Agents for inhibiting protein aggregation or modifying protein folding and their use

Abstract

To provide an agent for inhibiting protein aggregation or adjusting protein folding, to provide a kit for inhibiting protein aggregation or adjusting protein folding, comprising the agent, to provide a method for protein folding using the agent, to provide a method for inhibiting protein aggregation, and to provide a method for storing proteins.SOLUTION: Provided is an agent for inhibiting protein aggregation or adjusting protein folding, comprising a compound (A) in which at least one hydroxyl group of cyclodextrin is substituted with a group represented by the following formula (I). X represents a sulfur atom or a selenium atom, Y1 represents a divalent linking group, and * represents a bonding hand.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to agents for inhibiting protein aggregation or regulating protein folding, and to the use of the same. In particular, it relates to agents and kits for inhibiting protein aggregation or regulating protein folding. It also relates to methods for protein folding, methods for inhibiting protein aggregation, and methods for storing proteins. [Background technology]

[0002] Protein folding intermediates, which are deformed from their native structures, are the cause of neurodegenerative diseases, including amyloid fibril formation. Elucidating the chemical and biological properties of folding intermediates will lead to a better understanding of disease processes and the development of treatments and drugs. However, the structure of folding intermediates is unstable and difficult to control. Therefore, controlling the formation of folding intermediates is necessary for research into these intermediates.

[0003] The folding of proteins that have disulfide bonds in their natural structure involves the formation of disulfide bonds. One method for folding proteins with disulfide bonds involves introducing reducing and oxidizing agents into the protein folding process, repeatedly causing the formation and cleavage of disulfide bonds, and leading to the most stable natural structure. Typical reducing agents used in this process include glutathione (GSH), β-mercaptoethanol (β-ME), and dithiothreitol (DTT), while oxidized glutathione (GSSG) is used as an oxidizing agent.

[0004] Furthermore, Patent Document 1 proposes 1-(2-mercaptoethyl)guanidine as a reducing agent that can efficiently fold proteins. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2019-210258 [Overview of the project] [Problems that the invention aims to solve]

[0006] To elucidate the chemical and biological properties of folding intermediates, it is necessary to control the formation of these folding intermediates. Furthermore, for protein research, techniques for suppressing protein aggregation are required.

[0007] Therefore, the object of the present invention is to provide an agent for inhibiting protein aggregation or adjusting protein folding, a kit for inhibiting protein aggregation or adjusting protein folding containing the agent, a method for folding a protein using the agent, a method for inhibiting protein aggregation, and a method for storing a protein. [Means for solving the problem]

[0008] The present invention includes the following embodiments. [1] An agent for inhibiting protein aggregation or modifying protein folding, comprising compound (A) in which at least one hydroxyl group of cyclodextrin is substituted with a group represented by the following formula (I).

[0009] [ka] [In the formula, X represents a sulfur atom or a selenium atom, and Y 1 * represents a divalent linking group. * represents a bond.

[0010] [2] The agent for inhibiting protein aggregation or modifying protein folding according to [1], wherein the hydroxyl group is at least one hydroxyl group selected from the group consisting of a hydroxyl group at position 2 and a hydroxyl group at position 6. [3] The agent for inhibiting protein aggregation or modifying protein folding according to [1] or [2], wherein the cyclodextrin is β-cyclodextrin. [4] The agent for suppressing protein aggregation or adjusting protein folding according to any one of [1] to [3], wherein the group represented by the formula (I) is a group represented by the following formula (I-1).

[0011] [Chemical formula] [In the formula, X represents a sulfur atom or a selenium atom, and Y 11 represents a divalent linking group containing an amide bond or an alkylene group. * represents a bond. ]

[0012] [5] The agent for suppressing protein aggregation or adjusting protein folding according to [1], wherein the compound (A) is a compound represented by the following formula (I-1-2SH) or (I-1-6SH).

[0013] [Chemical formula]

[0014] [6] A kit for suppressing protein aggregation or adjusting protein folding, comprising the agent for suppressing protein aggregation or adjusting protein folding according to any one of [1] to [5] and a compound containing a hydrophobic group. [7] The kit for suppressing protein aggregation or adjusting protein folding according to [6], wherein the compound containing a hydrophobic group is a compound having a molecular weight of 50 to 1000. [8] The kit for suppressing protein aggregation or adjusting protein folding according to [6] or [7], wherein the hydrophobic group is a hydrocarbon group. [9] The kit for suppressing protein aggregation or adjusting protein folding according to [6], wherein the compound containing a hydrophobic group is adamantane.

[10] The kit for suppressing protein aggregation or adjusting protein folding according to any one of [6] to [9], further comprising an oxidizing agent. A method for protein folding, comprising the step of incubating a protein in the presence of an agent for protein aggregation inhibition or protein folding adjustment according to any one of [1] to [5].

[12] The method for protein folding according to

[11] , wherein the incubation is carried out in the co - presence of a compound containing a hydrophobic group.

[13] The method for protein folding according to

[11] or

[12] , wherein the incubation is carried out in the co - presence of an oxidizing agent.

[14] The method for protein folding according to any one of

[11] to

[13] , wherein the protein comprises at least one selected from the group consisting of an unfolded protein and a misfolded protein.

[15] A method for protein aggregation inhibition, comprising co - existing an agent for protein aggregation inhibition or protein folding adjustment according to any one of [1] to [5] in a solution containing a protein.

[16] A method for protein storage, comprising co - existing an agent for protein aggregation inhibition or protein folding adjustment according to any one of [1] to [5] in a solution containing a protein.

[17] A compound represented by the following formula (I - 1 - 2SH).

[0015] [Chemical formula] [Advantages of the Invention]

[0016] According to the present invention, there are provided an agent for protein aggregation inhibition or protein folding adjustment, a kit for protein aggregation inhibition or protein folding adjustment containing the agent, a method for protein folding using the agent, a method for protein aggregation inhibition, and a method for protein storage. [Brief Description of the Drawings]

[0017] [Figure 1]The 1H NMR spectral data of β-CD2SH (β-cyclodextrin-2SH) obtained in a synthesis example of β-CD2SH is shown. [Figure 2] The 1H NMR spectral data of β-CD6SH (β-CD6SH) obtained in a synthesis example of β-cyclodextrin-6SH is shown. [Figure 3] This shows the folding rate of RNase A. Adamantane(-): In the presence of β-CD2SH and oxidized glutathione (GSSG); Adamantane(+): In the presence of adamantane, β-CD2SH and oxidized glutathione (GSSG). [Figure 4] This diagram illustrates the formation of disulfide bonds during the RNase A folding reaction. GSH: in the presence of glutathione and GSSG; β-CD6SH: in the presence of β-CD6SH and GSSG; β-CD2SH: in the presence of β-CD2SH and GSSG. R: reduced RNase A; 1SS: RNase A folding intermediate (one disulfide bond); 2SS: RNase A folding intermediate (two disulfide bonds); 3SS: RNase A folding intermediate (three disulfide bonds); 4SS / N: mixture of the natural structure and the fully oxidized unnatural structure of RNase A. [Figure 5] This study demonstrates the inhibitory effect of β-CD2SH on lysozyme aggregation. Lysozyme was heated at 96°C and then cooled. 1.5(h): 1.5 hours after cooling; 6(h): 6 hours after cooling. β-CD2SH(+): In the presence of β-CD2SH; β-CD2SH(-): In the absence of β-CD2SH. [Modes for carrying out the invention]

[0018] <Agents for inhibiting protein aggregation or modifying protein folding> A first aspect of the present disclosure is an agent for inhibiting protein aggregation or modifying protein folding. The agent of this aspect comprises compound (A) in which at least one hydroxyl group of a cyclodextrin is substituted with a group represented by the following formula (I).

[0019] [ka] [In the formula, X represents a sulfur atom or a selenium atom, and Y 1 * represents a divalent linking group. * represents a bond.

[0020] (Compound (A)) Compound (A) is a compound in which at least one hydroxyl group of a cyclodextrin is substituted with a group represented by formula (I) (hereinafter also referred to as "group (I)"). Cyclodextrin is a cyclic oligosaccharide in which multiple D-glucose molecules are linked by α-1,4 glycosidic bonds. Examples of cyclodextrins include α-cyclodextrin (6 glucose molecules; α-CD), β-cyclodextrin (7 glucose molecules; β-CD), and γ-cyclodextrin (8 glucose molecules; γ-CD). Among these, β-CD is preferred as the cyclodextrin.

[0021] Compound (A) has a cyclodextrin skeleton, and at least one of the hydroxyl groups on the cyclodextrin skeleton is substituted with group (I). That is, compound (A) has a structure in which at least one of the hydroxyl groups on the D-glucose constituting the cyclodextrin is substituted with group (I). The number of hydroxyl groups to be substituted is not particularly limited. When the cyclodextrin is α-CD, the number of hydroxyl groups substituted with the group represented by formula (I) can be, for example, 1 to 18, 1 to 10, 1 to 6, 1 to 4, 1 to 3, 2, or 1. When the cyclodextrin is β-CD, the number of hydroxyl groups substituted with group (I) can be, for example, 1 to 21, 1 to 18, 1 to 10, 1 to 6, 1 to 4, 1 to 3, 2, or 1. When the cyclodextrin is γ-CD, the number of hydroxyl groups substituted with group (I) can be, for example, 1 to 24, 1 to 21, 1 to 18, 1 to 10, 1 to 6, 1 to 4, 1 to 3, 2, or 1. Preferably, the number of hydroxyl groups substituted with the group represented by formula (I) is 1.

[0022] The position of the hydroxyl group substituted by group (I) is not particularly limited. The hydroxyl group substituted by group (I) may be the hydroxyl group at position 1, the hydroxyl group at position 2, or the hydroxyl group at position 6. The hydroxyl group at position 1 and the hydroxyl group at position 2 may be substituted by group (I), the hydroxyl group at position 1 and the hydroxyl group at position 6 may be substituted by group (I), the hydroxyl group at position 2 and the hydroxyl group at position 6 may be substituted by group (I), or the hydroxyl group at position 1, the hydroxyl group at position 2, and the hydroxyl group at position 6 may be substituted by group (I). One hydroxyl group in a single D-glucose molecule may be substituted with group (I), or two or more hydroxyl groups in a single D-glucose molecule may be substituted with group (I). Only one hydroxyl group in a single D-glucose molecule may be substituted with group (I), or two or more hydroxyl groups in a single D-glucose molecule may be substituted with group (I).

[0023] The hydroxyl group substituted with group (I) is preferably at least one hydroxyl group selected from the group consisting of a hydroxyl group at position 2 and a hydroxyl group at position 6. In one embodiment, compound (A) is a compound in which one hydroxyl group at position 2 of cyclodextrin is substituted with group (I). In one embodiment, compound (A) is a compound in which one hydroxyl group at position 6 of cyclodextrin is substituted with group (I).

[0024] ≪Base represented by formula (I); Base (I)≫ In formula (I) above, X represents a sulfur atom or a selenium atom. A sulfur atom is preferred for X.

[0025] In equation (I) above, Y 1 This represents a divalent linking group. 1 Examples of divalent linking groups in this context include divalent hydrocarbon groups which may have substituents, and divalent linking groups which contain heteroatoms.

[0026] Y 1 The divalent hydrocarbon group in which substituents may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group, but an aliphatic hydrocarbon group is preferred. The aliphatic hydrocarbon group may be a saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group, but a saturated aliphatic hydrocarbon group is preferred. The aliphatic hydrocarbon group may be a linear aliphatic hydrocarbon group, a branched aliphatic hydrocarbon group, or a cyclic aliphatic hydrocarbon group. The linear aliphatic hydrocarbon group preferably has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, even more preferably 1 to 4 carbon atoms, and particularly preferably 2 to 3 carbon atoms. The branched aliphatic hydrocarbon group preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, even more preferably 2 to 4 carbon atoms, and particularly preferably 3 to 4 carbon atoms. The cyclic aliphatic hydrocarbon group preferably has 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms, even more preferably 3 to 6 carbon atoms, and particularly preferably 3 to 4 carbon atoms. The cyclic aliphatic hydrocarbon group may be a monocyclic or polycyclic group, but a monocyclic group is preferred.

[0027] The aliphatic hydrocarbon group may have substituents. The substituents are not particularly limited, but examples include amino groups, halogen atoms (fluorine, chlorine, bromine, or iodine atoms), hydroxyl groups, nitro groups, cyano groups, carboxyl groups, alkoxy groups, and the like.

[0028] An aromatic hydrocarbon group is a hydrocarbon group having at least one aromatic ring. The aromatic ring is not particularly limited as long as it is a cyclic conjugated system with 4n+2 π electrons, and may be monocyclic or polycyclic. The aromatic ring preferably has 5 to 30 carbon atoms, more preferably 5 to 20 carbon atoms, even more preferably 6 to 15 carbon atoms, and particularly preferably 6 to 12 carbon atoms. However, the number of carbon atoms does not include the number of carbon atoms in substituents. Specific examples of the aromatic ring include aromatic hydrocarbon rings such as benzene, naphthalene, anthracene, and phenanthrene; and aromatic heterocyclic rings in which some of the carbon atoms constituting the aromatic hydrocarbon ring are substituted with heteroatoms. Examples of the heteroatom in the aromatic heterocyclic ring include an oxygen atom, a sulfur atom, and a nitrogen atom. Specific examples of the aromatic heterocyclic ring include a pyridine ring and a thiophene ring.

[0029] The aromatic hydrocarbon group may have a substituent. The substituent is not particularly limited, and examples thereof include an alkyl group, an amino group, a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom), a hydroxyl group, a nitro group, a cyano group, a carboxy group, and an alkoxy group.

[0030] Y 1 The divalent linking group containing a heteroatom in Y is a divalent linking group containing a heteroatom in addition to carbon atoms and hydrogen atoms. Examples of the heteroatom include a nitrogen atom, an oxygen atom, and a sulfur atom. Examples of the divalent linking group containing a heteroatom include those in which a part of the methylene group (-CH2-) constituting an alkylene group is -O-, -C(=O)-O-, -O-C(=O)-, -C(=O)-, -O-C(=O)-O-, -C(=O)-NH-, -NH-, -NH-C(=NH)-, -C(=O)-NR 1 -, -NR 1 -, -NR 1 -C(=NR 2 )-(R 1 and R 2Examples of alkylene groups include groups substituted with at least one group selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, or an acyl group having 1 to 3 carbon atoms, -S-, -S(=O)2-, and -S(=O)2-O-. The alkylene group may be linear or branched, but linear is preferred. Examples of linear alkylene groups include those having 1 to 10 carbon atoms, preferably 1 to 6, more preferably 1 to 4, even more preferably 1 to 3, and particularly preferred 2 to 3. Examples of branched alkylene groups include those having 2 to 10 carbon atoms, preferably 2 to 6, more preferably 2 to 4, and even more preferably 2 to 3. The divalent group containing the heteroatom is preferably a linear alkylene group in which part of the methylene group is substituted with -NH-.

[0031] The group (I) is preferably the group represented by the following formula (I-1).

[0032] [ka] [In the formula, X represents a sulfur atom or a selenium atom, and Y 11 * represents a divalent linking group containing an amide bond, or an alkylene group with 1 to 6 carbon atoms. * represents a bond.

[0033] In equation (I-1) above, X is the same as X in equation (I) above.

[0034] In the above equation (I-1), Y 11 This represents a divalent linking group containing an amide bond (-C(=O)-NH-, or -NH-C(=O)-), or an alkylene group having 1 to 6 carbon atoms.

[0035] Examples of divalent linking groups containing amide bonds include groups in which a portion of the methylene group (-CH2-) constituting the alkylene group is replaced by an amide bond, and divalent linking groups containing peptides. The alkylene group may be linear or branched, but linear is preferred. Examples of linear alkylene groups include those having 1 to 10 carbon atoms, preferably 1 to 6, more preferably 1 to 4, even more preferably 1 to 3, and particularly preferred 2 to 3. Examples of branched alkylene groups include those having 2 to 10 carbon atoms, preferably 2 to 6, more preferably 2 to 4, and even more preferably 2 to 3. Examples of the divalent linking group containing the peptide include a linking group containing a peptide having 2 to 10 amino acid residues. The number of amino acid residues in the peptide is preferably 2 to 8, preferably 2 to 6, more preferably 2 to 4, and particularly preferably 2 to 3.

[0036] Y 11 The alkylene group in this compound may be linear or branched, but linear is preferred. Linear alkylene groups preferably have 1 to 4 carbon atoms, more preferably 1 to 3 carbon atoms, and even more preferably 2 to 3 carbon atoms. Branched alkylene groups have 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and more preferably 3 to 4 carbon atoms.

[0037] Specific examples of compound (A) include, but are not limited to, the compounds represented by the following formulas (I-1-2SH) or (I-1-6SH). The compound represented by the following formula (I-1-2SH) is β-CD2SH. The compound represented by the following formula (I-1-6SH) is β-CD6SH.

[0038] [ka]

[0039] Compound (A) may be in the form of a salt. The salt is not particularly limited and includes, for example, salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as magnesium and calcium; ammonium salts; or salts with inorganic acids such as hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, and sulfite; and salts with organic acids such as formic acid, acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, malic acid, mandelic acid, methanesulfonic acid, p-toluenesulfonic acid, and trifluoroacetic acid. Compound (A) may also be in the form of a solvate. The solvate is not particularly limited and includes, for example, hydrates; solvates with solvents such as alcohols (e.g., methanol, ethanol, propanol, isopropanol), acetone, tetrahydrofuran, dioxane, DMF, DMSO, etc.

[0040] ≪Method for synthesizing compound (A)≫ Compound (A) can be synthesized by combining known methods. For example, compound (A) can be synthesized as follows. First, a p-toluenesulfonyl group (tosyl group) is introduced to the hydroxyl group to be substituted with group (I) in cyclodextrin. Next, a compound having a disulfide bond or a diselenium bond is reacted with the tosyl group to introduce a group having a disulfide bond or a diselenium bond to the hydroxyl group. Then, compound (A) can be obtained by reducing the disulfide bond or diselenium bond with a reducing agent. An example of the synthesis of compound (A) is shown below, but is not limited to this example. The following synthesis example shows the case in which one of the hydroxyl groups at the 2 position of β-cyclodextrin is substituted with group (I).

[0041] [ka] [In the formula, X represents a sulfur atom or a selenium atom, and Y 1 represents a divalent linking group, R 1 This represents a monovalent organic group.

[0042] In the above synthesis example, compound (3) is obtained by reacting compound (2) with mono-2-O-(p-toluenesulfonyl)-β-cyclodextrin (1). Then, compound (A) is obtained by reducing the XX bond of compound (3) with a reducing agent.

[0043] (optional ingredient) The agent of this embodiment may contain optional components in addition to compound (A). The optional components are not particularly limited as long as they do not impair the function of compound (A). Examples of optional components include compounds containing hydrophobic groups (hereinafter also referred to as "hydrophobic group-containing compounds"), oxidizing agents, and the like. When the agent of this embodiment contains optional components in addition to compound (A), the agent of this embodiment may be a composition for inhibiting protein aggregation or regulating protein folding.

[0044] ≪Hydrophobic group-containing compound≫ The agent according to this embodiment may contain a hydrophobic group-containing compound in order to adjust the protein folding action of compound (A). The hydrophobic group-containing compound is not particularly limited as long as it is a compound that can function as a guest molecule of cyclodextrin. Cyclodextrin has a hydrophobic interior in its cyclic structure, and can encapsulate hydrophobic compounds within its cyclic structure through host-guest interactions. When a hydrophobic group-containing compound is encapsulated in compound (A) through host-guest interactions, the protein folding rate of compound (A) is suppressed. Examples of hydrophobic group-containing compounds include compounds with a molecular weight of 50 to 1000. The molecular weight of the hydrophobic group-containing compound is preferably 50 to 800, more preferably 50 to 500, even more preferably 50 to 300, and particularly preferably 50 to 200.

[0045] The hydrophobic group contained in the hydrophobic group-containing compound is not particularly limited, but a hydrocarbon group is preferred. Examples of hydrocarbon groups include hydrocarbon groups having 3 to 30 carbon atoms. Hydrocarbon groups with 6 to 20 carbon atoms are preferred, with 6 to 18 carbon atoms being more preferred, with 8 to 16 carbon atoms being even more preferred, and with 8 to 14 carbon atoms being particularly preferred. The hydrophobic group may be a monovalent group, a divalent group, or a trivalent or higher group.

[0046] The hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group. The aliphatic hydrocarbon group may be a saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group. The aliphatic hydrocarbon group may be a linear aliphatic hydrocarbon group, a branched unsaturated hydrocarbon group or a cyclic aliphatic hydrocarbon group. Examples of linear or branched aliphatic hydrocarbon groups include linear or branched alkyl or alkylene groups, linear or branched alkenyl or alkenylene groups, and linear or branched alkynyl or alkynylene groups. The cyclic aliphatic hydrocarbon group may be a monocyclic or polycyclic group. Examples of monocyclic groups include groups containing a monocycloalkane ring, a monocycloalkene ring, or a monocycloalkyne ring. Examples of monocycloalkanes include cyclopentane, cyclohexane, cycloheptane, cyclocyclooctane, cyclononane, and cyclodecane. Examples of monocycloalkenes include cyclopentene, cyclohexene, cycloheptene, cyclocyclooctene, and cyclononene, but are not limited to these. Examples of monocycloalkynes include cyclopentine, cyclohexine, cycloheptine, cyclocyclooctin, and cyclononine. Examples of polycyclic groups include groups containing a ring skeleton, such as adamantane, norbornane, norbornene, bicyclo[2.2.2]octatane, tricyclo[5.2.1.02,6]decane, tricyclo[3.3.1.13,7]decane, and tetracyclo[6.2.1.13,6.02,7]dodecane.

[0047] Aromatic hydrocarbon groups may be monocyclic or polycyclic. Aromatic hydrocarbon groups are preferably those containing an aromatic hydrocarbon ring. The number of aromatic rings in an aromatic hydrocarbon group can range from 1 to 6, preferably 1 to 5, more preferably 1 to 4, even more preferably 1 to 3, and particularly preferably 1 to 2. Examples of aromatic hydrocarbon groups include groups containing a ring skeleton, such as benzene, fluorene, naphthalene, anthracene, phenanthrene, and tetrahydronaphthalene.

[0048] When compound (A) has an α-cyclodextrin skeleton, examples of hydrophobic group-containing groups include n-alkyl groups having 3 to 18 carbon atoms. Specific examples include n-butyl groups, n-hexyl groups, n-octyl groups, and n-dodecyl groups. When compound (A) has a β-cyclodextrin skeleton, hydrophobic groups include polycyclic aliphatic hydrocarbon groups having 6 to 18 carbon atoms and molecular chain alkyl groups having 3 to 10 carbon atoms. Specific examples include adamantyl group, ethyladamantyl group, norbornyl group, isobornyl group, and tert-butyl group. When compound (A) has a γ-cyclodextrin skeleton, examples of hydrophobic group-containing groups include n-alkyl groups and cycloalkyl groups. Specific examples include n-octyl groups, n-dodecyl groups, and cyclododecyl groups.

[0049] The non-hydrophobic portion of a hydrophobic group-containing compound is not particularly limited, as long as the hydrophobic group-containing compound functions as a guest molecule of compound (A). The non-hydrophobic portion may be hydrophobic or hydrophilic. Examples of hydrophobic group-containing compounds include hydrocarbons. When compound (A) has an α-cyclodextrin skeleton, hydrophobic group-containing compounds include n-alkanes having 3 to 18 carbon atoms. Specific examples include n-butane, n-hexane, n-octane, and n-dodecane. When compound (A) has a β-cyclodextrin skeleton, hydrophobic group-containing compounds include polycyclic aliphatic hydrocarbons having 6 to 18 carbon atoms and molecular chain alkanes having 3 to 10 carbon atoms. Specific examples include adamantane, ethyladamantane, norbornane, isobornane, and isobutane. When compound (A) has a γ-cyclodextrin skeleton, examples of hydrophobic group-containing compounds include n-alkanes and cycloalkanes. Specific examples include n-octane, n-dodecane, and cyclododecane.

[0050] Oxidizing agents The agent of this embodiment may contain an oxidizing agent to oxidize thiol groups in proteins and form disulfide bonds. The reduction action of compound (A) and the oxidation action of the oxidizing agent repeatedly cause thiol-disulfide bond exchange reactions in the protein, inducing a change in protein structure and leading to the most stable natural structure.

[0051] The oxidizing agent can be any agent used for introducing disulfide bonds into proteins, without any particular limitations. Examples of oxidizing agents include oxidized glutathione, oxidized dithiothreitol, oxidized selenoglutathione, glutaredoxins, cystine, oxidized cystamine, oxidized selenocystamine, hydrogen peroxide, diamides, potassium ferricyanide, and the like. Among these, oxidized glutathione is preferred from the standpoint of its use in actual biological reactions.

[0052] The agent of this embodiment can be used to adjust protein folding. When protein folding is performed in the presence of the agent of this embodiment, the protein folding rate can be adjusted by adding a hydrophobic group-containing compound to the folding reaction solution. More specifically, the protein folding rate can be slowed down by increasing the amount of the hydrophobic group-containing compound relative to the amount of compound (A) present in the folding reaction solution. On the other hand, the protein folding rate can be accelerated by decreasing the amount of the hydrophobic group-containing compound relative to the amount of compound (A) present in the folding reaction solution. The molar ratio of compound (A) to the hydrophobic group-containing compound (compound (A):hydrophobic group-containing compound) can be varied, for example, between 50:1 and 1:50.

[0053] The agent of this embodiment can be used to suppress protein aggregation. Protein aggregation can be suppressed by coexisting the agent of this embodiment with protein. When the agent of this embodiment is used as a protein aggregation inhibitor, the amount of the agent used is, for example, 1 to 1000 times the molar amount of the protein. The amount of the agent used is preferably, for example, 50 to 800 times the molar amount of the protein, more preferably 100 to 600 times, and even more preferably 300 to 600 times.

[0054] The protein to which the agent of this embodiment is applied is not particularly limited, as long as it is a protein having disulfide bonds. The number of disulfide bonds in the protein is not particularly limited, and one or more is sufficient. Examples of the number of disulfide bonds in a protein include 1 to 20, 1 to 15, 1 to 10, or 1 to 5. The molecular weight of the protein is not particularly limited, but for example, it can range from 1,000 to 10,000,000, and is preferably 1,000 to 250,000.

[0055] <Kit for inhibiting protein aggregation or modifying protein folding> A second aspect of the present disclosure is a kit for inhibiting protein aggregation or regulating protein folding. The kit of this aspect comprises the agent of the first aspect and the hydrophobic group-containing compound.

[0056] The agent of the first embodiment is as described above. In the kit of this embodiment, the agent of the first embodiment may be one that does not contain a hydrophobic group-containing compound. In the kit of this embodiment, the agent of the first embodiment may be one that does not contain an oxidizing agent.

[0057] The hydrophobic group-containing compounds are as described above. When compound (A) contains a β-cyclodextrin skeleton, adamantane is preferred as the hydrophobic group-containing compound.

[0058] (Optional configuration) The kit of this embodiment may include, in addition to the agent and hydrophobic group-containing compound of the first embodiment, any other components. Examples of optional components include various reagents such as oxidizing agents and buffers, and instructions for use.

[0059] Examples of oxidizing agents include those listed in the section "<Agents for inhibiting protein aggregation or regulating protein folding>" above. Oxidized glutathione is preferred as the oxidizing agent.

[0060] The buffer can be used to dissolve proteins, compound (A), hydrophobic group-containing compounds, and oxidizing agents, etc., to carry out a protein folding reaction. The buffer may be a concentrated solution (e.g., 1 to 10 times concentrated solution) or may be diluted before use. Buffers commonly used in the field of biochemistry can be used without particular limitations. Examples of buffers include amine-based buffers such as Tris buffer, MES buffer, and trichine buffer; phosphate buffer; and Good's buffers. The pH of the buffer is typically between 4 and 10. A pH of 5 to 9 is preferred, 7 to 9 is more preferred, and 7 to 8 is even more preferred.

[0061] The kit according to this embodiment can be used to carry out a protein folding reaction. Since the kit according to this embodiment contains the agent of the first embodiment and a hydrophobic group-containing compound, the molar ratio of compound (A) to the hydrophobic group-containing compound in the folding reaction solution can be adjusted as appropriate. Therefore, the protein folding rate can be adjusted as appropriate by adjusting the molar ratio. The kit according to this embodiment may be used to suppress protein aggregation.

[0062] <Methods of protein folding> A third aspect of this disclosure is a method for folding a protein. The protein folding method of this aspect includes the step of incubating a protein in the presence of the agent of the first aspect.

[0063] (Protein incubation process; protein folding process) Protein incubation can be carried out by preparing a folding reaction solution by dissolving the protein and an agent of the first embodiment (e.g., compound (A)) in a suitable buffer solution and then incubating it. By performing this incubation, the protein folding reaction can be carried out.

[0064] As buffer solutions, those listed in the section "<Kits for inhibiting protein aggregation or adjusting protein folding>" above can be used.

[0065] The protein concentration in the folding reaction solution is not particularly limited, but for example, the final concentration in the folding reaction solution can be 0.1 to 100 μM. The protein concentration is preferably 0.5 to 50 μM, more preferably 1 to 20 μM, and even more preferably 1 to 10 μM.

[0066] The concentration of compound (A) in the folding reaction solution is not particularly limited, but for example, the final concentration in the folding reaction solution can be 0.01 to 100 mM. The concentration of compound (A) is preferably 0.05 to 50 mM, more preferably 0.1 to 20 mM, and even more preferably 0.5 to 10 mM.

[0067] The folding reaction solution may contain the above-mentioned hydrophobic group-containing compound in addition to the protein and the agent of the first embodiment. That is, the incubation of the protein may be carried out in the presence of compound (A) and the hydrophobic group-containing compound. The concentration of the hydrophobic group-containing compound in the folding reaction solution is not particularly limited, but for example, the final concentration in the folding reaction solution can be 0.001 to 100 mM. The concentration of the hydrophobic group-containing compound is preferably 0.001 to 50 mM, more preferably 0.01 to 20 mM, and even more preferably 0.05 to 10 mM. The molar ratio of compound (A) to the hydrophobic group-containing compound (compound (A):hydrophobic group-containing compound) can be varied, for example, between 50:1 and 1:50. Increasing the amount of the hydrophobic compound relative to compound (A) can slow down the folding speed. Decreasing the amount of the hydrophobic compound relative to compound (A) can speed up the folding speed.

[0068] The folding reaction solution may contain an oxidizing agent in addition to the protein and the agent of the first embodiment. That is, the incubation of the protein may be carried out in the presence of compound (A) and the oxidizing agent. The oxidizing agents listed above can be used. The concentration of the oxidizing agent in the folding reaction solution is not particularly limited, but for example, the final concentration in the folding reaction solution may be 0.001 to 100 mM. The concentration of the oxidizing agent is preferably 0.001 to 50 mM, more preferably 0.01 to 20 mM, and even more preferably 0.05 to 10 mM. The molar ratio of compound (A) to the oxidizing agent (compound (A):oxidizing agent) is preferably 20:1 to 1:1, and more preferably 10:1 to 2:1.

[0069] The folding reaction solution preferably contains a protein, the agent of the first embodiment, a hydrophobic group-containing compound, and an oxidizing agent. That is, the incubation of the protein is preferably carried out in the presence of compound (A), the hydrophobic group-containing compound, and the oxidizing agent.

[0070] The proteins used for incubation are not particularly limited, as long as they have one or more disulfide bonds. Examples of proteins are the same as those described above. Examples of proteins include unfolded proteins and misfolded proteins. An unfolded protein is a protein in which one or more disulfide bonds have been oxidized, resulting in the cleavage of one or more disulfide bonds. Protein unfolding can be carried out using a reducing agent. Compound (A) may be used as the reducing agent, or other reducing agents (e.g., glutathione, β-mercaptoethanol, dithiothreitol, etc.) may be used. A misfolded protein is a protein in which one or more disulfide bonds are formed in a structure other than the most stable native structure. Misfolded proteins can be produced, for example, by using an oxidizing agent and a reducing agent in combination.

[0071] The incubation temperature is not particularly limited, as long as it is a temperature at which the protein folding reaction can proceed. The incubation temperature can be appropriately selected depending on the type of protein. Examples of incubation temperatures include 10 to 50°C, preferably 20 to 40°C, and more preferably 25 to 35°C. The incubation time can be appropriately selected depending on the type of protein. For example, incubation times range from 1 to 600 minutes. The folding state can be observed over time, and incubation may be terminated when the most stable native structure is achieved. To observe the folding state, thiol group modifiers such as 4-Acetamido-4'-Maleimidylstilbene-2,2'-Disulfonic Acid (AMS) can be used.

[0072] (Optional process) The method of this embodiment may include optional steps in addition to the incubation step. Optional steps include, for example, a step of unfolding the protein and a step of isolating the folded protein.

[0073] ≪Protein unfolding process≫ The method according to this embodiment may include a step of unfolding the protein before the incubation step (protein folding step). The process of unfolding a protein can be carried out by incubating the protein in the presence of a reducing agent. The reducing agents listed above can be used. Protein denaturants (e.g., guanidine hydrochloride, urea, etc.) may also be used for protein unfolding. The protein unfolding reaction may also be carried out by preparing a protein unfolding reaction solution by dissolving the protein, an oxidizing agent, and a protein denaturing agent in a medium such as water, and then incubating it. As for the protein concentration, for example, the final concentration in the unfolding reaction solution can be 0.1 to 100 μM. The protein concentration is preferably 0.5 to 50 μM, more preferably 1 to 20 μM, and even more preferably 1 to 10 μM. The concentration of the reducing agent can be, for example, 0.1 to 1000 mM as the final concentration in the unfolding reaction solution. The concentration of the oxidizing agent is preferably 1 to 500 mM, more preferably 10 to 300 mM, and even more preferably 50 to 200 mM. The concentration of the denaturing agent can be, for example, 0.1 to 100 M as the final concentration in the unfolding reaction solution. The concentration of the denaturing agent is preferably 0.5 to 50 M, more preferably 1 to 30 M, and even more preferably 1 to 20 M. The pH of the unfolding reaction solution can be, for example, pH 5 to 10, preferably pH 6 to 9.5, more preferably pH 7 to 9, and even more preferably pH 8 to 9.

[0074] The incubation temperature is not particularly limited, as long as it is a temperature at which the protein unfolding reaction can proceed. The incubation temperature can be appropriately selected depending on the type of protein. Examples of incubation temperatures include 10 to 50°C, preferably 20 to 40°C, and more preferably 20 to 35°C. The incubation time can be appropriately selected depending on the type of protein. For example, incubation times can range from 1 to 600 minutes, preferably 10 to 500 minutes, more preferably 30 to 400 minutes, and even more preferably 60 to 300 minutes. The unfolding state can be observed over time, and incubation may be terminated when all disulfide bonds have been reduced.

[0075] After incubation, the unfolding reaction solution may be dialysis or otherwise performed to remove denaturing agents and reducing agents.

[0076] ≪Protein Isolation Process≫ The method according to this embodiment may include a step of isolating the folded protein after the incubation step (protein folding step). Known protein isolation methods can be used for protein isolation without particular limitations. Examples of protein isolation methods include salting out, dialysis, and column chromatography.

[0077] <Methods for inhibiting protein aggregation> A fourth aspect of this disclosure is a method for inhibiting protein aggregation. The method of this aspect includes coexisting the agent of the first aspect in a protein solution.

[0078] A protein solution can be prepared by dissolving protein in water or a buffer solution. Examples of buffer solutions include those described above. The pH of the protein solution is not particularly limited and can be appropriately selected depending on the type of protein. Examples of pH values ​​for protein solutions include pH 4 to 10. A pH of 5 to 9 is preferred, pH 7 to 9 is more preferred, and pH 7 to 8 is even more preferred. The protein concentration in the protein solution is not particularly limited. Examples of protein concentrations include 0.1 to 1000 μM. Preferably, the protein concentration is 1 to 500 μM, more preferably 5 to 300 μM, and even more preferably 10 to 100 μM.

[0079] The agent of the first embodiment (for example, compound (A)) can be made to coexist in the protein solution by adding it to the protein solution and dissolving it. The concentration of compound (A) in the protein solution can be, for example, 0.1 to 1000 mM as the final concentration in the protein solution. The concentration of compound (A) is preferably, for example, 1 to 500 mM, more preferably 5 to 300 mM, and even more preferably 5 to 100 mM. The molar concentration of compound (A) can be, for example, 1 to 1000 times the molar concentration of the protein. Preferably, the molar concentration of compound (A) is 50 to 800 times, more preferably 100 to 600 times, and even more preferably 300 to 600 times, compared to the molar concentration of the protein.

[0080] By adding the agent according to the first embodiment to a protein solution, the coexistence of the protein and compound (A) can be effectively suppressed.

[0081] <How to store protein> A fifth aspect of this disclosure is a method for storing proteins. The method of this aspect includes coexisting the agent of the first aspect in a protein solution.

[0082] The coexistence of the agent according to the first embodiment in a protein solution can be carried out in the same manner as described above. The protein solution can be stored by placing the protein solution containing the agent according to the first embodiment into a suitable container. Examples of suitable containers include plastic containers and glass containers. The storage temperature is not particularly limited, but examples include 0 to 40°C. The storage temperature is preferably 0 to 30°C, more preferably 0 to 20°C, and even more preferably 0 to 10°C.

[0083] By adding the agent according to the first embodiment to a protein solution and storing the protein and compound (A) together, the aggregation of the protein during storage can be suppressed.

[0084] <Compound> A sixth aspect of this disclosure is a compound represented by the following formula (I-1-2SH). The compound of this aspect can be used as an agent of the first aspect.

[0085] [ka] [Examples]

[0086] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0087] <Example of β-cyclodextrin-2SH synthesis> β-cyclodextrin-2SH (β-CD2SH) was synthesized using the following scheme.

[0088] [ka]

[0089] (Synthesis of compound (2)) Mono-2-O-(p-toluenesulfonyl)-β-cyclodextrin (Compound 1; 200.2 mg, 0.155 mmol, Tokyo Chemical Industry Co., Ltd.) was added to a round-bottom flask and dissolved in dimethyl sulfoxide (DMSO, 4 mL, Kishida Chemical). Next, cystamine dihydrochloride (350.2 mg, 1.55 mmol, Tokyo Chemical Industry Co., Ltd.) and sodium hydroxide (144.3 mg, 3.6075 mmol, Kishida Chemical) were dissolved in purified water (1 mL), and the resulting solution was added dropwise to the flask. After addition, the temperature was raised to 80°C and stirred for 48 hours. After cooling to room temperature, ethanol (Kishida Chemical) was added, and the precipitate was collected. The precipitate was washed with acetone and dried in a vacuum oven to obtain Compound 2. Yield: 158.5 mg, yield: 80.6%.

[0090] (Synthesis of β-CD2SH) Under a nitrogen atmosphere, compound 2 (113.8 mg, 0.0896 mmol) was dissolved in dehydrated N,N-dimethylformamide (DMF, 0.75 mL, Kanto Chemical). Next, dithiothreitol (DTT, 27.9 mg, 0.181 mmol, Nacalai Tesque) was dissolved in purified water (0.25 mL) that had been degassed beforehand by nitrogen bubbling, and the resulting solution was added to the flask. The mixture was then stirred at room temperature for 20 hours. Ethanol (Kishida Chemical) was added, and the precipitate was collected. The precipitate was washed with acetone and dried in a vacuum oven to obtain β-CD2SH. Yield: 16.5 mg, yield: 15.4%. The structure of the obtained β-CD2SH was confirmed by 1H NMR analysis (solvent: deuterium DMSO, measurement temperature: 25°C). The 1H NMR spectral data of β-CD2SH is shown in Figure 1.

[0091] <Synthesis of β-cyclodextrin-6SH> β-cyclodextrin-6SH (β-CD6SH) was synthesized using the following scheme.

[0092] [ka]

[0093] (Synthesis of Compound 5) Compound 3 (cystamine dihydrochloride, 681.5 mg, 3.03 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in 10 mL of 5 M sodium hydroxide aqueous solution (prepared using sodium hydroxide (Kishida Chemical Co., Ltd.)) and extracted with methylene chloride (10 mL, 5 times, AGC). Sodium sulfate (Kishida Chemical Co., Ltd.) was added to the recovered organic layer and filtered. The filtrate was removed by reduced pressure distillation and vacuum drying to obtain compound 4. Subsequently, mono-6-O-(p-toluenesulfonyl)-β-cyclodextrin (β-CD-6-OTs, 130.2 mg, 0.101 mmol, Tokyo Chemical Industry Co., Ltd.) was added to the round-bottom flask containing compound 4, and the temperature was raised to 70°C and stirred for 6 hours. After cooling to room temperature, acetone (Kanto Chemical Co., Ltd.) was added and the precipitate was collected. The precipitate was washed with acetone and dried in a vacuum oven to obtain compound 5. Yield: 49.9 mg, yield: 38.9%.

[0094] (Synthesis of β-CD6SH) Under a nitrogen atmosphere, compound 5 (49.9 mg, 0.0393 mmol) was dissolved in dehydrated N,N-dimethylformamide (DMF, 1.5 mL, Kanto Chemical). Next, dithiothreitol (DTT, 12.1 mg, 0.0784 mmol, Nacalai Tesque) was dissolved in purified water (0.5 mL) that had been degassed beforehand by nitrogen bubbling, and the resulting solution was added to the flask. The mixture was then stirred at room temperature for 10 hours. Acetone (Kanto Chemical) was added, and the precipitate was collected. The precipitate was washed with acetone and dried in a vacuum oven to obtain β-CD6SH. Yield: 27.7 mg, yield: 59.0%. The structure of the obtained β-CD6SH was confirmed by 1H NMR analysis (solvent: deuterium DMSO, measurement temperature: 25°C). The 1H NMR spectral data of β-CD6SH is shown in Figure 2.

[0095] <Production of reduced-denatured proteins> RNase A was employed as a model substrate (M.M. Lyles, H.F. Gilbert., Biochemistry. 1991 30(3):613-9.). RNase A is a protein with four disulfide bonds and is common as a model substrate in the art. To prepare a reduced variant of RNase A (sigma) (a reductant of four disulfide bonds), 16.0 mg of RNase A was incubated at pH 8.7 and 25 °C for 2 hours in the presence of 6 M guanidine hydrochloride and 100 mM DTT, and dialyzed against 10 mM HCl. Further, dialysis was performed twice to exchange the solution with 10 mM HCl, removing DTT etc. (M.M. Lyles, H.F. Gilbert., Biochemistry. 1991 30(3):613-9.).

[0096] <RNase A Activity Evaluation> (Folding Reaction of RNase A) To evaluate the folding promotion effect, the activity recovery of the reduced and denatured RNase A prepared above was measured. A buffer solution (50 mM Tris-HCl pH 7.5, 300 mM NaCl) containing 8 μM RNase A was incubated at 30 °C in the presence of 1 mM thiol compound (β-CD2SH) and 0.2 mM disulfide compound (oxidized glutathione; GSSG).

[0097] (RNase A Activity Measurement) The reaction solution was aliquoted over time and diluted with the same buffer containing cytidine 2’:3’-cyclic monophosphate (cCMP) as the substrate of RNase A to a final concentration of 8 μM RNase A and 0.6 mM cCMP. The change in absorbance at 284 nm was measured with a spectrophotometer to quantify the nuclease activity of RNase A folded into the native structure.

[0098] (Example 1) The folding reaction of RNase A was carried out under the following conditions. In condition (2), after preparing the folding reaction solution, the folding reaction solution was irradiated with ultrasound at 30°C for 3 minutes and 30 seconds. Then, the folding reaction solution was filtered. RNase A was added to the filtered folding reaction solution and incubated. (1) 1mM β-CD2SH / 0.2mM GSSG (2) 1 mM β-CD2SH / 0.2 mM GSSG / 0.1 mM Adamantane (Tokyo Chemical Industries) The reaction solution was sampled at different time points, and the activity of RNA A was measured as described above.

[0099] The results are shown in Figure 3. It was confirmed that the addition of adamantane delayed the folding of RNase A into its native structure. This result indicates that the rate of protein folding can be regulated by the addition of adamantane.

[0100] (Example 2) To observe the process of disulfide bond formation within the RNase A molecule, we selectively modified only the thiol groups that did not form disulfide bonds using the free thiol group modification reagent 4-Acetamido-4'-Maleimidylstilbene-2,2'-Disulfonic Acid (AMS, Invitrogen).

[0101] The folding reaction of RNase A was performed under the following conditions. (1) 1 mM glutathione (GSH) / 0.2 mM GSSG (2) 1mM β-CD6SH / 0.2mM GSSG (3) 1mM β-CD2SH / 0.2mM GSSG

[0102] The reaction solution was sampled at different time points, and the reaction was quenched by adding AMS to achieve a final concentration of 5 mM. The degree of disulfide bond introduction within RNase A was evaluated by subjecting these samples to polyacrylamide gel electrophoresis (SDS-PAGE).

[0103] The results are shown in Figure 4. Under all conditions (1) to (3), it was confirmed that the reduced form of RNase A (indicated as "R" in the figure) had almost completely disappeared after incubation for 30 minutes or more. This result indicates that the folding reaction of RNase A leads to the partial folding of RNase A, the formation of a partial folding intermediate of RNase A, and the disappearance of the reduced form of RNase A.

[0104] (Example 3) An aqueous solution containing 30 μM lysozyme and 12.75 mM β-CD2SH was heated at 96°C for 20 minutes. After cooling, the concentration of lysozyme dissolved in the water was measured at 1.5 hours and 6 hours later.

[0105] The results are shown in Figure 5. In the presence of β-CD2SH, the residual lysozyme concentration increased compared to the absence of β-CD2SH. This result indicates that β-CD2SH has an inhibitory effect on protein aggregation. [Industrial applicability]

[0106] The present invention provides an agent for inhibiting protein aggregation or regulating protein folding, a kit for inhibiting protein aggregation or regulating protein folding containing the agent, a method for folding a protein using the agent, a method for inhibiting protein aggregation, and a method for storing a protein. The agent provided by the present invention can be used as a protein folding regulator, a protein aggregation inhibitor, a protein folding promoter, and a protein folding rate modifier. The protein folding rate modifier is useful for analyzing the biochemical properties of protein folding intermediates. The protein aggregation inhibitor is useful for experiments using proteins and for storing protein reagents.

Claims

1. The compound (A) contains a cyclodextrin in which one hydroxyl group is replaced with a group represented by the following formula (I-1), The hydroxyl group is a hydroxyl group selected from the group consisting of a hydroxyl group at position 2 and a hydroxyl group at position 6. An agent for inhibiting protein aggregation or modifying protein folding. 【Chemistry 1】 [In the formula, X represents a sulfur atom, and Y11 represents an alkylene group having 1 to 6 carbon atoms, in which some of the methylene groups constituting the alkylene group may be substituted with amide bonds. * represents a bond.]

2. The agent for inhibiting protein aggregation or modifying protein folding according to claim 1, wherein the cyclodextrin is β-cyclodextrin.

3. The agent for inhibiting protein aggregation or modifying protein folding according to claim 1, wherein the compound (A) is a compound represented by the following formula (I-1-2SH) or (I-1-6SH). 【Chemistry 2】

4. A kit for inhibiting protein aggregation or modifying protein folding, comprising an agent for inhibiting protein aggregation or modifying protein folding according to any one of claims 1 to 3, and a compound containing a hydrophobic group.

5. The kit for inhibiting protein aggregation or adjusting protein folding according to claim 4, wherein the compound containing the hydrophobic group is a compound with a molecular weight of 50 to 1000.

6. The kit for inhibiting protein aggregation or modifying protein folding according to claim 4 or 5, wherein the hydrophobic group is a hydrocarbon group.

7. The kit for inhibiting protein aggregation or regulating protein folding according to claim 4, wherein the compound containing the hydrophobic group is adamantane.

8. A kit for inhibiting protein aggregation or modifying protein folding according to any one of claims 4 to 7, further comprising an oxidizing agent.

9. A method for folding a protein, comprising the step of incubating a protein in the presence of an agent for inhibiting protein aggregation or regulating protein folding according to any one of claims 1 to 3.

10. The protein folding method according to claim 9, wherein the incubation is carried out in the presence of a compound containing a hydrophobic group.

11. The protein folding method according to claim 9 or 10, wherein the incubation is carried out in the presence of an oxidizing agent.

12. The protein folding method according to any one of claims 9 to 11, wherein the protein comprises at least one selected from the group consisting of unfolded proteins and misfolded proteins.

13. A method for inhibiting protein aggregation, comprising coexisting a protein aggregation inhibitor or protein folding modifier agent according to any one of claims 1 to 3 in a protein-containing solution.

14. A method for storing proteins, comprising coexisting a protein aggregation inhibitor or protein folding modifier agent according to any one of claims 1 to 3 in a protein-containing solution.

15. A compound represented by the following formula (I-1-2SH). 【Chemistry 4】

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