Method for degrading target polypeptides

Abstract

To provide an AID-based targeted polypeptide degradation technology that allows control over the location at which the degradation of the target polypeptide occurs. [Solution] A method for degrading a target polypeptide, comprising irradiating cells containing a photodegradable auxin derivative, in which auxin or an auxin derivative is substituted with a photodegradable protecting group, and an auxin degron system with light.

Description

[Technical Field] 【0001】 This invention relates to a method for degrading a target polypeptide, etc. [Background technology] 【0002】 Target polypeptide degradation systems are widely used for gene function analysis. Among these, the auxin-inducible degron (AID) system can degrade target polypeptides in an auxin-dependent manner. In this system, the plant F-box protein Transport inhibitor response 1 (TIR1), known as the auxin receptor, is introduced into non-plant eukaryotes. TIR1 then forms an SCF complex with the endogenous Skp1-Cul1-Rbx1. In these organisms, the target polypeptide fused with the AID tag is polyubiquitinated in an auxin-dependent manner by SCF-TIR1 and degraded by the 26S proteasome. In vertebrates, TIR1 derived from Oriza sative (OsTIR1) is usually used. In recent years, several improved AID methods have been developed to reduce the cytotoxicity caused by auxin overdose (Patent Document 1, Non-Patent Document 1). In the ultra-high-sensitivity auxin-inducible degron (ssAID), OsTIR1 F74A The combination of mutants and 5-Adantyl-IAA (5-Ad-IAA), OsTIR1 F74G A combination of mutants and 5-Phenyl-IAA is used. In all cases, degradation of the target is sufficient with nM to pM degradation inducers. [Prior art documents] [Patent Documents] 【0003】 [Patent Document 1] International Publication No. 2013 / 073653 [Patent Document 2] International Publication No. 2018 / 164214 [Non-patent literature] 【0004】 [Non-Patent Document 1] Nucleic Acids Res. 2020 Oct 9;48(18):e108. doi: 10.1093 / nar / gkaa748. [Non-Patent Document 2] Bio Protoc. 2021 Jul 20;11(14):e4092. doi: 10.21769 / BioProtoc.4092. [Overview of the Initiative] [Problems that the invention aims to solve] 【0005】 Conventional target polypeptide degradation using AID systems is triggered by the addition of auxin or auxin derivatives. The inventors focused on the limitation that conventional methods have limitations in controlling the location where target polypeptide degradation occurs. While the location of auxin or auxin derivative addition can be controlled, it is difficult to control the extent to which it spreads in the culture medium or living organism. 【0006】 The present invention aims to provide an AID-based targeted polypeptide degradation technology that allows control over the location at which the degradation of the target polypeptide occurs. [Means for solving the problem] 【0007】 In view of the above problems, the inventors diligently conducted research and found that a method for degrading a target polypeptide, comprising irradiating cells containing a photodegradable auxin derivative (in which auxin or an auxin derivative is substituted with a photodegradable protecting group) and an auxin degron system with light, can solve the above problems. Based on this finding, the inventors furthered their research and completed the present invention. That is, the present invention encompasses the following aspects. 【0008】 Section 1. General formula (1): 【0009】 [ka] 【0010】 [In the formula, n and m are the same or different and each represents 0 or 1. R 1 and R 2 are the same or different and each represents a hydrogen atom, an optionally substituted adamantyl group, an optionally substituted aryl group, an optionally substituted alkyl group, or an optionally substituted heterocyclic group (however, the case where both R 1 and R 2 are simultaneously hydrogen atoms is excluded). One of R 3 and R 4 represents a carboxyalkyl group and the other represents a hydrogen atom. X represents -NH- or -CH=CH-.] A photodegradable auxin derivative in which a hydrogen atom in the carboxy group of R 3 or R 4 in the compound represented by is replaced with a photodegradable protecting group. 【0011】 Item 2. The photodegradable auxin derivative according to Item 1, wherein the photodegradable protecting group is a nitrobenzyl-type photodegradable protecting group. 【0012】 Item 3. A decomposition inducer for a target polypeptide, comprising a photodegradable auxin derivative in which an auxin or an auxin derivative is substituted with a photodegradable protecting group. 【0013】 Item 4. The decomposition inducer according to Item 3, wherein the photodegradable auxin derivative is the photodegradable auxin derivative according to Item 1 or 2. 【0014】 Item 5. A decomposition induction kit for a target polypeptide, comprising a photodegradable auxin derivative in which an auxin or an auxin derivative is substituted with a photodegradable protecting group, and a polynucleotide for introducing an auxin degron system. 【0015】 Item 6. The decomposition induction kit according to Item 5, wherein the photodegradable auxin derivative is the photodegradable auxin derivative according to Item 1 or 2. 【0016】 Item 7. A cell comprising a photodegradable auxin derivative, wherein auxin or an auxin derivative is substituted with a photodegradable protecting group, and an auxin degron system. 【0017】 Item 8. The cell according to item 7, wherein the photodegradable auxin derivative is the photodegradable auxin derivative according to item 1 or 2. 【0018】 Item 9. A method for degrading a target polypeptide, comprising irradiating cells containing a photodegradable auxin derivative, in which auxin or an auxin derivative is substituted with a photodegradable protecting group, and an auxin degron system with light. 【0019】 Item 10. The decomposition method according to item 9, wherein the photodegradable protecting group is a nitrobenzyl type photodegradable protecting group. 【0020】 Item 11. The decomposition method according to item 9 or 10, wherein the photodegradable auxin derivative is a photodegradable auxin derivative in which a hydrogen atom in the carboxyl group of the auxin or the auxin derivative is replaced with the photodegradable protecting group. 【0021】 Section 12. The auxin derivative is of general formula (1): 【0022】 [ka] 【0023】 [In the formula, n and m are the same or different, and represent 0 or 1. R 1 and R 2 These are the same or different, each representing a hydrogen atom, an optionally substituted adamantyl group, an optionally substituted aryl group, an optionally substituted alkyl group, or an optionally substituted heterocyclic group (however, R 1 and R 2 (Except when all are hydrogen atoms at the same time). 3 and R 4 One side represents a carboxyalkyl group, and the other represents a hydrogen atom. X represents -NH- or -CH=CH-. A compound represented by the terms, as described in any of items 9 to 11, and a method of decomposition. 【0024】 Item 13. The photodegradable auxin derivative is the R of the compound represented by the general formula (1). 3 or R 4 The decomposition method according to item 12, wherein the hydrogen atom in the carboxyl group of is replaced by the photodegradable protecting group, the photodegradable auxin derivative. 【0025】 Item 14. The degradation method according to any one of items 9 to 13, wherein the auxin degron system comprises polynucleotide A containing the coding sequence of a TIR1 family protein, and polynucleotide B containing the coding sequence of an Aux / IAA family protein or a partial peptide thereof. 【0026】 Item 15. The degradation method according to Item 14, further comprising a coding sequence for a target polypeptide or a coding sequence for a binding molecule to the target polypeptide, so that the polynucleotide B can be expressed by fusing it with the Aux / IAA family protein or a partial peptide thereof. 【0027】 Item 16. The degradation method according to item 14 or 15, wherein the TIR1 family protein is a mutant TIR1 family protein in which auxin-receptority is reduced, in which an amino acid residue that interacts with the benzene ring in the auxin indole ring is replaced with another amino acid residue. [Effects of the Invention] 【0028】 According to the present invention, it is possible to provide an AID-based targeted polypeptide degradation technology that allows control over the location at which the degradation of the target polypeptide occurs. [Brief explanation of the drawing] 【0029】 [Figure 1]A conceptual diagram of the AlissAID system is shown. The target protein is recognized and recruited by SCF-OsTIR1F74A via an AID binder fusion protein in a 5-adamantyl IAA-dependent manner. Subsequently, it undergoes polyubiquitination and is degraded by the 26S proteasome. [Figure 2] This shows that the GFP fusion protein is degraded by AlissAID vhhGFP4. (A) An overview of the AlissAID system targeting the GFP fusion protein is shown. The target protein is recognized by mAID-vhhGFP4. (B) Immunoblots of CDK1-GFP AlissAID mouse ES cells are shown. Cells were treated with 5 μM 5-Ad-IAA and 20 μM MG132. (C) Colony formation assay of this strain. Cells were cultured in medium containing 5 μM 5-Ad-IAA and stained with crystal violet. (D) Cell cycle analysis of this strain. Cells were treated with 5 μM 5-Ad-IAA for 16 hours. (E) Immunoblots of CENP-H-GFP AlissAID DT40 cells. Cells were treated with 5 μM 5-Ad-IAA and 20 μM MG132. (F) Cell cycle analysis of CENP-H-GFP AlissAID DT40 cells is shown. The cells were treated with 5 μM 5-Ad-IAA for 16 hours. [Figure 3] AlissAID LaM2 demonstrates the degradation of mCherry fusion proteins. (A) An overview of the AlissAID system targeting mCherry fusion proteins is shown. The target protein is recognized by mAID-LaM2. (B) Immunoblots of CENP-H-mCherry AlissAID DT40 cells are shown. LaM2, LaM4, or LaM8 were used as mCherry conjugates. Cells were treated with 5 μM 5-Ad-IAA and 20 μM MG132. (C) Cell cycle analysis of these strains is shown. Cells were treated with 5 μM 5-Ad-IAA for 16 hours. [Figure 4]This shows endogenous protein degradation by the AlissAID system. (A) Overview of the K55-AlissAID system. The target protein is recognized by mAID-DARPin K55. (B) Immunoblots of K55-AlissAID HeLa cells are shown. Cells were treated with 5 μM 5-Ad-IAA. (C) Overview of the K55-AlissAID system. The target protein is recognized by mAID-Monobody NS1. (D) Immunoblots of NS1-AlissAID HeLa cells are shown. Cells were treated with 5 μM 5-Ad-IAA. (E) Flow cytometry of GFP-Ras stable expression HEK293T cells transiently transfected with pAlissAID TT vhhGFP4 or NS1 is shown. 5 μM 5-Ad-IAA was added to induce degradation. GFP intensity is plotted on the x-axis and mCherry intensity on the y-axis, both on a logarithmic scale. [Figure 5] This paper shows the proteolytic degradation and phenotypic analysis induced by NS1-AlissAID. (A) Immunoblots of AlissAID T24 cells are shown. Cells were treated with 5 μM 5-Ad-IAA. (B) Colony formation assays of T24 original cells and NS1-AlissAID cells are shown. Cells were cultured in 5 μM 5-Ad-IAA-containing medium and stained with crystal violet. (C, D) Volcano plots (n = 3 biological replications) showing the log2-converted multiplicative changes in gene expression relative to the mean normalized count of each gene in T24 original cells and T24 NS1-AlissAID cells treated with DMSO (control) or 5 μM 5-Ad-IAA for 24 hours are shown. (E) TPM of each gene is shown. Error bars represent mean ± SD. [Figure 6]This shows KRas mutant-specific degradation by the AlissAID system. (A) Flow cytometry of GFP-KRas stably expressing HEK293T cells. pAlissAID TT vhhGFP4, 12VC1, or R11.1.6 was added. Degradation was induced by adding 5 μM 5-Ad-IAA. GFP intensity is plotted on the x-axis and mCherry intensity on the y-axis (both on a logarithmic scale). (B) Fluorescence observation of GFP-KRas stably expressing HEK293T cells transiently transfected with pAlissAID TT vhhGFP4 or 12VC1. Cells were treated with 5 μM 5-Ad-IAA for 24 hours. Scale bar = 10 μm. [Figure 7] This shows the degradation of targeted proteins in mouse embryos using the AlissAID system. (A) Overview of the degradation assay. (B) Time-lapse imaging of mouse embryos (E1.5). Embryos were treated with 5 μM 5-Ad-IAA. EGFP signals were normalized with mCherry signals and associated with time 0. [Figure 8] This shows photo-induced proteolysis by caged 5-Ad-IAA. (A) A conceptual diagram showing photoactivation of caged 5-Ad-IAA is shown. (B) This shows the inducer-dependent decrease in luciferase activity. Cells were treated with various concentrations of auxin and 365 nm light (red: 5-Ad-IAA, black: caged 5-Ad-IAA, dark, blue: caged 5-Ad-IAA and light). Luciferase activity was analyzed after 2 hours of treatment. Mean ± SD of bar graph (n = 3 biological replications). (C) This shows fluorescence observation of CENP-H-GFP in AlissAID DT40 cells. Cells were treated with 50 nM caged 5-Ad-IAA and light. Scale bar = 10 μm. (D) This shows cell cycle analysis of CENP-H-GFP AlissAID DT40 cells. Cells were treated with 50 nM caged 5-Ad-IAA and light. [Modes for carrying out the invention] 【0030】 In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.” 【0031】 The "identity" of amino acid sequences refers to the degree of agreement between two or more comparable amino acid sequences. Therefore, the higher the agreement between two amino acid sequences, the higher their identity or similarity. The level of amino acid sequence identity can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (S. Karlin, SF Altschul. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes." Proc. Natl. Acad. Sci. USA 87, 2264-2268 (1990), S. Karlin, SF Altschul. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc. Natl. Acad. Sci. USA 90, 5873-5877 (1993)). A program called BLASTX has been developed based on this BLAST algorithm. The specific methods for these analyses are publicly known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). Furthermore, the 'identity' of a nucleotide sequence is defined in accordance with the above. 【0032】 In this specification, "conservative substitution" means that an amino acid residue is substituted for an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, are considered conservative substitutions. Other examples of conservative substitutions include amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine. 【0033】 In this specification, polynucleotides include both natural and artificial ones. Polynucleotides such as DNA and RNA may be subjected to known chemical modifications, as exemplified below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues of each nucleotide can be replaced with chemically modified phosphate residues such as phosphorothioates (PS), methylphosphonates, and phosphorodithionates. The hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may also be replaced with -OR (where R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at position 5 of the pyrimidine base, or by substituting the carbonyl group at position 2 with a thiocarbonyl group. Furthermore, examples include, but are not limited to, those in which the phosphate or hydroxyl portion is modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group. In addition, BNA (LNA), in which the conformation of the sugar portion of the nucleotide is fixed to the N-type by cross-linking the 2' oxygen and 4' carbon atoms of the sugar portion, can also be used. Furthermore, peptide nucleic acids (e.g., PNA) can also be used. 【0034】 In this specification, a coding sequence is a nucleotide sequence consisting of a sequence of codons that encode the amino acid sequence of a protein / polypeptide. 【0035】 1. Methods for degrading target proteins In one aspect, the present invention relates to a method for degrading a target polypeptide (which may be referred to herein as "the degradation method of the present invention") comprising irradiating cells containing a photodegradable auxin derivative, in which auxin or an auxin derivative is substituted with a photodegradable protecting group, and an auxin degron system with light. 【0036】 Auxin is a plant hormone that regulates plant growth. While indole-3-acetic acid is a representative example of auxin, other substances with the above-mentioned effects include 4-chloroindole-3-acetic acid, naphthaleneacetic acid, α-naphthylacetamide, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid. 【0037】 The auxin derivative is not particularly limited, as long as it can bind to both TIR1 family proteins and Aux / IAA family proteins, thereby driving the auxin degron system and causing degradation of the target polypeptide. This binding affinity can be evaluated by the yeast two-hybrid assay shown in Figure 2 of Non-Patent Literature 1. 【0038】 Examples of auxin derivatives include compounds with improved or reduced binding affinity to wild-type TIR1 family proteins and / or wild-type Aux / IAA family proteins. In one aspect of the present invention, an auxin derivative is a compound (Patent Document 2) that has reduced binding affinity to endogenous auxin receptors (TIR1 family proteins) and good binding affinity to auxin-receptor-reduced TIR1 family proteins. More specifically, general formula (1): 【0039】 [ka] 【0040】 [In the formula, n and m are the same or different, and represent 0 or 1. R 1 and R 2 These are the same or different, each representing a hydrogen atom, an optionally substituted adamantyl group, an optionally substituted aryl group, an optionally substituted alkyl group, or an optionally substituted heterocyclic group (however, R 1 and R 2 (Except when all are hydrogen atoms at the same time).3 and R 4 One side represents a carboxyalkyl group, and the other represents a hydrogen atom. X represents -NH- or -CH=CH-. Examples of compounds represented by [the formula shown] are given. 【0041】 n is preferably 0. 【0042】 m is preferably 0. 【0043】 R 1 or R 2 The aryl group represented by is not particularly limited, but preferably has 6 to 50 carbon atoms, more preferably 6 to 30 carbon atoms, even more preferably 6 to 20 carbon atoms, even more preferably 6 to 12 carbon atoms, and most preferably 6 to 8 carbon atoms.Specific examples of such aryl groups include phenyl group, naphthyl group, phenylalkyl group (e.g., benzyl group, phenethyl group, etc.), biphenyl group, pentarenyl group, indenyl group, anthranyl group, tetracenyl group, pentacenyl group, pyrenyl group, perilenyl group, fluorenyl group, phenanthryl group, etc. Preferably phenyl group, naphthyl group, phenylalkyl group, biphenyl group, etc. More preferably phenyl group, naphthyl group, phenylalkyl group, etc., even more preferably phenyl group, benzyl group, etc., and even more preferably phenyl group. 【0044】 R 1 or R 2The substituents that the aryl group represented by may have are not particularly limited, but include, for example, optionally substituted alkyl groups, optionally substituted alkoxy groups, halogen atoms (F, Br, Cl, etc.), optionally substituted aryl groups, optionally substituted aryloxy groups, hydroxyl groups, heteroatom-containing groups, optionally substituted alkenyl groups, optionally substituted alkynyl groups, -COOR” (where R is a hydrogen atom or a hydrocarbon group), and so on. Preferably, these include optionally substituted alkyl groups, optionally substituted alkoxy groups, halogen atoms, optionally substituted aryl groups, optionally substituted aryloxy groups, and so on. More preferably, these include optionally substituted alkyl groups, optionally substituted alkoxy groups, halogen atoms, and so on. 【0045】 R 1 or R 2 The alkyl group that the aryl group represented by may have or may be substituted is not particularly limited, and examples include linear, branched, or cyclic alkyl groups having 1 to 20 carbon atoms, preferably 1 to 12, more preferably 1 to 6, even more preferably 1 to 3, and even more preferably 1 carbon atom, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, perfluoromethyl, perfluoroethyl, and cyclohexyl groups. 【0046】 R 1 or R 2The alkoxy group that the aryl group represented by may have or may be substituted is not particularly limited and includes linear or branched alkoxy groups having 1 to 20 carbon atoms, preferably 1 to 12, more preferably 1 to 6, even more preferably 1 to 3, and even more preferably 1 carbon atom, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted alkyl groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy, perfluoromethoxy, and perfluoroethoxy groups. 【0047】 R 1 or R 2 The halogen atoms that the aryl group represented by the symbol may have are preferably F, Cl, and the like. 【0048】 R 1 or R 2 The aryl group that the aryl group represented by may have or may be substituted is not particularly limited, and examples include aryl groups having 6 to 12 carbon atoms, preferably 6 to 8 carbon atoms, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted aryl groups include phenyl, naphthyl, benzyl, and phenethyl groups. 【0049】 R 1 or R 2The aryloxy group that the aryl group represented by may have or may be substituted is not particularly limited, and examples include aryloxy groups having 6 to 12 carbon atoms, preferably 6 to 8 carbon atoms, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted aryloxy groups include phenoxy, naphthoxy, benzyloxy, and phenethyloxy groups. 【0050】 R 1 or R 2 The heteroatom-containing group that the aryl group represented by may have is preferably a linear, branched, or cyclic group having at least one heteroatom such as a nitrogen atom (N), oxygen atom (O), sulfur atom (S), boron atom (B), phosphorus atom (P), or silicon atom (Si), particularly at least one of a nitrogen atom (N), oxygen atom (O), or sulfur atom (S). Specifically, examples include cyano(-CN) groups, nitro(-NO2) groups, amino groups, and groups obtained by removing one hydrogen atom from heterocycles such as furan rings, thiophene rings, pyrrole rings, silole rings, borol rings, phosphole rings, oxazole rings, thiazole rings, pyridine rings, pyridazine rings, pyrimidine rings, and pyrazine rings. Groups obtained by removing one hydrogen atom from fused rings (thienothiophene rings, quinoline rings, etc.) of the above heterocycles or benzene rings can also be used. 【0051】 R 1 or R 2The alkenyl group that the aryl group represented by may have or may be substituted is not particularly limited and includes linear, branched, or cyclic alkenyl groups having 2 to 20 carbon atoms, preferably 2 to 12, more preferably 2 to 6, and even more preferably 2 to 3 carbon atoms, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted alkenyl groups include vinyl, allyl, 1-propenyl, isopropenyl, butenyl, pentenyl, and hexenyl groups. 【0052】 R 1 or R 2 The alkynyl group that the aryl group represented by may have or may be substituted is not particularly limited and includes linear, branched, or cyclic alkynyl groups having 2 to 20 carbon atoms, preferably 2 to 12, more preferably 2 to 6, and even more preferably 2 to 3 carbon atoms, which may be substituted with halogen atoms (F, Br, Cl, I, etc.). The number of substituents is not particularly limited, preferably 0 to 6, more preferably 0 to 3, and even more preferably 0. Examples of such substituted alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and phenylacetylyl groups. 【0053】 R 1 or R 2 The aryl group represented by -COOR'' may have a hydrogen atom or a hydrocarbon group, and a hydrogen atom or the alkyl group described above is preferred. Specifically, examples of -COOR'' include -COOH, -COOCH3, -COOC2H5, -COOC3H7, -COOC(CH3)2, -COOC4H9, -COOCH(CH3)C2H5, -COOCH2CH(CH3)2, and -COOC(CH3)3. 【0054】 R 1 or R 2The number of substituents that the aryl group represented by may have is not particularly limited, for example, 0 to 6, preferably 1 to 3, and more preferably 1 to 2. In a preferred embodiment of the present invention, R 1 or R 2 When the substituents that the aryl group represented by may have are at least one selected from the group consisting of alkyl groups and halogen atoms, the number of substituents is preferably two, and when the substituent is an alkoxy group, the number of substituents is preferably one. 【0055】 R 1 or R 2 If the aryl group represented by has two or more substituents, two adjacent substituents may bond to each other to form a ring. Forming a ring means, for example, if the aryl group is a phenyl group, then, for example, in formula (1): 【0056】 [ka] 【0057】 [In the formula, R' and R'' are R 1 This indicates substituents that the aryl group represented by may have. n is the same as above. The base shown by is, for example, in formula: 【0058】 [ka] 【0059】 [In the formula, n is the same as above.] This means that it is the group represented by [the symbol]. 【0060】 R 1 or R 2 The adamantyl group represented by is not particularly limited, and examples include a 1-adamantyl group and a 2-adamantyl group. Among these, the 1-adamantyl group is preferred. 【0061】 R 1 or R2 The adamantyl group shown may have substituents such as R 1 or R 2 Examples of substituents that may be present on the aryl group shown can be found. 【0062】 R 1 or R 2 The number of substituents that the adamantyl group represented by may have is not particularly limited, for example, 0 to 6, preferably 0 to 3, and more preferably 0. 【0063】 R 1 or R 2 The alkyl group represented by is not particularly limited, but examples include linear, branched, or cyclic (preferably cyclic) alkyl groups having 1 to 20 carbon atoms, preferably 3 to 20, more preferably 4 to 15, and even more preferably 6 to 12 carbon atoms. Specific examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and cyclohexyl groups. 【0064】 R 1 or R 2 The substituents that the alkyl group shown may have include R 1 or R 2 Examples of substituents that may be present on the aryl group shown can be found. 【0065】 R 1 or R 2 The number of substituents that the alkyl group represented by may have is not particularly limited, for example, 0 to 6, preferably 0 to 3, and more preferably 0. 1 or R 2The heterocyclic group represented by is not particularly limited, but examples include groups obtained by removing one hydrogen atom from heterocyclic rings such as furan rings, thiophene rings, pyrrole rings, silole rings, borol rings, phosphole rings, oxazole rings, thiazole rings, pyridine rings, pyridazine rings, pyrimidine rings, and pyrazine rings. In addition, groups obtained by removing one hydrogen atom from fused rings of the above heterocyclic rings or from fused rings of these heterocyclic rings with benzene rings, etc. (such as benzothiephene rings, thienothiophene rings, and quinoline rings) can also be used. 【0066】 R 1 or R 2 The substituents that the heterocyclic group shown may have include R 1 or R 2 Examples of substituents that may be present on the aryl group shown can be found. 【0067】 R 1 or R 2 The number of substituents that the heterocyclic group represented by may have is not particularly limited, and is, for example, 0 to 6, preferably 0 to 3, and more preferably 0. 【0068】 R 1 -(O) n - and R 2 -(O) m - Preferably, one of them is a hydrogen atom. 2 -(O) m The one with the hyphen is a hydrogen atom. 【0069】 R 1 -(O) n -, R 2 -(O) m Specifically, in addition to hydrogen atoms, preferably 【0070】 [ka] 【0071】 [ka] 【0072】 【Chem.】 【0073】 etc. can be mentioned, and more preferably 【0074】 【Chem.】 【0075】 etc. are included. 【0076】 Also, as R 1 -(O) n -, preferably, in addition to a hydrogen atom, 【0077】 【Chem.】 【0078】 【Chem.】 【0079】 etc. can be mentioned, and as R 2 -(O) m -, preferably, in addition to a hydrogen atom, 【0080】 【Chem.】 【0081】 etc. are included. 【0082】 R 3 and R 4 one of them is a carboxyalkyl group and the other is a hydrogen atom. In a preferred embodiment, R 3 is a hydrogen atom and R 4is a carboxyalkyl group. In the carboxyalkyl group, the alkyl group is as described above for the alkyl group. That is, a carboxymethyl group (-CH2COOH), a carboxyethyl group (-C2H4COOH), a carboxypropyl group (-C3H6COOH), a carboxybutyl group (-C4H8COOH), a carboxypentyl group (-C5H 10 COOH), a carboxyhexyl group (-C6H 12 COOH), etc. are exemplified. 【0083】 X is preferably -NH-. 【0084】 In one aspect of the present invention, as the compound represented by the general formula (1), preferably the general formula (1A): 【0085】 【Chemical formula】 【0086】 [In the formula, n, m, R 1 , R 2 , R 3 , and R 4 are the same as described above.] The compound represented by is exemplified, and more preferably the general formula (1A1): 【0087】 【Chemical formula】 【0088】 [In the formula, n, m, R 1 , R 2 , and R 3 are the same as described above. R 4 is a carboxyalkyl group.] The compound represented by is exemplified, and even more preferably the general formula (1A1a): 【0089】 <00005​​​​​​ [In the formula, n and R 1 The same applies as above. R 4 It is a carboxyalkyl group. Examples of compounds represented by [the formula shown] are given. 【0091】 In another embodiment of the present invention, the compound represented by general formula (1) is as follows: R 2 -(O) m - is a hydrogen atom, and If n is 0, then R 1 teeth (a) an optional adamantyl group, (b) an aryl group which may be substituted with at least one substituent selected from the group consisting of aryl groups and aryloxy groups, or an alkyl group, alkoxy group, or halogen atom and at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups, (c) A C5-C20 alkyl group which may be substituted with at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups; or If n is 1, then R 1 teeth (d) Adamantyl group which may be substituted, (e) A benzyl group substituted with an optionally substituted phenyl group, an optionally substituted naphthyl group, or at least one substituent selected from the group consisting of alkyl groups, halogen atoms, aryl groups, and aryloxy groups, or (f) A C6-C20 alkyl group which may be substituted with at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups; or R 1 -(O) n - is a hydrogen atom, and If m is 0, then R 2 teeth (g) an optional adamantyl group, (h) At least one substituent selected from the group consisting of alkoxy groups, aryl groups, and aryloxy groups, or an aryl group substituted with an alkyl group or halogen atom and at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups, (i) an alkyl group having 4 to 20 carbon atoms, which may be substituted; or If m is 1, then R 2 teeth (j) Adamantyl group which may be substituted, (k) A phenyl group or naphthyl group which may be substituted with at least one substituent selected from the group consisting of alkyl groups, alkoxy groups, halogen atoms, aryl groups, and aryloxy groups, or (l) A C4-C20 alkyl group which may be substituted. The configuration is preferable. 【0092】 Compounds represented by general formula (1) can be produced, for example, in accordance with or in accordance with the method described in Patent Document 2. 【0093】 A photodegradable protecting group is a protecting group that is removed by light irradiation, and is not particularly limited in that respect. Photodegradable hydrophobic protecting groups are well known, and examples include nitrobenzyl-type photodegradable protecting groups (i.e., groups having a nitrobenzyl (particularly preferably 2-nitrobenzyl) derivative skeleton), benzophenone-type photodegradable protecting groups, bromocoumarin-type photodegradable protecting groups, and the like. 【0094】 Specifically, photodegradable hydrophobic protecting groups include, for example, (2,5-dimethoxyphenyl)(2-nitrobenzyl) ester (DMPNB) group, nitrobenzyl group, nitrophenyl ethyl ester group (NPE), dimethoxynitrobenzyl ester group (DMNB), bromohydroxycoumarin (Bhc) group, dimethoxybenzoin group, 2-nitropiperonyloxycarbonyl (NPOC) group, 2-nitroberatryloxycarbonyl (NVOC) group, 5'-(α-methyl-2-nitropiperonyl)oxycarbonyl (MeNPOC) group, 2-(2-nitro-4-ethyl-5-thiophenylphenyl)propyloxycarbonyl (PhSNPPOC) group, α-methyl-2-nitroberatryloxycarbonyl (MeNVOC) group, 2,6-dinitrobenzyloxycarbonyl (DNBOC) group, and α-methyl-2,6-dinitrobenzyl Examples include the oxycarbonyl (MeDNBOC) group, 1-(2-nitrophenyl)ethyloxycarbonyl (NPEOC) group, 1-methyl-1-(2-nitrophenyl)ethyloxycarbonyl (MeNPEOC) group, 9-anthracenylmethyloxycarbonyl (ANMOC) group, 1-pyrenylmethyloxycarbonyl (PYMOC) group, 3'-methoxybenzoinyloxycarbonyl (MBOC) group, 3',5'-dimethoxybenzoyloxycarbonyl (DMBOC) group, 7-nitroindolinyloxycarbonyl (NIOC) group, 5,7-dinitroindolinyloxycarbonyl (DNIOC) group, 2-anthraquinonylmethyloxycarbonyl (AQMOC) group, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl group, 5-bromo-7-nitroindolinyloxycarbonyl (BNIOC) group, and so on. 【0095】 The photodegradable hydrophobic protecting group is preferably a nitrobenzyl type photodegradable protecting group, and particularly preferably a DMPNB group, from the viewpoint of suppressing the degradation of the target polypeptide under non-light irradiation and / or improving the degradation of the target polypeptide under light irradiation. 【0096】 Photodegradable auxin derivatives are obtained by substituting auxin or an auxin derivative with a photodegradable protecting group, and are not particularly limited as long as the photodegradable protecting group is removed by light irradiation. The form of substitution may vary depending on the type of photodegradable protecting group, but specifically, examples include forms in which hydrogen atoms in the hydroxyl group, the hydroxyl group, the amide group, the amino group, or the thiol group of auxin or an auxin derivative are replaced with a photodegradable protecting group. 【0097】 The photodegradable auxin derivative is preferably a photodegradable auxin derivative in which a hydrogen atom in the carboxyl group of auxin or an auxin derivative is replaced with a photodegradable protecting group, from the viewpoint of suppressing the degradation of the target polypeptide under non-light irradiation and / or improving the degradation of the target polypeptide under light irradiation. In this embodiment, the carboxyl group of auxin or an auxin derivative is the carboxyl group of indole-3-acetic acid, or the carboxyl group of the corresponding site in other auxin or auxin derivatives. 【0098】 In a preferred embodiment of the present invention, the photodegradable auxin derivative is preferably a compound represented by general formula (1) with a carboxyl group (i.e., R 3 or R 4 This is a photodegradable auxin derivative in which a hydrogen atom in the carboxyl group of (1a) is replaced with a photodegradable protecting group. The photodegradable auxin derivative has the general formula (1a): 【0099】 [ka] 【0100】 [where n, m, R 1 , R 2 , and X are the same as above. R 3a and R 4a One of them is -R 5 -COOR 6 One side shows a hydrogen atom, the other shows a hydrogen atom. 5 R indicates an alkylene group. 6This indicates a photodegradable protecting group. It is a compound represented by [formula]. 【0101】 R 5 The alkylene group represented by may be linear or branched, but is preferably linear. The number of carbon atoms in the alkylene group is, for example, 1 to 8, preferably 1 to 6, more preferably 1 to 4, even more preferably 1 to 3, even more preferably 1 to 2, and particularly preferably 1. 【0102】 Photodegradable auxin derivatives may also be in the form of salts. The salts are not particularly limited and can be, for example, pharmaceutically acceptable salts. Specific examples of salts include salts with inorganic bases such as sodium salts, magnesium salts, potassium salts, calcium salts, and aluminum salts; salts with organic bases such as methylamine, ethylamine, and ethanolamine; salts with basic amino acids such as lysine, ornithine, and arginine, and ammonium salts. The salts may also be acid addition salts, and specific examples of such salts include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, malic acid, tartaric acid, fumaric acid, succinic acid, lactic acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, and ethanesulfonic acid; and acid addition salts with acidic amino acids such as aspartic acid and glutamic acid. 【0103】 The photodegradable auxin derivative may also be in the form of a solvate. The solvate is not particularly limited and examples include solvates with solvents such as water, ethanol, glycerol, and acetic acid. 【0104】 The photodegradable auxin derivative can be a single derivative or a combination of two or more derivatives. 【0105】 Photodegradable auxin derivatives can be obtained, for example, by introducing a photodegradable protecting group to auxin or an auxin derivative through an esterification reaction. 【0106】 The auxin-degron system is a protein degradation control technology that utilizes a plant-specific protein degradation system introduced by the plant hormone auxin. 【0107】 Specifically, this system involves introducing plant-derived TIR1 family proteins, which are F-box proteins that are subunits of the E3 ubiquitinating enzyme complex (SCF complex), and target polypeptides labeled with plant-derived Aux / IAA family proteins or peptides consisting of a subsequence thereof, into non-plant-derived eukaryotic cells. As a result, the TIR1 family proteins, which are auxin receptors, recognize Aux / IAA family proteins or peptides consisting of a subsequence thereof in an auxin-dependent manner, and degrade the target polypeptides using the ubiquitin / proteasome degradation system in non-plant-derived eukaryotic cells. 【0108】 The cells targeted by the degradation method of the present invention are not particularly limited, as long as they contain an auxin degron system. That is, the cells contain the components necessary to drive the auxin degron system so that it can be driven by irradiating a photodegradable auxin derivative with light to remove the photodegradable protecting group. 【0109】 The auxin degron system typically comprises polynucleotide A containing the coding sequence for a TIR1 family protein, and polynucleotide B containing the coding sequence for an Aux / IAA family protein or a partial peptide thereof. 【0110】 TIR1 family proteins are F-box proteins that form the E3 ubiquitinating enzyme complex (SCF complex) in the ubiquitin / proteasome system's protein degradation, and are unique to plants. TIR1 family proteins are receptors for the growth hormone auxin, and are known to recognize Aux / IAA family proteins, which are inhibitors of the auxin signaling pathway, and degrade target polypeptides upon receiving auxin. 【0111】 The genes encoding TIR1 family proteins are not limited to any specific type, as long as they are derived from plants. Furthermore, the type of plant from which the genes originate is also not limited; examples include Arabidopsis thaliana, rice, zinnia, pine, ferns, 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. 【0112】 The TIR1 family protein is preferably a protein expressed from the rice-derived TIR1 gene (NCBI Gene ID: 4335696) (for example, NCBI Reference Sequence: NP_001389282.1: SEQ ID NO: 1). 【0113】 TIR1 family proteins may be wild-type or mutant, insofar as they can drive the auxin degron system. TIR1 family proteins are proteins containing amino acid sequences that have 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% identity with the amino acid sequence shown in, for example, SEQ ID NO: 1. 【0114】 The TIR1 family protein is preferably an auxin-receptor-reducing TIR1 family protein. 【0115】 Auxin-receptor-reduced TIR1 family proteins are those in which mutations have been made in the amino acid sequence of wild-type TIR1 family proteins, and are not particularly limited as long as their binding affinity to endogenous auxin (e.g., indole-3-acetic acid) is reduced compared to wild-type TIR1 family proteins (e.g., reduced to 1 / 10 or less, 1 / 50 or less, 1 / 100 or less, 1 / 200 or less, 1 / 400 or less, 1 / 800 or less). 【0116】 Mutations that reduce auxin receptivity can be designed according to known information. For example, a previously published paper (Nature, Vol 446, 5 April 2007, pp640-645) analyzed the interaction region between auxin (indole-3-acetic acid) and its receptor (TIR1), and reported that the phenylalanine residue in loop 2 of TIR1 (for example, the 74th and 77th amino acid residues from the N-terminus in SEQ ID NO: 1 (phenylalanine residues)) interacts with the benzene ring in the auxin indole ring. Therefore, auxin receptivity can be reduced by substituting the amino acid residues in the auxin receptor that interact with the benzene ring in the auxin indole ring (preferably the phenylalanine residues in loop 2 of TIR1 (for example, the 74th and / or 77th amino acid residues from the N-terminus in SEQ ID NO: 1 (phenylalanine residues), or amino acid residues corresponding to the 74th and / or 77th amino acid residues from the N-terminus in SEQ ID NO: 1 in the ortholog or paralog of TIRI in SEQ ID NO: 1)) with other amino acid residues. 【0117】 Corresponding amino acid residues are those amino acid residues that are aligned at the same position when two amino acid sequences are compared using BLAST's default parameters. 【0118】 The "other amino acid residue" that is substituted is preferably an amino acid residue whose side chain molecular weight is smaller than the molecular weight of the side chain of the phenylalanine residue. Examples of such amino acid residues include hydrophobic amino acid residues such as glycine residues, alanine residues, valine residues, isoleucine residues, leucine residues, methionine residues, and cysteine ​​residues; serine residues, etc. Preferably, glycine residues, alanine residues, valine residues, isoleucine residues, serine residues, etc. are preferred, more preferably glycine residues, alanine residues, serine residues, etc., even more preferably alanine residues, serine residues, etc., and even more preferably alanine residues. 【0119】 As long as the Aux / IAA family protein is derived from a plant, there are no special limitations on its type. Specific examples of genes encoding Aux / IAA family proteins include, for example, IAA1, IAA2, IAA3, IAA4, IAA5, IAA6, IAA7, IAA8, IAA9, IAA10, IAA11, IAA12, IAA13, IAA14, IAA15, IAA16, IAA17, IAA18, IAA19, IAA20, IAA26, IAA27, IAA28, IAA29, IAA30, IAA31, IAA32, IAA33, and IAA34 proteins. 【0120】 For example, the Aux / IAA family genes derived from Arabidopsis thaliana are registered in TAIR (the Arabidopsis Information Resource), and the accession numbers for each gene are as follows: 【0121】 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), IAA14 gene (AT4G14550), IAA15 gene (AT1G8 IAA16 gene (AT3G04730), IAA17 gene (AT1G04250), IAA18 gene (AT1G51950), IAA19 gene (AT3G15540), IAA20 gene (AT2G46990), IAA26 gene (AT3G16500), IAA27 gene (AT4G29080), IAA28 gene (AT5G25890), IAA29 gene (AT4G32280), IAA30 gene (AT3G62100), IAA31 gene (AT3G17600), IAA32 gene (AT2G01200), IAA33 gene (AT5G57420), IAA34 gene (AT1G15050). 【0122】 Among these, the Arabidopsis thaliana IAA17 gene is preferred. 【0123】 Aux / IAA family proteins may be wild-type, mutant, or wild-type or mutant partial peptides, insofar as they can drive the auxin degron system. 【0124】 Examples of partial peptides include sequences consisting of regions containing at least two Lys residues each at the N-terminus and C-terminus of the domain II region of an Aux / IAA family protein, or sequences formed by linking two or more such sequences. Such partial peptides (mAI: Mini-auxin-inducible degron) have been reported in Patent Document 1, etc. For example, the amino acid sequence of mAID is represented by Sequence ID No. 2. 【0125】 The polynucleotide B preferably further contains the coding sequence of the target polypeptide or the coding sequence of a molecule that binds to the target polypeptide, so that it can be expressed in fusion with an Aux / IAA family protein or a partial peptide thereof (i.e., so that a fusion protein can be expressed). 【0126】 The target polypeptide is not particularly limited and may include, for example, enzyme proteins, structural proteins, muscle proteins, hormone proteins, metal ion and / or nutrient-binding proteins, receptor proteins, antibodies, luminescent proteins, tag proteins, or fragments thereof. The target polypeptide may be the full length of a protein or a combination of multiple protein fragments linked together. 【0127】 The binding molecule to the target polypeptide is a protein that has binding ability (preferably specific binding ability) to the target polypeptide, and is not particularly limited in that respect. Examples of binding molecules include monobodies, artificial antibodies based on protein scaffolds, humanized antibodies, antigen-binding fragments, single-chain antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F(ab')2 fragments, Fd, scFv, domain antibodies, VHH antibodies, multispecific antibodies, minibodies, scabs, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, or derivatives of the invariant region of an antibody. The molecular weight of the binding molecule is, for example, 500 kDa or less, preferably 200 kDa or less, more preferably 100 kDa or less, even more preferably 50 kDa or less, even more preferably 20 kDa or less, and particularly preferably 15 kDa or less. The lower limit is not particularly restricted and can be, for example, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, or 9kDa. 【0128】 In a fusion protein, the positional relationship between the Aux / IAA family protein or its partial peptide and the coding sequence of the target polypeptide or the binding molecule to the target polypeptide is not particularly limited; the former may be at the N-terminus, or the latter at the N-terminus. Furthermore, the two may be directly linked or linked via another polypeptide (e.g., a linker). 【0129】 In a preferred embodiment of the present invention, polynucleotide B further includes a coding sequence for a binding molecule to a target polypeptide so that it can be expressed in fusion with an Aux / IAA family protein or a partial peptide thereof (i.e., so that a fusion protein can be expressed). In this case, there is no need to construct cells that express a target protein to which an AID tag (Aux / IAA family protein or a partial peptide thereof) has been attached, and any endogenous target protein can be linked to the AID tag and degraded by a driven auxin degron system. 【0130】 Polynucleotide A and polynucleotide B may be linked together to form a single molecule, or they may be separate molecules. Furthermore, polynucleotide A and polynucleotide B may be integrated into the cell's genome, or they may not be integrated into the cell's genome. For example, polynucleotide A and / or polynucleotide B may be in the form of an expression vector. 【0131】 Polynucleotide A and / or polynucleotide B typically contain a promoter in order to transcribe mRNA from the above coding sequence. There are no particular limitations on the promoter, and it can be appropriately determined depending on, for example, the type of cell. Specific examples of promoters include the CMV promoter, SV40 promoter, EF1A promoter, and RSV promoter. In one embodiment, the promoter is derived from an organism of a different species than the organism from which the above coding sequence originates. 【0132】 The cells are not particularly limited as long as they are capable of forming an SCF complex with TIR1 family proteins, and eukaryotic cells (preferably non-plant eukaryotic cells) can be widely used. Examples of cells include those of animals, fungi, and protists. Examples of animals include mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, fish and amphibians such as zebrafish and African clawed frogs, and invertebrates such as C. elegans and fruit flies. Examples of fungi include budding yeast and fission yeast. The types of cells are also not particularly limited and include, for example, blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, egg cells), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinizing cells, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells. Furthermore, cell lines can also be used. Specifically, examples include cell lines derived from humans, mice, and chickens. More specifically, examples include human HCT116 cells, human HT1080 cells, human NALM6 cells, and chicken DT40 cells. 【0133】 The cells may be isolated cells or cells from a living organism (in one embodiment, the ecosystem of a non-human organism). 【0134】 Within cells, components of the auxin-degron system are expressed. 【0135】 Cells containing the auxin-degron system can be obtained according to or in accordance with known methods. For example, they can be obtained by introducing components of the auxin-degron system (e.g., polynucleotide A and polynucleotide B) into cells. Examples of introduction methods include calcium phosphate precipitation, DEAE dextran transfection, electroporation, lipofection, and viral vector methods. It is also possible to incorporate the polynucleotide components of the auxin-degron system into any position in the cell genome using a target-specific nuclease system such as the CRISPR / Cas system (Non-Patent Literature 2). 【0136】 The method for introducing photodegradable auxin derivatives into cells is not particularly limited, but can be carried out, for example, by contacting the photodegradable auxin derivative with the cells. The concentration of the photodegradable auxin derivative in the fluid surrounding the cells (e.g., culture medium) at the time of contact is not particularly limited and can be, for example, 0.1 nM to 1 mM. The lower limit is, for example, 1 nM, 5 nM, 10 nM, 20 nM, or 30 nM, and the upper limit is, for example, 100 μM, 10 μM, 1 μM, 500 nM, 200 nM, 100 nM, 70 nM, 60 nM, 50 nM, or 45 nM. 【0137】 During light irradiation, the wavelength and light intensity can be appropriately adjusted so that the photodegradable protecting group is removed from the photodegradable auxin derivative, depending on the photodegradable protecting group, the state of the cells (whether they are isolated cells or cells in vivo, and in the latter case, the location of the cells), etc. In one embodiment, for example, light with a wavelength of 250 to 450 nm (preferably 280 to 400 nm, more preferably 320 to 380 nm, and even more preferably 340 to 380 nm) can be irradiated, and the integrated light intensity can be 1 × 10⁻⁶4 ~1 × 10 6 μJ / cm 2 (preferably 2 × 10) 4 ~6×10 5 μJ / cm 2 , comfortable 5×10 4 ~3×10 5 μJ / cm 2 ) can be done as follows. 【0138】 This light irradiation can activate the auxin degron system, allowing for the degradation of the target polypeptide. Furthermore, if other polypeptides are bound to the target polypeptide, those other polypeptides can also be degraded. 【0139】 In some embodiments, the present invention, (A) A photodegradation inducer for a target polypeptide, comprising a photodegradable auxin derivative in which auxin or an auxin derivative is substituted with a photodegradable protecting group, (B) A kit for inducing the degradation of a target polypeptide, comprising a photodegradable auxin derivative obtained by substituting auxin or an auxin derivative with a photodegradable protecting group, and a polynucleotide for introducing an auxin degron system. (C) A photodegradable auxin derivative obtained by substituting auxin or an auxin derivative with a photodegradable protecting group, and a cell comprising an auxin degron system, and (D) R of compounds represented by general formula (1) 3 or R 4 A photodegradable auxin derivative obtained by replacing the hydrogen atom in the carboxyl group with a photodegradable protecting group, Regarding. 【0140】 In embodiment (A), the decomposition inducer may contain other components. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like. 【0141】 All embodiments (A) to (D) can be used in the decomposition method of the present invention. 【0142】 The degradation method of the present invention can be used for functional analysis of target polypeptides or proteins to which they bind, and for screening methods of drugs involving target polypeptides or proteins to which they bind (for example, drugs that bind to target polypeptides or proteins to which they bind, drugs that bind to proteins whose behavior (expression, degradation, etc.) is affected by target polypeptides or proteins to which they bind, etc.). 【0143】 Furthermore, photodegradable auxin derivatives can be used not only as reagents but also as pharmaceuticals. [Examples] 【0144】 The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. 【0145】 (1) Materials and Methods (1-1) Cell culture HeLa, U2OS, HEK293, HEK293T, and T24 cells were cultured in DMEM (Nacalai Tesque) supplemented with 10% FBS and 1% penicillin / streptomycin (Nacalai Tesque) at 37°C and 5% CO2. DT40 cells were cultured in DMEM (Nacalai Tesque) supplemented with 10% FBS, 1% penicillin / streptomycin (Nacalai Tesque), 1% chicken serum (Sigma), and 50 μM 2-mercaptoethanol (Sigma) at 38.5°C and 5% CO2. Feeder-free mouse embryonic stem cells (E14tg2a) were cultured on gelatin-coated dishes at 37°C and 5% CO2 in DMEM (Nacalai Tesque) supplemented with 10% FBS, 5% knockout serum, 2 mM l-glutamine (Nacalai Tesque), 1% penicillin / streptomycin, 1% non-essential amino acids (Nacalai Tesque), and LIF protein. 【0146】 (1-2) Auxin derivative stock solution 5-adamantyl IAA (Tokyo Chemical Industries: A3390) and caged 5-adamantyl IAA (developed in this study) were diluted in dimethyl sulfoxide (DMSO) to a concentration of 5 mM and stored at 30°C. When using, a solution 1000 times the final concentration of each was prepared and added to the culture medium at a concentration of 1 / 1000. 【0147】 (1-3) Plasmid construction For gene editing, plasmids containing GFP, mCherry-T2A-histidine D, or neomycin resistance genes were constructed between each 1kb homology arm of pBluescript II SL(-). In the pAlissAID plasmid, codon-optimized OsTIR1 -T2A-Brastcidin resistance gene (BSR)-Internal Ribosomal entry site (IRES)2-minimalized AID KR tag (lysin replaced with arginine) and conjugate (vhhGFP4) KR , LaM2 KR , LaM4 KR , LaM8 KRThe pT2 / HB plasmid was constructed using pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) and pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A) (pX335) (Addgene #42335). CRISPR / Cas9 vectors were constructed according to previously reported protocols using pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) and pX335-U6-Chimeric_BB-CBh-hSpCas9n (D10A) (pX335) (Addgene #42335). pX330-U6-Chimeric_BB-CBh-hSpCas9 and pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) were obtained from Feng Zhang (Addgene plasmid # 42230; http: / / n2t.net / addgene:42230; RRID:Addgene_42230) and (Addgene plasmid # 42335; http: / / n2t.net / addgene:42335; RRID:Addgene_42335). For GFP-tagged target gene expression, GFP-fusion target genes (ggCENP-I and ggCDK1) were constructed following the EF1a promoter-puro-IRES in the pT2 / HB plasmid. In the pAlissAID TT (transient transfection) plasmid, the BSR in the pAlissAID plasmid was replaced with mCherry. For pT2 / HB plasmid genome integration, the pCMV(CAT)T7-SB100 plasmid was used via co-introduction. The pCMV(CAT)T7-SB100 plasmid was obtained from Zsuzsanna Izsvak (Addgene plasmid # 34879; http: / / n2t.net / addgene:34879; RRID:Addgene_34879). 【0148】 (1-4) Transfection and Cloning Plasmid (total 4 μg) was diluted in 50 μl of DMEM. 18 μg of polyethyleneimine (PolySciences) was diluted in 50 μl of DMEM. The solutions were mixed and incubated for 30 minutes. This solution was added to cells (HeLa, E14tg2a) diluted in 300 μl of DMEM, vortexed for 5 seconds, and then allowed to stand for 30 minutes. The cells were then seeded in a culture dish and cultured at 37°C for 1 day. Selection was performed using a medium containing 10 μg / ml blasticidin S or 1 μg / ml puromycin. 【0149】 Plasmid (total 2 μg) was diluted in 50 μl of DMEM. 6 μg of polyethyleneimine was diluted in 50 μl of DMEM. The solutions were mixed and incubated at RT for 30 minutes. This solution was added to cells (U2OS, HEK293, HEK293T) and cultured at 37°C for 1 day. Selection was performed using a medium containing 10 μg / ml Blastcidin S or 1 μg / ml puromycin. 【0150】 Plasmid (total 10 μg) and DT40 cells were mixed in 200 μl of DMEM and electroporated (polling pulse: 150 V, 1 msec [pulse length], 50 msec [pulse interval], 5 pulses, 10% attenuation rate +; transfer pulse: 50 V, 50 msec [pulse length], 50 msec [pulse interval], 5 pulses, 40% attenuation rate ±). Subsequently, cells were seeded in a culture dish and cultured at 38.5°C for 1 day. Selection was performed using a medium containing 30 μg / ml Blastcidin S, 0.5 μg / ml puromycin, or 1 mg / ml D-Histidine. 【0151】 Plasmid (total 10 μg) and T24 cells were mixed in 100 μl of DMEM and electroporated (polling pulse: 125 V, 5 msec (pulse length), 50 msec (pulse interval), 2 pulses, 10% attenuation rate +; transfer pulse: 20 V, 50 msec (pulse length), 50 msec (pulse interval), 5 pulses, 40% attenuation rate ±). Subsequently, cells were seeded in a culture dish and cultured at 37°C for 1 day. Selection was performed using a medium containing 10 μg / ml Blastcidin S. 【0152】 (1-5) Imbublot Proteins were isolated by SDS-PAGE and transferred to a nitrocellulose membrane (Wako). This membrane was incubated with anti-GFP (1:2000 dilution; our laboratory), anti-RFP (1:2000 dilution; Chromo tek, 5f8-20 / 5f8-100), anti-TIR1 (1:2000 dilution; Medical and Biological Laboratories, PD048), anti-AID (1:2000 dilution; donated by Professor Karim Labib), and anti-Ras (1 / 5000 dilution; Abcam, ab206969). As secondary antibodies, HRP-labeled anti-rabbit IgG (1:5000 dilution; Sigma, A6154), peroxidase-labeled anti-sheep IgG (1:5000 dilution; 713-035-003, Jackson ImmnoResearch), or peroxidase-labeled anti-rat IgG (1:5000 dilution; 112-035-003, Jackson ImmnoResearch) were used. Immunodetection was performed using the Luminata Forte Western HRP Substrate system (Merck Millipore, Burlington, USA, 61-0206-81) or the Chemi-Lumi One L system (Nacalai Tes que, 07880) and a bioanalyzer (LAS4000 mini; GE Healthcare Biosciences). 【0153】 (1-6) Colony formation assay Cells (200 cells) were plated into 6-well plates and cultured for 1-2 weeks with or without auxin. Cells were fixed with cold 100% methanol for 2 minutes and air-dried. Cells were stained with Crystal Violette and washed with water. 【0154】 (1-7) Flow cytometry For cell cycle analysis, cells were fixed in 70% ethanol at -30°C for 1 day. The fixed cells were centrifuged and treated with 1 ml of stain mix (PBS, 1 mg / ml RNase, 5 μg / ml Propidium iodide) at 37°C for 30 minutes. Cells were obtained using an Attune Flow Cytometer (Thermo Fisher Scientific). 【0155】 For the degradation assay, pAlissAID TT plasmid was transfected into GFP-Ras-expressing HEK293T cells. After 24 hours, the medium was replaced with 5-Ad-IAA-containing medium. After another 24 hours, the cells were fixed with 4% PFA in PBS and measured using an Attune flow cytometer. 【0156】 (1-8) Cell proliferation assay DT40 cells (2 x 10 4 Cells (cells / ml) were cultured with or without the addition of an auxin derivative. Cell concentration was calculated using Countess II (Thermo Fisher) at the indicated time point after the addition of the auxin derivative. 【0157】 (1-9) Fluorescence Microscope Cells were grown in a culture medium containing 5 μM 5-Ad-IAA or 50 nM caged 5-Ad-IAA. After centrifugation, 4% formaldehyde in PBS was added and the cells were fixed at RT for 10 minutes. Cells were collected by centrifugation. After washing with PBS and staining with Hoechest 33342, the cells were resuspended in PBS and observed with a fluorescence microscope (AxioObserver Z1; Carl Zeiss, Oberkochen, Germany) equipped with a CCD camera (AxioCam MRm; Carl Zeiss). 【0158】 (1-10) Immunoprecipitation GFP-Ras stably expressing HEK293T cells were transiently transfected with the pAlissAID TT NS1 plasmid. After 48 hours, the cells were lysed in 1 ml PBS 0.2% Titon-X, centrifuged at 4°C, and the supernatant was collected in a new tube. 5 μl of Anti-GFP GFP-Trap Dynabeads (Proteintech) were added, and the tubes were rotated at 4°C for 2 hours. The beads were washed three times with cold PBS and eluted with SDS sample buffer. 【0159】 (1-11) Pull-down assay All proteins were expressed using E. coli BL21(DE3). Cells were sonicated, and cell lysates were incubated with GSH beads in PBS solution (GST-vhhGFP4). KR and GST-LaM2 KR Alternatively, the proteins were affinity-purified using His tags (His6-GFP, mClover3, venus, mScarlet, mCherry). GST-vhh-conjugated beads were dispensed, fluorescent proteins were added, and the mixture was rotated at room temperature for 1 hour. The beads were washed five times with PBS, and the proteins were eluted by adding sample buffer. 【0160】 (1-12) Luciferase assay OsTIR1 F74A and DT40 cells expressing mScarlet-mAID-luciferase (5 × 10 5The cells were cultured for 2 hours with various concentrations of inducing agents. Luciferase activity was measured using the Steady-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol. 【0161】 (1-13) RNA sequencing Cells were treated with 5-Ad-IAA or Mock for 24 hours, and RNA was extracted using NuclepSpin RNA plus (Takara). Library preparation was performed using the NEBNext Poly(A)mRNA Magnetic Isolation Module and the NEBNext Ultra II RNA Library Prep Kit for Illumina, and analysis was performed using the NextSeq 500 / 550 High Output Kit v2. Libraries were sequenced using the Nextseq550 system (Illumina). Low-quality reads were discarded using the fastq_quality_trimmer and fastq_quality_filter from the FASTX-Toolkit. Ribosomal RNA reads were removed by alignment with human rRNA sequences using STAR, and the remaining reads were aligned with the human transcriptome (GRCh38.p13) and human genome (hg38) using STAR. Multiple mappings were possible. Mapped reads were counted using featureCounts, and expression analysis was performed using DESeq2. Gene set enrichment analysis (GSEA) was performed using GSEApy version 0.9.9. 【0162】 (1-14) Time-lapse photography Animal rearing and experiments were conducted in accordance with the National Institutes of Natural Sciences guidelines for animal experimentation. Mouse embryos were obtained from ICR female mice (SLC). Embryos were cultured in KSOM + AA medium (Millipore) and observed under a microscope under conditions of 5% CO2 and 37°C. Time-lapse images were captured using a CV1000 system (Yokogawa Electric) at 5% CO2 and 37°C. 【0163】 (1-15) Electroporation OsTIR1 F74A The H2B-mCherry-P2A-mAID-vhhGFP4 and H2B-EGFP sequences were inserted into the IVTpro template vector (Takara). These were then cleaved with HIND3 and encapsulated in mMESSAGE mMACHINE. TM The T7 Transcription Kit (Invitrogen) was used as a template for in vitro transcription. The electroporation method was performed as previously reported. Briefly, each mRNA was dissolved in Opti-MEM (Gibco) to a concentration of 200 ng / μl, and the embryo and 5 μL of mRNA solution were placed on an electrode (LF501PT1-10, BEX). Electroporation was performed (25V, 6 times, 3ms ON ± 97ms OFF) (Genome Editor Plus, BEX), the embryo was returned to KSOM medium, and incubated at 5% CO2, 37°C. 【0164】 (1-16) Synthesis of Caged 5-Ad-IAA (1-16-1) General matters Unless otherwise noted, all materials, including the dry solvent, were obtained from commercially available suppliers and used without further purification. 2,5-Dimethoxy-2'-nitrobenzhydrol was prepared according to previously reported procedures. Unless otherwise noted, all reactions were carried out using dry solvents, under an argon atmosphere, and using flame-dried glassware in a standard vacuum line. All operations and purifications were performed in air using reagent-grade solvents. 【0165】 Analytical thin-layer chromatography (TLC) is visualized using UV light (254 nm) and ethanolic phosphomolybdic acid. Merck silica gel 60 F 254 The procedure was performed using pre-coated plates (0.25 mm). Silica gel for column chromatography was purchased from KANTO. 【0166】 High-resolution mass spectroscopy (HRMS) was obtained using Thermo Fisher Scientific Exactive (ESI). Nuclear magnetic resonance (NMR) spectra were obtained using JEOL ECA600II. 1 H 600 MHz, 13 Recorded using a C 150 MHz spectrometer. 1 The chemical shift of the 1H NMR spectrum is expressed as parts per million (ppm) relative to tetramethylsilane (δ 0.00 ppm). 13 The chemical shift of the 13C NMR spectrum is expressed in ppm relative to CDCl3 (δ 77.00 ppm). The data is reported as chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant (Hz), and integral value. 【0167】 (1-16-2) Synthesis method 【0168】 [ka] 【0169】 A 10 mL flask containing a magnetic stirring rod was flame-dried under vacuum, cooled to room temperature, and then filled with argon. 2-[5-(adamantan-1-yl)-1H-indole-3-yl]acetic acid (12.4 mg, 0.04 mmol), 2,5-dimethoxy-2'-nitrobenzhydrol (11.6 mg, 0.04 mmol), DMAP (6.1 mg, 0.05 mmol), dried CH2Cl2 (0.5 mL), and EDC (8.8 μL, 0.05 mmol) were added to the flask at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The mixture was quenched with water and extracted with ethyl acetate (three times). The combined extract was dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:Â=10:1→3:1) to obtain caged 5-Ad-IAA (12.5 mg, 54%) as a pale yellow oil. 1H NMR(600 MHz, CDCl3)δ 1.72(d, J = 11.4 Hz, 3H), 1.77(d, J = 11.4 Hz, 3H), 1.89(s, 6H), 2.06(s, 3H), 3.34(s, 3H), 3.66(s, 3H), 3.85(d, J = 15.6 Hz, 1H), 3.89(d, J = 15.6 Hz, 1H), 6.41(d, J = 3.0 Hz, 1H), 6.72-6.76(m, 2H), 7.12(d, J = 3.0 Hz, 1H), 7.24-7.29(m, 3H), 7.35-7.40(m, 2H), 7.54(s, 1H), 7.73(s, 1H), 7.91(dd, J = 7.8, 3.0 Hz, 1H), 8.00(s, 1H). 13 C NMR(150 MHz, CDCl3)δ 29.0, 31.4, 36.0, 36.8, 43.7, 55.2, 56.0, 68.0, 108.1, 110.6, 111.8, 112.8, 113.8, 114.4, 120.0, 123.3, 124.5, 127.0, 127.9, 128.5, 129.3, 132.7, 134.3, 143.1, 148.4, 150.9, 153.4, 170.2 (1 aryl carbon signal is obscured). HRMS(ESI)m / z calcd for C 35 H 36 O6N2Na [M+Na] + : 603.2466, found 603.2463. 【0170】 (1-17) Caged 5-Ad-IAA treatment and decaking Similar to 5-Ad-IAA, it was dissolved in DMSO at a concentration of 5 mM and stored at -30°C. For use, a solution 1000 times the final concentration was prepared and added to the culture medium. Uncaging was performed using an XL-1000 UV Crosslinker (UVP) equipped with a BLE 8T365, measuring 1.2 x 10⁻⁶. 5 μJ / cm 2 This was done by irradiating the cells. 【0171】 (2) Results (2-1) GFP fusion target proteins are degraded by the AlissAID system in vertebrate cell lines. OsTIR1 F74A The AlissAID system incorporating vhhGFP4 degrades GFP-fused target proteins in budding yeast. We also found that under the EF1α promoter, OsTIR1 F74A and minimalAID-vhhGFP4 KR We verified whether this technology works effectively in animal cultured cells by constructing mouse embryonic stem cells (mouseES) expressing (mAID-Nb) (Figures 1 and 2A). As in previous reports, SCF-OsTIR1 F74ATo avoid direct ubiquitination, all lysine residues in mAID-vhhGFP4 were replaced with arginine residues. GFP was fused to the C-terminus of endogenous Cdk1 (cytoplasmic protein) or CENP-H (nucleoprotein) via homology-directed repair (HDR) using clustered regularly interspaced palindromic repeat (CRISPR) / CRISPR-associated protein 9 (Cas9) genome editing. Immunoblotting revealed that both Cdk1-GFP and CENP-H-GFP were rapidly degraded in a 5-Ad-IAA-dependent manner. This degradation was effectively inhibited by the proteasome inhibitor MG132 (Figure 2B). Cdk1 is involved in M ​​phase progression, and CENP-H plays a crucial role in chromosome segregation. Both are essential for cell proliferation. Therefore, the viability of the strains in 5-Ad-IAA-containing medium was tested using a colony formation assay. The proliferation of these cell lines was significantly inhibited by the addition of 5 μM 5-Ad-IAA (Figure 2C). Furthermore, Cdk1-GFP was degraded in cells that accumulated in the G2 / M phase after 5-Ad-IAA treatment (Figure 2D). Similar experiments using chicken DT40 cells showed 5-Ad-IAA-dependent degradation of CENP-H-GFP (Figure 2E), growth impairment, and cell cycle arrest in the G2 / M phase (Figure 2F). AlissAID cell lines expressing Cdk1-GFP or GFP-CENP-I were also created. In these cells, endogenous Cdk1 and CENP-I were knocked out using CRISPR / Cas9. Furthermore, OsTIR1 F74A mAID-Nb, Cdk1-GFP, or GFP-CENP-I were exogenously expressed. Similar to the previous experimental results, a decrease in 5-Ad-IAA-dependent GFP fusion protein levels, a decrease in intracellular GFP signaling, and cell cycle arrest at the G2 / M phase were observed. These results suggest that the GFP-targeted AlissAID system is effective in various vertebrate cell lines. 【0172】 (2-2) AlissAID LaM2 effectively degrades mCherry fusion proteins. Next, we tested an AlissAID system targeting mCherry fusion proteins (Figure 3A). We constructed the AlissAID DT40 strain and used KR-substituted LaM nanobodies (LaM2, LaM4, LaM8) as mCherry conjugates. mCherry was fused to the C-terminus of endogenous CENP-H via HDR using CRISPR / Cas9 genome editing. In the AlissAID system using LaM2, CENP-H-mCherry was efficiently degraded in a 5-Ad-IAA-dependent manner, and the cells arrested in the G2 / M phase. On the other hand, LaM4 and LaM8 did not induce degradation of CENP-H-mCherry, and these cells did not show any abnormalities in the cell cycle (Figures 3B and C). 【0173】 While various fluorescent proteins are used in the biological field, the specificity of the nanobodies vhhGFP4 and LaM2 remains unclear. Therefore, we investigated the specificity of the binding affinity between several fluorescent proteins (GFP, mClover3, mVenus, mScarlet, and mCherry) and nanobodies using an in vitro pull-down assay. In particular, vhhGFP4 bound to GFP, mClover3, and mVenus, but not to mScarlet or mCherry. In contrast, LaM2 bound to mCherry and mScarlet, but not to GFP, mClover3, or mVenus. These results suggest that these fluorescent proteins can be used as degron tags in the AlissAID system. 【0174】 (2-3) The AlissAID system can induce the degradation of endogenous proteins. In the AlissAID system, target proteins are recognized by a single peptide antibody, such as a nanobody. Therefore, we verified that the AlissAID system can target endogenous proteins by replacing the nanobody with a specific antibody that binds to the target protein. Ras protein is a well-known GTPase and one of the most common oncogens in humans. Here, we investigated whether K55 DARPin and NS1 Monobody, which have been reported to date, can degrade endogenous Ras. K55 binds to all major Ras family members: HRas, KRas, and NRas. On the other hand, NS1 binds strongly to HRas, weakly to KRas, but not to NRas. First, OsTIR1 F74A and mAID KR K55-AlissAID HeLa cell lines expressing both -K55 and -K55 were created (Figure 4A). In this experiment, KR substitution was not performed because it may affect the binding affinity of K55 to the Ras protein. Immunoblot analysis showed that endogenous Ras protein was degraded in a 5-Ad-IAA-dependent manner (Figure 4B). Next, the effect of NS1-AlissAID on HeLa cells was tested. Immunoblot analysis showed that endogenous Ras protein was degraded in a 5-Ad-IAA-dependent manner (Figures 4C and D). In addition, NS1-AlissAID cells were created using HEK293, U2OS, and mouse embryonic stem cells. Degradation of Ras protein was observed in these cells. To determine the Ras protein targeted for degradation by the NS1-based AlissAID system, the binding of mAID-NS1 to HRas, KRas, and NRas was verified using immunoprecipitation (IP) assays. Consistent with previous reports, mAID-NS1 strongly bound to HRas, weakly bound to KRas, but did not bind to NRas. Next, the degradation of each Ras protein was evaluated by flow cytometry. HEK293T cells stably expressing GFP-HRas, GFP-KRas, and GFP-NRas were generated. Furthermore, OsTIR1 F74A, the pAlissAID TT plasmid containing the expression cassettes of mAID-binder and mCherry was constructed. The pAlissAIDTT plasmid was transiently transfected into the GFP-Ras expressing cell line, and the expression levels of OsTIR1 and mAID-binder were monitored by mCherry fluorescence, and the expression level of Ras was monitored by GFP fluorescence. In the AlissAID system using NS1, HRas was strongly degraded in a 5-Ad-IAA-dependent manner (Figure 4E). In contrast, KRas was hardly degraded even in cells with high mCherry expression. No degradation was observed for NRas at any expression level. In vhhGFP4-AlissAID used as a control, all GFP-Ras molecules were degraded. Therefore, NS1-AlissAID specifically degrades HRas, but does not degrade KRas and NRas. 【0175】 (2-4) The AlissAID system facilitates phenotypic analysis by endogenous proteolysis. In the previous section, it was shown that Ras degradation is induced by the AlissAID system using K55 and NS1, which are endogenous Ras-binding factors. However, it is unclear whether this system induces phenotypic changes through the degradation of Ras proteins. In T24 cells with the HRas(G12V) mutation, proliferation decreased due to RNA knockdown of HRas. Therefore, it was verified that a similar phenotype would occur when HRas was decreased using NS1-AlissAID in T2 four cells. First, OsTIR1 F74A and T24 cells stably expressing mAID-NS1 were constructed, and it was confirmed by immunoblotting that HRas was degraded in a 5-Ad-IAA-dependent manner (Figure 5A). Next, the proliferation of this cell line and T24 parental cells was examined. As a result, the proliferation of T24 AlissAID cells was significantly inhibited in the medium containing 5-Ad-IAA, whereas the inducer alone had no effect on the proliferation of the T24 parental cell line (Figure 5B). 【0176】 Next, we comprehensively investigated the transcriptional responses of cells to 5-Ad-IAA treatment and HRas degradation using RNA-seq. Treatment with 5 μM 5-Ad-IAA did not affect the transcriptional state of T24 parent cells (Figure 5C). We found that during NS1-A-mediated HRas degradation, the expression of factors regulated by the Ras pathway and factors involved in oncology, such as IDO1, BMF, and ATOH8, increased. Expression increased while HRas were degraded by NS1-AlissAID. On the other hand, factors that showed a decrease in transcription levels included the secretory factor ESM1, which promotes cancer cell proliferation, and FAM167A, which is involved in NF-κB activation (Figure 5D). Here, gene set enrichment analysis (GSEA) revealed that the transcription of E2F target genes and G2 / M checkpoint genes, which are related to G1 / S and cell cycle progression, decreased with HRas degradation. These factors are involved in the Ras pathway, oncology, and cell proliferation, indicating that NS1-AlissAID-mediated HRas degradation has appropriate effects on cells. Therefore, degradation of endogenous proteins using the AlissAID system reduces the target protein (POI) and affects the phenotypic outcome. 【0177】 (2-5) KRas mutant-specific degradation by the AlissAID system KRAS is one of the most frequently mutated cancer genes in various types of cancers, such as non-small cell lung cancer, colorectal cancer, and pancreatic ductal adenocarcinoma. KRAS mutations are observed in more than 80% of pancreatic ductal adenocarcinoma cases. G12 is a major point mutation site of KRas and is an important factor in cancer malignancy through changes in GTPase activity. The development of technologies to selectively control such point mutant variants is important, but it is difficult to recognize point mutations using small molecules. Among single-domain antibodies, some show low affinity for wild-type KRas but strongly bind to mutant KRas. Therefore, we investigated whether the AlissAID system using conjugates targeting KRas mutants could specifically knockdown mutant KRas. Notably, 12VC1 is a nanobody against KRas(G12C) and KRas(G12V), whereas R11.1.6 is a de novo conjugate of the KRas(G12D) mutant. HEK293T cells stably expressing GFP-KRas were transiently transfected with the pAlissAID TT plasmid containing these conjugates, and KRas knockdown was induced by 5-Ad-IAA treatment. 12VC1-AlissAID degraded KRas(G12C) and KRas(G12V), but not wild-type KRas and KRas(G12D), while R11.1.6-AlissAID did not induce the degradation of all KRas variants including KRas(G12D) (Figure 6A). This result was confirmed using a fluorescence microscope (Figure 6B). Overall, the AlissAID system was effective for mutant-specific knockdown, and 12VC1 was useful for KRas G12C and G12V. 【0178】 (2-6) Depletion of H2B-EGFP by the AlissAID system in mouse embryos Next, we evaluated the effectiveness of protein knockdown using AlissAID in early mouse embryos. While imaging techniques that track proteins using fluorescent proteins are widely used, conditional protein knockdown systems like the AID system are not widely used in early mouse embryos because establishing transgenic embryos with AID tags fused to the target protein is time-consuming and labor-intensive. Therefore, protein knockdown using the AlissAID system is a useful tool for analyzing early mouse embryos. 【0179】 In early mouse embryos, transient expression using mRNA electroporation and microinjection has been established. Among these methods, mRNA electroporation is less susceptible to the skill of the experimenter and is expected to have high reproducibility. We have developed a method to express OsTIR1 from pAlissAID or previously reported plasmids. F74A Three in vitro transcription plasmids were constructed, each containing either H2B-mCherry-P2A-mAID-Nb or H2B-EGFP expression cassettes. Protein expression and degradation were confirmed by transient transfection of these plasmids into HEK293T cells stably expressing GFP-KRas. Next, mRNA was synthesized by in vitro transcription using these plasmids as templates. Early-stage mouse embryos (E0.5) were obtained from spontaneously mated ICR mice, and OsTIR1 F74AmRNA for H2B-mCherry-P2A-mAID-vhhGFP4 and H2B-EGFP was introduced by electroporation (Figure 7A). Subsequently, time-lapse imaging was used to verify whether the expression level of H2B-EGFP changed after 5-Ad-IAA treatment. H2B-mCherry functioned as a non-degradable control and was used to normalize the H2B-EGFP signal. In the absence of 5-Ad-IAA, the H2B-EGFP signal did not change, but 5-Ad-IAA treatment reduced the H2B-EGFP signal (Figure 7B). This result suggests the effectiveness of conditional knockdown using the AlissAID system in early mouse embryos. Thus, the AlissAID system is an effective conditional knockdown approach in organisms such as mouse embryos where AID tagging is time-consuming. 【0180】 (2-7) Photoactivation of caged 5-adamantyl IAA to regulate protein degradation In recent years, optogenetics, which uses light to control biological phenomena, has come into use in various biological species. Optogenetics tools triggered by light irradiation can precisely control factors such as the site, timing, and degree of activation. Attaching a photoprotective group to the functional unit of a molecule is one way to impart photoregulatory properties. In a previous report, auxin was caged by attaching a photoprotective group to the carboxylic acid portion of auxin. We applied this method to the AlissAID system and developed a photogenetic control system that can control protein degradation by light irradiation. In the AlissAID system, 5-Ad-IAA is used as a degradation agent to induce protein degradation. To achieve photo-activated degradation, we synthesized caged 5Ad-IAA (Figure 8A). First, we verified the protein degradation-inducing activity of caged 5Ad-IAA using a luciferase assay in the ssAID system. Under non-light conditions (Dark conditions), caged 5Ad-IAA hardly induced luciferase degradation. However, when activated with blue light, caged 5-Ad-IAA successfully induced luciferase degradation. The degradation efficiency of luciferase was compared between 5-Ad-IAA and activated caged 5-Ad-IAA. No significant difference in degradation-inducing activity was observed at concentrations of 5-50 nM (Figure 8B). Next, caged 5-Ad-IAA was added to CENP-H-GFP AlissAID DT40 cells, and the fluorescence of CENP-H-GFP was observed by microscopy. Cells treated with caged 5-Ad-IAA showed reduced CENP-H-GFP fluorescence when irradiated with blue light compared to untreated cells. Conversely, no decrease in CENP-H-GFP fluorescence was observed in cells that were not irradiated with light (Figure 8C). Finally, the effectiveness of phenotypic analysis using caged 5-Ad-IAA was verified. After confirming that blue light irradiation does not affect the cell cycle, CENP-H-GFP AlissAID DT40 cells were treated with caged 5-Ad-IAA and irradiated with blue light. As a result, accumulation during the G2 / M phase of the cell cycle was observed only in cells treated with caged 5-Ad-IAA and irradiated with blue light (Figure 8D).This suggests that the AlissAID system, utilizing caged 5-Ad-IAA, is an effective optogenetic tool for inducing light-dependent protein degradation.

Claims

[Claim 1] General formula (1): 【Chemistry 1】 [In the formula, n and m are the same or different and represent 0 or 1. R 1 and R 2 These are the same or different, each representing a hydrogen atom, an optionally substituted adamantyl group, an optionally substituted aryl group, an optionally substituted alkyl group, or an optionally substituted heterocyclic group (however, R 1 and R 2 (Except when all are hydrogen atoms at the same time.) 3 and R 4 One side represents a carboxyalkyl group, and the other represents a hydrogen atom. X represents -NH- or -CH=CH-. The R of the compound represented by 3 or R 4 A photodegradable auxin derivative obtained by replacing the hydrogen atom in the carboxyl group with a photodegradable protecting group. [Claim 2] The photodegradable auxin derivative according to claim 1, wherein the photodegradable protecting group is a nitrobenzyl type photodegradable protecting group. [Claim 3] A degradation inducer for a target polypeptide, comprising a photodegradable auxin derivative in which auxin or an auxin derivative is substituted with a photodegradable protecting group. [Claim 4] The decomposition inducer according to claim 3, wherein the photodegradable auxin derivative is the photodegradable auxin derivative according to claim 1. [Claim 5] A kit for inducing the degradation of a target polypeptide, comprising a photodegradable auxin derivative in which auxin or an auxin derivative is substituted with a photodegradable protecting group, and a polynucleotide for introducing an auxin degron system. [Claim 6] The degradation induction kit according to claim 5, wherein the photodegradable auxin derivative is the photodegradable auxin derivative according to claim 1. [Claim 7] A cell comprising a photodegradable auxin derivative in which auxin or an auxin derivative is substituted with a photodegradable protecting group, and an auxin degron system. [Claim 8] The cell according to claim 7, wherein the photodegradable auxin derivative is the photodegradable auxin derivative according to claim 1. [Claim 9] A method for degrading a target polypeptide, comprising photoirradiating a cell containing an auxin degron system with light, a photodegradable auxin derivative in which auxin or an auxin derivative is substituted with a photodegradable protecting group, and auxin degron system. [Claim 10] The decomposition method according to claim 9, wherein the photodegradable protecting group is a nitrobenzyl type photodegradable protecting group. [Claim 11] The decomposition method according to claim 9, wherein the photodegradable auxin derivative is a photodegradable auxin derivative in which a hydrogen atom in the carboxyl group of the auxin or the auxin derivative is replaced with the photodegradable protecting group. [Claim 12] The aforementioned auxin derivative is general formula (1): 【Chemistry 2】 [wherein, n and m are the same or different and each represents 0 or 1. R 1 and R 2 are the same or different and each represents a hydrogen atom, an optionally substituted adamantyl group, an optionally substituted aryl group, an optionally substituted alkyl group, or an optionally substituted heterocyclic group (however, the case where both R 1 and R 2 are simultaneously hydrogen atoms is excluded). One of R 3 and R 4 represents a carboxyalkyl group and the other represents a hydrogen atom. X represents -NH- or -CH=CH-.] The decomposition method according to claim 9, wherein the compound is represented by the compound. [Claim 13] The photodegradable auxin derivative is the compound represented by the general formula (1) R 3 or R 4 The decomposition method according to claim 12, wherein the photodegradable auxin derivative is obtained by replacing the hydrogen atom in the carboxyl group with the photodegradable protecting group. [Claim 14] The degradation method according to claim 9, wherein the auxin degron system comprises polynucleotide A containing the coding sequence of a TIR1 family protein, and polynucleotide B containing the coding sequence of an Aux / IAA family protein or a partial peptide thereof. [Claim 15] The degradation method according to claim 14, further comprising a coding sequence for a target polypeptide or a coding sequence for a molecule that binds to a target polypeptide, so that the polynucleotide B can be expressed by fusing with the Aux / IAA family protein or a partial peptide thereof. [Claim 16] The degradation method according to claim 14, wherein the TIR1 family protein is a mutant TIR1 family protein in which auxin-receptority is reduced, in which an amino acid residue that interacts with the benzene ring in the auxin indole ring is replaced with another amino acid residue.

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