Virus infection inhibitor and method for inhibiting virus infection of cells

Abstract

To provide a viral infection inhibitor capable of inhibiting viral infection of cells.SOLUTION: A viral infection inhibitor contains a compound represented by a formula (1) and inhibits viral infection of cells by inhibiting adhesion of a virus 4 to a cell 1. (In the formula, n and m each independently represent an integer equal to or greater than 1; X is a hydrophobic site 2; Y is a hydrophilic site 3 being a polymer chain comprising a hydrophilic polymer having a chain length of 4.5 nm or more; if n+m is 2 then L is a single bond or divalent group, and if n+m is equal to or greater than 3 then L is an n+m-valent group.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a virus infection inhibitor and a method for inhibiting virus infection of cells.

Background Art

[0002] In cell cultures and samples collected from living organs and the like, there may be cases where a plurality of cells aggregate to form cell clumps. When measuring a sample containing such cell clumps using a flow cytometer or the like, it may be necessary to perform a treatment for dispersing the cell clumps and / or a treatment for maintaining the cells after the dispersion treatment so that they do not re-aggregate. Patent Document 1 describes a cell aggregation inhibitor containing a predetermined compound.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a virus infection inhibitor that can inhibit virus infection of cells. Another object of the present invention is to provide a method for inhibiting virus infection of cells.

Means for Solving the Problems

[0005] As a result of intensive studies to achieve the above problems, the present inventors have found that the above problems can be achieved by the following configuration.

[0006] [1] A virus infection inhibitor that contains a compound represented by Formula 1 described below and inhibits virus infection of cells by inhibiting virus attachment to cells. [2] The virus infection inhibitor according to [1], wherein the hydrophilic polymer of Y in Formula 1 described below is at least one selected from the group consisting of polyalkylene glycol and polyglycerin. [3] The virus infection inhibitor according to [1] or [2], wherein the weight average molecular weight of the hydrophilic polymer is 4400 or more. [4] The virus infection inhibitor according to any one of [1] to [3], wherein the chain length of the polymer chain of Y in Formula 1 described below is 4.9 nm or more. [5] A method for inhibiting virus infection to cells, comprising adding the virus infection inhibitor according to any one of [1] to [4] to an object containing at least one selected from the group consisting of a tissue composed of cells and an extracellular matrix and a liquid medium. [Advantages of the Invention]

[0007] According to the present invention, there can be provided a virus infection inhibitor capable of inhibiting virus infection to cells. Further, according to the present invention, there can also be provided a method for inhibiting virus infection to cells. [Brief Description of the Drawings]

[0008]

Figure 1

Figure 2

[0009] Hereinafter, the present invention will be described in detail. The description of the constituent elements described below may be made based on representative embodiments of the present invention, but the present invention is not limited to such embodiments. In the present specification, a numerical range represented by "~" means a range including the numerical values described before and after "~" as a lower limit value and an upper limit value.

[0010] In the notation of groups (atomic groups) in this specification, notations that do not indicate substitution or unsubstitution include both those having no substituent and those having a substituent as long as the effects of the present invention are not impaired. For example, the term "alkyl group" includes not only an alkyl group having no substituent (unsubstituted alkyl group) but also an alkyl group having a substituent (substituted alkyl group). This also applies to each compound. Also, in this specification, "(poly)oxyalkylene" represents both polyoxyalkylene and oxyalkylene, or either one.

[0011] [Virus infection inhibitor] The virus infection inhibitor according to an embodiment of the present invention contains a compound represented by Formula 1 described below (hereinafter also referred to as "specific compound").

[0012] [Chemical formula]

[0013] In Formula 1, n and m each independently represent an integer of 1 or more, X is a monovalent group obtained by removing an arbitrary hydrogen atom from at least one selected from the group consisting of sterols and sterol derivatives, a linear or branched alkyl group having 8 or more carbon atoms, a linear or branched alkenyl group having 8 or more carbon atoms, and a linear or branched alkynyl group having 8 or more carbon atoms, at least one group selected from the group consisting of; a plurality of Xs may be the same or different, and a plurality of Xs may be linked to each other to form a ring; Y is a polymer chain composed of a hydrophilic polymer having an effective length measured from the terminal fluorescence resonance transfer efficiency in an aqueous solution of 4.5 nm or more; when n + m is 2, L is a single bond or a divalent group; when n + m is 3 or more, L is an (n + m)-valent group.

[0014] The above virus infection inhibitor can be used by adding it to a tissue composed of cells and an extracellular matrix, and cells (hereinafter also referred to as "tissue, etc."). Among them, it is preferably added to a substance obtained by adding a liquid medium to the tissue, etc. Also, a method of previously dispersing the virus infection inhibitor in a liquid medium and adding the tissue, etc. thereto is also preferable.

[0015] Among specific compounds, the moiety represented by X in Formula 1 (hereinafter also referred to as "hydrophobic moiety") has a high affinity for the cell membrane possessed by cells. In other words, the above hydrophobic moiety is likely to adsorb to the cell membrane and / or is likely to bind to the cell membrane. That is, the specific compound is likely to be fixed to the cell membrane using the hydrophobic moiety as an anchor.

[0016] The specific compound also has a moiety represented by Y in Formula 1 (hereinafter also referred to as "hydrophilic moiety"). FIG. 1 is a schematic diagram showing a specific compound fixed to a cell. As shown in FIG. 1, when a specific compound having a hydrophobic moiety 2 and a hydrophilic moiety 3 is added to a mixture containing a liquid medium and cells 1, the hydrophobic moiety is fixed to the cell membrane side, and the hydrophilic moiety protrudes to the outside of the cell (liquid medium side).

[0017] When the effective length of this hydrophilic moiety measured from the fluorescence resonance energy transfer efficiency at the end in an aqueous solution is composed of a hydrophilic polymer of 4.5 nm or more, surprisingly, an effect of suppressing the attachment of virus 4 to the cell membrane can be obtained. On the other hand, if it is less than 4.5 nm, a sufficient effect cannot be obtained.

[0018] The above chain length means a value obtained by measuring the efficiency of fluorescence resonance energy transfer between a pair of fluorescent dyes having fluorescent characteristics capable of causing fluorescence resonance energy transfer, which are respectively bonded to both ends of the polymer in an aqueous solution, while changing the length of the polymer, and calculating the distance between the polymer ends from the theoretical calculation formula of the fluorescence resonance energy transfer efficiency.

[0019] The content of the specific compound in the virus infection inhibitor is not particularly limited, but in terms of obtaining a virus infection inhibitor having a more excellent effect of the present invention, generally 0.01 to 99.9% by mass is preferable based on the total mass of the virus infection inhibitor. Note that the virus infection inhibitor may contain one kind of the specific compound alone or may contain two or more kinds. When the virus infection inhibitor contains two or more kinds of specific compounds, it is preferable that the total content thereof is within the above numerical range.

[0020] In Formula 1, n and m are each independently an integer of 1 or more, and are not particularly limited, but are each independently preferably 1 to 5, more preferably 1 to 3, and still more preferably 1 or 2.

[0021] Among them, in terms of obtaining a virus infection inhibitor having a more excellent effect of the present invention, as the specific compound, Formula 1A: (X) n -L 1a -Y is preferably a compound represented by. In Formula 1A, n, X, and Y have the same meanings as the respective symbols in Formula 1. Also, L 1a is an (n + 1)-valent group, and its form is the same as L in Formula 1.

[0022] <Hydrophobic moiety> In Formula 1, the hydrophobic moiety represented by X is a monovalent group obtained by removing an arbitrary hydrogen atom from at least one selected from the group consisting of sterols and sterol derivatives (hereinafter also referred to as "substituent A"), a linear or branched alkyl group having 8 or more carbon atoms, a linear or branched alkenyl group having 8 or more carbon atoms, and a linear or branched alkynyl group having 8 or more carbon atoms. In terms of obtaining a virus infection inhibitor having a more excellent effect of the present invention, at least one selected from the group consisting of substituent A, a linear or branched alkyl group having 8 or more carbon atoms, and a linear or branched alkenyl group having 8 or more carbon atoms is preferable, at least one selected from the group consisting of substituent A and a linear or branched alkyl group having 8 or more carbon atoms is more preferable, and substituent A is still more preferable.

[0023] (Substituent A) Substituent A is a monovalent group obtained by removing an arbitrary hydrogen atom from at least one selected from the group consisting of sterols and sterol derivatives. In the present specification, the sterol derivative means a compound having a steroid nucleus, a hydroxy group bonded to the carbon atom at the 3-position of the steroid nucleus, and at least one of the hydrogen atoms bonded to the carbon atoms of the steroid nucleus being substituted with an arbitrary monovalent group. The monovalent group is not particularly limited, and examples include the substituent W described later. Among them, a hydrocarbon group, a hydroxy group, a halogen atom, etc. are preferable.

[0024] Substituent A is not particularly limited, but in terms of obtaining a virus infection inhibitor having a more excellent effect of the present invention, it is preferably a monovalent group obtained by removing one arbitrary hydrogen atom from the compound represented by the following formula 2.

[0025] [Chemical formula]

[0026] In formula 2, rings A to D represent a saturated or unsaturated steroid nucleus. In formula 2, R is a hydrogen atom or a monovalent group. The monovalent group is not particularly limited, and examples include the substituent W described later. Among them, in terms of obtaining a virus infection inhibitor having a more excellent effect of the present invention, a monovalent hydrocarbon group which may have a hetero atom is more preferable, a linear, branched, or cyclic alkyl group is more preferable, and the number of carbon atoms of the alkyl group is not particularly limited, but 1 to 20 is preferable, 2 to 10 is more preferable, and 3 to 8 is even more preferable. Further, the hydrogen atom bonded to the carbon atom of the steroid nucleus may be substituted with a monovalent group, and examples of the monovalent group include the substituent W described later.

[0027] More specifically, examples of the compound represented by Formula 2 include phytosterols such as campesterol, campestanol, brassicasterol, 22-dehydrocampesterol, stigmasterol, stigmasteranol, 22-dihydrositosterol, 22-dehydrostigmasteranol, 7-dehydrostigmasterol, sitosterol, tilucalol, oiyhol, fucosterol, isofucosterol, codisterol, clionasterol, polypherasterol, clerosterol, 22-dehydroclerosterol, fungisterol, condrillasterol, avenasterol, bernosterol, and polynastanol; zoosterols such as cholesterol, dihydrocholesterol, cholestanol, coprostanol, epicoprosterol, epicoprostanol, 22-dehydrocholesterol, desmosterol, 24-methylenecolesterol, lanosterol, 24,25-dihydrolanosterol, norlanosterol, spinasterol, dihydroagnosterol, agnosterol, lophenol, and latosterol; fungal sterols such as dehydroergosterol, 22,23-dihydroergosterol, epistesterol, ascosterol, and fecosterol; and the like can be mentioned.

[0028] The bonding position between the compound represented by Formula 2 and L of Formula 1 is not particularly limited, but a hydroxy group bonded to the carbon atom at the 3-position of the steroid nucleus is preferred. The bond between the hydroxy group and L can be easily formed, for example, by an ether bond, an ester bond, an amide bond, or the like. Although not particularly limited, as a specific compound when Y is a substituent A, for example, a compound represented by the following formula (3) is preferred.

[0029]

Chemical formula

[0030] In Formula 3, ring A to D, and R are synonymous with each symbol in Formula 2. Further, in Formula 3, Y and m are synonymous with each symbol in Formula 1. In Formula 3, L b is a single bond or -C(O)-, and L c is a single bond or an (m + 1)-valent group, and the form of the (m + 1)-valent group of L c is the same as L in Formula 1.

[0031] Among them, in terms of obtaining a virus infection inhibitor having a more excellent effect of the present invention, as the monovalent group obtained by removing an arbitrary hydrogen atom from at least one selected from the group consisting of sterols and sterol derivatives, the group represented by the following Formula 3 is preferable.

[0032]

Chemical formula

[0033] In Formula 4, R is a hydrogen atom or a monovalent group, and the monovalent group is not particularly limited, and examples thereof include the substituent W described later. Among them, in terms of obtaining a virus infection inhibitor having a more excellent effect of the present invention, a monovalent hydrocarbon group which may have a hetero atom is more preferable, a linear, branched or cyclic alkyl group is more preferable, and the number of carbon atoms of the alkyl group is not particularly limited, but 1 to 20 are preferable, 2 to 10 are more preferable, and 3 to 8 are still more preferable. Further, * represents the bonding position.

[0034] When the hydrophobic moiety is a linear or branched alkyl group having 8 or more carbon atoms, the number of carbon atoms is not particularly limited, but 10 or more is preferable, 12 or more is more preferable, 13 or more is still more preferable, 14 or more is particularly preferable, and 15 or more is most preferable. The upper limit of the number of carbon atoms is not particularly limited, but generally 30 or less is preferable, and 24 or less is more preferable. As the linear or branched alkyl group having 8 or more carbon atoms, a linear alkyl group having 8 or more carbon atoms is preferable.

[0035] When the hydrophobic moiety is a linear or branched alkenyl group having 8 or more carbon atoms, the number of carbon atoms is not particularly limited, but is preferably 10 or more, more preferably 12 or more, still more preferably 13 or more, particularly preferably 14 or more, and most preferably 15 or more. The upper limit of the number of carbon atoms is not particularly limited, but is generally preferably 30 or less, more preferably 24 or less. As the linear or branched alkenyl group having 8 or more carbon atoms, a linear alkenyl group having 8 or more carbon atoms is preferred.

[0036] When the hydrophobic moiety is a linear or branched alkynyl group having 8 or more carbon atoms, the number of carbon atoms is not particularly limited, but is preferably 10 or more, more preferably 12 or more, still more preferably 13 or more, particularly preferably 14 or more, and most preferably 15 or more. The upper limit of the number of carbon atoms is not particularly limited, but is generally preferably 30 or less, more preferably 24 or less. As the linear or branched alkynyl group having 8 or more carbon atoms, a linear alkynyl group having 8 or more carbon atoms is preferred.

[0037] <l> In Formula 1, when n + m (the sum of n and m) is 2, L is a single bond or a divalent group. When n + m is 3 or more, L is an (n + m)-valent group.

[0038] The divalent group of L is not particularly limited. For example, -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, -NR2- (R2 represents a hydrogen atom or a monovalent organic group), -O-P(O)(OH)-, -O-P(O)(O - M + )-, an alkylene group (preferably having 1 to 10 carbon atoms), a cycloalkylene group (preferably having 3 to 10 carbon atoms), an alkenylene group (preferably having 2 to 10 carbon atoms), and combinations thereof, etc. may be mentioned. Note that M + is a counter ion, and examples include Na + and NH4 + etc.

[0039] The group of L with a valence of 3 or more is not particularly limited. For example, groups represented by the following formulas (1a) to (1d) may be mentioned.

[0040]

Chemical formula

[0041] In Formula 1a, L3 represents a trivalent group. T3 represents a single bond or a divalent group, and the three T3s may be the same as or different from each other. Examples of L3 include a trivalent hydrocarbon group (preferably having 1 to 10 carbon atoms. Note that the hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group), or a trivalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group). The hydrocarbon group may contain a hetero atom (for example, -O-). Specific examples of L3 include a glycerin residue, a trimethylolpropane residue, a phloroglucinol residue, and a cyclohexanetriol residue, etc.

[0042] In Formula 1b, L4 represents a tetravalent group. T4 represents a single bond or a divalent group, and the four T4s may be the same as or different from each other. Preferred forms of L4 include a tetravalent hydrocarbon group (preferably having 1 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group.), and a tetravalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group). The hydrocarbon group may contain a heteroatom (e.g., -O-). Specific examples of L4 include a pentaerythritol residue and a ditrimethylolpropane residue.

[0043] In Formula 1c, L5 represents a pentavalent group. T5 represents a single bond or a divalent group, and the five T5s may be the same as or different from each other. Preferred forms of L5 include a pentavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group.), or a pentavalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group). The hydrocarbon group may contain a heteroatom (e.g., -O-). Specific examples of L5 include an arabinitol residue, a phloroglucinol residue, and a cyclohexanepentaol residue.

[0044] In Formula 1d, L6 represents a hexavalent group. T6 represents a single bond or a divalent group, and the six T6s may be the same as or different from each other. Preferred forms of L6 include a hexavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group.), or a hexavalent heterocyclic group (preferably a 6- to 7-membered heterocyclic group). The hydrocarbon group may contain a heteroatom (e.g., -O-). Specific examples of L6 include a mannitol residue, a sorbitol residue, a dipentaerythritol residue, hexahydroxybenzene, and a hexahydroxycyclohexane residue.

[0045] In Formulas 1a to 1d, specific examples and preferred forms of the divalent groups represented by T3 to T6 may be the same as those of the divalent groups of L already described. In addition, when L is a group with a valence of 7 or higher, a group formed by combining the groups represented by Formula 1a to Formula 1d can be used.

[0046] <Hydrophilic moiety> In Formula 1, Y is a hydrophilic moiety, which is a polymer chain composed of a hydrophilic polymer having an effective length measured from the fluorescence resonance energy transfer efficiency at the terminal in an aqueous solution of 4.5 nm or more. In the present specification, the "polymer chain composed of a hydrophilic polymer" means a partial structure in a specific compound obtained by bonding a hydrophilic polymer to L, and the bonding position of L in the hydrophilic polymer is not particularly limited, and examples thereof include the terminal of the hydrophilic polymer.

[0047] In the present specification, the hydrophilic polymer means a polymer having a hydrophilic group. The type of the hydrophilic group is not particularly limited, and examples thereof include a polyoxyalkylene group (for example, a polyoxyethylene group, a polyoxypropylene group, and a polyoxyalkylene group in which an oxyethylene group and an oxypropylene group are block- or randomly-bonded), an amino group, a carboxy group, an alkali metal salt of a carboxy group, a hydroxy group, an alkoxy group, an amide group, a carbamoyl group, a sulfonamide group, a sulfamoyl group, a sulfonic acid group, and an alkali metal salt of a sulfonic acid group.

[0048] The structure of the main chain of the hydrophilic polymer is not particularly limited, and examples thereof include polyurethane, poly(meth)acrylate, polystyrene, polyester, polyamide, polyimide, polyether, and polyurea. Note that poly(meth)acrylate is a concept including both polyacrylate and polymethacrylate.

[0049] Further, the hydrophilic polymer may be a protein (glycoprotein). The protein is not particularly limited as long as it does not have a function of inducing virus infection to cells, such as binding to a virus or activating molecules on the virus outer shell. That is, the protein that can be used as the hydrophilic polymer is different from the protein that induces virus binding and infection to cells. Even if it is a protein molecule that induces virus infection when expressed from its gene intracellularly, it is not limited as long as it does not induce virus infection when added to the cell culture medium as an amphiphilic molecule with a hydrophobic group added and binds to the cell membrane.

[0050] Generally, it is presumed that cell membrane-expressed molecules such as proteins are involved in virus infection to cells. The forms of this involvement include, for example, molecules such as specific cell surface proteins that have an affinity for and bind to proteins expressed on the virus outer shell, that is, receptors, and proteins that have a function of cleaving a part of the proteins expressed on the virus outer shell and deforming it into a form that can bind to cell surface proteins, that is, proteases; etc.

[0051] In any of the above cases, when the virus infection inhibitor according to the embodiment of the present invention is introduced, a structure is formed in which a hydrophilic polymer having a predetermined chain length protrudes three-dimensionally from the cell membrane surface, so that the distance between the adjacent cells and the virus surface is increased, and the frequency of molecules such as proteins on these cell membranes interacting and functioning with molecules on the virus outer shell is decreased. That is, it is presumed that a steric repulsive force is generated by the hydrophilic polymer protruding from the cell membrane surface.

[0052] As the hydrophilic polymer, for example, synthetic polymers, proteins (including sugar chain modification), peptides, oligo DNA, and sugar chains (not including proteins) etc. may be used.

[0053] More specifically, examples of the hydrophilic polymer include polyvinylpyrrolidone, polyalkylene glycol, polyglycerin, poly(meth)acrylate, cellulose derivatives (e.g., carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, etc.), starch derivatives (pullulan), polyvinyl alcohol, vinyl acetate, Eudragit, gelatin, polyacrylic acid, sodium polyacrylate, polyisobutylene maleic anhydride copolymer, alginic acid, sodium alginate, carrageenan, gum arabic, tragacanth, karaya gum, and polyvinyl methacrylate, etc.

[0054] As the hydrophilic polymer, at least one selected from the group consisting of polyalkylene glycol and polyglycerin is preferable in that a virus infection inhibitor having more excellent effects of the present invention can be obtained. It is presumed that polyalkylene glycol and polyglycerin have weak interactions with most biomolecules and also weak interactions with viruses. Therefore, it is presumed that a more excellent infection inhibitory effect can be easily obtained regardless of the type of virus.

[0055] The hydrophilic moiety represented by Y in Formula 1 is a partial structure (polymer chain) in a specific compound formed by the binding of the above hydrophilic polymer to L, and the effective length (chain length) of the hydrophilic polymer measured from the fluorescence resonance transfer efficiency at the terminal in an aqueous solution is 5.0 nm or more. The chain length is not particularly limited as long as it is 4.5 nm or more, but 4.9 nm or more is preferable, 7.0 nm or more is more preferable, 7.3 nm or more is still more preferable, 10 nm or more is particularly preferable, and 10.9 nm or more is most preferable in that a virus infection inhibitor having more excellent effects of the present invention can be obtained.

[0056] On the one hand, although the upper limit of the chain length is not particularly limited, when it is 20.0 nm or less, the hydrophilicity of the virus inhibitor is easily adjusted within an appropriate range, and it is preferable in that the virus inhibitor is likely to be fixed by the cell membrane of the cells to be inhibited from infection.

[0057] The above chain length is obtained by dispersing a sample obtained by binding a fluorescent dye having a fluorescence property capable of causing fluorescence resonance energy transfer to both ends of the hydrophilic polymer to be measured in an aqueous solution, measuring the efficiency of fluorescence resonance energy transfer, and calculating the distance between the polymer ends from the theoretical calculation formula of the fluorescence resonance energy transfer efficiency. According to the above method, the effective length of the polymer chain can be measured.

[0058] For example, when the hydrophilic polymer is a polyalkylene glycol, one end is biotinylated and the other end is maleimidated. The biotin side end is bound to a fluorescent (DyLight649) - labeled streptavidin bound on a biotin - immobilized plastic plate, and the maleimide side end is covalently bound to the cysteine group of another fluorescent - labeled protein (for example, IEkMCC labeled with Dylight549) to prepare a sample. When this sample is dispersed in water and the fluorescence intensity is measured, since the fluorescence spectrum of DyLight549 and the absorption spectrum of DyLight649 overlap, fluorescence resonance energy transfer (FRET) corresponding to the distance between the two dyes occurs, and the distance between the polymer ends can be calculated.

[0059] The above - mentioned measurement method is described in PLoS One. 2014 Nov 10;9(11):e112292, and the above content is incorporated herein. The method for adjusting the chain length of the hydrophilic polymer is not particularly limited, but it can be adjusted by the primary structure, molecular weight, etc. of the hydrophilic polymer, and the method is known to those skilled in the art.

[0060] The molecular weight of the hydrophilic polymer is not particularly limited, but in terms of obtaining a virus infection inhibitor having more excellent effects of the present invention, 4400 or more is preferable, 5000 or more is more preferable, 9400 or more is still more preferable, 10000 or more is particularly preferable, and 18000 or more is most preferable. The upper limit value of the molecular weight is not particularly limited, but generally, 200000 or less is preferable, and 100000 or less is more preferable. In addition, in this specification, the molecular weight means the weight average molecular weight measured by the gel permeation chromatography method.

[0061] The specific compound is not particularly limited, and examples thereof include compounds represented by the following formulas.

Chemical formula

[0062] In each formula, R represents a hydrogen atom or a monovalent group, R1 represents a polymer chain of Y, p represents an integer of 2 or more, q represents an integer of 0 to 2, and M represents a counter ion (for example, Na + and NH4 + etc. can be mentioned.). The specific compound can be synthesized by a known method, and commercially available products manufactured by Sigma, Nanocs, Biochempeg, and Avanti Polar Lipids can also be used.

[0063] 〔Other components〕 The virus infection inhibitor according to the embodiment of the present invention may contain other components other than the specific compound within the range where the effects of the present invention are exhibited. The other components are not particularly limited, and examples thereof include a buffering agent and a chelating agent.

[0064] <Buffering agent> The virus infection inhibitor according to the embodiment of the present invention may contain a buffering agent. The content of the buffering agent in the virus infection inhibitor is not particularly limited, but generally 0.001 to 99% by mass is preferable with respect to the total mass of the virus infection inhibitor in terms of obtaining a virus infection inhibitor having more excellent effects of the present invention. The virus infection inhibitor may contain one kind of buffering agent alone or two or more kinds thereof. When the virus infection inhibitor contains two or more kinds of buffering agents, it is preferable that the total content thereof is within the above numerical range.

[0065] The buffering agent is not particularly limited, and examples thereof include sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium acetate, and epsilon-aminocaproic acid.

[0066] <Chelating agent> The virus infection inhibitor according to an embodiment of the present invention may contain a chelating agent. The content of the chelating agent in the virus infection inhibitor is not particularly limited, but generally 0.001 to 99% by mass is preferable with respect to the total mass of the virus infection inhibitor in terms of obtaining a virus infection inhibitor having more excellent effects of the present invention. The virus infection inhibitor may contain one kind of chelating agent alone or two or more kinds thereof. When the virus infection inhibitor contains two or more kinds of chelating agents, it is preferable that the total content thereof is within the above numerical range.

[0067] The chelating agent is not particularly limited, and examples thereof include hydroxy acid-based such as citric acid, malic acid, lactic acid, tartaric acid, gluconic acid, ascorbic acid, etidronic acid, and dihydroxyethylglycine (DHEG); aminocarboxylic acid-based such as ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, hydroxyethylethylenediaminetriacetic acid, tetrasodium glutamate diacetate, glycol ether diamine tetraacetic acid, triethylenetetraminehexaacetic acid, hydroxyethyliminodiacetic acid, ethylenediamine-N,N'-disuccinic acid, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Ether carboxylic acids such as carboxymethyl tartronic acid (CMT) and carboxymethyloxysuccinic acid (CMOS); etc. can be mentioned.

[0068] [Use of virus infection inhibitor] The virus infection inhibitor according to the embodiment of the present invention can be used by adding it to a tissue or the like after dispersing the virus infection inhibitor in a liquid medium for the purpose of suppressing the attachment of the virus to cells. It can also be added to a tissue or the like to which a liquid medium has been added. Further, the virus infection inhibitor according to the embodiment of the present invention may be dispersed in a liquid medium in advance, and a tissue or the like may be added thereto. By using the virus infection inhibitor according to the embodiment of the present invention, the attachment of the virus to each cell surface can be suppressed.

[0069] In addition, when the virus infection inhibitor according to the embodiment of the present invention is used within an appropriate concentration and chain length range, it can function as a cell dispersant as another use (Japanese Patent Application Laid-Open No. 2020-048446). That is, the distance between adjacent and interacting cells can be expanded. Therefore, by adding the virus infection inhibitor according to the embodiment of the present invention, the attachment of the virus in the external solution to cells can be suppressed, and the cell-to-cell infection caused by the virus budding from the cells already infected with the virus to adjacent and adhering cells can be suppressed.

[0070] In particular, when the hydrophilic polymer is a polyalkylene glycol, it is more excellent in that it is non-toxic and easy to control the chain length. In the virus infection inhibitor according to the embodiment of the present invention, it is presumed that the polymer chain composed of the above hydrophilic polymer is not introduced into the cell. On the other hand, the hydrophobic site that is easy to bind and detach fixes the polymer chain on the cell membrane, holds the hydrophilic polymer in the solution (including cells, cell suspension) when necessary, and can be quickly removed by washing the solution when it becomes unnecessary.

[0071] (Substituent W) Examples of the substituent W include a halogen atom, an alkyl group (e.g., methyl, ethyl, propyl, isopropyl, tert-butyl, pentyl, hexyl, octyl, dodecyl, tridecyl, tetradecyl, pentadecyl, etc.), a cycloalkyl group (e.g., cyclopentyl, cyclohexyl, etc.), an alkenyl group (e.g., vinyl, allyl, etc.), an alkynyl group (e.g., ethynyl, propargyl, etc.), an aromatic hydrocarbon ring group (also referred to as an aromatic carbocyclic ring, aryl, etc., e.g., phenyl, p-chlorophenyl, mesityl, tolyl, xylyl, naphthyl, anthryl, azulenyl, acenaphthenyl, fluorenyl, phenanthryl, indenyl, pyrenyl, biphenylyl, etc.), and an aromatic heterocyclic group (a 5- or 6-membered aromatic heterocyclic group is preferred, and the ring-constituting heteroatoms are preferably a sulfur atom, a nitrogen atom, an oxygen atom, a silicon atom, boron, and a selenium atom, e.g., pyridyl, pyrimidinyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, pyrazolyl, pyrazinyl, triazolyl (e.g., 1,2,4-triazol-1-yl, and 1,2,3-triazol-1-yl, etc.), oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isothiazolyl, furazanyl, thienyl, quinolyl, benzofuryl, dibenzofuryl, benzothienyl, dibenzothienyl, indolyl, carbazolyl, carbolinyl, diazacarbazolyl (indicating that one of the carbon atoms constituting the carboline ring of the above carbolinyl group is replaced by a nitrogen atom), quinoxalinyl, pyridazinyl, triazinyl, quinazolinyl, phthalazinyl, borol, azaborine, etc.), a heterocyclic group (a non-aromatic heterocyclic group, which may be a saturated ring or an unsaturated ring, preferably a 5- or 6-membered ring, and the ring-constituting heteroatoms are preferably sulfur atoms, nitrogen atoms, oxygen atoms, silicon atoms, or selenium atoms, for example, pyrrolidyl, imidazolidyl, morpholyl, oxazolidyl, etc.), an alkoxy group (for example, methoxy, ethoxy, propyloxy, pentyloxy, hexyloxy, octyloxy, dodecyloxy, etc.), a cycloalkoxy group (for example, cyclopentyloxy, cyclohexyloxy, etc.), an aryloxy group (for example, phenoxy, naphthyloxy, etc.), an alkylthio group (for example, methylthio, ethylthio, propylthio, pentylthio, hexylthio, octylthio, dodecylthio, etc.), a cycloalkylthio group (for example, cyclopentylthio, cyclohexylthio, etc.), an arylthio group (for example, phenylthio, naphthylthio, etc.), an alkoxycarbonyl group (for example, methyloxycarbonyl, ethyloxycarbonyl, butyloxycarbonyl, octyloxycarbonyl, dodecyloxycarbonyl, etc.), an aryloxycarbonyl group (for example, phenyloxycarbonyl, naphthyloxycarbonyl, etc.), a sulfamoyl group (for example, aminosulfonyl, methylaminosulfonyl, dimethylaminosulfonyl, butylaminosulfonyl, hexylaminosulfonyl, cyclohexylaminosulfonyl, octylaminosulfonyl, dodecylaminosulfonyl, phenylaminosulfonyl, naphthylaminosulfonyl, 2-pyridylaminosulfonyl, etc.),,

[0072] Acyl groups (e.g., acetyl, ethylcarbonyl, propylcarbonyl, pentylcarbonyl, cyclohexylcarbonyl, octylcarbonyl, 2-ethylhexylcarbonyl, dodecylcarbonyl, acryloyl, methacryloyl, phenylcarbonyl, naphthylcarbonyl, pyridylcarbonyl, etc.), acyloxy groups (e.g., acetyloxy, ethylcarbonyloxy, butylcarbonyloxy, octylcarbonyloxy, dodecylcarbonyloxy, phenylcarbonyloxy, etc.), amide groups (e.g., methylcarbonylamino, ethylcarbonylamino, dimethylcarbonylamino, propylcarbonylamino, pentylcarbonylamino, cyclohexylcarbonylamino, 2-ethylhexylcarbonylamino, octylcarbonylamino, dodecylcarbonylamino, phenylcarbonylamino, naphthylcarbonylamino, etc.), carbamoyl groups (e.g., aminocarbonyl, methylaminocarbonyl, dimethylaminocarbonyl, propylaminocarbonyl, pentylaminocarbonyl, cyclohexylaminocarbonyl, octylaminocarbonyl, 2-ethylhexylaminocarbonyl, dodecylaminocarbonyl, phenylaminocarbonyl, naphthylaminocarbonyl, 2-pyridylaminocarbonyl, etc.), ureido groups (e.g., methylureido, ethylureido, pentylureido, cyclohexylureido, octylureido, dodecylureido, phenylureido, naphthylureido, 2-pyridylaminoureido, etc.), sulfinyl groups (e.g., methylsulfinyl, ethylsulfinyl, butylsulfinyl, cyclohexylsulfinyl, 2-ethylhexylsulfinyl, dodecylsulfinyl, phenylsulfinyl, naphthylsulfinyl, 2-pyridylsulfinyl, etc.), alkylsulfonyl groups (e.g., methylsulfonyl, ethylsulfonyl, butylsulfonyl, cyclohexylsulfonyl, 2-ethylhexylsulfonyl, dodecylsulfonyl, etc.), arylsulfonyl groups or heteroarylsulfonyl groups (e.g., phenylsulfonyl, naphthylsulfonyl, 2-pyridylsulfonyl, etc.), amino groups (including amino groups, alkylamino groups, alkenylamino groups, arylamino groups, heterocyclic amino groups, e.g., amino, ethylamino, dimethylamino,Butylamino, cyclopentylamino, 2-ethylhexylamino, dodecylamino, anilino, naphthylamino, 2-pyridylamino, etc.), cyano group, nitro group, hydroxy group, mercapto group, silyl group (for example, trimethylsilyl, triisopropylsilyl, triphenylsilyl, phenyldiethylsilyl, etc.), etc. are mentioned. Each of these groups may further have a substituent, and examples of this substituent include the above-mentioned substituents. For example, an aralkyl group in which an aryl group is substituted for an alkyl group, a hydroxyalkyl group in which a hydroxy group is substituted for an alkyl group, etc. are mentioned. In addition, when the substituent W further has a plurality of substituents, the plurality of substituents may be bonded to each other to form a ring.

[0073] [Virus infection suppression method] The virus infection suppression method according to an embodiment of the present invention is a method for suppressing virus infection to cells, in which the above-mentioned virus infection inhibitor is added to an object containing at least one selected from a tissue composed of cells and an intercellular matrix, and a group of cells, and a liquid medium. The tissue or the like to which the virus infection suppression method according to an embodiment of the present invention can be applied is not particularly limited, and it may be a tissue collected from a living organ (such as bronchus, nasal cavity, and pharynx), or a cultured cell or tissue, or the living organ itself.

[0074] The liquid medium is not particularly limited, but for example, water, a water-soluble organic solvent, and a mixed solvent of an aqueous solution and a water-soluble organic solvent can be used. Among them, water, or a mixed solvent of water and a water-soluble organic solvent is preferable.

[0075] The method for adding the virus infection inhibitor to the object is not particularly limited, and examples include a method of adding a predetermined amount of the virus infection inhibitor to the object (which may be stirred if necessary), and a method of previously mixing the virus infection inhibitor and the liquid medium and adding the obtained mixed solution to the object. In addition, the addition amount of the virus infection inhibitor is not particularly limited, but generally, in the object, 0.1 nM to 10 mM is preferable.

Example

[0076] The present invention will be described in more detail based on the following examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be appropriately changed as long as they do not deviate from the gist of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the examples shown below.

[0077] (Preparation of Specific Compounds) All specific compounds were purchased from Biochempeg. The specific compounds are compounds represented by the following formula, and the specific compounds used and their chain lengths are shown in Table 1. Hereinafter, each specific compound is collectively referred to as PEG-CLS, and depending on the weight average molecular weight of the polymer chain (PEG chain), it is called "PEG-2000CLS", etc.

Chemical formula

[0078]

Table 1

[0079] Note that the "chain length" in Table 1 is obtained by measuring the efficiency of fluorescence resonance energy transfer between a pair of fluorescent dyes having fluorescence characteristics that can cause fluorescence resonance energy transfer, each bonded to both ends of the polymer in an aqueous solution, while changing the length of the polymer, and calculating the distance between the polymer ends from the theoretical calculation formula of the fluorescence resonance energy transfer efficiency. In PEG-CLS, (chain length) = 0.035 × (weight average molecular weight of the PEG chain) 0.5797 The "chain length" calculated by this formula is obtained by rounding the second decimal place to the first decimal place.

[0080] (Preparation of Viruses) · Lentivirus pCAG-HIVgp, pCMV-VSV-G-RSV-Rev (where VSV-G was recombined with HCV E1E2), and pLV-eGFP (or pLenti CMV Puro LUC) were obtained from the RIKEN BioResource Center (RIKEN BRC) and Addgene, respectively. These plasmid vectors were transfected into HEK293T cells cultured in DMEM (Dulbecco’s Modified Eagle Medium) using polyethyleneimine, and lentiviruses were recovered from the culture medium solution 48 hours later. The recovered solution of lentivirus (or its recombinant) was filtered through a 0.22-μm filter to remove contaminants, and then concentrated by co-precipitation with PEG8000 or ultracentrifugation as needed, and stored at -80 °C in a deep freezer.

[0081] · Adeno-associated virus AAV-GFP, pAAV2 / 2, and pAdDeltaF6 were obtained from Addgene. These plasmid vectors were transfected into HEK293T cells cultured in DMEM using polyethyleneimine, and adeno-associated viruses were recovered from the culture medium solution and cell lysate 48 hours later.

[0082] · Adenovirus Ax1-CA-gfp was obtained from the RIKEN BRC. It was amplified by infecting HEK293T cells cultured in DMEM, and recovered from the culture medium solution and cell lysate 48 hours later. Adeno-associated virus and adenovirus were stored at -80 °C after purifying and concentrating each crude extract as it was or by density gradient centrifugation using cesium chloride as needed.

[0083] Among these prepared viruses, the lentivirus (with the G protein of VSV (vesicular stomatitis virus) on its outer shell) was infected into Hek293T cells and Jurkat cells. Those that express the E1-E2 molecule of HCV (hepatitis C virus) instead of VSV-G on the outer shell of the lentivirus were infected with cells into which the human Occludin gene was introduced by transfection in Hek293T cells. Adenovirus was infected with Hek293T cells. Adeno-associated virus was infected with Hek293T cells.

[0084] (Infection experiment method) · Suspended cells, or detached adherent cells Suspended cells such as Jurkat cells and adherent cells such as Hek293T cells suspended in PBS (phosphate-buffered saline) containing EDTA (ethylenediaminetetraacetic acid); adherent cells such as Hek293T cells suspended by short-term enzymatic treatment with trypsin; adherent cells such as Hek293T cells that were cultured in a microplate for 1 day, then the necessary genes were introduced by transfection of a plasmid vector and cultured for another 1 day until ~80% confluent and then detached in the presence of PBS containing EDTA; were each prepared and suspended at a concentration of 1.5×10 6 cells / mL.

[0085] In the presence of 5% CO2, PEG-CLS with a final concentration of 0.01 - 25 μM was added to the cell suspension, and it was held at 37°C for 30 minutes while gently rotating to bind PEG-CLS to the cell membrane. Incubation was carried out for 30 minutes to obtain a molecular binding density close to saturation.

[0086] After incubation, each virus at an arbitrary concentration was added to the cell suspension. Based on the previously measured titer of the virus stock, it was appropriately diluted so that the proportion of infected cells in the control sample (sample without PEG-CLS) was ~20%, ensuring a sufficient dynamic range.

[0087] The virus was infected while gently rotating at 37°C for 2 hours in the presence of 5% CO₂. After 2 hours, the virus and free PEG-CLS were removed from the culture medium by leaving the mixture as it was or by repeatedly washing the mixture with cell suspension in PBS and centrifugation, and then transferred to a cell culture plate and cultured for 2 days. After that, the virus infection of the cells was quantified by gene expression (fluorescence of GFP or activity of the expressed luciferase enzyme).

[0088] · Adherent cells Adherent cells such as Hek293T cells cultured in a 24-well microplate for 1 day; Adherent cells such as Hek293T cells that were cultured in a 24-well microplate for 1 day, then the necessary genes were introduced by transfection with a plasmid vector and cultured for another 1 day until ~80% confluent; After reducing the volume of the medium of these cell suspensions to 200 μL, PEG-CLS with a final concentration of 0.01 - 25 μM was added and incubated at 37°C for 30 minutes in the presence of 5% CO₂. Then, a virus appropriately diluted based on the titer of the pre-measured stock solution was added so that the proportion of infected cells became ~20%, and the cells were infected at 37°C for 3 hours in the presence of 5% CO₂. Thereafter, the medium containing the virus and PEG-CLS was removed, 500 μL of fresh medium was added, and the cells were cultured for 2 days. Then, the virus infection of the cells was quantified by gene expression (fluorescence of GFP or activity of the expressed luciferase enzyme).

[0089] (Measurement method) · Flow cytometry Cells infected with a virus containing the GFP gene were collected from the culture plate after the culture period, suspended in a PBS solution, and then the infection was measured by measuring the expression level of the GFP protein by flow cytometry. The average intensity of GFP fluorescence was measured as the infection amount, or the fluorescence value of non-infected cells was used as a threshold, and the proportion of cells showing a fluorescence value higher than that was measured as the infection amount as GFP-expressing cells, that is, infected cells.

[0090] (Luciferase assay) Adherent cells infected with a virus containing the luciferase gene were washed with PBS in the culture plate well while remaining in the adherent state to remove the culture medium solution, and then Passive Lysis Buffer (Promega Dual-Luciferase Reporter Assay System) containing a surfactant or the like was added to each, and incubated at room temperature for 15 minutes or more with shaking.

[0091] Suspended cells infected with a virus containing the luciferase gene were recovered from the culture plate, the culture medium was removed by centrifugation, and after further washing with PBS, Passive Lysis Buffer (Promega Dual-Luciferase Reporter Assay System) containing a surfactant or the like was added to each, suspended in the solution, and then incubated at room temperature for 15 minutes or more with shaking.

[0092] Thereafter, each lysate was collected, the cells were removed by centrifugation, and then up to 20 μL of the solution was mixed with 100 μL of LAR II solution (Promega Dual-Luciferase Reporter Assay System) containing luciferin and ATP, and the luciferase expression level in the cell solution was quantified by measuring the enzyme reaction with a luminometer.

[0093] ·Microscopic observation The deformation of cells into which PEG-CLS was introduced was observed using a scanning electron microscope (SEM). For Hek293T cells at 1.5×10 6 cells / mL, samples without PEG-CLS, 25 μM PEG-2000CLS, or samples containing PEG-20000CLS were incubated at 37 °C for 1 hour, fixed by the following method, and observed by SEM.

[0094] Fixation method: The cells were centrifuged to remove the PEG-CLS solution, and then suspended in PBS containing 2.5% glutaraldehyde and left at 4°C for 1 day for pre-fixation. Then, the glutaraldehyde was removed and the cells were resuspended in PBS. The suspension was dropped and aspirated onto a holder to adsorb the cells onto the membrane. The adsorbed cells were fixed with a post-fixative solution (1% osmium tetroxide - 0.1 M phosphate buffer 1:1) for 1 hour. Then, dehydration was performed with 50%, 70%, 80%, 90%, 95%, 100%, 100%, 100% ethanol for 10 minutes each. The dehydrated sample was immersed in 3-methylbutyl acetate for about 10 minutes and then dried with a critical point dryer. The dried sample was osmium vapor deposited (5 nm) with an osmium plasma coater.

[0095] · Fluorescence microscopy observation The deformation of cells into which PEG-CLS was introduced was observed using a fluorescence microscope. In RPMI medium in the presence of 1 μg / mL calcein-AM, 1.5×10 6 cells / mL of Jurkat cells were incubated with a sample without PEG-CLS, a sample containing 25 μM PEG-2000CLS, and a sample containing PEG-20000CLS at 37°C for 1 hour, and then the cells were photographed with a fluorescence microscope. Then, the cells were washed 3 times with RPMI medium at 15 minutes, 15 minutes, and 2 hours to reduce the concentration of PEG-CLS in the solution or bound to the cells, and then the cells were photographed again with a fluorescence microscope. Fluorescence microscope images were analyzed with ImageJ (software, NIH), and the Roundness of each cell was calculated as 4×area / (π X major-axis 2 )

[0096] As a result, it was found that the Roundness increased in both cells into which PEG-2000CLS and PEG-20000CLS were introduced. It was also found that the cells were swollen and spheroidized. This result was consistent with the SEM observation. It was also found that PEG-CLS was removed by washing and the cell state was restored.

[0097] (Results) Figure 2 shows the SEM image of Hek293T cells conjugated with PEG-CLS. The swelling state of the cells found from fluorescence microscopy analysis was observed at a high magnification. From the results in Figure 2, regardless of the introduction of PEG-CLS, no structure that could interfere with virus adhesion was observed on the cell surface. That is, it was suggested that virus infection suppression was achieved by a structure (molecule) smaller than the resolution of the electron microscope SEM. Also, for PEG-2000CLS and PEG-20000CLS with different chain lengths, no structural difference was found from the SEM images. In Figure 2, "2KPEG-CLS" represents "PEG-2000CLS" and 20KPEG-CLS represents "PEG-20000CLS".

[0098] Table 2 shows the results of adding PEG-CLS to Hek293T cells to a concentration of 25 μmol / L, infecting with lentivirus for 2 hours, removing the virus and PEG-CLS, culturing the cells for 48 hours, and then measuring the ratio of infected cells (GFP-expressing cells) by FACS, and normalizing with the expressing cells in the control cells without added PEG-CLS as a control. Values of 1.00 or less indicate that virus infection was suppressed.

[0099]

Table 2

[0100] Table 3 shows the results of adding PEG-CLS to Hek293T cells to a concentration of 25 μmol / L, infecting with adenovirus for 2 hours, removing the virus and PEG-CLS, culturing the cells for 48 hours, and then measuring the ratio of infected cells (GFP-expressing cells) by FACS, and normalizing with the expressing cells in the control cells without added PEG-CLS as a control. Values of 1.00 or less indicate that virus infection was suppressed.

[0101]

Table 3

[0102] From the results of Table 2 and Table 3, it was found that when the chain length of the PEG chain of PEG-CLS is 4.5 nm or more, it has an excellent virus infection inhibitory effect. On the other hand, when the chain length of PEG-CLS is less than 4.5 nm, no virus infection inhibitory effect was obtained.

[0103] Table 4 shows the results of infecting Jurkat cells with lentivirus in the presence of various concentrations of PEG-20000CLS for 48 hours and measuring the proportion of infected cells (GFP-expressing cells) by FACS, and normalizing the expressing cells in the cells without added PEG-CLS used as a control. From the results of Table 4, it was found that PEG-CLS has an infection inhibitory effect on lentivirus.

[0104]

Table 4

[0105] Table 5 shows the results of infecting Hek293T cells with adeno-associated virus in the presence of various concentrations of PEG-20000CLS for 2 hours, then removing the virus and PEG-CLS, culturing the cells for 48 hours, and measuring the proportion of infected cells (GFP-expressing cells) by FACS, and normalizing the expressing cells in the cells without added PEG-CLS used as a control. From the results of Table 5, it was found that PEG-CLS has an infection inhibitory effect on adeno-associated virus.

[0106]

Table 5

[0107] (Lentivirus cell uptake measurement experimental method) Suspended cells such as Jurkat cells, and cells obtained by suspending adherent cells such as Hek293T cells in PBS (phosphate-buffered saline) containing EDTA (ethylenediaminetetraacetic acid); adherent cells such as Hek293T cells that were suspended by short-term enzymatic treatment with trypsin; adherent cells such as Hek293T cells that were cultured in a microplate for 1 day, then genes necessary were introduced by transfection of a plasmid vector and cultured for another 1 day until they reached ~80% confluence and then detached in the presence of PBS containing EDTA; were each prepared and suspended at a concentration of 1.5×10 6 cells / mL.

[0108] In the presence of 5% CO2, PEG-CLS with a final concentration of 0.01 - 25 μM was added to the cell suspension, and it was held at 37°C for 30 minutes while gently rotating to bind PEG-CLS to the cell membrane. Incubation was carried out for 30 minutes to obtain a molecular binding density close to saturation.

[0109] After incubation, a lentivirus fluorescently labeled by pre-mixing DID (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine Perchlorate) into the cell solution was added.

[0110] In the presence of 5% CO2, the virus was allowed to infect while gently rotating at 37°C for 2 hours. After 2 hours, the mixture was either left as it was, or the virus and free PEG-CLS were removed from the culture medium by repeatedly washing the mixture by cell suspension with PBS and centrifugation, and then the amount of virus bound to and incorporated into the infected cells was evaluated by measuring the fluorescence intensity of DID by flow cytometry.

[0111] Table 6 shows the results of adding PEG-20000CLS to Hek293T cells at a concentration of 0.1 - 25 μmol / L, infecting them with lentivirus fluorescently labeled with DID for 2 hours, removing the virus and PEG-CLS immediately afterwards, measuring the percentage of cells that had taken up the virus (cells with the amount of DID fluorescence shown) by FACS, and normalizing to the expressing cells in the control cells without added PEG-CLS. Values of 1.00 or less indicate that virus infection was suppressed.

[0112] [Table 6]

Industrial Applicability

[0113] This virus infection inhibitor can be added to tissues etc. for the purpose of suppressing virus attachment to cells. By using this virus infection inhibitor, virus attachment to the surface of each cell can be suppressed.

Explanation of Symbols

[0114] 1 Cell 2 Hydrophobic region 3 Hydrophilic region 4 Virus< / l>

Claims

1. A virus infection inhibitor containing a compound represented by the following formula 1, 【Chemical 1】 In formula 1, n and m each independently represent an integer of 1 or more, X is a monovalent group obtained by removing the hydrogen atom of the hydroxy group bonded to the carbon atom at the 3-position of the steroid nucleus in the compound represented by formula 2, and a plurality of Xs may be the same or different, Y is a polymer chain composed of a hydrophilic polymer having an effective chain length measured from the terminal fluorescence resonance transfer efficiency in an aqueous solution of 4.5 nm or more, the hydrophilic polymer is a polyalkylene glycol, and a plurality of Ys may be the same or different, When n + m is 2, L is a single bond or a divalent group, A virus infection inhibitor, wherein when n + m is 3 or more, L is an (n + m)-valent group. 【Chemical 2】 In formula 2, rings A to D represent a saturated or unsaturated steroid nucleus, R is a hydrogen atom or a linear, branched or cyclic alkyl group having 3 to 8 carbon atoms, The hydrogen atom bonded to the carbon atom of the steroid nucleus may be substituted with a monovalent substituent selected from the group consisting of a hydrocarbon group, a hydroxy group, and a halogen atom.

2. The virus infection inhibitor according to claim 1, wherein in formula 1, n and m each represent 1, and L is a single bond or a divalent group.

3. The virus infection inhibitor according to claim 2, wherein in formula 1, L is selected from the group consisting of -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, -NR₂- (R₂ represents a hydrogen atom or a monovalent organic group), -O-P(O)(OH)-, -O-P(O)(O⁻M⁺)- (M⁺ represents Na⁺ or NH₄⁺), an alkylene group having 1 to 10 carbon atoms, a cycloalkylene group having 3 to 10 carbon atoms, an alkenylene group having 2 to 10 carbon atoms, and combinations thereof.

4. The virus infection inhibitor according to any one of claims 1 to 3, wherein X in formula 1 is a monovalent group represented by the following formula 4. [Chemical Formula 3] In formula 4, R is a hydrogen atom or a linear, branched or cyclic alkyl group having 3 to 8 carbon atoms, The hydrogen atom bonded to the carbon atom of the steroid nucleus may be substituted with a monovalent substituent selected from the group consisting of a hydrocarbon group, a hydroxy group, and a halogen atom.

5. The virus infection inhibitor according to any one of claims 1 to 4, wherein the compound represented by Formula 1 is one selected from the group consisting of compounds represented by the following formulas (i) to (iii). [Chemical Formula 4] 【Chemical Formula 5】 【Chemical Formula 6】 In formulas (i) to (iii), R represents a hydrogen atom or a monovalent group, and p and n each represent an integer of 2 or more.

6. The virus infection inhibitor according to any one of claims 1 to 5, wherein the weight average molecular weight of the hydrophilic polymer is 4400 or more.

7. The virus infection inhibitor according to any one of claims 1 to 6, wherein the chain length is 4.9 nm or more.

8. A method for inhibiting virus infection to cells, which comprises adding the virus infection inhibitor according to any one of claims 1 to 7 to an object (excluding humans) containing at least one selected from the group consisting of a tissue composed of cells and an intercellular matrix, and cells, and a liquid medium.

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