Protein degradation-controlling compound, agent, method, and kit

Abstract

To provide a compound that can control degradation of a target protein, an agent for controlling degradation of a target protein comprising the compound, a method for controlling degradation of a target protein using the compound, and a kit that contains the compound.SOLUTION: There are provided a compound or a salt thereof represented by the following formula [wherein R1 is a substituted or unsubstituted 6-10-membered aryl group or a 5- or 6-membered heteroaryl group; R2 is -OR3 or -N(R4)(R5); and R3, R4, and R5 are independently H or a C1-6 alkyl group], a target protein degradation-controlling agent comprising the compound of the formula or a salt thereof, and a protein degradation-controlling method and a kit using the compound of the formula or a salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a compound capable of controlling the degradation of a target protein, a target protein degradation regulator comprising said compound, a method for controlling target protein degradation using said compound, and a kit comprising said compound. [Background technology]

[0002] A technology for controlling protein degradation called the auxin-inducible degron (AID) system has been developed (Non-Patent Document 1). In this system, TIR1, which constitutes an auxin-responsive ubiquitin ligase, is introduced into eukaryotic cells such as yeast or animal cells, and the degradation of target proteins tagged with degradation tags (plant-derived Aux / IAA family proteins or their partial proteins; also called degrons) is controlled by adjusting the presence or absence and timing of auxin addition.

[0003] The aforementioned auxin degron method allows for rapid degradation of target proteins tagged with a degradation tag (degron) upon addition of auxin. However, this method makes it difficult to precisely control expression because weak degradation of the target protein occurs even in the absence of auxin. Furthermore, relatively high concentrations of auxin (over 100 μM) are used to induce degradation, raising concerns about the toxic effects of auxin itself, especially in multicellular animals.

[0004] In response to such concerns, an improved auxin-inducible degron (AID) system (also called the AID2 system) was developed, which combines a mutant TIR1 with an auxin analogue that has affinity for the mutant TIR1 (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-187958 [Patent Document 2] Japanese Patent Application Laid-Open No. 2023-30479 [Non-patent literature]

[0006] [Non-Patent Document 1] N. Nishimura, et al., Nature Method, 6, 917 (2009) Summary of the Invention [Problem to be solved by the invention]

[0007] Although the AID2 system improved the efficiency of target protein degradation, it was difficult to degrade only target proteins in cells located at a specific location among many other cells. Furthermore, a method for examining the function of target proteins over time involves periodically exchanging cells with a buffer solution containing or not containing an auxin analog. However, this method has the drawback of being discontinuous, making it impossible to observe the state of the cells during the buffer exchange (Patent Document 2). Furthermore, while perfusion allows observation while exchanging the solution, there is a risk of cell detachment due to the flow of the solution, or of physical stimuli affecting the state of the cells (Patent Document 2). [Means for solving the problem]

[0008] In order to solve the above problems, the inventors discovered that by using a compound that has affinity for mutant TIR1 family proteins and that undergoes a reversible change in three-dimensional structure upon the action of light, it is possible to control the degradation of target proteins in a site- and time-selective manner.

[0009] The present invention has been completed based on the above findings and includes the following aspects. [Section 1] Formula (I): [ka] (I) [In the formula, R 1 is an optionally substituted 6- to 10-membered aryl group or 5- or 6-membered heteroaryl group, R 2 -OR 3 or -N(R 4 )(R 5 ) and R 3 , R 4 and R 5 are each independently H or C 1-6 alkyl] or a salt thereof.

[0010] [Section 2] R 1 The compound or salt thereof according to [Item 1], wherein the -N=N- group is bonded to the 5th or 6th position of the indole ring.

[0011] [Section 3] R 1 The compound or salt thereof according to [Item 1] or [Item 2], wherein the -N=N- group is bonded to the 5-position of the indole ring.

[0012] [Section 4] The compound or salt thereof according to any one of [Item 1] to [Item 3], wherein the aryl group is phenyl.

[0013] [Section 5] The compound or salt thereof according to any one of [Item 1] to [Item 3], wherein the heteroaryl group is thiazole.

[0014] [Section 6] R 1 -CN, -CX3 (wherein each X is independently a halogen), -NO2, -COR 1a (where R 1a is R 11a OR 11a and R 11a is H or C 1-6 alkyl), -OR 1b (where R 1b is C1-6 alkyl), -N(R 1c )(R 1d ) (where R 1c and R 1d are each independently H or C 1-6 alkyl), and -SO2R 1e (where R 1e is OH or C 1-6 [Item 1] to [Item 5], wherein the aryl group or heteroaryl group is substituted with at least one group selected from the group consisting of alkyl, aryl, aryl group, aryl group, hetero ...

[0015] [Section 7] The compound or salt thereof according to any one of [Item 1] to [Item 4] or [Item 6], wherein the compound is 5-phenylazoindole-3-acetic acid or 6-phenylazoindole-3-acetic acid.

[0016] [Section 8] The compound is irradiated with light having a wavelength of 400 to 650 nm to form R 1 and the indole ring exhibits a trans configuration, or a salt thereof, according to any one of [Item 1] to [Item 7].

[0017] [Section 9] The compound is irradiated with light having a wavelength of 320 to 580 nm to form R 1 and the indole ring exhibits a cis configuration, or a salt thereof, according to any one of [Item 1] to [Item 8].

[0018] [Section 10] A target protein degradation regulator comprising the compound or salt thereof according to any one of [Item 1] to [Item 9].

[0019] [Section 11] The agent according to [Item 10], wherein the target protein is expressed intracellularly.

[0020] [Section 12] 1. A method for controlling the degradation of a target protein, comprising the steps of: operably linking a first nucleic acid encoding a degradation tag to a nucleic acid encoding a target protein; expressing the first nucleic acid and a second nucleic acid encoding a mutant TIR1 family protein in a cell; The cell in which the first and second nucleic acids have been expressed is contacted with the compound or salt thereof according to any one of [Item 1] to [Item 9], or the agent according to [Item 10] or [Item 11], and then a step of irradiating light onto the cells that have been contacted with the compound or its salt or agent to control degradation of the target protein. 1. A method comprising: the degradation tag comprises a full-length or partial protein of an Aux / IAA family protein and has affinity for the mutant TIR1 family protein-compound of formula (I) complex; The mutant TIR1 family protein is selected from the following (a) to (c): (a) an amino acid sequence in which phenylalanine at amino acid position 79 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine, alanine, or serine; (b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added at a site other than amino acid position 79 of (a); and (c) an amino acid sequence having 80% or more identity at a site other than amino acid position 79 of (a) and which binds to the degradation tag via the compound of formula (I) to guide the target protein to degradation.

[0021] [Section 13] The method according to [Item 12], wherein the phenylalanine at amino acid position 79 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine.

[0022] [Section 14] A kit for controlling degradation of a target protein, comprising the compound or salt thereof according to any one of [Item 1] to [Item 9], or the agent according to [Item 10] or [Item 11], a first nucleic acid encoding a degradation tag, and a second nucleic acid encoding a mutant TIR1 family protein, the degradation tag comprises a full-length or partial protein of an Aux / IAA family protein and has affinity for the mutant TIR1 family protein-compound of formula (I) complex; The mutant TIR1 family protein is selected from the following (a) to (c): (a) an amino acid sequence in which phenylalanine at amino acid position 79 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine; (b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added at a site other than amino acid position 79 of (a); and (c) an amino acid sequence having 80% or more identity at a site other than amino acid position 79 of (a) and a protein that binds to the degradation tag via the compound of formula (I) to guide the target protein to degradation. [Effects of the Invention]

[0023] According to the present invention, it is possible to repeatedly control the degradation of a target protein in a location-selective and time-selective manner using light of a specific wavelength. By selecting light of a specific wavelength, it is possible to limit the effects on a living organism or cells. By simply and quickly controlling the degradation of a target protein according to the present invention, it is expected that the present invention will be applied to the screening of target proteins in cultured cells and living cells, for example, nerve cells. [Brief explanation of the drawings]

[0024] [Figure 1]The upper panel of Figure 1 shows the state of green fluorescence from GFP in each cell at the start of culture in the experiment of Example 3. The lower panel of Figure 1 shows the state of cells after 4 hours of culture, with no 5-phenylazoindole-3-acetic acid (denoted as 1) added (upper left panel), with 5-phenylazoindole-3-acetic acid added (upper center panel), and with 5-phenylazoindole-3-acetic acid added (before irradiation with 365 nm light) (upper right panel). [Figure 2] Figures 2a and 2b show the compound concentration dependence of GFP intensity when 5-phenylazoindole-3-acetic acid (Compound 1) and 6-phenylazoindole-3-acetic acid (Compound 2), respectively, were used as auxins in the experiment of Example 4, without light irradiation or with 365 nm light or 405 nm light. [Figure 3] FIG. 3 shows the GFP fluorescence intensity at each timing when the procedure described in Example 5 was performed. [Figure 4] The upper left image of Figure 4 shows the GFP fluorescence observed under a fluorescence microscope in a cell group to which 60 nM of 5-phenylazoindole-3-acetic acid was added and which was then partially irradiated with 365 nm light in the experiment of Example 6. The upper right image of Figure 4 shows the same cell group observed under a bright field. The upper left image of Figure 4 shows the GFP fluorescence observed under a fluorescence microscope in a cell group to which 150 nM of 5-phenylazoindole-3-acetic acid was added and which was then partially irradiated with weak 365 nm light against 520 nm light as background light. The lower right image of Figure 4 shows the same cell group observed under a bright field. DETAILED DESCRIPTION OF THE INVENTION

[0025] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] As used herein, the term "alkyl" refers to a straight or branched chain saturated hydrocarbon group consisting solely of carbon and hydrogen atoms. An alkyl may have, for example, 1 to 6 carbon atoms (in which case C1-6 alkyl), for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, and the like.

[0027] The term "haloalkyl" as used herein means an alkyl group as defined above in which one or more hydrogen atoms have been replaced by halogen, such as trifluoromethyl, dichloromethyl, bromomethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.

[0028] The term "halogen" as used herein includes, for example, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0029] The term "alkoxy" as used herein means an alkyl, as defined above, attached to an oxygen atom and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexoxy, and the like.

[0030] As used herein, the term "aryl" means an aromatic monocyclic or aromatic polycyclic hydrocarbon ring group composed solely of hydrogen and carbon atoms and includes, for example, phenyl, naphthyl, and indenyl.

[0031] As used herein, the term "heteroaryl" refers to an aromatic ring group containing at least one heteroatom, where each heteroatom may be independently selected from oxygen, nitrogen, sulfur, or any combination thereof. Examples of heteroaryl include furanyl, thiazolyl, thienyl, pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, thiopyranyl, thiazinyl, and indolyl.

[0032] In one embodiment, the present invention provides a compound of formula (I): [ka] (I) or a salt thereof.

[0033] In certain embodiments, R in formula (I) 1 is an optionally substituted aryl group or heteroaryl group. In another embodiment, it is an optionally substituted 6- to 12-membered aryl group or 5- to 12-membered heteroaryl group. Preferably, it is an optionally substituted 6- to 10-membered aryl group or 5- or 6-membered heteroaryl group, and more preferably an optionally substituted 6-membered aryl group or 6-membered heteroaryl group. Examples of the aryl group include phenyl, naphthyl, and indenyl, and preferably phenyl. The heteroaryl group is a group in which a carbon atom of an aryl group is substituted with at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, and examples thereof include furanyl, thiazolyl, thienyl, pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, thiopyranyl, thiazinyl, and indolyl, and preferably thiazolyl.

[0034] R 1 The substituent of R in formula (I) can be formed by irradiating the compound of formula (I) with light of a specific wavelength. 1 A substituent that can contribute to converting the configuration of R and the indole ring into cis or trans is preferred. 1 Substituents of include, for example, halogen, alkyl, haloalkyl, alkoxy, nitro, cyano, hydroxy, N(R')(R''), carboxyl, formyl, -SO3H, acetyl, benzoyl, etc., where R' and R'' are each independently hydrogen or alkyl. In some embodiments, R 1 Examples of the substituent include -CN, -CX3 (wherein each X is independently a halogen), -NO2, -COR 1a (where R1a is R 11a OR 11a and R 11a is H or C 1-6 alkyl), -OR 1b (where R 1b is C 1-6 alkyl), -N(R 1c )(R 1d ) (where R 1c and R 1d are each independently H or C 1-6 alkyl), and -SO2R 1e (where R 1e is OH or C 1-6 In some embodiments, R in formula (I) is alkyl. 1 A preferred substituent of R is cyano. 1 When R is phenyl, it is preferably substituted at the 4-position. 1 The structure of R 1 Substituents (electron-withdrawing or electron-donating) of and R 1 Depending on the substitution position of the -N=N- group, the wavelength of the irradiated light can be adjusted, and the wavelength of the light can be shifted to the longer or shorter wavelength side.

[0035] In certain embodiments, R in formula (I) 1 The -N=N- group may be bonded to any of the 4- to 7-positions of the indole ring, preferably the 5- or 6-position of the indole ring, and more preferably the 5-position of the indole ring.

[0036] In certain embodiments, R in formula (I) 2 -OR 3 or -N(R 4 )(R 5 ) may also be used. 3 , R 4 and R 5 R may each independently be H, alkyl, alkoxy, hydroxy, carboxy, formyl, acetyl, benzoyl, etc., and each group may be appropriately substituted. 3 , R4 and R 5 are preferably each independently H or C 1-6 It is alkyl, more preferably H.

[0037] In certain embodiments, the compound of Formula (I) is 5-phenylazoindole-3-acetic acid or 6-phenylazoindole-3-acetic acid.

[0038] In one embodiment, the present invention may be a targeted protein degradation regulator comprising the compound of formula (I) or a salt thereof.

[0039] The compound of formula (I) of the present invention may be contained in the targeted protein degradation regulator in the form of a salt, which may be a salt derived from a variety of organic and inorganic counter ions known to those skilled in the art, such as salts derived from sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, or a salt of an organic or inorganic acid such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, or oxalate.

[0040] The compound of formula (I) of the present invention may be in the form of a salt, a solvate, a hydrate, an isotope-labeled compound, or a mixture thereof, and can be appropriately prepared by a person skilled in the art using a known method. 2 H(D), 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 18 O, etc.)

[0041] In certain embodiments, the compounds of formula (I) of the present invention comprise a compound of formula R 1 The configuration of the indole ring can be converted from cis to trans or from trans to cis by irradiation with light of a specific wavelength.

[0042] In the compounds of formula (I) of the present invention, R 1 When converting the configuration of the indole ring to trans, the light to be irradiated may vary depending on the compound used, but examples include light having a wavelength within the range of 400 to 650 nm, 405 to 500, or 500 to 600 nm, preferably light having a wavelength within the range of 500 to 550 nm, and more preferably light having a wavelength of 520 nm.

[0043] In the compounds of formula (I) of the present invention, R 1 When converting the configuration of the indole ring to cis, the wavelength of the light to be irradiated may vary depending on the compound used, but may be, for example, any wavelength within the range of 320 to 580 nm, 320 to 380 nm, or 400 to 500 nm, preferably any wavelength within the range of 350 to 405 nm, and more preferably a wavelength of 365 nm. To limit the effect on cells, light with a longer wavelength is preferred.

[0044] The wavelength of the light irradiated in the present invention may vary depending on the compound used. For example, in the compound of formula (I) of the present invention, R 1 is unsubstituted aryl, and R 1 When the -N=N- group is attached to the 5-position of the indole ring, R 1 The light irradiated to convert the configuration of R to the trans configuration of the indole ring has a wavelength in the range of 405 to 550 nm, and the light irradiated to convert the configuration to the cis configuration has a wavelength in the range of 320 to 380 nm. 1 is heteroaryl, and R 1 When the -N=N- group is attached to the 6-position of the indole ring, or when R 1 is an aryl substituted with a cyano group, an amino group, a dialkylamino group, or an alkoxy group, and R 1 When the -N=N- group is attached to the 5-position of the indole ring, R 1The light irradiated to convert the configuration of the indole ring to trans has a wavelength in the range of 500 to 650 nm, and the light irradiated to convert the configuration to cis has a wavelength in the range of 400 to 550 nm.

[0045] The amount of the compound of formula (I) of the present invention to be used can be appropriately determined by those skilled in the art depending on the conditions of use. For example, when used for a target protein expressed in cultured cells, the compound of formula (I) of the present invention is added to a culture medium, and the concentration of the compound contained in the culture medium may be, but is not limited to, for example, in the range of 1 nM to 100 μM, preferably 10 nM to 100 μM. Furthermore, when used for a target protein present in cells in a living body, the compound of formula (I) of the present invention may be used at, but is not limited to, for example, 0.1 mg to 100 mg per kg of animal body weight per day, preferably 0.2 mg to 50 mg, more preferably 0.5 mg to 20 mg.

[0046] In addition to the above-mentioned compounds, the proteolysis regulators of the present invention may contain other components, such as additives, as long as the effect of controlling the degradation of the target protein is not impaired. Examples of other components include, but are not limited to, additives, such as preservatives such as sodium azide, sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; diluents such as water, saline, and buffer solutions; and organic solvents such as methanol, ethanol, dimethyl sulfoxide, dimethylformamide, and glycerol. The proteolysis regulators of the present invention may be in any form, such as a liquid or solid, and may be used in the form of, for example, a powder, a solvent, or a gel.

[0047] The target protein degradation regulator of the present invention can target any protein as long as it is expressed in cells. Therefore, the cells are not particularly limited and may be cultured or present in vivo.

[0048] The cells used in the present invention may be eukaryotic cells, including cells derived from or existing in fungi (e.g., budding yeast, fission yeast, etc.) and protozoa, and may also be cells derived from or existing in animals (e.g., mammals such as humans, mice, rats, and rabbits; fish and amphibians such as zebrafish and Xenopus laevis; and invertebrates such as nematodes and fruit flies). From the perspective of drug evaluation performance, cells derived from or existing in rodents such as hamsters, guinea pigs, rats, and mice can also be used. Furthermore, the cells may also include established cultured cell lines, such as cells derived from animals, ES cells, and iPS cells. Examples of the cells include established human-derived cell lines, established mouse-derived cell lines, established chicken-derived cell lines, human ES cells, mouse ES cells, human iPS cells, and mouse iPS cells. For example, human HCT116 cells, human HT1080 cells, human NALM6 cells, human ES cells, human iPS cells, mouse ES cells, mouse iPS cells, and chicken DT40 cells can be used.

[0049] The cells that can be used in the present invention may be cells of any tissue derived from the animal or present within the animal. Examples of such tissues include various brain tissues such as nerves, liver, kidney, heart, stomach, bone marrow, thymus, and intestines, with nerves being preferred. The animal may also be suffering from any disease, such as Alzheimer's disease, epilepsy, autism spectrum disorder, cancer, autoimmune disease, or various genetic diseases.

[0050] In another aspect, the present invention relates to a method for controlling the degradation of a target protein. The method that can be used in the present invention comprises the following steps: operably linking a first nucleic acid encoding a degradation tag to a nucleic acid encoding a target protein; expressing in a cell a first nucleic acid encoding a degradation tag and a second nucleic acid encoding a mutant TIR1 family protein; The cells in which the first and second nucleic acids have been expressed are contacted with the compound of the present invention or a target protein degradation regulator, and then a step of irradiating the cells contacted with the control agent with light to control degradation of the target protein. The present invention is characterized by comprising:

[0051] In the method of the present invention, the step of operably linking a first nucleic acid encoding a degradation tag to a nucleic acid encoding a target protein may be performed at any time before expressing the first nucleic acid. For example, it may be performed before introducing the first nucleic acid into a cell, or it may be performed after introducing the first nucleic acid into a cell. When this step is performed before introducing the first nucleic acid into a cell, the nucleic acid encoding the target protein may be linked to the first nucleic acid using known genetic techniques. For example, the nucleic acid encoding the target protein can be linked to the first nucleic acid using ligation or the like. When this step is performed after introducing the first nucleic acid into a cell, it can be performed using known techniques for introducing a nucleic acid into a specific site, such as genome editing techniques (e.g., methods using CRISPR-Cas9), methods using nucleic acid aptamers, and transfection. As used herein, "operably linked" refers to a state in which a nucleic acid of interest is arranged with another nucleic acid in a sequence that allows it to exert a desired function.

[0052] The target protein contemplated by the present invention is not particularly limited, as long as it is a protein whose degradation is intended to be controlled using the method of the present invention. The target protein used in the present invention may be, for example, an exogenous protein intended to be expressed in a cell, or an endogenous protein in a cell used in the present invention. Furthermore, the nucleic acid encoding the target protein used in the present invention may be DNA containing exons and introns, or may be cDNA consisting of exons. Furthermore, the nucleic acid encoding the target protein used in the present invention may be, for example, a full-length sequence in genomic DNA or a full-length sequence in cDNA. Furthermore, the nucleic acid encoding the target protein used in the present invention may be a partial sequence in genomic DNA or a partial sequence in cDNA, to the extent that the expressed protein functions as a protein.

[0053] In the method of the present invention, the step of expressing a first nucleic acid encoding a degradation tag and a second nucleic acid encoding a mutant TIR1 family protein in a cell may be performed using any method that can express the first nucleic acid and the second nucleic acid in the cell. Known methods can be used to express the nucleic acids. For example, expression can be achieved by introducing the nucleic acid into a cell and then adding or contacting the cell with an expression-inducing factor (e.g., a drug). Methods for introducing the nucleic acid into the cell are well known, and include methods for introducing the nucleic acid into the cell via a vector or virus for genetic recombination (e.g., genetic recombination technology, gene editing technology, etc.), chemical methods for directly introducing the nucleic acid into the cell, and methods for directly delivering the nucleic acid into the cytoplasm or nucleus of the cell (e.g., electroporation, transfection, etc.). The first nucleic acid and the second nucleic acid may be introduced into the cell together or separately. Furthermore, the first nucleic acid and / or the second nucleic acid may be expressed in the cytoplasm as a vector or virus, or may be introduced into the chromosome of the cell and expressed. Furthermore, the first nucleic acid and / or the second nucleic acid may be modified so as to be constitutively and stably expressed in cells.

[0054] In the methods of the present invention, the step of contacting the cells expressing the first and second nucleic acids with the target protein degradation regulator may be carried out by any method that allows the agent to be taken up into the cells. For example, when the cells are cultured, the agent may be taken up into the cells by adding it to the culture medium. Furthermore, when the cells are present in vivo, the step may also be carried out by oral administration, injection (subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, etc.), sublingual administration, topical application (application to mucous membranes such as the skin, eyes, or vagina), or transdermal administration systems, allowing the agent to be taken up into the cells in vivo.

[0055] The conditions for the contact can be appropriately determined by those skilled in the art. For example, when cells are cultured, the concentration of the compound of formula (I) of the present invention added to the culture medium may be, but is not limited to, in the range of 1 nM to 100 μM, and preferably 10 nM to 100 μM. When cells are present in a living body, the compound of formula (I) of the present invention may be administered to the living body at, but is not limited to, for example, 0.1 mg to 100 mg per kg of animal body weight per day, preferably 0.2 mg to 50 mg, and more preferably 0.5 mg to 20 mg.

[0056] In the present invention, the step of controlling target protein degradation by irradiating the cells contacted with the agent with light can be carried out by irradiating the cells with light of two or more different wavelengths. The wavelength of light capable of degrading the target protein is in the range of 400 to 650 nm, 405 to 500 nm, or 500 to 600 nm, preferably in the range of 500 to 550 nm, and more preferably 520 nm. Irradiation with light of this wavelength converts the compound of formula (I) into a trans form, promoting ubiquitination of the degradation tag by ubiquitin ligase, thereby degrading the target protein. The wavelength of light capable of halting target protein degradation is in the range of 320 to 580 nm, 320 to 380 nm, or 400 to 500 nm, preferably in the range of 350 to 405 nm, and more preferably 365 nm. Irradiation with light of the wavelength converts the compound of formula (I) into a cis-isomer, thereby terminating the ubiquitination of the degradation tag by ubiquitin ligase. Considering the potential for cell damage due to irradiated light, it is preferable for the wavelength to be as long as possible. Therefore, irradiation with light of two or more different wavelengths can reversibly control the degradation of a target protein, which is advantageous in that the control can be achieved depending on the location and duration of light irradiation.

[0057] In the present specification, in controlling the degradation of a target protein, the R in the formula of the compound (I) of the present invention can be obtained by irradiating the compound with light having a wavelength within a range of 400 to 650 nm, 405 to 500, or 500 to 600 nm. 1 The configuration of R in the indole ring is converted to trans. The mutant TIR1 family protein expressed from the second nucleic acid can bind to the degradation tag expressed from the first nucleic acid via the compound of formula (I) converted to a trans form, and the degradation tag in this formed complex is ubiquitinated, and the target protein bound to the ubiquitinated degradation tag can be induced to undergo degradation by the ubiquitin / proteasome degradation system in cells. Furthermore, by irradiating light with a wavelength within the range of 320 to 580 nm, 320 to 380 nm, or 400 to 500 nm, the R in the compound of formula (I) can be converted to trans. 1 The configuration of the indole ring is converted to cis. The compound of formula (I) converted to a cis form does not bind to the mutant TIR1 family protein, and therefore the complex is not formed, and the degradation tag is not ubiquitinated, thereby terminating the degradation. Since the photoisomerization (cis-trans) of the compound of formula (I) can be reversibly repeated, it is possible to reversibly control the degradation of a target protein by irradiating it with light of two or more different wavelengths.

[0058] The irradiation time for degrading a target protein in the method of the present invention can be appropriately determined by those skilled in the art depending on the purpose and conditions. Typically, irradiation may be continued for the time required to degrade the target protein. The irradiation time may be, for example, 20 minutes to 24 hours, 30 minutes to 12 hours, or 1 hour to 6 hours, and can be selected depending on the time required for degradation. If the lifetime of the cis-isomer of the compound of formula (I) contained in the target protein degradation controller used is sufficiently longer than the time required to degrade the target protein, shorter irradiation times may be used. In such cases, irradiation times may be, for example, 1 nanosecond to 1 hour, 1 nanosecond to 30 minutes, 1 second to 30 minutes, or 1 minute to 15 minutes. However, it is desirable to use a light source with a sufficiently low intensity to take into account damage to cells.

[0059] The degradation of the target protein degraded in the method of the present invention is stopped by irradiation with light having a wavelength within the range of 320 to 580 nm, 320 to 380 nm, or 400 to 500 nm, and the target protein whose degradation has been stopped is re-expressed in the cells. The time until the target protein is re-expressed after the start of the light irradiation is usually 20 minutes to 24 hours, and can be, for example, 30 minutes to 20 hours, 1 hour to 10 hours, or 2 hours to 4 hours.

[0060] The method of controlling target protein degradation of the present invention may be carried out using a known auxin degron system (Patent Document 2), and those skilled in the art can appropriately modify or alter the auxin degron system to apply the method of the present invention. The "auxin degron system" is a technology for controlling protein degradation, which applies a plant-specific proteolytic system induced by the plant hormone auxin to non-plant eukaryotic cells. Specifically, this system involves introducing a plant-derived TIR1 family protein (an F-box protein, a subunit of the E3 ubiquitination complex (SCF complex)) and a target protein labeled with a plant-derived Aux / IAA family protein or a peptide consisting of a partial sequence thereof into non-plant eukaryotic cells. The TIR1 family protein, an auxin receptor, recognizes the Aux / IAA family protein or the peptide consisting of a partial sequence thereof in an auxin-dependent manner, and degrades the target protein using the ubiquitin / proteasome degradation system in non-plant eukaryotic cells.

[0061] The mutant TIR1 family proteins herein include, for example, the following (a) to (c): (a) an amino acid sequence in which phenylalanine at amino acid position 79 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine, alanine, or serine; (b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added at a site other than amino acid position 79 of (a); and (c) an amino acid sequence having 80% or more identity at a site other than amino acid position 79 of (a) and a protein that binds to the degradation tag via the compound of formula (I) to guide the degradation of a target protein, The phenylalanine at amino acid position 79 in the amino acid sequence represented by SEQ ID NO: 1 may be substituted with glycine as appropriate.

[0062] The second nucleic acid used in the method of the present invention encodes a mutant TIR1 family protein, which has a mutation in the auxin-binding site. The TIR1 family protein is an F-box protein, one of the subunits that form the E3 ubiquitination complex (SCF complex) in the protein degradation of the ubiquitin / proteasome system, and is a plant-specific protein. It is known that TIR1 family proteins act as receptors for the growth hormone auxin, and by receiving auxin, recognize Aux / IAA family proteins, which are inhibitors of the auxin signaling system, and degrade target proteins.

[0063] The gene encoding a TIR1 family protein may be any gene derived from a plant, as long as it encodes a TIR1 family protein. The type of plant from which the gene is derived is not limited, and examples include Arabidopsis thaliana, rice, zinnia, pine, fern, and Physcomitrella patens. Specific examples of genes encoding TIR1 family proteins include the TIR1 gene, AFB1 gene, AFB2 gene, AFB3 gene, FBX14 gene, and AFB5 gene. Particularly preferred are the AtTIR1 gene, which is a TIR1 gene derived from Arabidopsis thaliana, and rice-derived OsTIR1 (in OsTIR1, F74G corresponds to the "F79G mutation"). Examples of such genes include the gene registered with NCBI under accession number NM_116163 (Gene ID: 825473), and more specifically, a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 2.

[0064] The mutant TIR1 family protein used in the method of the present invention has a mutation in the auxin-binding site. Such a mutant protein is not particularly limited as long as it has affinity for the compound of formula (I). However, a mutant protein in which F at position 79 of AtTIR1 is mutated to A, G, or S is preferred, and a mutant protein in which F is mutated to G is more preferred.

[0065] More specifically, the mutant TIR1 family protein used in the method of the present invention may be a protein that has a sequence including any one of the following amino acid sequences and that binds to a degradation tag via a compound of formula (I) to guide the target protein to degradation. (a) an amino acid sequence in which phenylalanine at amino acid position 79 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine, alanine, or serine; (b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added at a site other than amino acid position 79 of (a); and (c) An amino acid sequence having 80% or more identity at a site other than amino acid position 79 of (a).

[0066] The number of deleted, inserted, substituted or added amino acids in the amino acid sequence (b) is preferably 1 to 120, more preferably 1 to 60, even more preferably 1 to 20, even more preferably 1 to 10, and even more preferably 1 to 5.

[0067] In order for the amino acid sequence (c) to be functionally equivalent to a protein consisting of a sequence containing the amino acid sequence (a), it must have an identity of 80% or more, preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more.

[0068] An example of the AtTIR1 F79G protein is one consisting of the amino acid sequence shown in SEQ ID NO:3.

[0069] In the present invention, the second nucleic acid encoding a mutant TIR1 family protein may be DNA containing exons and introns, or may be cDNA consisting of exons. The second nucleic acid encoding a mutant TIR1 family protein may be, for example, a full-length sequence in genomic DNA or a full-length sequence in cDNA. Furthermore, the second nucleic acid encoding a mutant TIR1 family protein may be a partial sequence in genomic DNA or a partial sequence in cDNA, as long as the expressed protein functions as a TIR1 protein. Here, "functioning as a TIR1 family protein" means, for example, recognizing a degradation tag (a full-length or partial protein of an Aux / IAA family protein) in the presence of an auxin analog. This is because if a TIR1 family protein can recognize a degradation tag, it can degrade a target protein labeled with the degradation tag.

[0070] In the present invention, the second nucleic acid encoding a mutant TIR1 family protein is preferably operably linked to the 5' end with a promoter sequence that controls the transcription of the second nucleic acid. This ensures reliable expression of the TIR1 family protein. Examples of the promoter include an inducible promoter, a viral promoter, a housekeeping gene promoter, and a tissue-specific promoter. In the cell of this embodiment, the promoter operably linked to the second nucleic acid encoding a mutant TIR1 family protein is preferably an inducible promoter, and specific examples include a CMV promoter, an SV40 promoter, an EF1a promoter, and an RSV promoter.

[0071] In the methods of the present invention, the second nucleic acid encoding a mutant TIR1 family protein and a promoter sequence operably linked to its 5' end can be used in the form of being inserted into a vector. The vector is preferably an expression vector. There are no particular limitations on the expression vector, and any expression vector suitable for the host cell can be used. The vector may have a polyadenylation signal, an NLS, a fluorescent protein marker gene, or the like operably linked to the 5' or 3' end of the second nucleic acid encoding the TIR1 family protein.

[0072] The first nucleic acid used in the method of the present invention comprises a full-length or partial protein of an Aux / IAA family protein and encodes a degradation tag having affinity for the mutant TIR1 family protein-compound of formula (I) complex. The gene encoding the Aux / IAA family protein used in the method of the present invention is not particularly limited, as long as it is an Aux / IAA family gene derived from a plant. Specific examples of genes encoding the Aux / IAA family proteins include the IAA1 gene, IAA2 gene, IAA3 gene, IAA4 gene, IAA5 gene, IAA6 gene, IAA7 gene, IAA8 gene, IAA9 gene, IAA10 gene, IAA11 gene, IAA12 gene, IAA13 gene, IAA14 gene, IAA15 gene, IAA16 gene, IAA17 gene, IAA18 gene, IAA19 gene, IAA20 gene, IAA26 gene, IAA27 gene, IAA28 gene, IAA29 gene, IAA30 gene, IAA31 gene, IAA32 gene, IAA33 gene, and IAA34 gene.

[0073] The first nucleic acid used in the method of the present invention may have the full-length or partial sequence of a gene encoding one or more of the Aux / IAA family proteins. For example, the sequence of an Aux / IAA family gene from Arabidopsis thaliana is described in TAIR (The Arabidopsis Information The gene accession numbers are as follows: IAA1 gene (AT4G14560), IAA2 gene (AT3G23030), IAA3 gene (AT1G04240), IAA4 gene (AT5G43700), IAA5 gene (AT1G15580), IAA6 gene (AT1G52830), IAA7 gene (AT3G23050), IAA8 gene (AT2G22670), IAA9 gene (AT5G65670), IAA10 gene (AT1G04100), IAA11 gene (AT4G28640), IAA12 gene (AT1G04550), IAA13 gene (AT2G33310), and IAA14 gene (AT4G14 550), IAA15 gene (AT1G80390), IAA16 gene (AT3G04730), IAA17 gene (AT1G04250), IAA18 gene (AT1G51950), IAA19 gene (AT3G15540), IAA20 gene (AT2G46990), IAA26 gene (AT3G16500), IAA27 gene IAA17 gene (AT4G29080), IAA28 gene (AT5G25890), IAA29 gene (AT4G32280), IAA30 gene (AT3G62100), IAA31 gene (AT3G17600), IAA32 gene (AT2G01200), IAA33 gene (AT5G57420), and IAA34 gene (AT1G15050). The Arabidopsis thaliana IAA17 gene is particularly preferred.

[0074] The degradation tag that can be used in the method of the present invention is not particularly limited as long as it binds to the complex of the mutant TIR1 family protein and the compound of formula (I) and leads to the degradation of the target protein. Among Aux / IAA family proteins, those containing the full-length or partial protein of mAID, or AID *Preferably, the mAID contains a full-length or partial protein of IAA17. Here, "mAID" is an abbreviation for "mini-auxin-inducible degron," and an example thereof is a protein consisting of a partial sequence of Arabidopsis thaliana IAA17, one of the Aux / IAA family proteins. The partial sequence is a sequence consisting of a region containing at least two Lys residues on the N-terminal and C-terminal sides of the domain II region of an Aux / IAA family protein, or a sequence consisting of two or more such sequences linked together. This mAID can be used as a degradation tag to label a target protein. For example, the amino acid sequence of mAID can be represented by SEQ ID NO: 4. "AID" * " is a protein consisting of a short amino acid sequence of 44 aa, which is derived from IAA17 and is different in length from mAID. For example, AID * The amino acid sequence of can be represented by SEQ ID NO:5.

[0075] The first nucleic acid used in the method of the present invention is preferably operably linked to a promoter sequence, similar to the second nucleic acid, and may be incorporated into an expression vector.

[0076] The cells used in the methods of the present invention are not particularly limited and may be cultured or present in vivo. The cells used in the methods of the present invention may be eukaryotic cells, including cells derived from or present in animals, fungi (e.g., budding yeast, fission yeast, etc.), protists, etc., such as cells derived from or present in mammals such as humans, mice, rats, and rabbits, fish and amphibians such as zebrafish and Xenopus laevis, and invertebrates such as nematodes and fruit flies. From the perspective of drug evaluation performance, cells derived from or present in rodents such as hamsters, guinea pigs, rats, and mice can also be used. Furthermore, the cells may include established cultured cell lines, such as cells derived from animals, ES cells, and iPS cells. Examples of the cells include established human-derived cell lines, established mouse-derived cell lines, established chicken-derived cell lines, human ES cells, mouse ES cells, human iPS cells, and mouse iPS cells. For example, human HCT116 cells, human HT1080 cells, human NALM6 cells, human ES cells, human iPS cells, mouse ES cells, mouse iPS cells, and chicken DT40 cells can be used. Cells that can be used in the present invention may be derived from any tissue within the animal or present within the animal. Examples of such tissues include various brain tissues such as nerves, liver, kidney, heart, stomach, and intestines, with nerves being preferred. The animal may also be suffering from any disease, such as Alzheimer's disease, epilepsy, autism spectrum disorder, cancer, autoimmune diseases, and various genetic diseases.

[0077] In yet another aspect, the present invention includes a kit for controlling target protein degradation, which comprises a target protein degradation regulator and optionally includes a first nucleic acid encoding a degradation tag and a second nucleic acid encoding a mutant TIR1 family protein.

[0078] When a target protein is defined, the kit of the present invention may include a third nucleic acid encoding the target protein linked upstream or downstream of the first nucleic acid. The first nucleic acid may be adjacent to either the 5'-end or the 3'-end of the third nucleic acid. Like the second nucleic acid, the fused nucleic acid comprising the first and third nucleic acids is preferably operably linked to a promoter sequence, and may be incorporated into an expression vector.

[0079] The kit of the present invention may include a cell or animal having a second nucleic acid on a chromosome. Preferably, such a cell or animal has the second nucleic acid at a safe harbor locus. Here, a "safe harbor locus" refers to a genetic region that is constitutively and stably expressed and that can sustain life even if the gene originally encoded in that region is deleted or modified. When inserting foreign DNA (in this embodiment, a gene encoding TIR1) into a safe harbor locus using the CRISPR system, it is preferable for the safe harbor locus to have a PAM sequence nearby. Examples of safe harbor loci include the GTP-binding protein 10 locus, the Rosa26 locus, the β-actin locus, and the AAVS1 (AAV integration site 1) locus. In particular, when using human-derived cells, it is preferable to insert foreign DNA into the AAVS1 locus.

[0080] General synthesis method (1) R in formula (I) 2 In compounds where is OH, R 1 Compounds in which -N=N- is bonded to the indole ring at position 5 can be produced according to the following synthesis method. Those skilled in the art can prepare the compounds of the present invention by appropriately changing or modifying the synthesis method. [ka] (A) Compound R 1 -I (where R 1is an optionally substituted aryl) is reacted with a hydrazine substituted with a protecting group in the presence of a catalyst and a base in a solvent to give an aryl-substituted hydrazine. (B) The obtained aryl-substituted hydrazine and ethyl 5-bromo-1-tetrahydropyranyl-1H-indole-3-acetate are heated under reflux in a solvent in the presence of a catalyst and a base to give 5-aryl-indolylhydrazine. (C) Further, the 5-aryl-indolylhydrazine is oxidized in the presence of a catalyst and an oxidizing agent in a solvent to give an indolyl azo compound: [ka] get. (D) The indolyl azo compound is treated with a first deprotecting agent in a first solvent to deprotect the ethyl ester moiety to give the free carboxylic acid, and then treated with a second protecting agent in a second solvent to deprotect the protecting group (e.g., tetrahydropyran), finally giving the desired 5-arylazoindole-3-acetic acid.

[0081] The reaction reagents, catalyst, reaction temperature, solvent, etc. used in this method may be appropriately modified according to methods known in the art. For example, in step (A), a tert-butoxycarbonyl group may be used as the protecting group, a combination of copper iodide and 1,10-phenanthroline may be used as the catalyst, cesium carbonate may be used as the base, dry dimethylformamide may be used as the solvent, and the reaction temperature may be 80°C. In the step (B), palladium acetate and tri-tert-butylphosphonium tetrafluoroborate can be used as catalysts, cesium carbonate can be used as base, and toluene can be used as solvent. In the step (C), pyridine may be used as the catalyst, N-bromosuccinimide may be used as the oxidizing agent, and methylene chloride may be used as the solvent. In the step (D), 2 M sodium hydroxide can be used as the first deprotecting agent, a tetrahydrofuran-methanol mixture can be used as the first solvent, trifluoroacetic acid can be used as the second deprotecting agent, and a water-chloroform mixture can be used as the second solvent.

[0082] R in formula (I) 2 In compounds where is OH, R 1 The compound in which is 4-cyanophenyl can be prepared by general synthesis method (1) using compound R 1 -I (where R 1 is optionally substituted aryl) as compound R 1 -I (where R 1 is 4-cyanophenyl).

[0083] General synthesis method (2) R in formula (I) 2 In compounds where is OH, R 1 The compound in which -N=N- is bonded to the indole ring at the 6-position can be obtained by using ethyl 6-bromo-1-tetrahydropyranyl-1H-indole-3-acetate instead of ethyl 5-bromo-1-tetrahydropyranyl-1H-indole-3-acetate in general synthesis method (1). [ka]

[0084] R in formula (I) 2 In compounds where is OH, R 1 Compounds in which is an optionally substituted heteroaryl can also be obtained by appropriately modifying the general synthesis method (1).

[0085] R in formula (I) 2 Ga-OC 1-6 Alkyl or -N(R 4 )(R 5) can be obtained by esterifying or amidating the compound obtained by the above synthesis method by a method known in the art. [Example]

[0086] Hereinafter, one embodiment of the present invention will be described, but this example is merely an example of how the present invention can be implemented and the present invention is not limited to this example.

[0087] [Example 1] [Synthesis of 5-phenylazoindole-3-acetic acid] A compound represented by the following formula (1) (also called 5-phenylazoindole-3-acetic acid) was synthesized.

[0088] [ka] (1) 2.2 ml of iodobenzene and 3.18 g of tert-butoxycarbonylhydrazine were heated in 15 ml of dry dimethylformamide at 80°C in the presence of 40 mg of copper iodide, 360 mg of 1,10-phenanthroline, and 9.12 g of cesium carbonate as catalysts to yield 6.1 g of 1-tert-butoxycarbonyl-1-arylhydrazine. 4.17 g of the resulting 1-tert-butoxycarbonyl-1-phenylhydrazine and 6.1 g of ethyl 5-bromo-1-tetrahydropyranyl-1H-indole-3-acetate were heated under reflux in 150 ml of toluene in the presence of 374 mg of palladium acetate, 484 mg of tri-tert-butylphosphonium tetrafluoroborate, and 10.88 g of cesium carbonate to yield 4.43 g of phenylindolylhydrazine. Furthermore, 4.43 g of phenyl-indolylhydrazine was oxidized in 125 ml of methylene chloride in the presence of 1.07 g of pyridine and 2.42 g of N-bromosuccinimide to give 420 mg of an indolyl azo compound. The indolyl azo compound was treated with a 2 M sodium hydroxide solution-tetrahydrofuran-methanol mixed solvent (1:1:2, 20 ml) to deprotect the ethyl ester moiety and derive the free carboxylic acid. Subsequently, the tetrahydropyran protecting group was deprotected by treatment with a trifluoroacetic acid-water-chloroform mixed solvent (1.0:0.3:2.0, 18 ml), and finally, 100 mg of the desired 5-phenylazoindole-3-acetic acid was obtained.

[0089] [Example 2] [Synthesis of 6-phenylazoindole-3-acetic acid] A compound represented by the following formula (2) (also called 6-phenylazoindole-3-acetic acid) was synthesized. [ka] (2)

[0090] 4.17 g of tert-butoxycarbonyl-1-phenylhydrazine and 6.10 g of 6-bromo-1-tetrahydropyranyl-1H-indole-3-ethyl acetate were heated under reflux in 150 ml of toluene in the presence of 374 mg of palladium acetate, 484 mg of tri-tert-butylphosphonium tetrafluoroborate, and 10.88 g of cesium carbonate to give 3.9 g of phenyl-indolylhydrazine. 3.90 g of phenyl-indolylhydrazine was further oxidized in 110 ml of methylene chloride in the presence of 0.95 g of pyridine and 2.13 g of N-bromosuccinimide to give 270 mg of an indolyl azo compound. The indolyl azo compound was treated in a 2 M sodium hydroxide solution-tetrahydrofuran-methanol mixed solvent (1:1:2, 20 ml) to deprotect the ethyl ester moiety and convert it to a free carboxylic acid. Subsequently, the compound was treated in a trifluoroacetic acid-water-chloroform mixed solvent (1.0:0.3:2.0, 18 ml) to deprotect the tetrahydropyran protecting group, finally obtaining 65 mg of the desired 6-phenylazoindole-3-acetic acid.

[0091] [Example 3] Isogenic HAP1 cells co-expressing OsTIR1F74G and mAID-EGFP-NLS using a plasmid obtained from Addgene were cultured at 1 × 10 5 Cells were seeded at a concentration of 1000 cells / mL and cultured for 24 hours. Cells were treated with 1 μg / mL doxycycline for 24 hours to induce OsTIR1F74G expression. Cells were then treated with a buffer containing 10 nM 5-phenylazoindole-3-acetic acid and cultured in the dark or under 365 nm light for 4 hours. Cells were then observed under a fluorescence microscope in the dark or under 405 nm light, and the intensity of GFP fluorescence was compared.

[0092] The top panel of Figure 1 shows the green fluorescence from GFP in each cell at the start of culture. Strong GFP fluorescence is observed in all cells, regardless of whether 5-phenylazoindole-3-acetic acid (1) was added (top left panel), added (top center panel), or added (before 365 nm light irradiation) (top right panel). The bottom panel of Figure 1 shows the cells after 4 hours of culture. Almost no GFP fluorescence was observed in cells cultured in the dark with 5-phenylazoindole-3-acetic acid (bottom center panel). In contrast, GFP fluorescence was observed in cells cultured with 5-phenylazoindole-3-acetic acid and exposed to 365 nm light for 4 hours (bottom right panel). These results demonstrate that the addition of 5-phenylazoindole-3-acetic acid can degrade GFP in cells in a 365 nm light-dependent manner.

[0093] [Example 4] To examine the concentration dependency of GFP degradation efficiency when 5-phenylazoindole-3-acetic acid and 6-phenylazoindole-3-acetic acid were added and the effect of light irradiation, the following procedure was performed using a flow cytometer.

[0094] Isogenic HAP1 cells co-expressing OsTIR1F74G and mAID-EGFP-NLS were seeded onto 35 mm dishes and cultured for 24 hours. These cells were treated with 1 μg / mL doxycycline for 24 hours to induce OsTIR1F74G expression. Subsequently, the cells were treated with a buffer solution containing various concentrations of 5-phenylazoindole-3-acetic acid and 6-phenylazoindole-3-acetic acid and cultured for 4 hours in the dark or under light. To detect EGFP signals, the cells were washed with DPBS and trypsinized with 0.05 w / v% trypsin-0.53 mmol / LEDTA·4Na solution. The suspended cells were then resuspended in DPBS and filtered through a cell strainer. GFP signal intensity was recorded using a flow cytometer, and the results were analyzed using software.

[0095] Figures 2a and b show the compound concentration dependence of GFP intensity when 5-phenylazoindole-3-acetic acid (compound 1) and 6-phenylazoindole-3-acetic acid (compound 2) were used as auxins, respectively, without light irradiation or with 365 nm or 405 nm light irradiation. For both compounds, the compound concentration required for GFP degradation upon light irradiation increased. Furthermore, 5-phenylazoindole-3-acetic acid induced GFP degradation at concentrations as low as 1 nM, and the concentration required for GFP degradation upon light irradiation increased significantly to 0.41 μM. Therefore, when 5-phenylazoindole-3-acetic acid was used at concentrations of 10–100 nM, GFP degradation could be completely switched between the presence and absence of 365 nm light irradiation.

[0096] [Example 5] To demonstrate that target proteins can be degraded at the desired timing by changing the light irradiation conditions without changing the medium, the following procedure was performed. 1 nM 5-phenylazoindole-3-acetic acid (Compound 1) was added to isogenic HAP1 cells co-expressing OsTIR1F74G and mAID-EGFP-NLS in multiple wells. The cells were then treated as in Example 4, and GFP intensity was measured using a flow cytometer. The GFP fluorescence intensity of the cells in the first well was measured immediately after the addition of 5-phenylazoindole-3-acetic acid, designated as Time = 0 h. The cells were then cultured in the dark for 12 hours (the second well) and measured at Time = 12 h. The GFP fluorescence intensity was then measured at Time = 24 h using the cells cultured under 365 nm light for another 12 hours (the third well). The cells were then cultured in the dark for another 12 hours and then under 365 nm light for another 12 hours, and the GFP fluorescence intensity was measured at Time = 36 h and Time = 48 h, respectively.

[0097] As a comparative procedure, the cells were treated by replacing the culture medium with one that did not contain auxin, a conventional method, and the GFP fluorescence intensity was measured 12 hours after the replacement.

[0098] Figure 3 shows the GFP fluorescence intensity at each timing when the above procedure was performed. In the dark, 5-phenylazoindole-3-acetic acid exhibits high auxin activity, and the target protein is degraded within 12 hours. Furthermore, under light irradiation, 5-phenylazoindole-3-acetic acid becomes inactive, and GFP is regenerated through re-expression. This change in light irradiation conditions alone has almost the same effect as replacing the medium with one that does not contain auxin. Furthermore, the degradation and regeneration of the target protein under dark or light irradiation conditions could be repeated.

[0099] [Example 6] To demonstrate the ability to site-selectively degrade the target protein, the following procedure was performed. 5 Isogenic HAP1 cells co-expressing mAID-EGFP-NLS and OsTIR1F74G (F74G corresponds to the "F79G mutation" in OsTIR1) were seeded into 35 mm wells and cultured for 24 hours. The cells were treated with 1 μg / mL doxycycline for 24 hours to induce OsTIR1F74G expression. A fixed concentration of 5-phenylazoindole-3-acetic acid was then added to the wells, and the cells were irradiated with 365 nm or 520 nm light, focused to a 350 μm diameter using a lens. When irradiating the 350 μm diameter area with 520 nm light, weak 365 nm light was used as background light.

[0100] The upper left image in Figure 4 shows the GFP fluorescence observed under a fluorescence microscope in a group of cells to which 60 nM 5-phenylazoindole-3-acetic acid had been added and which were then partially irradiated with 365 nm light. GFP was selectively degraded in cells other than those irradiated with 365 nm light. The upper right image in Figure 4 shows the same group of cells observed under bright field light. It can be seen that the cells are distributed almost uniformly throughout the group.

[0101] The bottom left image in Figure 4 shows the GFP fluorescence observed under a fluorescence microscope in a group of cells to which 150 nM 5-phenylazoindole-3-acetic acid had been added, and which was then irradiated entirely with weak 365 nm light, with partial 520 nm light as background light. GFP was selectively degraded only in the cells irradiated with 520 nm light. The bottom right image in Figure 4 shows the same group of cells observed under a bright field. It can be seen that the cells are distributed almost uniformly throughout the group. [Industrial Applicability]

[0102] The light-responsive auxin degron system of the present invention makes it possible to degrade target proteins in a location- or time-selective manner. [Sequence List Free Text]

[0103] SEQ ID NO: 1: Amino acid sequence of TIR1 protein from Arabidopsis thaliana MQKRIALSFPEEVLEHVFSFIQLDKDRNSVSLVCKSWYEIERWCRRKVFIGNCYAVSPATVIRRFPKVRSVELKGKPHFADFNLVPDGWGGYVYPWIEAMSSSYTWLEEIRLKRMVVTDDCLELIAKSFKNFKVLVLSSCEGFSTDGL AAIAATCRNLKELDLRESDVDDVSGHWLSHFPDTYTSLVSLNISCLASEVSFSALERLVTRCPNLKSLKLNRAVPLEKLATLLQRAPQLEELGTGGYTAEVRPDVYSGLSVALSGCKELRCLSGFWDAVPAYLPAVYSVCSRLTTLNLS YATVQSYDLVKLLCQCPKLQRLWVLDYIEDAGLEVLASTCKDLRELRVFPSEPFVMEPNVALTEQGLVSVSMGCPKLESVLYFCRQMTNAALITIARNRPNMTRFRLCIIEPKAPDYLTLEPLDIGFGAIVEHCKDLRRLSLSGLLTD KVFEYIGTYAKKMEMLSVAFAGDSDLGMHHVLSGCDSLRKLEIRDCPFGDKALLANASKLETMRSLWMSSCSVSFGACKLLGQKMPKLNVEVIDERGAPDSRPESCPVERVFIYRTVAGPRFDMPGFVWNMDQDSTMRFSRQIITTNGL SEQ ID NO: 2: SEQ ID NO: 3: DNA encoding the F79G mutant of the TIR1 protein derived from Arabidopsis thaliana SEQ ID NO: 3: Amino acid sequence of the F79G mutant of the TIR1 protein from Arabidopsis thaliana MQKRIALSFPEEVLEHVFSFIQLDKDRNSVSLVCKSWYEIERWCRRKVFIGNCYAVSPATVIRRFPKVRSVELKGKPHGADFNLVPDGWGGYVYPWIEAMSSSYTWLEEIRLKRMVVTDDCLELIAKSFKNFKVLVLSSCEGFSTDGL AAIAATCRNLKELDLRESDVDDVSGHWLSHFPDTYTSLVSLNISCLASEVSFSALERLVTRCPNLKSLKLNRAVPLEKLATLLQRAPQLEELGTGGYTAEVRPDVYSGLSVALSGCKELRCLSGFWDAVPAYLPAVYSVCSRLTTLNLS YATVQSYDLVKLLCQCPKLQRLWVLDYIEDAGLEVLASTCKDLRELRVFPSEPFVMEPNVALTEQGLVSVSMGCPKLESVLYFCRQMTNAALITIARNRPNMTRFRLCIIEPKAPDYLTLEPLDIGFGAIVEHCKDLRRLSLSGLLTD KVFEYIGTYAKKMEMLSVAFAGDSDLGMHHVLSGCDSLRKLEIRDCPFGDKALLANASKLETMRSLWMSSCSVSFGACKLLGQKMPKLNVEVIDERGAPDSRPESCPVERVFIYRTVAGPRFDMPGFVWNMDQDSTMRFSRQIITTNGL SEQ ID NO: 4: Amino acid sequence of mAID (mini Auxin Inducible Degron) KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVKVSMDGAPYLRKIDLRMYK SEQ ID NO: 5: AID * The amino acid sequence of PKDPAKPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK

Claims

1. Formula (I): 【Chemistry 1】 (I) [In the formula, R １ is an optionally substituted 6- to 10-membered aryl group or a 5- or 6-membered heteroaryl group; R ２ is -OR ３ or -N(R ４ ) (R ５ ) and R ３ , R ４ and R ５ are each independently H or C １－６ alkyl] or a salt thereof.

2. R １ 2. The compound or salt thereof according to claim 1, wherein the -N=N- group is attached at the 5- or 6-position of the indole ring.

3. R １ 3. The compound or salt thereof according to claim 1 or 2, wherein the -N=N- group is attached at the 5-position of the indole ring.

4. The compound or salt thereof according to any one of claims 1 to 3, wherein the aryl group is phenyl.

5. 4. The compound or salt thereof according to claim 1, wherein the heteroaryl group is thiazole.

6. R １ But -CN, -CX ３ (wherein each X is independently a halogen), —NO ２ , -COR １ａ (where R １ａ is R １１ａ OR １１ａ and R １１ａ is H or C １－６ alkyl), -OR １ｂ (where R １ｂ is C １－６ alkyl), —N(R １ｃ ) (R １ｄ ) (where R １ｃ and R １ｄ are each independently H or C １－６ alkyl), and —SO ２ R １ｅ (where R １ｅ is OH or C １－６ 6. The compound or salt thereof according to claim 1, wherein the aryl or heteroaryl group is substituted with at least one group selected from the group consisting of alkyl, aryl, aryl- ...

7. The compound or salt thereof according to any one of claims 1 to 4 or 6, wherein the compound is 5-phenylazoindole-3-acetic acid or 6-phenylazoindole-3-acetic acid.

8. The compound is irradiated with light having a wavelength of 400 to 650 nm to produce R １ The compound or salt thereof according to any one of claims 1 to 7, wherein the indole ring is in a trans configuration.

9. The compound is irradiated with light having a wavelength of 320 to 580 nm to produce R １ and the indole ring exhibits a cis configuration, or a salt thereof, according to any one of claims 1 to 8.

10. A target protein degradation regulator comprising the compound or salt thereof according to any one of claims 1 to 9.

11. The agent according to claim 10 , wherein the target protein is expressed intracellularly.

12. A method for controlling the degradation of a target protein, comprising the steps of: operably linking a first nucleic acid encoding a degradation tag to a nucleic acid encoding a target protein; expressing the first nucleic acid and a second nucleic acid encoding a mutant TIR1 family protein in a cell; The cells in which the first and second nucleic acids have been expressed are contacted with the compound or salt thereof according to any one of claims 1 to 9, or the agent according to claim 10 or 11, and then a step of irradiating light onto the cells that have been contacted with the compound or its salt or agent to control degradation of the target protein.

1. A method comprising: the degradation tag comprises a full-length or partial protein of an Aux / IAA family protein and has affinity for the mutant TIR1 family protein-compound of formula (I) complex; The mutant TIR1 family protein is one of the following (a) to (c): (a) an amino acid sequence in which phenylalanine at amino acid position 79 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine, alanine, or serine; (b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added at a site other than amino acid position 79 of (a); and (c) an amino acid sequence having 80% or more identity at a site other than amino acid position 79 of (a) and which binds to the degradation tag via the compound of formula (I) to guide the target protein to degradation.

13. The method according to claim 12, wherein phenylalanine at amino acid position 79 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine.

14. A kit for controlling degradation of a target protein, comprising the compound according to any one of claims 1 to 9 or a salt thereof, or the agent according to claim 10 or 11, a first nucleic acid encoding a degradation tag, and a second nucleic acid encoding a mutant TIR1 family protein, the degradation tag comprises a full-length or partial protein of an Aux / IAA family protein and has affinity for the mutant TIR1 family protein-compound of formula (I) complex; The mutant TIR1 family protein is one of the following (a) to (c): (a) an amino acid sequence in which phenylalanine at amino acid position 79 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with glycine; (b) an amino acid sequence in which one to several amino acids are deleted, inserted, substituted, or added at a site other than amino acid position 79 of (a); and (c) an amino acid sequence having 80% or more identity at a site other than amino acid position 79 of (a) and a protein that binds to the degradation tag via the compound of formula (I) to guide the target protein to degradation.

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