WO2022184056A1 - 与SARS-CoV-2刺突蛋白特异性结合的多肽、多肽组合物及其制备方法与应用 - Google Patents

与SARS-CoV-2刺突蛋白特异性结合的多肽、多肽组合物及其制备方法与应用 Download PDF

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WO2022184056A1
WO2022184056A1 PCT/CN2022/078627 CN2022078627W WO2022184056A1 WO 2022184056 A1 WO2022184056 A1 WO 2022184056A1 CN 2022078627 W CN2022078627 W CN 2022078627W WO 2022184056 A1 WO2022184056 A1 WO 2022184056A1
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polypeptide
kvp
protein
cov
sars
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PCT/CN2022/078627
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French (fr)
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许海燕
王琛
王涛
杨延莲
刘健
方小翠
温涛
孟洁
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中国医学科学院基础医学研究所
国家纳米科学中心
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Priority claimed from CN202110224800.1A external-priority patent/CN114989255B/zh
Priority claimed from CN202110224251.8A external-priority patent/CN114989245B/zh
Priority claimed from CN202110224252.2A external-priority patent/CN114989263B/zh
Priority claimed from CN202110224822.8A external-priority patent/CN114989264B/zh
Application filed by 中国医学科学院基础医学研究所, 国家纳米科学中心 filed Critical 中国医学科学院基础医学研究所
Publication of WO2022184056A1 publication Critical patent/WO2022184056A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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

Definitions

  • the present invention relates to the technical field of biomedicine, in particular to a polypeptide and a polypeptide composition specifically binding to SARS-CoV-2 spike protein (especially the SARS-CoV-2 surface spike S1 protein), as well as the polypeptide and Preparation method and application of polypeptide composition.
  • the pathogen of novel coronavirus pneumonia (referred to as new coronary pneumonia) is a new type of coronavirus SARS-CoV-2.
  • the current treatment drugs for novel coronavirus pneumonia are mainly broad-spectrum antiviral drugs and drugs to relieve symptoms, and there is still a lack of effective targeted drugs.
  • the main purpose of the present invention in the first aspect, is to provide a polypeptide composition that specifically binds to the SARS-CoV-2 spike protein, comprising one of polypeptide KVp-N, polypeptide KVp-R and polypeptide KVp-C or A variety of; polypeptide KVp-N contains the amino acid sequence shown in SEQ ID NO: 1, polypeptide KVp-R contains the amino acid sequence shown in SEQ ID NO: 3, and polypeptide KVp-C contains the amino acid sequence shown in SEQ ID NO: 5 amino acid sequence.
  • the ends of the polypeptide KVp-N, the polypeptide KVp-R and the polypeptide KVp-C are modified with a thiol group-containing peptide fragment; preferably, the amino acid sequence of the thiol group-containing peptide fragment is CCPPPP.
  • amino acid sequence of the polypeptide KVp-N modified with a thiol-containing peptide fragment is shown in SEQ ID NO: 2;
  • amino acid sequence of the polypeptide KVp-R modified with a thiol-containing peptide fragment is shown in SEQ ID NO: 4;
  • amino acid sequence of the polypeptide KVp-C modified with a thiol-containing peptide fragment is shown in SEQ ID NO: 6.
  • the polypeptide composition includes at least one polypeptide listed above, and when two or more polypeptides are included, the polypeptides are mixed in any proportion.
  • the three types of polypeptides of polypeptide KVp-N, polypeptide KVp-R and polypeptide KVp-C are mixed in an equimolar ratio.
  • the present invention provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, named polypeptide KVp-N, containing the amino acid sequence shown in SEQ ID NO: 1.
  • the present invention provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, named as polypeptide KVp-R, containing the amino acid sequence shown in SEQ ID NO:3.
  • a fourth aspect provides a polypeptide that specifically binds to the SARS-CoV-2 spike protein, named polypeptide KVp-C, containing the amino acid sequence shown in SEQ ID NO:5.
  • thiol group-containing peptide fragment preferably, the amino acid sequence of the thiol group-containing peptide fragment is CCPPPP.
  • amino acid sequence of the polypeptide KVp-N is shown in SEQ ID NO:1 or SEQ ID NO:2;
  • amino acid sequence of the polypeptide KVp-R is shown in SEQ ID NO:3 or SEQ ID NO:4;
  • amino acid sequence of the polypeptide KVp-C is shown in SEQ ID NO:5 or SEQ ID NO:6.
  • the present invention provides a method for preparing the above-mentioned polypeptide, which is synthesized using a standard solid-phase polypeptide synthesis method.
  • the present invention provides a method for preparing the above-mentioned polypeptide composition. After synthesizing the polypeptide using a standard solid-phase polypeptide synthesis method, the polypeptide is mixed to obtain the polypeptide composition.
  • the present invention provides the application of the above-mentioned polypeptide or polypeptide composition in the preparation of a medicine for treating novel coronavirus pneumonia.
  • the novel coronavirus is SARS-CoV-2.
  • the present invention provides the application of the above-mentioned polypeptide or polypeptide composition in the preparation of a blocking agent for blocking the binding of SARS-CoV-2 surface spike S1 protein to angiotensinase 2 (ACE2); preferably , the angiotensinase 2 (ACE2) exists on the surface of ciliated bronchial epithelial cells or type II pneumocytes.
  • ACE2 angiotensinase 2
  • ACE2 angiotensinase 2
  • polypeptide or the polypeptide composition is used to specifically block the binding of the SARS-CoV-2 surface spike S1 protein to angiotensinase 2 (ACE2).
  • ACE2 angiotensinase 2
  • polypeptide or polypeptide composition to specifically bind to the SARS-CoV-2 surface spike S1 protein to block the SARS-CoV-2 surface spike S1 protein from binding to angiotensinase 2 (ACE2); and/ or
  • polypeptide or polypeptide composition to specifically bind to the RBD domain of the SARS-CoV-2 surface spike S1 protein subunit to block the SARS-CoV-2 surface spike S1 protein and angiotensinase 2 ( ACE2) binding.
  • ACE2 angiotensinase 2
  • the polypeptide or polypeptide composition provided by the present invention that specifically binds to the S1 protein of the SARS-CoV-2 surface spike can block the SARS-CoV-2 virus by binding to the S1 protein.
  • Binding to the key target protein angiotensinase (ACE2) protein on the surface of its host cells has the potential to provide a feasible targeted drug for the treatment of new coronavirus pneumonia.
  • ACE2 angiotensinase
  • Figures 1A-1C are the detection curves of the binding ability of polypeptide KVp-N1, polypeptide KVp-R14, and polypeptide KVp-C15 to S1 protein;
  • Figures 2A-2C are respectively the detection curves of the binding ability of polypeptide KVp-N1, polypeptide KVp-R14, and polypeptide KVp-C15 to RBD protein;
  • Figures 3A-3C are the gel images of Western Blot detection of the ability of S1 protein to bind human lung cancer cell line A549 cells after co-incubation with different concentrations of polypeptide KVp-N1, polypeptide KVp-R14, and polypeptide KVp-C15;
  • Figure 3D is a gel image of Western Blot detection of the ability of S1 protein to bind human lung cancer cell line A549 cells after co-incubation with polypeptide KVp-N1, polypeptide KVp-R14, polypeptide KVp-C15 and the polypeptide composition;
  • Figures 4A-4C are the gel images of Western Blot detection of the ability of RBD protein to bind to A549 cells after co-incubating with different concentrations of polypeptide KVp-N1, polypeptide KVp-R14, and polypeptide KVp-C15;
  • Figures 5A-5C are the gel images of Western Blot detection of the ability of S1 protein to bind to 293T-ACE2 + cells after co-incubation with different concentrations of polypeptide KVp-N1, polypeptide KVp-R14, and polypeptide KVp-C15;
  • Figures 6A-6C are gel images of Western Blot detection of the ability of RBD protein to bind to 293T-ACE2 + cells after co-incubation with different concentrations of polypeptide KVp-N1, polypeptide KVp-R14, and polypeptide KVp-C15;
  • Figure 6D is a gel image of Western Blot detection of the ability of RBD protein to bind to 293T-ACE2 + cells after co-incubation with polypeptide KVp-N1, polypeptide KVp-R14, polypeptide KVp-C15 and the polypeptide composition;
  • Figure 7 is a bar graph showing the statistical results of the infection rate of SARS-CoV-2 pseudovirus on 293T-ACE2 + cells after co-incubation with peptides;
  • Panel A is the peptide KVp-N1 or peptide KVp-N1N after co-incubation
  • Panel B is the peptide After co-incubation with KVp-R14 or polypeptide KVp-R14N
  • panel C is after co-incubation with polypeptide KVp-C15 or polypeptide KVp-C15N;
  • Figure 7D is a bar graph of the statistical results of the infection rate of SARS-CoV-2 pseudovirus on 293T-ACE2 + cells after co-incubating with single polypeptide KVp-N1N, KVp-R14N, KVp-C15N and polypeptide composition respectively;
  • Figures 8A-8C are histograms of statistical results of the infection rate of SARS-CoV-2 pseudovirus on 293T-ACE2 + cells after co-incubation with different concentrations of polypeptide KVp-N1N, polypeptide KVp-R14N, and polypeptide KVp-C15N;
  • Figures 9A-9C are histograms of statistical results of the effects of different concentrations of polypeptide KVp-N1, polypeptide KVp-R14, and polypeptide KVp-C15 on the activity of 293T-ACE2 + cells;
  • Figures 10A-10C are histograms of statistical results of the effects of different concentrations of polypeptide KVp-N1N, polypeptide KVp-R14N, and polypeptide KVp-C15N on the activity of 293T-ACE2 + cells.
  • SARS-CoV-2 binds to angiotensinase 2 (ACE2) on the surface of ciliated bronchial epithelial cells and type II pneumocytes through the RBD domain of its surface spike protein S1 subunit, causing cell infection, thereby enabling people suffering from novel coronavirus pneumonia.
  • ACE2 angiotensinase 2
  • the invention designs a plurality of polypeptides that specifically bind to the SARS-CoV-2 surface spike S1 subunit or its RBD domain, and uses the polypeptides to block the SARS-CoV-2 virus and its host cell surface key target protein blood vessels Binding of tensinase (ACE2) protein; the polypeptides are named as polypeptide KVp-N, polypeptide KVp-R, and polypeptide KVp-C, respectively. These polypeptides are all prepared by artificial synthesis.
  • the polypeptide KVp-N contains the amino acid sequence GDGVYYPRDVFDSSVLDSTQR (denoted as KVp-N1N); or modified with the peptide fragment CCPPPP at the end of KVp-N1N, the amino acid sequence of the modified polypeptide KVp-N can be CCPPPPGDGVYYPRDVFDDSSVLDSTQR (denoted as KVp-N1) , the introduction of a thiol group (introduced by the peptide fragment CCPPPP) in KVp-N1, the purpose is to combine the polypeptide with gold-containing substances (such as gold nanoparticles) through the thiol group, which will help to expand the application of the polypeptide, such as Detect and identify peptides in applications.
  • gold-containing substances such as gold nanoparticles
  • the polypeptide KVp-R contains the amino acid sequence GDLFDDSNLDPFRDRISTRR (denoted as KVp-R14N); or modified with the peptide fragment CCPPPP at the end of KVp-R14N, the amino acid sequence of the modified polypeptide KVp-R can be CCPPPPGDLFDDSNLDPFRDRISTRR (denoted as KVp-R14), this
  • the invention introduces a sulfhydryl group (introduced by the peptide fragment CCPPPP) into KVp-R14, and the purpose is to combine the polypeptide with gold-containing substances (such as gold nanoparticles) through the sulfhydryl group, which will help to expand the application of the polypeptide, such as Detect and identify peptides in applications.
  • the polypeptide KVp-C contains the amino acid sequence GDGVYYPRDVFDDSSVLDSTQR (denoted as KVp-C15N); or modified with the peptide fragment CCPPPP at the end of KVp-C15N, the amino acid sequence of the modified polypeptide KVp-C can be CCPPPPGDGVYYPRDVFDDSSVLDSTQR (denoted as KVp-C15), this
  • the invention introduces a sulfhydryl group (introduced by the peptide fragment CCPPPP) into KVp-C15, and the purpose is to combine the polypeptide with gold-containing substances (such as gold nanoparticles) through the sulfhydryl group, which will help to expand the application of the polypeptide, such as Detect and identify peptides in applications.
  • the present invention also provides a polypeptide composition comprising at least one polypeptide that can specifically bind to the S1 subunit of the SARS-CoV-2 surface spike protein.
  • the present invention proves through experiments that the above-mentioned polypeptides and their combinations can block the binding of S1 protein and ACE2 protein, thereby preventing SARS-CoV-2 from infecting epithelial cells and lung cells, so that these polypeptides and their combinations can be used as novel targeted therapy Drugs for coronavirus pneumonia.
  • Example 1 Polypeptide, polypeptide composition and preparation thereof
  • polypeptides targeting the surface spike protein S1 of SARS-CoV-2 were prepared by standard solid-phase peptide synthesis methods, codenamed KVp-N1N and KVp-N1 (collectively referred to as polypeptides KVp-N), KVp-R14N and KVp-R14 (collectively referred to as polypeptide KVp-R), KVp-C15N and KVp-C15 (collectively referred to as polypeptide KVp-C), the amino acid sequences of these polypeptides are shown in Table 1 below.
  • KVp-N1N GDGVYYPRDVFDDSSVLDSTQR SEQ ID NO: 1
  • KVp-N1 CCPPPPGDGVYYPRDVFDSSSVLDSTQR SEQ ID NO: 2
  • KVp-R14N GDLFDDSNLDPFRDRISTRR SEQ ID NO: 3
  • KVp-R14 CCPPPPGDLFDDSNLDPFRDRISTRR SEQ ID NO:4
  • KVp-C15N GDGVYYPRDVFDDSSVLDSTQR (SEQ ID NO: 5)
  • KVp-C15 CCPPPPGDGVYYPRDVFDSSVLDSTQR (SEQ ID NO:6)
  • the obtained product was the target polypeptide in Table 1, and the six polypeptides were all white powders with a purity of ⁇ 98%.
  • the polypeptides code-named KVp-N1, KVp-R14 and KVp-C15 were modified by introducing CCPPPP at the ends of the polypeptides code-named KVp-N1N, KVp-R14N and KVp-C15N respectively.
  • a sulfhydryl group is introduced into KVp-R14 and KVp-C15, and the sulfhydryl group enables the polypeptides KVp-N1, KVp-R14 and KVp-C15 to bind to gold (eg, gold nanoparticles) (eg, Example 2 utilizes this binding to achieve the ability to bind polypeptides) detection).
  • gold eg, gold nanoparticles
  • the polypeptide composition of this embodiment is composed of at least two kinds of polypeptides in Table 1.
  • it can be a mixture of KVp-N1N and KVp-R14, or a mixture of KVp-N1, KVp-C15 and KVp-C15N, or a mixture of KVp-N1N and KVp-R14.
  • KVp-N1, KVp-R14 and KVp-C15 mixed KVp-R14, KVp-N1N, KVp-R14N and KVp-C15 mixed, KVp-N1, KVp-R14, KVp-C15 , KVp-N1N and KVp-C15N mixed, it can be mixed with KVp-N1, KVp-R14, KVp-C15, KVp-N1N, KVp-R14N and KVp-C15N; the mixing ratio is not limited.
  • each polypeptide was prepared into a stock solution with a concentration of 10 mM in a complete medium (commercially available), and the stock solutions of each polypeptide were mixed in a certain proportion to form a polypeptide composition.
  • each polypeptide was prepared into a polypeptide solution with a concentration of 0.5 mg/mL with double distilled water (ddH 2 O), and each polypeptide solution was mixed in a certain proportion to form a polypeptide composition solution. .
  • the quartz crystal microbalance method (QCM) is used to detect the polypeptides KVp-N1, KVp-R14, KVp-C15 (the thiol groups in these polypeptides need to be used in the detection) synthesized in Example 1 and SARS-CoV- 2
  • the binding ability of the S1 protein or its RBD segment both purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., which used Four-channel dissipative quartz microbalance was purchased from Biolin Scientific.
  • Figures 1A-1C show the binding curve of S1 protein and polypeptide KVp-N1 detected by QCM
  • Figure 1B shows the binding curve of S1 protein and polypeptide KVp-R14 detected by QCM
  • Figure 1C shows the detection of S1 protein and polypeptide KVp-R14 by QCM. Binding curve of polypeptide KVp-C15.
  • the oscillation frequency of the gold-coated quartz chip decreases, indicating that the S1 protein is bound to the polypeptides KVp-N1, KVp-R14, KVp-C15, namely the polypeptides KVp-N1, KVp-R14, KVp -C15 can bind to the S1 protein of SARS-CoV-2.
  • Experiment 2.2 was the same as that of Experiment 2.1 above, except that the RBD protein solution with a concentration of 20 nM prepared in PBS was used to replace the S1 protein solution as the mobile phase.
  • Figure 2A-2C shows the QCM detection results of the binding ability of polypeptides KVp-N1, KVp-R14 and KVp-C15 to the RBD segment, respectively.
  • Figure 2A shows the binding curve of the RBD segment and the polypeptide KVp-N1 detected by QCM.
  • 2B shows the binding curve of the RBD segment and the polypeptide KVp-R14 detected by QCM
  • FIG. 2C shows the binding curve of the RBD segment and the polypeptide KVp-C15 detected by QCM.
  • the oscillation frequency of the gold-coated quartz chip decreases, indicating that the RBD protein is bound to the polypeptides KVp-N1, KVp-R14, KVp-C15, namely the polypeptides KVp-N1, KVp-R14, KVp -C15 can bind to the RBD segment of SARS-CoV-2.
  • the experimental results show that the polypeptides KVp-N (KVp-N1N and KVp-N1), the polypeptides KVp-R (KVp-R14N and KVp-R14), and the polypeptides KVp-C (KVp-C15N and KVp-C15) can all interact with each other. Binding of the S1 protein of SARS-CoV-2 or its RBD segment.
  • Example 3 Polypeptide and its composition prevent the binding of S1 protein and RBD protein to target cells
  • the polypeptides KVp-N1, KVp-R14, KVp-C15 and their equimolar concentration mixtures are synthesized and prepared in Example 1.
  • composition as an example, taking human lung cancer cell line A549 cells (purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) and 293T cells (293T-ACE2 + ) overexpressing ACE2 protein (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. Ltd.) for target cells to verify that the mimic peptides KVp-N1, KVp-R14, KVp-C15 and their compositions prevent the binding effect of the S1 protein of SARS-CoV-2 or its RBD segment to target cells.
  • Experiment 3.1 Effects of polypeptides and their compositions on the binding ability of S1 protein to human lung cancer cell line A549 cells (1) A549 cells were seeded in a 6-well plate at a density of 2 ⁇ 10 5 cells/well, in a 37°C incubator Incubate overnight to allow cells to adhere;
  • the S1 protein was diluted with PBS to 1 ⁇ M S1 protein solution, and then it was mixed with three single polypeptides KVp-N1, KVp-R14, KVp-C15 (the concentration of each single polypeptide was 1 ⁇ M, in complete medium at room temperature) Dilution stock solution) and the polypeptide composition (the concentration of each polypeptide is 1 ⁇ M, and the stock solution is diluted with complete medium) for 1 h, and the S1 protein solution that does not co-incubate with the polypeptide is simultaneously set as the control group;
  • the intensity of the S1 protein signal was significantly reduced, and within the peptide concentration range of 1nM to 1000nM, with the peptide
  • concentrations of KVp-N1, KVp-R14, and KVp-C15 increased, the intensity of the positive signal of S1 protein became weaker, indicating that the polypeptides KVp-N1, KVp-R14 and KVp-C15 inhibited the binding of S1 protein to A549 cells.
  • the results of the polypeptide composition are shown in Figure 3D. It can be seen that the control group has a strong positive signal of S1 protein, indicating that the S1 protein is combined with A549 cells; -C15 and the polypeptide composition) were incubated for 1 h, and then added to the cell culture system, the intensity of the S1 protein signal was significantly reduced, indicating that the polypeptide inhibited the binding of the S1 protein to A549 cells, and the polypeptide composition inhibited the S1 protein to A549 cells.
  • the binding ability of the peptide is significantly stronger than that of the three single polypeptides, showing the joint effect of the three single polypeptides in the polypeptide composition (this effect will be further described in Example 4 below).
  • Experiment 3.2 The specific operation of Experiment 3.2 is the same as that of Experiment 3.1 above, the only difference is that the RBD protein solution is used to replace the S1 protein solution with 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, 100 ⁇ M of the polypeptides KVp-N1, KVp-R14, KVp-C15, and the polypeptide KVp, respectively. - Co-incubation of N1, KVp-R14, KVp-C15 and the polypeptide composition.
  • the content of RBD protein in the total protein was detected by Western Blot method.
  • the results of different concentrations of polypeptides KVp-N1, KVp-R14 and KVp-C15 are shown in Figure 4A-4C respectively. It can be seen that the control group has a strong positive signal of RBD protein.
  • control group had a strong positive signal for RBD protein, indicating that RBD protein was bound to cells, and when RBD protein was first incubated with polypeptides (KVp-N1, KVp-R14, KVp-C15 and the polypeptide composition) 1h, then added to the cell culture system, the intensity of RBD protein signal was significantly reduced, indicating that the polypeptide inhibited the binding of RBD protein to A549 cells, and the ability of the polypeptide composition to inhibit the binding of RBD protein to A549 cells was stronger than that of the three single polypeptide.
  • polypeptides KVp-N1, KVp-R14, KVp-C15 and the polypeptide composition
  • Experiment 3.3 The specific operation of Experiment 3.3 was the same as that of Experiment 3.2 above, except that A549 cells were replaced with 293T-ACE2 + cells as target cells.
  • the content of RBD protein in total protein was detected by Western Blot method.
  • the results of different concentrations of polypeptides KVp-N1, KVp-R14 and KVp-C15 are shown in Figures 6A-6C respectively. It can be seen that the control group has a strong positive signal of RBD protein.
  • polypeptide KVp-N KVp-N1 and KVp-N1N
  • polypeptide KVp-R KVp-R14 and KVp-R14N
  • polypeptide KVp-C KVp-C15 and KVp-C15N
  • target cells human lung cancer cell line A549 cells and human lung cancer cell line 293T-ACE2 + cells
  • the results of the polypeptide composition are shown in Figure 6D. It can be seen that the control group has a strong positive signal of RBD protein, indicating that the RBD protein is combined with the 293T-ACE2 + cells, and when the RBD protein is first associated with the polypeptides (KVp-N1, KVp -R14, KVp-C15 and polypeptide composition) were incubated for 1 h, and then added to the cell culture system, the intensity of RBD protein signal was significantly reduced, indicating that the polypeptide inhibited the binding of RBD protein to 293T-ACE2 + cells, and the polypeptide combination
  • the ability of the compound to inhibit the binding of the RBD protein to 293T-ACE2 + cells was significantly stronger than that of the three single polypeptides, indicating that the three single polypeptides in the polypeptide composition act together (the effect of this joint action will be further described in Example 4 below) .
  • Experiment 3.4 was the same as that of Experiment 3.1 above, except that A549 cells were replaced with 293T-ACE2 + cells as target cells.
  • the content of S1 protein in the total protein was detected by Western Blot method.
  • the results of different concentrations of polypeptides KVp-N1, KVp-R14 and KVp-C15 are shown in Figure 5A-5C respectively. It can be seen that the control group has a strong positive signal for S1 protein , indicating that the S1 protein was bound to 293T-ACE2 + cells; and when the S1 protein was incubated with the polypeptides KVp-N1, KVp-R14, KVp-C15 for 1 h, and then added to the cell culture system, the intensity of the S1 protein signal was obvious.
  • control group had a strong positive signal for S1 protein, indicating that the S1 protein bound to 293T-ACE2 + cells, and when the S1 protein first bound to the polypeptides (KVp-N1, KVp-R14, KVp-C15 and polypeptides)
  • the composition was incubated for 1 h, and then added to the cell culture system, the intensity of the S1 protein signal was significantly reduced, indicating that the polypeptide inhibited the binding of the S1 protein to 293T-ACE2 + cells, and the polypeptide composition inhibited the binding of the S1 protein to A549 cells.
  • the ability is stronger than three single peptides.
  • Example 4 Neutralizing ability of polypeptides and their compositions against SARS-CoV-2 pseudovirus
  • the pseudovirus neutralization method is used to detect the polypeptides KVp-N1N, KVp-R14N, KVp-C15N synthesized in Example 1 and their equimolar concentration mixtures (that is, the equimolar concentrations of polypeptides KVp-N1, KVp-R14 and KVp).
  • SARS-CoV-2 pseudovirus PSV
  • SARS-CoV-2 pseudovirus PSV
  • SARS-CoV-2 pseudovirus (PSV) used was purchased from Jiman Biotechnology (Shanghai) Co., Ltd., It is tagged with luciferase and green fluorescent protein (GFP). Specifically include the following operations:
  • polypeptides KVp-N1 and KVp-N1N were mixed with 10 ⁇ M polypeptides KVp-N1 and KVp-N1N (respectively composed of 10 mM KVp-N1 and KVp -N1N polypeptide stock solution prepared by dilution in complete medium), polypeptides KVp-R14 and KVp-R14N (respectively prepared by dilution of 10mM KVp-R14 and KVp-R14N polypeptide stock solution in complete medium), polypeptides KVp-C15 and KVp-C15N (respectively prepared from 10 mM KVp-C15 and KVp-C15N polypeptide stock solutions diluted in complete medium), mixed and incubated for 1 h, and mixed with polypeptide KVp-N1N with dose gradients of 100 ⁇ M, 10 ⁇ M, 1 ⁇ M, 100 n
  • step (3) Adding the mixed incubated pseudovirus polypeptide mixture to the cell culture system of step (1) to infect 293T-ACE2+ cells, infecting 293T-ACE2 + cells at 37°C for 1 hour, then changing to normal medium and continuing to culture for 47 hours. After culturing, cells were washed once with PBS, and chemiluminescence was detected by luciferase detection kit (Promega, Cat. No. E1500), and the infection rates of pseudoviruses in different groups were calculated accordingly.
  • luciferase detection kit Promega, Cat. No. E1500
  • polypeptide KVp-N1 and polypeptide KVp-N1N, polypeptide KVp-R14 and polypeptide KVp-R14N, polypeptide KVp-C15 and polypeptide KVp-C15N all have Strong and basically the same pseudovirus neutralization ability.
  • Figure 7D shows the infection rate of 293T-ACE2 + cells by SARS-CoV-2 pseudoviruses incubated with single polypeptide KVp-N1N, KVp-R14N, KVp-C15N and their mixed polypeptide compositions at a dose of 10 ⁇ M, respectively.
  • Figures 8A-8C show pseudovirus SARS-CoV-2 versus 293T-ACE2 after co-incubation with SARS-CoV-2 pseudovirus using peptides KVp-N1N, KVp-R14N, KVp-C15N in a dose range of 1 nM to 100 ⁇ M, respectively
  • the infection rate of + cells Figure 8A shows the infection rate of pseudovirus PSV on 293T-ACE2 + cells after co-incubation with PSV using the polypeptide KVp-N1N in the dose range of 1 nM to 100 ⁇ M
  • FIG. The infection rate of pseudovirus PSV on 293T-ACE2 + cells after co-incubation of polypeptide KVp-R14N with PSV
  • Fig. 8C shows the infection rate of pseudovirus PSV on 293T-ACE2 + cells after co-incubation of polypeptide KVp-C15N with PSV in a dose range of 1 nM to 100 ⁇ M Infection rate of cells.
  • 293T-ACE2 + cells were used to detect the cytotoxic effects of the polypeptides KVp-N1, KVp-N1N, KVp-R14, KVp-R14N, KVp-C15 and KVp-C15N prepared in Example 1, respectively, and the specific operations included the following operations.
  • CCK-8 working solution is composed of CCK8 stock solution and complete medium in a volume ratio of 1: 10 mixed to obtain, CCK8 stock solution purchased from Dojindo Molecular Technologies) 120 ⁇ L.
  • CCK-8 working solution is composed of CCK8 stock solution and complete medium in a volume ratio of 1: 10 mixed to obtain, CCK8 stock solution purchased from Dojindo Molecular Technologies
  • Figure 9A-9C and Figures 10A-10C show the results of Figures 9A-9C and Figures 10A-10C, wherein Figure 9A shows the effect of polypeptide KVp-N1 on the viability of 293T-ACE2 + cells within the tested dose range, and Figure 10A shows the polypeptide KVp-N1N in the tested dose range.
  • Figure 9B shows the effect of the polypeptide KVp-R14 on the viability of 293T-ACE2 + cells within the tested dose range
  • Figure 10B shows the effect of the polypeptide KVp-R14N on the viability of 293T-ACE2+ cells in the tested dose range.
  • Figure 9C shows the effect of the polypeptide KVp-C15 on the viability of 293T-ACE2 + cells within the tested dose range
  • Figure 10C shows the effect of the polypeptide KVp-C15N in the tested dose range. The effect on the viability of 293T-ACE2 + cells within the range of doses.
  • the polypeptides provided by the present invention and their compositions can bind to the S1 protein of SARS-CoV-2 virus and its RBD segment, and thus can block the S1 protein of SARS-CoV-2 virus and its RBD segment and its targets (such as ciliated bronchial epithelial cells and type II lungs)
  • the binding of angiotensinase 2 (ACE2)) on the cell surface and thus can prevent the SARS-CoV-2 virus from infecting epithelial cells and lung cells, and showed strong neutralization ability against SARS-CoV-2 pseudovirus
  • various polypeptides in the polypeptide composition work together, showing a significantly higher neutralization ability to the SARS-CoV-2 pseudovirus when using a single poly
  • 2019-nCoV pneumonia The infection mechanism of 2019-nCoV pneumonia is that SARS-CoV-2 virus binds to angiotensinase 2 (ACE2) of ciliated bronchial epithelial cells and type II pneumocytes through the RBD domain of its surface spike protein S1 subunit. Therefore, the polypeptide and its composition provided by the present invention can not only be used to prepare a blocker that blocks the binding of the S1 protein of the SARS-CoV-2 virus and its RBD segment to ACE2, but also can be used to prepare a treatment for novel coronavirus Drugs for viral pneumonia to achieve the purpose of targeted treatment of new coronavirus pneumonia.
  • ACE2 angiotensinase 2
  • the polypeptide and the polypeptide composition provided by the present invention have the characteristics of low molecular weight, high biological activity, and are not easy to accumulate in the body, and can not only be used as a drug by itself, but also can be used in combination with other drug molecules (such as anti-inflammatory drugs, anti-infective drugs, etc.) It can be used in combination, and there is less interaction with other drugs when compounding; at the same time, these polypeptides and their compositions also have the advantages of easy synthesis and molecular modification, and low production cost, so they can be produced and applied on a large scale. Industrial applicability
  • the polypeptide and the polypeptide composition provided by the present invention can block the binding of the SARS-CoV-2 virus and the key target protein angiotensinase (ACE2) protein on the surface of the host cell by combining with the S1 protein, thereby preventing the novel coronavirus pneumonia.
  • ACE2 angiotensinase
  • the treatment provides feasible targeted drugs suitable for industrial application.

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Abstract

本发明公开了一种与SARS-CoV-2刺突蛋白特异性结合的多肽、多肽组合物及其制备方法与应用。该多肽及多肽组合物通过与S1蛋白的结合，可以阻断SARS-CoV-2病毒与其宿主细胞表面的关键靶标蛋白血管紧张素酶(ACE2)蛋白的结合，从而为新型冠状病毒肺炎的治疗提供可行的靶向药物。

Description

与SARS-CoV-2刺突蛋白特异性结合的多肽、多肽组合物及其制备方法与应用 技术领域
本发明涉及生物医药技术领域，特别是涉及一种与SARS-CoV-2刺突蛋白(尤其是SARS-CoV-2表面刺突S1蛋白)特异性结合的多肽、多肽组合物，以及该多肽和多肽组合物的制备方法与应用。
背景技术
新型冠状病毒肺炎(简称新冠肺炎)的病原是一种新型冠状病毒SARS-CoV-2。当前对于新型冠状病毒肺炎的治疗药物主要是广谱的抗病毒药物和缓解症状的药物，尚缺少有效的靶向药。
发明内容
本发明的主要目的，第一方面，提供一种与SARS-CoV-2刺突蛋白特异性结合的多肽组合物，包含多肽KVp-N、多肽KVp-R和多肽KVp-C中的一种或多种；多肽KVp-N含有如SEQ ID NO:1所示的氨基酸序列，多肽KVp-R含有如SEQ ID NO:3所示的氨基酸序列，多肽KVp-C含有如SEQ ID NO:5所示的氨基酸序列。
多肽KVp-N、多肽KVp-R和多肽KVp-C的末端用含巯基的肽片段修饰；优选的，所述含巯基的肽片段的氨基酸序列为CCPPPP。
用含巯基的肽片段修饰的多肽KVp-N的氨基酸序列如SEQ ID NO:2所示；
用含巯基的肽片段修饰的多肽KVp-R的氨基酸序列如SEQ ID NO:4所示；
用含巯基的肽片段修饰的多肽KVp-C的氨基酸序列如SEQ ID NO:6所示。
所述多肽组合物包含以上所列至少一条多肽，当包含两条或两条以上多肽时，各多肽以任意比例混合。优选，多肽KVp-N、多肽KVp-R和多肽KVp-C三种类型多肽以等摩尔比混合。
第二方面，本发明提供一种与SARS-CoV-2刺突蛋白特异性结合的多肽，命名为多肽KVp-N，含有如SEQ ID NO:1所示的氨基酸序列。
第三方面，本发明提供一种与SARS-CoV-2刺突蛋白特异性结合的多肽，命名为多肽KVp-R，含有如SEQ ID NO:3所示的氨基酸序列。
第四方面，提供一种与SARS-CoV-2刺突蛋白特异性结合的多肽，命名为多肽KVp-C，含有如SEQ ID NO:5所示的氨基酸序列。
其末端用含巯基的肽片段修饰；优选的，所述含巯基的肽片段的氨基酸序列为CCPPPP。
所述多肽KVp-N的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示；
所述多肽KVp-R的氨基酸序列如SEQ ID NO:3或SEQ ID NO:4所示；
所述多肽KVp-C的氨基酸序列如SEQ ID NO:5或SEQ ID NO:6所示。
第五方面，本发明提供了制备上述多肽的方法，使用标准固相多肽合成方法合成得到。
第六方面，本发明提供了制备上述多肽组合物的方法，使用标准固相多肽合成方法合成得到多肽后，将多肽混合，即得到多肽组合物。
第七方面，本发明提供了上述多肽或多肽组合物在制备治疗新型冠状病毒肺炎药物中的应用，优选的，所述新型冠状病毒为SARS-CoV-2。
第八方面，本发明提供了上述多肽或多肽组合物在制备用于阻断SARS-CoV-2表面刺突S1蛋白与血管紧张素酶2(ACE2)结合的阻断剂中的应用；优选的，所述血管紧张素酶2(ACE2)存在于纤毛支气管上皮细胞或II型肺细胞表面。
该应用中，利用所述多肽或多肽组合物特异性地阻断SARS-CoV-2表面刺突S1蛋白与血管紧张素酶2(ACE2)结合。具体为：
利用所述多肽或多肽组合物特异性地与SARS-CoV-2表面刺突S1蛋白结合，以阻断SARS-CoV-2表面刺突S1蛋白与血管紧张素酶2(ACE2)结合；和/或
利用所述多肽或多肽组合物特异性地与SARS-CoV-2表面刺突S1蛋白亚基的RBD结构域结合，以阻断SARS-CoV-2表面刺突S1蛋白与血管紧张素酶2(ACE2)结合。
基于以上几方面内容，经实验证明，本发明提供的与SARS-CoV-2表面刺突S1蛋白特异性结合的多肽或多肽组合物通过与S1蛋白的结合，可以阻断SARS-CoV-2病毒与其宿主细胞表面的关键靶标蛋白血管紧张素酶(ACE2)蛋白的结合，从而有潜力为新型冠状病毒肺炎的治疗提供可行的靶向药物。
附图说明
图1A-1C分别为多肽KVp-N1、多肽KVp-R14、多肽KVp-C15对S1蛋白的结合能力检测曲线；
图2A-2C分别为多肽KVp-N1、多肽KVp-R14、多肽KVp-C15对RBD蛋白的结合能力检测曲线；
图3A-3C分别为与不同浓度的多肽KVp-N1、多肽KVp-R14、多肽KVp-C15共孵育后S1蛋白结合人肺癌细胞系A549细胞能力的Western Blot检测胶图；
图3D为与多肽KVp-N1、多肽KVp-R14、多肽KVp-C15以及多肽组合物共孵育后S1蛋白结合人肺癌细胞系A549细胞能力的Western Blot检测胶图；
图4A-4C分别为与不同浓度的多肽KVp-N1、多肽KVp-R14、多肽KVp-C15共 孵育后RBD蛋白结合A549细胞能力的Western Blot检测胶图；
图5A-5C分别为与不同浓度的多肽KVp-N1、多肽KVp-R14、多肽KVp-C15共孵育后S1蛋白结合293T-ACE2 +细胞能力的Western Blot检测胶图；
图6A-6C为与不同浓度的多肽KVp-N1、多肽KVp-R14、多肽KVp-C15共孵育后RBD蛋白结合293T-ACE2 +细胞能力的Western Blot检测胶图；
图6D为与多肽KVp-N1、多肽KVp-R14、多肽KVp-C15以及多肽组合物共孵育后RBD蛋白结合293T-ACE2 +细胞能力的Western Blot检测胶图；
图7为与多肽共孵育后SARS-CoV-2假病毒对293T-ACE2 +细胞的感染率统计结果柱状图；其中A幅为多肽KVp-N1或多肽KVp-N1N共孵育后，B幅为多肽KVp-R14或多肽KVp-R14N共孵育后，C幅为多肽KVp-C15或多肽KVp-C15N共孵育后；
图7D为分别与单一多肽KVp-N1N、KVp-R14N、KVp-C15N以及多肽组合物共孵育后SARS-CoV-2假病毒对293T-ACE2 +细胞的感染率统计结果柱状图；
图8A-8C分别为与不同浓度的多肽KVp-N1N、多肽KVp-R14N、多肽KVp-C15N共孵育后SARS-CoV-2假病毒对293T-ACE2 +细胞的感染率统计结果柱状图；
图9A-9C分别为不同浓度的多肽KVp-N1、多肽KVp-R14、多肽KVp-C15对293T-ACE2 +细胞活性影响的统计结果柱状图；
图10A-10C分别为不同浓度的多肽KVp-N1N、多肽KVp-R14N、多肽KVp-C15N对293T-ACE2 +细胞活性影响的统计结果柱状图。
具体实施方式
研究已经证实，SARS-CoV-2通过其表面刺突蛋白S1亚基的RBD结构域与纤毛支气管上皮细胞和II型肺细胞表面的血管紧张素酶2(ACE2)结合，致细胞感染，从而使人罹患新型冠状病毒肺炎。
本发明设计了多条与SARS-CoV-2表面刺突S1亚基或其RBD结构域特异性结合的多肽，利用该多肽来阻断SARS-CoV-2病毒与其宿主细胞表面的关键靶标蛋白血管紧张素酶(ACE2)蛋白的结合；该多肽分别命名为多肽KVp-N、多肽KVp-R、多肽KVp-C。这些多肽均是通过人工合成的方式制备得到。
其中，多肽KVp-N含有氨基酸序列GDGVYYPRDVFDSSVLDSTQR(记为KVp-N1N)；或者在KVp-N1N的末端用肽片段CCPPPP修饰，修饰后的多肽KVp-N氨基酸序列可为CCPPPPGDGVYYPRDVFDSSVLDSTQR(记为KVp-N1)，在KVp-N1中引入巯基(由肽片段CCPPPP引入的)，目的是通过巯基使多肽与含金元素的物质(例如金纳米颗粒)结合，这些结合将有助于扩展多肽的应用性，例如在应用中对多肽进行检测与识别。
多肽KVp-R含有氨基酸序列GDLFDDSNLDPFRDRISTRR(记为KVp-R14N)；或者在 KVp-R14N的末端用肽片段CCPPPP修饰，修饰后的多肽KVp-R氨基酸序列可为CCPPPPGDLFDDSNLDPFRDRISTRR(记为KVp-R14)，本发明在KVp-R14中引入巯基(由肽片段CCPPPP引入的)，目的是通过巯基使多肽与含金元素的物质(例如金纳米颗粒)结合，这些结合将有助于扩展多肽的应用性，例如在应用中对多肽进行检测与识别。
多肽KVp-C含有氨基酸序列GDGVYYPRDVFDSSVLDSTQR(记为KVp-C15N)；或者在KVp-C15N的末端用肽片段CCPPPP修饰，修饰后的多肽KVp-C氨基酸序列可为CCPPPPGDGVYYPRDVFDSSVLDSTQR(记为KVp-C15)，本发明在KVp-C15中引入巯基(由肽片段CCPPPP引入的)，目的是通过巯基使多肽与含金元素的物质(例如金纳米颗粒)结合，这些结合将有助于扩展多肽的应用性，例如在应用中对多肽进行检测与识别。
基于这些多肽，本发明还提供一种多肽组合物，该组合物包括至少一条能与SARS-CoV-2表面刺突蛋白S1亚基特异性结合的多肽。
本发明通过实验证明了上述多肽及其组合可阻断S1蛋白与ACE2蛋白的结合，从而阻止SARS-CoV-2感染上皮细胞和肺细胞，使得这些多肽及其组合物可用作靶向治疗新型冠状病毒肺炎的药物。
下面将结合实施例对本发明的实施方案进行详细描述。实施例中未注明具体技术或条件者，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。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。实施例中所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。如无特别说明，各实施例中相同名称的材料或试剂内容相同。
除非特别指明，以下实施例中所用的磷酸盐缓冲液均为1×PBS溶液(pH＝7.4)。
实施例1：多肽、多肽组合物及其制备
该实施例通过标准固相多肽合成方法制备得到靶向SARS-CoV-2表面刺突蛋白S1的多条多肽，代号分别为KVp-N1N和KVp-N1(统称多肽KVp-N)、KVp-R14N和KVp-R14(统称多肽KVp-R)、KVp-C15N和KVp-C15(统称多肽KVp-C)，这些多肽的氨基酸序列如下表1所示。
表1：多肽的氨基酸序列
代号 氨基酸序列
KVp-N1N GDGVYYPRDVFDSSVLDSTQR(SEQ ID NO:1)
KVp-N1 CCPPPPGDGVYYPRDVFDSSVLDSTQR(SEQ ID NO:2)
KVp-R14N GDLFDDSNLDPFRDRISTRR(SEQ ID NO:3)
KVp-R14 CCPPPPGDLFDDSNLDPFRDRISTRR(SEQ ID NO:4)
KVp-C15N GDGVYYPRDVFDSSVLDSTQR(SEQ ID NO:5)
KVp-C15 CCPPPPGDGVYYPRDVFDSSVLDSTQR(SEQ ID NO:6)
经高效液相色谱法和质谱的鉴定，确证得到的产品为表1中的目标多肽，六条多肽均为白色粉末，纯度均≥98％。其中代号为KVp-N1、KVp-R14和KVp-C15的多肽分别为在代号为KVp-N1N、KVp-R14N和KVp-C15N的多肽的末端引入CCPPPP修饰，这种修饰使得在多肽KVp-N1、KVp-R14和KVp-C15中引入了巯基，该巯基使多肽KVp-N1、KVp-R14和KVp-C15可以与金(例如金纳米颗粒)结合(例如实施例2利用该结合实现对多肽结合能力的检测)。
本实施例的多肽组合物由至少两种表1中的多肽组成，如可为KVp-N1N和KVp-R14混合而成，可为KVp-N1、KVp-C15和KVp-C15N混合而成，可为KVp-N1、KVp-R14和KVp-C15混合而成，可为KVp-R14、KVp-N1N、KVp-R14N和KVp-C15混合而成，可为KVp-N1、KVp-R14、KVp-C15、KVp-N1N和KVp-C15N混合而成，可为KVp-N1、KVp-R14、KVp-C15、KVp-N1N、KVp-R14N和KVp-C15N混合而成；混合比例不限。
以下实施例中，为了验证多肽及其组合物的生物学效应，用完全培养基(商购)将各多肽分别配制成浓度为10mM的母液，将各多肽的母液按一定比例混合形成多肽组合物的母液；为了检测多肽组合物的结合能力，用双蒸水(ddH 2O)将各多肽分别配制成浓度为0.5mg/mL的多肽溶液，将各多肽溶液按一定比例混合形成多肽组合物溶液。
实施例2：多肽的结合能力
该实施例以利用石英晶体微天平法(QCM)检测实施例1合成制备的多肽KVp-N1、KVp-R14、KVp-C15(该检测中需利用这些多肽中的巯基)分别与SARS-CoV-2的S1蛋白或其RBD段(均购自北京义翘神州科技股份有限公司)的结合能力，其中使用的
Figure PCTCN2022078627-appb-000001
四通道耗散型石英微天平购自Biolin Scientific。
实验2.1：多肽与S1蛋白的结合能力
(1)使用清洗液(浓硫酸和双氧水体积比3:1)清洗
Figure PCTCN2022078627-appb-000002
四通道耗散型石英微天平的金涂层石英芯片3次，每次三分钟，并用ddH 2O冲洗；
(2)按照
Figure PCTCN2022078627-appb-000003
四通道耗散型石英微天平操作说明，分别将浓度为0.5mg/mL的KVp-N1、KVp-R14、KVp-C15多肽溶液与金涂层石英芯片室温孵育30min使多肽KVp-N1、KVp-R14、KVp-C15分子分别通过巯基结合在金涂层石英芯片上；
(3)用ddH 2O冲洗芯片，并用氮气吹干。将芯片嵌入反应室，以PBS配制的浓度为13nM的S1蛋白为流动相，经过管路流经金涂层石英芯片，QCM检测S1蛋白分别 与多肽KVp-N1、KVp-R14、KVp-C15的结合。
结果如图1A-1C所示，其中图1A表示QCM检测S1蛋白与多肽KVp-N1的结合曲线，图1B表示QCM检测S1蛋白与多肽KVp-R14的结合曲线、图1C表示QCM检测S1蛋白与多肽KVp-C15的结合曲线。可见S1蛋白流动相流经管路时，金涂层石英芯片振荡频率降低，表明S1蛋白分别结合到了多肽KVp-N1、KVp-R14、KVp-C15上，即多肽KVp-N1、KVp-R14、KVp-C15均可以与SARS-CoV-2的S1蛋白结合。
实验2.2：多肽KVp-N1、KVp-R14、KVp-C15与RBD段蛋白的结合能力
实验2.2的具体操作与上述实验2.1的操作相同，不同之处仅在于使用以PBS配制的浓度为20nM的RBD蛋白溶液替换S1蛋白溶液作为流动相。
多肽KVp-N1、KVp-R14、KVp-C15分别与RBD段的结合能力的QCM检测结果分别如图2A-2C所示，其中图2A表示QCM检测RBD段与多肽KVp-N1的结合曲线，图2B表示QCM检测RBD段与多肽KVp-R14的结合曲线、图2C表示QCM检测RBD段与多肽KVp-C15的结合曲线。可见RBD蛋白流动相流经管路时，金涂层石英芯片振荡频率降低，表明RBD蛋白分别结合到了多肽KVp-N1、KVp-R14、KVp-C15上，即多肽KVp-N1、KVp-R14、KVp-C15均可以与SARS-CoV-2的RBD段结合。
综上实验结果证明多肽KVp-N(KVp-N1N和KVp-N1)、多肽KVp-R(KVp-R14N和KVp-R14)、以及多肽KVp-C(KVp-C15N和KVp-C15)均能与SARS-CoV-2的S1蛋白或其RBD段结合。
实施例3：多肽及其组合物阻止S1蛋白、RBD蛋白与靶细胞的结合
该实施例中以实施例1合成制备的多肽KVp-N1、KVp-R14、KVp-C15及其等摩尔浓度混合物(即由等摩尔浓度多肽KVp-N1、KVp-R14和KVp-C15组成的多肽组合物)为例，以人肺癌细胞系A549细胞(购自中国医学科学院基础医学研究所细胞资源中心)和过表达ACE2蛋白的293T细胞(293T-ACE2 +)(购自北京义翘神州科技股份有限公司)为靶细胞验证模拟多肽KVp-N1、KVp-R14、KVp-C15及其组合物阻止SARS-CoV-2的S1蛋白或其RBD段与靶细胞的结合效应。
实验3.1：多肽及其组合物对S1蛋白与人肺癌细胞系A549细胞结合能力的影响(1)将A549细胞以2×10 5cells/well的密度接种于6孔板中，37℃培养箱中过夜培养，使细胞贴壁；
(2)将S1蛋白用PBS稀释至1μM的S1蛋白溶液，再将其在室温下分别与0.1μM、1μM、10μM、100μM(分别由10mM的KVp-N1、KVp-R14、KVp-C15多肽母液经完全培养基稀释制备)的多肽KVp-N1、KVp-R14、KVp-C15共孵育1h，同步设置不与多肽KVp-N1、KVp-R14、KVp-C15共孵育的S1蛋白溶液作为对照组；
同时，将S1蛋白用PBS稀释至1μM的S1蛋白溶液，再将其在室温下与三种单一多肽KVp-N1、KVp-R14、KVp-C15(单一多肽的浓度均为1μM，用完全培养基配稀释母液)和多肽组合物(各多肽的浓度均为1μM，用完全培养基稀释母液)共孵育1h，同步设置不与多肽共孵育的S1蛋白溶液作为对照组；
(3)分别将上述各组的S1蛋白与多肽(KVp-N1、KVp-R14、KVp-C15和多肽组合物)的混合液和对照组的混合液加入到细胞培养体系中，使其中的S1蛋白终浓度均为10nM；
(4)共培养1h后，用PBS洗两遍，用RIPA裂解液裂解细胞抽提总蛋白，用Western Blot方法检测总蛋白中S1蛋白的含量。
不同浓度的多肽KVp-N1、KVp-R14、KVp-C15的结果分别如图3A-3C所示，可见对照组有较强的S1蛋白阳性信号，表明S1蛋白与A549细胞发生了结合；而当S1蛋白先分别与多肽KVp-N1、KVp-R14、KVp-C15孵育1h，再加入到细胞培养体系中，S1蛋白信号的强度明显降低，且在1nM至1000nM的多肽浓度范围内，随着多肽KVp-N1、KVp-R14、KVp-C15的浓度升高，S1蛋白阳性信号的强度就变弱，表明多肽KVp-N1、KVp-R14、KVp-C15抑制了S1蛋白对A549细胞的结合。
多肽组合物的结果如图3D所示，可见对照组有较强的S1蛋白阳性信号，表明S1蛋白与A549细胞发生了结合；而当S1蛋白先与多肽(KVp-N1、KVp-R14、KVp-C15和多肽组合物)孵育1h，再加入到细胞培养体系中，S1蛋白信号的强度均明显降低，表明多肽抑制了S1蛋白对A549细胞的结合，并且其中多肽组合物抑制S1蛋白对A549细胞的结合的能力显著强于三种单一的多肽，显示了多肽组合物中三种单一多肽共同作用效果(该效果还将在以下实施例4中进一步描述)。
实验3.2：多肽及其组合物对RBD蛋白与人肺癌细胞系A549细胞结合能力的影响
实验3.2的具体操作同上述实验3.1，不同之处仅在于用RBD蛋白溶液替换S1蛋白溶液分别与0.1μM、1μM、10μM、100μM的多肽KVp-N1、KVp-R14、KVp-C15，以及多肽KVp-N1、KVp-R14、KVp-C15和多肽组合物共孵育。
用Western Blot方法检测总蛋白中RBD蛋白的含量，不同浓度的多肽KVp-N1、KVp-R14、KVp-C15的结果分别如图4A-4C所示，可见对照组有较强的RBD蛋白阳性信号，表明RBD蛋白与A549细胞发生了结合；而分别与多肽KVp-N1、KVp-R14、KVp-C15孵育之后的RBD蛋白在A549细胞上的结合量显著少于对照组，表明多肽KVp-N1、KVp-R14、KVp-C15抑制了RBD蛋白对A549细胞的结合，且随着多肽浓度的增加，这种抑制作用越明显。
与图3D类似，对照组有较强的RBD蛋白阳性信号，表明RBD蛋白与细胞发生了结 合，而当RBD蛋白先与多肽(KVp-N1、KVp-R14、KVp-C15和多肽组合物)孵育1h，再加入到细胞培养体系中，RBD蛋白信号的强度均明显降低，表明多肽抑制了RBD蛋白对A549细胞的结合，并且其中多肽组合物抑制RBD蛋白对A549细胞的结合的能力强于三种单一多肽。
实验3.3：多肽及其组合物对RBD蛋白与人肺癌细胞系293T-ACE2 +细胞结合能力的影响
实验3.3的具体操作同上述实验3.2，不同之处仅在于用293T-ACE2 +细胞替换A549细胞作为靶细胞。
用Western Blot方法检测总蛋白中RBD蛋白的含量，不同浓度的多肽KVp-N1、KVp-R14、KVp-C15的结果分别如图6A-6C所示，可见对照组有较强的RBD蛋白阳性信号，表明RBD蛋白与293T-ACE2 +细胞发生了结合；而分别与多肽KVp-N1、KVp-R14、KVp-C15孵育之后的RBD蛋白在293T-ACE2 +细胞上的结合量显著少于对照组，表明多肽抑制了RBD蛋白对293T-ACE2 +细胞的结合，且随着多肽浓度的增加，这种抑制作用越明显。
综上实验结果，证明多肽KVp-N(KVp-N1和KVp-N1N)、多肽KVp-R(KVp-R14和KVp-R14N)、多肽KVp-C(KVp-C15和KVp-C15N)能阻止S1蛋白或其RBD蛋白与靶细胞(人肺癌细胞系A549细胞和人肺癌细胞系293T-ACE2 +细胞)的结合。
多肽组合物的结果如图6D所示，可见对照组有较强的RBD蛋白阳性信号，表明RBD蛋白与293T-ACE2 +细胞发生了结合，而当RBD蛋白先分别与多肽(KVp-N1、KVp-R14、KVp-C15和多肽组合物)孵育1h，再加入到细胞培养体系中，RBD蛋白信号的强度均明显降低，表明多肽抑制了RBD蛋白对293T-ACE2 +细胞的结合，并且其中多肽组合物抑制RBD蛋白对293T-ACE2 +细胞的结合的能力显著强于三种单一多肽，显示了多肽组合物中三种单一多肽共同作用(该共同作用的效果将在以下实施例4中进一步描述)。
实验3.4：多肽对S1蛋白与细胞系293T-ACE2 +细胞结合能力的影响
实验3.4的具体操作同上述实验3.1，不同之处仅在于用293T-ACE2 +细胞替换A549细胞作为靶细胞。
用Western Blot方法检测总蛋白中S1蛋白的含量，不同浓度的多肽KVp-N1、KVp-R14、KVp-C15的结果分别如图5A-5C所示，可见对照组有较强的S1蛋白阳性信号，表明S1蛋白与293T-ACE2 +细胞发生了结合；而当S1蛋白先分别与多肽KVp-N1、KVp-R14、KVp-C15孵育1h，再加入到细胞培养体系中，S1蛋白信号的强度明显降低，表明多肽KVp-N1、KVp-R14、KVp-C15抑制了S1蛋白对293T-ACE2 +细胞的结合，且随 着多肽浓度的增加，这种抑制作用越明显。
与图6D类似，对照组有较强的S1蛋白阳性信号，表明S1蛋白与293T-ACE2 +细胞发生了结合，而当S1蛋白先与多肽(KVp-N1、KVp-R14、KVp-C15和多肽组合物)孵育1h，再加入到细胞培养体系中，S1蛋白信号的强度明显降低，表明多肽抑制了S1蛋白对293T-ACE2 +细胞的结合，并且其中多肽组合物抑制S1蛋白对A549细胞的结合的能力强于三种单一多肽。
实施例4：多肽及其组合物对SARS-CoV-2假病毒的中和能力
该实施例利用假病毒中和法检测实施例1合成制备的多肽KVp-N1N、KVp-R14N、KVp-C15N及其等摩尔浓度混合物(即由等摩尔浓度多肽KVp-N1、KVp-R14和KVp-C15组成的多肽组合物)对SARS-CoV-2假病毒侵染细胞的阻断效应，其中所使用的SARS-CoV-2假病毒(PSV)购自吉满生物科技(上海)有限公司，其带有荧光素酶和绿色荧光蛋白(GFP)标签。具体包括以下操作：
(1)将293T-ACE2 +细胞接种于96孔板中，每孔2×10 4个细胞，过夜培养使其贴壁；
(2)将带有荧光素酶和绿色荧光蛋白(GFP)标签的假病毒(＞10 6copies/mL)分别与10μM的多肽KVp-N1和KVp-N1N(分别由10mM的KVp-N1和KVp-N1N多肽母液经完全培养基稀释制备)、多肽KVp-R14和KVp-R14N(分别由10mM的KVp-R14和KVp-R14N多肽母液经完全培养基稀释制备)、多肽KVp-C15和KVp-C15N(分别由10mM的KVp-C15和KVp-C15N多肽母液经完全培养基稀释制备)混合孵育1h，以及分别与剂量梯度为100μM、10μM、1μM、100nM、10nM、1nM的多肽KVp-N1N(分别由10mM的KVp-N1N多肽母液经完全培养基稀释制备)，剂量梯度为100μM、10μM、1μM、100nM、10nM、1nM的多肽KVp-R14N(分别由10mM的KVp-R14N多肽母液经完全培养基稀释制备)，剂量梯度为100μM、10μM、1μM、100nM、10nM、1nM的多肽KVp-C15N(分别由10mM的KVp-C15N多肽母液经完全培养基稀释制备)，以及三种单一多肽(KVp-N1N、KVp-R14N、KVp-C15N，剂量分别为10μM)溶液和多肽组合物(其中每种多肽的浓度均为10μM)溶液混合孵育1h；
(3)将混合孵育后的假病毒多肽混合液加入到步骤(1)的细胞培养体系中感染293T-ACE2 +细胞，37℃下感染1h后换用正常培养基继续培养47h。培养结束后，PBS洗一次细胞，采用荧光素酶检测试剂盒(普洛麦格公司，货号E1500)检测化学发光，并据此计算不同组别中假病毒的感染率。
多肽KVp-N1和KVp-N1N、多肽KVp-R14和KVp-R14N、多肽KVp-C15和KVp-C15N的结果如图7A-7C所示，其中PSV表示未与多肽孵育的SARS-CoV-2假病毒 的感染率，KVp-N1和KVp-N1N、KVp-R14和KVp-R14N、KVp-C15和KVp-C15N分别表示使用10μM的多肽与SARS-CoV-2假病毒混合孵育后SARS-CoV-2假病毒对293T-ACE2 +细胞的感染率，明显可见，同等剂量下，多肽KVp-N1和多肽KVp-N1N、多肽KVp-R14和多肽KVp-R14N、多肽KVp-C15和多肽KVp-C15N均具有较强的和基本相同的假病毒中和能力。
图7D表示分别与剂量10μM的单一多肽KVp-N1N、KVp-R14N、KVp-C15N及其混合的多肽组合物孵育的SARS-CoV-2假病毒对293T-ACE2 +细胞的感染率。
根据图7D所示结果，明显可见当将三种多肽(KVp-N1N、KVp-R14N、KVp-C15N)混合后成为多肽组合物，与SARS-CoV-2假病毒共孵育后可以显著降低SARS-CoV-2假病毒对293T-ACE2 +细胞的感染率(感染率仅为25％左右)，表明感染率的降低并非依赖三种肽组合后总浓度的提高，而是由于多肽组合物中各多肽靶向的位点不同，在抑制SARS-CoV-2的S1蛋白或其RBD蛋白与靶细胞的结合方面能共同发挥作用，使得在中和SARS-CoV-2假病毒感染能力方面效果显著。
图8A-8C分别示出了使用1nM～100μM的剂量范围的多肽KVp-N1N、KVp-R14N、KVp-C15N与SARS-CoV-2假病毒共孵育后假病毒SARS-CoV-2对293T-ACE2 +细胞的感染率，图8A表示使用1nM～100μM的剂量范围的多肽KVp-N1N与PSV共孵育后假病毒PSV对293T-ACE2 +细胞的感染率，图8B表示使用1nM～100μM的剂量范围的多肽KVp-R14N与PSV共孵育后假病毒PSV对293T-ACE2 +细胞的感染率，图8C表示使用1nM～100μM的剂量范围的多肽KVp-C15N与PSV共孵育后假病毒PSV对293T-ACE2 +细胞的感染率。
根据图8A-8C幅所示结果，证明多肽KVp-N(KVp-N1和KVp-N1N)、KVp-R(KVp-R14和KVp-R14N)、KVp-C(KVp-C15和KVp-C15N)均对SARS-CoV-2假病毒具有较强的中和能力，中和能力在50％左右。并且在检测的1nM～100μM剂量范围内，多肽抑制假病毒感染细胞的效率与其浓度之间并没有依赖性，表现出抑制SARS-CoV-2假病毒对293T-ACE2 +细胞的感染的效率基本一致。
实施例5：多肽及其组合物的细胞毒作用
该实施例利用293T-ACE2 +细胞分别检测实施例1制备的多肽KVp-N1、KVp-N1N、KVp-R14、KVp-R14N、KVp-C15和KVp-C15N的细胞毒作用，具体包括以下操作。
(1)取对数期生长的293T-ACE2 +细胞，用完全培养基调整细胞密度为10 5cells/mL，接种于96孔板中，每孔100μL；
(2)取多肽KVp-N1、KVp-N1N、KVp-R14、KVp-R14N、KVp-C15和KVp-C15N分别加入每孔的细胞培养体系中，使多肽终浓度分别为1nM、10nM、100nM、1μM、10 μM、100μM，37℃培养48h；
(3)培养结束后弃上清后用PBS洗一次，每孔加入cell counting kit-8(CCK-8)工作液(CCK-8工作液是由CCK8原液和完全培养基按体积比为1:10混合得到，CCK8原液购自Dojindo Molecular Technologies公司)120μL。混匀后37℃孵育3h后检测450nm及630nm(参比波长)处的吸光值，计算细胞活率。
结果如图9A-9C、图10A-10C所示，其中图9A为多肽KVp-N1在所检测的剂量范围内对293T-ACE2 +细胞活率的影响，图10A为多肽KVp-N1N在所检测的剂量范围内对293T-ACE2 +细胞活率的影响，图9B为多肽KVp-R14在所检测的剂量范围内对293T-ACE2 +细胞活率的影响，图10B为多肽KVp-R14N在所检测的剂量范围内对293T-ACE2 +细胞活率的影响，图9C为多肽KVp-C15在所检测的剂量范围内对293T-ACE2 +细胞活率的影响，图10C为多肽KVp-C15N在所检测的剂量范围内对293T-ACE2 +细胞活率的影响。可见六种多肽在所检测的剂量范围内对293T-ACE2 +细胞活率的影响均不大；结合图7A-7C、图8A-8C所示的结果，在所检测的1nM～100μM剂量范围内，单一多肽对293T-ACE2 +细胞均无细胞毒作用，且具有良好的SARS-CoV-2假病毒中和能力；那么由KVp-N1、KVp-N1N、KVp-R14、KVp-R14N、KVp-C15和KVp-C15N中的任两种以上多肽在所检测的1nM～100μM剂量范围内混合而成的多肽组合物也必然对293T-ACE2 +细胞均无细胞毒作用。
综上实施例结果可见，本发明提供的多肽及其组合物(例如KVp-N1、KVp-R14、KVp-C15、KVp-N1N、KVp-R14N和KVp-C15N中的任两种以上多肽混合而成)可以与SARS-CoV-2病毒的S1蛋白及其RBD段结合，并由此可以阻断SARS-CoV-2病毒的S1蛋白及其RBD段与其靶标(例如纤毛支气管上皮细胞和II型肺细胞表面的血管紧张素酶2(ACE2))的结合，并因此可以阻止SARS-CoV-2病毒感染上皮细胞和肺细胞，并表现出对SARS-CoV-2假病毒较强的中和能力，且多肽组合物中各种多肽共同发挥作用，呈现出显著高于使用单一多肽时对SARS-CoV-2假病毒的中和能力。
罹患新型冠状病毒肺炎的感染机理是SARS-CoV-2病毒通过其表面刺突蛋白S1亚基的RBD结构域与纤毛支气管上皮细胞和II型肺细胞的血管紧张素酶2(ACE2)结合导致细胞被感染，因此本发明提供的多肽及其组合物不仅仅可以用于制备阻断SARS-CoV-2病毒的S1蛋白及其RBD段与ACE2结合的阻断剂，还可以用于制备治疗新型冠状病毒肺炎的药物，实现靶向治疗新型冠状病毒肺炎的目的。
本发明提供的多肽及多肽组合物具有分子量较低、生物活性高、在体内不易产生蓄积等特点，不仅本身可以作为药物，还可用于与其他药物分子(如抗炎药物、抗感染药物等)复合使用，且复合时与其他药物的相互作用较少；同时这些多肽及其组合物还具有易于合成和分子修饰、生产成本较低等优势，因此可以大规模生产和应用。 工业应用性
本发明提供的多肽及多肽组合物通过与S1蛋白的结合，可以阻断SARS-CoV-2病毒与其宿主细胞表面的关键靶标蛋白血管紧张素酶(ACE2)蛋白的结合，从而为新型冠状病毒肺炎的治疗提供可行的靶向药物，适于工业应用。

Claims (16)

  1. 一种与SARS-CoV-2刺突蛋白特异性结合的多肽组合物，其特征在于，包含多肽KVp-N、多肽KVp-R和多肽KVp-C中的一种或多种；多肽KVp-N含有如SEQ ID NO:1所示的氨基酸序列，多肽KVp-R含有如SEQ ID NO:3所示的氨基酸序列，多肽KVp-C含有如SEQ ID NO:5所示的氨基酸序列。
  2. 根据权利要求1所述多肽组合物，其特征在于，多肽KVp-N、多肽KVp-R和多肽KVp-C的末端用含巯基的肽片段修饰；优选的，所述含巯基的肽片段的氨基酸序列为CCPPPP。
  3. 根据权利要求1或2所述多肽组合物，其特征在于，
    用含巯基的肽片段修饰的多肽KVp-N的氨基酸序列如SEQ ID NO:2所示；
    用含巯基的肽片段修饰的多肽KVp-R的氨基酸序列如SEQ ID NO:4所示；
    用含巯基的肽片段修饰的多肽KVp-C的氨基酸序列如SEQ ID NO:6所示。
  4. 根据权利要求1或2或3所述多肽组合物，其特征在于，各多肽以任意比例混合；优选多肽KVp-N、多肽KVp-R和多肽KVp-C以等摩尔比混合。
  5. 一种与SARS-CoV-2刺突蛋白特异性结合的多肽，命名为多肽KVp-N，其特征在于，含有如SEQ ID NO:1所示的氨基酸序列。
  6. 一种与SARS-CoV-2刺突蛋白特异性结合的多肽，命名为多肽KVp-R，其特征在于，含有如SEQ ID NO:3所示的氨基酸序列。
  7. 一种与SARS-CoV-2刺突蛋白特异性结合的多肽，命名为多肽KVp-C，其特征在于，含有如SEQ ID NO:5所示的氨基酸序列。
  8. 根据权利要求5-7任一所述多肽，其特征在于，其末端用含巯基的肽片段修饰；优选的，所述含巯基的肽片段的氨基酸序列为CCPPPP。
  9. 根据权利要求8所述多肽，其特征在于，所述多肽KVp-N的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示；
    所述多肽KVp-R的氨基酸序列如SEQ ID NO:3或SEQ ID NO:4所示；
    所述多肽KVp-C的氨基酸序列如SEQ ID NO:5或SEQ ID NO:6所示。
  10. 一种制备权利要求5-9任一所述多肽的方法，其特征在于，使用标准固相多肽合成方法合成得到。
  11. 一种制备权利要求1-4任一所述多肽组合物的方法，其特征在于，使用标准固相多肽合成方法合成得到多肽后，将多肽混合，即得到多肽组合物。
  12. 权利要求1-4任一所述多肽组合物或权利要求5-9任一所述多肽在制备治疗新型冠状病毒肺炎药物中的应用，优选的，所述新型冠状病毒为SARS-CoV-2。
  13. 权利要求1-4任一所述多肽组合物或权利要求5-9任一所述多肽在制备用于阻断SARS-CoV-2表面刺突S1蛋白与血管紧张素酶2(ACE2)结合的阻断剂中的应用；优选的，所述血管紧张素酶2(ACE2)存在于纤毛支气管上皮细胞或II型肺细胞表面。
  14. 根据权利要求12或13所述应用，其特征在于，利用所述多肽组合物或多肽特异性地阻断SARS-CoV-2表面刺突S1蛋白与血管紧张素酶2(ACE2)结合。
  15. 根据权利要求14所述应用，其特征在于，利用所述多肽组合物或多肽特异性地与SARS-CoV-2表面刺突S1蛋白结合，以阻断SARS-CoV-2表面刺突S1蛋白与血管紧张素酶2(ACE2)结合。
  16. 根据权利要求14所述应用，其特征在于，利用所述多肽组合物或多肽特异性地与SARS-CoV-2表面刺突S1蛋白亚基的RBD结构域结合，以阻断SARS-CoV-2表面刺突S1蛋白与血管紧张素酶2(ACE2)结合。
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