CN113336834A - Target for treating or preventing new coronavirus - Google Patents

Abstract

The invention provides a protein fragment, which is at least one of the following: A1) the amino acid sequence is shown as SEQ ID NO. 1; A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1; A3) a protein fragment which is obtained by substituting, deleting and adding one or more amino acid residues at any one or more of the 18 th, 19 th, 28 th and 29 th positions of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1); A4) the amino acid sequence is shown as SEQ ID NO. 2; A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2; A6) a protein fragment which is obtained by substituting, deleting and adding one or more amino acid residues of any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4) and has more than 90 percent of identity with the protein fragment shown in A4).

Description

Target for treating or preventing new coronavirus

Technical Field

The invention relates to the field of biological medicine, in particular to a target for treating or preventing new coronavirus.

Background

The COVID-19 pandemic caused by the new coronavirus (SARS-CoV-2) has caused a global public health crisis. To combat SARS-CoV-2, most therapeutic strategies have focused on vaccines or antiviral drugs directed against the Spike (S) proteins of the virus. However, viral evolution of RNA viruses is driven by a high mutation rate, which allows the virus to escape host immune attack. Despite the efforts of man to combat SARS-CoV-2 infection, many countries are now confronted with new outbreaks of new crown virus mutants and the incidence remains high. Therefore, the development of antiviral drugs or vaccines against the S protein may encounter the problem of drug resistance mutation. This is true also for the three vaccines tested against the b.1.351 mutant (Janssen, Novavax and AstraZeneca) with a reduced effect on preventing symptomatic infections, with estimates of effectiveness of 57%, 49% and a statistically even insignificant percentage, respectively. For the mRNA vaccines BNT162b2 (Pfizer) and mRNA-1273 (Moderna), the serum neutralizing activity against the B.1.351 mutant strain was reduced by 6.5-fold and 8.6-fold in the vaccinees. Therefore, there is an urgent need to develop broad spectrum therapeutic measures against SARS-CoV-2.

During viral infection, the S protein needs to be activated by host cell (e.g., human) proteases, such as furin, TMPRSS2 and cathepsin l (ctsl). CTSL is a member of the lysosomal cysteine proteases produced in the human body, whose main function is to proteolyze antigens produced by pathogens.

It has been reported that the level of CTSL in blood samples in SARS-CoV-2 infected patients is highly correlated with the severity and course of COVID-19. CTSL promotes SARS-CoV-2 infection by cleaving S and enhancing viral entry into the cell.

Disclosure of Invention

In a first aspect, the present invention provides a protein fragment, said protein fragment being any of protein fragments a1) -a4) as follows:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

In a second aspect, the use of a substance which does not cleave or has a reduced efficiency of cleavage by a protease of fragment 1 and/or fragment 2 of the S protein of a coronavirus in the preparation of a product; the coronavirus is SARS-CoV-2 or mutant strain thereof; the functions of the product are any one or more of the following:

(1) inhibiting cell fusion;

(2) preventing SARS-CoV-2 or its mutant strain infection;

(3) treating diseases caused by SARS-CoV-2 or its mutant strain infection;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

Alternatively, the protease is a cathepsin.

Optionally, the product is a medicament, vaccine, neutralizing antibody.

In a third aspect, the invention provides the use of a substance which mutates fragment 1 and/or fragment 2 of the S protein of a coronavirus in the preparation of a product; the coronavirus is SARS-CoV-2 or mutant strain thereof; the functions of the product are any one or more of the following:

(1) inhibiting cell fusion;

(2) preventing SARS-CoV-2 or its mutant strain infection;

(3) treating diseases caused by SARS-CoV-2 or its mutant strain infection;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

In a fourth aspect, the present invention provides a product, the active ingredient of which is a substance that prevents or reduces the efficiency of cleavage by a protease of fragment 1 and/or fragment 2 of the S protein of coronavirus; the functions of the product are any one or more of the following:

(1) inhibiting cell fusion;

(2) preventing SARS-CoV-2 or its mutant strain infection;

(3) treating diseases caused by SARS-CoV-2 or its mutant strain infection;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

Alternatively, the protease is a cathepsin.

Optionally, the cathepsin inhibitor is a cathepsin L inhibitor.

Optionally, the Cathepsin L inhibitor is Z-FY-CHO, Oxocarbazate, MDL-28170, K777, Cathepsin inhibitor1 and/or E64 d.

In a fifth aspect: the invention provides the application of S protein segment 1 and/or segment 2 of coronavirus as a drug target in screening products; the coronavirus is SARS-CoV-2 or mutant strain thereof;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4), and has more than 90% of identity with the protein fragment shown in A4);

the function of the product is any one or more of the following:

1) the S protein segment 1 and/or segment 2 of coronavirus is not cleaved by protease or the cleavage efficiency by protease is reduced;

2) inhibiting cell fusion;

3) preventing SARS-CoV-2 or its mutant strain infection;

4) treating diseases caused by infection of SARS-CoV-2 or its mutant strain.

In a sixth aspect, the invention provides the use of S protein fragment 1 and/or S protein fragment 2 of a coronavirus in the preparation of a product for the prevention and/or treatment of a disease caused by a coronavirus; the coronavirus is SARS-CoV-2 or mutant strain thereof;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

The product is a medicament.

The SARS-CoV-2 or its mutant strain caused diseases include respiratory tract system diseases, digestive tract system diseases or nervous system diseases.

Alternatively, the SARS-CoV-2 or mutant strain thereof causes a disease including, but not limited to, cold, fever, frontal sinusitis, otitis media, pharyngitis, chronic bronchitis, pneumonia, pleural effusion, respiratory syndrome, acute gastroenteritis, cardiopulmonary disease, immune hypofunction, repeated infection, lung injury or organ failure;

alternatively, the SARS-CoV-2 caused disease is novel coronavirus pneumonia (COVID-19);

optionally, the SARS-CoV-2 caused disease or infection is a respiratory disease; the respiratory system disease is one or more of simple infection, light pneumonia, severe pneumonia, acute respiratory infection, severe acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis or sepsis shock;

optionally, the simple infection is one or more of fever, cough, pharyngalgia, nasal obstruction, hypodynamia, headache, and myalgia or is not moderate;

optionally, the mild pneumonia is cough, dyspnea and/or tachypnea;

optionally, the severe pneumonia is respiratory frequency increase, severe respiratory failure or dyspnea, cyanosis of central type, hypersomnia, unconsciousness or convulsion, and air exhaust;

optionally, the acute respiratory distress syndrome is pulmonary edema;

alternatively, the sepsis is organ dysfunction.

Nucleotide and amino acid sequence table in the computer readable carrier SEQ ID NO.1 with three triplets representation GINITRFQTLCLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLK, nucleotide and amino acid sequence table in the computer readable carrier SEQ ID NO.2 with three triplets representation GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCL.

The technical scheme of the invention has the following advantages:

1. the present invention defines three new specific cleavage sites for CTSL in the S protein of SARS-CoV-2, which are highly conserved in all circulating SARS-CoV-2 mutants. The combination function experiment proves that the new cutting site of CTSL on the S protein of SARS-CoV-2 provides a promising therapeutic target for the development of drugs for overcoming virus mutation.

2. The invention proves that the CTSL inhibitor can prevent and treat SARS-CoV-2 infection through experiments.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1A is a schematic diagram of SARS-CoV-2S protein in example 1 of the present invention in which the functional domains and cleavage sites are highlighted, CTSL cleaves at CTSL cleavage site 1(CS-1) and CTSL cleavage site 2(CS-2), and Furin cleaves at S1/S2; NTD, N-terminal domain; RBD, receptor binding domain; CTD, C-terminal domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TD, transmembrane domain;

FIG. 1B shows the overall structure of the extracellular domain of SARS-2S protein in example 1 of the present invention (PDB code: 6 VXX);

FIG. 1C is an SDS-PAGE electrophoresis of CTSL cleaved S protein in example 1 of the present invention, the S protein ectodomain of purified SARS-CoV-2 was incubated with CTSL (2-8. mu.g/mL) at various concentrations, and the results of N-terminal sequencing of band 1 (60 kDa) and band 2 (100 kDa) after cleavage are shown below;

FIG. 1D shows that 6-Gly-mutated SARS-CoV-2S protein (i.e., Mutant obtained by mutating 6 amino acid residues, including CS-1A and CS-1B sites of wild-type S protein, P1 residue at CS-2 site, and P2 residue, to Gly) cannot be cleaved by CTSL into 60 kDa (lane 1) and 100 kDa (lane 2) fragments in example 1 of the present invention, wild-type S protein (1. mu.g) and 6-Gly-mutated SARS-CoV-2S protein (500 ng) were incubated with CTSL (a mixture of S protein and CTSL, with a final CTSL concentration of 8. mu.g/mL), and all samples were subjected to silver staining detection after SDS-PAGE; wild-type S protein is the S protein extracellular domain of SARS-CoV-2 in "first" of example 1, which is substituted with proline at K986 and V987 and substituted with "GSAS" at the furin cleavage site (S1/S2 site, R682 to R685) for enhanced stability, resulting in S protein mutant.

FIG. 1E shows that the polypeptide near the CS-1 and CS-2 cleavage site in example 1 of the present invention can be cleaved by CTSL, and the CS-1 polypeptide (i.e., CS-1 peptide in the figure) or the CS-2 polypeptide (i.e., CS-2 peptide in the figure) is incubated with CTSL for mass spectrometric detection. Wherein the CS-1 polypeptide can be cleaved at 249L (i.e., between positions 19 and 20 of SEQ ID NO. 1) and 259T (i.e., between positions 29 and 30 of SEQ ID NO. 1), and the CS-2 polypeptide can be cleaved at 636Y (i.e., between positions 36 and 37 of SEQ ID NO. 2).

FIG. 2A is an alignment of amino acid sequences of residues around CS-1 and CS-2 in SARS-CoV-2 mutant strain of example 1 according to the invention; the symbols "x" indicate amino acid residues that are conserved in all tested sequences, and the symbols "and" indicate the positions of heterogeneous amino acid residues with highly similar or similar biochemical properties;

FIG. 2B is an alignment of the amino acid sequences of the residues around CS-1 and CS-2 in SARS-CoV-2 and SARS-CoV-1-associated coronavirus derived from bat and pangolin in example 1 of the present invention; the symbols "x" indicate amino acid residues that are conserved in all tested sequences, and the symbols "and" indicate the positions of heterogeneous amino acid residues with highly similar or similar biochemical properties;

FIG. 2C is an alignment of the amino acid sequences of the residues around CS-1 and CS-2 in all coronaviruses capable of infecting humans according to example 1 of the present invention; the symbols "x" indicate amino acid residues that are conserved in all tested sequences, and the symbols "and" indicate the positions of heterogeneous amino acid residues with highly similar or similar biochemical properties;

FIG. 3A is a representation of mutants of the CS-1, CS-2 and S1/S2 cleavage sites of example 1 of the present invention;

FIG. 3B is a graph showing the evaluation of infectivity of pseudoviruses having different point mutations in CS-1 and CS-2 in LLC-MK2, Vero, 293T/hACE2 and Huh7 cells in example 1 of the present invention; pseudovirus infectivity was measured by luciferase assay and shown as starting luciferase activity (n =4), statistical significance assessed by one-way anova and Tukey test; the ordinate is luciferase activity;

FIG. 3C is a schematic diagram showing that luciferase gene expression is driven by the ERE promoter, and ESR1 (activator) binds to and activates the ERE promoter to up-regulate luciferase expression in example 1 of the present invention;

FIG. 3D is a schematic diagram showing the experimental principle of cell fusion in example 1 of the present invention; huh7 effector cells were co-transfected with plasmids expressing ERE-luciferase and various S proteins (detailed in fig. 3E); another dish of Huh7 target cells was transfected with a plasmid expressing ESR 1. After 24 hours, effector cells are isolated and added to target cells for 30-60 minutes. Then, the supernatant was removed and treated with PBS or CTSL for 20 minutes. The reaction was terminated by adding a medium, and the culture was continued for 24 hours to fuse the cells. ESR1 binds to and activates the ERE promoter to up-regulate luciferase expression when the target and effector cells fuse to form syncytia. Luciferase activity was then measured as a representative of the fusion rate.

FIG. 3E shows the results of the cell fusion experiment in example 1 of the present invention;

the abscissa WT, CS-1M, CS-2M or CS-1M +2M is plasmid of different S proteins, and after the Huh7 effector cells are transfected with the different plasmids respectively, cell fusion experiments are carried out according to the method D to obtain results; plasmid acquisition methods for different S proteins: comprises a plasmid which is obtained by inserting a coding gene of WT, CS-1M, CS-2M or CS-1M +2M protein into pcDNA3.1; WT is the S protein extracellular domain of SARS-CoV-2 in the first "of example 1 substituted with proline at K986 and V987, and furin cleavage site (S1/S2 site, R682 to R685) with" GSAS "for enhanced stability, resulting S protein mutant; CS-1M is a mutant obtained by replacing the amino acids at the 18 th, 19 th, 28 th and 29 th positions of the SEQ ID NO.1 of WT with glycine and keeping other amino acid residues unchanged; the CS-2 mutant is obtained by replacing 35 th and 36 th amino acids of SEQ ID NO.2 of the S protein pseudovirus (WT) with glycine and keeping other amino acid residues unchanged; the combined mutant is obtained by replacing amino acids at 18 th, 19 th, 28 th and 29 th positions of SEQ ID NO.1 and amino acids at 35 th and 36 th positions of SEQ ID NO.2 of an S protein pseudovirus (WT) with glycine, and keeping other amino acid residues unchanged; the ordinate is the fusion rate.

FIG. 3F shows the result of direct observation of syncytia formation by a bright field microscope in example 1 of the present invention; huh7 effector cells were co-transfected with a plasmid expressing ERE-luciferase (gecky gene) and a different S protein (WT, CS-1M, CS-2M or CS-1M + 2M), and another dish of Huh7 target cells was transfected with a plasmid expressing ESR 1. After 24 hours, effector cells are isolated and added to target cells for 30-60 minutes. Then, the supernatant was removed and treated with PBS or CTSL (4 μ g/mL, pH = 5.8) for 20 minutes. Images were taken after 10-16 hours of additional incubation in medium (scale bar, 50 μm), black arrows indicate syncytia, showing representative data from three independent experiments; the plasmids of different S proteins comprise plasmids obtained by inserting genes coding WT, CS-1M, CS-2M or CS-1M +2M proteins into pcDNA3.1; WT is the S protein extracellular domain of SARS-CoV-2 in the first "of example 1 substituted with proline at K986 and V987, and furin cleavage site (S1/S2 site, R682 to R685) with" GSAS "for enhanced stability, resulting S protein mutant; CS-1M is a mutant obtained by replacing the amino acids at the 18 th, 19 th, 28 th and 29 th positions of the SEQ ID NO.1 of WT with glycine and keeping other amino acid residues unchanged; the CS-2 mutant is obtained by replacing 35 th and 36 th amino acids of SEQ ID NO.2 of the S protein pseudovirus (WT) with glycine and keeping other amino acid residues unchanged; the combination mutant is obtained by replacing amino acids at 18 th, 19 th, 28 th and 29 th positions of SEQ ID NO.1 and amino acids at 35 th and 36 th positions of SEQ ID NO.2 of the S protein pseudovirus (WT) with glycine, and other amino acid residues are kept unchanged.

FIG. 3G is a graph showing that overexpression or knockdown of the CTSL gene in Huh7 cells in example 1 of the invention dose-dependently facilitated or inhibited infection by the wild-type and three furin mutated PsV mutants (FM-delta, FM-ARAA, and FM-GSAS), respectively. PsV infectivity was measured by luciferase assay and was shown as relative luciferase activity (n = 3); evaluating statistical significance by one-way analysis of variance and Tukey post-hoc test; both Control and scramblel were blank vectors (Control); the ordinate is luciferase activity; the abscissa siCTSL represents the concentration of the mixture of siRNA sequences used for knocking down CTSL, the mixture consisting of CTSL-1-CTSL-6 (the molar ratio of CTSL-1, CTSL-2, CTSL-3, CTSL-1-CTSL-4, CTSL-5 and CTSL-6 is 1:1: 1:1: 1); the sequence of CTSL-1-CTSL-6 is shown in Table 1, and the over-expression is obtained by inserting CTSL gene sequence into pcDNA3.1 plasmid.

FIG. 3H shows that CTSL promotes syncytia formation induced by the mutant S protein of FM-ARAA in example 1 of the present invention; effe-luciferase plasmid and FM-ARAA S or blank vector (control) were co-transfected into effector cells, ESR1 expression plasmid was used to transfect target cells, after which the effector and target cells were mixed, the supernatant was removed and treated with PBS or CTSL (8. mu.g/mL and 16. mu.g/mL). Luciferase activity was then measured and normalized to a control group (n = 4). Statistical significance was assessed by one-way anova and Tukey post hoc tests; the ordinate is the fusion rate.

FIG. 3I is a graph showing the effect of CTSL in syncytia formation caused by FM-ARAA mutant S protein in example 1 of the present invention;

the Huh7 cells were transfected with either the blank vector or the FM-ARAA mutant S protein expression plasmid, treated in the absence (PBS) or presence of CTSL (2 or 4. mu.g/mL), and cultured (scale bar)50 μm) for 10-16 additional hours. Black arrows indicate syncytia. Representative data from four independent experiments are shown; the FM-ARAA mutant S protein expression plasmid is obtained by inserting the encoding gene of the FM-ARAA mutant S protein into pcDNA3.1; the FM-ARAA mutant S protein is QTNSPRRARSVA in WT₆₈₂RRAR₆₈₅Residue replacement by₆₈₂ARAA₆₈₅Then, mutants obtained by leaving the other amino acid residues unchanged; data are expressed as mean ± SEM values,. P< 0.05，** P < 0.01，*** P < 0.001，*** P < 0.0001。

FIG. 4A shows that the six compounds of example 1 of the present invention were effective in blocking SARS-CoV-2 infection at non-cytotoxic doses; vero E6 cells were pretreated with increasing concentrations of each compound for 16 hours, then infected with SARS-CoV-2 at an MOI of 0.01, and 24 hours post-infection, viral RNA copies in the supernatant were quantified by RT-qPCR. Cell viability was assessed with CCK kit (TransGen Biotech) (n = 3). The ordinate is the relative infection percentage.

FIG. 4B is a graph of the antiviral efficacy of six compounds of example 1 of the present invention; vero E6 cells were pretreated with increasing concentrations of each compound for 16 hours, then infected with a different SARS-CoV-2 variant PsV; infectivity was measured by luciferase assay 24 hours post infection; statistical significance between the designated variants and WT PsV was assessed by two-way ANOVA with Dunnett's post-hoc test. P < 0.05, P < 0.01; the ordinate is the inhibition ratio (n = 3);

FIG. 5A is a schedule of prophylactic and therapeutic dosing regimens for E64d and Z-FY-CHO in example 1 of the invention; e64d and Z-FY-CHO were administered intraperitoneally at-2-3 dpi as a prophylactic treatment and at 0dpi with 10⁶ PFU SARS-CoV-2 euvirus infection of mice; treating both drugs at 0-3 dpi, and collecting tissue samples at 4 dpi;

fig. 5B is a copy of viral RNA in lung and turbinate tissues of mice (n =5 mice/group) in example 1 of the invention, with the dashed line representing the lower limit of detection (LOD), and statistical significance between the indicated group and the control group assessed by one-way anova and Tukey post hoc test;

FIG. 5C shows the results of SARS-CoV-2 infection in example 1 of the present inventionhACE2Representative images of lung histological analysis of transgenic mice, a magnified view of the boxed area in each image being shown below the respective image, indicating infiltration of inflammatory cells, degeneration of bronchiolar epithelial cells, thickening of alveolar spaces;

FIG. 5D shows the results of tissue scoring in mice according to example 1 of the present invention;

total scores were calculated by grading and summing the severity of bronchiolar epithelial cell injury (0-10 scores), alveolar injury (0-10 scores) and inflammatory cell infiltration (0-10 scores) in the vessels and bronchioles and semi-quantitatively histologically scoring these scores for each lung tissue, normal = 0 scores, indeterminate = 1-2 scores, mild = 3-4 scores, moderate = 5-7 scores, severe = 8-10 scores, (n =3) statistical significance between the assigned and control groups was assessed by one-way analysis of variance and Tukey post hoc test.

Detailed Description

Table 1: siRNA sequence mixture for human CTSL

Example 1

One, CTSL cuts SARS-CoV-2S protein at three new sites

The trimeric S protein on the surface of SARS-CoV-2 virus comprises S1 and S2 subunits (FIG. 1A and FIG. 1B). The S1 subunit binds to host cell receptors, while the S2 subunit is involved in the virus-cell membrane fusion process, which then releases viral genetic material into the host cell. In order to investigate the structural and functional changes of the S protein treated with CTSL, the present example expresses and purifies the extracellular domain (residues M1-Q1208) of the S protein of SARS-CoV-2 (amino acid sequence is shown in GenBank ID: MN 908947.3) using mammalian cells. The S protein extracellular segment of SARS-CoV-2 was substituted with proline at K986 and V987 and "GSAS" at the furin cleavage site (S1/S2 site, R682 to R685) for enhanced stability, and this S protein mutant was widely used for structural and functional analysis. The S protein mutant is cracked into two main fragments after being co-incubated with CTSL (0-8 mu g/mL) with different concentrations, and the molecular weights of the two main fragments are 60 kDa (band 1) and 100 kDa (band 2) in SDS-PAGE respectively. The cleavage efficiency was dose-dependent on CTSL concentration (fig. 1C). N-terminal amino acid sequencing of these two fragments, this example identified two new cleavage sites for CTSL on the SARS-CoV-2S protein, 259T (named CTSL cleavage site 1, or CS-1, position 29 of SEQ ID NO. 1: GINITRFQTLCLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLK) and 636Y (named CTSL cleavage site 2, or CS-2, position 36 of SEQ ID NO. 2: GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCL) (FIG. 1B). CS-1 is located at the N-terminal domain (NTD) of the S1 subunit, while CS-2 is located at the C-terminal domain (CTD) of the S1 subunit (FIG. 1A). Like the S1/S2 sites of furin, both CTSL cleavage sites are located in the amino acid loop at the exposed position of the S protein, and these sites are accessible to the protease (FIG. 1B).

To verify that CTSL can cleave the S protein at CS-1 and CS-2 sites, two polypeptides were designed based on the amino acid loop ranges of 259T and 636Y, based on the tertiary structure of the S protein of SARS-CoV-2 (FIG. 1E). After incubation with the CTSL enzyme, the CS-1 polypeptide (amino acid 245-262) can be cleaved at the 259T site and the CS-2 polypeptide (amino acid 618-641) can be cleaved at 636Y by mass spectrometric identification of the sequences. Surprisingly, this example found another novel 249L cleavage site, which is located upstream of the CS-1 cleavage site 259T. Considering that 249L and 259T are located relatively close to each other on the S protein, after CTSL treatment, the 250-636 fragment cleaved at the 249L site can be further cleaved into 260-636 fragment at the 259T site, so that the 249L site is difficult to use in N-terminal sequencing. Thus, there should be two CTSL cleavage sites in the CS-1 region, 249L (position 19 of SEQ ID NO. 1) and 259T, designated as CS-1A and CS-1B sites, respectively.

Since the recognition of CTSL and its substrate mainly depends on the cleavage site residue (named as P1 residue) and its upstream residue (named as P2 residue), the present example mutates the P1 and P2 residues of the polypeptides CS-1A, CS-1B and CS-2 into glycine, and then adds CTSL to cleave, the mass spectrum result shows that none of these mutated polypeptides can be cleaved by CTSL at the corresponding sites. In addition, this example prepared S proteins with double glycine mutations at these cleavage sites and cleaved them with CTSL. The results show that CTSL is unable to cleave the S protein when the cleavage site is replaced by glycine (fig. 1D). These results not only demonstrate that 249L (CS-1A), 259T (CS-1B) and 636Y (CS-2) of S are specific cleavage sites for CTSL, but also indicate that this example has identified all cleavage sites exhaustively.

Second, CTSL cleavage site is highly conserved in SARS-2 mutant strain

Since these three CTSL cleavage sites on the S protein have not been reported before, this example investigated their sequence conservation between different SARS-CoV-2 mutants. To date, many SARS-CoV-2 mutants have been found in different countries (FIG. 2A). These mutants may possess three characteristics of enhanced transmission, pathogenicity, or immune escape, and are the predominant circulating mutants in most countries. Many neutralizing antibody targets are located in the RBD region of the S protein, and these targets are mutated in these mutants. However, 249L (P2 site) and 250T (P1 site) at CS-1A, 258W (P2 site) and 259T (P1 site) at CS-1B, and 635V (P2 site) and 636Y (P1 site) at CS-2 were highly conserved among all mutants of SARS-CoV-2 of interest (FIG. 2A). It indicates that CTSL cleavage is highly resistant to viral mutational escape, suggesting that these CTSL cleavage sites are critical for the SARS-CoV-2 life cycle.

It was reported that SARS-CoV-2 may be derived from bat coronavirus, such as RaTG13, and thus this example performed sequence alignment of amino acid sequences around the cleavage point of CTSL in coronaviruses found in bat and pangolin. As shown in FIG. 2B, the CS-2 site is highly conserved among most SARS-CoV and SARS-CoV-2 associated coronavirus, while the CS-1B site is conserved only among SARS-CoV-2 associated coronavirus, but not among SARS-CV associated coronavirus. More interestingly, the CS-1A site is variable in all these coronaviruses, and is only present in RaTG13 and Pangolin CoV Guingxi. This suggests that these three CTSL cleavage points may have different roles in the evolution of different animal hosts. The CS-2 site appears to be most important to the viral life cycle, while CS-1B appears probably to have evolved from SARS-CoV to SARS-CoV-2 to enhance viral infection, while CS-1A probably has evolved the latest. In addition, this example compares the conservation of three CTSL cleavage sites in 7 coronaviruses known to infect humans. CS-2 is present in SARS-CoV and SARS-CoV-2, while CS-1A and CS-1B are present only in SARS-CoV-2 (FIG. 2C), suggesting that CTSL may play a unique and important role in the SARS-CoV-2 infection process.

Thirdly, CTSL cleavage site is crucial to SARS-CoV-2 infection and cell fusion

Since CTSL is effective in cleaving the S protein of SARS-CoV-2 in vitro, this example investigates the function of CTSL during viral infection. In this example, several SARS-CoV-2S pseudovirus (PsV) mutants were prepared based on three cleavage sites, including CS-1 mutant (CS-1M), CS-2 mutant (CS-2M) and their combination mutant (CS-1M + 2M) (FIG. 3A), wherein the CS-1 mutant is obtained by replacing amino acids at positions 18, 19, 28 and 29 of SEQ ID NO.1 of S pseudovirus (WT) with glycine, and the other amino acid residues are kept unchanged; the CS-2 mutant is obtained by replacing 35 th and 36 th amino acids of SEQ ID NO.2 of the S protein pseudovirus (WT) with glycine and keeping other amino acid residues unchanged; the combination mutant is obtained by replacing amino acids at 18 th, 19 th, 28 th and 29 th positions of SEQ ID NO.1 and amino acids at 35 th and 36 th positions of SEQ ID NO.2 of the S protein pseudovirus (WT) with glycine, and other amino acid residues are kept unchanged. As an appropriate control, the present example prepared three furin mutated PsV mutants, which can escape furin cleavage and were designated as Furin Mutant (FM) -delta (residue 682-685 deletion, residue QTNSPRRARSVA of S protein pseudovirus, respectively₆₈₂RRAR₆₈₅Deletion, mutant in which other amino acid residues are left unchanged), FM-ARAA (QTNSPRRARSVA in S protein pseudovirus)₆₈₂RRAR₆₈₅Residue replacement by₆₈₂ARAA₆₈₅Later, mutants in which other amino acid residues remained unchanged) and FM-GSAS (i.e., QTNSPRRARSVA in S protein pseudovirus)₆₈₂RRAR₆₈₅Residue replacement by₆₈₂GSAS₆₈₅Later, mutants in which other amino acid residues remain unchanged). Next, this example compares the viral infectivity of Wild Type (WT) and mutant PsVs using four human and monkey cell lines (LIC-MK 2, Vero, HEK-293T/hACE2 (293T/hACE 2 for short), Huh7, respectively, which have been described in the prior art as having the highest susceptibility to SARS-CoV-2 infection. The infectivity of FM-delta, FM-ARAA and FM-GSAS was not reduced much compared to the WT group. However, once the CTSL cleavage site was mutated to glycine, these PsV mutants lost most of the infectious capacity in all four cell lines. When all three CTSL cleavage sites were mutated, viral infectivity was reduced 100-700 fold compared to WT PsV (fig. 3B). The results indicate that three new CTSL cleavage sites for CS-1 and CS-2 are critical for SARS-CoV-2 infection. Furthermore, the CS-2 site appears to play a more important role in the entry of SARS-CoV-2 virus than the CS-1 site. Similar results were observed at different PsV concentrations.

The S protein of SARS-CoV-2 can cause cell fusion, which is a feature of SARS-CoV-2 infection. A large number of syncytia were found in the lung tissue of patients with COVID-19, suggesting that this effect plays a role in the pathogenic process of the disease. This example quantifies syncytia formation by the ESR1-ERE transcriptional activation system (FIGS. 3C, 3D and 3E, FIGS. 3C-3E are quantitative analyses of syncytia formation induced by the CS mutant SARS-CoV-2S protein), and direct observation of syncytia formation by bright field microscopy (FIG. 3F). The CS-1M group significantly reduced syncytium formation, the CS-2M group inhibited syncytium formation more severely than the control group, and little syncytium formation was observed in the CS-1M +2M group (FIGS. 3E and 3F). More importantly, the inhibition of these mutants could not be rescued by adding the CTSL enzyme to the system (fig. 3E and 3F), confirming that CTSL enhances syncytia formation by cleaving the CS-1 and CS-2 sites found in this example. In summary, this example demonstrates that CTSL plays a critical role in SARS-CoV-2 infection by cleaving the viral S protein at CS-1 and CS-2.

Fourthly, the effect of CTSL on promoting SARS-CoV-2 infection is independent of furin

Since the CS-2 region is very close to the furin cleavage site, this example next compares the function of the two proteases with different Furin Mutated (FM) PsVs. PsV infection experiments were performed in the human Huh7 cell line in the case of over-expression or knockdown of CTSL. The results show that overexpression of the CTSL gene significantly increases the infection of SARS-CoV-2 PsV, while the knock-down of the CTSL gene results in a significant decrease in the infection efficiency (FIG. 3G), confirming that CTSL plays a key role in the SARS-CoV-2 infection process. However, similar to WT PsV, overexpression or knockdown of the CTSL gene also significantly affected the level of infection of the three FM mutant PsVs in a dose-dependent manner (fig. 3G). This indicates that CTSL enhances entry of SARS-CoV-2 virus independently of furin action.

It has been reported that the furin cleavage site is important for the cell fusion by SARS-CoV-2S protein. The same cell fusion system (FIG. 3D) was used here to evaluate the role of CTSL in syncytia formation caused by FM mutant S proteins. FM-ARAA mutant S protein induced little syncytia formation at levels similar to control (FIG. 3H), the mutant S protein of FM-ARAA being at the QTNSPRRARSVA sequence in WT₆₈₂RRAR₆₈₅Residue replacement by₆₈₂ARAA₆₈₅Thereafter, other amino acid residues were kept unchanged to obtain a mutant, WT was a mutant of S protein obtained in the first "of example 1 in which the S protein extracellular domain of SARS-CoV-2 was substituted with proline at K986 and V987 and with" GSAS "at the furin cleavage site (S1/S2 site, R682 to R685) for enhanced stability. However, once CTSL was added to the mutant S proteome, a large number of syncytia began to form, and this rescue effect was dose-dependent on the concentration of CTSL (fig. 3H and 3I). This suggests that CTSL can replace furin to activate viral S proteins during viral infection.

CTSL inhibitor for preventing SARS-CoV-2 infection

Since CTSL plays a key role in mediating SARS-CoV-2 infection, 6 compounds (Z-FY-CHO, Oxocarbazate, MDL-28170, K777, Cathepsin inhibitor1 and E64 d) that inhibit CTSL activity by more than 90% at a concentration of 50mM were selected in this example to evaluate their efficacy in preventing SARS-CoV-2 infection, with Z-FY-CHO and Oxocarbazate being selective CTSL inhibitors and the other 4 compounds being broad Cathepsin inhibitors.

First, using the live SARS-CoV-2 virus in vitro, all six compounds were found to be effective in blocking SARS-CoV-2 infection at non-cytotoxic doses, EC₅₀Varying from 35 nM to 10.89mM (FIG. 4A). At the maximum non-toxic dose, all compounds reduced the viral RNA load in VeroE6 cells by about 10³To 10⁴And (4) doubling. This finding confirms that CTSL plays a key role in SARS-CoV-2 infection.

Next, this example further investigated the antiviral effects of these six compounds using the recently emerged S protein PsVs of the SARS-CoV-2 mutant strain (FIG. 4B). As expected, these six compounds also effectively prevented infection of all mutant S protein PsVs, with E64d showing higher efficiency for p.1, while MDL-28170 and Oxocarbazate were more effective for b.1.429. This indicates that CTSL inhibitors can protect against the immune escape of mutants including b.1.351, while b.1.351 reduces the efficacy of most current vaccines.

Finally, this example investigates CTSL inhibitors in humansACE2（hACE2) Antiviral action in transgenic mice infected with SARS-CoV-2 Euvirus (Harbin veterinary institute). This example selects two compounds, E64d and Z-FY-CHO, that have been shown to be safe for in vivo studies. E64d and Z-FY-CHO were given prophylactic and therapeutic dosing regimens and mice were treated with appropriate doses. Each mouse was perfused intranasally at 0dpi 10⁶ SARS-CoV-2 euviral infection of PFU. Tissue samples were collected at 4dpi (one day post infection) (FIG. 5A), when viral load peaked and there were distinct groupsHistopathological changes. Both compounds resulted in a significant reduction in the number of copies of viral RNA in lung tissue, with no detectable viral load (below the lower limit of detection, LOD) in lung tissue from both 2 mice treated with Z-FY-CHO and 4 mice treated with E64d (FIG. 5B). Among them, the preventive regimen provides better protection against viral infections, while the therapeutic regimen also shows significant efficacy. At the same time, both drugs also significantly reduced the viral load in the turbinate (fig. 5B). The lung pathology results showed that the control mice showed moderate pathological changes, large areas of lung septum thickening, inflammatory cell infiltration, and bronchial epithelial cell degeneration. In contrast, the lungs of E64D-and Z-FY-CHO treated mice exhibited a more normal morphology and less penetration (FIGS. 5C and 5D). Taken together, these results indicate that CTSL inhibitors are effective in blocking SARS-CoV-2 infection in vitro and in vivo, and are resistant to escape of viral mutations.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Sequence listing

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Claims (7)

1. A protein fragment, wherein the protein fragment is at least one of the following protein fragments A1) -A6):

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

2. Use of a substance which does not cleave or has a reduced efficiency of cleavage by a protease of fragment 1 and/or fragment 2 of the S protein of a coronavirus in the preparation of a product; the coronavirus is SARS-CoV-2 or mutant strain thereof; the functions of the product are any one or more of the following:

(1) inhibiting cell fusion;

(2) preventing SARS-CoV-2 or its mutant strain infection;

(3) treating diseases caused by SARS-CoV-2 or its mutant strain infection;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

3. Use of a substance which mutates fragment 1 and/or fragment 2 of the S protein of a coronavirus in the preparation of a product; the coronavirus is SARS-CoV-2 or mutant strain thereof; the functions of the product are any one or more of the following:

(1) inhibiting cell fusion;

(2) preventing SARS-CoV-2 or its mutant strain infection;

(3) treating diseases caused by SARS-CoV-2 or its mutant strain infection;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

4. A product characterized by comprising an active ingredient which is a substance that prevents or reduces the efficiency of cleavage by a protease of fragment 1 and/or fragment 2 of the S protein of coronavirus; the functions of the product are any one or more of the following:

(1) inhibiting cell fusion;

(2) preventing SARS-CoV-2 or its mutant strain infection;

(3) treating diseases caused by SARS-CoV-2 or its mutant strain infection;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

5. The product according to claim 4, characterized in that the active ingredient is in particular any one of the following:

1) a substance which prevents or reduces the efficiency of cleavage by a protease at the 18 th, 19 th, 28 th and/or 29 th positions of the S protein fragment 1 having the amino acid sequence of coronavirus as shown in SEQ ID NO. 1;

2) a substance which prevents or reduces the efficiency of cleavage by a protease at the 35 th and/or 36 th positions of the S protein fragment 2 having the amino acid sequence of coronavirus as shown in SEQ ID NO. 2;

3) a combination of the substance of 1) and the substance of 2) above.

6. The application of the S protein segment 1 and/or segment 2 of coronavirus as a drug target in screening products; the coronavirus is SARS-CoV-2 or mutant strain thereof;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4), and has more than 90% of identity with the protein fragment shown in A4);

the function of the product is any one or more of the following:

1) the S protein segment 1 and/or segment 2 of coronavirus is not cleaved by protease or the cleavage efficiency by protease is reduced;

2) inhibiting cell fusion;

3) preventing SARS-CoV-2 or its mutant strain infection;

4) treating diseases caused by infection of SARS-CoV-2 or its mutant strain.

7. The application of S protein segment 1 and/or S protein segment 2 of coronavirus in preparing a product for preventing and/or treating diseases caused by coronavirus; the coronavirus is SARS-CoV-2 or mutant strain thereof;

the S protein fragment 1 is any one of the following substances:

A1) the amino acid sequence is shown as SEQ ID NO. 1;

A2) the amino acid sequence is shown as 12 th to 34 th positions of SEQ ID NO. 1;

A3) a protein fragment which is obtained by substituting and/or deleting and/or adding one or more amino acid residues at any one or more of positions 18, 19, 28 and 29 of the protein fragment of A1) and has more than 90% of identity with the protein fragment shown in A1);

the S protein fragment 2 is any one of the following:

A4) the amino acid sequence is shown as SEQ ID NO. 2;

A5) the amino acid sequence is shown as 32 th to 41 th positions of SEQ ID NO. 2;

A6) a protein fragment which has more than 90 percent of identity with the protein fragment shown in A4) and is obtained by substituting and/or deleting and/or adding one or more amino acid residues for any 1 or 2 amino acid residues at 35 th and 36 th positions of the protein fragment of A4).

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