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WO2024192698A1 - Antagoniste polypeptidique ciblant la glycoprotéine de fusion du virus respiratoire syncytial humain - Google Patents

Antagoniste polypeptidique ciblant la glycoprotéine de fusion du virus respiratoire syncytial humain Download PDF

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Publication number
WO2024192698A1
WO2024192698A1 PCT/CN2023/082969 CN2023082969W WO2024192698A1 WO 2024192698 A1 WO2024192698 A1 WO 2024192698A1 CN 2023082969 W CN2023082969 W CN 2023082969W WO 2024192698 A1 WO2024192698 A1 WO 2024192698A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
respiratory syncytial
syncytial virus
rsv
human respiratory
Prior art date
Application number
PCT/CN2023/082969
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English (en)
Chinese (zh)
Inventor
朱进
董服民
陈昌斌
Original Assignee
中科牧维(南京)生物科技有限公司
渥太华Hdl药物研发公司
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Application filed by 中科牧维(南京)生物科技有限公司, 渥太华Hdl药物研发公司 filed Critical 中科牧维(南京)生物科技有限公司
Priority to PCT/CN2023/082969 priority Critical patent/WO2024192698A1/fr
Publication of WO2024192698A1 publication Critical patent/WO2024192698A1/fr

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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/03—Herpetoviridae, e.g. pseudorabies virus
    • C07K14/05—Epstein-Barr virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention belongs to the field of biomedicine, and in particular, relates to a polypeptide and an application thereof in inhibiting the infection and reproduction of human respiratory syncytial virus.
  • RSV Human respiratory syncytial virus
  • Symptoms of RSV infection can include fever, cough, runny nose and wheezing, and in severe cases can cause bronchiolitis, pneumonia or other respiratory complications.
  • the immune response to RSV after a person is infected with RSV does not prevent RSV infection again. There is currently no specific drug to treat RSV infection, and there is no preventive vaccine.
  • Antiviral drugs such as ribavirin (a drug used for chronic hepatitis C virus infection) and remdesivir (a broad-spectrum antiviral drug), are often used in high-risk groups that have already been infected with RSV, such as premature infants and children with certain diseases.
  • ribavirin has limited effects and may have serious side effects, including anemia and respiratory distress.
  • the effectiveness of remdesivir remains uncertain.
  • a common preventive measure is to inject AstraZeneca's monoclonal antibody Palivizumab, but Palivizumab is only used for preventive administration and is applicable to a small group. Therefore, it is very necessary to develop highly effective and specific drugs for human respiratory syncytial virus.
  • the surface protein F (fusion glycoprotein) of human respiratory syncytial virus is responsible for the fusion of virus and host cell membranes, as well as the formation of syncytia between virus particles.
  • Surface protein F exists in the form of a trimer on human respiratory syncytial virus. After binding to the host cell surface, surface protein F undergoes conformational changes and inserts itself into the host cell membrane, causing the virus and host cell membrane to fuse. The final conformation of surface protein F is transformed into a more stable post-fusion protein structure. Whether the surface protein F of human respiratory syncytial virus can smoothly undergo structural rearrangement and protein conformational changes before and after fusion with the host cell plays a very important role in viral infection. By using small molecule compounds, antibodies, and peptides, it is possible to affect the conformational changes of the surface protein F of human respiratory syncytial virus, thereby preventing viral infection and reproduction.
  • Peptides have been used as clinical drugs for a long time, and the famous ones include insulin, oxytocin, somatostatin, cortisol, gonadotropin, calcitonin, parathyroid hormone, adrenocorticotropic hormone, etc.
  • Peptide drugs are used in the fields of diabetes, cancer, immune diseases, osteoporosis, multiple sclerosis, HIV, etc.
  • a polypeptide consisting of 16 amino acid residues matching the amino acid sequence of human respiratory syncytial virus surface protein F was designed to detect its effect of inhibiting human respiratory syncytial virus.
  • the inventor believes that the polypeptide antagonist has high specificity, can affect the conformational changes of human respiratory syncytial virus surface protein F, and inhibit the fusion of human respiratory syncytial virus and host cell membrane, thus completing the present invention.
  • One object of the present invention is to provide a polypeptide that can be used as a polypeptide antagonist for inhibiting human respiratory syncytial virus, in particular a polypeptide antagonist targeting human respiratory syncytial virus fusion glycoprotein.
  • Another object of the present invention is to provide the use of the polypeptide.
  • the present invention provides a polypeptide comprising a polypeptide fragment consisting of 16-18, preferably 16-17, or most preferably 16 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence of SEQ ID NO: 1 is as follows:
  • the polypeptide has the activity of inhibiting human respiratory syncytial virus.
  • the polypeptide of the present invention comprises a peptide segment consisting of an amino acid sequence selected from any one of SEQ ID NO: 2 and SEQ ID NO: 3. More preferably, the polypeptide of the present invention consists of a peptide segment consisting of an amino acid sequence selected from any one of SEQ ID NO: 2 and SEQ ID NO: 3.
  • one or more amino acid residues in the polypeptide of the present invention may be in the form of the D-enantiomer. More preferably, all amino acid residues in the polypeptide are in the form of the D-enantiomer.
  • polypeptides of the present invention may be further modified.
  • polypeptide of the present invention can be modified by various well-known technical means.
  • Polypeptide modification includes but is not limited to N-terminal modification, C-terminal modification, side chain modification, amino acid replacement, peptide backbone modification, binding to other polypeptides or proteins, and reverse polypeptide (reverse polypeptide) fragments consisting of 16-18, preferably 16-17, and most preferably 16 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 1.
  • Polypeptide modification can optimize the physicochemical properties of the polypeptide, improve its water solubility, prolong the in vivo action time, change its in vivo distribution, eliminate immunogenicity, and reduce toxic side effects.
  • modification methods include, but are not limited to, amidation modification, acetylation modification, biotinylation modification, fluorescent labeling modification, polyethylene glycol modification, isoprenylation modification, myristoylation and palmitoylation modification, phosphorylation modification, glycosylation modification, polypeptide conjugate modification, special amino acid modification, etc.
  • the modification comprises N-terminal acetylation, and/or C-terminal amidation.
  • the present invention provides use of the above polypeptide in preparing a drug.
  • the drug may be a polypeptide antagonist that inhibits human respiratory syncytial virus, particularly an antagonist that targets surface protein F of human respiratory syncytial virus.
  • the polypeptide when used as a prevention and treatment target, can be used to prevent and treat human respiratory syncytial virus.
  • the drug is a drug for preventing or treating human respiratory syncytial virus infection.
  • the present invention provides a method for preventing or treating human respiratory syncytial virus infection in a subject, the method comprising administering a therapeutically effective amount of the polypeptide of the present invention to a subject in need thereof.
  • a therapeutically effective amount refers to an amount that has a therapeutic effect and can be used to prevent or treat a specific disease, disorder or condition described herein.
  • a “therapeutically effective amount” may refer to an amount required to provide a therapeutic or desired effect on a subject being treated.
  • a therapeutically effective amount may vary depending on the route of administration, the use of excipients, and the possibility of co-administration with other therapies.
  • the present invention designs a novel mimetic polypeptide with reference to the protein structure of human respiratory syncytial virus surface protein F.
  • Cell experiments have shown that the novel mimetic polypeptide pep16 can inhibit the infection and reproduction of human respiratory syncytial virus.
  • FIG. 1A and FIG. 1B are graphs showing that the polypeptide of the present invention can inhibit the infection and reproduction of human respiratory syncytial virus by detecting the relative content of human respiratory syncytial virus L gene in human lung epithelial cells by quantitative PCR (qPCR).
  • qPCR quantitative PCR
  • Figures 2A to 2D show that the polypeptide of the present invention can inhibit the infection and reproduction of human respiratory syncytial virus by using the method of DAPI staining of the cell nucleus and the expression of fluorescent protein (GFP) after GFP-RSV virus infection.
  • GFP fluorescent protein
  • Figure 2A is (1) a blank control group (NC);
  • Figure 2B is (2) a GFP-RSV infection group;
  • Figure 2C is (3) a GFP-RSV+pep16 group, in which GFP-RSV and pep16 After co-incubation, the cells were infected, and then the culture medium contained the peptide pep16 and the culture was continued for 24 hours;
  • Figure 2D is (4) GFP-RSV+pep16 group, GFP-RSV was not co-incubated with pep16, the cells were infected, and then the culture medium contained the peptide pep16 and the culture was continued for 24 hours.
  • the peptides were synthesized by a peptide synthesis company (Zhejiang Paipeptide Biological Co., Ltd., No. 291, Fucheng Road, Xiasha Street, Jianggan District, Hangzhou) using the solid phase method.
  • the peptide SEQ ID NO:3 was synthesized and acetylated at the N-terminus and amidated at the C-terminus to become the peptide pep16.
  • the peptide was purified by reverse phase chromatography to a purity of >95%.
  • the purity of the peptide was determined by high performance liquid chromatography (HPLC), and the molecular weight of the peptide was determined by mass spectrometry (MALDI-TOF).
  • the peptide pep16 was dissolved in 2.5% volume of DMSO (D2650, Sigma-Aldrich), and then 1 ⁇ PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.2-7.4) (absin, abs962, Shanghai, China) buffer was added to a final concentration of 1 mM.
  • Experimental Example 1 Detection of the relative content of human respiratory syncytial virus L gene in human lung epithelial cells.
  • Human lung epithelial cells BEAS-2B (ECACC 95102433, Shanghai, China) were cultured in DMEM medium (Gibco, Cat#C11995500BT) containing 10% FBS (Gibco, 10270-106) and 1% penicillin-streptomycin (Gibco, 15140-122) in a humidified incubator with 5% CO 2 at 37°C until approximately 80% of the area was covered. After the cells were separated, they were inoculated into 12-well tissue culture plates at a number of 1 ⁇ 10 5 cells per well, with 2 ml of cell culture medium per well. The cells were cultured for 20 hours, and the culture medium was aspirated. The cell culture medium DMEM used in the subsequent experiments did not contain FBS.
  • RSV virus was grown in HEp-2 cells The experiment was divided into (1) blank control group (NC), (2) RSV infection group (RSV), and (3) RSV infection + peptide group (RSV+pep16).
  • the experimental cells were then incubated for 2 hours, the culture medium was aspirated, the cells were washed with 1 ⁇ PBS, and serum-free culture medium was added again. Only the culture medium of the (3) RSV+pep16 group contained 10 ⁇ M pep16, and the culture was continued for 24 hours.
  • the RSV-L gene was detected using the same method as above to quantify the relative content of RSV virus.
  • the experimental results are shown in Table 2 and Figure 1B.
  • the results show that after the virus and cells were exposed to the peptide pep16, the infection and reproduction of the virus were inhibited by 36% (experiment RSV+pep16). If only the virus was exposed to the peptide pep16, the infection and reproduction of the virus would also be weakened by 27% (experiment A). In the case where the virus has already infected the cell, the peptide pep16 still has a protective effect on the cell, which is manifested in less cell death [experiment B, see the experiment (4) group in experiment 2 below].
  • Experimental Example 2 Detection of human lung epithelial cell nuclei and GFP-RSV fluorescent protein.
  • Human lung epithelial cells BEAS-2B (ECACC 95102433, Shanghai, China) were cultured in DMEM medium (Gibco, Cat#C11995500BT) containing 10% FBS (Gibco, 10270-106) and 1% penicillin-streptomycin (Gibco, 15140-122) at 37°C in a humidified incubator with 5% CO2 until the cells covered about 80% of the area. After separation, the cells were seeded into 12-well tissue culture plates at a number of 1 ⁇ 105 cells per well. Each well of the culture plate contained 2 ml of cell culture medium. The cells were cultured for 20 hours and the culture medium was removed.
  • DMEM medium Gibco, Cat#C11995500BT
  • FBS Gibco, 10270-106
  • penicillin-streptomycin Gabco, 15140-122
  • GFP-RSV virus was isolated after propagation in HEp-2 cells (ATCC CCL-23, Shanghai, China).
  • the cell culture medium DMEM used in the following experiments did not contain FBS.
  • the experiments were divided into (1) blank control group (NC), (2) GFP-RSV infection group (GFP-RSV), and (3) GFP-RSV infection + peptide group (GFP-RSV+pep16).
  • the experimental cells were then incubated for 2 hours, the culture medium was aspirated, the cells were washed with 1 ⁇ PBS, and serum-free culture medium was added again. Only the culture medium of the GFP-RSV+pep16 groups (3) and (4) contained 10 ⁇ M pep16, and the culture was continued for 24 hours.
  • the culture medium was removed, and the cells were washed three times with 1 ⁇ PBS for five minutes each time, stained with anti-fluorescence quenching mounting medium (containing DAPI) for 10 minutes, and then observed and photographed under a confocal microscope.
  • the experimental results are shown in Figures 2A to 2D.
  • the BEAS-2B cell nucleus is blue, and the GFP protein expression is green.
  • Figure 2A is (1) a blank control group (NC).
  • Figure 2B is (2) a GFP-RSV infection group.
  • Figure 2C is (3) a GFP-RSV+pep16 group, in which GFP-RSV was incubated with pep16 and then infected with cells, and then the culture medium contained the polypeptide pep16 and the culture was continued for 24 hours.
  • Figure 2D is (4) a GFP-RSV+pep16 group, in which GFP-RSV was not incubated with pep16 and then infected with cells, and then the culture medium contained the polypeptide pep16 and the culture was continued for 24 hours.
  • the experimental results showed that compared with (1) the blank control group (NC), the number of cell nuclei in the (2) GFP-RSV infection group was significantly reduced, and the GFP protein expression was obvious. (3) The number of cell nuclei in the GFP-RSV+pep16 group was close to that in the (1) blank control group (NC), and the GFP protein expression was significantly reduced compared with the (2) GFP-RSV infection group because of the peptide's inhibition of viral infection and reproduction and its protective effect on cells.
  • GFP-RSV had infected cells and GFP protein expression was high
  • the number of cell nuclei in the (4) GFP-RSV+pep16 group was significantly higher than that in the (2) GFP-RSV infection group, and close to that in the (1) blank control group (NC), indicating that the peptide pep16 has a protective effect on cells infected with the virus.

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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Tropical Medicine & Parasitology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention appartient au domaine de la biomédecine. La présente invention concerne un antagoniste polypeptidique ciblant une glycoprotéine de fusion du virus respiratoire syncytial humain. Le polypeptide de la présente invention comprend un fragment polypeptidique constitué de 16 à 18 acides aminés continus dans la séquence d'acides aminés représentée dans SEQ ID NO : 1. Lorsque la glycoprotéine de fusion du virus respiratoire syncytial humain est utilisée en tant que protéine cible thérapeutique, le polypeptide peut être utilisé pour inhiber l'infection par le virus respiratoire syncytial humain et la multiplication de celui-ci.
PCT/CN2023/082969 2023-03-22 2023-03-22 Antagoniste polypeptidique ciblant la glycoprotéine de fusion du virus respiratoire syncytial humain WO2024192698A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105246910A (zh) * 2013-03-14 2016-01-13 姆科希斯有限责任公司 热稳定的呼吸道合胞病毒融合前f蛋白寡聚物及其在免疫组合物中的用途
CN106119287A (zh) * 2016-08-29 2016-11-16 广东华南联合疫苗开发院有限公司 一种表达呼吸道合胞病毒f蛋白的重组载体及方法
CN114644708A (zh) * 2020-12-18 2022-06-21 珠海泰诺麦博生物技术有限公司 呼吸道合胞病毒特异性结合分子
CN115786369A (zh) * 2023-01-04 2023-03-14 北京交通大学 一种呼吸道合胞病毒抗原蛋白的编码基因、抗原蛋白、重组载体、疫苗及应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105246910A (zh) * 2013-03-14 2016-01-13 姆科希斯有限责任公司 热稳定的呼吸道合胞病毒融合前f蛋白寡聚物及其在免疫组合物中的用途
CN107029227A (zh) * 2013-03-14 2017-08-11 姆科希斯有限责任公司 热稳定的呼吸道合胞病毒融合前 f 蛋白寡聚物及其在免疫组合物中的用途
CN106119287A (zh) * 2016-08-29 2016-11-16 广东华南联合疫苗开发院有限公司 一种表达呼吸道合胞病毒f蛋白的重组载体及方法
CN114644708A (zh) * 2020-12-18 2022-06-21 珠海泰诺麦博生物技术有限公司 呼吸道合胞病毒特异性结合分子
CN115786369A (zh) * 2023-01-04 2023-03-14 北京交通大学 一种呼吸道合胞病毒抗原蛋白的编码基因、抗原蛋白、重组载体、疫苗及应用

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