制活性提高了大约8倍。
同时，发明人发现脂肽IPB29活性的提高的原因在于刚性接头EAAAK显著增加了此脂肽的螺旋含量，同时提高了稳定性。并且，这种脂肽IPB29能够抑制多种不同类型的新冠病毒，包括但不限于SARS-CoV-2及其各种突变株、SARS-CoV、MERS-CoV、HCoV-229E，HCoV-OC43和HCoV-NL63等冠状病毒。
具体的，以本公开使用具有α-螺旋结构的刚性接头EAAAK序列作为多肽序列和脂类化合物之间的连接臂，制备了新的化合物，其特征具有显著增加的α-螺旋结构、稳定性和抗病毒活性。
本公开提供了化合物或其药用盐或其衍生物；
所述化合物为式(I)所示的化合物或式(Ⅱ)所示的化合物；
Figure PCTCN2022094001-appb-000001
式(I)和式(Ⅱ)中，X 1为氨基端保护基团；
式(I)和式(Ⅱ)中，X 2为多肽，氨基酸序列为(EAAAK)n或A[(EAAAK)n]A；n为5以下的自然数，表示EAAAK序列的重复次数；
式(I)中，X 3为赖氨酸或半胱氨酸或2,3-二氨基丙酸(Dap)或鸟氨酸(Orn)或2,4-二氨基丁酸(Dab)或2,7-二氨基庚酸(Dah)；
式(I)中，X 4为修饰于X 3的亲脂性化合物基团；
式(Ⅱ)中，X 4为修饰于X 2中的K上的亲脂性化合物基团；
式(I)和式(Ⅱ)中，X 5为羧基端保护基团。
示例性的，X 1为乙酰基(Ac)、氨基(NH 2)、马来酰基、琥珀酰基、叔丁氧羰基或苄氧或其他疏水基团或大分子载体基团中的任一基团。
示例性的，X 5为氨基(NH 2)、羧基、羟基、酰胺基或叔丁氧羰基或其他疏水基团或大分子载体基团中的任一基团。
示例性的，所述亲脂性化合物为胆固醇琥珀酸单酯、2-胆固醇乙酸、2-胆固醇丙酸、3-胆固醇丙酸、2-胆固醇丁酸、2-胆固醇异丁酸、3-胆固醇丁酸、3-胆固醇异丁酸、4-胆固醇丁酸、2-胆固醇戊酸、2-胆固醇异戊酸、3-胆固醇戊酸、5-胆固醇戊酸、2-胆固醇己酸、6-胆固醇己酸、2-胆固醇庚酸、7-胆固醇庚酸、2-胆固醇辛酸、8-胆固醇辛酸、溴乙酸胆固醇酯、含8到20个碳原子的脂肪酸(如十八烷酸)、二氢(神经)鞘氨醇、维生素E等脂类化合物。
示例性的，所述亲脂性化合物为硬脂酰氯。
示例性的，所述化合物为脂肽IPB29。脂肽IPB29为式(I)所示化合物，X 1为Ac，X 2为EAAAK，X 3为赖氨酸，亲脂性化合物为胆固醇琥珀酸单酯，X 5为NH 2。
示例性的，所述化合物为脂肽IPB30。脂肽IPB30为式(I)所示化合物，X 1为Ac，X 2为EAAAK，X 3为赖氨酸，亲脂性化合物为硬脂酰氯，X 5为NH 2。
EAAAK即(EAAAK)n中n等于1的形式。
本公开还保护一种多聚体，为如下(a1)或(a2)或(a3)：
(a1)由以上任一所述化合物形成的多聚体；
(a2)由以上任一所述药用盐形成的多聚体；
(a3)由以上任一所述衍生物形成的多聚体。
本公开还保护以上任一所述化合物或其药用盐或其衍生物的应用，为如下(b1)或(b2)或(b3)或(b4)：
(b1)在制备冠状病毒膜融合抑制剂中的应用；
(b2)在制备用于预防和/或治疗冠状病毒所致疾病的药物中的应用；
(b3)作为冠状病毒膜融合抑制剂的应用；
(b4)在预防和/或治疗冠状病毒所致疾病中的应用。
本公开还保护一种产品，包括以上任一所述化合物或其药用盐或其衍生物；所述产品的功能为如下(c1)或(c2)：
(c1)作为冠状病毒膜融合抑制剂；
(c2)预防和/或治疗冠状病毒所致疾病。
本公开还保护接头多肽在制备增强病毒膜融合抑制剂的抗病毒活性和/或稳定性的产品中的应用；所述接头多肽的氨基酸序列为(EAAAK)n或A[(EAAAK)n]A；n为5以下的自然数。
本公开还保护增强病毒膜融合抑制剂的抗病毒活性和/或稳定性的方法，包括如下步骤：将接头多肽连接至病毒膜融合抑制剂；所述接头多肽的氨基酸序列为(EAAAK)n或A[(EAAAK)n]A；n为5以下的自然数。
本公开还保护一种改造后的病毒膜融合抑制剂的制备方法，包括如下步骤(d1)或步骤(d2)：
(d1)将氨基酸序列为(EAAAK)n或A[(EAAAK)n]A的接头多肽作为连接臂连接改造前的病毒膜融合抑制剂和X 3(X 4)基团，得到脂肽；X 3(X 4)基团中，X 4修饰于X 3；X 3为赖氨酸或半胱氨酸或2,3-二氨基丙酸(Dap)或鸟氨酸(Orn)或2,4-二氨基丁酸(Dab)或2,7-二氨基庚酸(Dah)；X 4为亲脂性化合物基团；n为5以下的自然数；
(d2)将氨基酸序列为(EAAAK)n或A[(EAAAK)n]A的接头多肽作为连接臂连接改造前的病毒膜融合抑制剂和X 4基团，得到脂肽；X 4为亲脂性化合物基团，修饰于接头多肽中的K上；n为5以下的自然数；
所述脂肽即为改造后的病毒膜融合抑制剂。
本公开还保护一种改造后的病毒膜融合抑制剂，为如下(e1)或(e2)：
(e1)将氨基酸序列为(EAAAK)n或A[(EAAAK)n]A的接头多肽作为连接臂连接改造前的病毒膜融合抑制剂和X 3(X 4)基团得到的脂肽；X 3(X 4)基团中，X 4修饰于X 3；X 3为赖氨酸或半胱氨酸或2,3-二氨基丙酸(Dap)或鸟氨酸(Orn)或2,4-二氨基丁酸(Dab)或2,7- 二氨基庚酸(Dah)；X 4为亲脂性化合物基团；n为5以下的自然数；
(e2)将氨基酸序列为(EAAAK)n或A[(EAAAK)n]A的接头多肽作为连接臂连接改造前的病毒膜融合抑制剂和X 4基团得到的脂肽；X 4为亲脂性化合物基团，修饰于接头多肽中的K上；n为5以下的自然数；
所述脂肽即为改造后的病毒膜融合抑制剂。
本公开还保护药用化合物，为以上任一所述化合物或其药用盐或其衍生物或者以上任一所述多聚体。
所述药用化合物的用途为如下(f1)或(f2)或(f3)或(f4)或(f5)或(f6)：
(f1)作为冠状病毒膜融合抑制剂；
(f2)预防和/或治疗冠状病毒所致疾病；
(f3)用于抗冠状病毒；
(f4)用于抑制冠状病毒进行细胞融合；
(f5)用于抑制冠状病毒侵入细胞；
(f6)用于抑制冠状病毒复制；
本公开还保护治疗或/和预防冠状病毒感染动物的方法，包括给受体动物施用所述药用化合物以抑制冠状病毒感染动物。
以上任一所述X 2为刚性接头。
以上任一所述接头多肽为刚性接头多肽。
以上任一所述X 2为具有α-螺旋结构的刚性接头。
以上任一所述接头多肽为具有α-螺旋结构的刚性接头多肽。
以上任一所述n可为1或2或3或4或5。
以上任一所述X 2起到连接臂的作用，可以显著增加α-螺旋结构，进而提高所述化合物或其药用盐或其衍生物的稳定性和抗病毒活性。
以上任一所述接头多肽起到连接臂的作用，可以显著增加α-螺旋结构，进而提高病毒膜融合抑制剂的稳定性和抗病毒活性。
当X 3为赖氨酸时，通过其侧链的氨基连接脂类化合物。
当X 3为半胱氨酸时，通过其侧链的巯基连接脂类化合物。
当X 3为赖氨酸时，优选的亲脂性化合物为胆固醇琥珀酸单酯，通过酰胺化反应连接于赖氨酸的侧链。
在X 4为半胱氨酸时，优选的亲脂性化合物为溴乙酸胆固醇酯。
式(I)和式(Ⅱ)中，氨基酸的缩写具有本领域公知的含义，例如S为丝氨酸、V为缬氨酸、N为天冬酰胺、I为异亮氨酸、Q为谷氨酰胺、K为赖氨酸、E为谷氨酸、D为天冬氨酸、R为精氨酸、L为亮氨酸、A为丙氨酸、G为甘氨酸、Y为酪氨酸、C为半胱氨酸等。
所述氨基酸可为L型氨基酸。
多肽中的一个或多个(如2-5个、2-4个或2-3个)氨基酸也可以用构象为D型的氨基酸、人工修饰的氨基酸、自然界存在的稀有氨基酸等进行替换，以提高多肽的生物利用度、稳定性和/或抗病毒活性。
D型氨基酸是指与组成蛋白质的L型氨基酸相对应的氨基酸。
人工修饰的氨基酸指经过甲基化、磷酸化等修饰的组成蛋白质的常见L型氨基酸。
自然界存在的稀有氨基酸包括组成蛋白质的不常见氨基酸和不组成蛋白质的氨基酸，例如5-羟基赖氨酸、甲基组氨酸、γ氨基丁酸、高丝氨酸等。
本公开提供了一种设计病毒膜融合抑制剂脂肽的新方法，采用此方法或策略所制备的脂肽抑制剂具有显著增加的螺旋结构特征以及显著增加的抗病毒活性。该化合物所形成的可药用的盐、溶剂化物、螯合物或非共价复合物，基于该化合物基础上的药物前体，或上述形式的任意混合物亦是本公开的内容。
本公开提供了含本公开化合物在预防和治疗冠状病毒所致疾病的方法。
本公开还提供了包括根据本公开化合物的用于制备预防和治疗冠状病毒所致疾病的药物组合物。作为优选，所述药物组合物在预防和治疗冠状病毒所致疾病的用途。
本公开所述的冠状病毒包括但不限于在实施例中体现的各种冠状病毒。例如，SARS-CoV-2(SARS-CoV-2野毒株及其各种突变株，所述野毒株的全基因组序列参见GenBank:MN908947.3,所述突变株可为阿尔法、贝塔、伽马、德尔塔和奥密克戎等毒株)、SARS-CoV、MERS-CoV、以及其他人冠状病毒(例如HCoV-229E、HCoV-OC43和HCoV-NL63等)。例如，蝙蝠来源的冠状病毒和穿山甲来源的冠状病毒等。
所述化合物包括不同的光学异构体、消旋体和/或它们的混合物。上述情况下，其中单一的对映异构体或非对映异构体，如有旋光的异构体，可以用不对称合成的方法或消旋体拆分的方法获得。消旋体的拆分可用不同的方法实现，如常规的用助拆分的试剂重结晶，或用色谱方法。
所述化合物包括不同的带双键的顺式和/或反式的异构体。
化合物的衍生物可为化合物的溶剂化物、化合物的络合物、化合物的螯合物或化合物的非共价复合物。化合物的衍生物还可为该化合物基础上的药物前体(例如化合物的酯或者酰胺衍生物)。
在制备药物时，可采用以上化合物的任意形式的混合物。
在制备的药物中，可含有以上化合物的任意形式的混合物。
采用本公开的方法制备的改造后的病毒膜融合抑制剂，具有显著增加的螺旋结构特征，从而抗病毒活性和/或稳定性显著增加。
本公开所涉及到的更多内容在以下有详细描述，或者有些也可以在本公开的实施例中体现。除非另有所指，本文中所用来表示不同成分的数量、反应条件，在任意情况下都可解读为“大致的”、“大约的”意思。相应的，除有明确的特指外，在下述以及权利要求中所引用的数字 参数都是大致的参数，在各自的实验条件下由于标准误差的不同，有可能会得到不同的数字参数。
本文中，当一个化合物的化学结构式和化学名称有分歧或疑义时，以化学结构式确切定义此化合物。本文所描述的化合物有可能含有一个或多个手性中心，和/或者双键以及诸如此类的结构，也可能存在立体异构体，包括双键的异构体(比如几何异构体)、旋光对映异构体或者非对映异构体。相应的，在本文描述范围内的任意化学结构，无论是部分或整体结构中含有上述类似结构，都包括了此化合物的所有可能的对映异构体和非对映异构体，其中也包括了单纯的任一种立体异构体(如单纯的几何异构体、单纯的对映异构体或者单纯的非对映异构体)以及这些异构体的任意一种混合物。这些消旋异构体和立体异构体的混合物由本领域技术人员利用手性分离技术或手性分子合成的方法也可进一步被拆分成其组成成分的对映异构体或立体异构体。
在实际应用中，可以将本公开的药物直接给予病人、或者与适宜的载体或赋形剂混合后给予病人，以达到治疗和/或预防冠状病毒感染的目的。这里的载体材料包括但不限于水溶性载体材料(如聚乙二醇、聚乙烯吡咯烷酮、有机酸等)、难溶性载体材料(如乙基纤维素、胆固醇硬脂酸酯等)、肠溶性载体材料(如醋酸纤维素酞酸酯和羧甲乙纤维素等)。其中优选的是水溶性载体材料。使用这些材料可以制成多种剂型，包括但不限于片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、口含片、栓剂、冻干粉针剂等。可以是普通制剂、缓释制剂、控释制剂及各种微粒给药系统。为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯、山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。为了将单位给药剂型制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等；粘合剂如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。为了将单位给药剂型制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。为了将单位给药剂型制成 注射用制剂，如溶液剂、乳剂、冻干粉针剂和混悬剂，可以使用本领域常用的所有稀释剂，例如，水、乙醇、聚乙二醇、1,3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。使用上述剂型可以经注射给药，包括皮下注射、静脉注射、肌肉注射和腔内注射等；腔道给药，如经直肠和阴道；呼吸道给药，如经鼻腔；粘膜给药。上述给药途径优选的是注射、雾化吸入、鼻喷或滴鼻给药。
本公开的药物的给药剂量取决于许多因素，例如所要预防或治疗疾病的性质和严重程度，患者或动物的性别、年龄、体重及个体反应，所用的具体活性成分，给药途径及给药次数等。上述剂量可以单一剂量形式或分成几个，例如二、三或四个剂量形式给药。
本公开的药物可以直接单独用于冠状病毒感染者的治疗和预防，也可以与一种或多种其他抗病毒药物联合使用，以达到提高整体治疗效果的目的。这些抗病毒药物包括但不限于中和抗体、蛋白酶抑制剂、RNA依赖的RNA聚合酶(RdRp)抑制剂、病毒侵入抑制剂等。上述的中和抗体可以是安巴韦单抗(BRII-196)、罗米司韦单抗(BRII-198)、卡西瑞单抗(Casirivimab)、伊德单抗(Imdevimab)、索罗维单抗(Sotrovimab)、巴姆拉尼单抗(Bamlanivimab)等的一种或几种；所述蛋白酶抑制剂可以是帕昔洛韦(Paxlovid)、达芦那韦(darunavir)、洛匹那韦/利托那韦(Lopinavir/Ritonavir)等的一种或几种；所述RdRp抑制剂可以是莫努匹拉韦(Molnupiravir)、法匹拉韦(Favipiravir)、瑞德西韦(Remdesivir)、索磷布韦(Sofosbuvir)等的一种或几种；所述病毒侵入抑制剂可以是阿比朵尔(Arbidol)、羟氯喹(hydroxychloroquine)等的一种或几种。
对于任何具体的患者，具体的治疗有效剂量水平须根据多种因素而定，所述因素包括所治疗的障碍和该障碍的严重程度；所采用的具体活性成分的活性；所采用的具体组合物；患者的年龄、体重、一般健康状况、性别和饮食；所采用的具体活性成分的给药时间、给药途径和排泄率；治疗持续时间；与所采用的具体活性成分组合使用或同时使用的药物；及医疗领域公知的类似因素。例如，本领域的做法是，活性成分的剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。
本公开的发明人首次发现：具有刚性接头EAAAK的式(I)化合物IPB29和IPB30，尤其是IPB29，相对于没有此接头的化合物，抑制新冠病毒的活性提高了至少70倍，相对于具有柔性接头(例如PEG或者GSGSG)的化合物抑制活性提高了大约8倍。
同时，本公开的发明人发现脂肽IPB29活性的提高的原因在于刚性接头EAAAK显著增加了此脂肽的螺旋含量，同时提高了稳定性。并且，这种脂肽IPB29能够抑制多种不同类型的新冠病毒。
本公开化合物性质稳定，是一种高效的、广谱的新型冠状病毒膜融合抑制剂，用于制备 预防和治疗冠状病毒所致疾病的药物组合物，所述药物组合物用于预防和治疗冠状病毒所致疾病。
附图说明
图1为作为病毒膜融合抑制剂的脂肽的α-螺旋含量(左图)和热稳定性(右图)。
图2为作为病毒膜融合抑制剂的脂肽与靶序列多肽复合物的α-螺旋含量(左图)和热稳定性(右图)。
图3为脂肽抑制SARS-CoV-2感染293T/ACE2细胞(左图)或Huh-7细胞(右图)的活性。
图4为脂肽对各种SARS-CoV-2突变株感染293T/ACE2细胞的抑制活性。
图5为脂肽对各种SARS-CoV-2突变株感染Huh-7细胞的抑制活性。
图6为脂肽抑制其他冠状病毒的活性。
图7为脂肽的体外细胞毒性检测。
图8为脂肽IBP24和IPB29稳定性分析。
图9为新型脂肽对SARS-CoV-2的S蛋白介导细胞膜融合的抑制作用。左图靶细胞为293T/ACE2细胞，右图靶细胞为Huh-7细胞。
具体实施方式
本公开科研团队一直致力于病毒膜融合抑制剂药物的研究与开发，设计出基于脂肽的广谱冠状病毒膜融合抑制剂，对SARS-CoV-2及其突变株具有较强的抑制活性(参考文献2-5)。
在设计基于脂肽的病毒膜融合抑制剂中，通常需要在多肽序列和脂类基团(如脂肪酸和胆固醇等)之间加入一个接头(linker)起到连接臂的作用。脂类基团的预期结合位点为病毒或细胞膜，而多肽在靶点区富集，因此脂类基团和多肽的结合位点不同。由于多肽倾向于形成稳定的二级结构，具有较强的结构刚性，因此多肽和脂类基团之间通常选用柔性接头(flexible linker)连接，使多肽和脂类基团能够分别形成合适的构象，结合到各自的结合位点。同时，在充分发挥各自的作用的同时，避免因空间位阻等原因而相互影响。常见柔性接头有甘氨酸(G)和丝氨酸(S)的组合，如(GGGGS)n或(GSGSG)n等，通过改变n的大小可以将结构域之间的距离放大和减小；另一常见的柔性接头为小分子聚乙二醇(PEG)n,其中n多在2至24之间。目前文献报道的冠状病毒膜融合抑制剂脂肽也都是采用柔性接头，如IBP02V1～IBP02V5均为PEG8(参考文献2)，IPB24～IPB27分别为PEG 4、PEG 5、PEG 6和PEG 8(参考文献4)，EKL1C为GSG(参考文献6),EK1C4为串联的GSGSG和PEG 4(参考文献7)，[SARS HRC-PEG 4] 2-chol为PEG 4(参考文献8)等。制备融合蛋白常见的刚性接头(rigid linker)有能够形成α-螺旋的(EAAAK)n序列，其具有内部氢键和密切联系肽链骨干，刚性且稳定；另一种类型的刚性接头具有Pro-rich序列(XP)n，其中X可指定任何氨基酸，优选丙 氨酸、赖氨酸或谷氨酸，(XP)n序列没有螺旋结构，但其中脯氨酸可以增加骨架硬度，并且有效分离结构域。
下面结合具体实施方式对本公开进行进一步的详细描述，给出的实施例仅为了阐明本公开，而不是为了限制本公开的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本公开的限制。本领域技术人员可以借鉴本文内容，适当改进相关参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本公开范围内。本公开的方法已经通过较佳实施例进行了描述，相关人员明显能在不脱离本公开内容、精神和范围内对本文所述的化合物和制备方法进行改动或适当变更与组合，来实现和应用本公开技术。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、脂肽的制备
分别制备如下脂肽：脂肽IPB29、脂肽IPB30、脂肽IPB20、脂肽IPB24、脂肽IPB28。
脂肽IPB29和脂肽IPB30中均具有EAAAK(SEQ ID NO：4)刚性接头。脂肽IPB29和脂肽IPB30的氨基酸部分均如SEQ ID NO：1所示。
SEQ ID NO：1：SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAKK。
脂肽IPB20(其中不具有作为连接臂的接头)、脂肽IPB24(其中具有作为连接臂的柔性接头PEG 4，由Fmoc-NH-PEG 4-CH 2CH 2COOH为合成原料衍生)、脂肽IPB28(其中具有作为连接臂的柔性接头GSGSG(SEQ ID NO：5))均作为对照。
脂肽IPB29和脂肽IPB30均满足如下通式：
Figure PCTCN2022094001-appb-000002
脂肽IPB29，X 1为氨基端保护基团Ac，X 2为EAAAK刚性接头，X 3为赖氨酸残基，X 4为修饰于X 3上的胆固醇琥珀酸单酯基团，X 5为羧基端保护基团NH 2。
脂肽IPB30，X 1为氨基端保护基团Ac，X 2为EAAAK刚性接头，X 3为赖氨酸残基，X 4为修饰于X 3上的硬脂酰氯基团，X 5为羧基端保护基团NH 2。
5个脂肽的序列结构见表1。五个脂肽中，Ac代表乙酰基，NH 2代表氨基，EAAAK代表氨基酸序列为EAAAK的短肽。
表1：5个脂肽的序列结构
Figure PCTCN2022094001-appb-000003
5个脂肽之间的结构区别如表2所示。
表2：5个脂肽的结构区别
Figure PCTCN2022094001-appb-000004
一、制备过程中所需的化学试剂
所有的化学试剂如各种Fmoc氨基酸、N,N'-二异丙基碳二亚胺(DIC)、1-羟基苯并三唑(HOBt)、N,N-二甲基甲酰胺(DMF)、哌啶(PIPE)、茚三酮、乙酸酐(Ac 2O)、N,N-二异丙基乙胺(DIEA)、水合肼、胆固醇单琥珀酸酯、硬脂酰氯、三氟乙酸(TFA)、乙二硫醇(EDT)、苯甲硫醚(TA)、三异丙基硅烷(TIPS)、苯酚、N-芴甲氧羰基-四聚乙二醇-羧酸(Fmoc-NH-PEG 4-CH 2CH 2COOH)等均从主要化学试剂供应商购买，使用前未经过进一步的提纯。
多肽合成过程中使用的保护氨基酸原料包括Fmoc-Lys(Dde)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc-Ala-OH、Fmoc-Ser(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Ile-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH。其中的缩写具有公知的 定义：Fmoc为9-芴甲氧羰基，Dde为1-(4,4-二甲基-2,6-二氧代环己亚基)乙基，Boc为叔丁氧羰酰基，tBu为叔丁基，OtBu为叔丁氧基，Trt为三苯甲基，Pbf为(2,3-二氢-2,2,4,6,7-五甲基苯并呋喃-5-基)磺酰基。
二、肽树脂的合成
使用Rink Amide MBHA树脂为载体树脂，通过去Fmoc保护和偶联反应，依次与多肽氨基酸序列相应的保护氨基酸偶联，制得肽树脂。
1、接入主链第1个保护氨基酸
取0.3mmol第1个保护氨基酸Fmoc-Lys(Dde)-OH和0.3mmol HOBt，用适量DMF溶解；另取0.3mmol DIC，振荡下慢慢加入至保护氨基酸DMF溶液中，于室温环境中振荡反应5分钟，得到活化后的保护氨基酸溶液，备用。
取0.1mmol的Rink Amide MBHA树脂(0.35mmol/g*0.3g)，采用25％PIPE/DMF溶液(体积比)去保护20分钟(两次)，洗涤过滤得到去Fmoc的树脂。
将活化后的第1个保护氨基酸溶液加入到已去Fmoc的树脂中，偶联反应60分钟，过滤洗涤，得含第1个保护氨基酸Fmoc-Lys(Dde)的树脂。
2、接入主链其他保护氨基酸
采用上述接入主链第1个保护氨基酸同样的方法，依次接入多肽对应的其他保护氨基酸，得含主链氨基酸的树脂。最后用0.3mmol Ac 2O+0.6mmol DIEA对N端进行乙酰化封端，完成主链的合成。以上每步反应后都通过Kaiser Test检测对反应进行控制，若某个氨基酸缩合反应不完全，则重复缩合一次，直至得到所需的目标肽段。
3、侧链的接入
(1)用尽量小体积的2％水合肼/DMF溶液(体积比)处理树脂去除C端赖氨酸侧链的Dde保护基(10分钟，两次)，过滤洗涤，得到去Dde的树脂备用。
(2)多肽的胆固醇修饰：取0.3mmol胆固醇琥珀酸单酯和0.3mmol HOBt，用适量DMF溶解；另取0.3mmol DIC，慢慢加入至含胆固醇琥珀酸单酯和HOBt的溶液中，于室温环境中振荡反应5分钟。将制备的含胆固醇琥珀酸单酯、HOBt和DIC溶液加入到步骤(1)获得的去Dde的树脂中，偶联反应60分钟，过滤、洗涤和干燥，得到肽树脂。
(3)多肽的硬脂酰化：取0.3mmol硬脂酰氯和0.6mmol DIEA溶于适量DMF中，慢慢加入步骤(1)获得的去Dde的树脂中，于室温环境中振荡反应60分钟，过滤、洗涤和干燥，得到肽树脂。
三、粗品的制备
取上述肽树脂，加入裂解试剂(裂解试剂15mL/克树脂)，混合均匀后于30℃下振荡反应3小时，将目标多肽从树脂上裂解下来并除去侧链保护基。收集反应混合物滤液，树脂再用少量TFA/DCM洗涤3次，合并滤液后加入无水乙醚沉淀，离心。滤饼再用冷的无水乙醚洗涤沉淀2次，抽干得类白色粉末即为脂肽粗品。
裂解试剂的组成如下：三氟乙酸：1,2-乙二硫醇：苯甲硫醚：苯酚：H 2O：三异丙基硅烷＝68.5:10:10:5:3.5:1(体积比)。
四、纯品的制备
取上述脂肽粗品，加水/乙腈搅拌溶解，离心除去不溶物后备用。采用反相高效液相色谱法进行纯化。所用色谱柱型号为Agela C18(10μm,
Figure PCTCN2022094001-appb-000005
50×250mm)，流动相由流动相A(0.05％TFA和2％乙腈的水溶液)和流动相B(90％乙腈/水溶液)组成。流动相流速为每分钟25mL。紫外检测波长为220纳米。取粗品溶液上样于色谱柱中，进行梯度洗脱，收集对应纯化组分，直接冷冻干燥除去溶剂，即得到蓬松状态的三氟乙酸盐多肽纯品。
将三氟乙酸盐多肽纯品用水和乙腈重新溶解，加入大量的阴离子交换树脂(醋酸根形式)后搅拌3个小时。过滤并用水/乙腈混合溶剂冲洗离子交换树脂后，合并滤液冻干，得到蓬松状态的多肽醋酸盐纯品(即表1中的脂肽)。
表1中的脂肽的化学结构由MALDI-TOF质谱进行表征，其纯度则由分析型高效液相色谱仪(Agela C18-4.6×250mm，流速每分钟1mL)给出。结果表明，所合成脂肽的纯度均大于95％。
实施例2、脂肽的结构特征及其与靶序列的相互作用分析
采用圆二色谱(CD)技术测定供试脂肽的二级结构(α-螺旋)和热稳定性以及供试脂肽与靶序列模拟多肽之间的相互作用，具体方法参照本发明人发表的论文(参考文献4和参考文献5)。靶序列模拟多肽N52，源自于SARS-CoV-2刺突蛋白S2亚基的HR1序列。N52的序列结构如下：
Ac-FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQ-NH 2。(N52所含氨基酸序列如SEQ ID NO：6所示)
供试脂肽：实施例1制备的脂肽IPB29、脂肽IPB30、脂肽IPB20、脂肽IPB24或脂肽IPB28。供试复合物：供试脂肽与N52的混合物。
1、使用磷酸盐缓冲液(PBS,pH 7.2)将供试脂肽(或供试复合物)配制成10μM的溶液(对于供试复合物来说，10μM指的是供试脂肽和N52的浓度均为10μM)，于37℃水浴锅中放置30分钟。
2、将步骤1得到的溶液移至相应比色皿中，使用Jasco分光偏振仪(型号J-815)扫描195-270nm波长范围内溶液摩尔椭圆率[θ]λ的变化情况，典型α-螺旋结构可在208nm和222nm处出现最大负峰，减去PBS空白对照来校正谱值，计算过程中以峰值为-33000degree.cm2.dmol-1作为α-螺旋含量100％的标准，根据溶液在222nm处的摩尔椭圆率计算α-螺旋含量的百分比。
3、将步骤1得到的溶液加入热稳定性检测比色皿中，调整CD温控模块以每分钟2℃的速度扫描20-98℃时溶液[θ]222随温度变化情况。对熔解曲线进行平滑处理，利用Origin软 件计算热解离转变的中点温度值(Tm)以反映螺旋热稳定程度。
供试脂肽的CD结果如图1所示。不含柔性或刚性接头的IPB20的α-螺旋含量为51％；引入柔性接头的IPB24和IPB28的α-螺旋含量分别为19％和20％，显示柔性接头的引入提高了脂肽的整体转动自由度，增加了脂肽的整体分子熵，对稳定多肽二级结构不利；引入刚性接头的IPB29和IPB30的α-螺旋含量分别为74％和62％，说明引入EAAAK序列显著增加了脂肽的螺旋结构，可能是由于EAAAK接头中的E-K盐桥与多肽α-螺旋二级结构中的盐桥发生共轭，稳定其二级结构，同时相比G，A更倾向形成α-螺旋结构。相对较高的自身α-螺旋含量可有效减少脂肽的多肽部分与靶点的结合熵，增加结合常数，提高活性；相对稳定的二级结构也有利于多肽抗体内蛋白酶水解，提高体内稳定性，也对抗病毒活性有利。
供试复合物的CD结果如图2所示。各脂肽可以与靶序列模拟多肽相互作用形成复合物，具有典型的α螺旋结构，其中IPB20-N52复合物、IPB24-N52复合物和IPB28-N52复合物的α-螺旋含量分别为66％、48％和37％，IPB29-N52复合物和IPB30-N52复合物的α-螺旋含量分别为68％和53％(图2的左图)。IPB20-N52复合物、IPB24-N52复合物和IPB28-N52复合物的Tm值分别为90℃、90℃和89℃，IPB29-N52复合物和IPB30-N52复合物的Tm值分别为86℃和78℃(图2的右图)。这显示引入EAAAK接头的脂肽在增加与细胞膜和病毒膜结合能力的同时，能保持与靶标的专一性稳定结合。
实施例3、脂肽对新冠病毒SARS-CoV-2及其突变体的抑制作用
293T细胞为美国模式培养物集存库产品(ATCC，目录号CRL-3216)。Huh-7细胞为国家实验细胞资源共享服务平台产品。293T/ACE2细胞记载于参考文献2。
供试细胞：293T/ACE2细胞或Huh-7细胞。
一、脂肽对新冠病毒SARS-CoV-2的抑制作用
供试脂肽：实施例1制备的脂肽IPB29、脂肽IPB30、脂肽IPB20、脂肽IPB24或脂肽IPB28。
1、SARS-CoV-2假病毒的制备
表达SARS-CoV-2的S蛋白的质粒(定义为pCoV2-S)，该质粒记载于参考文献5的材料和方法部分的“Single-cycle infection assay”中的“a plasmid expressing the S protein of SARS-CoV-2”。HIV骨架质粒pNL4-3.luc.RE由美国国立卫生研究院艾滋病试剂和参照物项目提供(目录号3418)。
将pCoV2-S和pNL4-3.luc.RE按1:1共转染293T细胞，在37℃、5％CO 2细胞培养箱中培养48小时后收取含有SARS-CoV-2假病毒的上清液，过滤后于-80℃保存备用。
制备得到的SARS-CoV-2假病毒即为参考文献2中的“SARS-CoV-2pseudovirus(SARS-CoV-2PV，以下又称SARS-CoV-2WT)”。
2、脂肽对SARS-CoV-2的抑制作用
(1)采用去离子水溶解供试脂肽并测定浓度，然后用DMEM培养基将供试脂肽稀释到起始浓度，在96孔细胞培养板中进行3倍倍比稀释，最终，每个孔中含有50μL脂肽溶液。设置9个稀释度，每个稀释度3个复孔。设置加入DMEM培养基(每孔50μL)的对照孔。
(2)完成步骤(1)后，每孔加入50μL(病毒量为500TCID 50)步骤1制备的假病毒，然后于室温孵育30分钟。
(3)将预先培养的供试细胞调整浓度为10×10 4个细胞/mL的细胞悬液，加入DEAE-dextran并使其浓度为15μg/mL，然后加入至完成步骤(2)的96孔板中(100μL/孔)，于37℃、5％CO 2细胞培养箱中培养48小时。
(4)完成步骤(3)后，弃除上清，然后按30μL/孔加入细胞裂解液，室温裂解15分钟，然后加入荧光素酶底物(Promega公司)，用微孔板化学发光检测仪测定相对荧光单位(RLU)，并计算制作抑制率曲线和药物半数抑制浓度(IC 50)。
结果见图3。IPB20、IBP24、IBP28、IBP29和IBP30抑制SARS-CoV-2感染293T/ACE2细胞的IC 50值分别为63.85nM、5.51nM、6.91nM、0.57nM和4.19nM；IPB20、IBP24、IBP28、IBP29和IBP30抑制SARS-CoV-2感染Huh-7细胞的IC 50值分别为39nM、2.43nM、2.88nM、0.53nM和2.77nM。IPB29与IPB20的区别在于，IPB29含有刚性接头EAAAK，IPB20无刚性或柔性接头；与IPB20相比，IPB29在293T/ACE2细胞的抗病毒活性提高了约112倍，IPB29在Huh-7细胞的抗病毒活性提高了约74倍。IPB29与IPB24的区别在于，IPB29含有刚性接头EAAAK，IPB24含有柔性接头PEG 4；与IPB24相比，IPB29在293T/ACE2细胞的抗病毒活性提高了约10倍，IPB29在Huh-7细胞的抗病毒活性提高了约5倍。IPB29与IPB28的区别在于，IPB29含有刚性接头EAAAK，IPB28含有柔性接头GSGSG；与IPB28相比，IPB29在293T/ACE2细胞的抗病毒活性提高了约12倍，IPB29在Huh-7细胞的抗病毒活性提高了约5倍。实验结果表明EAAAK接头显著提高了脂肽作为抑制剂的抑制活性，例如IPB29和IPB30抑制SARS-CoV-2的活性。
二、脂肽对新冠病毒SARS-CoV-2的突变体的抑制作用
供试脂肽：实施例1制备的脂肽IPB29、脂肽IPB30、脂肽IPB24或脂肽IPB28。
1、SARS-CoV-2的突变体的假病毒的制备
分别制备各种新冠病毒SARS-CoV-2的突变体的假病毒，突变体具体见图4。
方法参见步骤一的1。差别仅在于将表达SARS-CoV-2的S蛋白的质粒替换为表达SARS-CoV-2的突变体(单点突变或代表性流行毒株)的S蛋白的质粒。
制备得到的SARS-CoV-2的D614G突变体的假病毒即为参考文献2中的“D614G PV”。
2、脂肽对SARS-CoV-2的突变体的抑制作用
方法同步骤一的2。
供试细胞为293T/ACE2细胞时的结果见图4。在293T/ACE2细胞中，IPB24、IPB28、IPB29和IPB30抑制Delta毒株的IC 50值分别为4.94nM、6.30nM、0.79nM和3.57nM，IPB24、 IPB28和IPB30的IC 50值分别约为IPB29的6倍、8倍和4倍。在293T/ACE2细胞中，IPB24、IPB28、IPB29和IPB30抑制Omicron毒株的IC 50值分别为4.51nM、4.51nM、0.47nM和1.78nM，IPB24、IPB28和IPB30的IC 50值分别约为IPB29的10倍、10倍和4倍。
供试细胞为Huh-7细胞时的结果见图5。在Huh-7细胞中，IPB24、IPB28、IPB29和IPB30抑制Delta毒株感染的IC 50值分别为3.46nM、4.34nM、0.56nM和2.17nM，IPB24、IPB28和IPB30的IC 50值分别约为IPB29的6倍、8倍和4倍。在Huh-7细胞中，IPB24、IPB28、IPB29和IPB30抑制Omicron毒株感染的IC 50值分别为2.56nM、2.46nM、0.46nM和1.46nM，IPB24、IPB28和IPB30的IC 50值分别约为IPB29的6倍、5倍和3倍。
结果表明，新型脂肽(例如IPB29和IPB30)作为病毒膜融合抑制剂，对各种SARS-CoV-2的突变体(突变株)均具有良好的抑制活性。
实施例4、脂肽对其他冠状病毒的抑制作用
供试脂肽：实施例1制备的脂肽IPB29、脂肽IPB30、脂肽IPB24或脂肽IPB28。
供试细胞：293T/ACE2细胞或Huh-7细胞。
1、其他冠状病毒假病毒的制备
分别制备各种其他冠状病毒假病毒，其他冠状病毒具体如下：蝙蝠来源的冠状病毒(bat RaTG13)、穿山甲来源的冠状病毒(PCoV-GD或PCoV-GX)、SARS-CoV、MERS-CoV、HCoV-NL63和HCoV-229E。
方法参见实施例3的步骤一的1。差别仅在于将表达SARS-CoV-2的S蛋白的质粒替换为表达其他冠状病毒的S蛋白的质粒。
制备得到的SARS-CoV假病毒即为参考文献2中的“SARS-CoV PV”。
制备得到的MERS-CoV假病毒即为参考文献2中的“MERS-CoV PV”。
制备得到的HCoV-NL63假病毒即为参考文献2中的“HCoV-NL63PV”。
制备得到的HCoV-229E假病毒即为参考文献2中的“HCoV-229E PV”。
2、脂肽对其他冠状病毒的抑制作用
方法同实施例3的步骤一的2。
结果见图6。结果表明，新型脂肽能够有效抑制上述七种病毒的感染。在293T/ACE2细胞中，IPB24、IPB28和IPB30抑制bat RaTG13毒株、PCoV-GD毒株和PCoV-GX毒株感染的IC 50值最高分别约可达到IPB29的4倍、6倍和5倍。在Huh-7细胞中，IPB24、IPB28和IPB30抑制SARS-CoV毒株、MERS-CoV毒株、HCoV-NL63毒株和HCoV-229E毒株感染的IC 50值最高分别约可达到IPB29的11倍、7倍、9倍和7倍。由此可知，脂肽IPB29和IPB30对其他冠状病毒均有较强抑制作用，尤其是IPB29；与其他脂肽相比，IPB29对SARS-CoV-2密切相关的SARS-CoV、PCoV-GD和PCoV-GX仍表现为最强的抑制活性。
实施例5、脂肽的体外细胞毒性和治疗指数分析
供试脂肽：实施例1制备的脂肽IPB29、脂肽IPB30、脂肽IPB24或脂肽IPB28。
供试细胞：293T/ACE2细胞或Huh-7细胞。
供试脂肽的体外细胞毒性检测采用CCK-8细胞增殖/毒性检测试剂盒(厂家：Abbkine，货号：KTC011001)进行。具体步骤：(1)在96孔细胞培养板中将供试脂肽进行3倍梯度稀释，最终，每个孔中含有100μL脂肽溶液；设置9个稀释度，每个稀释度3个复孔。设置加入DMEM培养基(每孔100μL)的对照孔；(2)将10×10 4个细胞/mL的供试细胞悬液加入到完成步骤(1)的96孔细胞培养板中，100μL/孔，于37℃、5％CO 2条件下培养48小时；(3)完成步骤(2)后，每孔加入20μL CCK-8溶液，继续将培养板在培养箱孵育2小时，然后用酶标仪测定在450nm处的吸光度(OD450)。采用GraphPad Prism软件制作抑制率曲线和计算药物半数细胞毒性浓度(CC 50)。
结果见图7。IBP24、IBP28、IBP29和IBP30四个脂肽，对293T/ACE2细胞的CC 50值分别是14.36μM、12.28μM、23.94μM和45.46μM，对Huh-7细胞的CC 50值分别是15.02μM、15.97μM、22.75μM和44.43μM。比较而言，IBP29和IBP30具有相对较低的细胞毒性，尤其是硬脂酰氯基团修饰的IPB30。
以CC 50/IC 50分析可知(IC 50数据是实施例3中获得的数据)，四个多肽均具有极高的选择治疗指数(TI)。例如，对Omicron突变株感染293T/ACE2细胞的抑制活性，IPB24、IPB28、IPB29和IPB30的TI值分别高达约3184、2723、50936和25539，IPB29的TI值分别约为IPB24、IPB28和IPB30的16倍、19倍和2倍。例如，对Omicron突变株感染Huh-7细胞的抑制活性，IPB24、IPB28、IPB29和IPB30的TI值分别高达约5867、6492、49457和30432，IPB29的TI值分别约为IPB24、IPB28和IPB30的8倍、8倍和1.6倍。可见，IBP29和IBP30比IBP24和IBP28具有较高的治疗指数，因此成药性更高。
实施例6、脂肽抑制剂的稳定性研究
本实施例中，发明人从多个角度比较分析了代表性脂肽IPB24和IPB29的稳定性，包括蛋白酶的消化处理、肝微粒体的消化处理、与人血清的孵育及37℃长期放置。
供试脂肽：实施例1制备的脂肽IPB29、脂肽IPB24。
检测脂肽的抗病毒活性的方法同实施例3的步骤一(供试细胞：293T/ACE2细胞)。
一、蛋白酶的消化
供试蛋白酶：蛋白酶K、胰蛋白酶和α-胰凝乳蛋白酶。蛋白酶K、胰蛋白酶和α-胰凝乳蛋白酶均购自Sigma-Aldrich，产品货号分别为P2308、T4799和C4129。
将供试脂肽与供试蛋白酶分别按终浓度2mg/mL和0.1mg/mL混合，于37℃分别孵育0、30、60、120或180分钟，然后检测脂肽的抗病毒活性。
二、肝微粒体的消化
Ⅰ相代谢稳定性试剂盒人肝微粒体(混合)试剂购自北京汇智泰康医药技术有限公司，货号为0111A1.03。实验方法按厂家提供的说明书进行。首先将试剂盒中A液10μL、B液2μL和0.1M PBS缓冲液28μL均匀混合，于37℃预孵育5分钟后40μL/管分装，于37℃水浴中温浴，配制成预孵育液备用。将0.1M PBS缓冲液154μL、肝微粒体5μL和浓度为4mM的供试脂肽溶液1μL混合，然后加入40μL预孵育液，立即置于37℃水浴中进行孵育并计时。设定不同的孵育时间点，向孵育体系中加入预冷乙腈200μL终止反应，然后检测脂肽的抗病毒活性。
三、人血清稳定性实验
将含有20％人血清和终浓度为150μM的供试脂肽混合，于37℃分别孵育0、5、30、60、120或180分钟，然后检测脂肽的抗病毒活性。
四、温度稳定性实验
将浓度为300μM的供试脂肽水溶液置于37℃不同时间，然后检测其抗病毒活性的变化情况。
五、结果分析
实验结果见图8。
与未处理的脂肽(即孵育时间为0时刻的处理组)相比，蛋白酶K、胰蛋白酶或α-胰凝乳蛋白酶处理的IPB24和IPB29的抗病毒活性没有明显的变化，说明单一酶消化对脂肽的稳定性影响有限。然而，人肝微粒体的处理72和96小时后，IPB24对SARS-CoV-2感染293T/ACE2细胞的抑制活性明显下降，其IC 50值分别升高了约8和14倍，而对IPB29的抗病毒活性影响有限。肝微粒体中包含了大部分Ⅰ相酶，其中最重要的是以CYP450为主要成分的微粒体混合功能氧化酶系统，其中影响IPB24活性的成分有待深入研究。
IPB24和IPB29对20％人血清的处理比较敏感，尤其是IPB24在孵育5分钟后抗病毒活性即下降了约24倍；在孵育30、60、120和180分钟后IPB24抗病毒活性则分别下降了约32、36、37和42倍。比较而言，IPB29对人血清则表现出明显改善的抗性，在上述5到180分钟各时间点其抗病毒活性分别下降约4、10、12、13和13倍。
IPB24在37℃分别放置3、7、14、21和28天后的抗病毒活性随着时间延长逐渐下降，尤其在28天后下降了5倍多。相较而言，IPB29的抗病毒活性在同样放置条件下则无变化或者小得多。更长期的脂肽的温度稳定性有待进一步研究。
总之，IPB29比IPB24表现出明显的稳定性优势，稳定性大大增强，进一步支持携带EAAAK序列的IPB29螺旋脂肽的更优成药性。
实施例7 新型脂肽对SARS-CoV-2的S蛋白介导细胞-细胞膜融合的抑制作用
供试脂肽：实施例1制备的脂肽IPB29、脂肽IPB30、脂肽IPB20。
供试细胞：293T/ACE2细胞、Huh-7细胞。
为进一步评价新型脂肽抑制剂的抗SARS-CoV-2活性，本公开进行了基于DSP系统的细胞-细胞融合抑制实验，具体方法参见参考文献4和5中的Cell-cell fusion assay部分。步骤如下：
(1)将293T效应细胞悬液(1.5×10 4个/100μL/孔)铺于96孔板中，同时将293T/ACE2或Huh-7靶细胞悬液(1.5×10 5个/mL)铺于10-cm细胞培养皿中，置于37℃、5％CO 2条件下进行培养。
(2)培养16小时后，将pCoV2-S质粒和pDSP 1-7质粒共转染293T效应细胞，同时将pDSP 8-11质粒转染293T/ACE2或Huh-7靶细胞，然后继续培养细胞。
(3)于24小时后，将多肽在96孔板中进行3倍梯度稀释，设置3个复孔和9个稀释梯度。将稀释的多肽加到效应细胞，于37℃、5％CO2细胞培养箱中孵育1小时。
(4)将DMEM完全培养基预热并按1：4000比例加入EnduRen活细胞底物(Promega公司)，然后用于重悬离心收集的293T/ACE2或Huh-7靶细胞，调整细胞浓度为3×10 5/mL，于37℃、5％CO 2条件下孵育30分钟。
(5)将293T/ACE2或Huh 7靶细胞按100μL/孔加入到293T效应细胞中，然后400g离心1分钟以便使效应细胞和靶细胞充分接触，然后培养混合后的细胞2小时。
(6)于微孔板光度计中读取其荧光素酶活性(RLU)，并计算抑制率和IC 50值。
结果见图9。当靶细胞为293T/ACE2时(左图)，IPB20、IBP29和IBP30抑制SARS-CoV-2S蛋白介导的细胞-细胞膜融合的IC 50值分别为3.65nM、0.2nM和0.44nM；但靶细胞为Huh-7时(右图)，IPB20、IBP29和IBP30抑制SARS-CoV-2S蛋白介导的细胞-细胞膜融合的IC 50值分别为4.01nM、0.31nM和0.45nM。实验结果表明新型膜融合抑制剂IPB29和IPB30对SARS-CoV-2的S蛋白介导的细胞-细胞融合的抑制活性较强。
以上对本公开进行了详述。对于本领域技术人员来说，在不脱离本公开的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本公开。虽然本公开给出了特殊的实施例，应该理解为，可以对本公开作进一步的改进。总之，按本公开的原理，本申请欲包括任何变更、用途或对本公开的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
参考文献清单如下：
1.Xue J,Chong H,Zhu Y,Zhang J,Tong L,Lu J,Chen T,Cong Z,Wei Q,He Y.2022.Efficient treatment and pre-exposure prophylaxis in rhesus macaques by an HIV fusion-inhibitory lipopeptide.Cell 185:131-144e18.
2.Zhu Y,Yu D,Hu Y,Wu T,Chong H,He Y.2021.SARS-CoV-2-derived fusion inhibitor  lipopeptides exhibit highly potent and broad-spectrum activity against divergent human coronaviruses.Signal Transduct Target Ther 6:294.
3.Yu D,Zhu Y,Yan H,Wu T,Chong H,He Y.2021.Pan-coronavirus fusion inhibitors possess potent inhibitory activity against HIV-1,HIV-2,and simian immunodeficiency virus.Emerg Microbes Infect 10:810-821.
4.Yu D,Zhu Y,Jiao T,Wu T,Xiao X,Qin B,Chong H,Lei X,Ren L,Cui S,Wang J,He Y.2021.Structure-based design and characterization of novel fusion-inhibitory lipopeptides against SARS-CoV-2 and emerging variants.Emerg Microbes Infect 10:1227-1240.
5.Zhu Y,Yu D,Yan H,Chong H,He Y.2020.Design of Potent Membrane Fusion Inhibitors against SARS-CoV-2,an Emerging Coronavirus with High Fusogenic Activity.J Virol 94:e00635-20.
6.Zhou J,Xu W,Liu Z,Wang C,Xia S,Lan Q,Cai Y,Su S,Pu J,Xing L,Xie Y,Lu L,Jiang S,Wang Q.2021.A highly potent and stable pan-coronavirus fusion inhibitor as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases.Acta Pharm Sin B doi:10.1016/j.apsb.2021.07.026.
7.Xia S,Liu M,Wang C,Xu W,Lan Q,Feng S,Qi F,Bao L,Du L,Liu S,Qin C,Sun F,Shi Z,Zhu Y,Jiang S,Lu L.2020.Inhibition of SARS-CoV-2(previously 2019-nCoV)infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion.Cell Res 30:343-355.
8.de Vries RD,Schmitz KS,Bovier FT,Predella C,Khao J,Noack D,Haagmans BL,Herfst S,Stearns KN,Drew-Bear J,Biswas S,Rockx B,McGill G,Dorrello NV,Gellman SH,Alabi CA,de Swart RL,Moscona A,Porotto M.2021.Intranasal fusion inhibitory lipopeptide prevents direct-contact SARS-CoV-2 transmission in ferrets.Science 371:1379-1382.

Claims (10)

  1. 化合物或其药用盐或其衍生物；
    所述化合物为式(I)所示的化合物或式(Ⅱ)所示的化合物；
    Figure PCTCN2022094001-appb-100001
    式(I)和式(Ⅱ)中，X 1为氨基端保护基团；
    式(I)和式(Ⅱ)中，X 2为多肽，氨基酸序列为(EAAAK)n或A[(EAAAK)n]A；n为5以下的自然数，表示EAAAK序列的重复次数；
    式(I)中，X 3为赖氨酸或半胱氨酸或2,3-二氨基丙酸或鸟氨酸或2,4-二氨基丁酸或2,7-二氨基庚酸；
    式(I)中，X 4为修饰于X 3的亲脂性化合物基团；
    式(Ⅱ)中，X 4为修饰于X 2中的K上的亲脂性化合物基团；
    式(I)和式(Ⅱ)中，X 5为羧基端保护基团。
  2. 如权利要求1所述的化合物或其药用盐或其衍生物，其特征在于：所述化合物为式(I)所示化合物，X 1为Ac，X 2为EAAAK，X 3为赖氨酸，亲脂性化合物为胆固醇琥珀酸单酯，X 5为NH 2。
  3. 如权利要求1所述的化合物或其药用盐或其衍生物，其特征在于：所述化合物为式(I)所示化合物，X 1为Ac，X 2为EAAAK，X 3为赖氨酸，亲脂性化合物为硬脂酰氯，X 5为NH 2。
  4. 多聚体，为如下(a1)或(a2)或(a3)：
    (a1)由权利要求1至3中任一所述化合物形成的多聚体；
    (a2)由权利要求1至3中任一所述药用盐形成的多聚体；
    (a3)由权利要求1至3中任一所述衍生物形成的多聚体。
  5. 权利要求1至3中任一所述化合物或其药用盐或其衍生物的应用，为如下(b1)或(b2)或(b3)或(b4)：
    (b1)在制备冠状病毒膜融合抑制剂中的应用；
    (b2)在制备用于预防和/或治疗冠状病毒所致疾病的药物中的应用；
    (b3)作为冠状病毒膜融合抑制剂的应用；
    (b4)在预防和/或治疗冠状病毒所致疾病中的应用。
  6. 一种产品，包括权利要求1至3中任一所述化合物或其药用盐或其衍生物；所述产品的功能为如下(c1)或(c2)：
    (c1)作为冠状病毒膜融合抑制剂；
    (c2)预防和/或治疗冠状病毒所致疾病。
  7. 接头多肽在制备增强病毒膜融合抑制剂的抗病毒活性和/或稳定性的产品中的应用；所述接头多肽的氨基酸序列为(EAAAK)n或A[(EAAAK)n]A；n为5以下的自然数。
  8. 增强病毒膜融合抑制剂的抗病毒活性和/或稳定性的方法，包括如下步骤：将接头多肽连接至病毒膜融合抑制剂；所述接头多肽的氨基酸序列为(EAAAK)n或A[(EAAAK)n]A；n为5以下的自然数。
  9. 一种改造后的病毒膜融合抑制剂的制备方法，包括如下步骤(d1)或步骤(d2)：
    (d1)将氨基酸序列为(EAAAK)n或A[(EAAAK)n]A的接头多肽作为连接臂连接改造前的病毒膜融合抑制剂和X 3(X 4)基团，得到脂肽；X 3(X 4)基团中，X 4修饰于X 3；X 3为赖氨酸或半胱氨酸或2,3-二氨基丙酸或鸟氨酸或2,4-二氨基丁酸或2,7-二氨基庚酸；X 4为亲脂性化合物基团；n为5以下的自然数；
    (d2)将氨基酸序列为(EAAAK)n或A[(EAAAK)n]A的接头多肽作为连接臂连接改造前的病毒膜融合抑制剂和X 4基团，得到脂肽；X 4为亲脂性化合物基团，修饰于所述接头多肽中的K上；n为5以下的自然数；
    所述脂肽即为改造后的病毒膜融合抑制剂。
  10. 一种改造后的病毒膜融合抑制剂，为如下(e1)或(e2)：
    (e1)将氨基酸序列为(EAAAK)n或A[(EAAAK)n]A的接头多肽作为连接臂连接改造前的病毒膜融合抑制剂和X 3(X 4)基团得到的脂肽；X 3(X 4)基团中，X 4修饰于X 3；X 3为赖氨酸或半胱氨酸或2,3-二氨基丙酸或鸟氨酸或2,4-二氨基丁酸或2,7-二氨基庚酸；X 4为亲脂性化合物基团；n为5以下的自然数；
    (e2)将氨基酸序列为(EAAAK)n或A[(EAAAK)n]A的接头多肽作为连接臂连接改造前的病毒膜融合抑制剂和X 4基团得到的脂肽；X 4为亲脂性化合物基团，修饰于所述接头多肽的K上；n为5以下的自然数；
    所述脂肽即为改造后的病毒膜融合抑制剂。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116987152A (zh) * 2023-09-27 2023-11-03 中国科学院微生物研究所 一种新冠病毒环肽抑制剂
CN116987152B (zh) * 2023-09-27 2024-01-02 中国科学院微生物研究所 一种结合沙贝冠状病毒s蛋白rbd结构域的环肽及其应用

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