Inhibitors of viral cell entry

Abstract

To provide an inhibitory agent for cell invasion by a virus, the agent inhibiting the virus from invading cells.SOLUTION: The present invention includes an inhibitory agent for cell invasion by a virus, the agent comprising at least one component (A) selected from α-glucosylrutin, α-glucosylhesperidin, and α-glucosylnaringin, where the virus has a spike protein that binds to an angiotensin-converting enzyme 2 (ACE2).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an inhibitor of viral cell entry. [Background technology]

[0002] Coronaviruses have long been prevalent in human society as the cause of the common cold. However, in recent years, attention has been focused on coronaviruses that can cause global pandemics, such as SARS-CoV, which causes SARS (Severe Acute Respiratory Syndrome), and SARS-CoV-2, which causes COVID-19.

[0003] Coronaviruses are classified by the International Committee on Taxonomy of Viruses (ICTV) as members of the Coronaviridae family within the suborder Cornidoviridae of the order Nidoviridae, which is further divided into the subfamily Retrovirinae and the subfamily Orthocoronavirinae, which contains four genera: αcoronaviruses, βcoronaviruses, γcoronaviruses, and δcoronaviruses.

[0004] Coronaviruses typically have spike proteins (also called S proteins), nucleocapsid proteins (also called N proteins), membrane proteins (also called M proteins), envelopes, envelope proteins (also called E proteins), and RNA as components.

[0005] For coronaviruses to infect host cells, the spike protein must bind to and adsorb to a receptor on the host cell surface, allowing the genomic RNA to enter the cell. The host cell receptor to which the coronavirus spike protein binds varies depending on the virus type. For example, HCoV-NL63, a cold virus classified as an alpha coronavirus, and SARS-CoV and SARS-CoV-2, classified as beta coronaviruses, bind to the host cell receptor angiotensin-converting enzyme 2 (ACE2).

[0006] Furthermore, after the spike protein binds to ACE2, the virus must fuse with the host cell membrane in order to invade the cell. It is believed that cleavage of the spike protein by the protease transmembrane protease serin 2 (TMPRSS2) is crucial during this process. Therefore, inhibiting TMPRSS2 activity is thought to inhibit membrane fusion and prevent the virus from invading the host cell. Drugs that inhibit TMPRSS2 activity and effectively block viral entry include nafamostat and camostat.

[0007] Meanwhile, Patent Document 1 discloses an antiviral agent containing a cold water extract of hops, which are used in brewing sparkling alcoholic beverages such as beer, as an active ingredient, as an antiviral agent containing a specific flavonoid glycoside, which is presumed to adsorb to the hemagglutinin protein of influenza viruses and inhibit the binding of the influenza viruses to cell surface receptors, thereby preventing the influenza viruses from entering cells.

[0008] Non-Patent Document 1 discloses that virtual screening, a type of computer-based drug target evaluation, has shown that hesperidin has the potential to bind to the receptor binding domain (RBD) of the spike protein of SARS-CoV-2.

[0009] Furthermore, Non-Patent Document 2 discloses that the affinity of natural substances such as flavonoid glycosides with ACE2 was evaluated by virtual screening using nafamostat and captopril, an angiotensin-converting enzyme inhibitor, as control reagents, and that resveratrol, quercetin, luteonin, naringenin, etc., exhibit high affinity with ACE2.

[0010] However, in both Non-Patent Documents 1 and 2, the effects of these natural products on SARS-CoV-2 have not been confirmed even in vitro, and their effects have not actually been verified. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2007 / 099915 [Non-patent literature]

[0012] [Non-Patent Document 1] Canrong Wu et al. Acta Pharmaceutica Sinica B 2020;10(5):766-768 [Non-patent document 2] Vimal K Maurya et al. Virusdisease. 2020 Jun;31(2):179-193 Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention addresses the problem of providing a virus cell entry inhibitor that inhibits virus entry into cells. [Means for solving the problem]

[0014] The present inventors have conducted extensive research to solve the above problems, and as a result have found that a virus cell entry inhibitor having the following composition can solve the above problems, thereby completing the present invention. That is, the present invention includes the following [1] to [6].

[0015] [1] A viral cell entry inhibitor comprising at least one component (A) selected from α-glucosyl rutin, α-glucosyl hesperidin, and α-glucosyl naringin, wherein the virus has a spike protein that binds to angiotensin-converting enzyme 2 (ACE2). [2] The virus cell entry inhibitor described in [1], wherein the virus is any one selected from SARS-CoV, SARS-CoV-2, and HCoV-NL63. [3] An inhibitor of viral cell entry described in [1] or [2], wherein the virus is SARS-CoV-2. [4] A viral cell entry inhibitor according to any one of [1] to [3], wherein the component (A) comprises at least one selected from α-monoglucosyl rutin, α-monoglucosyl hesperidin and α-monoglucosyl naringin. [5] The virus cell entry inhibitor according to any one of [1] to [4], wherein the content of the component (A) is 50% by mass or more. [6] The virus cell entry inhibitor according to any one of [1] to [5], wherein the content of the component (A) is 65% by mass or more. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide an inhibitor of viral cell entry that inhibits viral entry into cells. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 is a graph showing the amount of luminescence due to luciferase activity when pseudo-SARS-CoV-2 enters Caco-2 cells. [Figure 2] Figure 2 is a graph showing the amount of luminescence due to luciferase activity when pseudo-SARS-CoV-2 enters ACE2-expressing A549 cells. DETAILED DESCRIPTION OF THE INVENTION

[0018] Preferred embodiments for carrying out the present invention will be described below. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0019] The present invention is an inhibitor of viral cell entry, comprising at least one component (A) selected from α-glucosyl rutin, α-glucosyl hesperidin and α-glucosyl naringin, wherein the virus has a spike protein that binds to angiotensin-converting enzyme 2 (ACE2).

[0020] <Component (A)> The viral cell entry inhibitor of the present invention contains at least one component (A) selected from α-glucosylrutin, α-glucosyl hesperidin, and α-glucosylnaringin. Component (A) may contain one or two arbitrarily selected components of α-glucosylrutin, α-glucosyl hesperidin, and α-glucosylnaringin, or may contain all three components of α-glucosylrutin, α-glucosyl hesperidin, and α-glucosylnaringin. Among these, components containing α-glucosylrutin or α-glucosylnaringin are preferred as component (A) because they have a high inhibitory effect on viral cell entry even at relatively low concentrations.

[0021] From the viewpoint of the effect of inhibiting viral cell invasion, component (A) preferably contains at least one selected from α-monoglucosyl rutin, α-monoglucosyl hesperidin, and α-monoglucosyl naringin.

[0022] The content of component (A) contained in the viral cell entry inhibitor of the present invention is not particularly limited. For example, the lower limit of the content of component (A) contained in the viral cell entry inhibitor of the present invention can be, for example, 30% by mass, 40% by mass, 45% by mass, 50% by mass, 60% by mass, 70% by mass, or 80% by mass. Furthermore, the upper limit of the content of component (A) contained in the viral cell entry inhibitor of the present invention can be, for example, 100% by mass, 99% by mass, 98% by mass, 95% by mass, 90% by mass, or 85% by mass. The content of component (A) contained in the viral cell entry inhibitor of the present invention can be set to any range that combines the above lower and upper limits, such as 30 to 100% by mass, 60 to 100% by mass, or 60 to 90% by mass. From the viewpoint of the viral cell entry inhibitory effect, the content of component (A) contained in the viral cell entry inhibitor of the present invention is preferably 50% by mass or more, and more preferably 65% ​​by mass or more.

[0023] [α-Glucosylrutin] α-Glucosylrutin (also called α-glucosylrutin) is a general term for compounds in which one or more glucose molecules are added to a glucose residue in a rutinose residue of rutin via an α1→4 bond. The α-glucosylrutin of the present invention may consist of a single type of compound having such a structure, or a mixture of two or more types.

[0024] α-Glucosylrutin can be represented by the following formula (1): In formula (1), n ​​is 0 or an integer of 1 or more, for example, an integer of 1 to 19.

[0025] [ka]

[0026] α-Glucosylrutin is a compound found as the main component in a product known as "enzyme-treated rutin" (sometimes called "transferred rutin"). α-Glucosylrutin with only one glucose bonded is called "α-monoglucosylrutin," while α-polyglucosylrutin with two or more glucose bonded is called "α-polyglucosylrutin." In other words, in formula (1), α-monoglucosylrutin is a compound where n is 0, and α-polyglucosylrutin is generally a compound where n is 1 to 19.

[0027] Enzyme-treated rutin is a collection of compounds produced by enzymatic treatment of the sugars of rutin, and typically includes a mixture of compounds with different numbers of glucose units bound to rutin, such as a mixture of α-monoglucosylrutin and α-polyglucosylrutin. Furthermore, since enzyme-treated rutin is generally produced by enzymatic treatment, it may also contain unreacted rutin or other derivatives, such as isoquercitrin. Isoquercitrin (sometimes called "isoquercitrin") is a compound in which β-D-glucose is bound to the hydroxyl group at the 3-position of the quercetin skeleton; in other words, a compound in which the rhamnose residue in the rutinose residue of rutin is cleaved.

[0028] Enzyme-treated rutin is a product (referred to in this specification as "first enzyme-treated rutin") obtained by, for example, allowing a glycosyltransferase (an enzyme that has the function of adding glucose to rutin, such as cyclodextrin glucanotransferase (CGTase, EC2.4.1.19)) to act on rutin in the presence of an α-glucosyl sugar compound (cyclodextrin, partial starch hydrolysate, etc.).

[0029] The first enzyme-treated rutin is a composition containing various α-glucosylrutins with different numbers of bound glucose atoms, i.e., an aggregate consisting of α-monoglucosylrutin and α-polyglucosylrutin, and unreacted rutin. If necessary, the first enzyme-treated rutin can be purified using, for example, a porous synthetic adsorbent and an appropriate eluent to remove sugar donors and other impurities, further reducing the rutin content and obtaining first enzyme-treated rutin (purified α-glucosylrutin) with increased purity of α-glucosylrutin.

[0030] Alternatively, first enzyme-treated rutin can be treated with an enzyme having glucoamylase activity that cleaves α-1,4-glucosidic bonds at glucose units, such as glucoamylase (EC 3.2.1.3), to cleave the glucose residues in α-glucosylrutin to which multiple glucose residues have been added, leaving only one glucose residue directly attached to the glucose residue (in the rutinose residue) of the rutin itself, thereby obtaining enzyme-treated rutin (referred to herein as "second enzyme-treated rutin") that contains a large amount of α-monoglucosylrutin. This enzyme treatment does not cleave the glucose residue in the rutinose residue that is directly attached to the quercetin skeleton from the quercetin skeleton.

[0031] In the viral cell entry inhibitor of the present invention, taking into consideration the effects of the present invention, it is preferable to use enzyme-treated rutin, which is a composition containing α-glucosylrutin, and a composition containing either the first enzyme-treated rutin or the second enzyme-treated rutin may be used.

[0032] Considering the effects of the present invention, the enzyme-treated rutin is preferably a mixture containing at least α-glucosylrutin and further containing isoquercitrin. Such a mixture can be produced by the following procedures: (i) preparing the above-mentioned first enzyme-treated rutin, (ii) treating the first enzyme-treated rutin with an enzyme having glucoamylase activity to convert almost all of the α-glucosylrutin into α-monoglucosylrutin, and (iii) simultaneously treating it with an enzyme having rhamnosidase activity to convert almost all of the unreacted rutin into isoquercitrin.

[0033] Commercially available enzyme-treated rutin products include "αG Rutin PS," "αG Rutin P," and "αG Rutin H," all manufactured by Toyo Sugar Refining Co., Ltd. "αG Rutin PS" is a composition containing 65% by mass of α-monoglucosylrutin and 15% by mass of isoquercitrin. "αG Rutin P" is a composition containing 60% by mass of α-glucosylrutin, 10% by mass of rutin, and 1% by mass of isoquercitrin.

[0034] As the α-glucosylrutin, α-monoglucosylrutin is preferred, because the molecular weight of α-monoglucosylrutin is smaller than that of α-polyglucosylrutin, and therefore the number of molecules per unit mass of α-monoglucosylrutin is greater, which is thought to be advantageous in terms of action and effect.

[0035] The presence of various α-glucosyl rutins and other components contained in enzyme-treated rutin can be confirmed by HPLC chromatograms, and the content of each component or the purity of a specific desired component can be calculated from the peak area of ​​the chromatogram.

[0036] The method for producing α-glucosylrutin is not particularly limited, and known methods can be used. As described above, production by enzymatic treatment of rutin is preferred because it has a good yield and is easy to produce. The method for obtaining and preparing rutin is not particularly limited, and compounds that are generally manufactured and sold as reagents or purified products may be used, or compounds prepared by extraction from raw materials such as the peels of citrus fruits (tangerines, oranges, etc.) or buckwheat seeds may be used.

[0037] [α-Glucosylhesperidin] α-Glucosyl hesperidin (also called α-glucosyl hesperidin) is a general term for compounds in which one or more glucose molecules are attached to the hydroxyl group in the rutinose unit of hesperidin via an α-1,4 bond. The α-glucosyl hesperidin of the present invention may consist of a single compound having such a structure, or a mixture of two or more compounds. Hesperidin is a compound in which β-rutinose (6-O-α-L-rhamnosyl-β-D-glucose) is attached to the hydroxyl group at the 7-position of hesperetin, i.e., a hesperetin glycoside.

[0038] α-Glucosyl hesperidin can be represented by the following formula (2): In formula (2), n is 0 or an integer of 1 or more, for example, an integer of 1 to 19.

[0039] [ka]

[0040] α-Glucosyl hesperidin is a compound contained as the main component in a material known as "enzyme-treated hesperidin" (sometimes called "transglycosylated hesperidin"). α-Glucosyl hesperidin with only one glucose bond is called "α-monoglucosyl hesperidin," while α-polyglucosyl hesperidin with two or more glucose bonds is called "α-polyglucosyl hesperidin." In other words, in formula (2), α-monoglucosyl hesperidin is a compound where n is 0, and α-polyglucosyl hesperidin is generally a compound where n is 1 to 19.

[0041] Enzyme-treated hesperidin is a collection of compounds produced by enzymatic treatment of the sugars of hesperidin, and typically includes a mixture of compounds with different numbers of glucose units bound to hesperidin, such as a mixture of α-monoglucosyl hesperidin and α-polyglucosyl hesperidin. In addition to α-glucosyl hesperidin, it may also contain unreacted hesperidin or hesperidin derivatives other than α-glucosyl hesperidin, such as 7-glucosyl hesperitin (also referred to as other hesperidin derivatives). However, it is preferred that the enzyme-treated hesperidin does not contain hesperetin.

[0042] An example of the enzyme treatment of the sugars of hesperidin is as follows. (1) Hesperidin is treated with a glycosyltransferase in the presence of a glycosyl donor, and glucose is added to the glucose unit of hesperidin via an α-1,4 bond to produce α-glucosylhesperidin, resulting in a composition containing unreacted hesperidin and α-glucosylhesperidin (first enzyme-treated hesperidin). (2) The α-glucosyl hesperidin produced by (1) above is treated with glucoamylase or the like to cleave all but one glucose molecule from the glucose chain bound to the glucose unit of hesperidin, thereby producing α-monoglucosyl hesperidin, and a composition containing unreacted hesperidin and α-monoglucosyl hesperidin is obtained (second enzyme-treated hesperidin). (3) The unreacted hesperidin from (2) above is treated with α-L-rhamnosidase to cleave the rhamnose contained in the rutinose unit of hesperidin, thereby producing 7-glucosylhesperetin, and a composition containing 7-glucosylhesperetin and α-monoglucosylhesperidin is obtained (third enzyme-treated hesperidin).

[0043] An example of the first enzyme treatment is to treat hesperidin with a glycosyltransferase (e.g., an enzyme that has the function of adding glucose to hesperidin, such as cyclodextrin glucanotransferase (CGTase, EC2.4.1.19)) in the presence of an α-glucosyl sugar compound (e.g., cyclodextrin, partial starch hydrolysate).

[0044] In the viral cell entry inhibitor of the present invention, taking into consideration the effects of the present invention, it is preferable to use enzyme-treated hesperidin, which is a composition containing α-glucosyl hesperidin, and any of the first enzyme-treated hesperidin, second enzyme-treated hesperidin, and third enzyme-treated hesperidin compositions may be used.

[0045] Considering the effects of the present invention, the enzyme-treated hesperidin is preferably a mixture that contains at least α-glucosylhesperidin and further contains either or both of hesperidin and 7-glucosylhesperetin.

[0046] Commercially available enzyme-treated hesperidin products include "αG hesperidin PS-CC" and "αG hesperidin PA-T," both manufactured by Toyo Sugar Refining Co., Ltd. "αG hesperidin PS-CC" contains 80% by mass or more of α-monoglucosyl hesperidin and 7-glucosyl hesperetin. "αG hesperidin PA-T" contains 75% by mass or more of α-monoglucosyl hesperidin and also contains hesperidin.

[0047] As the α-glucosyl hesperidin, α-monoglucosyl hesperidin is preferred, because the molecular weight of α-monoglucosyl hesperidin is smaller than that of α-polyglucosyl hesperidin, and therefore the number of molecules per unit mass of α-monoglucosyl hesperidin is greater, which is thought to be advantageous in terms of action and effect.

[0048] α-Monoglucosyl hesperidin can be produced by treating α-polyglucosyl hesperidin with a glycosidase to cleave all but one glucose bond to hesperidin (second enzyme-treated hesperidin). Examples of glycosidases include enzymes with glucoamylase activity that cleave α-1,4-glucosidic bonds in glucose units, such as glucoamylase (EC 3.2.1.3). The proportion of α-monoglucosyl hesperidin in α-glucosyl hesperidin can be adjusted by adjusting the temperature and time of the enzyme treatment with glucoamylase. Furthermore, methods for purifying and separating α-monoglucosyl hesperidin from a mixture of enzyme-treated hesperidin are also known.

[0049] The presence of various α-glucosylhesperidins, hesperidin, and other components contained in enzyme-treated hesperidin can be confirmed by HPLC chromatogram, and the content of each component or the purity of a specific desired component can be calculated from the peak area of ​​the chromatogram.

[0050] The method for producing α-glucosyl hesperidin is not particularly limited, and known methods can be used. Because of its high yield and ease of production, it is preferably produced by enzymatic treatment of hesperidin. The method for obtaining and preparing hesperidin is not particularly limited, and compounds that are generally manufactured and sold as reagents or purified products may be used, or compounds prepared by extraction from raw materials such as the peels of citrus fruits (e.g., mandarins, oranges, etc.).

[0051] [α-Glucosylnaringin] α-Glucosyl naringin (also called α-glucosyl naringin) is a general term for compounds in which one or more glucose molecules are attached to the hydroxyl group of naringin. Naringin is a type of flavonoid in which neohesperidose (L-rhamnosyl-(α1→2)-D-glucose) is β-bonded to the hydroxyl group at the 7-position of the naringenin (5,7,4'-trihydroxyflavanone) skeleton. α-Glucosyl naringin has a structure in which one or more α-glucose molecules are attached to at least one of the hydroxyl group at the 3-position (3" position) of the glucose residue in the neohesperidose residue and the hydroxyl group at the 4-position (4' position) of the phenyl group in the naringenin skeleton. α-Glucosyl naringin in the present invention may consist of one or more compounds having such a structure, or may be a mixture of two or more.

[0052] α-Glucosylnaringin can be represented by the following formula (3): 1 m and R 2 The n's represent the number of α-glucose residues attached to the 3' and 4' positions, respectively, and are mutually independent integers of 0 or more and usually 25 or less. However, for formula (3) to represent "α-glucosylnaringin", it is necessary that m + n ≥ 1 is satisfied, that is, at least one molecule of α-glucose is linked to naringin (when m = n = 0, that is, R 1 , R 2 When both are -H, formula (3) represents "naringin".

[0053] [ka]

[0054] α-Glucosyl naringin is a compound found as the main component of a material known as "enzyme-modified naringin" (sometimes called "transglycosylated naringin"). α-Glucosyl naringin with only one glucose bond is called "α-monoglucosyl naringin," while α-glucosyl naringin with two or more glucose bonds is called "α-polyglucosyl naringin."

[0055] Enzyme-treated naringin is, for example, a product (referred to herein as "first enzyme-treated naringin") obtained by reacting a mixture of naringin and a sugar donor (e.g., dextrin) with a glycosyltransferase (e.g., cyclodextrin glucosyltransferase), and is a collection of various compounds in which one or more glucose molecules are added to the hydroxyl groups of naringin. Therefore, enzyme-treated naringin usually includes a mixture of compounds with different numbers of glucose atoms bound to naringin, for example, a mixture consisting of α-monoglucosyl naringin and α-polyglucosyl naringin. Furthermore, in addition to α-glucosyl naringin, it may also contain unreacted naringin and naringin derivatives other than α-glucosyl naringin, such as 7-glucosyl naringenin (also referred to as other naringin derivatives).

[0056] For the basic method of producing first enzyme-treated naringin, see, for example, Japanese Patent Application Laid-Open No. 4-13691. If necessary, the first enzyme-treated naringin can be purified using, for example, a porous synthetic adsorbent and an appropriate eluent to remove sugar donors and other impurities, further reducing the naringin content and obtaining first enzyme-treated naringin (purified α-glucosyl naringin) with increased purity of α-glucosyl naringin.

[0057] As α-glucosyl naringin, naringin having one α-glucose molecule linked to the 3'' position and / or one α-glucose molecule linked to the 4' position is preferred, i.e., at least one type of α-glucosyl naringin selected from 3''-α-monoglucosyl naringin (in formula (3) m=1, n=0), 4'-α-monoglucosyl naringin (also m=0, n=1), and 3''-4'-α-diglucosyl naringin (also m=1, n=1), with 3''-α-monoglucosyl naringin being more preferred. This is because the molecular weight of α-monoglucosyl naringin is smaller than that of α-polyglucosyl naringin, and therefore the number of molecules per unit mass of α-monoglucosyl naringin is greater, which is thought to be advantageous in terms of action and effect.

[0058] Enzyme-treated naringin containing a large amount of the three types of α-monoglucosyl naringin (referred to herein as "second enzyme-treated naringin") can be obtained, for example, by treating the above-mentioned first enzyme-treated naringin with an enzyme having glucoamylase activity, and cleaving the sugar chain in which two or more molecules of α-glucose are linked by α-1,4 bonds, which have been transferred to the 3'- and / or 4'-positions of naringin by the glycosyltransferase, leaving only the α-glucose residue equivalent to one molecule at the base. Furthermore, by treating the second enzyme-treated naringin with an enzyme having α-glucosidase activity and cleaving the α-glucose residue equivalent to one molecule directly bonded to the hydroxyl group at the 4'-position, 3'-α-monoglucosyl naringin remains, and enzyme-treated naringin containing little or no 4'-α-monoglucosyl naringin and 3'-4'-α-diglucosyl naringin (referred to herein as "third enzyme-treated naringin") can be obtained. For the basic production method of the second and third enzyme-treated naringin, reference can be made to, for example, Japanese Patent Application Laid-Open No. 2002-199896.

[0059] Furthermore, α-L-rhamnosidase is applied to the third enzyme-treated naringin to cleave the rhamnose contained in the rutinose units of naringin, thereby producing 7-glucosylnaringenin, and enzyme-treated naringin containing 7-glucosylnaringenin and α-monoglucosylnaringin (referred to as "fourth enzyme-treated naringin" in this specification) is obtained.

[0060] Furthermore, by treating the first-enzyme-treated naringin with transglucosidase, transglucosidase has both glucoamylase and α-glucosidase activities, and thus a single-step treatment, rather than the two-step treatment described above, can yield a third-enzyme-treated naringin enriched in 3′-α-monoglucosyl naringin. Furthermore, if necessary, an enzyme with β-glucosidase activity may be treated with the third-enzyme-treated naringin (or may be treated with the first-enzyme-treated naringin simultaneously with transglucosidase) to cleave the neohesperidose residue (unmodified by glycosyltransferase) present in a small amount of unreacted naringin dissolved in aqueous solution from its aglycone, naringenin. The naringenin produced by such treatment has a lower solubility than naringin and therefore forms a precipitate that can be easily removed from the aqueous solution, enabling the recovery of 3′-α-monoglucosyl naringin from the aqueous solution with a higher purity.

[0061] In the viral cell entry inhibitor of the present invention, taking into consideration the effects of the present invention, it is preferable to use enzyme-treated naringin, which is a composition containing α-glucosyl naringin, and any of the following compositions may be used: first enzyme-treated naringin, second enzyme-treated naringin, third enzyme-treated naringin, and fourth enzyme-treated naringin.

[0062] Considering the effects of the present invention, the enzyme-treated naringin is preferably a mixture that contains at least α-glucosylnaringin and further contains either or both of naringin and 7-glucosylnaringenin.

[0063] The presence of various α-glucosyl naringin, naringin, and other components contained in enzyme-treated naringin can be confirmed by HPLC chromatograms, and the content of each component, or the purity of a specific desired component, can be calculated from the peak area of ​​the chromatogram.

[0064] The method for producing α-glucosyl naringin is not particularly limited, and known methods can be used. Because of its high yield and ease of production, it is preferably produced by enzymatic treatment of naringin. The method for obtaining or preparing naringin is not particularly limited, and a compound that is generally manufactured and sold as a reagent or purified product may be used, or a compound prepared by extraction from a raw material such as the peel of citrus fruits (e.g., mandarin oranges, grapefruit, etc.).

[0065] <Virus> In the present invention, the virus having a spike protein that binds to angiotensin-converting enzyme 2 (hereinafter referred to as "ACE2") is not particularly limited, as long as it can invade a host cell with its genomic DNA or genomic RNA via binding of the viral spike protein to ACE2, a host cell receptor, and examples thereof include SARS-CoV, SARS-CoV-2, HCoV-NL63, BatCoV-WIV1, BatCoV-WIV16, BatCoV-RS4231, and BatCoV-RsSHC014. Of these, SARS-CoV, SARS-CoV-2, or HCoV-NL63 are preferred, with SARS-CoV-2 being more preferred, in terms of the inhibitory effect of the present invention.

[0066] The spike protein (also called S protein or spike glycoprotein) is a glycoprotein that usually penetrates the envelope on the surface of the virus. The spike protein is not particularly limited as long as it can bind to ACE2, but preferably has a subunit or domain having a receptor binding domain (RBD) that binds to ACE2 and a subunit or domain having a fusion peptide for fusing with the host cell membrane.

[0067] The amino acid sequence of the spike protein and its nucleotide sequence information can be obtained from databases such as GenBank and GISAID (Global Initiative on Sharing All Influenza Data). Examples of the amino acid sequence of the spike protein include SEQ ID NO: 1 (SARS-CoV), SEQ ID NO: 2 (SARS-CoV-2), and SEQ ID NO: 3 (HCoV-NL63).

[0068] As long as the spike protein can bind to ACE2, it may be a mutant spike protein in which 1 to 3, preferably 1 to 2, and more preferably 1 amino acid has been deleted, substituted, or added in its amino acid sequence.

[0069] The structure of the spike protein is not particularly limited as long as it can bind to ACE2. For example, if the receptor-binding domain has a down-type structure or an up-type structure, it is preferable that the receptor-binding domain has an up-type structure in terms of affinity with ACE2.

[0070] Viruses with spike proteins that bind to ACE2 also include mutant strains in which the spike proteins have been mutated. For example, SARS-CoV-2 mutant strains include N501Y, E484K, L452R, E484Q, K417N, H417T, N439K, D614G, A222V, Y453F, P681H, A570D, T716I, S982A, A1708D, A701V, D80A, L18F, R246I, D215G, delH69V70, delY144, Q27stop, and L242_244L.

[0071] The cells into which the virus invades are not particularly limited as long as they have ACE2 on the cell surface, and examples thereof include cells of the brain, heart, aorta, lung, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, kidney, skeletal muscle, spleen, thymus, trachea, placenta, bladder, uterus, prostate, testes, ovaries, pancreas, adrenal gland, thyroid, salivary gland, tongue, gums, mammary gland, bone marrow, blood vessels, etc. The origin of the host cells is not particularly limited, but is preferably from humans, bats, pangolins, pigs, cats, dogs, cows, mice, rats, or chickens, and more preferably from humans.

[0072] <Virus cell entry inhibitor> The viral cell entry inhibitor may contain at least one component (A) selected from α-glucosyl rutin, α-glucosyl hesperidin, and α-glucosyl naringin, and may consist solely of component (A), or may further contain known optional components such as excipients, stabilizers, humectants, emulsifiers, etc., as long as they do not interfere with the viral cell entry inhibitory effect of component (A). Furthermore, the viral cell entry inhibitor may be a composition containing component (A) and at least one selected from enzyme-treated rutin, enzyme-treated hesperidin, and enzyme-treated naringin, or may contain at least one selected from enzyme-treated rutin, enzyme-treated hesperidin, and enzyme-treated naringin.

[0073] The viral cell entry inhibitor may have the effect of binding and adsorbing a virus having a spike protein that binds to ACE2 to the cellular ACE2, thereby inhibiting the entry of the viral genomic DNA or genomic RNA into the cell. The viral cell entry inhibitor's effect of inhibiting viral cell entry can be confirmed by a change in luminescence value due to luciferase activity of the virus that has entered a host cell containing ACE2 in an in vitro cell entry inhibition test using a pseudovirus, as performed in the Examples. That is, if the luminescence value due to luciferase activity is lower in the group using the viral cell entry inhibitor compared to the group not using the viral cell entry inhibitor, this indicates that the virus has inhibited entry into the host cell by the viral cell entry inhibitor.

[0074] Although there are no particular limitations on the rate of inhibition of viral cell entry, in a cell entry inhibition test using a pseudovirus, if the rate of inhibition when no inhibitor of viral cell entry of the present invention is added is taken as 0%, the rate of inhibition when an inhibitor of viral cell entry of the present invention is added is preferably 10% or more, more preferably 50% or more. The rate of inhibition of viral cell entry can be expressed by the following formula.

[0075] Viral cell entry inhibition rate (%) = 100 × {1 - (luminescence value of the group with viral cell entry inhibitor added) / (luminescence value of the group without viral cell entry inhibitor added)}

[0076] Pseudoviruses can be prepared by known methods, or commercially available products such as Lenti-X™ SARS-CoV-2 (manufactured by Takara Bio Inc.), SARS-CoV pseudovirus, and HCov-NL63 pseudovirus (manufactured by Vector Builder) can be used.

[0077] [Application] The uses of viral cell entry inhibitors are not particularly limited, but because viral cell entry inhibitors have the effect of inhibiting viral entry into cells, they can be used to prevent viral infections, for example, infections with SARS-CoV, SARS-CoV-2, and HCoV-NL63.

[0078] The dosage of the viral cell entry inhibitor may be appropriately selected depending on the type of viral infection and the age, sex, race, etc. of the recipient. For example, when the viral cell entry inhibitor is orally administered, the daily dose of component (A) is preferably 10 mg to 1000 mg, and more preferably 100 mg to 300 mg. The viral cell entry inhibitor may be administered once or multiple times, and the daily administration frequency may be, for example, 1 to 3 divided doses per day, or may be administered in two or three divided doses. The administration period of the viral cell entry inhibitor may be appropriately selected depending on the type of viral infection and the age, sex, race, etc. of the recipient. From the viewpoint of the viral cell entry inhibitory effect, it is preferably 1 to 90 days, more preferably 14 to 60 days.

[0079] [formulation] The virus cell entry inhibitor may be administered to a living body as it is, or may be administered as a formulation in which an effective amount of the virus cell entry inhibitor is formulated with a pharmaceutically acceptable carrier. Examples of the formulation include foods and beverages, pharmaceuticals, and quasi-drugs. The administration method is not particularly limited, and may be oral or parenteral. The formulation is preferably administered orally because it is easy to administer.

[0080] In the case of oral administration, the preparation may be a food or drink (including health functional foods such as specified health foods, nutritional functional foods, and functional food products, as well as other so-called health foods and supplements), a medicine, a quasi-drug, etc.

[0081] Oral preparations can be in solid or liquid form (including paste form). There are no limitations on the dosage form, and specific examples of solid preparations include powders, granules, tablets, capsules, and troches. Examples of liquid preparations include oral liquids, suspensions, emulsions, syrups, and drinks. These and other dosage forms can be appropriately selected depending on the purpose.

[0082] In the case of parenteral administration, the preparation may be in the form of an injection, suppository, or the like.

[0083] The formulation can be produced by adding a viral cell entry inhibitor according to a method commonly used for these formulations. The viral cell entry inhibitor may be added at the beginning, middle, or end of the production process of the formulation, and the method of addition may be selected from mixing, kneading, dissolving, immersion, sprinkling, spraying, coating, etc., as appropriate depending on the form of the formulation.

[0084] The component (A), which is the active ingredient of the virus cell entry inhibitor, has good water solubility, so that it can be dissolved or dispersed uniformly even when added to water or a preparation with a high water content.

[0085] The amount of the viral cell entry inhibitor to be incorporated into the formulation may be selected appropriately depending on the type of disease and the severity of symptoms in which the viral cell entry inhibitory activity is involved, but from the viewpoint of ease of administration and stability in the formulation, the amount of component (A) is preferably 5 to 98% by mass, more preferably 20 to 70% by mass, and even more preferably 30 to 50% by mass. [Example]

[0086] The present invention will be described in more detail below based on examples. However, the present invention is not limited to these examples and can be practiced with appropriate modifications within the scope of the present invention.

[0087] [Preparation Example 1] As described below, we prepared a pseudo-SARS-CoV-2 that has the SARS-CoV-2 spike protein on its envelope surface and exhibits luciferase activity after entering host cells. (1) Preparation of lentiviral vector plasmid (pCDH-fLuc) for fLuc expression The pCDH-EF1-MCS-(PGK-copGFP-T2A-Puro) plasmid (System Biosciences) was purified using the PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit (Thermo Fisher Scientific) and then cleaved with EcoRI (EcoRI-HF: New England Biolabs) and BamHI (BamHI-HF: New England Biolabs) to prepare a linearized plasmid vector.

[0088] The column-purified linear plasmid vector was ligated to the synthetic fLuc gene (SEQ ID NO: 4) using the In-Fusion (registered trademark) HD ​​cloning kit (manufactured by Takara Bio Inc.) according to the instructions attached to the kit, and the resulting product was used to transform Escherichia coli.

[0089] Colony PCR was performed on the drug-resistant E. coli using various primers (CD813_F1 (sequence number 5), CD813_R1 (sequence number 6)), and amplification of an approximately 1.8 kbp product containing the target gene was confirmed. The amplified product was sequenced using various primers (CD813_F1, CD813_R1), and it was confirmed that the sequence of pCDH-fLuc was as designed.

[0090] E. coli harboring this vector plasmid for preparing lentivirus was cultured in liquid culture, and a plasmid (pCDH-fLuc) was prepared using Maxi-prep.

[0091] (2) Preparation of a plasmid (pCD5spCoV2) expressing SAR-CoV-2 A linearized plasmid vector was prepared by cleaving the pcDNA3.1 / Hygro plasmid (Thermo Fisher Scientific) with NheI (NheI-HF: New England Biolabs) and AflII (New England Biolabs). The linearized plasmid vector was purified using a column. The purified linearized plasmid vector was then ligated with a synthetic gene (SEQ ID NO: 9) ligated with the CD5 signal sequence (SEQ ID NO: 7) and the SARS-CoV-2 S gene (SEQ ID NO: 8) using an In-Fusion HD cloning kit (Takara Bio Inc.). The resulting product was used to transform E. coli.

[0092] Colony PCR was performed on the drug-resistant E. coli using plasmid-specific primers (pcDNA3_F1 (SEQ ID NO: 10) and pcDNA3_R2 (SEQ ID NO: 11)), and amplification of an approximately 4 kbp product containing the target gene was confirmed. The amplified product was sequenced using primers CMV_F4 (SEQ ID NO: 12), SARS-CoV-2_Seq1 (SEQ ID NO: 13), SARS-CoV-2_Seq2 (SEQ ID NO: 14), SARS-CoV-2_Seq3 (SEQ ID NO: 15), and pcDNA3_R2, confirming that pCD5spCoV2 had the sequence as designed.

[0093] E. coli harboring this plasmid vector was cultured in liquid, and the plasmid (pCD5spCoV2) was prepared using Maxi-prep.

[0094] (3) Preparation of pseudo-coronavirus Lenti-X 293T cells (Takara Bio Inc.) were cultured in Opti-MEM (Thermo Fisher Scientific) containing 5% FBS and 1 mM sodium pyrubate as the basal medium. Then, Lenti-X 293T cells (1.8 × 10 7 The cells were suspended in 34 mL of the above basal medium and seeded onto a 15 cm dish.

[0095] The next day, transformation was carried out using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) as follows (the composition of the mixture below is for one 15-cm dish, and transformation was carried out in three 15-cm dishes).

[0096] A mixture was prepared by mixing 3.9 mL of Opti-MEM (Thermo Fisher Scientific), 14.4 μg of pPACKH1-GAG (a component of the pPACKH1 HIV Lentivector Packaging Kit [System Biosciences]), 7.2 μg of pPACKH1-REV (a component of the pPACKH1 HIV Lentivector Packaging Kit [System Biosciences]), 11.2 μg of the above pCDH-fLuc, 12.2 μg of the above pCD5spCoV2, and 91 μL of P3000 reagent (a component of Lipofectamine 3000 Transfection Reagent [Thermo Fisher Scientific]). A mixture of 3.9 mL of Opti-MEM and 107 μL of Lipofectamine 3000 reagent was added to the mixture and incubated at room temperature for 10 minutes to allow the formation of DNA / Lipofectamine 3000 complexes.

[0097] The complex solution (7.8 mL) was then added to Lenti-X 293T cells. Six hours after addition, the medium was replaced with 21 mL of Opti-MEM containing 5% FBS and 1 mM sodium pyrubate. After 24 hours of culture, the culture supernatant (1) was collected and stored at 4°C.

[0098] After collecting the culture supernatant (1), the medium was replaced with 21 mL of Opti-MEM containing 5% FBS and 1 mM sodium pyrubate, and the cells were cultured for 24 hours, after which the culture supernatant (2) was collected.

[0099] The mixture of culture supernatant (1) and culture supernatant (2) was used to obtain pseudo-SARS-CoV-2, which was then used in the following experiments. The pseudo-SARS-CoV-2 was stored at -80 °C until use.

[0100] [Example 1] [Cell invasion inhibition test of pseudo-SARS-CoV-2 using Caco-2 cells] The cell invasion inhibitory effect of pseudo-SARS-CoV-2 when cultured with Caco-2 cells (human colon cancer-derived cells) in a medium containing αG hesperidin PA-T, αG rutin PS, or αG naringin PS (fourth enzyme-treated naringin) was verified.

[0101] [Method] As samples, αG rutin PS (manufactured by Toyo Seito Co., Ltd.) as α-glucosyl rutin, αG hesperidin PA-T (manufactured by Toyo Seito Co., Ltd.) as α-glucosyl hesperidin, and αG naringin PS (fourth enzyme-treated naringin; 75% by mass of α-monoglucosyl naringin, 10% by mass of 7-glucosylnarigenin) as α-glucosyl naringin were used.

[0102] Caco-2 cells (manufactured by ATCC) were prepared with Eagle's minimum essential medium (composition: MEM non-essential amino acid solution) to a density of 6×10 3 cells / well, seeded in a 96-well plate, and cultured at 37 °C in a 5% CO2 environment. 24 hours after seeding, 10 μL of each sample at the concentrations shown in Fig. 1 (final concentration in the medium) was added to each well.

[0103] 2 hours after adding the sample, 140 μL of pseudo-SARS-CoV-2 was added to each well, and the Caco-2 cells were cultured for 46 hours. After culturing, the medium was changed, the pseudo-SARS-CoV-2 and the sample were removed, and the cells were further cultured for 24 hours.

[0104] After culturing, the luminescence value due to the luciferase activity of pseudo-SARS-CoV-2 that had invaded the cells was measured.

[0105] [Results] The results of the luminescence values are shown in Figure 1. The cell invasion inhibition rate calculated from the luminescence values is shown in Table 1. The cell invasion inhibition rate was calculated as follows. Cell invasion inhibition rate (%) = 100 × {1 - (luminescence value with sample) / (luminescence value without sample)}. The number of N per experimental condition was 3, and the p-value was calculated by t-test with the sample without virus addition (0 ppm) as the control, and p < 0.05 was considered to have a significant difference. From the above results, it was shown that at any concentration of each sample, there was an effect of inhibiting the cell invasion of pseudo-SARS-CoV-2.

[0106] [Comparative Example 1] The luminescence value due to luciferase activity was measured and the cell invasion inhibition rate (%) was calculated in the same manner as in Example 1, except that nafamostat (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of the above sample. The results are shown in Figure 1 and Table 1.

[0107] [Table 1]

[0108] [Example 2]<00004​​​​​​​​​​​​​​​​​​​​The pcDNA3.1 / Hygro plasmid (Thermo Fisher Scientific) was cleaved with NheI (NheI-HF: New England Biolabs) and AflII (New England Biolabs), and column purified. Each purified product was ligated with the ACE2 synthetic gene (SEQ ID NO: 16) using the In-Fusion HD cloning kit (Takara Bio Inc.) according to the instructions provided with the kit, and the resulting products were used to transform Escherichia coli.

[0111] Colony PCR was performed on the drug-resistant E. coli, and an approximately 2.8 kbp amplification product containing the target gene was confirmed. The amplification product was sequenced using primers CMV_F4 (SEQ ID NO: 12), Hs_ACE2_Seq1 (SEQ ID NO: 17), and pcDNA3_R2 (SEQ ID NO: 11), confirming the sequence as designed (pcDNA3.1-Hyg-ACE2). E. coli harboring the plasmid vector was cultured in liquid culture, and the pcDNA3.1-Hyg-ACE2 plasmid was prepared using Maxi-prep, which served as DNA for transfection.

[0112] (2) Preparation of ACE2-expressing A549 cells The pcDNA3.1-Hyg-ACE2 plasmid was transfected into A549 cells (JCRB Cell Bank) and selected in growth medium containing 50 mg / mL hygromycin B (Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain cells stably transfected with the plasmid (A549-ACE2). Thirty-two clones were isolated by limiting dilution. RNA was extracted and purified from the cloned cells and a negative control (untransfected A549), followed by cDNA synthesis. The A549-ACE2 clones were quantitatively analyzed for ACE2 mRNA expression by real-time PCR using primers Hs_ACE2_RTF1 (SEQ ID NO: 18) and Hs_ACE2_RTR1 (SEQ ID NO: 19).

Claims

1. An inhibitor of viral cell entry, comprising at least one component (A) selected from α-monoglucosyl rutin, α-monoglucosyl hesperidin and α-monoglucosyl naringin, wherein the virus has a spike protein that binds to angiotensin-converting enzyme 2 (ACE2).

2. The virus cell entry inhibitor according to claim 1, wherein the virus is any one selected from SARS-CoV, SARS-CoV-2, and HCoV-NL63.

3. The virus cell entry inhibitor according to claim 1 or 2, wherein the virus is SARS-CoV-2.

4. The virus cell entry inhibitor according to any one of claims 1 to 3, wherein the content of component (A) is 50 mass% or more.

5. The virus cell entry inhibitor according to any one of claims 1 to 4, wherein the content of component (A) is 65 mass% or more.

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