Polypeptide for inhibiting new coronavirus infection, polypeptide inhibitor and application

Abstract

The invention discloses a polypeptide for inhibiting new coronavirus infection, a polypeptide inhibitor and application. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The polypeptide can significantly inhibit infection of new coronavirus, can inhibit more than 95% of viruses in the early stage of infection, can be applied to preparation of drugs for treating or preventing SARS-CoV-2 infection, and is high in safety, suitable for wide application and wide in adaptive population through strict safety evaluation; the half-life period is long, frequent administration is not needed, and the compliance of a patient is improved; the specificity is high, the treatment accuracy is improved, and the influence on host cells is reduced.

Description

Technical Field

[0001] The present application belongs to the technical field of polypeptide inhibitors, and specifically relates to a polypeptide, polypeptide inhibitor and application for inhibiting novel coronavirus infection. Background Art

[0002] International drug development for SARS-CoV-2 involves multiple targets and mechanisms of action, and has made some progress, but further verification of safety and effectiveness is still needed. Studies have reported that Remdesivir can shorten the hospitalization time of COVID-19 patients, but has limited effect on reducing mortality, while the results of clinical trials recently published by Chloroquine, Ritonavir, Arbidol, Darunavir, etc. have shown that they are not effective in treating COVID-19.

[0003] Given that pregnant women and postpartum women need to use small molecule drugs with caution, and that the elderly, frail people, and those with underlying diseases are sensitive to drug toxicity and side effects, there is an urgent need to develop safer and more effective anti-SARS-CoV-2 drugs. Peptide drugs are highly safe, relatively low cost, and highly modifiable, and have become a new direction for drug development. However, the current peptide drugs for the new coronavirus have problems such as narrow targeting, low bioavailability, limited indications, and high frequency of administration. Summary of the invention

[0004] The purpose of the present application is to provide a polypeptide, polypeptide inhibitor and application for inhibiting novel coronavirus infection, so as to solve the technical problems that small molecule drugs are limited in effect and have a limited applicable population among the existing drugs for SARS-CoV-2 in the prior art, and polypeptide drugs with higher safety performance have narrow targeting, low bioavailability, limited indications and high frequency of administration.

[0005] In order to achieve the above-mentioned objectives, the first aspect of the present application provides a polypeptide for inhibiting infection by the new coronavirus, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0006] In one or more embodiments, the polypeptide targets the transmembrane-associated region of the spike protein of SARS-CoV-2.

[0007] In one or more embodiments, the polypeptide inhibits SARS-CoV-2 from entering a cell.

[0008] In order to achieve the above-mentioned purpose, the second aspect of the present application provides a polypeptide inhibitor against the new coronavirus, including the polypeptide described in any of the above embodiments.

[0009] In one or more embodiments, the polypeptide inhibitor is used to inhibit the novel coronavirus from crossing the membrane and entering the cell.

[0010] In order to achieve the above-mentioned objectives, the third aspect of the present application provides a use of a polypeptide as described in any of the above embodiments in the preparation of a drug for preventing or treating new coronavirus infection.

[0011] In one or more embodiments, the novel coronavirus is SARS-CoV-2.

[0012] In order to achieve the above-mentioned objectives, the fourth aspect of the present application provides an application of a polypeptide as described in any of the above embodiments in the preparation of a drug for inhibiting the transmembrane entry of the new coronavirus into cells.

[0013] In one or more embodiments, the novel coronavirus is SARS-CoV-2.

[0014] Different from the prior art, the beneficial effects of this application are:

[0015] The polypeptide of the present application can significantly inhibit the infection of SARS-CoV-2. In the early stage of SARS-CoV-2 infection, the polypeptide of the present application can inhibit more than 95% of the virus and can be used to prepare drugs for treating or preventing SARS-CoV-2 infection;

[0016] The polypeptide of the present application can effectively inhibit viral infection in the early stage of viral infection and reduce the risk of disease progression;

[0017] The design of the polypeptide of the present application can reduce immunogenicity and reduce the risk of allergic reactions and immune rejection;

[0018] The peptides of this application have undergone rigorous safety assessments and are highly safe, suitable for wide application and applicable to a wide range of populations;

[0019] The peptides of the present application have a long half-life and do not require frequent administration, thus improving patient compliance;

[0020] The polypeptide of the present application has high specificity, which improves the accuracy of treatment and reduces the impact on host cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1It is a sequence analysis diagram of the S protein in this application;

[0023] Figure 2 This is a data graph of the relative expression levels of SARS-CoV-2S gene fragments in different groups in Example 2 of the present application;

[0024] Figure 3 This is a data graph of the virus infection inhibition rate of polypeptide P6b at different infection time points in Example 3 of the present application;

[0025] Figure 4 This is a data graph showing the inhibitory effect of polypeptide P6b on infection of different viruses in Example 4 of the present application;

[0026] Figure 5 This is a data graph showing the effect of polypeptide P6b in Example 5 of the present application on the cell activity of different cells. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.

[0028] This application designs and develops peptide drugs that inhibit SARS-CoV-2 based on the S protein sequence of SARS-CoV-2. In addition to targeting the HR1-HR2 related regions, peptides are also designed for non-related regions. The targeting range is wide, which is conducive to the rapid development of new peptide drugs with high activity, good safety, and effective clinical treatment of COVID-19.

[0029] Specifically, the present application provides a polypeptide that inhibits infection by the new coronavirus, whose amino acid sequence is: GYHLMSFPQSAPHGVVFLHVTY (SEQ ID NO.1).

[0030] The polypeptide of the present application inhibits SARS-CoV-2 from crossing the membrane and entering the cell by targeting the transmembrane-related region of the spike protein of SARS-CoV-2, thereby achieving the purpose of inhibiting SARS-CoV-2 infection.

[0031] The present invention has no particular limitation on the method for preparing the above polypeptide, and preferably the polypeptide is prepared by artificial synthesis.

[0032] The present application also provides a polypeptide inhibitor against the new coronavirus, which includes the above-mentioned polypeptide. The form of the polypeptide inhibitor in the present application is not particularly limited. Preferably, the polypeptide inhibitor can be a powder, tablet, granule, capsule, solution, emulsion, suspension, etc., which can be administered orally. In other embodiments, the polypeptide inhibitor can also be an injection.

[0033] In addition, in different pharmaceutical dosage forms of the polypeptide inhibitor, the polypeptide inhibitor also includes pharmaceutically acceptable excipients.

[0034] The polypeptides provided in the present application are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1. Design of polypeptide sequence

[0036] Based on the hexamer-forming region structures HR1 and HR2 in the amino acid sequence of the S protein of SARS-CoV-2, peptides were designed to supplement the non-hexamer-forming regions.

[0037] The above designed regions were analyzed using MPEx3.2 software, and the regions that may interact with lipid membranes were selected as targets. Figure 1 , Figure 1 It is a sequence analysis diagram of the S protein in the present application, wherein the horizontal line areas marked with P1a / P1b, P2, P3, P4, P5, P6a / P6b, and P7 are targets selected by the polypeptides of the present application, and the horizontal line marked areas above the targets are areas that may interact with the lipid membrane.

[0038] Peptides were designed for P1a / P1b, P2, P3, P4, P5, P6a / P6b, and P7 targets respectively, and finally a polypeptide targeting P6b target was selected for the next step of effect verification. The polypeptide was named polypeptide P6b, and its amino acid sequence was: GYHLMSFPQSAPHGVVFLHVTY (SEQ ID NO.1).

[0039] Example 2: Verification of the inhibitory activity of polypeptide P6b against SARS-CoV-2 pseudovirus (hereinafter referred to as SARS-CoV-2 or virus) infection in 293T-ACE2 cells

[0040] SARS-CoV-2 entry into host cells is mediated by the interaction between the spike protein and the angiotensin-converting enzyme II (ACE2) receptor. In order to verify the inhibitory activity of the polypeptide P6b of Example 1 on SARS-CoV-2 infection, a SARS-CoV-2 infection experiment was carried out using 293T-ACE2 cells, wherein the 293T-ACE2 cells were homemade by a stable cell line method.

[0041] The specific experimental process is as follows:

[0042] S100, use 2% FBS DMED culture medium to dilute the polypeptide P6b in the well plate, so that the final concentration is 200μM, 100μM, 50μM, 25μM, 12.5μM, 6.25μM, the volume of each well is 500μL, and each concentration is set to 3 replicates.

[0043] At the same time, the scrambled rearranged peptide P6b-Scr of the peptide P6b was diluted to the same concentration, and 3 replicates were set for each concentration;

[0044] S200, SARS-CoV-2 was diluted with DMED culture medium containing 2% FBS to make the virus infection multiplicity 8 MOI, and 500 μL of virus solution was added to each well in the S100 polypeptide P6b culture medium, which was recorded as the experimental group;

[0045] A positive control group and a negative control group were set up at the same time, wherein the positive control group was added with a virus solution but without the polypeptide P6b, and the negative control group was added with a virus solution and a scrambled rearranged polypeptide P6b-Scr. The experimental group, the positive control group and the negative control group were cultured at 37° C. for 2 hours to obtain a culture solution;

[0046] S300, use trypsin to digest 293T-ACE2 cells, and spread the cells into 12-well plates with DMED culture medium containing 10% FBS. Adjust the cell density to 10 6 After overnight culture, the culture medium was discarded, and 500 μL of the culture medium prepared by S200 was added to each well. The cells were cultured at 37°C and 5% CO2 for 6 h, and then the supernatant was discarded, the cells were washed twice with PBS, and the culture medium was replaced with DMED culture medium containing 2% FBS and cultured for another 48 h.

[0047] S400, extract the total RNA of the cells after culture in the above groups, measure the concentration with Nanodrop, take 1 μg of total RNA from each sample for reverse transcription to obtain cDNA products, select β-ACTIN gene as the internal reference gene, detect the expression level changes of SARS-CoV-2S gene (spike gene) fragments to reflect the inhibition of viral intracellular replication by polypeptides, and process the experimental data with Graphpadprism 8.4.2 software to obtain Figure 2 , Figure 2 This is a data graph of the relative expression levels of SARS-CoV-2S gene fragments in different groups of Example 2 of the present application.

[0048] like Figure 2As shown, compared with the positive control group (the leftmost side in the figure), the 200μM, 100μM, 50μM, 25μM, and 12.5μM experimental groups of peptide P6b can effectively inhibit the expression of SARS-CoV-2S gene fragments and the infection of SARS-CoV-2, and its half-inhibitory concentration IC 50 About 23.25μM;

[0049] However, the scrambled rearranged peptide P6b-Scr in the negative control group had no inhibitory effect on the expression of the SARS-CoV-2S gene fragment.

[0050] Example 3: Verification of the inhibitory effect of polypeptide P6b at different stages of SARS-CoV-2 infection

[0051] S100, 293T-ACE2 cells were plated in 12-well plates, 10 cells per well. 6 cells were cultured at 37°C and 5% CO2 for 12 h;

[0052] S200, adding SARS-CoV-2 or polypeptide P6b to the cell culture medium of S100 at different time points to simulate the effect of polypeptide P6b at different infection stages, specifically including:

[0053] a. Incubate the polypeptide P6b and the virus at 37°C for 2 hours and then add them to the cell culture medium. Record this time point as -2 hours.

[0054] b. Add the polypeptide P6b and the virus into the cell culture medium at the same time, and record this time point as 0h;

[0055] c. Add the virus to the cell culture medium for co-incubation, and add the polypeptide P6b at 2, 8, 16, and 24 hours of incubation, and record the time points as 2h, 8h, 16, and 24h respectively;

[0056] A positive control group and a negative control group were set up at the same time, wherein the positive control group did not add polypeptide P6b, and the negative control group did not add virus and polypeptide P6b;

[0057] The S300 and S200 groups were cultured at 37°C and 5% CO2 for 24 h, and then the total cell RNA was extracted. 1 μg of total cell RNA was taken from each sample for reverse transcription to obtain cDNA products. The β-ACTIN gene was selected as the internal reference gene, and the expression level changes of the SARS-CoV-2S gene (spike gene) fragment were detected and compared with the positive control group. The infection inhibition rate was calculated, and the experimental data were processed with Graphpad prism 8.4.2 software to obtain Figure 3 , Figure 3This is a data graph of the virus infection inhibition rate of polypeptide P6b at different infection time points in Example 3 of the present application.

[0058] like Figure 3 As shown, in the early stage of viral infection, the infection inhibition rate of polypeptide P6b is the highest. It can be seen that the polypeptide P6b of the present application acts on the early stage of viral infection, wherein more than 95% of the virus can be inhibited after the virus and polypeptide are co-incubated for 2 hours and then added to the cell culture medium; adding the polypeptide at different time points after viral infection can still play a significant infection inhibition role. After 8 hours of viral infection, polypeptide P6b can still inhibit nearly 60% of the virus, and after 16 hours of viral infection, polypeptide P6b still has a significant effect of inhibiting the virus.

[0059] It can be seen from the above experiments that the polypeptide P6b of the present application plays a role in the early stage of SARS-CoV-2 infection and can still play a significant role in inhibiting infection within 24 hours after infection.

[0060] Example 4: Study on the specificity of polypeptide P6b

[0061] S100, Vero cells were plated in 12-well plates, 10 cells per well. 6 Cells were cultured at 37°C and 5% CO2 for 12 h before use;

[0062] S200, using 2% FBS DMED culture medium to dilute the polypeptide P6b in a 12-well plate to a final concentration of 200 μM, 50 μM, and 12.5 μM, with a drug volume of 500 μL per well, and 3 replicates for each concentration;

[0063] S300, use 2% FBS DMEM culture medium to dilute SFTSV, ZIKV, and EV71 viruses to 8 MOI, then add them to the S200 well plate, 500 μL per well, mix well, and culture for 2 h at 37°C and 5% CO2, then add them to the S100 cell culture medium;

[0064] A positive control group and a negative control group were set up at the same time. The positive control group did not add peptide P6b, and the negative control group did not add virus or peptide P6b.

[0065] S400, incubate the cell culture medium at 37°C and 5% CO2 for 2 h, then discard the supernatant, wash twice with PBS, replace with DMED culture medium containing 2% FBS and continue culturing for 24 h;

[0066] S500, extract the total RNA of the cells after culture in the above groups, measure the concentration with Nanodrop, take 1 μg of total RNA from each sample for reverse transcription to obtain cDNA products, select β-ACTIN gene as the internal reference gene, detect the expression level changes of each viral gene fragment, and process the experimental data with Graphpad prism 8.4.2 software to obtain Figure 4 , Figure 4 This is a data graph showing the inhibitory effect of polypeptide P6b on infection of different viruses in Example 4 of the present application.

[0067] like Figure 4 As shown, polypeptide P6b has no obvious inhibitory effect on SFTSV, ZIKV and EV-A71 virus infections, indicating that the polypeptide P6b of the present application has specificity in inhibiting SARS-CoV-2 infection.

[0068] Example 5: Biological toxicity detection of polypeptide P6b

[0069] S100, 293T-ACE2, 293T, Vero and HMC3 cells were plated in 96-well plates, 10 cells per well. 4 Cells were cultured at 37°C and 5% CO2 for 6 h before use;

[0070] S200, using 2% FBS DMEM or MEM culture medium to dilute the polypeptide P6b to a concentration of 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM, with 3 replicates for each concentration, recorded as the drug group;

[0071] A control group and a drug-free and cell-free group were set up at the same time, wherein the control group was supplemented with 3.5 μM fludarabine (Flu), and the drug-free and cell-free group was not supplemented with either cell culture or polypeptide P6b, i.e., pure DMEM or MEM culture medium;

[0072] S300, add the above groups into the cell culture medium of S100, add 100 μL to each well, culture for 48 hours at 37°C and 5% CO2, and observe the cell status under a microscope;

[0073] S400, discard the supernatant of the 96-well plate, add 10 μL of CCK8 reagent and 90 μL of 2% FBS DMEM or MEM mixture to each well, culture for 2 h at 37°C, 5% CO2, measure the absorbance value of each well at OD 450 on a microplate reader, and calculate the cell activity of each experimental group based on the calculation formula: cell activity = (experimental group wells - cell-free group wells) × 100 / (drug-free group wells - cell-free group wells). Figure 5 , Figure 5This is a data graph showing the effect of polypeptide P6b in Example 5 of the present application on the cell activity of different cells.

[0074] like Figure 5 As shown, after different concentrations of polypeptide P6b were co-incubated with 293T-ACE2, 293T, Vero and HMC3 cells, the cell survival rates were all above 99%, while the control group (Flu in the figure) added with fludarabine (a purine analogue reported to inhibit SFTSV infection) had a cell survival rate of only about 60% at a concentration of 3.5 μM.

[0075] The above data show that the polypeptide P6b has no obvious toxicity to cells, and the polypeptide P6b of the present application has good biocompatibility and safety.

[0076] It is obvious to those skilled in the art that the present application is not limited to the details of the exemplary embodiments described above, and that the present application can be implemented in other specific forms without departing from the spirit or essential features of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting in all respects, and the scope of the present application is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present application. Any reference numeral in a claim should not be regarded as limiting the claim to which it relates.

[0077] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A polypeptide for inhibiting infection by the new coronavirus, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The polypeptide according to claim 1, characterized in that The polypeptide targets the transmembrane-associated region of the spike protein of SARS-CoV-2.

3. The polypeptide according to claim 1, characterized in that The polypeptide inhibits SARS-CoV-2 from entering cells.

4. A polypeptide inhibitor against novel coronavirus, characterized in that: The invention comprises the polypeptide according to any one of claims 1 to 3.

5. The polypeptide inhibitor according to claim 4, characterized in that The polypeptide inhibitor is used to inhibit the novel coronavirus from crossing the membrane and entering the cell.

6. Use of a polypeptide as described in any one of claims 1 to 3 in the preparation of a medicament for preventing or treating novel coronavirus infection.

7. The use according to claim 6, characterized in that: The new coronavirus is SARS-CoV-2.

8. Use of a polypeptide as described in any one of claims 1 to 3 in the preparation of a drug for inhibiting the transmembrane entry of the new coronavirus into cells.

9. The use according to claim 8, characterized in that: The new coronavirus is SARS-CoV-2.

Images