WO2021155798A1 - Use of immune polypeptide in antiviral drug - Google Patents

Abstract

Provided are the use of an immune polypeptide in an antiviral drug and the use of an immune polypeptide or a derivative thereof in the preparation of a drug for inhibiting the replication of human infectious coronaviruses. Further provided is the use of an immune polypeptide or a derivative thereof in the preparation of a drug for treating pneumonia, wherein the pneumonia is caused by coronavirus infection. The solution is of significant value for the research on the mechanism of further effectively inhibiting the replication of coronaviruses, and the development of clinical drugs and treatment schemes for related diseases caused by coronavirus infection.

Description

Application of immune peptides in antiviral drugs Technical field

The present invention belongs to the field of medical technology. Specifically, the present invention relates to the application of immunogenic peptides and derivatives thereof in antiviral drugs, and in particular to the application of the peptides or derivatives thereof in preparing drugs for inhibiting human coronavirus.

Background technique

The peptide with the sequence of GQTYTSG (herein referred to as 7P) is an immunogenic peptide originally designed based on the hepatitis C virus. The present inventors disclosed in Chinese patents CN1194986C and CN1216075C as the immunogenicity of hepatitis C virus. Peptide 7P and its derivatives. In addition, in the PCT application WO/2007/137456, the inventors also proposed that 7P and its derivatives can also be used to prevent and treat liver damage, especially to prevent or treat immune liver damage and hepatotoxic chemicals. Liver damage. After that, the inventors proved that 7P also has a certain therapeutic effect on nephritis, and it was disclosed in Chinese patent application CN101559217A.

Coronavirus has a huge impact on human health, and their genetic recombination may lead to the emergence of new strains, research and development of new antiviral strategies are crucial. However, there is no molecular entity that can inhibit human coronavirus infection. Therefore, it is a new topic to study the efficacy of immune polypeptides containing the above-mentioned 7P in suppressing viral activity, especially the suppression of human coronavirus.

Summary of the invention

The technical problem solved by the present invention is to provide a new application of immune polypeptides in suppressing viruses, so as to provide a basis for antiviral research and strategy implementation.

In one aspect of the present invention, an application of an immune polypeptide or its derivative in the preparation of a drug for inhibiting human coronavirus replication is proposed. The immune polypeptide or its derivative refers to the peptide represented by the following formula I or its pharmaceutically acceptable The salt or ester:

Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (Formula I)

in,

Xaa1 is deletion, Ala, Gly, Val, Leu or Ile,

Xaa2 is Thr or Ser,

Xaa3 is Tyr, Phe or Trp, and

Xaa4 is deletion, Ala, Gly, Val, Leu, Ile, or Pro.

In the solution of the present invention, the inhibition of human coronavirus replication is achieved by combining the immune polypeptide with the 3CL protease protease, especially by competing for binding to the active center of the protease, thereby inhibiting rapid virus replication.

Common signs of people infected with coronavirus include respiratory symptoms, fever, cough, shortness of breath and dyspnea. Due to the different strains of the virus, the disease caused may be the common cold, or it may be the Middle East Respiratory Syndrome (MERS) and severe acute Respiratory syndrome (SARS) and other more serious diseases. Coronavirus is a large virus family. Whenever a new strain appears, it poses new challenges to suppression and treatment technologies. The global epidemic in 2020 is due to a previous The unknown coronavirus (known as the "new coronavirus") spreads widely and infects many people, which can be said to far exceed other similar viruses. Therefore, in the solution of the present invention, the human coronavirus may especially be the above-mentioned new coronavirus and coronaviruses of similar nature. For example, the new coronavirus (COVID-19) that caused pneumonia in 2020 also includes The mutated virus that may appear due to COVID-19.

In the present invention, the inhibitory activity of the 7P peptide with the sequence GQTYTSG on human coronaviruses was studied, and it was proved that it can inhibit the rapid replication of the coronavirus, which can suggest that 7P or its derivatives are the effective ingredients of the corresponding anti-coronavirus drugs. , And can be used as the only active ingredient of the drug.

The present invention provides the application of the above-mentioned immune polypeptide or its derivative in the preparation of a medicine for the treatment of pneumonia. The pneumonia is caused by a coronavirus, and the immune polypeptide or its derivative is the peptide represented by the following formula I or its pharmacologically Acceptable salt or ester:

Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (Formula I)

in,

Xaa1 is deletion, Ala, Gly, Val, Leu or Ile,

Xaa2 is Thr or Ser,

Xaa3 is Tyr, Phe or Trp, and

Xaa4 is deletion, Ala, Gly, Val, Leu, Ile, or Pro.

The pneumonia caused by coronavirus infection targeted by the present invention specifically includes pneumonia caused by new coronaviruses such as COVID-19 and coronaviruses of similar nature or mutations thereof, for example, due to COVID-19 in 2020 The pneumonia epidemic caused by -19 infection, the pneumonia caused by COVID-19 is also called the new type of coronavirus pneumonia.

The active ingredient of the medicine for treating pneumonia provided by the present invention may contain or only use the above-mentioned immune polypeptide as the only effective ingredient, for example, 7P peptide or its derivative is the only effective ingredient, thereby providing a novel anti-coronavirus drug.

As an extension of the idea of the present invention, another aspect of the present invention also provides a drug, which is a drug for inhibiting the replication of human coronavirus and/or a drug for treating pneumonia caused by coronavirus infection, wherein the drug includes An immune polypeptide or a derivative thereof, wherein the immune polypeptide or a derivative thereof is a polypeptide represented by the following formula I or a pharmaceutically acceptable salt or ester thereof:

Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (Formula I)

in,

Xaa1 is deletion, Ala, Gly, Val, Leu or Ile,

Xaa2 is Thr or Ser,

Xaa3 is Tyr, Phe or Trp, and

Xaa4 is deletion, Ala, Gly, Val, Leu, Ile, or Pro.

According to some embodiments of the present invention, the human coronavirus is COVID-19, or its mutant virus; the inhibition of human coronavirus replication is through the combination of immune polypeptide and 3CL protease protease, thereby inhibiting virus replication; the immune polypeptide has the following sequence The 7P peptide: GQTYTSG; this 7P peptide is the only effective ingredient in drugs that inhibit human coronavirus replication.

In another aspect of the present invention, there is also provided a method for the treatment of respiratory diseases or pneumonia caused by coronavirus infection, which comprises administering to a patient a drug containing the polypeptide represented by the following formula I or a pharmaceutically acceptable salt or ester thereof as an active ingredient:

Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (Formula I)

in,

Xaa1 is deletion, Ala, Gly, Val, Leu or Ile,

Xaa2 is Thr or Ser,

Xaa3 is Tyr, Phe or Trp, and

Xaa4 is deletion, Ala, Gly, Val, Leu, Ile, or Pro.

According to some embodiments of the present invention, the respiratory disease or pneumonia is caused by a coronavirus infection; the coronavirus is COVID-19, or a variant virus thereof; the immune polypeptide is a 7P peptide with the following sequence: GQTYTSG; the 7P peptide is the The only active ingredient in the medicine.

Therefore, the implementation of the present invention has significant value for further effectively inhibiting the mechanism of coronavirus replication, as well as for the development of clinical medications and treatment plans for related diseases caused by coronavirus infection.

Description of the drawings

Figure 1 shows the high-resolution crystal structure of COVID-19 coronavirus 3CL hydrolase (Mpro).

Figure 2 shows the effect of the binding mode of the ligand molecule in the crystal structure of the 3CL hydrolase protein. In the figure:

A shows the binding mode of the natural ligand and 3CL protease in the protein crystal structure;

B shows the docking conformational binding mode of the short peptide 7P in the protein crystal structure, which ranks first in the docking score;

C shows the superimposing effect of the docking conformational binding mode of the short peptide 7P in the protein crystal structure with the binding mode ranked first in the docking score and the binding mode of the ligand in the protein crystal structure.

Figure 3 shows the docking conformation distribution diagram of the top 10 binding modes of the short peptide 7P docking score in the experimental example.

Specific implementation plan

The present invention is a new achievement based on the existing technology and research against the coronavirus. In the process of coronavirus replication, the large open reading frame (ORF) 1a/1ab gene located at the 5'end of the genome is responsible for the expression of two large replicase polyproteins (pp). These virus-encoded 3CL ^pro and papain-like protease (Papain-like Protease) cooperate or cleave after translation to produce 16 non-structural proteins responsible for virus replication. 3CL ^{pro is} also called the main protease because it has 11 cleavage sites in pp1a and pp1ab, while papain-like protease has only 3 cleavage sites.

3CL ^pro has three different domains. Domains I and II are largely composed of several anti-parallel β-barrels and long loop regions connected to domain III. Domain III is composed of several globular alpha helices (α-helices) and functioning protein dimerization. The active site is located in the chymotrypsin-like fold between the I and II domains. It contains the catalytic dimer of His41, which acts as a proton acceptor, and Cys144/5 nucleophilic attack on the carbonyl carbon of the substrate.

All coronavirus 3CL ^pro have high sequence homology, and the main chain structure and substrate are conserved. The substrate binding site of 3CL ^pro has two deeply buried S1 and S2 subsites, and the shallowly buried S1, S3 and S4 subsites have varying degrees of solvent exposure. The substrate specificity of coronavirus 3CL ^pro is mainly determined by P1, P2 and P1'. In theory, since these viruses have highly homologous binding sites and substrate conservation, it is reasonable to predict that broad-spectrum inhibitor strategies may be successful. Therefore, this study of the present invention will provide further evidence for the identification of ^{broad-spectrum 3CL pro inhibitors.}

References: Michael Berry, Burtram Fielding, Junaid Gamieldien. Human coronavirus OC43 3CL protease and the potential of ML188 as a broad-spectrum lead compound: Homology modelling and molecular number 15 Biology dynamic studies. BMC Volume number, 2015 .

3CL ^{pro is} also known as the main protease (Mpro), which plays a key role in the virus life cycle, including the processing of polyproteins and virus replication. The polyproteins pp1a and pp1ab were subsequently cleaved into 16 non-structural functional proteins (nsp1 to nsp16). These proteins are mainly involved in the proteolytic process and viral RNA synthesis, including genome replication and subgenomic mRNA synthesis. The remaining genome contains 9 orf, which encode 4 structural proteins-ear protein (S), nucleocapsid protein (N), membrane protein (M) and envelope protein (E), and 5 accessory proteins (3a -c, 7a and 7b).

Prior to the present invention, ^{inhibitors targeting 3CL pro} , including pyridine n-oxide derivatives, peptidomimetic analogs, covalent inhibitors and peptidyl compounds, have been evaluated. Natural products of herbal extracts may become the main resource for the development of antiviral drugs. Previous studies have shown that natural compounds ^{have antiviral activity against 3CL pro} of SARs-CoV and interfere with virus replication. There is currently no antiviral drug ^{for 3CL pro.}

References: Sirin Theerawatanasirikul, Chih Jung Kuo, Nanthawan Phetcharat, Porntippa Lekcharoensuk.Insilico and in Vitro analysis of small molecules and natural compounds. Targeting, the 3CL virus. Protease of Feline, February, 2020. Antivirus.

Using the structure-based drug design method, the drug screening work against the new coronavirus COVID-19 was carried out. Studies have shown that the new pneumonia coronavirus 3CL hydrolase may become a research target for anti-new coronavirus drugs. ^{The similarity of the new coronavirus 3CL hydrolase (3C-like Proteinase, 3CL protease, 3CL pro} ) and SARS reaches 96%. Through this strategy, it is suggested that potential 3CL hydrolase inhibitors can be obtained.

According to the inventor’s research, the growth and reproduction of coronaviruses in the host relies on the 3CL protease protease that is synthesized and cleaved. Therefore, 3CL protease is an important target for research on inhibiting the replication and reproduction of coronaviruses. The results of this molecular simulation docking show that artificially synthesized immune peptides derived from the E1-C end of the hepatitis C virus HCV envelope, such as GQTYTSG synthetic peptide, combined with the active center of 3CL protease protease, showing that the synthetic peptide and 3CL protease protein The competitive binding of hydrolase (3CL ^pro ) substrates is expected to inhibit the replication and reproduction of coronaviruses (such as COVID-19), and it is possible to treat such coronavirus infections.

The immune polypeptide studied in the present invention can also be called immune 7 peptide, especially 7P peptide, which itself is a previously disclosed product. Therefore, the definition of the immune polypeptide, the method of obtaining it, and related drugs are all You can refer to the published content. For ease of reading, a brief explanation is as follows.

"Pharmaceutically acceptable ester" refers to an ester suitable for contact with human or animal tissues without excessive toxicity, irritation, or allergic reactions. Generally, esterification modification can reduce the hydrolysis of peptides by proteases in the body. The terminal amino group, carboxyl group or side chain group of the peptide of the present invention can be modified to form a pharmaceutically acceptable ester. Modifications to amino acid side chain groups include, but are not limited to, the esterification reaction of threonine and serine side chain hydroxyl groups with carboxylic acids. Preferably, the terminal group is protected with a protective group known to those skilled in the field of protein chemistry, such as acetyl, trifluoroacetyl, Fmoc (9-fluorenyl-methoxycarbonyl), Boc (tert-butoxycarbonyl), Alloc (allyloxycarbonyl), C1-3 alkyl, C6-12 aralkyl, etc. In the specific embodiment of the present invention, we have found that 7P is sufficient for treatment under physiological conditions without modification, so we can choose not to modify the N-terminal amino group and the C-terminal carboxyl group and amino acid side chain groups of the polypeptide of formula I. That is, the N-terminal chemical group is still the α-amino group (-NHB _2B ) on the first amino acid, and the C-terminal chemical group is the C-terminal amino acid carboxyl group (-COOH), so it is also called amino acid 7 peptide.

"Pharmaceutically acceptable salt" refers to a salt suitable for contact with human or animal tissues without excessive toxicity, irritation, or allergic reactions. Pharmaceutically acceptable salts are well known in the art. This salt can be prepared during the final separation and purification of the polypeptide of the present invention, or the peptide can be separately prepared by reacting the peptide with a suitable organic or inorganic acid or base. Representative acid addition salts include, but are not limited to, acetate, dihexanoate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate , Camphorate, camphorsulfonate, glycerophosphate, hemisulfate, heptanoate, caproate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate Acid salt, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, 3-phenylpropionate, propionate, succinate, tartrate , Phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. The preferred acids that can be used to form pharmaceutically acceptable salts are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, oxalic acid, maleic acid, succinic acid, and citric acid. Cations in pharmaceutically acceptable base addition salts include, but are not limited to, alkali metal or alkaline earth metal ions such as lithium, sodium, potassium, calcium, magnesium, and aluminum, and non-toxic quaternary ammonium cations such as ammonium, tetramethylammonium, Tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, diethylamine, ethanolamine, diethanolamine, piperidine, piperazine, etc. Preferred base addition salts include phosphate, tris and acetate. These salts generally can increase the solubility of the polypeptide, and the formed salt does not substantially change the activity of the polypeptide. The polypeptide of the present invention can be used alone or in the form of a pharmaceutically acceptable salt.

In this article, "drug", "pharmaceutical preparation", "pharmaceutical preparation" and the abbreviated "preparation" can be used interchangeably unless otherwise specified, and refer to a product containing active ingredients of a drug, and optionally it also contains A pharmaceutically acceptable carrier. Among them, "pharmaceutically acceptable carrier" refers to non-toxic solid, semi-solid or liquid fillers, diluents, adjuvants, wrapping materials or other preparation excipients. The carrier used can be compatible with the corresponding administration form, and can be formulated into injections, (for injection) freeze-dried powders, sprays, oral solutions, oral suspensions, tablets, capsules, etc., using carriers known to those skilled in the art. Enteric-coated tablets, pills, powders, granules, sustained release or delayed release formulations. In the specific embodiment of the present invention, the administration is by injection, and it is dissolved in physiological saline as a carrier. Therefore, the drug of the present invention may be an injection or a freeze-dried powder for injection.

In order to facilitate administration, the drug may be a unit preparation, that is, a preparation that satisfies the active ingredients required for one administration. Common unit preparations such as a unit (tablet) tablet, a unit (needle) injection or powder injection, etc., where the active ingredient is the amount required for one administration. The amount required for one administration can be conveniently calculated by multiplying the weight of the subject and the dose per unit body weight required for one administration of the subject (referred to as "dose" herein). For example, in the process of preparing medicines, the weight of an adult is usually set as 60 kg, and this weight value can be used for calculation. The unit body weight dose of different subjects can be calculated through the equivalent dose conversion relationship. For example, according to the equivalent dose conversion relationship between experimental animals and humans known to those of ordinary skill in the art (usually refer to the guidance of FDA, SFDA and other drug regulatory agencies, and also refer to "Huang Jihan et al. Animals and Animals in Pharmacological Tests" Conversion of equivalent dose with human body. Chinese Clinical Pharmacology and Therapeutics, 2004 Sep; 9(9): 1069-1072”) The effective human dose can be derived from the dose of experimental animals. Unless otherwise specified, the "dose" herein refers to the dose for rats. A factor of 6 can usually be used to convert human and rat doses. According to the dosage, the pharmaceutical manufacturer can convert the content of the active ingredient in the unit preparation for application in its pharmaceutical process. Comprehensive medication safety, cost and efficacy, the dose of 7P or its derivatives in a unit preparation can be 30-300μg/kg rat dose, or 40-180μg/kg rat dose, such as 174, 87, or 43.5μg/kg Rat dose. According to the equivalent dose conversion relationship and the normal body weight of a person, the unit preparation contains 300-3000μg 7P or its derivatives, preferably 400-1800μg 7P or its derivatives, such as 1740, 870, or 435μg 7P or its derivatives.

In the following experimental examples, based on the research of the present invention, the inventors used a molecular docking method to specifically study the binding mode of COVID-19 virus target protein 3CL protease and 7P peptide (GQTYTSG), and further studied the effect of 7P peptide (GQTYTSG) on 3CL. Inhibition of protease activity.

Experimental Example 1 Immune peptide docking experiment

1. Materials and methods

1.1 Protein structure preparation

The crystal structure of the COVID-19 coronavirus 3CL hydrolase complex analyzed and determined by the research team of Shanghai University of Science and Technology Rao Zihe is to delete the water molecules and small molecular compounds, leaving the protein structure, and the high-resolution crystal structure obtained Figure 1. The coordinates of the crystal structure are from the PDB Protein Data Bank (PDB) (ID: 6LU7).

包裹整个活性位点。 Select the center of the ligand structure of the enzyme activity center in the crystal structure as the center of the box, and set the box size to

Encapsulate the entire active site.

1.2 Short peptide docking

构象搜索exhaustiveness值设为100，展开分子对接。 Using the MDOCK software package, the above-mentioned protein structure is used as the receptor, and the 7P peptide sequence is input. The following parameters are used: three peptide structure models are generated, and the peptide conformation RMSD value is set to

The exhaustiveness value of the conformation search is set to 100, and the molecular docking is expanded.

2. Immune peptide docking results and analysis

Results of 7P molecular docking

MDOCK uses a statistical function of basic knowledge to evaluate the binding ability between short peptides and proteins. The binding ability is reflected logarithmically as an evaluation score (matching scoring). Generally speaking, the score value for docking is a negative number. The lower the score value, the stronger the bonding ability or the greater the possibility of bonding.

Table 1 shows the docking scores of the top 10 binding modes (Model 1 to Model 1) of short peptide 7P and 3CL protein binding ability. Among them, the binding mode (Model 1) ranked No. 1 is very similar to the binding mode of the ligand in the crystal structure of the 3CL protein. Refer to Figure 2 A, B, C, and A shows the binding mode of natural ligands, which are represented by a blue stick-shaped model in the color diagram (A); The purple-red stick model represents (B), and it can be seen that the binding conformation of the natural ligand in A is very similar; the effect of superimposing the two also illustrates this point (C).

Display all the top 10 binding modes (refer to Figure 3). The 3CL protein is represented by a ribbon model (shown in green in the color picture), and the natural ligand in the crystal structure is represented by a relatively thick stick model (color picture) (Shown in blue in the middle), the 7P docking conformation is represented by a relatively thin rod-shaped model (shown in purple in the color picture), and it is obviously observed that they are more concentrated.

It is found that most of the docking conformations are concentrated in the enzyme active pocket region, and only 3 conformations are distributed in other regions. Therefore, it can be speculated that the short peptide 7P may bind to the enzyme active pocket region of 3CL protease and compete with the natural substrate to occupy this pocket region.

Table 1 The top 10 binding modes of short peptide 7P and 3CL protease

Combined mode name Docking scoring Model 1 -117.8 Model 2 -104.9 Model 3 -104.9 Model 4 -104.5 Model 5 -97.6 Model 6 -95.8 Model 7 -93.3 Model 8 -93.0 Model 9 -91.7 Model 10 -91.2

4 Conclusion

Using MDOCK and AutoDock Vina respectively, the target molecule is docked to the structure of the target 3CL protease. The 7P peptide has a docking conformation that binds to the active pocket region of 3CL protease. It can be considered that the short peptide 7P binds to the active pocket region of 3CL protease and competes with the natural substrate to occupy the pocket region.

Experimental example 2 3CL protease enzyme activity influence experiment

This experimental example investigates the effect of 7P peptide and control peptide on the enzyme activity of 3C-like Proteinase. The main principle is: both the fluorescent group EDANS and the quenching group DABCYL are connected to ^{the natural substrate of 3CL Pro} protease (such as Dabcyl-KTSAVLQSGFRKME- Edans), when the substrate is not cut off, DABCYL can quench the fluorescence of EDANS, so that no fluorescence can be detected; when the substrate is ^{cut off by 3CL Pro} , the fluorescence of EDANS is no longer quenched by DABCYL, and then EDANS can be detected. Fluorescence, therefore, the weaker the fluorescence intensity, the lower the degree to which the ^{natural substrate is cleaved by 3CL Pro , and the lower the enzyme activity of 3CL Pro} , which proves that the protease inhibitor inhibits the enzyme activity ^{of 3CL Pro more strongly.} Therefore, the effectiveness of protease inhibitors (such as 7P peptide) can be monitored by the changes in the fluorescence intensity of EDANS.

1. Reagents and sample preparation

(1) Buffer: 20mM Tris-HCl buffer, pH 7.0;

(2) Standard enzyme solution: 3C-like Proteinase dissolved in deionized water to form an enzyme solution with a concentration of 1.2ug/ul;

(3) Substrate solution: Dabcyl-KTSAVLQSGFRKME-Edans (coronavirus main protease fluorescent substrate, hereinafter referred to as substrate) is stored in deionized water at a concentration of 3mM.

(4) Test substance: a) The 7P peptide was dissolved in distilled water to a 1mg/ml 7P peptide solution; b) The control polypeptide was dissolved in distilled water into a 1mg/ml control polypeptide solution; the control polypeptide was obtained by chemical synthesis, and its sequence is RKNHVGL is close to the 7P peptide amino acid number and sequence to exclude the non-specificity of the peptide sequence.

(5) Testing equipment: Microplate reader, using 96-well plate/enzyme plate.

2. Test process

2.1 The effect of 7P peptide on 3C-like Proteinase activity

(1) Draw a standard curve: take the standard enzyme solution and dilute it in a 2-fold gradient with buffer to obtain the first dilution with an enzyme amount of 6000pmol and the second dilution with a half-fold of the enzyme amount of the first dilution. Solution... The amount of enzyme is one-half times of the 10th dilution of the 11th dilution (a total of 11 sets of dilutions are obtained). Use a microplate reader to test the absorbance of each dilution. The measurement process is as follows: each take 100μL The 1st dilution to the 11th dilution were placed in the wells of the 96-well plate/enzyme standard plate (100μL per well), excited at the wavelength of 340nm, and measured the absorbance value (RFU value) at the wavelength of 535nm. The enzyme amount in is the abscissa and the RFU value is the ordinate to draw a standard curve, and fit the slope of the standard curve.

(2) Measurement process

test group:

(A) Take 4 parts of the above-mentioned substrate solution, each of which has the same volume; use the above-mentioned buffer solution to dilute the 4 parts of the substrate solution into a third mixed solution with a substrate concentration of 60μM, and add to each third Add 7P peptide (inhibitor) solution to the mixed solution and distilled water to dilute 4 parts of the third mixed solution to 90 μl, so that the 7P peptide concentration in the 4 parts of the third mixed solution after constant volume is 50 μM, 25 μM, 12.5μM, 6.25μM, four fourth mixtures with 7P peptide concentrations of 50μM, 25μM, 12.5μM, 6.25μM were obtained;

(B) Incubate the above 4 parts of the fourth mixture at 37°C for 5 min; then add 10 μL of the above enzyme solution to each part of the fourth mixture, and mix well to obtain 4 parts of the fifth mixture (enzyme coefficient multiples are 10 times);

(C) Excite each fifth mixed solution at a wavelength of 340nm, measure the fluorescence intensity change of each fifth mixed solution within 10 minutes at a wavelength of 535nm, read the RFU value every 1 minute, and calculate the fluorescence intensity per minute Change value RFU ^Test (automatically read by testing instrument);

Blank control group: Take 10 μL of the enzyme solution in the above-mentioned buffer replacement step (B) to perform the above process, and measure the change in fluorescence intensity per minute RFU ^{Blank of the} blank control group;

The calculation method of Specific Activity is as follows:

Among them, △RFU=RFU ^{Test- RFU Blank} ; dilution factor=10; enzyme amount=10μL×1.2ug/ul.

2.2 The effect of control peptide (RKNHVGL) on 3C-like Proteinase activity

Replace the 7P peptide solution in the above 2.1 experimental group with the control peptide solution, and determine the effect of the control peptide on the 3C-like protein enzyme activity according to the experimental procedure in 2.1. The enzyme activity measured according to the third mixed solution of different control peptide concentrations Table 2.

Table 2 3C-like Proteinase activity corresponding to different inhibitor concentrations

It can be seen from the test results that the control peptide has basically no effect on the 3C-like Proteinase activity, while the 7P peptide has a significant inhibitory effect on the 3C-like Proteinase activity.

It should be particularly pointed out that the specific examples and drawings are only for illustration and do not constitute a limitation on the scope of the present invention. Obviously, those of ordinary skill in the art can make various modifications and changes to the present invention within the scope of the present invention based on the description herein, and these modifications and changes are also included in the scope of the present invention.

Claims (19)

The application of the immune polypeptide or its derivative in the preparation of a medicine for inhibiting the replication of human coronavirus, the immune polypeptide or its derivative is the polypeptide represented by the following formula I or a pharmaceutically acceptable salt or ester thereof: Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (Formula I) in, Xaa1 is deletion, Ala, Gly, Val, Leu or Ile, Xaa2 is Thr or Ser, Xaa3 is Tyr, Phe or Trp, and Xaa4 is deletion, Ala, Gly, Val, Leu, Ile, or Pro. The application according to claim 1, wherein the human coronavirus is COVID-19, or its mutant virus. The application according to claim 1, wherein the inhibition of human coronavirus replication is through the combination of the immune polypeptide and 3CL protease protease, thereby inhibiting virus replication. The application according to any one of claims 1 to 3, wherein the immune polypeptide is a 7P peptide having the following sequence: GQTYTSG. The application according to claim 4, wherein the 7P peptide is the only effective ingredient in the drug for inhibiting human coronavirus replication. The application of an immune polypeptide or its derivative in the preparation of a medicine for treating pneumonia, the pneumonia is caused by a coronavirus infection, and the immune polypeptide or its derivative is the polypeptide represented by the following formula I or a pharmaceutically acceptable salt thereof or ester: Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (Formula I) in, Xaa1 is deletion, Ala, Gly, Val, Leu or Ile, Xaa2 is Thr or Ser, Xaa3 is Tyr, Phe or Trp, and Xaa4 is deletion, Ala, Gly, Val, Leu, Ile, or Pro. The application according to claim 6, wherein the coronavirus is COVID-19, or its mutant virus. The application according to claim 6 or 7, wherein the immune polypeptide is a 7P peptide having the following sequence: GQTYTSG. The application according to claim 8, wherein the 7P peptide is the only active ingredient in the medicine for treating pneumonia. A medicine which is a medicine for inhibiting the replication of human coronavirus and/or a medicine for treating pneumonia caused by coronavirus infection, wherein the medicine includes an immune polypeptide or a derivative thereof, and the immune polypeptide or a derivative thereof It is a polypeptide represented by the following formula I or a pharmaceutically acceptable salt or ester thereof: Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (Formula I) in, Xaa1 is deletion, Ala, Gly, Val, Leu or Ile, Xaa2 is Thr or Ser, Xaa3 is Tyr, Phe or Trp, and Xaa4 is deletion, Ala, Gly, Val, Leu, Ile, or Pro. The medicine according to claim 10, wherein the human coronavirus is COVID-19, or a mutant virus thereof. The medicament according to claim 10, wherein the inhibition of human coronavirus replication is through the combination of the immune polypeptide and 3CL protease protease, thereby inhibiting virus replication. The medicine according to any one of claims 10-12, wherein the immune polypeptide is a 7P peptide having the following sequence: GQTYTSG. The drug according to claim 13, wherein the 7P peptide is the only effective ingredient in the drug for inhibiting human coronavirus replication. The method for treating respiratory disease or pneumonia caused by coronavirus infection includes administering to the patient a drug containing the polypeptide represented by the following formula I or a pharmaceutically acceptable salt or ester thereof as an active ingredient: Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (Formula I) in, Xaa1 is deletion, Ala, Gly, Val, Leu or Ile, Xaa2 is Thr or Ser, Xaa3 is Tyr, Phe or Trp, and Xaa4 is deletion, Ala, Gly, Val, Leu, Ile, or Pro. The treatment method according to claim 15, wherein the respiratory disease or pneumonia is caused by coronavirus infection. The treatment method according to claim 16, wherein the coronavirus is COVID-19, or a mutant virus thereof. The treatment method according to any one of claims 15-17, wherein the polypeptide is a 7P peptide having the following sequence: GQTYTSG. The treatment method according to claim 18, wherein the 7P peptide is the only active ingredient in the medicine.

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