CN112316152A - Method for inhibiting coronavirus by acid anhydride modified protein - Google Patents

Abstract

The present invention provides methods of inhibiting the growth of a coronavirus, preventing or controlling infection of a cell by a coronavirus, in vitro, with an anhydride-modified protein or a composition comprising an anhydride-modified protein. The invention also provides the use of an anhydride-modified protein or a composition comprising an anhydride-modified protein in the manufacture of a medicament. The protein is selected from albumin, beta lactoglobulin and RNase, and the acid anhydride is selected from 3-hydroxyphthalic anhydride, succinic anhydride or maleic anhydride. The anhydride modified protein can effectively inactivate coronavirus in vivo or in vitro, is nontoxic to cells, and provides a method for controlling the epidemic situation of coronavirus.

Description

Method for inhibiting coronavirus by acid anhydride modified protein

Technical Field

The present invention relates to the field of biomedicine, and in particular to a method for preventing and controlling novel coronavirus by using acid anhydride modified protein.

Background

The World Health Organization (WHO) announced that the outbreak of a novel coronavirus disease (COVID-19) was an international emergent Public Health Event (PHEIC). Related studies have shown that the clinical manifestations of the novel coronavirus disease (COVID-19) range from asymptomatic or mild upper respiratory disease to moderate and severe disease, rapidly progressive pneumonia, respiratory failure, acute respiratory distress syndrome and multiple organ failure and are fatal.

At present, the prevention and control of the new coronavirus epidemic situation mainly depends on diagnosis and isolation, and symptomatic support treatment is adopted for patients. Specific drugs and vaccines against the disease are in preclinical or clinical trials. The prior drugs which show good antiviral activity at the cellular level do not perform well in clinical trials, and the related effectiveness is not yet effectively proved. At least 4 anti-inflammatory and antiviral drugs are available for clinical use in 2019 at the beginning of the SARS-CoV-2 pandemic, which may have therapeutic effects on COVID-19, respectively: hydroxychloroquine, corticosteroids, Reineckvir and lopinavir/ritonavir. These drugs are not very effective for patients in clinical trials. The results of the specific clinical trials are (a) hydroxychloroquine treatment was not beneficial to hospitalized COVID-19 patients; (b) dexamethasone treatment reduced the mortality rate by one third in patients with mechanically ventilated COVID-19, and by one fifth in patients receiving oxygen only treatment; (c) the combination of lopinavir and ritonavir was ineffective in reducing the mortality of COVID-19 hospitalized patients.

From these clinical test results, it is known that the specific drugs against the novel coronavirus are not available at present. Therefore, the development of a safe and effective drug is very important for controlling the possible future emergence of new coronavirus epidemic situations.

Disclosure of Invention

Before the invention, the protein modified by acid anhydride has better anti-HPV, HIV and HSV and other anti-virus activities, although the anti-virus activities are more broad-spectrum, in the past, the protein also has weaker MERS-CoV activity on middle east respiratory syndrome, and different modification strategies and acid anhydride modifications of different proteins have larger difference, as shown in the example of FIG. 7, and meanwhile, the protein has larger activity difference between different acid anhydride modifications aiming at different viruses. The invention firstly identifies that the protein modified by anhydride has better anti-novel coronavirus activity in pseudoviruses and live viruses, and the related modification strategy and antiviral activity result provide basis for related drug development.

The invention aims to provide a novel biological medicine preparation for preventing and controlling the infection of novel coronavirus aiming at the deficiency and insufficiency of the novel coronavirus. The active acid anhydride is used for modifying and blocking amino acid with positive charge on the surface of the purified protein, so that the modified protein has the activity of blocking the target cell infected by the novel coronavirus, and becomes a candidate medicine for resisting the novel coronavirus. Unexpectedly, the inventors have found that anhydride-modified proteins are effective in controlling the novel coronavirus SARS-CoV-2.

In one aspect, the invention provides a method of inhibiting the growth of a coronavirus, preventing or controlling infection of a cell by a coronavirus, or a composition comprising an anhydride-modified protein, in vitro, the method comprising the step of contacting the anhydride-modified protein or the composition comprising an anhydride-modified protein with an article or sample containing, suspected of containing or at risk of containing a coronavirus.

In one embodiment, the protein is selected from albumin, beta lactoglobulin, and rnase.

In one embodiment, the anhydride is selected from 3-hydroxyphthalic anhydride, succinic anhydride, or maleic anhydride.

In one embodiment, the protein is fully anhydrified as determined by the ratio of lysine and arginine modifications to the protein.

In one embodiment, the sample is selected from the group consisting of stool, saliva, blood, and serum.

In one embodiment, the article is selected from the group consisting of sanitary napkins, pantiliners, wet wipes, and paper towels; the paper towel comprises paper handkerchiefs, removable toilet paper, napkins or paper towels.

In one embodiment, the coronavirus is SARS-CoV-2.

In one embodiment, the albumin is selected from serum albumin or ovalbumin. In one embodiment, the serum albumin is selected from human serum albumin, bovine serum albumin or chicken ovalbumin. In one embodiment, the beta lactoglobulin is bovine beta lactoglobulin. In one embodiment, the rnase is selected from rnase 1 or rnase 7.

In one embodiment, the composition is in the form of a solution, tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel, or aerosol.

In one embodiment, the anhydride-modified protein is prepared by mixing the anhydride with the protein.

In another aspect, the invention provides the use of an anhydride-modified protein or a composition comprising an anhydride-modified protein in the manufacture of a medicament for treating or preventing a coronavirus infection or a disease caused by a coronavirus infection in a subject.

In one embodiment, the protein is selected from albumin, beta lactoglobulin, and rnase.

In one embodiment, the anhydride is selected from 3-hydroxyphthalic anhydride, succinic anhydride, or maleic anhydride.

In one embodiment, the protein is fully anhydrified as determined by the ratio of lysine and arginine modifications of the protein.

In one embodiment, the coronavirus is SARS-CoV-2.

In one embodiment, the albumin is selected from serum albumin or ovalbumin. In one embodiment, the serum albumin is selected from human serum albumin, bovine serum albumin or chicken ovalbumin. In one embodiment, the beta lactoglobulin is bovine beta lactoglobulin. In one embodiment, the rnase is selected from rnase 1 or rnase 7.

In one embodiment, the anhydride-modified protein is prepared by mixing the anhydride with the protein.

In one embodiment, the medicament is in the form of a tablet, capsule, powder, nasal drop or aerosol, or in the form of a solution, tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel or aerosol.

Drawings

FIG. 1 shows the entry inhibition curves of 3HP-HSA, 3HP- β -LG and 3HP-OVA against the novel coronavirus pseudovirus.

FIG. 2 shows the entry inhibition curves of 3HP-HSA, 3HP- β -LG and 3HP-OVA against live viruses of the novel coronavirus.

FIG. 3 shows the results of cytotoxicity experiments of 3HP-HSA, 3HP- β -LG and 3HP-OVA against 293T-ACE2 cells.

FIG. 4 shows the results of cytotoxicity experiments with 3HP-HSA, 3HP- β -LG and 3HP-OVA against VERO-E6 cells.

FIG. 5 shows the binding of 3HP-HSA and 3HP- β -LG to the novel coronavirus S protein.

FIG. 6 shows the binding of 3HP-OVA to the novel coronavirus S protein (RBD).

FIG. 7 shows that anhydride-modified proteins are not effective in inhibiting MERS-CoV pseudovirus. In this figure, SU-HSA is succinic anhydride modified human serum albumin; ML-BLG is maleic anhydride modified beta lactoglobulin; ML-HSA is beta lactoglobulin modified by maleic anhydride; HP-BLG is beta lactoglobulin modified by 3-hydroxy-phthalic anhydride; HP-OVA is 3-hydroxy-phthalic anhydride modified ovalbumin; anti-MERS-CoV peptide (amino acid sequence SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL) see patent application No.: 201610070216.4, synthesized from gill biochemistry.

Detailed Description

The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. The following description is provided to aid in understanding the invention.

As used herein, "anhydride-modified proteins" refers to anhydride-derivatized/modified albumins and beta lactoglobulins. The degree of anhydride modification of a protein can be determined by the ratio of arginine and/or lysine modifications to the protein. The preparation of anhydride-modified proteins and the degree of anhydride modification are known to those skilled in the art. See, for example, chinese patent applications CN201210066696.9 and CN 201810691093.5. Herein, the degree of anhydride modification of a protein as measured by the arginine and/or lysine modification ratio of the protein may be independently 100%, 99%, 95%, 93%, 92%, 90%, 85%, 80%, etc. The ratio of each component can be appropriately adjusted by those skilled in the art according to the anhydride modification/derivatization ratio. Preferably, the degree of anhydride modification of the protein may be 100%. Herein, the anhydride-modified protein, preferably 3-hydroxy-phthalic anhydride-modified human serum albumin or 3-hydroxy-phthalic anhydride-modified bovine beta lactoglobulin, may be prepared as follows: (1) preparing 0.1M disodium hydrogen phosphate, adjusting the pH to 8.5, and filtering and sterilizing through a filter membrane of 0.2 mu M; (2) dissolving Human Serum Albumin (HSA) or bovine beta lactoglobulin (beta-LG) powder in a prepared disodium hydrogen phosphate solution to a concentration of 20 mg/ml; (3) acid anhydrides (3-hydroxy-phthalic anhydride (3HP), maleic anhydride (ML), succinic anhydride (SU)) were dissolved in DMSO to a concentration of 1M; (4) adding acid anhydride with the final concentration of 12mM into a solution of human serum albumin and bovine beta lactoglobulin; mixing evenly immediately; (5) adjusting pH with 5M sodium hydroxide solution to 8.5-9; (6) standing at room temperature for 20 min; (7) repeating the step 4-6 four times to make the final adding concentration of the acid anhydride be 60 mM; (8) after the last time of adding the anhydride, standing for 2 hours at room temperature to ensure that the anhydride fully reacts; (9) filling the modified anhydrified protein into a dialysis bag (7.5kDa), and putting the dialysis bag into PBS for dialysis for 48 hours, wherein the PBS buffer solution is replaced for three times; (10) and (3) carrying out ultrafiltration concentration on the dialyzed protein by using a 10kDa ultrafiltration tube.

As used herein, a "coronavirus" is an enveloped, linearly single-stranded plus-stranded RNA virus whose genome is characterized by the presence of spinous processes on the envelope, and the entire virus, like coronas, is a large class of viruses that are widespread in nature. SARS-CoV-2 is the 7 th coronavirus known to infect humans, the remaining 6 are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing middle east respiratory syndrome), respectively.

Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions for administration to a subject suffering from a coronavirus infection. Any suitable route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, topical, or oral administration. The therapeutic formulation may be in the form of a liquid solution or suspension; for oral administration, the formulation may be in the form of a tablet or capsule; for intranasal formulations, they may be in the form of powders, nasal drops or aerosols.

Methods for preparing formulations well known in the art can be found, for example, in "Remington's Pharmaceutical Sciences" (19 th edition), A.Gennaro eds, 1995, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water or saline, poly (alkylene) glycols such as polyethylene glycol, vegetable oils or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems include ethylene vinyl acetate particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration may contain excipients, for example lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or as a colloid.

Herein, the anhydride-modified protein may be prepared as a composition, for example, in the form of a solution, tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel, or aerosol; such as solutions, lotions, sprays, toilet lotions and dry powders.

The present invention provides compositions, preferably biologicals, for preventing or controlling coronavirus infection, which contain anhydride-modified proteins. The anhydride-modified protein is a mixture of an anhydride and a protein. Proteins used herein may include Human Serum Albumin (HSA), bovine beta-lactoglobulin (beta-LG), bovine serum albumin, chicken egg albumin. Preferably, the proteins used herein are human serum albumin and bovine beta lactoglobulin. The biological agent can be prepared using 3-hydroxyphthalic anhydride (3HP), maleic anhydride (ML), and succinic anhydride (SU).

In this context, the anhydride solution is the anhydride powder dissolved in DMSO and the concentration may be between 0.5 and 10M, preferably 1M.

The protein solution may be prepared by dissolving the protein in a phosphate buffer. Phosphate buffers include, but are not limited to, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, and the like. Disodium hydrogen phosphate is preferred. The phosphate buffer pH may be 7-10, preferably 8.5. The protein concentration may be 5mg/ml to 100mg/ml, preferably 20 mg/ml. The pH of the anhydride-treated protein should be greater than 7, preferably 8.5, to allow sufficient anhydride modification.

The composition may be in a dosage form selected from: solutions, lotions, sprays, lotions and dry powders.

Provided herein are methods of inhibiting infection of a coronavirus in vitro using an anhydridized protein, wherein the selected protein comprises: albumin, preferably human serum albumin and lactoglobulin, preferably bovine beta lactoglobulin.

Coronaviruses can be isolated from feces, saliva, blood, serum, and surfaces of objects.

Herein, the acid anhydride may include 3-hydroxy-phthalic acid anhydride, maleic anhydride, and succinic anhydride. The anhydride solution may be prepared by dissolving anhydride powder in DMSO and the concentration may be 0.5-10M, preferably 1M. Phosphate buffers may include, but are not limited to, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, and the like. Disodium hydrogen phosphate is preferred. The phosphate buffer pH may be 7-10, preferably 8.5.

In the present invention, the used acid-anhydride-modified protein is completely acid-anhydride-modified by measuring the modification rate of arginine and lysine.

Examples

Example 1: preparation of acid anhydride-modified proteins

Preparation of 3HP-HSA

(1) Preparing 3-hydroxy-phthalic anhydride (3HP) into mother liquor with the concentration of 1M by using dimethyl sulfoxide (DMSO);

(2) preparing 0.1M disodium hydrogen phosphate solution (pH8.5), adding human serum albumin to obtain HSA solution with HSA concentration of 20 mg/ml;

(3) adding the anhydride solution prepared in the step (1) into the HSA solution to be modified to ensure that the final concentration of the added anhydride is 12mM, fully and uniformly mixing, adjusting the pH value of the protein solution to 9.0 by using a 4M NaOH solution, and incubating for 20min at 25 ℃;

(4) repeating the step (3) for five times, wherein the final concentration of the anhydride is 12mM, 24mM, 36mM, 48mM and 60mM respectively, namely the final concentration of the anhydride in the solution is 60mM, and incubating for 2h at 25 ℃ to complete the anhydride reaction;

(5) putting the anhydride protein solution into a dialysis bag with a cut-off of 7.5KDa, putting the dialysis bag into a phosphate buffer solution with the pH of 7.4 for dialysis and fluid change (4 ℃), dialyzing for 48 hours, replacing the dialyzed external fluid in the middle once, filtering the anhydride protein solution after fluid change by using a 0.45-micron microporous filter membrane, and storing at 4 ℃ to obtain finished products of various dosage forms according to a conventional method.

Preparation of 3 HP-beta-LG

(1) Preparing 3-hydroxy-phthalic anhydride (3HP) into mother liquor with the concentration of 1M by using dimethyl sulfoxide (DMSO);

(2) preparing a disodium hydrogen phosphate solution (pH8.5) with the concentration of 0.1M, adding bovine beta-lactoglobulin (beta-LG) to prepare a beta-LG solution, wherein the concentration of the beta-LG is 20 mg/ml;

(3) adding the anhydride solution prepared in the step (1) into the beta-LG solution to be modified to ensure that the final concentration of the added anhydride is 12mM, fully and uniformly mixing, adjusting the pH value of the protein solution to 9.0 by using a 4M NaOH solution, and incubating for 20min at 25 ℃;

(4) repeating the step (3) for five times, wherein the final concentration of the anhydride is 12mM, 24mM, 36mM, 48mM and 60mM respectively, namely the final concentration of the anhydride in the solution is 60mM, and incubating for 2h at 25 ℃ to complete the anhydride reaction;

(5) putting the anhydride protein solution into a dialysis bag with a cut-off of 7.5KDa, putting the dialysis bag into a phosphate buffer solution with the pH of 7.4 for dialysis and fluid change (4 ℃), dialyzing for 48 hours, replacing the dialyzed external fluid in the middle once, filtering the anhydride protein solution after fluid change by using a 0.45-micron microporous filter membrane, and storing at 4 ℃ to obtain finished products of various dosage forms according to a conventional method.

Preparation of 3HP-OVA

(1) Preparing 3HP into a mother solution with the concentration of 1M by using dimethyl sulfoxide (DMSO);

(2) preparing 0.1M disodium hydrogen phosphate solution (pH8.5), adding ovalbumin (OVALbumin, abbreviated as OVA) to obtain OVA solution with concentration of 20 mg/ml;

(3) adding the anhydride solution prepared in the step (1) into an OVA solution to be modified to ensure that the final concentration of the added anhydride is 12mM, fully and uniformly mixing, adjusting the pH value of a protein solution to 9.0 by using a 4M NaOH solution, and incubating for 20min at 25 ℃;

(4) repeating the step (3) for five times, wherein the final concentration of the anhydride is 12mM, 24mM, 36mM, 48mM and 60mM respectively, namely the final concentration of the anhydride in the solution is 60mM, and incubating for 2h at 25 ℃ to complete the anhydride reaction;

(5) putting the anhydride protein solution into a dialysis bag with a cut-off of 7.5KDa, putting the dialysis bag into a phosphate buffer solution with the pH of 7.4 for dialysis and fluid change (4 ℃), dialyzing for 48 hours, replacing the dialyzed external fluid in the middle once, filtering the anhydride protein solution after fluid change by using a 0.45-micron microporous filter membrane, and storing at 4 ℃ to obtain finished products of various dosage forms according to a conventional method.

SU-HAS, ML-BLG and ML-HAS were prepared similarly except that either succinic anhydride or maleic anhydride was used in place of 3HP, or BLG was used in place of OVA.

Determination of the concentration of an anhydride-modified protein

The prepared anhydride protein sample is used for measuring the concentration according to the operation steps of the BCA protein concentration analysis kit, and the specific method comprises the following steps:

(1) diluting protein standard (BSA) with PBS solution to obtain BSA final concentrations of 0, 0.1, 0.2, 0.4, 1, 2 mg/mL;

(2) adding 20 μ l of each concentration standard and 20 μ l of the sample to be tested (diluted with PBS) into a 96-well plate;

(3) preparing a BCA working solution, and mixing the solution A and the solution B according to the ratio of 1: mixing at a ratio of 50;

(4) adding 200 μ l of working solution into each well, mixing, and incubating at 37 deg.C for 20 min;

(5) after cooling to room temperature, reading each blank absorbance at 562nm by using an enzyme-labeling instrument;

(6) according to the OD reading of the protein standard, a protein concentration standard curve is drawn by using Excel and GraphPad Prism 8 software, and the concentration of the anhydrified protein sample is calculated according to the standard curve and a corresponding formula.

Example 2: effect of anhydride-modified proteins on live coronavirus

Preparation of pseudoviruses

(1) The envelope protein (S protein) gene sequence of the novel coronavirus SARS-CoV-2 was codon-optimized and subjected to gene synthesis, and the information was as follows.

SARS-CoV-2S protein gene sequence (for synthesis by Kinry Biotechnology, Inc.):

ATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTT CCGTGCTAGCCCAGTGCGTGAACCTGACCACAAGGACCCAGCTGCCCCCTGCCTATACCAATTC CTTCACACGGGGCGTGTACTATCCCGACAAGGTGTTTAGAAGCTCCGTGCTGCACTCTACACAG GATCTGTTTCTGCCTTTCTTTAGCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCA ATGGCACAAAGCGGTTCGACAATCCAGTGCTGCCCTTTAACGATGGCGTGTACTTCGCCTCTAC CGAGAAGAGCAACATCATCAGAGGCTGGATCTTTGGCACCACACTGGACTCCAAGACACAGTC TCTGCTGATCGTGAACAATGCCACCAACGTCGTGATCAAGGTGTGCGAGTTCCAGTTTTGTAAT GATCCTTTCCTGGGCGTGTACTATCACAAGAACAATAAGAGCTGGATGGAGTCCGAGTTTCGCG TGTATTCTAGCGCCAACAATTGCACATTTGAGTACGTGTCCCAGCCATTCCTGATGGACCTGGA GGGCAAGCAGGGCAATTTCAAGAACCTGAGGGAGTTCGTGTTTAAGAATATCGATGGCTACTT CAAGATCTACTCTAAGCACACCCCAATCAACCTGGTGCGCGACCTGCCACAGGGCTTCAGCGCC CTGGAGCCACTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCC TGCACAGAAGCTACCTGACACCTGGCGACTCCTCTAGCGGATGGACCGCAGGAGCTGCCGCCT ACTATGTGGGCTATCTGCAGCCAAGGACCTTCCTGCTGAAGTACAACGAGAATGGCACCATCA CAGACGCAGTGGATTGCGCACTGGACCCCCTGAGCGAGACCAAGTGTACACTGAAGTCCTTTA CCGTGGAGAAGGGCATCTATCAGACATCCAATTTCAGGGTGCAGCCCACCGAGTCTATCGTGC GCTTTCCCAATATCACAAACCTGTGCCCTTTTGGCGAGGTGTTCAACGCAACCAGGTTCGCAAG CGTGTACGCATGGAATAGGAAGCGGATCAGCAACTGCGTGGCCGACTATAGCGTGCTGTACAA CTCCGCCTCTTTCAGCACCTTTAAGTGCTATGGCGTGTCCCCCACAAAGCTGAATGACCTGTGCT TTACCAACGTGTACGCCGATTCTTTCGTGATCAGGGGCGACGAGGTGCGCCAGATCGCACCAG GACAGACAGGCAAGATCGCAGACTACAATTATAAGCTGCCTGACGATTTCACCGGCTGCGTGA TCGCCTGGAACAGCAACAATCTGGATTCCAAAGTGGGCGGCAACTACAATTATCTGTACCGGCT GTTTAGAAAGTCTAATCTGAAGCCATTCGAGAGGGACATCTCTACAGAGATCTACCAGGCAGG CAGCACCCCATGCAATGGAGTGGAGGGCTTTAACTGTTATTTCCCTCTGCAGAGCTACGGCTTC CAGCCAACAAACGGCGTGGGCTATCAGCCCTACCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGC ACGCACCTGCAACAGTGTGCGGACCAAAGAAGTCCACCAATCTGGTGAAGAACAAGTGCGTGA ACTTCAACTTCAACGGACTGACCGGCACAGGCGTGCTGACCGAGTCCAACAAGAAGTTCCTGC CCTTTCAGCAGTTCGGCAGGGACATCGCAGATACCACAGACGCCGTGCGCGACCCTCAGACCC TGGAGATCCTGGACATCACACCATGCTCTTTCGGCGGCGTGAGCGTGATCACACCTGGCACCAA TACAAGCAACCAGGTGGCCGTGCTGTATCAGGACGTGAATTGTACCGAGGTGCCCGTGGCAAT CCACGCAGATCAGCTGACCCCTACATGGCGGGTGTACAGCACCGGCTCCAACGTGTTCCAGAC AAGAGCCGGATGCCTGATCGGAGCAGAGCACGTGAACAATTCCTATGAGTGCGACATCCCTAT CGGCGCCGGCATCTGTGCCTCTTACCAGACCCAGACAAACTCTCCAAGGAGAGCCCGGAGCGT GGCATCCCAGTCTATCATCGCCTATACAATGAGCCTGGGCGCCGAGAACAGCGTGGCCTACTCT AACAATAGCATCGCCATCCCTACCAACTTCACAATCTCCGTGACCACAGAGATCCTGCCAGTGT CCATGACCAAGACATCTGTGGACTGCACAATGTATATCTGTGGCGATTCTACCGAGTGCAGCAA CCTGCTGCTGCAGTACGGCAGCTTTTGTACCCAGCTGAATAGAGCCCTGACAGGCATCGCCGTG GAGCAGGACAAGAACACACAGGAGGTGTTCGCCCAGGTGAAGCAGATCTACAAGACCCCACC CATCAAGGACTTTGGCGGCTTCAATTTTTCCCAGATCCTGCCCGATCCTTCCAAGCCTTCTAAGC GGAGCTTTATCGAGGACCTGCTGTTCAACAAGGTGACCCTGGCCGATGCCGGCTTCATCAAGCA GTATGGCGATTGCCTGGGCGACATCGCCGCCAGAGACCTGATCTGTGCCCAGAAGTTTAATGGC CTGACCGTGCTGCCTCCACTGCTGACAGATGAGATGATCGCACAGTACACAAGCGCCCTGCTGG CAGGCACCATCACATCCGGATGGACCTTCGGCGCAGGAGCCGCCCTGCAGATCCCCTTCGCTAT GCAGATGGCCTATCGGTTCAACGGCATCGGCGTGACCCAGAATGTGCTGTACGAGAACCAGAA GCTGATCGCCAATCAGTTTAACTCCGCCATCGGCAAGATCCAGGACAGCCTGTCCTCTACAGCC TCCGCCCTGGGCAAGCTGCAGGATGTGGTGAATCAGAACGCCCAGGCCCTGAATACCCTGGTG AAGCAGCTGAGCTCCAACTTCGGCGCCATCTCTAGCGTGCTGAATGACATCCTGAGCCGGCTGG ACAAGGTGGAGGCAGAGGTGCAGATCGACCGGCTGATCACAGGCAGACTGCAGTCTCTGCAGA CCTACGTGACACAGCAGCTGATCAGGGCAGCAGAGATCAGGGCAAGCGCCAATCTGGCAGCAA CCAAGATGTCCGAGTGCGTGCTGGGCCAGTCTAAGAGAGTGGACTTTTGTGGCAAGGGCTATC ACCTGATGTCCTTCCCACAGTCTGCCCCTCACGGAGTGGTGTTTCTGCACGTGACCTACGTGCC AGCCCAGGAGAAGAACTTCACCACAGCACCAGCAATCTGCCACGATGGCAAGGCACACTTTCC CAGGGAGGGCGTGTTCGTGAGCAACGGCACCCACTGGTTTGTGACACAGCGCAATTTCTACGA GCCTCAGATCATCACCACAGACAATACATTCGTGTCCGGCAACTGTGACGTGGTCATCGGCATC GTGAACAATACCGTGTATGATCCTCTGCAGCCAGAGCTGGACAGCTTTAAGGAGGAGCTGGAT AAGTACTTCAAGAATCACACCTCCCCAGACGTGGATCTGGGCGACATCAGCGGCATCAATGCC TCCGTGGTGAACATCCAGAAGGAGATCGACAGGCTGAACGAGGTGGCCAAGAATCTGAACGA GAGCCTGATCGATCTGCAGGAGCTGGGCAAGTATGAGCAGTACATCAAGTGGCCCTGGTATAT CTGGCTGGGCTTCATCGCCGGCCTGATCGCTATCGTGATGGTGACCATCATGCTGTGCTGTATG ACATCCTGCTGTTCTTGCCTGAAGGGCTGCTGTAGCTGTGGCTCCTGCTGTAAGTTTGATGAGG ACGATTCCGAGCCAGTGCTGAAGGGCGTGAAGCTGCACTACACCGGCGGCACCGAGACATCTC AGGTGGCCCCCGCCTAA

(2) the synthetic gene was loaded on the multiple cloning site of pcDNA3.1(Invitrogen, available from Kinsley Biotechnology Ltd.) expression vector using molecular cloning (restriction sites: BamH1 and Xho1), and the correctly identified recombinant plasmid was stored in Top10 E.coli strain, and the resulting plasmid was pcDNA3.1-SARS-CoV-2.

(3) Human embryonic kidney cells HEK293T were cultured in T75 cell culture flasks using DMEM medium with 10% final fetal bovine serum. When the cell growth conjugation degree is about 70%, the serum-free DMEM medium is replaced, and the culture is continued for 1h at 37 ℃. After mixing the transfection reagent (5. mu.g/. mu.l) with 15. mu.g of pcDNA3.1-SARS-CoV-2 (prepared in the laboratory) plasmid and 15. mu.g of pNL4-3 Luc. R-E-plasmid (provided by NIH AIDS reagent and reference, catalog No. 3418, storage of the experiment) were incubated with HEK293T cells in T75 flasks at 37 ℃ for 6 h. Changing DMEM culture medium, culturing at 37 deg.C for 60 hr, collecting culture medium supernatant, filtering with 0.45 μm microporous membrane, packaging, and storing at-80 deg.C. The supernatant contained the novel coronavirus pseudovirus, which was initially identified by assessing its infectivity on target cells expressing human ACE 2. The relevant information of pcDNA3.1-SARS-CoV-2 and pNL4-3 Luc. R-E-can also be found in Chinese patent application 202010667795.7.

(5) MERS-CoV pseudovirus packaging: the experimental procedures were as above, and the plasmids used were pcDNA3.1-MERS-CoV (stock made in this laboratory) and pNL4-3 Luc. R-E-plasmid (stock made in this experiment). See also, in particular, MERS-CoV pseudovirus packaging of Chinese patent application 202010667795.7.

Antiviral activity

1. The entry inhibition effect of the anhydride protein on the novel coronavirus is researched by using the coronavirus pseudovirus. 293T-ACE2 cells were cultured in T75 cell culture flasks using DMEM medium with 10% final fetal bovine serum. 293T-ACE2 cells were plated in 96-well plates after trypsinization, and when the cell growth density approached 80%, for SARS-CoV-2 pseudovirus, 3HP anhydride-modified HSA-anhydrified protein (3HP-HSA) solution, 3HP anhydride-modified beta-lactoglobulin (3 HP-beta-LG) and 3HP anhydride-modified ovalbumin (3HP-OVA) were used; for MERS-CoV pseudovirus, 2-fold gradient dilutions were performed using DMEM medium with SU-HAS, ML-BLG, ML-HAS, 3HP- β -LG (HP-BLG), 3HP-OVA, anti-MERS-CoV peptide, and 50 μ l of each concentration gradient of the diluted anhydrified protein was mixed with 50 μ l of a pseudovirus (SARS-COV-2 or MERS-CoV) solution and incubated at room temperature for 0.5 h. 100 μ l of each gradient mixture was added to a single well of a 96-well plate, with three wells for each drug concentration. Unmodified HSA, beta-LG and OVA solutions diluted by the same concentration gradient are used as a drug negative control group and are also mixed with pseudoviruses; an infection positive control group to which only the pseudovirus was added without adding the anhydridized protein and an infection negative control group to which neither the anhydridized protein nor the pseudovirus was added were set at the same time. Culturing the cells at 37 ℃ until 12h after infection, and replacing with fresh culture medium; and continuously culturing the cells until 72h after infection, washing the cells once by using a PBS solution, adding 50 mu l of cell lysate into each hole, cracking the cells at room temperature for 1h, taking 40 mu l of cell cracking mixture from each hole, adding 50 mu l of luciferase reaction substrate solution, and reading the fluorescence value of each hole by using a fluorescence reader. The entry inhibition rate of each concentration gradient drug against pseudoviruses was calculated using the following calculation formula:

the virus entry inhibition (%) was [ (% infection positive control fluorescence reading mean-experimental group well fluorescence reading)/(infection positive control fluorescence reading mean-infection negative control fluorescence reading mean) ]. 100.

According to the inhibition rate of the virus entering at each concentration, a graph (shown in figure 1) of the inhibition rate and the drug concentration is made by using GraphPad Prism 8 software; calcusyn software was used to calculate the half inhibitory concentrations (IC50 values) of 3HP-HAS and 3HP- β -LG that inhibited entry of the novel coronavirus pseudovirus. The results are shown in FIG. 1. As shown in FIG. 1, IC50(μ M) of 3HP-HSA, 3HP- β -LG and 3HP-OVA were 1.06. + -. 0.302, 1.27. + -. 0.620 and 0.79. + -. 0.490, respectively, in 293T-ACE2 cells; whereas the IC50(μ M) for undiluted β -LG, HSA and OVA was greater than 15. The data in FIG. 1 show that the anhydride-modified protein is effective in inhibiting SARS-CoV-2 pseudovirus. FIG. 7 shows that anhydride-modified proteins are not effective in inhibiting MERS-CoV pseudoviruses. Specifically, MERS-CoV pseudoviruses have IC50 greater than 200 μm for MERS-CoV pseudoviruses with SU-HAS, ML-BLG, ML-HAS, 3HP- β -LG (HP-BLG) and 3 HP-OVA.

The above results indicate that the acid anhydride-modified protein does not have an inhibitory effect on all coronaviruses, but has an inhibitory activity against SARS-CoV-2. This is an unexpected result.

2. The inhibition effect of the anhydride protein on the novel coronavirus is researched by utilizing the novel coronavirus live virus. The experiment needs to be carried out in a biological safety 3-grade laboratory, and the experiment carries out inhibition analysis on SARS-CoV-2 live virus in the Wuhan institute and the biological safety level 3(BSL3) facility of a new-release and repeated infectious disease animal model key laboratory in Beijing. The inhibitory activity of the protein on SARS-CoV-2 was determined by plaque reduction assay. Specifically, the method comprises the following steps: chemically modified proteins with varying dilution concentrations were mixed with SARS-CoV-2(100TCID50) for 30 minutes and then added to monolayers of VERO-E6 cells. After adsorption at 37 ℃, the supernatant was removed and 0.9% methylcellulose was overlaid on the cells. After 72h, plates were fixed and stained. Plaques were counted by fixation with 4% paraformaldehyde and staining with 0.1% crystal violet. The results are shown in FIG. 2. IC50 (. mu.M) for 3HP-HSA, 3HP- β -LG and 3HP-OVA in VERO-E6 cells was 0.432. + -. 0.62, 2.31. + -. 1.31 and 1.21. + -. 0.15, respectively; whereas the IC50(μ M) for undiluted β -LG, HSA and OVA was greater than 15. The data in FIG. 2 indicate that the anhydride-modified protein is effective in inhibiting SARS-CoV-2 live virus, with 3HP-HSA having the lowest IC50 (. mu.M), indicating the best inhibitory effect on live virus.

Cytotoxicity assays

Cytotoxicity of anhydride-modified proteins the cytotoxicity assays of 3HP-HSA or 3HP- β -LG were investigated. The method comprises the following steps:

(1) VERO-E6 or 293T-ACE2 cells at 1X 10⁵Uniformly spreading the mixture in a ratio of each well in a 96-well cell culture plate, and culturing overnight at 37 ℃;

(2) respectively carrying out multiple dilution on a 3HP-HSA sample to be detected, a3 HP-beta-LG or a 3HP-OVA sample and unmodified HSA or beta-LG by using a DMEM culture medium, wherein the initial concentration of the samples is 100 mu M;

(3) aspirating the culture solution of the overnight cultured cells, adding the diluted sample into VERO-E6 cells at a volume of 100. mu.l/well, setting a cytotoxicity control group containing 1% TritonX 100 and a cell control group added with DMEM medium only, making 5 duplicate wells for each dilution, and continuing culturing the cells at 37 ℃ for 48 h;

(4) sucking out the culture solution from each well, adding 100 μ l of DMEM culture solution containing 3 μ l of CCK8 reagent into each well, and continuously incubating for 3h at 37 ℃;

(5) the absorbance values (OD) were read at 450nm using a microplate reader and the cell viability was calculated for each well using the following formula: active cell ratio (%) - (OD 450nm sample-OD 450nm cytotoxicity control group mean)/(OD 450nm cell control group mean-OD 450nm cytotoxicity control group mean) ]. 100.

A cytomotility map (as shown in FIGS. 3 and 4) showing the relationship between cytotoxicity and dose of 3HP-HSA or 3HP- β -LG as a biopharmaceutical agent was prepared using GraphPad Prism 8 software. As shown in FIGS. 3 and 4, the 3HP-HSA, 3HP- β -LG or 3HP-OVA samples had no toxic effect on cell viability of VERO-E6 or 293T-ACE2 cells.

3HP-HSA, 3 HP-beta-LG or 3HP-OVA binding to novel coronavirus S proteins

1. Coating quilt

ELISA 96-well plates were coated with 3HP-HSA or 3HP- β -LG diluted in 10 μ M gradient.

2. Sealing of

Add 300. mu.l of blocking solution to each well and incubate with shaking at room temperature for 2 h.

S protein binding

Commercial neo-coronas protein (containing FC tag with RBD) (sine Biological, Cat: 40592-V05H) was used to incubate with pre-plated 3HP-HSA or 3HP- β -LG for 1 hour at 37 ℃.

4. First antibody (HRP mouse anti-human FC)

After PBST washing three times, incubation was performed with HRP-labeled anti-human FC antibody (Fortebio, 1811062) at 37 ℃ for 0.5 hour.

5 detection

The binding profiles were prepared using GraphPad Prism 8 software with color development and read using a microplate reader.

The results are shown in FIGS. 5 and 6

In FIGS. 5 and 6, it is shown that 3HP-HSA, 3HP- β -LG or 3HP-OVA binds to the novel coronavirus S protein, and that three secondary wells per concentration, HSA, β -LG or OVA, are negative controls, and that the results show dose dependence, indicating that the anhydride-modified protein is capable of specifically binding to the novel coronavirus S protein.

Sequence listing

<110> Shanxi brocade biomedical products Ltd

FUDAN University

SOUTHERN MEDICAL University

GUANGZHOU INSTITUTE OF RESPIRATORY HEALTH

<120> method for inhibiting coronavirus using acid anhydride-modified protein

<130> C20P7809

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 3888

<212> DNA

<213> Artificial sequence

<220>

<223> SARS-CoV-2S protein gene

<400> 1

atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60

tccgtgctag cccagtgcgt gaacctgacc acaaggaccc agctgccccc tgcctatacc 120

aattccttca cacggggcgt gtactatccc gacaaggtgt ttagaagctc cgtgctgcac 180

tctacacagg atctgtttct gcctttcttt agcaacgtga cctggttcca cgccatccac 240

gtgagcggca ccaatggcac aaagcggttc gacaatccag tgctgccctt taacgatggc 300

gtgtacttcg cctctaccga gaagagcaac atcatcagag gctggatctt tggcaccaca 360

ctggactcca agacacagtc tctgctgatc gtgaacaatg ccaccaacgt cgtgatcaag 420

gtgtgcgagt tccagttttg taatgatcct ttcctgggcg tgtactatca caagaacaat 480

aagagctgga tggagtccga gtttcgcgtg tattctagcg ccaacaattg cacatttgag 540

tacgtgtccc agccattcct gatggacctg gagggcaagc agggcaattt caagaacctg 600

agggagttcg tgtttaagaa tatcgatggc tacttcaaga tctactctaa gcacacccca 660

atcaacctgg tgcgcgacct gccacagggc ttcagcgccc tggagccact ggtggatctg 720

cccatcggca tcaacatcac ccggtttcag acactgctgg ccctgcacag aagctacctg 780

acacctggcg actcctctag cggatggacc gcaggagctg ccgcctacta tgtgggctat 840

ctgcagccaa ggaccttcct gctgaagtac aacgagaatg gcaccatcac agacgcagtg 900

gattgcgcac tggaccccct gagcgagacc aagtgtacac tgaagtcctt taccgtggag 960

aagggcatct atcagacatc caatttcagg gtgcagccca ccgagtctat cgtgcgcttt 1020

cccaatatca caaacctgtg cccttttggc gaggtgttca acgcaaccag gttcgcaagc 1080

gtgtacgcat ggaataggaa gcggatcagc aactgcgtgg ccgactatag cgtgctgtac 1140

aactccgcct ctttcagcac ctttaagtgc tatggcgtgt cccccacaaa gctgaatgac 1200

ctgtgcttta ccaacgtgta cgccgattct ttcgtgatca ggggcgacga ggtgcgccag 1260

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acagagatct accaggcagg cagcacccca tgcaatggag tggagggctt taactgttat 1500

ttccctctgc agagctacgg cttccagcca acaaacggcg tgggctatca gccctaccgc 1560

gtggtggtgc tgagctttga gctgctgcac gcacctgcaa cagtgtgcgg accaaagaag 1620

tccaccaatc tggtgaagaa caagtgcgtg aacttcaact tcaacggact gaccggcaca 1680

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tgctctttcg gcggcgtgag cgtgatcaca cctggcacca atacaagcaa ccaggtggcc 1860

gtgctgtatc aggacgtgaa ttgtaccgag gtgcccgtgg caatccacgc agatcagctg 1920

acccctacat ggcgggtgta cagcaccggc tccaacgtgt tccagacaag agccggatgc 1980

ctgatcggag cagagcacgt gaacaattcc tatgagtgcg acatccctat cggcgccggc 2040

atctgtgcct cttaccagac ccagacaaac tctccaagga gagcccggag cgtggcatcc 2100

cagtctatca tcgcctatac aatgagcctg ggcgccgaga acagcgtggc ctactctaac 2160

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agcaacctgc tgctgcagta cggcagcttt tgtacccagc tgaatagagc cctgacaggc 2340

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aagaccccac ccatcaagga ctttggcggc ttcaattttt cccagatcct gcccgatcct 2460

tccaagcctt ctaagcggag ctttatcgag gacctgctgt tcaacaaggt gaccctggcc 2520

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atcgcacagt acacaagcgc cctgctggca ggcaccatca catccggatg gaccttcggc 2700

gcaggagccg ccctgcagat ccccttcgct atgcagatgg cctatcggtt caacggcatc 2760

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gatgtggtga atcagaacgc ccaggccctg aataccctgg tgaagcagct gagctccaac 2940

ttcggcgcca tctctagcgt gctgaatgac atcctgagcc ggctggacaa ggtggaggca 3000

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cccagggagg gcgtgttcgt gagcaacggc acccactggt ttgtgacaca gcgcaatttc 3360

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gccaagaatc tgaacgagag cctgatcgat ctgcaggagc tgggcaagta tgagcagtac 3660

atcaagtggc cctggtatat ctggctgggc ttcatcgccg gcctgatcgc tatcgtgatg 3720

gtgaccatca tgctgtgctg tatgacatcc tgctgttctt gcctgaaggg ctgctgtagc 3780

tgtggctcct gctgtaagtt tgatgaggac gattccgagc cagtgctgaa gggcgtgaag 3840

ctgcactaca ccggcggcac cgagacatct caggtggccc ccgcctaa 3888

Claims (10)

1. A method of inhibiting the growth of a coronavirus, preventing or controlling infection of a cell by a coronavirus, in vitro, or a composition comprising an anhydride-modified protein, the method comprising the step of contacting the anhydride-modified protein or the composition comprising an anhydride-modified protein with an article or sample containing, suspected of containing, or at risk of containing a coronavirus;

wherein the protein is selected from albumin, beta lactoglobulin, and RNase, and the acid anhydride is selected from 3-hydroxyphthalic anhydride, succinic anhydride, or maleic anhydride;

preferably wherein said protein is fully anhydrified as determined by a lysine to arginine modification ratio of said protein;

preferably wherein the sample is selected from the group consisting of stool, saliva, blood and serum;

preferably wherein the article is selected from the group consisting of sanitary napkins, pantiliners, wet wipes and paper towels; the paper towel comprises paper handkerchiefs, removable toilet paper, napkins or paper towels.

2. The method of claim 1, wherein the coronavirus is SARS-CoV-2.

3. The method according to claim 1 or 2, wherein the albumin is selected from serum albumin or ovalbumin, preferably human serum albumin, bovine serum albumin or chicken ovalbumin; the beta lactoglobulin is bovine beta lactoglobulin; and the rnase is selected from rnase 1 or rnase 7.

4. The method of any one of claims 1-3, wherein the composition is in the form of a solution, tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel, or aerosol.

5. The method of any one of claims 1-4, wherein the anhydride-modified protein is prepared by mixing the anhydride with the protein.

6. Use of an anhydride-modified protein or a composition comprising an anhydride-modified protein in the manufacture of a medicament for treating or preventing a coronavirus infection or a disease caused by a coronavirus infection in a subject,

wherein the protein is selected from albumin, beta lactoglobulin, and RNase, and the acid anhydride is selected from 3-hydroxyphthalic anhydride, succinic anhydride, or maleic anhydride;

preferably, wherein said protein is fully anhydrified as determined by a lysine and arginine modification ratio of said protein.

7. The use of claim 6, wherein the coronavirus is SARS-CoV-2.

8. Use according to claim 6 or 7, wherein the albumin is selected from serum albumin or ovalbumin, preferably human serum albumin, bovine serum albumin or chicken ovalbumin; the beta lactoglobulin is bovine beta lactoglobulin; and the rnase is selected from rnase 1 or rnase 7.

9. The use of any one of claims 6-8, wherein the anhydride-modified protein is prepared by mixing the anhydride with the protein.

10. The use according to any one of claims 6-9, wherein the medicament is in the form of a tablet, capsule, powder, nasal drop or aerosol, or in the form of a solution, tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel or aerosol.

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