CN116804061A - Polymer for inhibiting human coronavirus membrane fusion for long time and application thereof - Google Patents

Polymer for inhibiting human coronavirus membrane fusion for long time and application thereof Download PDF

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Publication number
CN116804061A
CN116804061A CN202210261212.XA CN202210261212A CN116804061A CN 116804061 A CN116804061 A CN 116804061A CN 202210261212 A CN202210261212 A CN 202210261212A CN 116804061 A CN116804061 A CN 116804061A
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human coronavirus
membrane fusion
polymer
polypeptide
long
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Inventor
王瑞
徐巍
姜世勃
陆路
段倩玉
夏帅
王茜
黄金
里卡多·罗塞约
卢水秀
滕灵燕
姚雪莲
史孟君
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BEIJING PROSPEROUS BIOPHARM CO LTD
Fudan University
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BEIJING PROSPEROUS BIOPHARM CO LTD
Fudan University
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Priority to CN202210261212.XA priority Critical patent/CN116804061A/en
Priority to PCT/CN2023/080301 priority patent/WO2023174122A1/en
Publication of CN116804061A publication Critical patent/CN116804061A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Abstract

The application provides a macromolecule capable of inhibiting human coronavirus membrane fusion for a long time and application thereof, and relates to the field of medicines. A macromolecule for inhibiting human coronavirus membrane fusion for a long time, comprising the following two parts: a polypeptide inhibiting human coronavirus membrane fusion and a serum albumin targeting pseudo-antibody, wherein the polypeptide and the pseudo-antibody are connected through a connecting molecule. The polymer can effectively interfere or block the membrane fusion process of the human coronavirus entering the host cell, thereby achieving the effect of inhibiting the human coronavirus infection and having lasting in vivo activity. The polymer has a virus-inhibiting effect even when it has a nonfunctional tag sequence which facilitates production and purification. The polymer has the characteristics that the polymer has good drug properties, and can be further developed into an economic and applicable anti-novel coronavirus drug.

Description

Polymer for inhibiting human coronavirus membrane fusion for long time and application thereof
Technical Field
The application relates to the field of medicines, in particular to a polymer for inhibiting fusion of human coronavirus membranes for a long time and application thereof.
Background
Coronaviruses can be divided into four genera, namely α -, β -, γ -and δ -, β -coronaviruses are further divided into four lineages: A. b, C and D. Currently, there are 7 coronaviruses that can infect humans, including HCoV-229E and HCoV-NL63 of the α -coronavirus family, HCoV-OC43 and HCoV-HKU1 in the β -coronavirus A line, SARS-CoV and SARS-CoV-2 in the β -coronavirus B line, and MERS-CoV in the β -coronavirus C line. Since month 12 of 2019, a new form of coronavirus disease (covd-19) caused by SARS-CoV-2 infection has spread worldwide. In particular, the recent emergence of a range of SARS-CoV-2 mutants (VOCs), including Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and Omicron (B.1.1.529), has led to enhanced transmissibility and reduced sensitivity to current COVID-19 vaccines and antibody therapies.
Many membrane-fused polymers against human coronaviruses are polypeptides or proteins, and polypeptide or protein drugs face the problem of degradation after administration. Some conventional long-lasting techniques may partially solve the degradation problem described above to some extent. However, by using conventional polypeptide long-acting techniques (such as PEG chemical modification and serum albumin/Fc fusion techniques), it is necessary to introduce an irrelevant group or protein molecule that is 10 times or more larger than the volume of the anti-human coronavirus membrane-fused polymer, which results in loss of activity of the anti-human coronavirus membrane-fused polymer, and the long-acting effect is still limited.
Content of the application
The application aims to provide a long-acting human coronavirus membrane fusion polymer which can effectively inhibit the human coronavirus membrane fusion process, thereby achieving the effect of inhibiting human coronavirus infection and having long activity in vivo.
It is another object of the present application to provide the use of a long-acting human coronavirus membrane-fused polymer which can be used for the preparation of a medicament for the treatment of human coronavirus infections.
The application solves the technical problems by adopting the following technical scheme.
In one aspect, the embodiment of the application provides a polymer for inhibiting human coronavirus membrane fusion for a long time, which comprises the following two parts: a polypeptide inhibiting human coronavirus membrane fusion and a serum albumin targeting pseudo-antibody, wherein the polypeptide and the pseudo-antibody are connected through a connecting molecule.
On the other hand, the embodiment of the application provides an application of a macromolecule capable of inhibiting human coronavirus membrane fusion for a long time in preparing a medicament for treating human coronavirus infection.
Compared with the prior art, the embodiment of the application has at least the following advantages or beneficial effects:
1. the high polymer for inhibiting the membrane fusion of the human coronavirus for a long time can effectively interfere or block the membrane fusion process of the human coronavirus entering host cells, thereby achieving the effect of inhibiting the infection of the human coronavirus and having lasting in vivo activity.
2. The long-acting human coronavirus membrane fused polymer provided by the application can be transferred into escherichia coli by utilizing a genetic engineering technology for mass production, and is more economical and economical.
3. The long-acting human coronavirus membrane fusion polymer provided by the application still has a virus inhibition effect even under the condition of having a nonfunctional tag sequence which is convenient for production and purification.
4. The long-acting human coronavirus membrane fused polymer provided by the application has good drug properties, and can be further developed into an economic and applicable anti-new coronavirus drug.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of SDS-PAGE of FL-EK1 and a quasi-antibody AB after purification in experimental example 1 of the present application;
FIG. 2 shows the results of the test of FL-EK1 in Experimental example 1 of the present application on the formation of six helices (6-HB) of HR1P and HR 2P;
FIG. 3 shows FL-EK1 mediated inhibition of SARS-CoV-2S and HCoV-OC43S proteins, respectively, in Experimental example 2 of the present application;
FIG. 4 shows FL-EK1 mediated inhibition of SARS-CoV-2 and HCoV-OC43S pseudoviruses, respectively, in Experimental example 2 of the present application;
FIG. 5 shows FL-EK1 mediated inhibition of SARS-CoV-2 and HCoV-OC43S live virus infection, respectively, in Experimental example 2 of the present application;
FIG. 6 shows FL-EK1 mediated inhibition of SARS-CoV-2Delta mutant live virus infection in Experimental example 2 of the present application;
FIG. 7 shows the results of the anti-SARS-CoV-2 pseudovirus activity assay of serum samples collected at various time points in Experimental example 3 of the present application;
FIG. 8 shows the concentrations and respective half-lives of FL-EK1 and EK1 in the serum samples of mice estimated in Experimental example 3 of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other.
The present application relates to a polymer capable of inhibiting human coronavirus in a long-acting manner, which comprises a moiety capable of inhibiting human coronavirus and a moiety capable of prolonging the inhibition effect of the polymer. The moiety that inhibits human coronavirus is typically one or more polypeptides or proteins that may interfere with or block a step of human coronavirus entry into a host cell, such as a membrane fusion step. Human coronaviruses complete a membrane fusion process mediated by envelope glycoproteins when they invade target cells. In this process, the S1 subunit recognizes and adsorbs to the receptor, and the S2 subunit mediates mainly fusion of the viral envelope and cell membrane. The 2 heptad repeats (HR 1 and HR 2) on the S2 subunit play an important role in this process. When human coronavirus infects target cells, the conformation of the S2 subunit changes in a series, eventually forming a hairpin trimer (or six helix bundle) structure, i.e. 3 HR2 helices attach in antiparallel fashion to the groove of the central trimer formed by polymerization of 3 HR1 helices. It is speculated that there is an intermediate hairpin precursor where the HR1 and HR2 regions are exposed before membrane fusion occurs, but do not form the final hairpin structure. In the intermediate state, the HR1 and HR2 regions bind to exogenously added HR1, HR2 or similar polypeptides, which prevents interaction between the HR1 and HR2 of the S2 subunit itself in the intermediate state of the hairpin precursor, resulting in the ultimate inability of the S2 subunit to convert to the hairpin trimer structure, i.e., inhibition of membrane fusion.
EK1 is an inhibitor for membrane fusion of the pan-coronavirus, wherein EK1 contains 36 amino acid sequences, has high antiviral capability as the membrane fusion inhibitor, and can be widely used for resisting various human coronaviruses. However, since EK1 belongs to a long polypeptide, the production process is complicated and expensive, and does not have a protein tertiary structure with thermal stability, the half-life in vivo may be short, and thus clinical application of EK1 may be limited. Therefore, the development of long-acting inhibitors of human coronavirus membrane fusion is an urgent problem we are faced with. In addition to the inhibition of human coronavirus, the polymer of the present application also includes a small protein with serum albumin targeting function, i.e., the aforementioned pseudo-antibody AB. In recent years, a polypeptide long-acting technical route based on targeting serum albumin is paid attention to. The technology adopts the artificial reconstruction, and the small protein molecule (generally only about 100 residues) with serum albumin targeting is recombined with the polypeptide gene with the half life needing to be prolonged, so that the original activity of the polypeptide is not greatly reduced due to the larger probability after further rational design. After the obtained fusion protein medicine enters the blood circulation system, most of the fusion protein medicine is adsorbed on serum albumin, and the fusion protein medicine is kept in a free state in a small part. The adsorbed fusion protein drug is released from serum albumin gradually as the free fusion protein drug is consumed or cleared, thereby maintaining the concentration of the drug in the blood and maintaining the efficacy for a long period of time by virtue of reversible binding to human serum albumin (half-life: 19-20 days (avoiding degradation or excretion. The prototype proteins useful for producing such artificial targeting proteins include Staphylococcus aureus A domain protein (US 5831012, EP 0739353), human fibronectin (US 6818418 EP 1266025), and the like.
The present application will be described in detail with reference to specific examples.
A polymer for inhibiting human coronavirus membrane fusion for a long time, which is characterized by comprising the following two parts: a polypeptide that inhibits fusion of human coronavirus membrane and a mimetic antibody that targets serum albumin, said polypeptide and said mimetic antibody being linked by a linker molecule.
In the embodiment of the application, the pseudo-antibody, the connecting molecule and the polypeptide are connected together in the form of fusion protein, and the amino acid sequence of the polymer is SEQ ID NO:4.
in the embodiment of the application, the polymer is also provided with an additional nonfunctional label. After the high polymer is added into the label, the production efficiency is improved and the production cost is reduced.
In an embodiment of the present application, the non-functional tag includes a plurality of histidines, and the amino acid sequence of the polymer with the non-functional tag is SEQ ID NO:5.
in an embodiment of the present application, the above-mentioned pseudo-antibody is a polypeptide or protein composed of natural amino acids or unnatural amino acids and derivatives thereof, which have the ability to target serum albumin.
In an embodiment of the application, the above-mentioned pseudo-antibody is a mutant derived from human fibronectin domain FN 3.
In an embodiment of the application, the polypeptide is derived from EK1 or a derivative thereof.
In an embodiment of the application, the linking molecule is a natural amino acid or an unnatural amino acid with a molecular weight between 300Da and 5500 Da. The molecular weight is too large or too small to be easily linked, and the molecular weight of the present linking molecule is in this range more easily linked to the polypeptide EK1 and the mimetic antibody AB.
An isolated nucleic acid molecule encoding a polypeptide or protein of any one of the above-described macromolecules that long-acting inhibits human coronavirus membrane fusion.
A recombinant vector comprising the above nucleic acid molecule.
A genetically engineered bacterium comprising the above nucleic acid molecule.
A pharmaceutical composition comprising any one of the above-described polymers that inhibits membrane fusion of human coronaviruses for a long period of time.
The application of a macromolecule capable of inhibiting human coronavirus membrane fusion for a long time in preparing a medicament for treating human coronavirus infection is provided.
The sequence comparison table is shown in Table 1.
TABLE 1 sequence comparison Table
As shown in table 1, wherein SEQ ID NO:1 is the amino acid sequence of polypeptide EK 1; SEQ ID NO:2 is the amino acid sequence of a Linker molecule (Linker); SEQ ID NO:3 is the amino acid sequence of the quasi-antibody AB; SEQ ID NO:4 is the amino acid sequence of the polymer AB-EK 1; SEQ ID NO:5 is the amino acid sequence of the polymer FL-EK1 with a nonfunctional tag.
The features and capabilities of the present application are described in further detail below in connection with the examples.
Example 1
A preparation method of a macromolecule for inhibiting human coronavirus membrane fusion for a long time comprises the following steps:
1. construction and expression of FL-EK1 genetically engineered bacteria
1.1, experimental materials and reagents
The quasi-antibody AB is obtained by screening through special technology in Beijing Hua Jinrui company, and the polypeptide EK1 and the connecting molecule are obtained by screening through the compound denier university. Both the gene of interest (i.e., the gene-linked fragment from the pseudoantibody AB, the linker molecule with the non-functional tag and the polypeptide EK 1) and the plasmid vector (pET-28 a) were supplied by the company beijing-a Jin Ruiqing; coli BL21 (DE 3) was competent purchased from Tiangen, beijing.
1.2 Experimental procedures
The plasmid vector carrying the target gene, namely plasmid pET-28a-FL-EK1, is transformed into BL21 (DE 3) competence, cultured for 12-16h, 4-5 clone strains are selected and added into LB culture medium containing kanamycin resistance for overnight culture. The following day, 5ml of overnight culture broth was added to 500ml of LB medium, then cultured at 30℃and 220rpm/min for about 4 hours, when the OD value reached 0.6, 0.2mM of IPTG was added to induce protein expression, induction was continued at 16℃for about 12 hours, and the cultured broth was centrifuged at 6000rpm/min for 10 minutes to collect the cells. The collected cells were resuspended in 30ml of PBS buffer, centrifuged again, and the supernatant was discarded, and the cells were frozen in a-80℃refrigerator.
2. Protein purification
2.1, experimental materials and reagents
Sterilizing water: high pressure deionized water
Plasmid: pET-28a-FL-EK1 plasmid was supplied by Beijing Hua Jinrui clear Co
30% bis-Acr polyacrylamide gel: purchased from Bio-rad Co
Ni purifying column: purchased from Qiagen Inc
Coli BL21 (DE 3) competent purchased from Tiangen, beijing
2.2 Experimental procedures
The purification process of the protein by the Ni column is as follows:
(1) The frozen bacterial solution was allowed to stand at room temperature until thawing, and purified reagent binding buffer (50 mM NaH) 2 PO 4 pH8.0, 300mM NaCl,10mM imidazole) 30ml of resuspended cells, vortexing for 10min with a vortexing shaker, adding 150. Mu.l of 10% Titron100-PBS, shaking upside down, and placing in an ice-water mixture.
(2) Ultrasonic crushing, wherein the ultrasonic crushing conditions are as follows: the ultrasonic power is 300W, the working time is 3s, the interval is 5s, and the total ultrasonic treatment is 30min. Centrifuge at 10000rpm/min for 30min.
(3) The supernatant of the centrifuged and crushed bacterial liquid was filtered with a 0.45 μm filter membrane. And taking out the balanced Ni purification column, mixing 1ml of Ni column material into the supernatant, and horizontally vibrating on ice for 45min.
(4) And adding the protein into a protein purification column, gradually settling the Ni column agarose particles in the solution, and passing the supernatant through the column for at least 2 times so that the protein is fully combined with the affinity purification column.
(5) Using a washing buffer (50 mM NaH) 2 P0 4 Eluting the hybrid protein with 300mM NaCI,60mM imidazole solution at pH8.0, eluting volume about50ml.
(6) Using a string buffer (50 mM NaH) 2 P0 4 The protein of interest was eluted at pH8.0, 300mM NaCI,300mM imidazole. Collecting the eluted components, dialyzing with PBS overnight in a refrigerator at 4deg.C, and freezing in a refrigerator at-80deg.C.
(7) The used Ni purification column was treated with 5ml of 6M guanidine hydrochloride to strip off the proteins on the column until the binding buffer was equilibrated, and then 20% ethanol solution was added to store in a refrigerator at 4 ℃.
It should be noted here that, in order to better purify the protein of the polymer, a section of tag is added in front of the connecting molecule sequence of the polymer, and the tagged polymer sequence table is SEQ ID NO:5, the tagged linker is named FL, and the untagged macromolecule sequence table
Is SEQ ID NO:4. in the experimental examples of the present application, the labeled polymer was used
Row experiments, i.e. pET-28a-FL-EK1.
Example 2
This embodiment is substantially the same as embodiment 1, except for the following points: 1. the ultrasonic power is 250W, the working time is 2s, the interval is 4s, and the total ultrasonic time is 25min. Centrifuging at 8000rpm/min for 35min; 2. the washing buffer was composed of 40mM NaH 2 P0 4 250mM NaCl and 55mM imidazole; string buffer is composed of 40M NaH 2 PO 4 250mM NaCl and 250mM imidazole; 3. the pH of the washing buffer and the stirring buffer was 7.5.
Example 3
This embodiment is substantially the same as embodiment 1, except for the following points: 1. the ultrasonic power is 250W, the working time is 4s, the interval is 6s, and the total ultrasonic treatment is 35min. Centrifuging at 12000rpm/min for 25min; 2. the washing buffer consists of 60mM NaH 2 P0 4 350mM NaCl and 65mM imidazole; string buffer is composed of 60M NaH 2 PO 4 350mM NaCl and 350mM imidazole; 3. the pH of the washing buffer and the stirring buffer was 8.5.
Example 4
This embodiment is substantially the same as embodiment 1 except that: washing buffer from 45mM NaH 2 P0 4 300mM NaCl and 60mM imidazole; stringing buffer is composed of 50M NaH 2 PO 4 280mM NaCl and 280mM imidazole.
Experimental example 1
Non-denaturing polyacrylamide gel electrophoresis buffer (Native buffer) was purchased from beijing tianzenze company; HR1P, HR P polypeptide was synthesized by the biotechnology company of nanjie.
The effect of FL-EK1 on hexaspiral formation was examined by FN-PAGE (non-denaturing polyacrylamide gel electrophoresis), and the protein materials used in this and all subsequent examples were the purified proteins of example 1, and the results of SDS-PAGE of the purified FL-EK1 and the quasi-antibody AB were shown in FIG. 1, which were identified as FL-EK1 and the quasi-antibody AB in the experiments of this example.
The FN-PAGE experimental procedure was as follows:
(1) Preparing 18% of separating gel and 5% of concentrated gel
(2) HR1P, FL-EK1 (4.2. Mu.M), HR1P/FL-EK1 mixtures (final concentration of FL-EK 1: 4.2. Mu.M, 16.7. Mu.M, and 66.7. Mu.M, respectively) were formulated to give final HR1P concentrations of 120. Mu.M. Placed at 37℃for 1h.
(3) HR2P (40. Mu.M) was formulated, HR2P was added to the HR1P/FL-EK1 mixture and the reaction was continued for 30min at 37 ℃.
(4) And adding a special loading buffer for N-PAGE, and carrying out electrophoresis for 3h in an ice-water bath under the condition of 125V.
(5) After staining the N-PAGE gel with Coomassie brilliant blue for 30min, decolorizing the decolorized solution.
(6) Gray value analysis was performed using ImageJ software, and the results are shown in fig. 2.
As can be seen from FIG. 2, the first lane is HR1P, the second lane is HR2P, the third lane is +H2P, the fourth lane is FL-EK1, the fifth, sixth and seventh lanes are FL-EK1+H2, the difference is that the FL-EK1 concentration is different, and the FL-EK1 concentration of the fifth, sixth and seventh lanes is gradually increased. As can be seen from the bands, the formation of the six-helical bundle (6-HB) formed by HR1P and HR2P was disturbed as the concentration of FL-EK1 increased. The experimental result shows that: FL-EK1 can bind to HR1P, compete with HR2P to affect 6-HB formation, and the amount of 6-HB formed gradually decreases as the concentration of FL-EK1 increases.
Experimental example 2
Cell-cell fusion experiments for SARS-CoV-2, HCoV-OC43 and pseudovirus/live virus inhibition experiments.
The experimental materials were as follows:
and (3) cells: 293T cells, hul-7 (SARS-CoV-2 cell fusion target cells), calu-3 (SARS-CoV-2 target cells), RD cells (HCoV-OC 43 target cells).
Culture medium: DMEM with 10% fbs.
96-well flat-bed plates (corning), 96-well round-bottomed plates (corning), 5 Xcell lysates (Promega), luciferase Assay System (Promega), vigofect transfection reagent, easyPure Viral DNA/RNA Kit, one Step PrimeScript RT-PCR Kit (Perfect Real Time, taKaRa).
Virus: SARS-CoV-2 pseudovirus, live virus, SARS-CoV-2 mutant strain Delta live virus.
The cell-cell fusion experiment was as follows:
(1) 292T cells are spread in a six-hole plate until the length reaches 70% -80% after 24 hours, the plasmid pAAV-IRES-EGFP encoding EGFP and a Vigofect transfection reagent are used for co-transfecting 293T cells, fresh culture medium is replaced after 12 hours, and the 293T cells expressing green fluorescence are collected as effector cells after 24 hours of culture.
(2) Regulating Hul-7 cells and RD cells to 2X 10 one night in advance 4 Mu.l of cells were added to each well of a 96-well flat bottom plate and placed at 37℃in 5% CO 2 Is cultured in an incubator.
(3) The FL-EK1, polypeptide EK1, and quasi-antibody AB were subjected to multiple dilution in 96-well round bottom plates, while cell control wells and virus (effector) control wells were set.
(4) Collecting the effector cells expressing green fluorescence, adding into a drug dilution plate in equal volume, and placing at 37deg.C with 5% CO 2 Is incubated for 30min.
(5) 100 μl/well of the mixture was pipetted into a 96 Kong Ba cell plate, cultured for 2-6h, and the cell fusion was observed under a fluorescence microscope and counted.
The pseudovirus inhibition experiment steps are as follows:
(1) Regulating Calu-3 cells to 1X 10 4 RD cells were adjusted to 8000 cells/well, 100. Mu.l cells were added to each well in a 96 well flat bottom plate, and the plate was placed at 37℃with 5% CO 2 Overnight in the incubator of (a).
(2) The FL-EK1, EK1 and the quasi-antibody AB were subjected to multiple dilution in 96-well round bottom plates, while cell control wells and virus control wells were set.
(3) Fully and uniformly mixing the thawed virus strains at the temperature of minus 80 ℃, and 1:1 equivalent amount of the mixture is added into a medicine dilution plate and placed at 37 ℃ and contains 5 percent of CO 2 Is incubated for 30min.
(4) 100. Mu.l/well of the medium was aspirated into 96 Kong Ba cell plates, the original medium was discarded after 12 hours of culture, new DMEM medium containing 10% FBS was added, and the culture was continued at 37℃for 2 days.
(5) Removing the supernatant, adding 50 μl of diluted cell lysate for 1 hr, sucking 35 μl of the supernatant into 96-well white plate, adding 35 μl of Luciferase, and detecting OD 450 Absorbance values of (2).
The live virus inhibition experiment steps are as follows:
(1) Cell preparation and drug preparation were as in pseudovirus inhibition experiments (1), (2).
(2) Mixing the thawed virus strain at-80deg.C, and mixing the live virus with 100 times TCID 50 Value (i.e., 50% tissue infection dose) 1:1 equivalent addition of drug dilution plate containing 5% CO 2 The mixture was placed in an incubator at 37℃for 2 hours. The supernatant from the 96-well cell plate was discarded, DMEM with 2% fbs was added and placed in a solution containing 5% co 2 Culturing is continued for 4 hours at 37 ℃.
(3) For SARS-CoV-2 live virus cell plates, sample RNA was extracted according to the EasyPure Viral DNA/RNA Kit. The copy number of the N gene of the SARS-CoV-2 live virus and the SARS-CoV-2 mutant strain Delta (B.1.617.2) were detected by the probe method and the One Step PrimeScript RT-PCR Kit (Perfect Real Time) (RT-PCR Kit).
(4) For HCoV-OC43 viable virus cell plate, the original supernatant was discarded, CCK8 (100. Mu.L/well) was added, and the mixture was incubated at 37℃for about 2 hours,detection of OD per well 450 Absorbance.
The FL-EK1 mediated inhibition effect on SARS-CoV-2S protein and HCoV-OC43S protein mediated cell-cell fusion is shown in FIGS. 3A and 3B, respectively;
the FL-EK1 mediated inhibition of SARS-CoV-2S pseudovirus and HCoV-OC43S pseudovirus is shown in FIGS. 4A and 4B, respectively;
the FL-EK1 mediated inhibition of SARS-CoV-2S live virus and HCoV-OC43S live virus is shown in FIGS. 5A and 5B, respectively;
the FL-EK1 mediated inhibition effect on the active virus of SARS-CoV-2Delta mutant strain is shown in FIG. 6;
from FIGS. 3A and 3B, it can be seen that FL-EK1 and EK 1-mediated cell-cell fusion of SARS-CoV-2S protein IC 50 68.5nM and 393.5nM, respectively, indicating that FL-EK1 and EK1 both have an inhibitory effect on SARS-CoV-2S protein-mediated cell-cell fusion, and FL-EK1 has a stronger inhibitory effect on SARS-CoV-2S protein-mediated cell-cell fusion than EK1 has on SARS-CoV-2S protein-mediated cell-cell fusion; FL-EK1 and IC of EK1 to HCoV-OC43S protein mediated cell-cell fusion 50 398.2nM and 246.4nM, respectively, indicate that FL-EK1 and EK1 have an inhibitory effect on HCoV-OC43S protein-mediated cell-cell fusion, and that EK1 has a stronger inhibitory effect on HCoV-OC43S protein-mediated cell-cell fusion than FL-EK1 has on HCoV-OC43S protein-mediated cell-cell fusion.
From FIGS. 4A and 4B, it can be seen that FL-EK1 and EK1 pair SARS-CoV-2S pseudovirus IC 50 90.6nM and 491.9nM, respectively, indicating that FL-EK1 and EK1 both have inhibitory effect on SARS-CoV-2S pseudovirus, and that FL-EK1 has stronger inhibitory effect on SARS-CoV-2S pseudovirus than EK1 has on SARS-CoV-2S pseudovirus; FL-EK1 and EK1 versus HCoV-OC43S pseudovirus IC 50 1592.6nM and 3261.2nM, respectively, indicate that FL-EK1 and EK1 both have an inhibitory effect on HCoV-OC43S pseudoviruses, and that FL-EK1 has a stronger inhibitory effect on HCoV-OC43S pseudoviruses than EK1.
From FIGS. 5A and 5B, it can be seen that FL-EK1 and EK1 have an IC against SARS-CoV-2S live virus 50 25.3nM and respectively96.0nM, indicating that FL-EK1 and EK1 both have inhibitory effect on SARS-CoV-2S live virus, and FL-EK1 has stronger inhibitory effect on SARS-CoV-2S live virus than EK1 has on SARS-CoV-2S live virus; FL-EK1 and IC of EK1 against HCoV-OC43S live virus 50 29.4nM and 66.8nM, respectively, indicating that FL-EK1 and EK1 both have an inhibitory effect on HCoV-OC43S live virus, and that FL-EK1 has a stronger inhibitory effect on HCoV-OC43S live virus than EK1 has on HCoV-OC43S live virus.
From FIG. 6, it can be seen that FL-EK1 and EK1 vs. SARS-CoV-2Delta mutant live virus IC 50 281.2nM and 38.9nM, respectively, indicate that FL-EK1 and EK1 have inhibitory effects on the live virus of the SARS-CoV-2Delta mutant strain, and that EK1 has stronger inhibitory effects on the live virus of the SARS-CoV-2Delta mutant strain than FL-EK1.
Experimental example 3
Half-life test in mice
Experimental materials: BALB/C mice, FL-EK1 recombinant proteins, EK1 polypeptides, caco-2 cells and SARS-CoV-2 pseudovirus.
The experimental procedure was as follows:
the FL-EK1 and EK1 are injected into the abdominal cavity of the mouse, and the drug metabolism process of the mice in the body of the mouse is detected respectively.
(1) Negative serum was used as negative control prior to intraperitoneal injection.
(2) Medicine is injected into abdominal cavity: the drug injections of FL-EK1 and EK1 were 40mg/kg and 8.25mg/kg, respectively.
(3) Mouse blood was collected from the orbits 8min, 0.5h, 1h, 3h, 7h, 12h, 8min, 0.5h, 1h, 3h, 7h, 24h, 72h, 96h after intraperitoneal injection of EK1.
(4) After the sample was left at room temperature for 1h, serum was isolated and frozen in a-80℃refrigerator.
(5) Placing for 30min at 56 ℃, and inactivating complement and enzymes in serum, wherein the steps are respectively 1: 40. 1: 80. 1: 160. 1: 320. 1: 640. 1: after 1280 times dilution, a pseudo-virus inhibition test was performed in the same manner as in experimental example 2.
(6) Mouse serum resulting in 50% inhibition of SARS-CoV-2 pseudovirus was calculated by inhibition rateDilution times (IC) 50 )。
(7) IC for inhibiting SARS-CoV-2 pseudovirus in vitro based on dilution factor of mouse serum resulting in 50% inhibition of SARS-CoV-2 pseudovirus and FL-EK1 and EK1 calculated in example 2 50 The concentration of FL-EK1 and EK1 in serum was estimated.
(8) The in vivo half-life and other pharmacokinetic parameters of FL-EK1 and EK1 were calculated from the concentration of FL-EK1 and EK1 in serum at various time points using MODIT software.
As discussed in the previous application, binding to serum albumin can greatly increase the half-life of a polypeptide or protein. In this experimental example, the pharmacokinetics of FL-EK1 and EK1 in mice were examined, and the serum inhibitory activity against SARS-CoV-2 pseudovirus of serum samples collected at various time points after intraperitoneal injection of FL-EK1 and EK1 in mice is shown in FIG. 7. The estimated concentrations of FL-EK1 and their half-lives in the serum samples of mice are shown in FIG. 8, showing that FL-EK1 has a half-life of 30 hours and EK1 has a half-life of 1.8 hours, showing that FL-EK1 has a half-life about 15.7 times longer than EK1. It should be noted in particular that, because the half-life of human serum albumin (19 to 21 days) is much longer than that of mouse serum albumin, the half-life of the fusion protein (FL-EK 1) in humans is likely to be longer than that of FL-EK1 measured on mice.
In summary, the polymer for inhibiting human coronavirus membrane fusion for a long time, and the preparation method and the application thereof in the embodiment of the application, wherein the polymer comprises a part EK1 capable of inhibiting human coronavirus, a part of quasi-antibody AB for prolonging the inhibiting effect of the polymer, and a connecting molecule, and the polypeptide EK1 and the quasi-antibody AB are connected through the connecting molecule. The polymer can effectively interfere or block the membrane fusion process of the human coronavirus entering the host cell, thereby achieving the effect of inhibiting the human coronavirus infection, and the inhibition time is long, as the polymer can be used for preparing long-acting membrane fusion polypeptide medicines for treating the human coronavirus infection.
FL-EK1 and EK1 have inhibition effects on both SARS-CoV-2S protein and HCoV-OC43S protein-mediated cell-cell fusion, and FL-EK1 has stronger inhibition effects on SARS-CoV-2S protein-mediated cell-cell fusion than EK1 has on SARS-CoV-2S protein-mediated cell-cell fusion; the inhibition effect of EK1 on HCoV-OC43S protein mediated cell-cell fusion is stronger than that of FL-EK1 on HCoV-OC43S protein mediated cell-cell fusion. FL-EK1 and EK1 have inhibition effects on SARS-CoV-2S pseudovirus and HCoV-OC43S pseudovirus, and FL-EK1 has stronger inhibition effects on SARS-CoV-2S pseudovirus and HCoV-OC43S pseudovirus than EK1 has on SARS-CoV-2S pseudovirus HCoV-OC43S pseudovirus. FL-EK1 and EK1 have inhibition effects on SARS-CoV-2S live virus and HCoV-OC43S live virus, and FL-EK1 has stronger inhibition effects on SARS-CoV-2S live virus and HCoV-OC43S live virus than EK1 has on SARS-CoV-2S live virus and HCoV-OC43S live virus. In addition, the pharmacokinetic results of FL-EK1 and EK1 in mice showed that the half-life of FL-EK1 in mice was 30h and the half-life of EK1 was 1.8h, showing that FL-EK1 had a half-life that was about 15.7 times longer than EK1. The half-life of human serum albumin (19 to 21 days) is much longer than that of mouse serum albumin, and therefore the half-life of fusion protein (FL-EK 1) in humans is likely to be much longer than that of EK1.
The preparation method of the long-acting human coronavirus membrane fusion polymer provided by the application has the advantages that after a series of rational design is carried out on the polymer through a genetic engineering technology, the biological activity of the polymer can be reserved, and the inhibition effect of the polymer on the human coronavirus membrane fusion process is exerted to the maximum extent; in addition, the preparation method has simple process and convenient operation, and can maximally exert the inhibition effect of the long-acting human coronavirus membrane fusion polymer.
The embodiments described above are some, but not all embodiments of the application. The detailed description of the embodiments of the application is not intended to limit the scope of the application, as claimed, but is merely representative of selected embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Sequence listing
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Fudan University
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Claims (13)

1. A polymer for inhibiting human coronavirus membrane fusion for a long time, which is characterized by comprising the following two parts: a polypeptide that inhibits human coronavirus membrane fusion and a mimetic antibody that targets serum albumin, said polypeptide and said mimetic antibody being linked by a linker molecule.
2. The macromolecule for long-acting inhibition of membrane fusion of human coronavirus according to claim 1, wherein the pseudo-antibody, the linker molecule and the polypeptide are linked together in the form of a fusion protein, and the amino acid sequence of the macromolecule is SEQ ID NO:4.
3. a long-acting human coronavirus membrane fusion inhibiting polymer according to claim 2, wherein said polymer further carries an additional nonfunctional tag.
4. A long-acting human coronavirus membrane fusion inhibiting polymer according to claim 3, wherein said nonfunctional tag comprises a plurality of histidines, and the amino acid sequence of said nonfunctional tag-bearing polymer is SEQ ID NO:5.
5. the polymer for inhibiting membrane fusion of human coronavirus for a long time according to claim 2, wherein the quasi-antibody is a polypeptide or protein composed of natural amino acid or non-natural amino acid and its derivative having the ability of targeting serum albumin.
6. The macromolecule of claim 5, wherein the mimetic antibody is a mutant from human fibronectin domain FN 3.
7. The macromolecule for long-acting inhibition of membrane fusion of human coronavirus according to claim 1, wherein the polypeptide is derived from EK1 or a derivative thereof.
8. The macromolecule for long-acting inhibition of membrane fusion of human coronavirus according to claim 1, wherein the linker molecule is composed of natural or unnatural amino acids with a molecular weight between 300Da and 5500 Da.
9. An isolated nucleic acid molecule encoding a polypeptide or protein of a macromolecule according to any one of claims 2-8 that inhibits human coronavirus membrane fusion for a long duration.
10. A recombinant vector comprising the nucleic acid molecule of claim 9.
11. A genetically engineered bacterium comprising the nucleic acid molecule of claim 9.
12. A pharmaceutical combination comprising a macromolecule of any one of claims 1-8 that inhibits membrane fusion of human coronavirus for a long duration.
13. Use of a macromolecule as defined in any one of claims 1 to 12 for the preparation of a medicament for the treatment of human coronavirus infection with a long-acting inhibition of human coronavirus membrane fusion.
CN202210261212.XA 2022-03-16 2022-03-16 Polymer for inhibiting human coronavirus membrane fusion for long time and application thereof Pending CN116804061A (en)

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CN103333255A (en) * 2013-06-28 2013-10-02 复旦大学 Long-acting HIV-1 (Human Immunodeficiency Virus-1) membrane fusion inhibitor
CN107022008B (en) * 2016-01-30 2021-04-23 山西锦波生物医药股份有限公司 Polypeptide for broad-spectrum inhibition of human coronavirus infection and application thereof
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