CN116392504A - Application of marine organism-derived natural sulfated polysaccharide as anti-coronavirus and disease-causing drug - Google Patents

Application of marine organism-derived natural sulfated polysaccharide as anti-coronavirus and disease-causing drug Download PDF

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CN116392504A
CN116392504A CN202310510656.7A CN202310510656A CN116392504A CN 116392504 A CN116392504 A CN 116392504A CN 202310510656 A CN202310510656 A CN 202310510656A CN 116392504 A CN116392504 A CN 116392504A
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sulfated polysaccharide
coronavirus
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于广利
刘婵娟
蔡超
王鑫
李国云
蒋昊
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Ocean University of China
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Abstract

The invention belongs to the field of biological medicine, and in particular relates to application of marine organism-derived natural sulfated polysaccharide as a medicine for resisting coronaviruses and diseases caused by coronaviruses. Use of a natural sulfated polysaccharide of marine origin, a pharmaceutically acceptable salt, for the preparation of a medicament for binding to Spike protein (Spike), for the preparation of an inhibitor of main protease (Mpro), or for the preparation of a papain-like (PLpro) inhibitor. The natural sulfated polysaccharide from marine organisms provided by the invention has obvious effect of inhibiting the proliferation of coronaviruses, can inhibit the binding of Spike protein (Spike) on the surface of the coronaviruses and host cells, and can inhibit the activities of main protease (Mpro) and papain-like (PLpro), so that the coronaviruses are effectively blocked from infecting the host cells.

Description

Application of marine organism-derived natural sulfated polysaccharide as anti-coronavirus and disease-causing drug
The application is a divisional application of Chinese invention patent application, and the application date of the original application is: 8 months 17 days 2020, application number: 202010825078 . 2, the invention creates the name: application of marine organism source natural sulfated polysaccharide as anti-coronavirus and disease drug, publication No.: CN111773240a; in the process of patent examination, the marine organism source natural sulfated polysaccharide in the original application claim 1 comprises a plurality of parallel technical schemes, and the parallel technical schemes have no specific technical characteristics and have no singleness with each other, so that the divisional application is proposed.
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of marine organism-derived natural sulfated polysaccharide as a medicine for resisting coronaviruses and diseases caused by coronaviruses.
Background
SARS-CoV-2 is an RNA virus having an envelope structure. The spike glycoprotein on the lipid membrane of the viral particle is responsible for adhesion to the host receptor. Viral genomic RNA within the lipid membrane may encode replicase multimeric proteins that can be cleaved by 3C-like proteases (3 CLpro, also known as the main protease Mpro) and papain-like (PLpro) to produce functional polypeptides, such as RNA-dependent RNA polymerase (RdRp) and helicase (Hel), that are involved in viral RNA transcription and replication. Thus, the spike glycoprotein associated with SARS-CoV-2 infection of host cells, the primary protease associated with SARS-CoV-2 transcriptional replication, and papain-like are important targets for anti-SARS-CoV-2 drug development.
Marine organism resources are abundant, and natural saccharide compounds derived from marine organisms, especially acidic saccharide compounds, can bind with proteins with positive charges of viruses to prevent viruses from replicating or infecting fineAnd (5) cells. In addition, the preparation method has the advantages of low toxicity, difficulty in causing virus to generate drug resistance and the like, and has wide prospect in antiviral drug development. Literature searches have found that natural sulfated polysaccharides of marine origin, such as complex structural fucan sulfates of brown algae origin, have an anti-multiple influenza virus effect (publication No. CN 103880975A; patent No. ZL201410132608. X), the backbone structure of which consists of alpha-1, 2-mannose (Man) and beta-1, 4-glucuronic acid (GlcA), contain branched alpha-1, 3-linked-sulfated fucose residues at the mannose C3 position (Jiandong Wu, J Carbohy Chem,34:303-317;Wei Wang,Sci.Rep.2017,7:40760), and that a fucoidan (RPI-27 and RPI-128) of similar structure with different molecular weights (100 kD and 12 kD) has been recently reported to have an activity of inhibiting VID-COV-2 coronavirus Spike protein, which contains alpha-1, 2-mannose (Man) and beta-1, 4-glucuronic acid (GlcA) backbones, and branched alpha-1, 2/3-linked-fucose residues at the mannose C3 position (Cell, 6:50, cup); fucoidan derived from undaria pinnatifida (Hayashi, k., int. Immunopharmacol.2008,8,109) was found to have anti-HSV activity. Studies on other types of structural fucose sulfates against SARS-CoV-2 have not been reported. Carrageenan from red algae has a broad spectrum of anti-H1N 1, HSV, HPV activity (Cui Hao, rev Med Virol.2019, 29, e 2043), and iota-carrageenan also has anti-Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) activity (Chunhe Guo, anti-vir Ther,2019, 24 (4): 261-270), but no anti-neocoronavirus activity has yet been found. Rhamnoglycans sulfate derived from Chlorella has anti-children hand-foot-and-mouth virus (EV 71) activity (Shuyao Wang, carbohydrate Polym,2018, 200:43-53) and Newcastle Disease Virus (NDV) activity (Aguilar-
Figure SMS_1
Drugs,2015,13,697-712), but no anti-novel coronavirus activity was found.
Disclosure of Invention
The invention aims to provide an application of marine organism source natural sulfated polysaccharide in preparing medicines for resisting coronaviruses and diseases caused by the coronaviruses, and provides an effective medicine for treating coronaviruses, especially novel coronavirus infection (COVID-19) and a composition thereof, wherein specific marine plants and marine animals are taken as raw materials, serial marine source natural sulfated polysaccharides are obtained through extraction and separation, and the activity of the marine organism source natural sulfated polysaccharide in inhibiting coronaviruses, especially novel coronaviruses (SARS-COV-2) is screened.
The technical scheme of the invention is as follows:
an application of marine organism source natural sulfated polysaccharide in preparing medicament for resisting coronavirus and diseases caused by coronavirus, an application of marine organism source natural sulfated polysaccharide and pharmaceutically acceptable salt in preparing medicament for combining with Spike protein (Spike), preparing inhibitor of main protease (Mpro) or preparing papain-like (PLpro) inhibitor, wherein the marine organism source natural sulfated polysaccharide has any one of the following structural characteristics, and the molecular weight range is 3-600 kDa, and the application comprises the following steps:
(1) Fucoidan sulfate (also known as fucoidan) has one of the following structural features: (a) Fucoidan of type I structure, the backbone consisting of α -1, 3-linked sulfated fucose; (b) Fucoidin with a II type structure, wherein the main chain consists of sulfated fucose with alpha-1, 3 and alpha-1, 4 alternately connected; (c) Fucoidin with III structure, wherein the main chain consists of beta-1, 4-glucuronic acid and alpha-1, 2-mannose, and the branch structure consists of alpha-1, 3-sulfated fucoidin; (d) Other types of fucoidan, the backbone consisting of α -1,2 and α -1,3 and/or α -1,4 linked sulfated fucose;
(2) Fucosylated chondroitin sulfate, its structural features are as follows: chondroitin sulfate disaccharide repeating units with main chains formed by alternately connecting glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through beta-1, 3 and beta-1, 4 glycosidic bonds, wherein hydroxyl groups at C4, C6 or C4 and C6 of GalNAc contain sulfate groups, and sulfated fucooligosaccharide branches are connected at C3 position of GlcA through alpha-1, 3 glycosidic bonds;
(3) The structural characteristics of the sulfated galactan are as follows: galactose (Gal) consisting of a beta-1, 3-linkage and an alpha-1, 4-linkage, or a galactose (AnG) consisting of a beta-1, 3-linkage and an alpha-1, 4-linkage-3, 6-endo-ether, and having different sulfate groups at the C2, C4 or C6 positions of the galactose;
(4) Rhamnoglycan sulfate: the structural characteristics are as follows: is formed by alternately connecting rhamnose (Rha) and glucuronic acid through alpha-1, 4 and beta-1, 4 glycosidic bonds; or alternatively connecting Rha and iduronic acid via alpha-1, 4 glycosidic bond; or alternatively connecting rhamnose (Rha) and xylose (Xyl) through alpha-1, 4 and beta-1, 4 glycosidic bonds; or alternatively, the alpha-1, 2 and the alpha-1, 3-rhamnose are connected alternately; and the C2 and/or C3 position of the structural Rha contains sulfate ester groups.
The fucoidin with the I-type structure also has other sugar residue branched chain structures; in the type II structural fucoidan, other sugar residue branched chain structures are also provided.
The coronavirus is one or more than two selected from SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
The natural sulfated polysaccharide is derived from marine plants or marine animals.
The natural sulfated polysaccharide can be used as an inhibitor for inhibiting the infection cells of coronaviruses by combining with Spike protein (Spike) of the coronaviruses, and can inhibit the replication and amplification of the viruses by inhibiting the activity of main protease (Mpro) and/or papain-like protease (PLpro) of the coronaviruses.
The application of the natural sulfated polysaccharide and the pharmaceutically acceptable salt in preparing coronavirus inhibitors.
The application of the natural sulfated polysaccharide and the pharmaceutically acceptable salt in preparing medicaments for preventing and/or treating coronavirus infection or preventing and/or treating diseases or symptoms related to coronavirus infection.
The application of the natural sulfated polysaccharide, a kit or a reagent for combining pharmaceutically acceptable salts and Spike protein (Spike), a kit or a reagent for preparing a main protease (Mpro) inhibitor, or a kit or a reagent for preparing a papain-like protease (PLpro) inhibitor.
The natural sulfated polysaccharide, the pharmaceutically acceptable salt and one or more than two other active agents for preventing and/or treating coronavirus infection are mixed to prepare a pharmaceutical composition for preventing and/or treating coronavirus infection.
The design idea of the invention is as follows:
the natural sulfated polysaccharide from marine organisms provided by the invention can inhibit the binding of Spike protein (Spike) on the surface of coronaviruses and host cells, so that the coronaviruses are effectively blocked from infecting the host cells, and the activities of main protease (Mpro) and papain-like protease (PLpro) of the coronaviruses can be inhibited, so that the replication and amplification processes of the viruses are inhibited. Therefore, the marine natural sulfated polysaccharide provided by the invention has great potential for developing anti-coronavirus and disease-causing drugs.
The invention has the advantages and beneficial effects that:
(1) The natural sulfated polysaccharide from marine organisms provided by the invention can inhibit Spike protein (Spike) on the surface of coronaviruses from being combined with host cells, and the inhibition activity level of the natural sulfated polysaccharide is superior to that of clinical drugs heparin and low-molecular heparin.
(2) The natural sulfated polysaccharide from marine organisms provided by the invention has the activity of inhibiting main protease (Mpro) and papain-like protease (PLpro), and the inhibition activity level is superior to that of heparin.
(3) In the whole, the anti-coronavirus activity and the better than clinical drugs heparin and low molecular heparin of the natural sulfated polysaccharide derived from marine organisms can be used in the preparation and application fields of anti-coronavirus and related diseases.
Drawings
FIG. 1 inhibitory activity of fucan sulfate, galactan sulfate, fucosylated chondroitin sulfate, rhamno sulfate, and heparin on SARS-CoV-2 spike protein. In the figure, the abscissa Log Dose represents the logarithm of the compound concentration (. Mu.M), and the ordinate Relative Luminescence represents the relative luminous intensity (%).
FIG. 2 inhibition curves of the SARS-CoV-2 main protease by galactan sulfate, fucan sulfate, fucosylated chondroitin sulfate. In the figure, the abscissa Time represents Time(s), the ordinate Intensity represents fluorescence Intensity, blank represents Blank, iota-carageenan represents Iota-carrageenan, FCS represents fucosylated chondroitin sulfate, and Fucoidan represents Fucoidan sulfate.
FIG. 3 inhibition of SARS-CoV-2 main protease IC by fucan sulfate 50 The curve is determined. In the figure, log concentration on the abscissa represents Log concentration (nM), IC 50 Representing half the inhibitory concentration.
Detailed Description
In a specific implementation process, the invention relates to application of marine-source natural sulfated polysaccharide and pharmaceutically acceptable salt in preparation of coronavirus inhibition inhibitor, wherein the natural sulfated polysaccharide has any one of the following structures:
(1) Fucoidan sulfate (also known as fucoidan), comprising: (a) Type I fucoidin, the main chain of which consists of alpha-1, 3-linked sulfated fucoidin, and can also have other sugar residue branched chain structures; (b) Type II fucoidin, the main chain is composed of sulfated fucoidin with alpha-1, 3 and alpha-1, 4 alternately connected, and can also have other sugar residue branched chain structure; (c) Type III fucoidin, wherein the main chain consists of beta-1, 4-glucuronic acid and alpha-1, 2-mannose, and the branch structure consists of alpha-1, 3-sulfated fucoidin; (d) Other types of fucoidan have a backbone consisting of alpha-1, 2 and alpha-1, 3 and/or alpha-1, 4 linked sulfated fucose.
Figure SMS_2
(2) Fucosylated chondroitin sulfate, its structural features are as follows: chondroitin sulfate disaccharide repeating units with main chains formed by alternately connecting glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through beta-1, 3 and beta-1, 4 glycosidic bonds, and sulfated fuco-oligosaccharide branched chains are connected at the C3 position of GlcA through alpha-1, 3 glycosidic bonds.
Figure SMS_3
(3) The structural characteristics of the sulfated galactan are as follows: galactose (Gal) consisting of beta-1, 3-linkage and alpha-1, 4-linkage, or galactose (AnG) consisting of beta-1, 3-linkage and alpha-1, 4-linkage-3, 6-endo-ether, and having different sulfate groups at the C2, C4 or C6 positions of galactose.
Figure SMS_4
(4) The rhamnoglycan sulfate is characterized by comprising the following structural characteristics: is formed by alternately connecting rhamnose (Rha) and glucuronic acid through alpha-1, 4 and beta-1, 4 glycosidic bonds; or alternatively connecting Rha and iduronic acid via alpha-1, 4 glycosidic bond; or alternatively connecting rhamnose (Rha) and xylose (Xyl) through alpha-1, 4 and beta-1, 4 glycosidic bonds; or alternatively, the alpha-1, 2 and the alpha-1, 3-rhamnose are connected alternately; and the C2 and/or C3 position of the structural Rha contains sulfate ester groups.
Figure SMS_5
The invention also provides the use of a marine organism-derived natural sulfated polysaccharide having any of the following structures, a pharmaceutically acceptable salt thereof, for the preparation of a medicament for binding to Spike protein (Spike), for the preparation of a main protease (Mpro) inhibitor, or for the preparation of a papain-like protease (PLpro) inhibitor:
(1) Fucoidan sulfate (also known as fucoidan), comprising: (a) Fucoidan of type I structure, the main chain of which consists of alpha-1, 3-linked sulfated fucose, can also have branched chain structure of other sugar residues; (b) Fucoidan with II type structure, wherein the main chain consists of sulfated fucose with alternately connected alpha-1, 3 and alpha-1, 4, and can also have branched chain structure of other sugar residues; (c) Fucoidin with a III type structure, wherein the main chain consists of beta-1, 4-glucuronic acid and alpha-1, 2-mannose, and the branch structure consists of alpha-1, 3-sulfated fucoidin; (d) Other types of fucoidan have a backbone consisting of alpha-1, 2 and alpha-1, 3 and/or alpha-1, 4 linked sulfated fucose.
Figure SMS_6
(2) Fucosylated chondroitin sulfate, its structural features are as follows: chondroitin sulfate disaccharide repeating units with main chains formed by alternately connecting glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through beta-1, 3 and beta-1, 4 glycosidic bonds, and sulfated fucose branches are connected at the C3 position of GlcA through alpha-1, 3 glycosidic bonds.
Figure SMS_7
(3) The structural characteristics of the sulfated galactan are as follows: galactose (Gal) consisting of beta-1, 3-linkage and alpha-1, 4-linkage, or galactose (AnG) consisting of beta-1, 3-linkage and alpha-1, 4-linkage-3, 6-endo-ether, and having different sulfate groups at the C2, C4 or C6 positions of galactose.
Figure SMS_8
(4) Rhamnoglycan sulfate: the structural characteristics are as follows: is formed by alternately connecting rhamnose (Rha) and glucuronic acid through alpha-1, 4 and beta-1, 4 glycosidic bonds; or alternatively connected by Rha and iduronic acid (IdoA) through alpha-1, 4 glycosidic bond; or alternatively connecting rhamnose (Rha) and xylose (Xyl) through alpha-1, 4 and beta-1, 4 glycosidic bonds; or alternatively, the alpha-1, 2 and the alpha-1, 3-rhamnose are connected alternately; and the C2 and/or C3 position of the structural Rha contains sulfate ester groups.
Figure SMS_9
For the above-described applications, it is preferable that one or two or more of Spike protein (Spike), main protease (Mpro), papain-like protease (PLpro) in coronavirus; more preferably, the coronavirus is selected from one or more of the group consisting of COVID-19 (SARS-CoV-2), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
The invention also provides application of the marine organism source natural sulfated polysaccharide and pharmaceutically acceptable salt in preparation of coronavirus inhibitor.
The invention also provides application of the marine organism-derived natural sulfated polysaccharide and pharmaceutically acceptable salt in preparing medicaments for preventing and/or treating coronavirus infection or preventing and/or treating diseases or symptoms related to coronavirus infection.
For use as described above, preferably the coronavirus is selected from the group consisting of COVID-19 (SARS-CoV-2), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
The invention also provides application of the marine organism-derived natural sulfated polysaccharide and pharmaceutically acceptable salt in preparing a kit or reagent for binding with Spike protein (Spike), a kit or reagent for preparing a main protease (Mpro) inhibitor, or a kit or reagent for preparing a papain-like protease (PLpro) inhibitor.
The invention also provides a pharmaceutical composition for preventing and/or treating coronavirus infection, which comprises the marine organism-derived natural sulfated polysaccharide, pharmaceutically acceptable salt and one or more than two other active agents for preventing and/or treating coronavirus infection.
Preferably, the molecular weight of the marine organism-derived natural sulfated polysaccharide as described above ranges from 3 to 600kDa.
The experimental screening research shows that the natural sulfated polysaccharides from marine organisms have better inhibition effect on coronaviruses, especially Spike protein (Spike) of novel coronavirus (SARS-CoV-2), main protease (Mpro or 3CL pro) and PLpro. The invention provides the application potential of marine organism source natural sulfated polysaccharide in resisting coronavirus, especially SARS-CoV-2, and proves that the marine organism source natural sulfated polysaccharide has good clinical development and application prospect.
The marine organism source natural sulfated polysaccharide provided by the invention is preferably characterized by comprising the following structural characteristics:
(1) Fucoidan sulfate (also known as fucoidan), comprising: (a) Fucoidan of type I structure, the main chain of which consists of alpha-1, 3-linked sulfated fucose, can also have branched chain structure of other sugar residues; (b) Fucoidan with II type structure, wherein the main chain consists of sulfated fucose with alternately connected alpha-1, 3 and alpha-1, 4, and can also have branched chain structure of other sugar residues; (c) Fucoidin with a III type structure, wherein the main chain consists of beta-1, 4-glucuronic acid and alpha-1, 2-mannose, and the branch structure consists of alpha-1, 3-sulfated fucoidin; (d) Other types of fucoidan have a backbone consisting of alpha-1, 2 and alpha-1, 3 and/or alpha-1, 4 linked sulfated fucose.
Figure SMS_10
(2) Fucosylated chondroitin sulfate, its structural features are as follows: chondroitin sulfate disaccharide repeating units with main chains formed by alternately connecting glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through beta-1, 3 and beta-1, 4 glycosidic bonds, and sulfated fucose branches are connected at the C3 position of GlcA through alpha-1, 3 glycosidic bonds.
Figure SMS_11
(3) The structural characteristics of the sulfated galactan are as follows: consists of beta-1, 3-linked and alpha-1, 4-linked galactose (Gal), or of beta-1, 3-linked galactose and alpha-1, 4-linked-3, 6-endo-ether galactose (AnG), with the C2, C4 or C6 position of galactose containing different sulphate groups.
Figure SMS_12
(4) Rhamnoglycan sulfate: the structural characteristics are as follows: is formed by alternately connecting rhamnose (Rha) and glucuronic acid through alpha-1, 4 and beta-1, 4 glycosidic bonds; or alternatively connecting Rha and iduronic acid via alpha-1, 4 glycosidic bond; or alternatively connecting rhamnose (Rha) and xylose (Xyl) through alpha-1, 4 and beta-1, 4 glycosidic bonds; or alternatively, the alpha-1, 2 and the alpha-1, 3-rhamnose are connected alternately; and the C2 and/or C3 position of the structural Rha contains sulfate ester groups.
Figure SMS_13
The marine organism-derived sulfated polysaccharides of the invention have the effect of inhibiting coronaviruses, especially novel coronaviruses (SARS-CoV-2). Experimental results show that the sulfated polysaccharides from various marine organisms provided by the invention have remarkable anti-novel coronavirus effect and inhibit Spike IC 50 IC with value between 0.01 and 30 mu M for inhibiting Mpro 50 IC with a value between 1nM and 100nM for inhibiting papain-like protease (PLpro) 50 The value is 0.001-30 mu M. IC for inhibiting Mpro by fucoidan 50 IC that inhibits Spike protein at 5.68nM 50 IC inhibiting papain-like protease (PLpro) at 0.06. Mu.M 50 7.65nM, and the effect is significantly better than heparin.
As a preferred embodiment, the use as described above, the coronaviruses include, but are not limited to, novel coronaviruses (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing middle east respiratory syndrome).
The application of the marine organism source natural sulfated polysaccharide in resisting novel coronaviruses can prepare the marine organism source natural sulfated polysaccharide into spray, injection, capsule, ointment, cream, gel, liniment.
The therapeutically effective amount of the marine organism-derived natural sulfated polysaccharide, pharmaceutically acceptable salt, or stereoisomer of the invention is preferably administered to a patient in need of such treatment in accordance with the usual route of administration and formulated according to methods known in the art in conventional pharmaceutical compositions comprising an effective amount of the active ingredient and a suitable pharmaceutically acceptable carrier and dosage form.
By "therapeutically effective amount" is meant an amount which, when administered, is sufficient to prevent the development of, or to alleviate to some extent, one or more symptoms of the disease for which it is intended. The specific dosage of the compounds administered according to the present invention will be determined by the specific circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and similar considerations. In particular, a "therapeutically effective amount" of a compound refers to an amount of the compound that is sufficient to prevent or to some extent ameliorate one or more coronavirus infections.
The individual doses and daily doses will further vary depending on the type and severity of the coronavirus infection to be treated and the response of the particular patient to the drug treatment. Thus, an accurate single dose will be determined according to standard medical principles under the direction of a physician.
The human effective daily dosage of the marine-derived natural sulfated polysaccharide, pharmaceutically acceptable salt of the invention for use in treating coronavirus-derived diseases is 1mg to about 500mg, about 5mg to about 1000mg, or about 10mg to about 2000mg of an injectable or other active agent oral dosage form.
The marine-derived natural sulfated polysaccharides and pharmaceutically acceptable salts of the present invention may be used alone or in combination with other therapeutic agents.
In one embodiment of the invention, the marine-derived natural sulfated polysaccharide, pharmaceutically acceptable salt, is for use in the prevention and/or treatment of coronavirus infection, wherein the prevention or treatment comprises administration as the sole active ingredient.
In another embodiment of the invention, the marine organism-derived natural sulfated polysaccharide, pharmaceutically acceptable salt described above, is for use in the prevention and/or treatment of coronavirus infection, wherein the prevention or treatment comprises use in combination therapy with a therapeutic agent selected from the group consisting of other therapeutic agents.
As will be apparent to those skilled in the art, the combination of the present invention comprising a marine-derived natural sulfated polysaccharide of the present invention, a pharmaceutically acceptable salt, and an additional therapeutic agent is effective not only when these active ingredients are used in a single composition, but also when they are used in two different compositions (administered simultaneously, sequentially, or separately after a period of time). Furthermore, it will be appreciated by those skilled in the art that the marine-derived natural sulfated polysaccharides, pharmaceutically acceptable salts of the present invention may be formulated for use with other active ingredients in combination therapies to prevent and/or treat coronavirus infections.
In a particular embodiment, the combination therapy comprises administering to a subject the marine organism-derived natural sulfated polysaccharide of the invention, a pharmaceutically acceptable salt, and an additional therapeutic agent, simultaneously, sequentially, or separately. Alternatively, the combination therapy comprises administering to a subject the marine organism-derived natural sulfated polysaccharide of the invention, a pharmaceutically acceptable salt or stereoisomer, and an additional therapeutic agent in a single composition.
In one embodiment of the present invention, the marine-derived natural sulfated polysaccharide, pharmaceutically acceptable salt, or stereoisomer of the present invention may be conveniently administered to a patient. Thus, the compounds for use in the present invention may be in the form of a pharmaceutical composition comprising an effective amount of the marine-derived natural sulfated polysaccharide of the invention, a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable excipient or carrier. This aspect may also be expressed as a composition comprising an effective amount of a marine organism-derived natural sulfated polysaccharide, a pharmaceutically acceptable salt of the invention in combination with a pharmaceutically acceptable excipient or carrier for use in the prevention and/or treatment of coronavirus infection.
In one embodiment of the invention, the compounds used may be administered orally, by injection, subcutaneously, respiratory tract, transdermally, parenterally, rectally, topically, intravenously, intramuscularly or by other means in a dosage unit formulation comprising conventional pharmaceutical carriers. The pharmaceutical composition may be formulated in any pharmaceutically acceptable form, such as: tablets, granules, injections, gels, pills, capsules, suppositories, implants, nano-preparations and powder injections. Some dosage forms, such as tablets and capsules, may be subdivided into suitable dosage unit forms containing an appropriate amount of the active component, such as an amount effective to achieve the desired end.
In another embodiment, the marine-derived natural sulfated polysaccharide, pharmaceutically acceptable salt for use in the present invention is an injectable formulation to be administered to a patient to be treated, and the injectable formulation suitable for use in the present invention refers to a sterilized or sterile solution, emulsion or suspension of a drug with a suitable solvent or dispersion medium for injection into a human body, and a powder sterile formulation for formulation into a solution or suspension prior to use. The injection includes injection (where large volume injection for intravenous drip is also called intravenous infusion), sterile powder for injection and concentrated solution for injection.
The carrier includes excipients and diluents and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient to be treated. The carrier may be inert or it may have a pharmaceutical benefit itself.
The types of carriers include, but are not limited to: diluents such as fillers and bulking agents, binders, lubricants, anti-caking agents, disintegrants, sweeteners, buffers, preservatives, solubilizers, isotonic agents, suspending and dispersing agents, wetting or emulsifying agents, flavoring and perfuming agents, thickening agents and vehicles.
Alternative active agents may be included in the pharmaceutical compositions that do not substantially affect the activity of the compounds of the present invention.
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified. The marine organism source natural sulfated polysaccharide has the following structure:
(1) Fucoidan sulfate: the backbone consisted of alpha-1, 3-linked sulfated fucose with a molecular weight of 600kDa.
(2) Iota-carrageenan: consists of a beta-1, 3-linked galactose and alpha-1, 4-linked-3, 6-lacto-galactose repeating disaccharide unit, the C4 position of galactose residue and the C2 position of lacto-galactose residue are replaced by sulfate radical, and the molecular weight is 200kDa.
(3) Fucosylated chondroitin sulfate: chondroitin sulfate disaccharide repeating units with main chains formed by alternately connecting glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through beta-1, 3 and beta-1, 4 glycosidic bonds, and fucose branched chains with C2 and C3 replaced by sulfate radicals at C3 position of GlcA through alpha-1, 3 glycosidic bonds, wherein the molecular weight is 3kDa and 50kDa.
(4) Rhamnoglycan sulfate: is formed by alternately connecting rhamnose (Rha) and glucuronic acid through alpha-1, 4 and beta-1, 4 glycosidic bonds, wherein the C2 and C3 positions of the Rha contain sulfate groups; the molecular weight is 200kDa.
Example-inhibition of SARS-CoV-2 spike protein by marine-derived Natural sulfated polysaccharide
Based on the SARS-CoV-2-spike protein virus detection system, the effect of fucoidan (derived from brown algae), iota-carrageenan (derived from red algae), fucosylated chondroitin sulfate (derived from sea cucumber) and rhamnosan sulfate (derived from green algae) on blocking SARS-CoV-2 infected cells was evaluated.
The sulfated polysaccharide samples employed in the present invention may be extracted using reagents, methods and apparatus conventional in the art.
1) Cell resuscitating culture: the 293T/17 human embryo kidney cell strain is cultured by mixing 10% fetal calf serum, 1% diabody and the balance of DMEM culture medium according to weight percentage, and the cells are cultured for two generations after resuscitating for later use.
2) Viroid preparation: 293T/17 human embryonic kidney cells were plated into 6-well cell culture plates, when cell confluency reached about 60%, 3. Mu.g of the plasmid was subjected to pack transfection with a transfection reagent (Lipofiter 3.0), and then the 6-well cell culture plates were placed in a carbon dioxide incubator (37 ℃,5% CO) 2 ) Culturing, collecting viroid supernatant solution after 48h transfection, and preserving at-80 ℃.
3) Transient transfection of cells: 293T/17 human embryonic kidney cells were plated into 6-well cell culture plates, and when cell confluency reached about 70%, 3. Mu.g of plasmid (ACE 2-pcDNA3.1) was transfected into 293T17 cells using a transfection reagent (lipofilter 3.0), and the 6-well cell culture plates were incubated in a carbon dioxide incubator for 48h.
4) Cell digestion: the transiently transfected 293T/17 human embryonic kidney cells are digested by pancreatin, resuspended in complete medium, counted by a cell counter, plated on 96-well cell culture plates with 12000 cells per well, and cultured for 6-8 h.
5) Preparation of natural sulfated polysaccharides from marine organisms with different concentrations: the compound to be tested was prepared as a stock solution with a molar concentration of 1mM, and according to the difference in the concentration detected on the cell line, 9 dilutions with different concentration gradients were prepared in the molar concentration range of 0.00015 to 1.00mM, and stored in 1.5mL clear EP tubes, respectively, at-20 ℃. The prepared 9 concentration gradient compounds to be tested are respectively diluted by 10 times by using a complete culture medium, and simultaneously, the same volume of dimethyl sulfoxide (DMSO) solvent is used as a control.
6) And (3) paving: taking out 96-well white cell culture plate adhered with wall for 6-8 hr in incubator, taking out 60 μl of each well, adding 10 μl of the test substance into the culture plate containing 40 μl of cell volume, each concentration gradient of 2 multiple wells, and culturing in incubator (37deg.C, 5% CO) 2 ) After 1 hour of medium culture, 50 mu L of collected viroid supernatant solution is added into each hole, wherein the final detection concentration of the compound to be detected is 9 concentration gradients with the molar concentration range of 0.00152-10.00 mu M; after the plates were placed in an incubator for 24 hours of incubation, 100. Mu.L of medium was replaced for further incubation for 48 hours and detection.
7) Reading a plate: renilla luciferase detection reagent was allowed to stand at room temperature, the cell culture plate was removed and allowed to stand for 10 minutes to equilibrate to room temperature, 15. Mu.L of detection reagent was added to each well, the plate was shaken on an orbital shaker for 2 minutes to induce cell lysis, the plate was allowed to stand at room temperature for 10 minutes, and the luminescence signal was measured on an MD (Molecular Devices, mei Gu molecule) SpectraMax Paradigm plate reader.
8) Data analysis: using SpectraMax Paradigm readings, the corresponding fluorescence per well values RLU were obtained. The data is processed using the following formula: RLU (%) = (RLU) Drug )/(RLU DMSO ) 100%. Calculating cell viability corresponding to the different concentrations of the compounds in EXCEL, and then plotting with GraphPad 7.0Prism software to calculate IC for each compound 50 Values. The results are shown in the following table,
compounds of formula (I) IC 50 (μM)
Heparin 10.0
Fucoidan sulfate 0.06
iota-carrageenan 0.27
Fucosylated chondroitin sulfate 0.25
Rhamnoglycans sulfate 0.44
As shown in figure 1, the series of marine organism source natural sulfated polysaccharides have obvious inhibition effect on blocking new coronavirus (SARS-CoV-2) infected cells, and the effect of the compounds fucoidan sulfate, carrageenan, fucosylated chondroitin sulfate, rhamnoides sulfate and other marine organism source natural sulfated polysaccharides on blocking virus infected cells is stronger than that of Heparin (Heparin).
EXAMPLE two inhibition of SARS-CoV-2 Main protease by marine-derived Natural sulfated polysaccharide
SARS-CoV-2 main protease can hydrolyze the fluorescent polypeptide substrate to cause the substrate to fluoresce. The activity of the main protease is inhibited by marine natural sulfated polysaccharide, and the fluorescence intensity is reduced compared with that of a blank control. Among them, the main protease and the fluorescent polypeptide substrate can be obtained by the report method of document W.Dai et al, science 10.1126/Science. Abb4489 (2020).
(1) TE buffer solution preparation
100. Mu.L of an aqueous solution of disodium ethylenediamine tetraacetate (EDTA-2 Na) having a molar concentration of 0.5M was added to 2.5mL of a 1M Tris-HCl buffer, the volume was adjusted to 50mL, and the solution was filtered through a filter membrane having a pH of 7.3,0.22. Mu.m, followed by storage at-4 ℃.
(2) Screening of main protease inhibition activity by marine organism-derived sulfated polysaccharide
Adding 87 mu L TE buffer solution into 96-well cell culture plate, adding 1 mu L main protease with molar concentration of 19.7 mu M respectively, mixing well, and adding H 2 O (2. Mu.L), seaweed-derived fucoidan sulfate aqueous solution (2. Mu.L, molar concentration 15. Mu.M; molecular weight 260.1 kDa), eucheuma-derived iota-carrageenan (2. Mu.L, molar concentration 15. Mu.M; molecular weight 200 kDa), shark-derived keratan sulfate (2. Mu.L, molar concentration 15. Mu.M, molecular weight 45.98 kDa) and sea cucumber-derived fucosylated chondroitin sulfate (2. Mu.L, molar concentration 15. Mu.M, molecular weight 42 kDa) were sequentially and respectively added to each well, shaking was carried out, and after 30min of reaction at room temperature, 10. Mu.L of a substrate having a molar concentration of 20. Mu.M was added to each well, and fluorescence intensity was rapidly detected by an enzyme-labeling instrument. The sulfated polysaccharide samples employed in the present invention may be extracted using reagents, methods and apparatus conventional in the art.
(3) Detection condition of enzyme-labeled instrument
Continuously detecting the fluorescence intensity of each hole within 10min under the conditions of the excitation wavelength of 320nm, the emission wavelength of 405nm and the detection temperature of 27 ℃ to obtain a time-fluorescence intensity dynamic curve.
(4) Data processing
Performing linear fitting based on the obtained time-fluorescence intensity dynamic curve to obtain the slope k of the corresponding curve of the natural sulfated polysaccharides from each ocean source and the slope k of the corresponding curve of the blank control 0
Inhibition of the main protease by the naturally sulfated polysaccharide (molar concentration 300 nM) = (1-k/k) 0 )×100％
Compounds of formula (I) Inhibition/%
Heparin 72.2
Fucoidan sulfate 92.3
iota-carrageenan 62.8
Fucosylated chondroitin sulfate 65.9
Rhamnoglycans sulfate 75.0
As shown in figure 2, the inhibition rate of brown algae source fucoidan sulfate, red algae source carrageenan and sea cucumber source fucosylated chondroitin sulfate on SARS-CoV-2 main protease activity is all higher than 50%.
EXAMPLE three inhibition of SARS-CoV-2 Main protease by fucoidan at different concentrations
(1) Inhibition experiment of fucoidan on Main protease Activity
Adding 87 μL TE buffer solution into 96-well cell culture plate, adding 1 μL main protease with molar concentration of 19.7 μM, mixing, and adding 2 μLH respectively 2 The fucoidan sulfate aqueous solution of seaweed source with the molar concentration range of 0.5 nM-45 mu M of O and 9 concentration gradients is sequentially and respectively added into each hole, and is uniformly shaken, after 30min reaction at room temperature, 10 mu L of substrate with the molar concentration of 20 mu M is added into each hole, and the fluorescence intensity is rapidly detected by an enzyme-labeled instrument. The sulfated polysaccharide samples employed in the present invention may be extracted using reagents, methods and apparatus conventional in the art.
(2) Detection condition of enzyme-labeled instrument
Continuously detecting the fluorescence intensity of each hole within 10min under the conditions of the excitation wavelength of 320nm, the emission wavelength of 405nm and the detection temperature of 27 ℃ to obtain a time-fluorescence intensity dynamic curve.
(3) Data processing
Based on acquisitionLinear fitting is carried out on the obtained time-fluorescence intensity dynamic curve to obtain the slope k of the corresponding curve of the fucoidan sulfate and the slope k of the corresponding curve of the blank control under different concentrations 0
Inhibition rate of fucoidan to main protease by different concentrations= (1-k/k) 0 )×100％
(4)IC 50 Calculation of
The IC was obtained by plotting the logarithm of the concentration against the inhibition rate using the software origin 8.1 and by using a nonlinear fit 50 Values.
(5) Results
As shown in FIG. 3, marine-derived fucoidan has good inhibitory effect on the activity of main protease, and its corresponding IC 50 Value = 5.68 ± 0.43nM (nmol/L)
Experimental results show that the marine organism-derived natural sulfated polysaccharide provided by the invention has obvious effect of inhibiting the proliferation of coronaviruses, can inhibit the binding of Spike protein (Spike) on the surface of coronaviruses and host cells, and can inhibit the activities of main protease (Mpro) and papain-like (PLpro), so that the infection of coronaviruses to host cells is effectively blocked.

Claims (8)

1. The application of marine organism source natural sulfated polysaccharide as coronavirus and disease-causing medicine is characterized in that the application of marine organism source natural sulfated polysaccharide and pharmaceutically acceptable salt in preparing medicine combined with Spike protein (Spike), preparing main protease (Mpro) inhibitor or papain-like (PLpro) inhibitor is provided, and the marine organism source natural sulfated polysaccharide is fucosylated chondroitin sulfate, and the structural characteristics are as follows: the main chain is composed of chondroitin sulfate disaccharide repeating units formed by alternately connecting glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through beta-1, 3 and beta-1, 4 glycosidic bonds, and a sulfated fuco-oligosaccharide branched chain is connected at the C3 position of GlcA through alpha-1, 3 glycosidic bonds, and the molecular weight range is 3-600 kDa.
2. The use according to claim 1, wherein the coronavirus is selected from one or more of SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
3. The use according to claim 1, wherein the naturally sulfated polysaccharide is derived from a marine plant or a marine animal.
4. Use according to claim 1, wherein the natural sulfated polysaccharide, by binding to Spike protein (Spike) of coronavirus, acts as an inhibitor of its invading cells, inhibiting viral replication and amplification by inhibiting coronavirus main protease (Mpro) and/or papain-like protease (PLpro) activity.
5. The use according to claim 1, wherein the natural sulfated polysaccharide, pharmaceutically acceptable salt is used in the preparation of coronavirus inhibitors.
6. The use according to claim 1, wherein the natural sulfated polysaccharide, pharmaceutically acceptable salt is used for the preparation of a medicament for preventing and/or treating coronavirus infection, or for preventing and/or treating diseases or symptoms associated with coronavirus infection.
7. The use according to claim 1, wherein the natural sulfated polysaccharide, the pharmaceutically acceptable salt, and the Spike protein (Spike) are combined in a kit or reagent, a kit or reagent for preparing a main protease (Mpro) inhibitor, or a kit or reagent for preparing a papain-like protease (PLpro) inhibitor.
8. The use according to claim 1, wherein the natural sulfated polysaccharide, the pharmaceutically acceptable salt, and the additional one or more than two agents for preventing and/or treating coronavirus infection are mixed to prepare a pharmaceutical composition for preventing and/or treating coronavirus infection.
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