Polypeptide, application thereof and inhibitor

Abstract

The invention provides a polypeptide, application thereof and an inhibitor. The sequence of the polypeptide is shown as SEQ ID NO: 1 or SEQ ID NO: 2. The invention also provides a fusion protein or conjugate of the polypeptide. The invention also provides a lipid derivative of the polypeptide, and the C terminal of the polypeptide in the lipid derivative is a palmitoylation modifier or a cholesterol modifier. The invention also provides a nucleic acid molecule for coding the polypeptide, a vector containing the nucleic acid molecule, and a host cell containing the vector. The invention also relates to an application of any one of the polypeptide, the fusion protein or the conjugate, the lipid derivative, the nucleic acid molecule, the carrier or the host cell in preparation of a product. The polypeptide provided by the invention has relatively high antiviral activity, such as Nipah virus, Hendea virus or other Henipah virus, and is high in safety. The polypeptide also has high stability and druggability.

Description

Technical Field

[0002] The present invention relates to the field of polypeptide drugs, and in particular to a polypeptide and its application and inhibitor. Background Art

[0003] Nipah virus (NiV) is a newly emerged zoonotic virus belonging to the Henipavirus genus of the Paramyxoviridae family. Since the first outbreak in Malaysia in 1998, Nipah virus has caused more than 260 cases and more than 100 deaths in Malaysia, Singapore, Bangladesh and India. The World Health Organization has identified it as a priority disease that requires special attention. However, no drugs or vaccines specifically for Nipah virus infection have been approved, and treatment is mostly limited to critical supportive care. Hendra virus (HeV), formerly known as equine measles virus, is a virulent zoonotic pathogen that has been listed as a biosafety level 4 pathogen by the World Organization for Animal Health. Nipah virus and Hendra virus belong to the Henipavirus genus of the Paramyxoviridae family. Today, the emergence of other new Henipavirus viruses also poses a potential burden to people's health. Therefore, the development of broad-spectrum specific antiviral drugs targeting conserved sites of Nipah virus and other Henipavirus viruses is an important task that humans need to solve urgently.

[0004] Nipah virus is an enveloped virus that contains two glycoproteins: receptor binding protein (G) and fusion protein (F), which play a vital role in its entry process. G protein-mediated receptor attachment triggers irreversible conformational rearrangement of F protein, allowing the fusion peptide (FP) to insert into the cell membrane. Subsequently, the two heptad repeat sequences HR1 and HR2 are exposed and interact with each other to form a six-helix structure (6-HB), which brings the virus and cell membranes closer and realizes the membrane fusion process. Therefore, blocking the formation of 6-HB is expected to block the fusion of Nipah virus with the cell membrane, thereby blocking the virus from invading cells.

[0005] Studies have shown that peptides derived from the heptapeptide repeat sequence (HR2) of the F protein of human parainfluenza virus 3 (HPIV3) have anti-Nipah virus activity, and their activity is greatly improved after further modification with lipids and polyethylene glycol (PEG). However, PEGylation in drugs is still controversial. Studies have shown that it may induce specific anti-PEG antibodies, and repeated use will accelerate drug blood clearance and reduce the therapeutic effect. In addition, PEG will accumulate in the human body, and it still poses a safety hazard. Summary of the invention

[0006] The purpose of the present invention is to provide a polypeptide and its application and inhibitor to obtain a novel, potent, highly stable, PEG-free anti-Henipavirus polypeptide.

[0007] The present invention provides a polypeptide, the sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2. The amino acid sequence of the polypeptide is as follows:

[0008] SEQ ID NO:1:VALDPIDISIVLNKIKSDLEESKEWIRRSNKILDSIGNWHQ,

[0009] SEQ ID NO:2: VKLDPEDIKKVLEEIKKDLEESKEWIERSNKILEEIGKKHQ.

[0010] The present invention also provides a fusion protein or conjugate of the above polypeptide.

[0011] The present invention also provides a lipid derivative of the above-mentioned polypeptide, wherein the C-terminus of the polypeptide in the lipid derivative is a palmitoylated modified substance or a cholesterol modified substance.

[0012] In some embodiments, the C-terminus of the polypeptide is the palmitoylation modification.

[0013] The present invention also provides a nucleic acid molecule encoding the above polypeptide.

[0014] The present invention also provides a vector comprising the above nucleic acid molecule.

[0015] The present invention also provides a host cell comprising the above-mentioned vector.

[0016] The present invention also provides use of any of the above polypeptides, or the above fusion proteins or conjugates, or the above lipid derivatives, or the above nucleic acid molecules, or the above vectors, or the above host cells in preparing products; the use of the products is to prevent or treat diseases caused by Henipavirus.

[0017] In some embodiments, the Henipavirus comprises Nipah virus or Hendra virus.

[0018] The present invention also provides an inhibitor, comprising any of the above polypeptides whose sequence is shown in SEQ ID NO: 2. The inhibitor is used for preventing or treating diseases caused by Henipavirus.

[0019] The polypeptide provided by the present invention has high antiviral activity, such as Nipah virus, Hendra virus or other Henipa viruses, and is highly safe.

[0020] The polypeptide of the present invention has not only high antiviral activity, but also high stability and drugability, which is specifically reflected in having higher thermal stability, good safety, longer in vivo half-life and anti-protease activity, such as the polypeptide of SEQID NO:2 sequence.

[0021] The present invention can be well applied to scientific research and clinical practice for preventing or treating diseases caused by Nipah virus, Hendra virus or other Henipa viruses.

[0022] The present invention provides a more efficient and stable polypeptide membrane fusion inhibitor. The invention can inhibit a variety of Henipaviruses in vitro and in vivo with ultra-high efficiency, and has higher solubility, safety, anti-protease activity, thermal stability and in vivo half-life. The present invention can be used as a candidate drug for preventing and treating the epidemic of existing or other new Henipaviruses in the future, and has important strategic drug reserve significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the implementation of the present invention, a brief introduction will be given below in conjunction with the accompanying drawings. Obviously, the following drawings are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative labor.

[0024] Figure 1 : Schematic diagram of the design principle of dePEGylated peptides.

[0025] Figure 2 : The in vitro inhibitory activity of the polypeptide in Example 2 against Nipah virus Malaysian strain (NiV-M) pseudovirus.

[0026] Figure 3 : The in vitro inhibitory activity of the polypeptide in Example 2 against Nipah virus Bangladesh strain (NiV-B) pseudovirus.

[0027] Figure 4 : In vitro inhibitory activity of the polypeptide in Example 3 on NiV-M cell fusion.

[0028] Figure 5 : In vitro inhibitory activity of the polypeptide in Example 3 on NiV-B and HeV cell fusion.

[0029] Figure 6 : In vivo inhibitory activity of the polypeptide in Example 5 against NiV-M and NiV-B pseudoviruses (wherein, A, B: inhibition of brain infection by NiV-M and NiV-B pseudoviruses; C, D: inhibition of lung infection by NiV-M and NiV-B pseudoviruses; E, F: quantification of fluorescence intensity at the infection site).

[0030] Figure 7 : Safety of the polypeptide in Example 6 on different target cells (A: Caco-2; B: Huh-7; C: U87; D: Vero).

[0031] Figure 8 : Analysis of the tolerance of the polypeptide to proteases in Example 7 by pseudovirus detection (wherein A: trypsin; B: proteinase K).

[0032] Fig. 9 : Analysis of the tolerance of the polypeptide to proteases in Example 8 by LC-MS / MS detection (wherein A: trypsin; B: proteinase K).

[0033] Fig.10 : Analysis of thermal stability of the polypeptide in Example 9 (wherein, A: 4°C; B: RT; C: 37°C).

[0034] Fig.11 : Analysis of the in vivo half-life of the polypeptide in Example 10 (wherein, A: plasma polypeptide concentration determination; B: pseudovirus inhibition analysis). DETAILED DESCRIPTION

[0035] The following specific implementations are only used to illustrate the present application and should not be considered to limit the scope of the present application. The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0036] During the invasion of target cells by Nipah virus (NiV) and Hendra virus (HeV), receptor binding protein (G protein) binds to target cell receptors - ephrin B2 (B2) or ephrin B3 (B3), and the stem of G protein is completely exposed and activates fusion protein (F protein). The conformation of F protein changes, the fusion peptide on F protein is exposed and inserted into the target cell membrane, and the two heptad repeat sequences HR1 and HR2 interact and form a hexahelix, which brings the virus membrane and cell membrane closer and mediates membrane fusion. Therefore, preventing the formation of hexahelix and thus preventing NiV and HeV-mediated fusion is an important target for drug design. At present, studies have reported that peptide fusion inhibitors derived from the HR2 region of parainfluenza virus type 3 (HPIV3) have good inhibitory activity against NiV and HeV. On this basis, the antiviral activity of peptides is greatly improved by coupling lipid structures to peptides and modifying them with polyethylene glycol (PEG). However, some studies have found that certain PEG-modified proteins can cause the body to produce specific anti-PEG antibodies. Repeated use of such drugs will accelerate the body's blood clearance of drugs, thereby reducing the drug's effectiveness. In addition, PEG can accumulate in the body, and its safety is still unclear. Moreover, PEG modification of the C-terminus of the peptide will reduce the peptide's tolerance to enzymatic hydrolysis, which is not conducive to the long-term application and in vivo storage of PEG peptides. However, the anti-NiV or HeV virus peptides currently studied all use PEG-modified designs, and their changes are relatively small compared to the original HPIV3 HR2 sequence, and their antiviral activity and stability have much room for improvement.

[0037] The present invention provides a polypeptide, whose sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0038] The present invention also provides a lipid derivative of the above polypeptide, wherein the C-terminus of the polypeptide is a palmitoylated modification. The polypeptide is connected to the palmitic acid portion via a linker at the C-terminus, and the linker may be (GSG)n or (GSGSG)n, wherein n is 1 or 2.

[0039] The present invention first uses the amino acid sequence of the PEG polypeptide VIKI-PEG4-Chol as a template, replaces cholesterol with palmitic acid to synthesize the PEG polypeptide VIKI-PEG4-C16, and removes PEG on this basis to obtain the polypeptide VIKI-C16. Subsequently, the de-PEG polypeptide is shortened and derived according to the prototype HPIV3 F protein sequence, and the polypeptide VQ-P1-C16 with significantly enhanced anti-Nipah virus activity is screened (its amino acid sequence is shown in SEQ ID NO: 1, and the C-terminus is connected to palmitic acid through the linker GSG-K), and its sequence is further optimized by EK and ER mutations, and the helix is ​​made more stable by increasing the EK and ER salt bridges inside the polypeptide, thereby achieving the purpose of increasing the antiviral activity and solubility of the polypeptide.

[0040] The present invention also provides a de-PEG polypeptide anti-henipavirus membrane fusion inhibitor with higher stability. In the present invention, stability is mainly reflected in thermal stability, resistance to enzymatic degradation and in vivo half-life. The results of thermal stability experiments show that after storing for the same time at the same temperature (4°C, room temperature, 37°C), the pseudovirus inhibition activity of the polypeptide is detected, and the activity of the polypeptide designed by the present invention is significantly less reduced than that of the original PEG polypeptide. The results of the resistance to enzymatic degradation experiment show that under the treatment of trypsin or proteinase K under the same treatment conditions, the polypeptide designed by the present invention is less degraded and has stronger resistance to enzymatic degradation by high performance liquid chromatography / mass spectrometry (LC-MS / MS). The results of the in vivo half-life experiment show that the half-life of the polypeptide designed by the present invention in mice is twice that of the original PEG polypeptide. Therefore, the above results show that in addition to the extremely high antiviral activity, the polypeptide designed by the present invention also shows extremely strong stability and has a better application prospect as a reserve drug.

[0041] According to the existing peptide VIKI-PEG4-Chol, palmitic acid lipid was replaced, PEG was removed on this basis, and the N segment and C segment of the peptide were truncated and extended according to the original sequence of the HR2 region of the parainfluenza type 3 virus. After the optimal length peptide was screened, the EK / R mutation of the peptide part was performed to obtain the corresponding peptide. The specific sequences of these peptides are shown in Table 1.

[0042] Table 1

[0043]

[0044]

[0045] pAAV-IRES-EGFP plasmid was purchased from Agilent Technologies, USA. pcDNA3.1-NiV-MF plasmid and pcDNA3.1-NiV-MG plasmid were synthesized by Jiepide Biotechnology Co., Ltd. pcDNA3.1-NiV-BF plasmid, pcDNA3.1-NiV-BG plasmid, pcDNA3.1-HeV-F plasmid, and pcDNA3.1-HeV-G were synthesized by GenScript Biotech. The pMD2.G-VSV-G plasmid was stored in the inventor's laboratory.

[0046] Target cells expressing Nipah virus receptors include U87 cells, Vero cells, Huh-7 cells, and Caco-2 cells, all of which were purchased from the American Type Culture Collection (ATCC).

[0047] Example 1: Preparation of NiV-M and NiV-B pseudoviruses

[0048] Experimental methods:

[0049] 1. 24h before transfection, 2×10 6 293T cells were plated in a 10 cm tissue culture dish and cultured at 37°C with 5% CO2.

[0050] 2. During transfection, add 500μL 0.9% NaCl solution to two 1.5mL EP tubes respectively. Add a total of 24μg of NiV-M or NiV-B envelope plasmid to tube 1, in which the plasmid expressing F protein (pcDNA3.1-NiV-MF plasmid or pcDNA3.1-NiV-BF plasmid) and the plasmid expressing G protein (pcDNA3.1-NiV-MG plasmid or pcDNA3.1-NiV-BG plasmid) are added in a ratio of 1:1. Add 10μL of transfection reagent vigofect to tube 2, add the liquid in tube 2 dropwise to tube 1, mix well and let stand at room temperature for 15 minutes, and then add the mixed liquid evenly to the culture dish.

[0051] 3. After culturing for 6 h at 37°C and 5% CO2, 100 μl of VSV-ΔG rLuc pseudovirus supplemented with VSV-G was added for infection to provide the VSV virus backbone.

[0052] 4. After 2 hours of infection, discard the cell supernatant, rinse the cell surface with PBS 3 times to remove the free VSV-ΔG rLuc pseudovirus particles in the supernatant as much as possible, add DMEM containing 10% FBS and 5% CO2, and culture at 37°C.

[0053] 5. After 48 hours, collect the supernatant containing pseudovirus; centrifuge at 3000 rpm for 10 min to remove cell debris, filter with a 0.45 μm sterile filter, aliquot and store at -80°C for later use.

[0054] Example 2: Determination of the inhibitory activity of polypeptides against NiV-M and NiV-B pseudoviruses

[0055] Experimental methods:

[0056] 1. Dissolve the peptide drug in DMSO and measure its concentration.

[0057] 2. Select U87 cells expressing human Nipah virus receptor Ephrin-B2 / B3 as target cells and adjust the cell concentration to 1×10 4 The cells were cultured in 96-well cell culture plates at 37°C with 5% CO2 for 12 h.

[0058] 3. Dilute the peptide in multiple ratios with DMEM in a 96-well U-bottom plate. The diluted peptide drug is reacted with the same volume of NiV-M or NiV-B pseudovirus at 37°C for 30 minutes to allow the drug and virus to fully act.

[0059] 4. Discard the supernatant of the cell culture plate, add the drug and virus mixture to the target cells, culture at 37°C in 5% CO2 for 12 hours, and then replace with DMEM medium containing 10% FBS.

[0060] 5. After culturing for 48 h at 37°C and 5% CO2, the RLU was detected using the Renilla luciferase detection system from Promega, and the drug inhibition rate curve was prepared to calculate the half-maximal inhibitory concentration (IC50) of the drug.

[0061] The calculation formula of the inhibitory activity is: inhibition rate = (virus control well value - drug well value) / (virus control well value - cell well value) * 100%; the virus control wells are wells without adding drugs, and the cell wells are wells without adding viruses.

[0062] Results and Analysis:

[0063] The antiviral activity of VQ-P1-EK3-C16 series peptides was tested on NiV-M and NiV-B pseudovirus systems. Figure 2 and Figure 3 As shown, the peptides designed by the present invention have a good inhibitory effect on NiV-M and NiV-B pseudoviruses. Compared with other designed peptides, the VQ-P1-EK3-C16 peptide exhibits better antiviral activity, and its IC50 is lower than 0.001nM. Its inhibitory activity is increased by hundreds of times compared with the original modified peptide VIKI-PEG4-C16.

[0064] Example 3: Inhibition of NiV-M, NiV-B, HeV envelope protein-mediated cell-cell fusion experiment (cell-cell fusion)

[0065] Experimental methods:

[0066] 1. Use 293T as effector cells and culture at 2×10 6 The cells were plated in a 10 cm tissue culture dish at a concentration of 5% CO2 and cultured at 37°C for 16 h until the cell monolayer covered the bottom of the dish and then transfected using vigofect.

[0067] 2. Eukaryotic expression plasmids encoding F protein and fluorescent protein (GFP) of NiV-M, NiV-B or HeV (pAAV-IRES-GFP-NiV-MF or pAAV-IRES-GFP-NiV-BF or pAAV-IRES-GFP-HeV-F) and eukaryotic expression plasmids encoding G protein (pcDNA3.1-NiV-MG or pcDNA3.1-NiV-BG or pcDNA3.1-HeV-G) were co-transfected into 293T cells (293T / NiV-M / EGFP or 293T / NiV-B / EGFP or 293T / HeV / EGFP) at a ratio of 1:1. Only pAAV-IRES-GFP-NiV-MF or pAAV-IRES-GFP-NiV-BF or pAAV-IRES-GFP-HeV-F were transfected as negative controls.

[0068] 3. 8 hours after transfection, discard the cell supernatant, continue to culture with DMEM containing 10% FBS for 8 hours, digest and centrifuge, resuspend with fresh DMEM medium containing 10% FBS, and adjust the cell concentration to 3×10 5 / mL. Add equal volumes to the gradient diluted peptide drugs to allow the two to fully contact. Incubate at 37°C for 30 minutes. Cells without drug addition are used as positive control cells.

[0069] 4. Take 100 μL of the above peptide and cell mixture and add it to the target U87 cells plated on the 96-well plate 12 hours ago (U87 needs to cover the bottom of the well with a single layer). Incubate for 4 hours at 5% CO2 and 37°C, and observe the cell fusion under an inverted microscope. When the wells without peptide are obviously fused, use 4% paraformaldehyde to terminate the fusion and record the cell fusion.

[0070] The calculation formula of fusion inhibition activity is: inhibition rate = (number of fusion cells in positive wells - number of fusion cells in drug wells) / (number of fusion cells in positive wells - number of fusion cells in negative wells) * 100.

[0071] Results and Analysis:

[0072] like Figure 4 and Figure 5 As shown, consistent with the pseudovirus results, the polypeptide designed by the present invention has a good inhibitory effect on cell-cell fusion mediated by the F protein of NiV-M, NiV-B and HeV. Compared with other designed polypeptides, the VQ-P1-EK3-C16 polypeptide also showed the best ability to inhibit cell-cell fusion mediated by NiV and HeV envelope proteins. These data show that the VQ-P1-EK3-C16 polypeptide has the potential to broadly inhibit Henipavirus viruses.

[0073] Example 4: Construction of NiV-M and NiV-B pseudovirus models

[0074] Since NiV RNA can be detected in the brain and lungs of suckling mice after they are infected with NiV, and intracranial infection can lead to their death, the present invention uses suckling mice of ICR mice as an animal model of NiV pseudovirus infection.

[0075] Experimental methods:

[0076] 1. Package NiV-M and NiV-B pseudoviruses as in Example 1, harvest the pseudoviruses by ultracentrifugation at 25,000 rpm, 4°C for 3 h, discard the supernatant, dilute the pseudoviruses in 1 mL PBS, aliquot and store at -80°C for later use.

[0077] 2. Three-day-old ICR mice were randomly divided into groups and anesthetized with isoflurane. 10 μL of NiV-M pseudovirus or PBS (negative control) were injected into the anesthetized mice through the intracranial or intrathoracic route for brain or lung infection.

[0078] 3. Three days after infection with NiV-M or NiV-B pseudovirus, add 20uL ViviRen TM In vivo Renilla luciferase substrate was injected into the infection site of suckling mice.

[0079] 4. 5 minutes after injection, the mice were anesthetized and imaged using a small animal in vivo imaging system (IVIS luminaK seriesⅢ) to collect NiV rLuc fluorescence data.

[0080] Example 5: Determination of in vivo inhibitory activity of polypeptides against NiV-M and NiV-B pseudovirus infection

[0081] Experimental methods:

[0082] 1. Package NiV-M and NiV-B pseudoviruses as in Example 1, harvest the pseudoviruses and ultracentrifuge them at 25,000 rpm, 4° C. for 3 h, discard the supernatant, dilute the pseudoviruses in 1 mL PBS, aliquot and store at -80° C. for later use.

[0083] 2. Three-day-old ICR suckling mice were randomly divided into groups and injected intraperitoneally with VQ-P1-EK3-C16 and the positive control peptide VIKI-PEG4-C16 at a dose of 5 mg / kg 30 minutes before infection. PBS was injected intraperitoneally as a virus control.

[0084] 3. The suckling mice were anesthetized with isoflurane, and 10 μL of NiV-M or NiV-B pseudovirus or PBS (negative control) was injected into the anesthetized suckling mice through the intracranial or intrathoracic route for brain or lung infection.

[0085] 4. Three days after infection, add 20uL ViviRen TM In vivo Renilla luciferase substrate was injected into the infection site of suckling mice.

[0086] 5. 5 minutes after injection, the suckling mice were anesthetized and imaged using a small animal in vivo imaging system (IVIS lumina K seriesⅢ) to collect NiV rLuc fluorescence data and evaluate the in vivo activity of the peptide in preventing NiV-M and NiV-B pseudovirus infection.

[0087] Results and Analysis:

[0088] In the NiV-M and NiV-B pseudovirus infection models of suckling mice, a prevention study was conducted by intraperitoneal injection of VQ-P1-EK3-C16. Figure 6 As shown, mice treated with VQ-P1-EK3-C16 can more effectively prevent NiV-M and NiV-B pseudovirus infection in the lungs and brain compared with the control peptide VIKI-PEG4-C16. Compared with the virus group, the viral load of NiV-M or NiV-B in different infection sites was reduced by 100 times, with significant differences, and the P value was less than 0.001. This shows that VQ-P1-EK3-C16 has a good in vivo preventive effect on NiV-M and NiV-B pseudoviruses.

[0089] Example 6: Peptide cytotoxicity analysis

[0090] Experimental methods:

[0091] 1. Caco-2, U87, Huh-7 and Vero were selected as target cells, with 1×10 4 The cells were plated in 96-well cell plates at a density of 100 cells / well and cultured at 37°C with 5% CO2 for 12 h.

[0092] 2. Discard the cell supernatant, add the gradient diluted peptide VQ-P1-EK3-C16 and the control peptide VIKI-PEG4-C16 to the target cells, and incubate at 37°C for 48 hours.

[0093] 3. Discard the supernatant, add 100ul of 10-fold diluted CCK8 reagent, incubate at 37°C for 4h, measure the absorbance at 450nm (OD450) with an enzyme reader, and calculate the survival rate of the target cells.

[0094] Results and Analysis:

[0095] Peptide cytotoxicity results Figure 7 As shown in the figure: VQ-P1-EK3-C16 peptide still did not show obvious cytotoxicity to target cells Caco-2, U87, Huh-7 and Vero at a concentration as high as 40 μM, that is, CC50 was greater than 40 μM. Since its IC50 for NiV-M and NiV-B was less than 0.001 nM, its selectivity index (SI = CC50 / IC50) was > 10 7 .

[0096] Example 7: Analysis of the antiviral activity of peptides treated with protease

[0097] The antiviral effect of the peptides after the action of trypsin and proteinase K was determined by pseudovirus method.

[0098] Experimental methods:

[0099] 1. Antiviral activity of peptides treated with trypsin

[0100] 1. Incubate at 37°C with 1ug of peptide (VQ-P1-EK3-C16, VQ-P1-C16, VIKI-PEG4-C16) and 1ug of trypsin.

[0101] 2. After incubation for 10 min, 30 min, 1 h, 2 h, 4 h and 6 h, take out a small amount of enzyme-treated polypeptide, add the corresponding volume of 10% FBS to terminate the action of trypsin, and then freeze the mixture to -80°C for use.

[0102] 3. Take out the digested polypeptide, dilute it with DMEM medium, add an equal volume of NiV-M pseudovirus to make the final concentration of the polypeptide 1 μM, and detect the inhibition of NiV-M pseudovirus infection by the polypeptide at this concentration (as in Example 3), thereby determining that the activity of the polypeptide is affected by the action of trypsin.

[0103] 2. Antiviral activity of peptides treated with proteinase K

[0104] 1. Incubate the peptides (VQ-P1-EK3-C16, VQ-P1-C16, VIKI-PEG4-C16) with proteinase K at a ratio of 1:50 at 37°C.

[0105] 2. After incubation for 10 min, 30 min, 1 h, 2 h, 4 h and 6 h, take out a small amount of enzyme-treated polypeptide, centrifuge at 12000 rpm for 5 min to remove proteinase K, terminate digestion, and freeze the mixture to -80 °C for later use.

[0106] 3. Take out the digested polypeptide, dilute it with DMEM medium, add an equal volume of NiV-M pseudovirus to make the final concentration of the polypeptide 1 μM, and detect the inhibition of NiV-M pseudovirus infection by the polypeptide at this concentration (as in Example 3), thereby determining that the activity of the polypeptide is affected by the action of proteinase K.

[0107] Results and Analysis:

[0108] The results are as follows Figure 8 As shown, after proteinase K and trypsin treatment, the antiviral activities of polypeptides VQ-P1-C16 and VIKI-PEG4-C16 decreased compared to the initial activities, while the antiviral activity of VQ-P1-EK3-C16 of the present invention did not decrease. It could still inhibit 90% of NiV-M pseudovirus infection after digestion for 6 hours at a concentration of 1 μM, indicating that it still maintained good antiviral activity under the action of protease.

[0109] Example 8: Quantitative analysis of peptide tolerance to proteases

[0110] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to determine the resistance of the peptides to trypsin and proteinase K, respectively.

[0111] Experimental methods:

[0112] 1. Prepare the peptides (VQ-P1-EK3-C16, VQ-P1-C16, VIKI-PEG4-C16) into 10 μM working concentration solutions, and mix them with an equal volume of 40 μg / mL trypsin solution or 40 μg / mL proteinase K solution in a 1.5 mL ep tube.

[0113] 2. Take out a small amount of enzyme-treated polypeptides at 10 min, 30 min, 1 h, 2 h, 4 h and 6 h of incubation. For the polypeptide solution digested with trypsin, add the corresponding volume of 10% FBS to terminate the trypsin action. For the polypeptide solution digested with proteinase K, centrifuge at 2000 rpm for 5 min to remove proteinase K and terminate the digestion. Then freeze the mixture to -80 °C for later use.

[0114] 3. The LC-MS / MS system consists of Shimadzu Nexera LC-40 ultra-high performance liquid chromatograph and AB SCIEX triple quadrupole mass spectrometer 6500, the chromatographic column selected was Fei Nuo Mei 00B-4424-B0 liquid chromatography column (50mm×2mm inner diameter), and solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid) were used as mobile phases. The gradient elution program was optimized as follows: 0.00-0.50min, 5% B phase; 0.50-3.00min, 5%-95% B phase; 3.00-4.00min, 95% B phase; 4.00-4.10min, 95%-5% B phase; 4.10-5.00min, 5% B phase, and the flow rate was 0.6mL / min. The column temperature was maintained at 40°C, the autosampler temperature was maintained at 15°C, and the injection volume was set to 2μL.

[0115] 4. Dissolve VQ-P1-EK3-C16 and VIKI-PEG4-C16 in water to prepare 1 mg / mL standard stock solutions, then dilute with methanol / water (1 / 1, v / v) to 0.01, 0.1, 1, 10 μg / mL standards to establish a standard curve. Select 1002.5 / 1142.8 as the ion pair of VIKI-PEG4-C16 with a retention time of 2 min; select 695.6 / 742.3 as the ion pair of VQ-P1-EK3-C16 with a retention time of 2 min.

[0116] 5. Pipette 50 μL of the previous incubation mixture into a test tube containing 50 μL of ice-cold methanol, and then centrifuge at 5000×g for 10 min at 4°C to terminate the reaction. Transfer the supernatant to an autosampler vial and analyze according to the above analytical method.

[0117] Results and Analysis:

[0118] like Fig. 9 As shown, after proteinase K or trypsin digestion, the content of the control polypeptide VIKI-PEG4-C16 decreased significantly, and only 20% remained after 6 hours. However, 90% of the polypeptide VQ-P1-EK3-C16 of the present invention was not degraded after 6 hours of trypsin digestion, and more than 70% was not degraded after 6 hours of proteinase K digestion. This suggests that the de-PEG polypeptide VQ-P1-EK3-C16 of the present invention has higher protease resistance than the prototype PEG polypeptide VIKI-PEG4-C16.

[0119] Example 9: Peptide thermal stability analysis

[0120] Experimental methods:

[0121] 1. Dissolve the peptides VQ-P1-EK3-C16 and VIKI-PEG4-C16 in ddH2O, set the storage concentration to 200 μM, and store at 4°C, room temperature (RT), and 37°C, respectively.

[0122] 2. Take out the peptides at different time points (30, 60, 90 days) and freeze them at -80°C.

[0123] 3. Uniformly measure NiV-B pseudovirus inhibition, detect peptide inhibitory activity, and determine the thermal stability of peptides at different temperatures.

[0124] Results and Analysis:

[0125] The thermal stability test results are as follows Fig.10 As shown, when stored at 4°C, room temperature (RT), and 37°C for the same time, the activity of the dePEG polypeptide VQ-P1-EK3-C16 of the present invention decreased less than that of the prototype PEG polypeptide VIKI-PEG4-C16, proving that VQ-P1-EK3-C16 has higher thermal stability.

[0126] Example 10: Peptide Half-Life Determination

[0127] Experimental methods:

[0128] 1. 6-week-old Balb / c female mice were randomly divided into 2 groups, with 3 mice in each group. VIKI-PEG4-C16 and VQ-P1-EK3-C16 were dissolved in PBS and injected into the mice intraperitoneally at a dose of 5 mg / kg.

[0129] 2. Before injection (0h) or 0.5h, 1h, 2h, 3h, 6h, 9h, 12h, 15h and 24h after injection, collect mouse blood with a micro capillary through the retro-orbital venous plexus of mice, let it stand at room temperature for 3h, centrifuge at 6000rpm, 4℃ for 10min, collect serum in a sterile 1.5mL eppendorf tube, and inactivate it in a 56℃ metal bath for 30min.

[0130] 3. An equal amount of methanol was added to a portion of the serum, mixed, and allowed to stand at 4°C for 30 min. The supernatant was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected for subsequent mass spectrometry detection. The mass spectrometry method was the same as in Example 8 to obtain the content of the polypeptide in the serum. The half-life of the polypeptide in mice was evaluated using MODFIT software.

[0131] 4. Another part of the serum was diluted with DMEM in multiple ratios, and an equal volume of NiV-M pseudovirus was added to detect the inhibition of NiV-M pseudovirus infection by mouse serum at different dilution multiples.

[0132] Results and Analysis:

[0133] like Fig.11 As shown, the polypeptide VQ-P1-EK3-C16 of the present invention significantly improves the half-life, which is 2 times longer than the prototype PEG polypeptide VIKI-PEG4-C16, and has better value for clinical application.

[0134] discuss

[0135] In order to improve the thermal stability and resistance to enzymatic degradation of the peptide, the PEG modification of the original peptide was removed, and it was found that the antiviral activity of the peptide was reduced, which was speculated to be caused by the steric hindrance effect. In order to avoid steric hindrance, the inventor proposed an innovative idea to extend the peptide sequence with the natural amino acid sequence of its own source to increase the distance between the main binding site and the lipid structure, such as Figure 1 As shown. Studies have shown that the membrane proximal region (MPER) between HR2 and the TM region is also involved in the membrane fusion process. Therefore, the peptide is extended to the MPER region, and the amino acids in the MPER region are used to replace PEG, hoping to shorten the distance between the main binding site and the cell membrane while targeting the new target site in the proximal membrane region between HR1 and the fusion peptide (FP) (such as Figure 1 After screening out the strongest anti-henipavirus peptide by truncation and extension, in order to further improve the peptide solubility and antiviral activity, we continued the previous optimization method of EK / R mutation, introduced glutamic acid (E) and lysine (K) or arginine (R) mutations at the corresponding sites, increased EK and ER salt bridges, and increased peptide helicity. A series of peptides were designed as shown in Table 1.

[0136] The pseudovirus and membrane fusion system of Henipa virus were established respectively, and the screening results were obtained through them together. It was found that the polypeptide VQ-P1-EK3-C16 of SEQ ID NO: 1 had the best antiviral activity. Its IC50 for the pseudovirus inhibition activity of Nipah virus Malaysian strain and Nipah virus Hendra strain was less than 1 picomolar, and it had extremely strong antiviral activity (such as Figures 2 to 5 shown).

[0137] The present invention also constructs a pseudovirus infection model of Henipa virus in vivo. Through the in vivo imaging of ICR suckling mice, it is found that the intraperitoneal injection of the polypeptide VQ-P1-EK3-C16 of SEQ ID NO: 1 can effectively protect suckling mice from pseudovirus lung and brain infection of Nipah virus Malaysia strain and Nipah virus Bangladesh strain ( Figure 6 ), indicating that the peptide still has a good protective effect in vivo. On different target cells, 40μM VQ-P1-EK3-C16 did not show cytotoxicity, indicating that VQ-P1-EK3-C16 also has good safety and has good clinical drug guidance significance.

[0138] Furthermore, the stability of the peptides was analyzed. The resistance of the optimized dePEGylated peptides to proteases (trypsin and proteinase K) was evaluated. Figure 8 and Fig. 9As shown, under the same enzyme incubation time, the antiviral activity of the de-PEG polypeptide VQ-P1-EK3-C16 decreased significantly less than that of the control polypeptide VIKI-PEG4-C16, and the polypeptide content after the action of protease was tested by LC-MS. It was found that after 6 hours of action, the de-PEG polypeptide VQ-P1-EK3-C16 still maintained a content greater than 80%, which shows that VQ-P1-EK3-C16 has good tolerance to enzymatic hydrolysis. Nipah virus mostly breaks out in tropical areas. If it is to be used as a long-term reserve drug, high requirements must be placed on its thermal stability. The present invention evaluated the thermal stability of the polypeptide through a pseudovirus inhibition experiment, and the results are as follows Fig.10 As shown in the figure, when stored at different temperatures (4°C, RT, 37°C) for different time periods (30, 60, 90 days), the activity of the polypeptides decreased, but the decrease in VQ-P1-EK3-C16 was less, which indicates that the polypeptides of the present invention have higher thermal stability. Subsequently, the present invention detected the half-life of the polypeptides in mice, and the results are as follows: Fig.11 As shown, the half-life of the de-PEGylated peptide VQ-P1-EK3-C16 obtained after the optimized design of this patent is increased by 2 times, and it can still exert a good anti-Nipah pseudovirus effect when stored in the blood 24 hours after administration.

[0139] In summary, the C-segment extension and PEG-free novel polypeptide designed by the present invention has good inhibitory activity against various henipavirus pseudovirus infections. In addition, the VQ-P1-EK3 polypeptide has good stability and drugability, and is expected to be further developed into a candidate drug for the prevention and treatment of Hendra virus, Nipah virus and other henipaviruses that may break out in the future.

[0140] Although the present invention has been described in detail above with general descriptions and specific implementation modes, it is obvious to those skilled in the art that some modifications or improvements may be made based on the present invention.

Claims

1. A polypeptide, characterized in that Its sequence is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. A fusion protein or conjugate of the polypeptide according to claim 1.

3. The lipid derivative of the polypeptide according to claim 1, characterized in that in, The C-terminus of the polypeptide is a palmitoylation modification or a cholesterol modification.

4. The lipid derivative according to claim 3, characterized in that The C-terminus of the polypeptide is the palmitoylation modification.

5. A nucleic acid molecule encoding the polypeptide of claim 1.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the vector of claim 6.

8. Use of the polypeptide according to claim 1, or the fusion protein or conjugate according to claim 2, or the lipid derivative according to claim 3 or 4, or the nucleic acid molecule according to claim 5, or the vector according to claim 6, or the host cell according to claim 7 in preparing a product; the product is used for preventing or treating diseases caused by Henipavirus.

9. The use according to claim 8, characterized in that The Henipa virus includes Nipah virus or Hendra virus.

10. An inhibitor, characterized in that It includes the polypeptide according to claim 1 whose sequence is shown in SEQ ID NO: 2.

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