Application of a natural sulfated polysaccharide derived from marine organisms in the preparation of anti-SARS-CoV-2 drugs

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to the application of a natural sulfated polysaccharide derived from marine organisms in the preparation of anti-SARS-CoV-2 drugs. The application of a natural sulfated polysaccharide derived from marine organisms and a pharmaceutically acceptable salt thereof in the preparation of a drug that binds to the spike protein (Spike), in the preparation of an inhibitor of the main protease (Mpro), or in the preparation of a papain-like protease (PLpro) inhibitor. The natural sulfated polysaccharide derived from marine organisms provided by the present invention has an obvious effect of inhibiting the proliferation of coronaviruses. It can not only inhibit the binding of the spike protein (Spike) on the surface of coronaviruses to host cells, but also inhibit the activities of the main protease (Mpro) and the papain-like protease (PLpro), thereby effectively blocking the infection of host cells by coronaviruses.

Description

[0001] This application is a divisional application of a Chinese invention patent application. The filing date of the original application is August 17, 2020, and the application number is 202010825078. . 2. Invention title: Application of natural sulfated polysaccharides derived from marine organisms in the preparation of drugs against coronavirus and the diseases caused thereby. Publication number: CN111773240A. During the patent examination process, since the natural sulfated polysaccharides derived from marine organisms in claim 1 of the original application include multiple parallel technical solutions, and there is no specific technical feature between the parallel technical solutions and they do not have unity, this divisional application is filed. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to the application of natural sulfated polysaccharides derived from marine organisms in the preparation of drugs against novel coronavirus. Background Art

[0003] The World Health Organization named the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as coronavirus disease 2019 (COVID-19), and on January 30, 2020, the World Health Organization declared it a public health emergency of international concern. COVID-19 has spread rapidly worldwide, posing a great threat to human health and social public health security. However, there is currently no specific drug for this virus, and clinically, it mainly focuses on supportive treatment and symptomatic treatment to improve symptoms. The research and development of anti-SARS-CoV-2 drugs is extremely urgent. A large number of drug screening experiments have been carried out globally for the novel coronavirus, but no drug with good therapeutic effects has been found yet.

[0004] SARS-CoV-2 is an RNA virus with an envelope structure. The spike glycoprotein on the lipid membrane of the virus particle is responsible for the adhesion to the host receptor. The viral genomic RNA inside the lipid membrane can encode replicase polyproteins, and these polyproteins can be cleaved by 3C-like protease (3CLpro, also known as the main protease Mpro) and papain-like protease (PLpro) to produce functional polypeptides, such as RNA-dependent RNA polymerase (RdRp) and helicase (Hel), which are involved in viral RNA transcription and replication. Therefore, the spike glycoprotein related to the infection of host cells by SARS-CoV-2, and the main protease and papain-like protease related to the transcription and replication of SARS-CoV-2 are important targets for the research and development of anti-SARS-CoV-2 drugs.

[0005] Marine biological resources are abundant. Natural saccharide compounds, especially acidic saccharide compounds, derived from marine organisms can bind to the positively charged proteins of viruses, thereby preventing virus replication or cell infection. In addition, they have the advantages of low toxicity and being less likely to cause drug resistance in viruses, showing broad prospects in the development of antiviral drugs. Literature retrieval reveals that natural sulfated polysaccharides from marine organisms, such as fucan sulfate with a complex structure derived from brown algae, have antiviral effects against a variety of influenza viruses (Publication No. CN 103880975A; Patent No.: ZL201410132608.x). Its main chain structure is composed of α-1,2-mannose (Man) and β-1,4-glucuronic acid (GlcA), and it contains a branched α-1,3-linked sulfated fucose residue at the C3 position of mannose (Jiandong Wu, J Carbohy Chem, 34:303-317; Wei Wang, Sci.Rep.2017,7:40760). Moreover, it has recently been reported that fucoidans (RPI-27 and RPI-128) with a similar structure and different molecular weights (100kD and 12kD) have inhibitory activity against the Spike protein of the COVID-COV-2 coronavirus. This polysaccharide contains a main chain of α-1,2-mannose (Man) and β-1,4-glucuronic acid (GlcA), and a branched α-1,2 / 1,3-linked sulfated fucose residue at the C3 position of mannose (PaulS.Kwon, Cell Discovery, 2020, 6:50); it has been found that fucoidan from Undaria pinnatifida (Hayashi, K., Int.Immunopharmacol.2008,8,109) has antiviral activity against HSV. There is still a lack of reports on the research of other types of fucus sulfate esters in terms of anti-SARS-CoV-2. Carrageenan from red algae has broad antiviral activities against H1N1, HSV, and HPV (Cui Hao, Rev Med Virol.2019, 29, e2043), and iota-carrageenan also has antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV) (Chunhe Guo, Antivir Ther, 2019, 24(4):261-270), but no antiviral activity against the novel coronavirus has been found. Sulfated rhamnan from green algae has antiviral activities against enterovirus 71 (EV71) in children with hand, foot, and mouth disease (Shuyao Wang, Carbohydr Polym, 2018, 200:43-53) and Newcastle disease virus (NDV) ( Mar.Drugs, 2015,13,697-712), but no antiviral activity against the novel coronavirus has been found either. Summary of the Invention

[0006] The object of the present invention is to provide an application of a natural sulfated polysaccharide derived from marine organisms in the preparation of an anti-SARS-CoV-2 drug. By using specific marine plants and marine animals as raw materials, a series of natural sulfated polysaccharides derived from the ocean are obtained through extraction and separation. The activity of inhibiting coronaviruses, especially the novel coronavirus (SARS-CoV-2), is screened, and effective drugs and their compositions for treating coronaviruses, especially coronavirus disease 2019 (COVID-19), are provided.

[0007] The technical solution of the present invention is as follows:

[0008] An application of a natural sulfated polysaccharide derived from marine organisms in the preparation of an anti-SARS-CoV-2 drug, an application of the natural sulfated polysaccharide derived from marine organisms and a pharmaceutically acceptable salt thereof in the preparation of a drug that binds to the spike protein (Spike), the preparation of an inhibitor of the main protease (Mpro), or the preparation of a papain-like protease (PLpro) inhibitor. The natural sulfated polysaccharide derived from marine organisms has any of the following structural characteristics, and its molecular weight ranges from 3 to 600 kDa, including:

[0009] (1) Fucoidan (also known as fucan), having one of the following structural characteristics: (a) Type I fucoidan, the main chain is composed of α-1,3-linked sulfated fucose; (b) Type II fucoidan, the main chain is composed of alternately linked α-1,3 and α-1,4 sulfated fucose; (c) Type III fucoidan, the main chain is composed of β-1,4-glucuronic acid and α-1,2-mannose, and the branched structure is composed of α-1,3-sulfated fucooligosaccharides; (d) Other types of fucoidan, the main chain is composed of α-1,2 and α-1,3 and / or α-1,4-linked sulfated fucose;

[0010] (2) Fucosylated chondroitin sulfate, with the following structural characteristics: The main chain is composed of chondroitin sulfate disaccharide repeating units in which glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) are alternately linked by β-1,3 and β-1,4 glycosidic bonds. The C4, C6 or C4 and C6 hydroxyl groups of GalNAc contain sulfate ester groups, and a sulfated fucooligosaccharide branch chain is linked to the C3 position of GlcA through an α-1,3 glycosidic bond;

[0011] (3) Sulfated galactan, with the following structural characteristics: It is composed of β-1,3-linked and α-1,4-linked galactose (Gal), or composed of β-1,3-linked and α-1,4-linked 3,6-anhydrogalactose (AnG), and the C2, C4 or C6 position of galactose contains different sulfate ester groups;

[0012] (4) Rhamnan sulfate: Its structural characteristics are as follows: It is formed by the alternating connection of rhamnose (Rha) and glucuronic acid through α-1,4 and β-1,4 glycosidic bonds; or by the alternating connection of Rha and iduronic acid through α-1,4 glycosidic bonds; or by the alternating connection of rhamnose (Rha) and xylose (Xyl) through α-1,4 and β-1,4 glycosidic bonds; or by the alternating connection of α-1,2 and α-1,3-rhamnose; and the C2 and / or C3 positions of Rha in the above structure contain sulfate ester groups.

[0013] In the described type I fucoidan, there are also other sugar residue branched chain structures; in the type II fucoidan, there are also other sugar residue branched chain structures.

[0014] The described coronavirus is selected from one or more of SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV.

[0015] The described natural sulfated polysaccharide is derived from marine plants or marine animals.

[0016] The described natural sulfated polysaccharide binds to the spike protein (Spike) of the coronavirus and serves as an inhibitor to inhibit its infection of cells. By inhibiting the activities of the coronavirus main protease (Mpro) and / or papain-like protease (PLpro), it inhibits the replication and amplification of the virus.

[0017] Use of the described natural sulfated polysaccharide, pharmaceutically acceptable salt in the preparation of a coronavirus inhibitor.

[0018] Use of the described natural sulfated polysaccharide, pharmaceutically acceptable salt in the preparation of a drug for preventing and / or treating coronavirus infection, or preventing and / or treating diseases or symptoms related to coronavirus infection.

[0019] Use of the described natural sulfated polysaccharide, pharmaceutically acceptable salt in a kit or reagent for binding to the 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.

[0020] The described natural sulfated polysaccharide, pharmaceutically acceptable salt, and one or two or more additional active agents for preventing and / or treating coronavirus infection are mixed to form a pharmaceutical composition for preventing and / or treating coronavirus infection.

[0021] The design concept of the present invention is:

[0022] The natural sulfated polysaccharides of marine biological origin provided by the present invention can not only inhibit the binding of the spike protein (Spike) on the surface of the coronavirus to host cells, thereby effectively blocking the coronavirus from infecting host cells, but also inhibit the activities of the main protease (Mpro) and papain-like protease (PLpro) of the coronavirus, and further inhibit the replication and amplification process of the virus. Therefore, the natural marine sulfated polysaccharides provided by the present invention have great potential for developing into drugs against coronaviruses and the diseases caused by them.

[0023] The advantages and beneficial effects of the present invention are as follows:

[0024] (1) The natural sulfated polysaccharides of marine biological origin provided by the present invention can inhibit the binding of the spike protein (Spike) on the surface of the coronavirus to host cells, and its inhibitory activity level is superior to that of the clinical drugs heparin and low molecular weight heparin.

[0025] (2) The natural sulfated polysaccharides of marine biological origin provided by the present invention have the activity of inhibiting the main protease (Mpro) and papain-like protease (PLpro), and its inhibitory activity level is superior to that of heparin.

[0026] (3) Generally speaking, the anti-coronavirus activity of the natural sulfated polysaccharides of marine biological origin provided by the present invention is superior to that of the clinical drugs heparin and low molecular weight heparin, and it can be used in the preparation and application fields of drugs related to anti-coronaviruses and the diseases caused by them. Description of the Drawings

[0027] Figure 1 Inhibitory activities of fucoidan, galactan sulfate, fucosylated chondroitin sulfate, rhamnan sulfate and heparin on the spike protein of SARS-CoV-2. In the figure, the abscissa Log Dose represents the logarithm of the compound concentration (μM), and the ordinate Relative Luminescence represents the relative luminescence intensity (%).

[0028] Figure 2 Inhibitory effect curves of galactan sulfate, fucoidan and fucosylated chondroitin sulfate on the main protease of SARS-CoV-2. In the figure, the abscissa Time represents time (s), the ordinate Intensity represents fluorescence intensity, Blank represents the blank control, Iota-carrageenan represents iota-carrageenan, FCS represents fucosylated chondroitin sulfate, and Fucoidan represents fucoidan.

[0029] Figure 3 IC of fucoidan inhibiting the main protease of SARS-CoV-2 50 Determination curve. In the figure, the abscissa Log concentration represents the logarithm of the concentration (nM), and IC 50 represents the half-maximal inhibitory concentration. Detailed implementation mode

[0030] In the specific implementation process, the present invention relates to the application of marine-derived natural sulfated polysaccharides and pharmaceutically acceptable salts thereof in the preparation of coronavirus inhibitors, and the natural sulfated polysaccharides have any of the following structures:

[0031] (1) Fucoidan, including: (a) Type I fucoidan, the main chain is composed of α-1,3-linked sulfated fucose and may also have other sugar residue branched chain structures; (b) Type II fucoidan, the main chain is composed of α-1,3- and α-1,4-alternately linked sulfated fucose and may also have other sugar residue branched chain structures; (c) Type III fucoidan, the main chain is composed of β-1,4-glucuronic acid and α-1,2-mannose, and the branched structure is composed of α-1,3-sulfated fucooligosaccharides; (d) Other types of fucoidan, the main chain is composed of α-1,2 and α-1,3 and / or α-1,4-linked sulfated fucose.

[0032]

[0033] (2) Fucosylated chondroitin sulfate, the structural characteristics of which are as follows: the main chain is composed of chondroitin sulfate disaccharide repeating units formed by glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) alternately linked by β-1,3 and β-1,4 glycosidic bonds, and a sulfated fucooligosaccharide branched chain is linked to the C3 position of GlcA through an α-1,3 glycosidic bond.

[0034]

[0035] (3) Galactan sulfate, the structural characteristics of which are as follows: composed of β-1,3-linked and α-1,4-linked galactose (Gal), or composed of β-1,3-linked and α-1,4-linked 3,6-anhydrogalactose (AnG), and different sulfate ester groups are contained at the C2, C4 or C6 positions of galactose.

[0036]

[0037] (4) Rhamnan sulfate, the structural characteristics of which are as follows: composed of rhamnose (Rha) and glucuronic acid alternately linked by α-1,4 and β-1,4 glycosidic bonds; or composed of Rha and iduronic acid alternately linked by α-1,4 glycosidic bonds; or composed of rhamnose (Rha) and xylose (Xyl) alternately linked by α-1,4 and β-1,4 glycosidic bonds; or composed of α-1,2 and α-1,3-rhamnose alternately linked; and the C2 and / or C3 positions of the above structure Rha contain sulfate ester groups.

[0038]

[0039] The present invention also provides the use of a natural sulfated polysaccharide derived from marine organisms and a pharmaceutically acceptable salt thereof in the preparation of a drug that binds to the Spike protein, the preparation of a main protease (Mpro) inhibitor, or the preparation of a papain-like protease (PLpro) inhibitor. The sulfated polysaccharide derived from marine organisms has any of the following structures:

[0040] (1) Fucoidan, including: (a) type I fucoidan, the main chain of which is composed of α-1,3-linked sulfated fucose and may also have other sugar residue branched-chain structures; (b) type II fucoidan, the main chain of which is composed of α-1,3- and α-1,4-alternately linked sulfated fucose and may also have other sugar residue branched-chain structures; (c) type III fucoidan, the main chain of which is composed of β-1,4-glucuronic acid and α-1,2-mannose, and the branched structure is composed of α-1,3-sulfated fucooligosaccharides; (d) other types of fucoidan, the main chain of which is composed of α-1,2 and α-1,3 and / or α-1,4-linked sulfated fucose.

[0041]

[0042] (2) Fucosylated chondroitin sulfate, the structural characteristics of which are as follows: the main chain is composed of chondroitin sulfate disaccharide repeating units formed by glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) alternately linked by β-1,3 and β-1,4 glycosidic bonds, and a sulfated fucose branch chain is linked to the C3 position of GlcA through an α-1,3 glycosidic bond.

[0043]

[0044] (3) Galactan sulfate, the structural characteristics of which are as follows: it is composed of β-1,3-linked and α-1,4-linked galactose (Gal), or composed of β-1,3-linked and α-1,4-linked 3,6-anhydrogalactose (AnG), and different sulfate ester groups are contained at the C2, C4 or C6 positions of galactose.

[0045]

[0046] (4) Rhamnan sulfate: Its structural characteristics are as follows: It is formed by the alternating connection of rhamnose (Rha) and glucuronic acid through α-1,4 and β-1,4 glycosidic bonds; or by the alternating connection of Rha and iduronic acid (IdoA) through α-1,4 glycosidic bonds; or by the alternating connection of rhamnose (Rha) and xylose (Xyl) through α-1,4 and β-1,4 glycosidic bonds; or by the alternating connection of α-1,2 and α-1,3-rhamnose; and the C2 and / or C3 positions of Rha in the above structure contain sulfate groups.

[0047]

[0048] For the application as described above, preferably, it is one or more of the spike protein (Spike), main protease (Mpro), and papain-like protease (PLpro) in coronaviruses; more preferably, the coronavirus is selected from one or more of COVID-19 (SARS-CoV-2), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV.

[0049] The present invention also provides the application of the natural sulfated polysaccharide derived from marine organisms and the pharmaceutically acceptable salt as described above in the preparation of coronavirus inhibitors.

[0050] The present invention also provides the application of the natural sulfated polysaccharide derived from marine organisms and the pharmaceutically acceptable salt as described above in the preparation of drugs for preventing and / or treating coronavirus infection, or preventing and / or treating diseases or symptoms related to coronavirus infection.

[0051] For the use as described above, preferably, the coronavirus is selected from COVID-19 (SARS-CoV-2), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV.

[0052] The present invention also provides the application of the natural sulfated polysaccharide derived from marine organisms and the pharmaceutically acceptable salt as described above in the preparation of a kit or reagent for binding to the spike protein (Spike), the preparation of a kit or reagent for the main protease (Mpro) inhibitor, or the preparation of a kit or reagent for the papain-like protease (PLpro) inhibitor.

[0053] The present invention also provides a pharmaceutical composition for preventing and / or treating coronavirus infection, which contains the natural sulfated polysaccharide derived from marine organisms, the pharmaceutically acceptable salt, and one or more other active agents for preventing and / or treating coronavirus infection.

[0054] Preferably, the molecular weight range of the natural sulfated polysaccharide derived from marine organisms as described above is 3 - 600 kDa.

[0055] Experimental screening and research of the present invention show that a series of natural sulfated polysaccharides derived from marine organisms have good inhibitory effects on the spike protein (Spike), main protease (Mpro or 3CL pro), and PLpro of coronaviruses, especially the novel coronavirus (SARS-CoV-2). The present invention provides the application potential of natural sulfated polysaccharides derived from marine organisms in anti-coronavirus, especially SARS-CoV-2, and confirms its good clinical development and application prospects.

[0056] The natural sulfated polysaccharide derived from marine organisms provided by the present invention, preferably, has the following structural characteristics:

[0057] (1) Fucoidan, including: (a) type I fucoidan, the main chain is composed of α-1,3-linked sulfated fucose and may also have other sugar residue branched chain structures; (b) type II fucoidan, the main chain is composed of alternately linked α-1,3- and α-1,4-sulfated fucose and may also have other sugar residue branched chain structures; (c) type III fucoidan, the main chain is composed of β-1,4-glucuronic acid and α-1,2-mannose, and the branched structure is composed of α-1,3-sulfated fucose oligosaccharides; (d) other types of fucoidan, the main chain is composed of α-1,2 and α-1,3- and / or α-1,4-linked sulfated fucose.

[0058]

[0059] (2) Fucosylated chondroitin sulfate, with the following structural characteristics: the main chain is composed of chondroitin sulfate disaccharide repeating units formed by alternately linking glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) through β-1,3 and β-1,4 glycosidic bonds, and a sulfated fucose branch chain is linked to the C3 position of GlcA through an α-1,3 glycosidic bond.

[0060]

[0061] (3) Sulfated galactan, with the following structural characteristics: composed of β-1,3-linked and α-1,4-linked galactose (Gal), or composed of β-1,3-linked galactose and α-1,4-linked 3,6-anhydrogalactose (AnG), and different sulfate ester groups are contained at the C2, C4, or C6 positions of galactose.

[0062]

[0063] (4) Rhamnan sulfate: Its structural characteristics are as follows: It is formed by the alternating connection of rhamnose (Rha) and glucuronic acid through α-1,4 and β-1,4 glycosidic bonds; or by the alternating connection of Rha and iduronic acid through α-1,4 glycosidic bonds; or by the alternating connection of rhamnose (Rha) and xylose (Xyl) through α-1,4 and β-1,4 glycosidic bonds; or by the alternating connection of α-1,2 and α-1,3-rhamnose; and the C2 and / or C3 positions of Rha in the above structure contain sulfate ester groups.

[0064]

[0065] The marine biological source sulfated polysaccharides of the present invention all have the effect of inhibiting coronaviruses, especially the novel coronavirus (SARS-CoV-2). The experimental results show that all kinds of marine biological source sulfated polysaccharides provided by the present invention have a significant anti-novel coronavirus effect, and the IC 50 value for inhibiting Spike is between 0.01 and 30 μM, and the IC 50 value for inhibiting Mpro is between 1 nM and 100 nM, and the IC 50 value for inhibiting papain-like protease (PLpro) is between 0.001 and 30 μM. For example, the IC 50 of fucoidan sulfate for inhibiting Mpro is 5.68 nM, the IC 50 for inhibiting Spike protein is 0.06 μM, and the IC 50 for inhibiting papain-like protease (PLpro) is 7.65 nM, and the effects are all significantly better than heparin.

[0066] As a preferred embodiment, in the above-mentioned application, the coronaviruses include but are not limited to the novel coronavirus (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).

[0067] The application of the marine biological source natural sulfated polysaccharides provided by the present invention in anti-novel coronavirus can prepare the marine biological source natural sulfated polysaccharides into sprays, injections, capsules, ointments, creams, gels, liniments, and paints.

[0068] Preferably, a therapeutically effective amount of the marine biological source natural sulfated polysaccharides, pharmaceutically acceptable salts or stereoisomers of the present invention are formulated and administered to a patient in need of such treatment according to the usual routes of administration and according to methods known in the art in a conventional pharmaceutical composition (the pharmaceutical composition contains an effective amount of the active ingredient and a suitable pharmaceutical carrier) and dosage form.

[0069] The "therapeutically effective amount" means that when administered, it is sufficient to prevent the development of one or more symptoms of the disease targeted, or to alleviate to some extent said one or more symptoms. The specific dose of the compound administered according to the present invention will be determined by the specific circumstances surrounding the case, said circumstances including the compound administered, the route of administration, the specific medical condition being treated, and similar considerations. In particular, a "therapeutically effective amount of a compound" means an amount of the compound sufficient to prevent or alleviate to some extent one or more coronavirus infections.

[0070] Furthermore, depending on the type and severity of the coronavirus infection to be treated and the response of the specific patient to the drug treatment, the individual dose as well as the daily dose vary. Therefore, the exact individual dose will be determined according to standard medical principles under the guidance of a doctor.

[0071] The effective daily human dose of the natural marine - derived sulfated polysaccharide, pharmaceutically acceptable salt of the present invention for use in treating diseases caused by coronaviruses is an oral dosage form of 1 mg to about 500 mg, about 5 mg to about 1000 mg, or about 10 mg to about 2000 mg of an injection or other active agent.

[0072] The natural marine - derived sulfated polysaccharide, pharmaceutically acceptable salt of the present invention can be used alone, in combination, or in combination therapy with other therapeutic agents.

[0073] In one embodiment of the present invention, the natural marine - derived sulfated polysaccharide, pharmaceutically acceptable salt is used for preventing and / or treating coronavirus infections, wherein said prevention or treatment comprises administration as the sole active ingredient.

[0074] In another embodiment of the present invention, the above - mentioned natural marine - derived sulfated polysaccharide, pharmaceutically acceptable salt is used for preventing and / or treating coronavirus infections, wherein said prevention or treatment comprises use in combination therapy with a selected other therapeutic agent.

[0075] As will be apparent to those skilled in the art, the combinations of the present invention comprising the natural marine - derived sulfated polysaccharide, pharmaceutically acceptable salt of the present invention and additional therapeutic agents are effective not only when these active ingredients are used in a single composition, but also when used in two different compositions (administered simultaneously, sequentially, or separately after a period of time). Furthermore, those skilled in the art will understand that the natural marine - derived sulfated polysaccharide, pharmaceutically acceptable salt of the present invention can be prescribed for use together with other active ingredients in combination therapy to prevent and / or treat coronavirus infections.

[0076] In a particular embodiment, the combination therapy comprises administering to a subject the marine organism-derived natural sulfated polysaccharide of the present 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 present invention, a pharmaceutically acceptable salt or stereoisomer, and an additional therapeutic agent in a single composition.

[0077] In one embodiment of the present invention, the marine organism-derived natural sulfated polysaccharide of the present invention, a pharmaceutically acceptable salt or stereoisomer can be conveniently administered to a patient. Thus, the compound for use in the present invention can be in the form of a pharmaceutical composition comprising an effective amount of the marine organism-derived natural sulfated polysaccharide of the present invention, a pharmaceutically acceptable salt in combination with a pharmaceutical excipient or carrier. This aspect can also be expressed as a composition comprising an effective amount of the marine organism-derived natural sulfated polysaccharide of the present invention, a pharmaceutically acceptable salt in combination with a pharmaceutical excipient or carrier for use in the prevention and / or treatment of coronavirus infection.

[0078] In one embodiment of the present invention, the compound used can be administered orally, by injection, subcutaneously, via the respiratory tract, transdermally, parenterally, rectally, topically, intravenously, intramuscularly, or by other means in dosage unit formulations comprising conventional pharmaceutical carriers. The pharmaceutical composition can be formulated into any pharmaceutical form, such as tablets, granules, injections, gels, pills, capsules, suppositories, implants, nano-formulations, lyophilized powders. Some dosage forms such as tablets and capsules can be further divided into appropriate dosage unit formulations comprising an appropriate amount of the active ingredient such as an effective amount to achieve the desired purpose.

[0079] In another embodiment, the marine organism-derived natural sulfated polysaccharide of the present invention, a pharmaceutically acceptable salt for use in the present invention is an injection preparation to be administered to a patient to be treated. An injection suitable for the present invention refers to a sterile or aseptic solution, emulsion or suspension of a drug and a suitable solvent or dispersion medium for injection into the human body, as well as a sterile powder preparation for use in preparing a solution or suspension before use. The injections include injection solutions (wherein the large-volume injection solutions for intravenous drip are also called intravenous infusions), sterile powders for injection, and concentrated solutions for injection.

[0080] The carrier includes excipients and diluents and must have a sufficiently high purity and a very low toxicity such that they are suitable for administration to the patient to be treated. The carrier can be inert or it can itself have a pharmaceutical benefit.

[0081] 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 aromatic agents, thickening agents, and vehicles.

[0082] Optional active agents can be included in the pharmaceutical composition, which basically do not affect the activity of the compounds of the present invention.

[0083] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available. The natural sulfated polysaccharides derived from marine organisms used have the following structures:

[0084] (1) Fucoidan: The main chain is composed of α-1,3-linked sulfated fucose, with a molecular weight of 600 kDa.

[0085] (2) ι-Carrageenan: It is composed of a repeating disaccharide unit of β-1,3-linked galactose and α-1,4-linked 3,6-anhydrogalactose. The C4 position of the galactose residue and the C2 position of the anhydrogalactose residue are substituted by sulfate groups, with a molecular weight of 200 kDa.

[0086] (3) Fucosylated chondroitin sulfate: The main chain is composed of a chondroitin sulfate disaccharide repeating unit formed by alternating β-1,3 and β-1,4 glycosidic bonds between glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), and a fucose branch chain with sulfate groups at the C2 and C3 positions of GlcA is linked through an α-1,3 glycosidic bond, with molecular weights of 3 kDa and 50 kDa.

[0087] (4) Rhamnan sulfate: It is composed of rhamnose (Rha) and glucuronic acid alternatingly linked by α-1,4 and β-1,4 glycosidic bonds, and the C2 and C3 positions of the above-mentioned structure Rha contain sulfate ester groups; the molecular weight is 200 kDa.

[0088] Example 1 Inhibitory effect of natural sulfated polysaccharides derived from marine organisms on SARS-CoV-2 spike protein

[0089] Based on the SARS-CoV-2-spike protein-like virus detection system, the effects of fucoidan (derived from brown algae), ι-carrageenan (derived from red algae), fucosylated chondroitin sulfate (derived from sea cucumbers), and rhamnan sulfate (derived from green algae) in blocking SARS-CoV-2 from infecting cells were evaluated.

[0090] The sulfated polysaccharide samples used in the present invention can be extracted using conventional reagents, methods and equipment in the technical field.

[0091] 1) Cell resuscitation and culture: For the 293T / 17 human embryonic kidney cell line, a mixture of 10% fetal bovine serum, 1% double antibody, and the remaining DMEM medium is used for culture by weight percentage. After cell resuscitation, the cells are cultured for two generations and then reserved.

[0092] 2) Preparation of virus-like particles: The 293T / 17 human embryonic kidney cells are seeded into a 6-well cell culture plate. When the cell confluence reaches about 60%, 3 μg of plasmid is packaged and transfected using a transfection reagent (Lipofiter 3.0). Then, the 6-well cell culture plate is cultured in a carbon dioxide incubator (37 °C, 5% CO2). After 48 h of transfection, the virus-like particle supernatant solution is collected and stored at -80 °C.

[0093] 3) Transient transfection of cells: The 293T / 17 human embryonic kidney cells are seeded into a 6-well cell culture plate. When the cell confluence reaches about 70%, 3 μg of plasmid (ACE2-pcDNA3.1) is transfected into the 293T17 cells using a transfection reagent (Lipofiter 3.0). The 6-well cell culture plate is cultured in a carbon dioxide incubator for 48 h.

[0094] 4) Cell digestion: The transiently transfected 293T / 17 human embryonic kidney cells are digested with trypsin. After resuspending the cells with complete medium, they are counted using a cell counter and seeded into a 96-well cell culture plate, with 12,000 cells per well, and cultured for 6 - 8 h.

[0095] 5) Preparation of natural sulfated polysaccharides from marine organisms at different concentrations: The test compound is prepared into a stock solution with a molar concentration of 1 mM. According to different detection concentrations on the cell line, 9 different concentration gradients of dilutions are prepared in the range of 0.00015 - 1.00 mM and stored in 1.5 mL transparent EP tubes at -20 °C. The 9 prepared concentration gradients of the test compound are each diluted 10 times with complete medium, and at the same time, an equal volume of dimethyl sulfoxide (DMSO) solvent is used as a control.

[0096] 6) Seeding: Take out the 96-well white cell culture plate that has been adherent in the incubator for 6 - 8 h, take out 60 μL from each well, and then add 10 μL of the test substance to the above culture plate containing 40 μL of cell volume. There are 2 replicates for each concentration gradient. After culturing in the incubator (37 °C, 5% CO2) for 1 hour, 50 μL of the collected virus-like particle supernatant solution is added to each well. The final detection concentration of the test compound is 9 concentration gradients in the molar concentration range of 0.00152 - 10.00 μM; after placing the culture plate in the incubator and incubating for 24 hours, 100 μL of the medium is replaced and the cells are continued to be cultured for 48 h before detection.

[0097] 7) Read the plate: Let the Renilla luciferase detection reagent reach room temperature. Take out the cell culture plate and place it for 10 minutes to equilibrate to room temperature. Add 15 μL of the detection reagent to each well. Shake the culture plate on an orbital shaker for 2 minutes to induce cell lysis. Leave the culture plate at room temperature for 10 minutes, and measure the luminescence signal on an MD (Molecular Devices) SpectraMax Paradigm plate reader.

[0098] 8) Data analysis: Use the SpectraMax Paradigm to read the values and obtain the corresponding fluorescence value RLU per well. The data is processed using the following formula: RLU(%) = (RLU Drug ) / (RLU DMSO ) * 100%. Calculate the cell viability corresponding to different concentrations of the compound in EXCEL, and then use GraphPad 7.0 Prism software to create a curve graph and calculate the IC 50 value of each compound. The results are shown in the following table,

[0099] Compound <![CDATA[IC 50 (μM)]]> Heparin 10.0 Fucoidan 0.06 iota-Carrageenan 0.27 Fucosylated chondroitin sulfate 0.25 Rhamnan sulfate 0.44

[0100] As Figure 1 shown, a series of natural marine - derived sulfated polysaccharides all have obvious inhibitory effects on blocking the infection of cells by the novel coronavirus (SARS - CoV - 2), and the natural marine - derived sulfated polysaccharides such as fucoidan, carrageenan, fucosylated chondroitin sulfate, and rhamnan sulfate have stronger effects on blocking virus - infected cells than Heparin.

[0101] Example 2: Inhibitory effect of natural marine - derived sulfated polysaccharides on SARS - CoV - 2 main protease

[0102] The SARS - CoV - 2 main protease can hydrolyze a fluorescent polypeptide substrate to produce fluorescence. The activity of the main protease is inhibited by natural marine - derived sulfated polysaccharides, and the fluorescence intensity decreases compared to the blank control. Among them, the main protease and the fluorescent polypeptide substrate can be obtained by the method reported in the literature W. Dai et al., Science 10.1126 / science.abb4489 (2020).

[0103] (1) Preparation of TE buffer solution

[0104] Add 100 μL of an aqueous solution of disodium ethylenediaminetetraacetate (EDTA - 2Na) with a molar concentration of 0.5 M to 2.5 mL of a Tris - HCl buffer solution with a molar concentration of 1 M, make up the volume to 50 mL, adjust the pH to 7.3, filter through a 0.22 μm filter membrane, and store at - 4°C.

[0105] (2) Screening of the inhibitory activity of marine organism-derived sulfated polysaccharides against the main protease

[0106] Add 87 μL of TE buffer solution to a 96-well cell culture plate, and then add 1 μL of the main protease with a molar concentration of 19.7 μM. After mixing, add H2O (2 μL), fucoidan sulfate aqueous solution derived from seaweed (2 μL, molar concentration 15 μM; molecular weight 260.1 kDa), iota-carrageenan derived from Eucheuma (2 μL, molar concentration 15 μM; molecular weight 200 kDa), keratan sulfate derived from shark bone (2 μL, molar concentration 15 μM, molecular weight 45.98 kDa), and fucosylated chondroitin sulfate derived from sea cucumber (2 μL, molar concentration 15 μM, molecular weight 42 kDa) to each well in sequence. Shake well, and after reacting at room temperature for 30 min, add 10 μL of the substrate with a molar concentration of 20 μM to each well, and quickly detect the fluorescence intensity using a microplate reader. The sulfated polysaccharide samples used in the present invention can be extracted using conventional reagents, methods, and equipment in this technical field.

[0107] (3) Microplate reader detection conditions

[0108] Continuously detect the fluorescence intensity of each well within 10 min under the conditions of an excitation wavelength of 320 nm, an emission wavelength of 405 nm, and a detection temperature of 27 °C to obtain a time-fluorescence intensity dynamic curve.

[0109] (4) Data processing

[0110] Based on the obtained time-fluorescence intensity dynamic curve, perform linear fitting to obtain the slope k of the curve corresponding to each natural sulfated polysaccharide from marine sources and the slope k0 of the curve corresponding to the blank control.

[0111] Inhibition rate of natural sulfated polysaccharide (molar concentration 300 nM) against the main protease = (1 - k / k0) × 100%

[0112] Compound Inhibition rate / % Heparin 72.2 Fucoidan 92.3 iota-Carrageenan 62.8 Fucosylated chondroitin sulfate 65.9 Rhamnan sulfate 75.0

[0113] As Figure 2 shown, the inhibition rates of fucoidan sulfate derived from brown algae, carrageenan derived from red algae, and fucosylated chondroitin sulfate derived from sea cucumber against the activity of SARS-CoV-2 main protease are all over 50%.

[0114] Example 3 Inhibitory effect of fucoidan sulfate at different concentrations on SARS-CoV-2 main protease

[0115] (1) Experiment on the inhibitory activity of fucoidan sulfate against the main protease activity

[0116] Add 87 μL of TE buffer solution to a 96-well cell culture plate, and then add 1 μL of the main protease with a molar concentration of 19.7 μM. After mixing, add 2 μL of H2O and aqueous solutions of fucoidan sulfate from seaweed with 9 concentration gradients in the molar concentration range of 0.5 nM to 45 μM to each well in sequence. Shake well. After reacting at room temperature for 30 min, add 10 μL of the substrate with a molar concentration of 20 μM to each well, and quickly detect the fluorescence intensity using a microplate reader. The sulfuric polysaccharide sample used in the present invention can be extracted using conventional reagents, methods, and equipment in this technical field.

[0117] (2) Microplate reader detection conditions

[0118] Continuously detect the fluorescence intensity of each well within 10 min under the conditions of an excitation wavelength of 320 nm, an emission wavelength of 405 nm, and a detection temperature of 27 °C to obtain a time-fluorescence intensity dynamic curve.

[0119] (3) Data processing

[0120] Based on the obtained time-fluorescence intensity dynamic curve, perform linear fitting to obtain the slope k of the curve corresponding to fucoidan sulfate at different concentrations and the slope k0 of the curve corresponding to the blank control.

[0121] The inhibition rate of fucoidan sulfate at different concentrations on the main protease = (1 - k / k0) × 100%

[0122] (4) IC 50 Calculation

[0123] Using the software origin 8.1, plot the inhibition rate against the logarithm of the concentration and perform non-linear fitting to obtain the IC 50 value.

[0124] (5) Results

[0125] As Figure 3 shown, fucoidan sulfate from marine organisms has a good inhibitory effect on the activity of the main protease, and its corresponding IC 50 value = 5.68 ± 0.43 nM (nmol / L)

[0126] The experimental results show that the natural sulfuric polysaccharide from marine organisms provided by the present invention has an obvious effect of inhibiting the proliferation of coronaviruses. It can not only inhibit the binding of the spike protein (Spike) on the surface of coronaviruses to host cells, but also inhibit the activities of the main protease (Mpro) and papain-like protease (PLpro), thereby effectively blocking the infection of host cells by coronaviruses.

Claims

1. Use of a natural sulfated polysaccharide derived from marine organisms in the preparation of a drug against novel coronavirus, characterized in that, Use of a natural sulfated polysaccharide derived from marine organisms or a pharmaceutically acceptable salt thereof in the preparation of a drug that binds to the spike protein Spike, in the preparation of an inhibitor of the main protease Mpro, or in the preparation of an inhibitor of the papain-like protease PLpro, wherein the natural sulfated polysaccharide derived from marine organisms is fucosylated chondroitin sulfate, and its structural characteristics are as follows: the main chain is composed of chondroitin sulfate disaccharide repeating units formed by alternating linkage of glucuronic acid GlcA and N-acetylgalactosamine GalNAc through β-1,3 and β-1,4 glycosidic bonds, and a sulfated fucose oligosaccharide branch chain is linked to the C3 position of GlcA through an α-1,3 glycosidic bond, with a molecular weight range of 3 to 600 kDa, and the novel coronavirus is selected from SARS-CoV-2.

2. The application according to claim 1, characterized in that The natural sulfated polysaccharide is derived from marine plants or marine animals.

3. The application according to claim 1, wherein The natural sulfated polysaccharide binds to the spike protein Spike of the coronavirus and serves as an inhibitor to inhibit its infection of cells. By inhibiting the activities of the main protease Mpro and / or the papain-like protease PLpro of the coronavirus, the replication and amplification of the virus are inhibited.

4. The application according to claim 1, characterized in that, Use of a natural sulfated polysaccharide or a pharmaceutically acceptable salt thereof in the preparation of a coronavirus inhibitor.

5. The application according to claim 1, wherein Use of a natural sulfated polysaccharide or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating coronavirus infection, or preventing and / or treating diseases or symptoms associated with coronavirus infection.

6. The application according to claim 1, characterized in that A natural sulfated polysaccharide or a pharmaceutically acceptable salt thereof, and one or more other active agents for preventing and / or treating coronavirus infection are mixed to form a pharmaceutical composition for preventing and / or treating coronavirus infection.

Images