JP7478412B2 - Compounds localized in cell membranes or Golgi apparatus and uses thereof - Google Patents

Description

Applicable under Article 30, Paragraph 2 of the Patent Act. Published in the Abstracts of the 98th Spring Annual Meeting of the Chemical Society of Japan on March 6, 2018 (presentation numbers 2D3-05, 2D3-07, 2D3-42, poster presentation 2PB-143)

Application of Article 30, Paragraph 2 of the Patent Act. Presented at the 98th Spring Annual Meeting of the Chemical Society of Japan on March 21, 2018 (presentation numbers 2D3-05, 2D3-07, 2D3-42, poster presentation 2PB-143)

Application of Article 30, Paragraph 2 of the Patent Act. Presented at the 82nd Chubu Branch Meeting and Symposium of the Japanese Biochemical Society on May 19, 2018 (Poster presentations P-09, P-11, P-48)

Application of Article 30, Paragraph 2 of the Patent Act. July 19, 2018, Presentation at the 30th Summer School of the Young Researchers' Group of Biofunctional Chemistry (Poster Presentations P-01, P-02)

Application of Article 30, Paragraph 2 of the Patent Act. Presented at the 12th Bio-Related Chemistry Symposium on September 9, 2018 (Poster Presentation 1P-055)

Application of Article 30, Paragraph 2 of the Patent Act. November 3, 2018, presented at the 49th Chubu Chemical Academic Association Branch Joint Autumn Meeting (Poster presentation 1 PA30, Oral presentation 2 K08)

Application of Article 30, Paragraph 2 of the Patent Act November 8, 2018, presented at the 2018 Frontier Research Institute Symposium - Invitation to Interdisciplinary Research and Doctoral Research (Poster Presentation 2P-15)

Application of Article 30, Paragraph 2 of the Patent Act January 7, 2019, Presentation at the 6th Study Group on Future-oriented Molecular Design from Structure and Function Analysis of Biomolecules (Poster presentations P10 and P13)

Application of Article 30, Paragraph 2 of the Patent Act January 28, 2019, Published in the Abstracts of the International Symposium on "Optobiotechnology" (Invited Talk)

Application of Article 30, Paragraph 2 of the Patent Act February 5, 2019, Presentation at the International Symposium on "Optobiotechnology" (Invited Talk)

The present invention relates to a compound that localizes to the cell membrane or Golgi apparatus and uses thereof. More specifically, the present invention relates to a novel compound, an agent for localizing a tagged protein to the cell membrane or Golgi apparatus, a method for controlling the localization of a target protein in a cell, and a kit for controlling the localization of a tagged protein in a cell.

The technology of activating specific signal molecules using small molecule compounds is an important tool in life research. Since intracellular signal transduction is initiated from specific localized regions such as the cell membrane, technology of controlling the localization of signal molecules is an effective method of artificially controlling intracellular signals. Under physiological conditions, signal activity is reversible, and the activation time is an important factor that determines the function and fate of cells. Therefore, technology of reversibly controlling the localization of signal molecules using small molecule compounds and arbitrarily controlling the ON-OFF of intracellular signals is expected to become a powerful research tool for detailed analysis and elucidation of the relationship between the activation time of signals and cell functions. Such technology of controlling the localization of signal molecules is also expected to be applied as a fundamental technology for controlling the function and fate of cells.

The inventors have previously synthesized a compound (mgcTMP) that combines trimethoprim (TMP), a ligand for Escherichia coli-derived dihydrofolate reductase (eDHFR), with a motif for localization to the cell membrane or Golgi apparatus (see, for example, Non-Patent Document 1). By introducing mgcTMP into cells, a fusion protein of Escherichia coli-derived dihydrofolate reductase (eDHFR) expressed in the cytoplasm can be localized to the cell membrane or Golgi apparatus. The chemical formula of trimethoprim is shown below in formula (A1).

The chemical formula of mgcTMP is shown in the following formula (A2). The portion of mgcTMP consisting of a myristoyl group, a glycine residue, and a cysteine residue functions as a motif for localization to the cell membrane or the Golgi apparatus.

Ishida M., et al., Synthetic self-localizing ligands that control the spatial location of proteins in living cells., J Am Chem Soc., 135(34), 12684-12689, 2013.

However, the inventors discovered that when eDHFR fusion protein is localized to the cell membrane or Golgi apparatus using mgcTMP, the localization of the eDHFR fusion protein to the cell membrane or Golgi apparatus spontaneously disappears. The present invention aims to provide a new technology for localizing a target protein to the cell membrane or Golgi apparatus.

［式（１）中、Ｚは下記式（２）で表される基を表し、Ｌは、－Ｃ－が、－Ｏ－、－ＣＯ－、－ＮＨ－又は下記式（３）で表される基に置換されることにより中断されていてもよい炭素数１～５０の２価の炭化水素基を表し、Ｒ^１はタグタンパク質のリガンドとして機能する基又は前記タグタンパク質のリガンドとして機能する基に光分解性保護基が更に結合した基を表し、下記式（２）中、Ｒ^２は炭素数９以上で飽和又は不飽和の直鎖状炭化水素基を表す。

］
［２］前記タグタンパク質及び前記タグタンパク質のリガンドとして機能する基の組み合わせが、大腸菌ジヒドロ葉酸還元酵素（ｅＤＨＦＲ）タンパク質及び下記式（４）で表される基の組み合わせ、ＦＫ５０６結合タンパク質及び下記式（５）で表される基の組み合わせ、炭酸脱水酵素Ｉタンパク質若しくは炭酸脱水酵素ＩＩタンパク質及び下記式（６）で表される基の組み合わせ、ＳＮＡＰ－ｔａｇ（登録商標）タンパク質及び下記式（７）若しくは下記式（８）で表される基の組み合わせ、ＨａｌｏＴａｇ（登録商標）タンパク質及び下記式（９）で表される基の組み合わせ、又は、光活動性黄色タンパク質及び下記式（１０）で表される基の組み合わせである、［１］に記載の化合物。

［３］前記光分解性保護基が、下記式（１１）～（１９）のいずれかで表される基である、［１］又は［２］に記載の化合物。

［４］［１］～［３］のいずれかに記載の化合物からなる、前記タグタンパク質の細胞膜又はゴルジ体への局在化剤。
［５］細胞内における対象タンパク質の局在を制御する方法であって、前記細胞の細胞内に、［４］に記載の局在化剤であって、前記Ｒ^１が前記タグタンパク質のリガンドとして機能する基である局在化剤を導入し、その結果、前記局在化剤の前記リガンドとして機能する基と前記タグタンパク質とが結合し、前記タグタンパク質が前記対象タンパク質と共に細胞膜又はゴルジ体に局在する工程（ａ）を含み、前記細胞は、前記対象タンパク質と前記タグタンパク質との融合タンパク質を発現した細胞である、方法。
［６］前記工程（ａ）の後に、前記細胞内に前記タグタンパク質のリガンドを導入する工程（ｂ）を更に含み、前記工程（ｂ）において、前記リガンドとして機能する基と前記リガンドが競合し、細胞膜又はゴルジ体に局在した前記タグタンパク質の少なくとも一部において前記リガンドとして機能する基と前記リガンドが置換し、その結果、前記対象タンパク質が細胞膜又はゴルジ体から移動する、［５］に記載の方法。
［７］細胞内における対象タンパク質の局在を制御する方法であって、前記細胞の細胞内に、［４］に記載の局在化剤であって、前記Ｒ^１が前記タグタンパク質のリガンドとして機能する基に光分解性保護基が更に結合した基である局在化剤を導入する工程（ａ１）と、前記工程（ａ１）の後に前記細胞に光を照射し、その結果、前記局在化剤から前記光分解性保護基が脱離し、前記局在化剤の前記リガンドとして機能する基と前記タグタンパク質とが結合し、前記タグタンパク質が前記対象タンパク質と共に細胞膜又はゴルジ体に局在する工程（ａ２）と、を含み、前記細胞は、前記対象タンパク質と前記タグタンパク質との融合タンパク質を発現した細胞である、方法。
［８］前記工程（ａ２）の後に、前記細胞内に前記タグタンパク質のリガンドを導入する工程（ｂ）を更に含み、前記工程（ｂ）において、前記リガンドとして機能する基と前記リガンドが競合し、細胞膜又はゴルジ体に局在した前記タグタンパク質の少なくとも一部において前記リガンドとして機能する基と前記リガンドが置換し、その結果、前記対象タンパク質が細胞膜又はゴルジ体から移動する、［７］に記載の方法。
［９］前記融合タンパク質が、前記融合タンパク質のＮ末端若しくはＣ末端又は前記タグタンパク質の内部に、リジン残基が４～１０個連続したアミノ酸配列を有する、［５］～［８］のいずれかに記載の方法。
［１０］前記対象タンパク質がシグナル伝達タンパク質である、［５］～［９］のいずれかに記載の方法。
［１１］前記対象タンパク質が内在性のタンパク質であり、前記細胞が、前記対象タンパク質をコードする遺伝子座に、前記タグタンパク質をコードする遺伝子断片をノックインした細胞である、［５］～［１０］のいずれかに記載の方法。
［１２］［４］に記載の局在化剤を含む、細胞内におけるタグタンパク質の局在制御用キット。
［１３］対象タンパク質と前記タグタンパク質との融合タンパク質を発現した細胞、前記対象タンパク質と前記タグタンパク質との融合タンパク質の発現ベクター、又は、前記対象タンパク質と前記タグタンパク質との融合タンパク質作製用ベクターを更に含む、［１２］に記載のキット。
［１４］前記タグタンパク質のリガンドを更に含む、［１２］又は［１３］に記載のキット。 The present invention includes the following aspects.
[1] A compound represented by the following formula (1):

[In formula (1), Z represents a group represented by formula (2) below; L represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by substitution of -C- with -O-, -CO-, -NH-, or a group represented by formula (3) below; R ¹ represents a group which functions as a ligand for a tagged protein or a group in which a photodegradable protecting group is further bonded to a group which functions as a ligand for the tagged protein; and in formula (2) below, R ² represents a saturated or unsaturated linear hydrocarbon group having 9 or more carbon atoms.

］
[2] The compound according to [1], wherein the combination of the tag protein and the group functioning as a ligand for the tag protein is a combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and a group represented by the following formula (4), a combination of FK506 binding protein and a group represented by the following formula (5), a combination of carbonic anhydrase I protein or carbonic anhydrase II protein and a group represented by the following formula (6), a combination of SNAP-tag (registered trademark) protein and a group represented by the following formula (7) or (8), a combination of HaloTag (registered trademark) protein and a group represented by the following formula (9), or a combination of photoactive yellow protein and a group represented by the following formula (10).

[3] The compound according to [1] or [2], wherein the photodegradable protecting group is a group represented by any one of the following formulas (11) to (19):

[4] An agent for localizing the tagged protein to a cell membrane or a Golgi apparatus, comprising the compound according to any one of [1] to [3].
[5] A method for controlling localization of a target protein in a cell, the method comprising: a step (a) of introducing into the cell the localization agent according to [4], wherein the ^R1 is a group that functions as a ligand for the tag protein, so that the group that functions as a ligand of the localization agent binds to the tag protein, and the tag protein is localized together with the target protein in a cell membrane or a Golgi apparatus; the cell is a cell that expresses a fusion protein of the target protein and the tag protein.
[6] The method according to [5], further comprising, after the step (a), a step (b) of introducing a ligand of the tagged protein into the cell, in which in the step (b), the group functioning as the ligand competes with the ligand, and the ligand replaces the group functioning as the ligand in at least a part of the tagged protein localized in the cell membrane or the Golgi apparatus, resulting in the target protein being translocated from the cell membrane or the Golgi apparatus.
[7] A method for controlling localization of a target protein in a cell, the method comprising: a step (a1) of introducing into the cell the localization agent according to [4], wherein the R ¹ is a group in which a photolabile protecting group is further bound to a group that functions as a ligand of the tagged protein; and a step (a2) of irradiating the cell with light after the step (a1), so that the photolabile protecting group is detached from the localization agent, the group that functions as a ligand of the localization agent is bound to the tagged protein, and the tagged protein is localized in a cell membrane or a Golgi apparatus together with the target protein, wherein the cell is a cell that expresses a fusion protein of the target protein and the tagged protein.
[8] The method according to [7], further comprising, after the step (a2), a step (b) of introducing a ligand of the tagged protein into the cell, in which in the step (b), the group functioning as the ligand competes with the ligand, and the ligand replaces the group functioning as the ligand in at least a part of the tagged protein localized in the cell membrane or the Golgi apparatus, resulting in the target protein being translocated from the cell membrane or the Golgi apparatus.
[9] The method according to any of [5] to [8], wherein the fusion protein has an amino acid sequence of 4 to 10 consecutive lysine residues at the N-terminus or C-terminus of the fusion protein, or inside the tag protein.
[10] The method according to any one of [5] to [9], wherein the target protein is a signal transduction protein.
[11] The method according to any of [5] to [10], wherein the target protein is an endogenous protein, and the cell is a cell in which a gene fragment encoding the tag protein has been knocked in to a gene locus encoding the target protein.
[12] A kit for controlling the localization of a tagged protein in a cell, comprising the localization agent according to [4].
[13] The kit described in [12], further comprising a cell expressing a fusion protein of a target protein and the tag protein, an expression vector for the fusion protein of the target protein and the tag protein, or a vector for producing the fusion protein of the target protein and the tag protein.
[14] The kit according to [12] or [13], further comprising a ligand for the tag protein.

The present invention provides a new technology for localizing a target protein to the cell membrane or Golgi apparatus.

FIG. 1(a) is a schematic diagram showing the structure of the fusion protein expressed in Experimental Example 1. FIG. 1(b) to (d) are confocal laser microscope photographs showing the results of Experimental Example 1. 6(a) to 6(f) are confocal laser microscope photographs showing the results of Experimental Example 2. 6(a) to 6(h) are confocal laser microscope photographs showing the results of Experimental Example 3. 6(i) is a graph in which the results of 6(a) to 6(h) are quantified. 6(a) to (l) are confocal laser microscope photographs showing the results of Experimental Example 4. 1A and 1B are schematic diagrams showing the structure of the fusion protein expressed in Experimental Example 5. 1C to 1F are confocal laser microscope photographs showing the results of Experimental Example 5. 1A is a schematic diagram showing the structure of the fusion protein expressed in Experimental Example 6. 1B to 1G are confocal laser microscope photographs showing the results of Experimental Example 6. 1A is a schematic diagram showing the structure of the fusion protein expressed in Experimental Example 7. 1B and 1C are confocal laser microscope photographs showing the results of Experimental Example 7. 1A is a schematic diagram showing the structure of the fusion protein expressed in Experimental Example 8. 1B and 1C are confocal laser microscope photographs showing the results of Experimental Example 8. 1A is a schematic diagram showing the structure of the fusion protein expressed in Experimental Example 9. 1B and 1C are confocal laser microscope photographs showing the results of Experimental Example 9. 1A is a schematic diagram showing the structure of the fusion protein expressed in Experimental Example 10. 1B and 1C are confocal laser microscope photographs showing the results of Experimental Example 10. 1A and 1B are schematic diagrams showing the structure of the fusion protein expressed in Experimental Example 11. 13( a ) to 13 ( f ) are confocal laser microscope photographs showing the results of Experimental Example 11. 13( a ) to 13 ( f ) are confocal laser microscope photographs showing the results of Experimental Example 12. 13 is a graph showing the results of Experimental Examples 11 and 12. (a) is a schematic diagram showing the structure of the fusion protein expressed in Experimental Example 14. (b) to (d) are confocal laser microscope photographs showing the results of Experimental Example 14. (e) is a graph showing the results of Experimental Example 14. ¹³ is a graph showing the results of measuring the absorption spectrum of ^mDcNVOC TMP in Experimental Example 15. 13 is a graph showing the results of Experimental Example 15. 14(a) to 14(h) are confocal laser microscope photographs showing the results of Experimental Example 16. FIG. 14(i) is a graph showing the results of Experimental Example 16. (a) to (d) are confocal laser microscope photographs showing the results of Experimental Example 17. (e) is an image showing areas where the cell area has increased or decreased in Experimental Example 17. (a) to (d) are confocal laser microscope photographs showing the results of Experimental Example 18. (e) is an image showing areas where the cell area has increased or decreased in Experimental Example 18. 13(a) to (d) are images showing areas where the cell area has increased or decreased in Experimental Example 19. Graph (a) shows the amount of increase in the area of a protruding region (area where the area has increased) of a cell in Experimental Example 19. Graph (b) shows the amount of decrease in the area of a retracting region (area where the area has decreased) of a cell in Experimental Example 19. ¹³ is a graph showing ^{the results of measuring the absorption spectra of mDcNVOC TMP and mDcDEACM TMP in} Experimental Example 20. 13A and 13B are confocal laser microscope photographs showing the results of Experimental Example 21. 13C and 13D are graphs showing the results of Experimental Example 21. (a) is a schematic diagram showing the structure of a donor vector prepared in Experimental Example 22. (b) is a schematic diagram showing the structure of genomic DNA of a cell into which an insertion cassette has been knocked in as intended in Experimental Example 22. (c) is a schematic diagram showing the structure of a fusion protein expressed by a cell into which an insertion cassette has been knocked in as intended in Experimental Example 22. 1A is a photograph of agarose electrophoresis showing typical PCR results and a schematic diagram showing the structure of genomic DNA in Experimental Example 22. 1B is a photograph showing typical Western blotting results and a schematic diagram showing the structure of a fusion protein in Experimental Example 22. 1(a) is a schematic diagram showing the structure of cRaf-GDiK6 used in Experimental Example 23. FIG. 1(b) is a schematic diagram showing the structure of mCherry-ERK used in Experimental Example 23. 14(a) to (d) are representative confocal laser microscope photographs showing the results of Experimental Example 23. 13 is a graph showing the numerical results of Experimental Example 23. 1 is a graph showing the results of Western blotting in Experimental Example 24. 1 is a graph showing the relative phosphorylated cRaf level and the relative phosphorylated ERK level measured in Experimental Example 24.

［式（１）中、Ｚは下記式（２）で表される基を表し、Ｌは、－Ｏ－、－ＣＯ－、－ＮＨ－又は下記式（３）で表される基で中断されていてもよい炭素数１～５０の２価の炭化水素基を表し、Ｒ^１はタグタンパク質のリガンドとして機能する基又は前記タグタンパク質のリガンドとして機能する基に光分解性保護基が更に結合した基を表し、下記式（２）中、Ｒ^２は炭素数９以上で飽和又は不飽和の直鎖状炭化水素基を表す。

］ [Compound]
In one embodiment, the present invention provides a compound represented by formula (1):

[In formula (1), Z represents a group represented by formula (2) below; L represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by -O-, -CO-, -NH- or a group represented by formula (3) below; ^R1 represents a group which functions as a ligand for a tagged protein or a group which functions as a ligand for the tagged protein to which a photodegradable protecting group is further bonded; and in formula (2) below, ^R2 represents a saturated or unsaturated linear hydrocarbon group having 9 or more carbon atoms.

］

As described later in the Examples, the compound of this embodiment can be used to localize a tagged protein to the cell membrane (inner cell membrane) or the Golgi apparatus. In addition, by expressing a tagged protein in a cell as a fusion protein with a target protein, any target protein can be localized together with the tagged protein to the cell membrane or the Golgi apparatus.

In addition, as described later in the Examples, when a target protein is localized to a cell membrane or Golgi apparatus using conventional mgcTMP, the target protein spontaneously loses its localization. In contrast, by using the compound of this embodiment, the spontaneous loss of localization of the target protein can be suppressed, and the target protein can be maintained in localization for a longer period of time.

As described later in the Examples, the structure of the group represented by the above formula (2) is particularly important for the compound of this embodiment to localize in the cell membrane or Golgi apparatus. The group represented by the above formula (2) has a structure obtained by modifying the portion of mgcTMP described above consisting of myristoyl group-glycine residue-cysteine residue, and is a structure obtained by removing the glycine residue from the portion of mgcTMP consisting of myristoyl group-glycine residue-cysteine residue and replacing the L-cysteine residue with a D-cysteine.

Furthermore, as described later in the Examples, the inventors have clarified that when the carbon number of the group represented by ^R2 is 9 or more, it can be localized in the cell membrane or the Golgi apparatus. There is no particular upper limit to the carbon number of ^R2 , and it has been confirmed that R2 functions even if it is at least 17. Furthermore, ^R2 may contain a double bond.

In the compound represented by formula (1) above, L is not particularly limited as long as it is a group that functions as a linker connecting the group represented by formula (2) above with a group that functions as a ligand for the tag protein. More specifically, L represents a divalent hydrocarbon group having 1 to 50 carbon atoms, which may be interrupted by replacing -C- with -O-, -CO-, -NH-, or a group represented by formula (3) above.

In the compound of this embodiment, the combination of the tag protein and the group that functions as a ligand for the tag protein is not particularly limited, and any combination can be used.

Specific examples of combinations of tag proteins and groups that function as ligands for the tag proteins include a combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and a group represented by the following formula (4), a combination of FK506 binding protein and a group represented by the following formula (5), a combination of carbonic anhydrase I protein or carbonic anhydrase II protein and a group represented by the following formula (6), a combination of SNAP-tag (registered trademark) protein and a group represented by the following formula (7) or (8), a combination of HaloTag (registered trademark) protein and a group represented by the following formula (9), and a combination of photoactive yellow protein and a group represented by the following formula (10).

The tag protein and the group that functions as a ligand for the tag protein may be modified as long as they can bind to each other. For example, the tag protein may have an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to the amino acid sequence of the tag protein described above. Here, several may be, for example, 10, for example, 5, or for example, 3. In addition, the group that functions as a ligand for the tag protein may have a substituent in the above group.

The compound of this embodiment may further have a photolabile protecting group bound to the group that functions as a ligand for the tagged protein. Such a compound is called a caged compound.

As described later in the Examples, in the compound of this embodiment, when a photolabile protecting group is further bound to a group that functions as a ligand for a tagged protein, the function can be controlled by irradiation with light, etc.

When a photolabile protecting group is further bound to the group that functions as a ligand for the tagged protein, the tagged protein and the group that functions as a ligand for the tagged protein do not bind to each other. However, when the photolabile protecting group is deprotected by, for example, irradiating the protein with light of a specific wavelength, the tagged protein and the group that functions as a ligand for the tagged protein can bind to each other.

The photodegradable protecting group is not particularly limited, and any type can be used. Specific photodegradable protecting groups include, for example, a 6-nitroveratryloxycarbonyl (NVOC) group represented by the following formula (11), a 4,5-dimethoxy-2-nitrobenzyl (DMNB) group represented by the following formula (12), an α-methyl-6-nitropiperonyloxymethyl (MNPOM) group represented by the following formula (13), a (7-diethylaminocoumarin-4-yl)methyloxycarbonyl (DEACM) group represented by the following formula (14), a (7-diethylaminothiocoumarin-4-yl)methyloxycarbonyl (DEACM) group represented by the following formula (15), and a (7-diethylaminothiocoumarin-4-yl)methyloxycarbonyl (DEACM) group represented by the following formula (16). Examples of the methyloxycarbonyl (DEATCOC) group include a (6-bromo-7-hydroxycoumarin-4-yl)methyloxycarbonyl (BHCOC) group represented by the following formula (16), a (8-bromo-7-hydroxyquinolin-2-yl)methyloxycarbonyl (BHQOC) group represented by the following formula (17), a (3-nitrodibenzofuran-2-yl)methyloxycarbonyl (NDBFOC) group represented by the following formula (18), and a 3-nitrodibenzofuran-2-ylmethyl (NDBF) group represented by the following formula (19).

[Agent for localizing tagged proteins to the plasma membrane or Golgi apparatus]
In one embodiment, the present invention provides an agent for localizing a tagged protein to a cell membrane or a Golgi apparatus, comprising the above-mentioned compound. As described later in the Examples, the above-mentioned compound can be used as an agent for localizing a tagged protein to a cell membrane or a Golgi apparatus.

[Method for controlling intracellular localization of a target protein]
First Embodiment
The method of the first embodiment is a method for controlling the localization of a target protein in a cell, comprising the step (a) of introducing into the cell the above-mentioned localization agent, in which the R ¹ is a group that functions as a ligand for the tag protein, so that the group that functions as a ligand of the localization agent binds to the tag protein, and the tag protein is localized in the cell membrane or Golgi apparatus together with the target protein, and the cell is a cell that expresses a fusion protein of the target protein and the tag protein. The method of the first embodiment is a method for controlling the localization of a target protein in a cell using a localization agent that does not have a photocleavable protecting group.

In the method of the first embodiment, the cells used express a fusion protein of a target protein and a tag protein. The cells are preferably eukaryotic cells. Examples of eukaryotic cells include animal cells, insect cells, plant cells, yeast cells, etc.

The fusion protein may have one target protein or two or more. The fusion protein may have one tag protein or two or more. When there are multiple target proteins, the target proteins may be one type or two or more types. When there are multiple tag proteins, the tag proteins may be one type or two or more types.

In addition, in the fusion protein, the order in which the target protein and the tag protein are arranged is not particularly limited, and either may be present on the N-terminus side. For example, the target protein and the tag protein may be arranged in that order from the N-terminus side, or the tag protein and the target protein may be arranged in that order from the N-terminus side. In addition, when there are multiple target proteins and multiple tag proteins, the order in which they are arranged in the fusion protein is not particularly limited.

For example, as described later in the Examples, the fusion protein may have one tag protein and two target proteins. The target proteins are not particularly limited and may be fluorescent proteins, signaling proteins, enzymes, etc.

In the method of the first embodiment, in step (a), the above-mentioned localization agent is introduced into the cell expressing the fusion protein of the target protein and the tag protein. The localization agent can be introduced, for example, by adding the localization agent to the cell culture medium. Here, the cell may be a cell into which an expression vector for the fusion protein of the target protein and the tag protein has been transiently or stably introduced. Alternatively, the target protein may be an endogenous protein, and the cell may be a cell into which a gene fragment encoding the tag protein has been knocked in to a gene locus encoding the target protein. Here, the knock-in of the gene fragment encoding the tag protein may be performed by genome editing.

As described later in the Examples, when a localization agent is introduced into a cell, a group that functions as a ligand for the localization agent binds to the tagged protein, and the tagged protein is localized to the cell membrane or Golgi apparatus together with the target protein.

As described later in the Examples, the use of the above-mentioned localization agent can suppress spontaneous loss of localization of the target protein, allowing the target protein to be maintained localized for a longer period of time.

The method of the first embodiment may further include, after step (a), step (b) of introducing a ligand of the tagged protein into the cell. In this case, in step (b), the group functioning as the ligand competes with the ligand, and the ligand is substituted for the group functioning as the ligand in at least a part of the tagged protein localized in the cell membrane or Golgi apparatus, resulting in the target protein being moved from the cell membrane or Golgi apparatus. In other words, the localization of the target protein is eliminated. Step (b) makes it possible to control and eliminate the localization of the localized target substance.

Second Embodiment
The method of the second embodiment is a method for controlling the localization of a target protein in a cell, comprising: a step (a1) of introducing into the cell the above-mentioned localization agent, wherein the R ¹ is a group formed by further binding a photolabile protecting group to a group functioning as a ligand of the tagged protein; and a step (a2) of irradiating the cell with light after the step (a1), so that the photolabile protecting group is detached from the localization agent, the group functioning as a ligand of the localization agent is bound to the tagged protein, and the tagged protein is localized in the cell membrane or Golgi apparatus together with the target protein, wherein the cell is a cell expressing a fusion protein of the target protein and the tagged protein. The method of the second embodiment is mainly different from the method of the first embodiment in that a localization agent that is a caged compound is used.

In the method of the second embodiment, in step (a1), a localization agent, which is a caged compound, is introduced into a cell expressing a fusion protein of a target protein and a tag protein. In the method of the second embodiment, the method of introducing the cell and the localization agent into the cell is the same as in the method of the first embodiment.

Next, after step (a1), step (a2) is carried out in which the cells are irradiated with light. The light irradiated in step (a2) may be light that satisfies the conditions for deprotecting the photolabile protecting group of the caged compound, and may be appropriately adjusted depending on the photolabile protecting group used. For example, as described later in the Examples, when an NVOC group is used as the photolabile protecting group, light with a wavelength of 405 nm may be irradiated. When a DEACM group is used as the photolabile protecting group, light with a wavelength of 405 nm, light with a wavelength of 445 nm, or the like may be irradiated.

Upon irradiation with light, the photolabile protecting group is released from the localization agent. The group that functions as a ligand of the localization agent then binds to the tagged protein, and the tagged protein is localized together with the target protein in the cell membrane or Golgi apparatus.

As described later in the Examples, the method of the second embodiment makes it possible to control the localization of tagged proteins at the single-cell level or in specific regions on a single cell.

The method of the second embodiment may further include, after step (a2), step (b) of introducing a ligand for the tagged protein into the cell. Step (b) in the method of the second embodiment is similar to step (b) in the method of the first embodiment.

In this case, in step (b), the group functioning as a ligand competes with the ligand, and the ligand is substituted for the group functioning as a ligand in at least a portion of the tagged protein localized in the cell membrane or Golgi apparatus, resulting in the target protein being moved from the cell membrane or Golgi apparatus. In other words, the target protein is delocalized. Step (b) makes it possible to control and delocalize the localized target substance.

In the method of the first embodiment or the method of the second embodiment described above, the fusion protein of the target protein and the tag protein may have an amino acid sequence of 4 to 10 consecutive lysine residues at the N-terminus or C-terminus of the fusion protein or inside the tag protein. Here, the number of lysine residues may be 4 to 8, 4 to 7, or 6 (KKKKKK, SEQ ID NO: 1).

As described later in the Examples, when an amino acid sequence of 4 to 10 consecutive lysine residues is located at the N-terminus or C-terminus of the fusion protein or inside the tag protein, the fusion protein tends to be specifically localized to the cell membrane.

The N-terminus here may be near the N-terminus, for example, an amino acid sequence in which 4 to 10 consecutive lysine residues are located a few amino acids from the N-terminus. Similarly, the C-terminus may be near the C-terminus, for example, an amino acid sequence in which 4 to 10 consecutive lysine residues are located a few amino acids from the C-terminus. Here, a few amino acids may be, for example, 1 to 10 amino acids, for example, 1 to 5 amino acids, or for example, 1 to 3 amino acids. Furthermore, when an amino acid sequence in which 4 to 10 consecutive lysine residues are present inside the tag protein, the position is not particularly limited as long as the tag protein maintains its function of binding to its ligand.

[kit]
In one embodiment, the present invention provides a kit for controlling intracellular localization of a tagged protein, comprising the above-mentioned localization agent. By using the kit of this embodiment, the localization of the tagged protein in cells can be controlled.

In the kit of this embodiment, the localization agent may or may not have a photolabile protecting group.

The kit of this embodiment may further include cells expressing a fusion protein of the target protein and a tag protein, an expression vector for the fusion protein of the target protein and a tag protein, or a vector for producing the fusion protein of the target protein and a tag protein.

In the kit of this embodiment, the cells may be the same as those described above. When the kit of this embodiment includes cells expressing a fusion protein of a target protein and a tag protein, the localization of the tag protein within the cells can be controlled by introducing the localization agent described above into the cells.

When the kit of this embodiment includes an expression vector for a fusion protein of a target protein and a tag protein, the expression vector can be introduced into a desired cell to express the fusion protein, thereby preparing a cell expressing the fusion protein of the target protein and the tag protein. By introducing the above-mentioned localization agent into this cell, the localization of the tag protein within the cell can be controlled.

An example of a vector for producing a fusion protein of a target protein and a tag protein is a vector that includes a gene encoding the tag protein and a multi-cloning site downstream of a promoter. Either the gene encoding the tag protein or the multi-cloning site may be located on the promoter side. An expression vector for a fusion protein of a target protein and a tag protein can be prepared by cloning a gene encoding a desired target protein into the multi-cloning site of a vector for producing a fusion protein.

Then, the expression vector can be introduced into a desired cell to express the fusion protein of the target protein and the tag protein, thereby preparing a cell that expresses the fusion protein of the target protein and the tag protein. By introducing the above-mentioned localization agent into this cell, the localization of the tag protein within the cell can be controlled.

The kit of this embodiment may further include a ligand for the tagged protein. As described later in the Examples, after localizing the tagged protein, the localization of the tagged protein can be eliminated by introducing the ligand for the tagged protein into the cell.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
Spontaneous delocalization by mgcTMP
A fusion protein of a target protein and a tag protein was expressed in HeLa cells, a cell line derived from human cervical cancer. EGFP was used as the target protein, and eDHFR was used as the tag protein. FIG. 1(a) is a schematic diagram showing the structure of the expressed fusion protein. eDHFR was placed on the N-terminus side of the fusion protein, and EGFP was placed on the C-terminus side. Furthermore, a K6 tag was placed on the N-terminus of the fusion protein. The amino acid sequence of the expressed fusion protein is shown in SEQ ID NO: 2. Hereinafter, this fusion protein may be referred to as "K6-DG".

Next, mgcTMP was added to the medium of HeLa cells expressing the fusion protein (K6-DG) (hereinafter sometimes referred to as "K6-DG expressing cells") to a final concentration of 10 μM. Then, EGFP fluorescence was observed using a confocal laser microscope.

Figures 1(b) to (d) are confocal laser microscope photographs. Figure 1(b) is a photograph of K6-DG expressing cells before the addition of mgcTMP. Figure 1(c) is a photograph of K6-DG expressing cells taken 10 minutes after the addition of mgcTMP. Figure 1(d) is a photograph of K6-DG expressing cells taken 40 minutes after the addition of mgcTMP.

As a result, it was found that EGFP was localized to the cell membrane 10 minutes after the addition of mgcTMP. However, it was found that EGFP localization spontaneously disappeared 40 minutes after the addition of mgcTMP.

[Experimental Example 2]
(Examination of localization by mg ^D cTMP)
A localization agent in which the cysteine residue of mgcTMP was replaced with the D form (hereinafter, sometimes referred to as "mg ^D cTMP") was synthesized, and its ability to localize tagged proteins was examined.

Synthesis of mg- ^D- cTMP
First, mg ^-D- cTMP was synthesized according to the following scheme (1).

Specifically, mg ^D cTMP was synthesized on Rink amide resin following standard solid phase peptide synthesis protocols utilizing the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.

Fmoc deprotection was performed with 20% piperidine in N,N-dimethylformamide (DMF) for 15 min at room temperature. Amino acid coupling reactions were performed with a mixture of Fmoc-protected amino acid (4.1 equiv.), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 4.0 equiv.), 1-hydroxybenzotriazole (HOBt, 4.0 equiv.) and N,N-diisopropylethylamine (DIPEA, 8.0 equiv.) in DMF at room temperature. All Fmoc deprotection and coupling steps were monitored by Kaiser test. All washing procedures were performed with DMF.

First, Rink amide resin (0.65 mmol/g) (185 mg, 120 μmol) was Fmoc-deprotected and washed. Then, Fmoc-Lys(ivDde)-OH was coupled to the resin and washed with DMF.

Next, Fmoc deprotection and coupling reactions were repeated using Fmoc-Adox-OH, Fmoc-D-Cys(Trt)-OH, and Fmoc-Gly-OH as building blocks.

Alternatively, the N-terminus was myristoylated using a mixture of myristic acid (4.1 equiv.), HBTU (4.0 equiv.), HOBt (4.0) and DIPEA (8.0 equiv.) in DMF/CH ₂ Cl ₂ (1:1).

The ivDde group was then selectively deprotected by treatment with DMF containing 5% hydrazine.

Subsequently, a mixture of compound X1 (3.1 equivalents), HBTU (3.0 equivalents), HOBt (3.0 equivalents) and DIPEA (8.0 equivalents) was reacted in DMF to attach compound X1 to the side chain of the lysine residue.

Trt deprotection and cleavage from the resin was carried out with TFA containing 5% ethanedithiol (EDT) and 2.5% water. The crude product was then precipitated with diethyl ether and purified by reversed-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA to give mg ^D cTMP as a white solid.

^1H NMR (400MHz, _CD3 OD): δ 0.89 (3H,t), 1.28 (22H,m), 1.40 (2H,m), 1.53 (2H,m), 1.62 (2H,m), 1.71 (2H,m), 1.80 (2H,m), 2.26 (4H,m), 2.86 (2H,m), 3.18 (2H,t), 3.41 (2H,m), 3.45 (4H,m), 3.59 (6H,m), 3.66 (14H,m), 3.80 (6H,s), 3.88 (2H,s,s), 3.92 (2H,t), 4.00 (4H,s), 4.03 (2H,s), 4.42 (1H,m), 4.50 (1H,m), 6.56 (2H,s), 7.22 (1H,s).

<<Examination of localization by ^mgDcTMP >>
^mgDcTMP was added to the medium of K6-DG expressing cells to a final concentration of 10 μM, and EGFP fluorescence was observed using a confocal laser microscope. For comparison, a group in which mgDcTMP was added to the medium of K6-DG expressing cells at a final concentration of 10 μM was also prepared.

Figures 2(a) to (f) are confocal laser microscope photographs. Figure 2(a) is a photograph of K6-DG expressing cells before the addition of mgcTMP. Figure 2(b) is a photograph of K6-DG expressing cells taken 10 minutes after the addition of mgcTMP. Figure 2(c) is a photograph of K6-DG expressing cells taken 90 minutes after the addition of mgcTMP.

Fig. 2(d) is a photograph of K6-DG expressing cells before the addition of mg ^D cTMP, Fig. 2(e) is a photograph of K6-DG expressing cells taken 10 minutes after the addition of mg ^D cTMP, and Fig. 2(f) is a photograph of K6-DG expressing cells taken 90 minutes after the addition of mg ^D cTMP.

As a result, similar to the results of Experimental Example 1, it was found that EGFP was localized to the cell membrane 10 minutes after the addition of mgcTMP. However, it was found that the localization of EGFP spontaneously disappeared 90 minutes after the addition of mgcTMP.

Furthermore, it was revealed that EGFP was localized to the cell membrane 10 minutes after the addition of mg ^-D- cTMP, but that the localization of EGFP spontaneously disappeared 90 minutes after the addition of mg ^-D- cTMP.

These results demonstrated that the spontaneous loss of EGFP localization could not be prevented even when mg ^D cTMP was used instead of mgcTMP.

[Experimental Example 3]
(Examination of localization by ^mDcTMP )
A compound was synthesized by removing the glycine residue from mgcTMP and further substituting the cysteine residue with the D-form (hereinafter sometimes referred to as " ^mDcTMP "), and its ability to localize tagged proteins was examined.

Synthesis of ^mDcTMP
First, m ^D cTMP was synthesized according to the following scheme (2). Specifically, after coupling Fmoc-D-Cys(Trt)-OH to a resin, the same reaction as in scheme 1 in Experimental Example 2 was carried out, except that the N-terminus was myristoylated without coupling Fmoc-Gly-OH. The product was then purified by reversed-phase HPLC to obtain m ^D cTMP as a white solid.

^1H NMR (400MHz, _CD3OD ): δ 0.89 (3H,t), 1.28 (22H,m), 1.40 (2H,m), 1.53 (2H,m), 1.61 (2H,m), 1.71 (2H,m), 1.82 (2H,m), 2.25 (4H,m), 2.84 (2H,m), 3.18 (2H,t), 3.46 (6H,m), 3.59 (6H,m), 3.66 (14H,m), 3.80 (6H,s), 3.92 (2H,t), 4.00 (4H,s), 4.03 (2H,s), 4.44 (2H,m), 6.56 (2H,s), 7.22 (1H,s).

<<Examination of localization by ^mDcTMP >>
To the medium of K6-DG expressing cells, ^mDcTMP was added to a final concentration of 10 μM, and the fluorescence of EGFP was observed with a confocal laser microscope. 120 minutes after the addition of ^mDcTMP , a ligand was further added to the medium at a final concentration of 100 μM, and the fluorescence of EGFP was observed with a confocal laser microscope. Free trimethoprim (hereinafter sometimes referred to as "TMP") was used as the ligand.

3(a) to (c) are confocal laser microscope photographs showing the results for comparison, in which mgcTMP was added to the medium of K6-DG expressing cells at a final concentration of 10 μM. Also, FIG. 3(d) to (g) are confocal laser microscope photographs showing the results of adding m ^D cTMP to the medium of K6-DG expressing cells at a final concentration of 10 μM. Also, FIG. 3(h) is a confocal laser microscope photograph showing the results of further adding TMP 120 minutes after adding m ^D cTMP to the medium of K6-DG expressing cells.

Figure 3(a) is a photograph of K6-DG expressing cells before the addition of mgcTMP. Figure 3(b) is a photograph of K6-DG expressing cells taken 10 minutes after the addition of mgcTMP. Figure 3(c) is a photograph of K6-DG expressing cells taken 90 minutes after the addition of mgcTMP.

FIG. 3(d) is a photograph of K6-DG expressing cells before the addition of m ^D cTMP. FIG. 3(e) is a photograph of K6-DG expressing cells taken 10 minutes after the addition of m ^D cTMP. FIG. 3(f) is a photograph of K6-DG expressing cells taken 90 minutes after the addition of m ^D cTMP. FIG. 3(g) is a photograph of K6-DG expressing cells taken 120 minutes after the addition of m ^D cTMP. FIG. 3(h) is a photograph of K6-DG expressing cells taken 20 minutes after the addition of TMP 120 minutes after the addition of m ^D cTMP, that is, 140 minutes after the addition of m ^D cTMP.

Figure 3(i) is a graph that quantifies the results of Figures (a) to (c) and (d) to (h). In Figure 3(i), the vertical axis represents the standardized intracytoplasmic fluorescence intensity (relative value), and the horizontal axis represents the time (minutes) after addition of the localization agent.

As a result, it was found that EGFP localization spontaneously disappeared 90 minutes after the addition of mgcTMP, whereas EGFP localization was maintained at least 120 minutes after the addition of ^mDcTMP . Furthermore, it was found that the EGFP localization could be restored at the desired timing by adding free TMP.

[Experimental Example 4]
(Investigation of the structure of the myristoyl group of ^mDcTMP )
The number of carbon atoms and the number of double bonds in the myristoyl group of ^mDcTMP were modified to examine the localization ability of the tagged protein. First, compounds in which the group represented by ^R3 in the compound represented by the following formula (20) was variously modified were synthesized.

Specifically, by appropriately modifying Scheme 1 in Experimental Example 2, compounds in which R ³ in the above formula (20) is a nonanoyl group shown in the following formula (21), a decanoyl group shown in the following formula (22), a lauroyl group shown in the following formula (23), a palmitoyl group shown in the following formula (24), a stearoyl group shown in the following formula (25), or an oleoyl group shown in the following formula (26) were synthesized.

Next, each compound was added to the culture medium of K6-DG-expressing cells to a final concentration of 10 μM, and EGFP fluorescence was observed using a confocal laser microscope. Figures 4(a) to (l) are confocal laser microscope photographs showing the results of adding each compound to the culture medium of K6-DG-expressing cells at a final concentration of 5 μM.

4(a) and (b) are photographs of K6-DG expressing cells before and 60 minutes after the addition of a compound in which R ³ in formula (20) is a nonanoyl group represented by formula (21), respectively.

4(c) and (d) are photographs of K6-DG expressing cells before and 60 minutes after the addition of a compound in which R ³ in formula (20) is a decanoyl group represented by formula (22), respectively.

4(e) and (f) are photographs of K6-DG expressing cells before and 60 minutes after the addition of a compound in which R ³ in formula (20) is a lauroyl group represented by formula (23), respectively.

4(g) and (h) are photographs of K6-DG expressing cells before and 60 minutes after the addition of a compound in which R ³ in formula (20) is a palmitoyl group represented by formula (24), respectively.

4(i) and (j) are photographs of K6-DG expressing cells before and 60 minutes after the addition of a compound in which R ³ in formula (20) is a stearoyl group represented by formula (25), respectively.

4(k) and (l) are photographs of K6-DG expressing cells before and 60 minutes after the addition of a compound in which R ³ in formula (20) is an oleoyl group represented by formula (26), respectively.

As a result, it was found that the efficiency of localization of EGFP to the cell membrane is significantly reduced in the compound in which R ³ in the above formula (20) is a nonanoyl group shown in the above formula (21).In contrast, the localization of EGFP to the cell membrane was observed in the compound in which R ³ in the above formula (20) is a decanoyl group shown in the above formula (22), a lauroyl group shown in the above formula (23), a palmitoyl group shown in the above formula (24), a stearoyl group shown in the above formula (25), and an oleoyl group shown in the above formula (26).

From the above results, it was revealed that in the compound represented by the above formula (20), if ^R3 is an acyl group having a saturated or unsaturated linear hydrocarbon group with 10 or more carbon atoms, the compound has the ability to localize a tagged protein. In other words, it was revealed that in the compound represented by the following formula (27), if ^R2 is a saturated or unsaturated linear hydrocarbon group with 9 or more carbon atoms, the compound has the ability to localize a tagged protein.

[Experimental Example 5]
(Effect of adding K6 tag to fusion protein 1)
A fusion protein of a target protein and a tag protein was expressed in HeLa cells. EGFP was used as the target protein, and eDHFR was used as the tag protein. FIG. 5(a) is a schematic diagram showing the structure of the expressed fusion protein. eDHFR was placed on the N-terminus side of the fusion protein, and EGFP was placed on the C-terminus side. Hereinafter, this fusion protein may be referred to as "DG". The amino acid sequence of DG is shown in SEQ ID NO:3.

HeLa cells expressing a fusion protein in which a K6 tag was placed at the N-terminus of DG (hereinafter, sometimes referred to as "K6-DG") were also prepared. Figure 5(b) is a schematic diagram showing the structure of K6-DG. The amino acid sequence of K6-DG is shown in SEQ ID NO:2.

Next, mgcTMP was added to the medium of HeLa cells expressing DG (hereinafter sometimes referred to as "DG expressing cells") and HeLa cells expressing K6-DG (hereinafter sometimes referred to as "K6-DG expressing cells") to a final concentration of 10 μM. Next, EGFP fluorescence was observed using a confocal laser microscope.

Figures 5(c) to (f) are confocal laser microscope photographs. Figure 5(c) is a photograph of DG-expressing cells before the addition of mgcTMP. Figure 5(d) is a photograph of DG-expressing cells taken 10 minutes after the addition of mgcTMP. Figure 5(e) is a photograph of K6-DG-expressing cells before the addition of mgcTMP. Figure 5(f) is a photograph of K6-DG-expressing cells taken 10 minutes after the addition of mgcTMP.

As a result, it was found that in DG-expressing cells, EGFP was localized in the cell membrane and Golgi apparatus 10 minutes after the addition of mgcTMP. On the other hand, in K6-DG-expressing cells, EGFP was localized in the cell membrane 10 minutes after the addition of mgcTMP.

These results demonstrate that adding a K6 tag to the N-terminus of the fusion protein improves the specificity of localization to the cell membrane.

[Experimental Example 6]
(Effect of adding K6 tag to fusion protein 2)
A fusion protein of a target protein and a tag protein was expressed in HeLa cells. EGFP was used as the target protein, and eDHFR was used as the tag protein. EGFP was placed on the N-terminus side of the fusion protein, and eDHFR was placed on the C-terminus side. In addition, a K6 tag was placed between EGFP and eDHFR. FIG. 6(a) is a schematic diagram showing the structure of the expressed fusion protein. Hereinafter, this fusion protein may be referred to as "G-K6-D". The amino acid sequence of G-K6-D is shown in SEQ ID NO:4.

Next, mDcTMP was added to the medium of HeLa cells expressing G-K6-D (hereinafter sometimes referred to as "G-K6-D expressing cells"), DG expressing cells similar to those in Experimental Example 5, and K6- ^DG expressing cells to a final concentration of 10 μM. Next, EGFP fluorescence was observed with a confocal laser microscope.

6(b) to (g) are confocal laser microscope photographs. FIG. 6(b) is a photograph of DG-expressing cells before the addition of m ^D cTMP. FIG. 6(c) is a photograph of DG-expressing cells taken 90 minutes after the addition of m ^D cTMP. FIG. 6(d) is a photograph of K6-DG-expressing cells before the addition of m ^D cTMP. FIG. 6(e) is a photograph of K6-DG-expressing cells taken 90 minutes after the addition of m ^D cTMP. FIG. 6(f) is a photograph of G-K6-D-expressing cells before the addition of m ^D cTMP. FIG. 6(g) is a photograph of G-K6-D-expressing cells taken 90 minutes after the addition of m ^D cTMP.

As a result, it was revealed that in DG expressing cells, EGFP was localized in the cell membrane and Golgi apparatus 90 minutes after the addition of m ^D cTMP. Also, in K6-DG expressing cells, EGFP was localized in the cell membrane 90 minutes after the addition of m ^D cTMP. Also, in G-K6-D expressing cells, EGFP was localized in the cell membrane and Golgi apparatus 90 minutes after the addition of m ^D cTMP.

These results demonstrate that adding a K6 tag to the inside of the fusion protein does not improve the specificity of localization to the cell membrane.

[Experimental Example 7]
(Effect of adding K6 tag to fusion protein 3)
A fusion protein of a target protein and a tag protein was expressed in HeLa cells. EGFP was used as the target protein, and FKBP12 (F36V) was used as the tag protein. EGFP was placed at the N-terminus of the fusion protein, and FKBP12 (F36V) was placed at the C-terminus. In addition, a K6 tag was placed at the C-terminus of FKBP12 (F36V). FIG. 7(a) is a schematic diagram showing the structure of the expressed fusion protein. Hereinafter, this fusion protein may be referred to as "EGFP-FKBP-K6". The amino acid sequence of EGFP-FKBP-K6 is shown in SEQ ID NO:5.

Synthesis of ^{mDcSLF *}
m ^D cSLF ^* was synthesized according to the following scheme (3). Specifically, the same reaction as in Scheme 1 in Experimental Example 2 was carried out, except that a compound represented by the following formula (X2) was used instead of compound X1, Fmoc-D-Cys(Trt)-OH was coupled to the resin and then the N-terminus was myristoylated without coupling Fmoc-Gly-OH, the number of couplings of Fmoc-Adox-OH was different, and the resin and the conditions for cleavage from the resin were different. Subsequently, the product was purified by reverse phase HPLC to obtain m ^D cSLF ^* as a white solid.

^1H NMR (400MHz, _CD3OD ): δ 0.83-0.91 (6H, m), 1.25-1.38 (20H, m), 1.59-2.10 (16H, m), 2.26-2.37 (4H, m), 2.50 (2H, m), 2.69-2.85 (3H, m), 3.42-3.46 (10H, m), 3.57-3.69 (39H, m), 3.80-3.82 (10H, m), 4.46-4.52 (4H, m), 5.39 (1H, m), 5.58 (1H, m), 6.54 (1H, d), 6.58 (2H, s), 6.68 (1H, d), 6.73 (1H, d), 6.79 (1H, s), 6.84-6.87 (2H,m), 7.19 (1H,m).

<<Examination of localization using ^{mDcSLF *} >>
^{mDcSLF *} was added to the medium of HeLa cells expressing EGFP-FKBP-K6 (hereinafter sometimes referred to as "EGFP-FKBP-K6 expressing cells") to a final concentration of 5 μM. Subsequently, the fluorescence of EGFP was observed with a confocal laser microscope.

Figures 7(b) and (c) are confocal laser microscope photographs. Figure 7(b) is a photograph of EGFP-FKBP-K6 expressing cells before the addition of ^{mDcSLF *} . Figure 7(c) is a photograph of EGFP-FKBP-K6 expressing cells taken 120 minutes after the addition of ^{mDcSLF *} . As a result, it was revealed that EGFP was localized in the cell membrane 120 minutes after the addition of ^{mDcSLF *} .

These results demonstrate that adding a K6 tag to the C-terminus of the fusion protein can improve the specificity of localization to the cell membrane.

It was also revealed that the tagged protein can be localized even when the combination of the tagged protein and the group functioning as a ligand is a combination other than the combination of TMP and eDHFR.

[Experimental Example 8]
(Effect of adding K6 tag to fusion protein 4)
A fusion protein of a target protein and a tag protein was expressed in HeLa cells. The target protein was mCherry, a fluorescent protein, and the tag protein was FKBP12 (F36V). FKBP12 (F36V) was placed on the N-terminus of the fusion protein, and mCherry was placed on the C-terminus. FIG. 8(a) is a schematic diagram showing the structure of the expressed fusion protein. Hereinafter, this fusion protein may be referred to as "FKBP-mCherry." The amino acid sequence of FKBP-mCherry is shown in SEQ ID NO:6.

^{mDcSLF *} was added to the medium of HeLa cells expressing FKBP-mCherry (hereinafter sometimes referred to as "FKBP-mCherry-expressing cells") to a final concentration of 5 μM. Subsequently, the fluorescence of mCherry was observed with a confocal laser microscope.

Figures 8(b) and (c) are confocal laser microscope photographs. Figure 8(b) is a photograph of FKBP-mCherry-expressing cells before the addition of ^{mDcSLF *} . Figure 8(c) is a photograph of FKBP-mCherry-expressing cells taken 60 minutes after the addition of ^{mDcSLF *} . The results demonstrated that mCherry was localized in the Golgi apparatus 60 minutes after the addition of ^{mDcSLF *} .

These results demonstrate that fusion proteins without a K6 tag tend to localize to the Golgi apparatus.

It was also revealed that the tag protein can be localized even when the combination of the target protein and tag protein is a combination other than the combination of EGFP and FKBP12 (F36V).

[Experimental Example 9]
(Examination of localization by ^mDcSA 1)
A fusion protein of a target protein and a tag protein was expressed in HeLa cells. EGFP was used as the target protein, and carbonic anhydrase I protein (hereinafter sometimes referred to as "CAI") was used as the tag protein. CAI was positioned at the N-terminus of the fusion protein, and EGFP was positioned at the C-terminus. Figure 9(a) is a schematic diagram showing the structure of the expressed fusion protein. Hereinafter, this fusion protein will be referred to as "CAI-EGFP". The amino acid sequence of CAI-EGFP is shown in SEQ ID NO:7.

Synthesis of ^mDcSA
m ^D cSA was synthesized according to the following scheme (4). Specifically, the same reaction as in scheme 1 in Experimental Example 2 was carried out, except that a compound represented by the following formula (X3) was used instead of compound X1, Fmoc-D-Cys(Mmt)-OH was coupled to the resin and then the N-terminus was myristoylated without coupling Fmoc-Gly-OH, the number of couplings of Fmoc-Adox-OH was different, and the resin and the conditions for cleavage from the resin were different. The product was then purified by reverse phase HPLC to obtain m ^D cSA as a white solid.

^1H NMR (400MHz, _CD3OD ): δ 0.88-0.91 (3H, t, J = 6.0Hz), 1.28-1.31 (22H, m), 1.47-1.67 (7H, m), 2.26 (2H, t, J = 8.0Hz), 2.73-2.88 (2H, m), 3.37-3.46 (12H, m), 3.54-3.61 (10H, m), 3.65-3.67 (20H, m), 3.99-4.02 (10H, m), 4.43-4.48 (2H, m), 7.94-7.99 (4H, m).

<<Examination of localization by ^mDcSA >>
^mDcSA was added to the medium of HeLa cells expressing CAI-EGFP (hereinafter sometimes referred to as "CAI-EGFP expressing cells") to a final concentration of 100 μM. Subsequently, the fluorescence of EGFP was observed with a confocal laser microscope.

9(b) and (c) are confocal laser microscope photographs. Fig. 9(b) is a photograph of CAI-EGFP expressing cells before the addition of ^mDcSA . Fig. 9(c) is a photograph of CAI-EGFP expressing cells taken 60 minutes after the addition of ^mDcSA . The results demonstrated that EGFP was localized in the Golgi apparatus 60 minutes after the addition of ^mDcSA .

These results further support the idea that fusion proteins without a K6 tag tend to localize to the Golgi apparatus.

It was further supported that the tagged protein can be localized even when the combination of the tagged protein and the group functioning as a ligand is a combination other than the combination of TMP and eDHFR.

[Experimental Example 10]
(Examination of localization by ^mDcSA 2)
A fusion protein of a target protein and a tag protein was expressed in HeLa cells. EGFP was used as the target protein, and carbonic anhydrase II protein (hereinafter sometimes referred to as "CAII") was used as the tag protein. CAII was positioned at the N-terminus of the fusion protein, and EGFP was positioned at the C-terminus. FIG. 10(a) is a schematic diagram showing the structure of the expressed fusion protein. Hereinafter, this fusion protein will be referred to as "CAII-EGFP". The amino acid sequence of CAII-EGFP is shown in SEQ ID NO:8.

^mDcSA was added to the medium of HeLa cells expressing CAII-EGFP (hereinafter sometimes referred to as "CAII-EGFP expressing cells") to a final concentration of 10 μM. Subsequently, the fluorescence of EGFP was observed with a confocal laser microscope.

Figures 10(b) and (c) are confocal laser microscope photographs. Figure 10(b) is a photograph of CAII-EGFP expressing cells before the addition of ^mDcSA . Figure 10(c) is a photograph of CAII-EGFP expressing cells taken 60 minutes after the addition of ^mDcSA . The results demonstrated that EGFP was localized in the Golgi apparatus 60 minutes after the addition of ^mDcSA .

These results further support the idea that fusion proteins without a K6 tag tend to localize to the Golgi apparatus.

It was further supported that the tagged protein can be localized even when the combination of the tagged protein and the group functioning as a ligand is a combination other than the combination of TMP and eDHFR.

[Experimental Example 11]
(Control of intracellular signaling using ^mDcTMP 1)
Intracellular signaling was controlled using ^mDcTMP . First, a fusion protein of a target protein and a tag protein was prepared. EGFP and cRaf were used as the target proteins. eDHFR was used as the tag protein. eDHFR, EGFP, and cRaf were arranged in order from the N-terminus of the fusion protein. In addition, a K6 tag was further arranged on the N-terminus of eDHFR.

Figure 11(a) is a schematic diagram showing the structure of the fusion protein used. Hereinafter, this fusion protein may be referred to as "K6-DG-cRaf." The amino acid sequence of K6-DG-cRaf is shown in SEQ ID NO:9.

We also prepared a fusion protein of mCherry and ERK (hereinafter sometimes referred to as "mCherry-ERK"). Figure 11(b) is a schematic diagram showing the structure of mCherry-ERK. The amino acid sequence of mCherry-ERK is shown in SEQ ID NO: 10.

Next, K6-DG-cRaf and mCherry-ERK were expressed in HeLa cells. Next, mDcTMP was added to the medium of the HeLa cells expressing K6-DG-cRaf and mCherry-ERK (hereinafter, sometimes referred to as "K6- ^DG -cRaf and mCherry-ERK expressing cells") to a final concentration of 10 μM. Next, the fluorescence of EGFP and the fluorescence of mCherry were observed using a confocal laser microscope.

When cRaf is localized to the cell membrane, it can activate MEK, which in turn activates ERK. ERK is localized in the cytoplasm in the inactive state, but translocates into the nucleus when activated.

By observing the fluorescence of EGFP, the localization of K6-DG-cRaf can be detected. In addition, by observing the fluorescence of mCherry, the localization of mCherry-ERK can be detected.

12(a) to (f) are confocal laser microscope photographs. FIG. 12(a) and (d) are photographs of K6-DG-cRaf and mCherry-ERK expressing cells before the addition of m ^D cTMP. FIG. 12(b) and (e) are photographs of K6-DG-cRaf and mCherry-ERK expressing cells taken 10 minutes after the addition of m ^D cTMP. FIG. 12(c) and (f) are photographs of K6-DG-cRaf and mCherry-ERK expressing cells taken 70 minutes after the addition of m ^D cTMP.

Figures 12(a) to (c) show the results of observing EGFP fluorescence in K6-DG-cRaf, and Figures 12(d) to (f) show the results of observing mCherry fluorescence in mCherry-ERK.

As a result, it was revealed that K6-DG-cRaf was localized to the cell membrane and mCherry-ERK was translocated into the nucleus 10 minutes after the addition of m ^D cTMP. This state was maintained even 70 minutes after the addition of m ^D cTMP. This result indicates that intracellular signals can be controlled using m ^D cTMP.

[Experimental Example 12]
(Control of intracellular signaling using ^mDcTMP 2)
To the medium of K6-DG-cRaf and mCherry-ERK expressing cells as in Experimental Example 11, m ^D cTMP was added to a final concentration of 10 μM. In addition, 20 minutes after the addition of m ^D cTMP, a ligand was further added to a final concentration of 100 μM. Free trimethoprim (hereinafter sometimes referred to as "TMP") was used as the ligand. Subsequently, the fluorescence of EGFP and the fluorescence of mCherry were observed with a confocal laser microscope.

13(a) to (f) are confocal laser microscope photographs. FIG. 13(a) and (d) are photographs of K6-DG-cRaf and mCherry-ERK expressing cells before the addition of m ^D cTMP. FIG. 13(b) and (e) are photographs of K6-DG-cRaf and mCherry-ERK expressing cells taken 10 minutes after the addition of m ^D cTMP. FIG. 13(c) and (f) are photographs of K6-DG-cRaf and mCherry-ERK expressing cells taken 70 minutes after the addition of m ^D cTMP.

Figures 13(a) to (c) show the results of observing EGFP fluorescence in K6-DG-cRaf, and Figures 13(d) to (f) show the results of observing mCherry fluorescence in mCherry-ERK.

As a result, it was revealed that 10 minutes after the addition of m ^D cTMP, K6-DG-cRaf was localized to the cell membrane, and mCherry-ERK was translocated into the nucleus. It was also revealed that the addition of TMP eliminated the localization of K6-DG-cRaf to the cell membrane, which resulted in mCherry-ERK becoming inactive and the localization of mCherry-ERK in the nucleus being eliminated. This result indicates that intracellular signals can be reversibly controlled using m ^D cTMP.

Fig. 14 is a graph showing the results of Experimental Examples 11 and 12. In Fig. 14, "m ^D cTMP" indicates the result of Experimental Example 11, "m ^D cTMP→TMP" indicates the result of Experimental Example 12, "Normalized nucleus/cytoplasm ratio of mCherry-ERK" indicates the standardized value of the nucleus/cytoplasm ratio of the fluorescence intensity of mCherry, and "Normalized cytosolic fluorescence intensity of K6-DG-cRaf" indicates the standardized value of the fluorescence intensity of EGFP in the cytoplasm.

[Experimental Example 13]
(Synthesis of ^{mDcNVOC TMP} )
A caged localization agent was synthesized. Specifically, m ^D c ^NVOC TMP, a compound in which a photodegradable protecting group is further bonded to the trimethoprim group of m ^D cTMP, was synthesized. The NVOC group represented by the above formula (11) was used as the photodegradable protecting group. The NVOC group can be deprotected by, for example, irradiating light with a wavelength of 405 nm. The synthesis of ^{m D} c ^NVOC TMP was carried out according to the following scheme (5).

^1H NMR (400MHz, d-DMSO): δ 0.89 (t, 3H), 1.43-1.25 (m, 22H), 1.86-1.49 (m, 10H), 2.28-2.23 (m, 4H), 2.78-2.72 (m, 1H), 2.88-2.83 (m, 1H), 3.18 (t, 2H), 3.70-3.34 (m, 24H), 3.76 (s, 6H), 4.04-3.84 (m, 12H), 4.47-4.40 (m, 2H), 5.60 (s, 2H), 6.54 (s, 2H), 7.20 (s, 1H), 7.70 (s, 1H), 7.77 (s, 1H).

[Experimental Example 14]
(Localization induction by light irradiation 1)
A fusion protein of a target protein and a tag protein was expressed in HeLa cells. The target protein was a fluorescent protein, mScarlet I, and the tag protein was eDHFR. A K6 tag was placed at the N-terminus of eDHFR.

Figure 15(a) is a schematic diagram showing the structure of the expressed fusion protein. Hereinafter, this fusion protein may be referred to as "K6-DS." The amino acid sequence of K6-DS is shown in SEQ ID NO:11.

Next, ^mDcNVOC TMP was added to the medium of HeLa cells expressing K6-DS (hereinafter sometimes referred to as "K6-DS expressing cells") to a final concentration of 10 μM. Then, 5 minutes after the addition of ^mDcNVOC TMP, the cells were washed. For comparison, ^K6 -DS expressing cells to which ^mDcNVOC TMP was not added were also prepared. Next, the cells were irradiated with light having ^a wavelength of 405 nm for 2.6 seconds, and the fluorescence of mScarlet I was observed with ^a confocal laser microscope.

15(b) to (d) are confocal laser microscope photographs of K6-DS expressing cells to which m ^D c ^NVOC TMP was added. FIG. 15(b) is a photograph of K6-DS expressing cells before light irradiation. FIG. 15(c) is a photograph of K6-DS expressing cells 10 seconds after the start of the experiment. FIG. 15(d) is a photograph of K6-DS expressing cells 60 seconds after the start of the experiment. FIG. 15(e) is a graph in which the experimental results are quantified. In FIG. 15(e), the horizontal axis indicates time (seconds) and the vertical axis indicates the fluorescence intensity ratio of the cytoplasm, with "+m ^D c ^NVOC TMP" indicating the results of K6-DS expressing cells to which m ^D c ^NVOC TMP was added and "-m ^D c ^NVOC TMP" indicating the results of K6-DS expressing cells to which m ^D c ^NVOC TMP was not added.

As a result, it was revealed that in K6-DS expressing cells ^{to which mDcNVOC TMP} had been added, mScarlet I was localized in the cell membrane immediately after light irradiation.

These results demonstrate that the use of a compound with a photolabile protecting group makes it possible to control the induction of localization of tagged proteins by light irradiation.

[Experimental Example 15]
(Localization induction by light irradiation 2)
16 is a graph showing the results of measuring the absorption spectrum of ^mDcNVOC TMP. As shown in FIG ^{. 16, it was revealed that mDcNVOC TMP absorbs} light with a wavelength of 405 nm, but hardly absorbs light with a wavelength of 445 nm.

Next, K6-DS expressing cells were treated with various combinations of ^mDcNVOC TMP, irradiation with light having a wavelength of 405 nm, irradiation with light having ^a wavelength of 445 nm, and free trimethoprim (hereinafter sometimes referred to as "TMP"), and the fluorescence of mScarlet I was observed with a confocal laser microscope. Next, the localization and translocation efficiency of the tagged protein was measured based on the confocal laser microscope photographs. ^mDcNVOC TMP was added at a final concentration of 10 μM, irradiation with light having a wavelength of 405 nm for 2.6 seconds, irradiation with light having a wavelength of 445 nm for 2.6 seconds, and ^TMP was added at a final concentration of 100 μM.

17 is a graph showing the results of measuring the localization efficiency of tagged proteins, in which "-" indicates that ^mDcNVOC TMP or TMP was not added, or that irradiation with light having a wavelength of 405 nm or 445 nm was not performed, and "+" indicates that ^{mDcNVOC TMP} or TMP was added, or that irradiation with light having ^a wavelength of 405 nm or 445 nm was performed.

The vertical axis of the graph represents the localization efficiency calculated by the following formula (F1).
Localization efficiency=1−(fluorescence intensity in the cytoplasm 5 minutes after the start of the experiment/fluorescence intensity in the cytoplasm at the start of the experiment (0 minutes)) (F1)

As a result, it was revealed that mScarlet I was localized in the cell membrane when ^mDcNVOC TMP was added and light irradiation with a wavelength of 405 ^nm was performed, and that the localization of mScarlet I in the cell membrane was suppressed in the presence of TMP.

[Experimental Example 16]
(Localization induction by light irradiation 3)
The induction of localization by light irradiation at the single cell level was examined. First, ^mDcNVOC TMP was added to the medium of K6-DS expressing cells at a final concentration of 10 μM, and the cells were observed with a confocal laser microscope. FIG. 18(a) is a confocal laser microscope photograph at the start ^of the experiment. Next, using the function of the confocal laser microscope, only the cell indicated as "Cell ¹ " in FIG. 18(a) was irradiated with light of a wavelength of 405 nm for 2.6 seconds. FIG. 18(a) is a confocal laser microscope photograph 60 seconds after the start of the experiment. As a result, it was revealed that mScarlet I was localized to the cell membrane in the cell indicated as "Cell ¹ ".

Fig. 18(c) is a confocal laser microscope photograph taken 600 seconds after the start of the experiment. Next, using the function of the confocal laser microscope, only the cell shown as "Cell ² " in Fig. 18(c) was irradiated with light having a wavelength of 405 nm for 2.6 seconds. Fig. 18(d) is a confocal laser microscope photograph taken 660 seconds after the start of the experiment. As a result, it was revealed that mScarlet I was localized in the cell membrane in the cell shown as "Cell ² ".

FIG. 18(e) is a confocal laser microscope photograph taken 1200 seconds after the start of the experiment. Next, using the function of the confocal laser microscope, only the cell indicated as "Cell ³ " in FIG. 18(e) was irradiated with light having a wavelength of 405 nm for 2.6 seconds. FIG. 18(f) is a confocal laser microscope photograph taken 1260 seconds after the start of the experiment. As a result, it was revealed that mScarlet I was localized in the cell membrane in the cell indicated as "Cell ³ ". FIG. 18(f) also shows the position of the cell indicated as "Cell ⁴ ".

Fig. 18(g) is a confocal laser microscope photograph taken 1800 seconds after the start of the experiment. Subsequently, free TMP was added to the cell medium at a final concentration of 100 μM. Fig. 18(h) is a confocal laser microscope photograph taken 2400 seconds after the start of the experiment. As a result, it was revealed that the localization of mScarlet I to the cell membrane was eliminated in the cells indicated as "Cell ¹ ", "Cell ² ", and "Cell ³ ".

Fig. 18(i) is a graph showing the numerical values of the change in the fluorescence intensity ratio of the cytoplasm in the cells indicated as "Cell ¹ ,""Cell ² ,""Cell ³ ," and "Cell ⁴ " in Fig. 18(a) to (h). The horizontal axis indicates the time (seconds) from the start of the experiment.

[Experimental Example 17]
(Localization induction by light irradiation 4)
The control of cell motility signals by light irradiation was examined. First, a fusion protein of a target protein and a tag protein was prepared. mScarlet I and Tiam1 were used as the target proteins. eDHFR was used as the tag protein. eDHFR, mScarlet I, and Tiam1 were arranged in this order from the N-terminus of the fusion protein. In addition, a K6 tag was further arranged on the N-terminus of eDHFR. Hereinafter, this fusion protein may be referred to as "K6-DS-Tiam1". The amino acid sequence of K6-DS-Tiam1 is shown in SEQ ID NO:12.

A fusion protein of Lifeact and mNeonGreen (hereinafter sometimes referred to as "Lifeact-mNeonGreen") was also prepared. The amino acid sequence of Lifeact-mNeonGreen is shown in SEQ ID NO: 13. Lifeact is a peptide that specifically binds to F-actin.

Next, K6-DS-Tiam1 and Lifeact-mNeonGreen were expressed in NIH3T3 cells, which are mouse embryo fibroblasts. Next, mDcNVOC TMP was added to the medium of the NIH3T3 cells expressing K6-DS-Tiam1 and Lifeact-mNeonGreen (hereinafter, sometimes referred to as " ^{K6-DS-Tiam1 and Lifeact-mNeonGreen expressing cells") to a final concentration of 10 μM. Next, 5 minutes after the addition of mDcNVOC TMP , the} cells were washed.

Then, the fluorescence of mScarlet I and mNeonGreen were observed using a confocal laser microscope. Figures 19(a) and (c) are confocal laser microscope photographs taken at the start of the experiment. Next, using the functions of the confocal laser microscope, the area enclosed by the square in Figure 19(a), i.e., the entire cell, was irradiated with light having a wavelength of 405 nm for 2.6 seconds.

Figures 19(b) and (d) are confocal laser microscope photographs taken 300 seconds after the start of the experiment. Figure 19(e) is an image showing areas of increased or decreased area, comparing K6-DS-Tiam1 and Lifeact-mNeonGreen expressing cells at the start of the experiment with those at 300 seconds after the start of the experiment. In Figure 19(e), "increased" indicates areas of increased area compared to the start of the experiment, and "decreased" indicates areas of decreased area compared to the start of the experiment.

As a result, it was found that the area of K6-DS-Tiam1 and Lifeact-mNeonGreen expressing cells changed when exposed to light with a wavelength of 405 nm. These results demonstrated that the use of a compound with a photodegradable protecting group makes it possible to control cell motility signals by exposure to light.

[Experimental Example 18]
(Localization induction by light irradiation 5)
^{mDcNVOC TMP} was added to the culture medium of K6-DS-Tiam1 and Lifeact-mNeonGreen expressing cells to a final concentration of 10 μM.

Then, the fluorescence of mScarlet I and mNeonGreen were observed using a confocal laser microscope. Figures 20(a) and (c) are confocal laser microscope photographs taken at the start of the experiment. Next, using the functions of the confocal laser microscope, the area circled in Figure 20(a), i.e., a part of the cell, was locally irradiated with light of a wavelength of 405 nm for 2.6 seconds.

Figures 20(b) and (d) are confocal laser microscope photographs taken 300 seconds after the start of the experiment. Figure 20(e) is an image showing areas of increased or decreased area, comparing K6-DS-Tiam1 and Lifeact-mNeonGreen expressing cells at the start of the experiment with those at 300 seconds after the start of the experiment. In Figure 20(e), "increased" indicates areas of increased area compared to the start of the experiment, and "decreased" indicates areas of decreased area compared to the start of the experiment.

As a result, it was found that the area of K6-DS-Tiam1 and Lifeact-mNeonGreen expressing cells changed upon irradiation with light at a wavelength of 405 nm. This result further supports the idea that the use of compounds with photolabile protecting groups makes it possible to control cell motility signals through light irradiation.

[Experimental Example 19]
(Localization induction by light irradiation 6)
^mDcNVOC TMP was added to the medium of NIH3T3 cells expressing K6-DS-Tiam1 (hereinafter sometimes referred to as "K6-DS-Tiam1-expressing cells") to a final concentration of 10 μM. For comparison, ^mDcNVOC TMP was also added to the medium of NIH3T3 cells expressing K6- ^DS (hereinafter sometimes referred to as "K6-DS ^- expressing cells") to a final concentration of 10 μM.

Then, the fluorescence of mScarlet I was observed using a confocal laser microscope. Next, the whole cell or a part of the cell was locally irradiated with light of 405 nm wavelength for 2.6 seconds using the function of the confocal laser microscope. The changes in the cell morphology were then observed using the confocal laser microscope.

Figures 21(a)-(d) are images showing areas that have increased or decreased in area, comparing K6-DS-Tiam1 expressing cells or K6-DS expressing cells at the start of the experiment with those at 300 seconds after the start of the experiment. In Figures 20(a)-(d), "increased" indicates areas that have increased in area compared to the start of the experiment, and "decreased" indicates areas that have decreased in area compared to the start of the experiment.

FIG. 21(a) shows a representative result of irradiating the entire cell with light of 405 nm wavelength, and FIG. 21(b)-(d) show a representative result of irradiating a part of the cell locally with light of 405 nm wavelength. In FIG. 21(a)-(d), "+m ^D c ^NVOC TMP" indicates that m ^D c ^NVOC TMP was added, "-m ^D c ^NVOC TMP" indicates that m ^D c ^NVOC TMP was not added, "+K6-DS-Tiam1" indicates the result of K6-DS-Tiam1 expressing cells, and "+K6-DS" indicates the result of K6-DS expressing cells. FIG. 21(c) and (d) show the result of the negative control. In addition, in FIG. 21(b)-(d), the circled area indicates the area irradiated with light of 405 nm wavelength.

22(a) is a graph showing the increase in the area of the protruding region (region with increased area) of the cell, and FIG. 22(b) is a graph showing the decrease in the area of the retracted region (region with decreased area) of the cell. In addition, in FIGS. 22(a) and (b), "whole" indicates the result of irradiating the whole cell with light of 405 nm wavelength, "local" indicates the result of irradiating a part of the cell locally with light of 405 nm wavelength, "+" indicates the result of adding m ^D c ^NVOC TMP, the result of K6-DS-Tiam1 expressing cells, or the result of K6-DS expressing cells, and "-" indicates the result of not adding m ^D c ^NVOC TMP, the result of not using K6-DS-Tiam1 expressing cells, or the result of not using K6-DS expressing cells.

As a result, it was found that the area of K6-DS-Tiam1 expressing cells changed upon irradiation with light at a wavelength of 405 nm. This result further supports the idea that the use of compounds with photolabile protecting groups makes it possible to control cell motility signals through light irradiation.

[Experimental Example 20]
(Synthesis of ^{mDcDEACM TMP} )
A caged localization agent was synthesized that has a wavelength different from that of ^mDcNVOC TMP for deprotecting a photolabile protecting group. Specifically, ^mDcDEACM TMP was synthesized according to the following scheme (6), which is a compound in which a photolabile protecting group is further bonded to the trimethoprim group of ^mDcTMP . ^{The DEACM} group represented by the above formula (14) was used as the photolabile protecting group. The DEACM group can be deprotected by irradiation with light having a wavelength of, for example, 445 nm.

^1H NMR (400MHz, d-DMSO): δ 0.85 (t, 3H), 1.12 (t, 6H), 1.18-1.41 (m, 22H), 1.45-1.75 ( m, 10H), 2.03-2.22 (m, 5H), 2.58-2.80 (m, 2H), 2.99 (q, 2H), 3.50-3.62 (m, 15H), 3 ． 69 (s, 6H), 3.75 (t, 2H), 3.80 (s, 2H), 3.85-3.95 (m, 6H), 4.22-4.36 (m, 2H) , 5.37 (s, 2H), 6.15 (s, 1H), 6.51 (s, 2H), 6.56 (d, 1H), 6.69 (dd, 1H), 7.09 ( s, 1H), 7.46 (d, 1H), 7.49 (s, 1H), 7.55 (d, 1H), 7.69 (q, 2H), 7.74 (t, 1H), 7.99 (t, 2H), 8.03 (t, 1H), 10.15 (brs, 1H).

23 is a graph showing the results of measuring the absorption spectra of ^mDcNVOC TMP and ^mDcDEACM TMP. As shown in FIG. 23, it was revealed that ^mDcNVOC TMP does not absorb light ^with a wavelength of 445 nm, ^{but mDcDEACM} TMP can absorb light with ^a wavelength of 445 ^nm .

[Experimental Example 21]
(Light-induced localization 7)
To the medium of HeLa cells expressing K6-DS (hereinafter sometimes referred to as "K6-DS expressing cells"), ^mDcDEACM TMP was added to a final concentration of 10 μM. Then, 5 minutes after the addition of ^mDcDEACM TMP, the cells were washed. Then, the cells were irradiated with light having a wavelength of 445 nm ^for 2.6 seconds, and the fluorescence of mScarlet I was observed with ^a confocal laser microscope.

24(a) and (b) are confocal laser microscope photographs of K6-DS expressing cells to ^{which mDcDEACM TMP} was added. Fig. 24(a) is a photograph of the K6-DS expressing cells before light irradiation. Fig. 24(b) is a photograph of the K6-DS expressing cells after light irradiation.

As a result, it was revealed that in ^K6 -DS expressing cells to which ^mDcDEACM TMP had been added, localization of the tagged protein could be induced by irradiation with light having a wavelength of 445 nm.

FIG. 24(c) is a graph showing the results of measuring the localization efficiency of the tagged protein after irradiating K6-DS expressing cells containing ^mDcNVOC TMP with light having a wavelength of 405 ^nm or 445 nm.

FIG. 24(d) is a graph showing the results of measuring the localization efficiency of tagged proteins after irradiating K6-DS expressing cells containing ^mDcDEACM TMP with light having a wavelength of ⁴⁰⁵ nm or 445 nm.

In Figures 24(c) and (d), the horizontal axis of the graph represents the intensity (%) of the laser output, and the vertical axis of the graph represents the localization transfer efficiency calculated by the following formula (F1).
Localization efficiency=1−(fluorescence intensity in the cytoplasm 5 minutes after the start of the experiment/fluorescence intensity in the cytoplasm at the start of the experiment (0 minutes)) (F1)

As a result, it ^{was revealed that in K6-DS expressing cells containing mDcNVOC TMP} , the tagged protein was localized to the cell membrane by irradiation with 405 nm light, but the localization of the tagged protein was hardly changed by irradiation with ^{445 nm light. On the other hand, it was revealed that in K6-DS expressing cells containing mDcDEACM TMP} , the tagged protein was localized to the cell membrane by both irradiation with 405 nm light and 445 nm light.

These results demonstrate that it is possible to control the induction of localization of tagged proteins by using compounds with photodegradable protecting groups with different absorption wavelengths and controlling the wavelength of the irradiated light.

[Experimental Example 22]
(Preparation of cells expressing a fusion protein of a target protein and a tag protein)
By using genome editing, a gene fragment encoding a tag protein was knocked into the cRaf locus of HeLa cells, and cells expressing a fusion protein of the target protein (cRaf) and the tag protein were generated.

<<Preparation of expression vectors for Cas proteins and guide RNAs>>
First, a vector (pX459: pSpCas9(BB)-2A-Puro V2.0) containing a wild-type Cas9 expression cassette whose expression is controlled by a CMV enhancer and an avian β-actin promoter was prepared. The pX459 vector has an expression cassette in which, from the 5' to the 3' side, a U6 promoter, two BbsI recognition sites, and a tracer RNA sequence are arranged in this order.

Then, the pX459 vector was cleaved with BbsI, and a double-stranded DNA containing the target sequence of the guide RNA was inserted to obtain an expression vector that expresses the Cas protein and the guide RNA. The above double-stranded DNA was produced by annealing two single-stranded DNAs. The base sequence of the target sequence of the guide RNA is shown in SEQ ID NO: 14. This target sequence recognizes the area surrounding the coding region of the stop codon of the cRaf gene.

<<Construction of donor vector>>
25(a) is a schematic diagram showing the structure of the prepared donor vector. The donor vector had, from the 5' to 3' end, a first recombination arm (L.arm, a base sequence corresponding to 700 bp upstream from the cleavage site), an insertion cassette, and a second recombination arm (R.arm, a base sequence corresponding to 700 bp downstream from the cleavage site), in this order.

The insertion cassette contained, in order from the 5' to the 3' end, a base sequence encoding the fluorescent protein mClover3, a base sequence encoding the tag protein eDHFR (69K6), a base sequence encoding a triple repeat sequence of the HA tag, a P2A sequence that induces ribosome skipping, and a neomycin resistance gene that is a cell selection marker.

eDHFR (69K6) is a tag protein that contains an amino acid sequence of six consecutive lysine residues inside eDHFR. The amino acid sequence of eDHFR (69K6) is shown in SEQ ID NO: 15. SEQ ID NO: 16 shows the base sequences of the first recombination arm, the insertion cassette, and the second recombination arm in the donor vector.

Figure 25(b) is a schematic diagram showing the structure of the genomic DNA of a cell in which the insertion cassette was knocked in as intended. As shown in Figure 25(c), this cell expresses a fusion protein in which three repeats of cRaf, mClover3, eDHFR (69K6), and the HA tag are linked in this order.

Genome editing
HeLa cells were seeded in a 24-well plate, and 1 μg of expression vectors for Cas protein and guide RNA and 50 ng of donor vector were introduced using polyethyleneimine "Max" (Polysciences). Three days after transfection, the cells were seeded in a 60 mm dish and treated with 1 mg/mL G418 for 7 days or more. Subsequently, cells selected with G418 were isolated by limiting dilution.

Next, genomic DNA from the isolated cells was extracted and analyzed by PCR amplification. Figure 26(a) shows a photograph of agarose electrophoresis showing a representative PCR result and a schematic diagram showing the structure of genomic DNA. As shown in Figure 26(a), an amplified fragment of 3,837 bp was obtained from the clone in which the desired genome editing had occurred, and an amplified fragment of 1,597 bp was obtained from the wild-type clone.

The isolated cells were then subjected to Western blotting using anti-cRaf and anti-HA antibodies to examine the expression of the fusion protein. Figure 26(b) shows a photograph showing a representative Western blotting result and a schematic diagram showing the structure of the fusion protein. As shown in Figure 26(b), the clone in which the desired genome editing had occurred expresses a 126 kDa fusion protein, while the wild-type clone expresses a 73 kDa cRaf. As a result, it was confirmed that a clone in which the desired genome editing had occurred was obtained.

[Experimental Example 23]
(Control of intracellular proteins using ^mDcTMP 1)
Intracellular proteins were controlled using ^mDcTMP . The cells used were those prepared in Experimental Example 22. As described above, these cells express a fusion protein in which three repeats of cRaf, mClover3, eDHFR (69K6), and HA tag are linked in this order (hereinafter, sometimes referred to as "cRaf-GDiK6"). Figure 27(a) is a schematic diagram showing the structure of cRaf-GDiK6.

An expression vector for the mCherry and ERK fusion protein (mCherry-ERK) used in Experimental Example 11 was also prepared. Figure 27(b) is a schematic diagram showing the structure of mCherry-ERK. The amino acid sequence of mCherry-ERK is shown in SEQ ID NO: 10. Next, mCherry-ERK was expressed in HeLa cells encoding cRaf-GDiK6 in the genome.

Next, ^mDcTMP was added to the medium of HeLa cells expressing cRaf-GDiK6 and mCherry-ERK (hereinafter, sometimes referred to as "cRaf-GDiK6 and mCherry-ERK expressing cells") to a final concentration of 5 μM. Then, the fluorescence of mClover3 and the fluorescence of mCherry were observed using a confocal laser microscope.

Figures 28(a) to (d) are representative confocal laser microscope photographs. Figures 28(a) and (c) are photographs of cRaf-GDiK6- and mCherry-ERK-expressing cells before the addition of ^mD- cTMP. Figures 28(b) and (d) are photographs of cRaf-GDiK6- and mCherry-ERK-expressing cells taken 10 minutes after the addition of ^mD- cTMP.

Figures 28(a) and (b) show the results of observing the fluorescence of mClover3 in cRaf-GDiK6, and Figures 28(c) to (d) show the results of observing the fluorescence of mCherry in mCherry-ERK.

When cRaf is localized to the cell membrane, it can activate MEK, which in turn activates ERK. ERK is localized in the cytoplasm in the inactive state, but translocates into the nucleus when activated.

By observing the fluorescence of mClover3, the localization of cRaf-GDiK6 can be detected. In addition, by observing the fluorescence of mCherry, the localization of mCherry-ERK can be detected.

As a result, it was revealed that cRaf-GDiK6 was localized to the cell membrane and mCherry-ERK was translocated into the nucleus 10 minutes after the addition of ^mDcTMP , indicating that ^mDcTMP can be used to control target proteins encoded in the genome and intracellular signals.

29 is a graph showing the numerical results of the above-mentioned confocal laser microscope observation performed on a plurality of cells (n≧4). For comparison, similar analysis was performed on cRaf-GDiK6 and mCherry-ERK expressing cells to which epidermal growth factor (EGF) was added instead of ^mD- cTMP to a final concentration of 50 ng/mL, and on cRaf-GDiK6 and mCherry-ERK expressing cells to which dimethyl sulfoxide (DMSO) was added instead of ^mD -cTMP to a final concentration of 0.05% (n≧4).

In Figure 29, "Normalized nucleus/cytoplasm ratio of mCherry-ERK" indicates the normalized nuclear/cytoplasmic ratio of mCherry fluorescence intensity.

[Experimental Example 24]
(Control of intracellular proteins using ^mDcTMP 2)
Intracellular proteins were regulated using ^mDcTMP . HeLa cells encoding cRaf-GDiK6 in the genome, as prepared in Experimental Example 22, were used.

First, ^mDcTMP (final concentration 5 μM), EGF (final concentration 50 ng/mL) or DMSO (final concentration 0.05%) was added to the medium of HeLa cells encoding cRaf-GDiK6 in their genome, and incubated for 5 or 15 minutes.

For comparison, ^mDcTMP (final concentration 5 μM) or EGF (final concentration 50 ng/mL) was added to the medium of wild-type HeLa cells that had not been subjected to genome editing, and incubated for 5 or 15 minutes.

Next, cell extracts were prepared from each cell line, and the levels of total cRaf, activated cRaf (phosphorylated cRaf), total ERK, activated ERK (phosphorylated ERK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin were analyzed by Western blotting.

Figure 30 is a graph showing the results of Western blotting. In Figure 30, "KI clone #5" indicates the results for HeLa cells (clone 5) that encode cRaf-GDiK6 in their genome, "Intact HeLa cells" indicates the results for wild-type HeLa cells that have not been subjected to genome editing, "pcRaf" indicates the results for phosphorylated cRaf, and "pERK1/2" indicates the results for phosphorylated ERK1/2. "+" indicates that the substance was added, and "-" indicates that the substance was not added.

Figure 31 also shows the relative phosphorylated cRaf levels (pcRaf/Raf) normalized to the total cRaf levels in each cell extract, and the relative phosphorylated ERK levels (pERK/ERK) normalized to the total ERK levels, based on the results of three independent experiments.

In Figure 31, "Clone #5" indicates the results for HeLa cells (clone 5) that encode cRaf-GDiK6 in their genome, and "Intact HeLa cells" indicates the results for wild-type HeLa cells that have not undergone genome editing. In addition, "+" indicates that the gene was added, and "-" indicates that the gene was not added.

As a result, it was revealed that cRaf-GDiK6 and ERK were phosphorylated 5 and 15 minutes after addition of ^mDcTMP in HeLa cells encoding cRaf-GDiK6 in the genome. This result indicates that endogenous target proteins and their downstream signaling proteins can be regulated by ^mDcTMP .

The present invention provides a new technology for localizing a target protein to the cell membrane or Golgi apparatus.

Claims (25)

A compound represented by the following formula (1):

[In formula (1), Z represents a group represented by formula (2) below; L represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by substitution of -C- with -O-, -CO-, -NH-, or a group represented by formula (3) below; R ¹ represents a group which functions as a ligand for a tagged protein or a group in which a photodegradable protecting group is further bonded to a group which functions as a ligand for the tagged protein; and in formula (2) below, R ² represents a saturated or unsaturated linear hydrocarbon group having 9 or more carbon atoms.

］ The compound according to claim 1, wherein the combination of the tag protein and the group functioning as a ligand for the tag protein is a combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and a group represented by the following formula (4), a combination of FK506 binding protein and a group represented by the following formula (5), a combination of carbonic anhydrase I protein or carbonic anhydrase II protein and a group represented by the following formula (6), a combination of SNAP-tag (registered trademark) protein and a group represented by the following formula (7) or the following formula (8), a combination of HaloTag (registered trademark) protein and a group represented by the following formula (9), or a combination of photoactive yellow protein and a group represented by the following formula (10).

The compound according to claim 1 or 2, wherein the photodegradable protecting group is a group represented by any one of the following formulas (11) to (19):

An agent for localizing the tagged protein to a cell membrane or Golgi apparatus, comprising the compound according to any one of claims 1 to 3. A method for controlling intracellular localization of a target protein, comprising:
The method includes the step (a) of introducing into the cell a localization agent according to claim 4, wherein the ^R1 is a group that functions as a ligand for the tagged protein, so that the group that functions as a ligand of the localization agent binds to the tagged protein, and the tagged protein is localized to a cell membrane or a Golgi apparatus together with the target protein;
The method, wherein the cell is a cell that expresses a fusion protein of the target protein and the tag protein. The method further comprises, after the step (a), a step (b) of introducing a ligand for the tagged protein into the cell,
The method according to claim 5, wherein in step (b), the group functioning as a ligand competes with the ligand, and the ligand replaces the group functioning as a ligand in at least a portion of the tagged protein localized in the cell membrane or the Golgi apparatus, resulting in the target protein being translocated from the cell membrane or the Golgi apparatus. A method for controlling intracellular localization of a target protein, comprising:
A step (a1) of introducing into the cell the localization agent according to claim 4, wherein the R ¹ is a group in which a photolabile protecting group is further bonded to a group that functions as a ligand for the tagged protein;
and step (a2) of irradiating the cell with light after step (a1), thereby causing the photolabile protecting group to be cleaved from the localization agent, causing the group of the localization agent that functions as a ligand to bind to the tagged protein, and causing the tagged protein to localize together with the target protein in a cell membrane or a Golgi apparatus,
The method, wherein the cell is a cell that expresses a fusion protein of the target protein and the tag protein. The method further comprises, after the step (a2), a step (b) of introducing a ligand for the tagged protein into the cell,
The method according to claim 7, wherein in step (b), the group functioning as a ligand competes with the ligand, and the ligand replaces the group functioning as a ligand in at least a portion of the tagged protein localized in the cell membrane or the Golgi apparatus, resulting in the target protein being translocated from the cell membrane or the Golgi apparatus. The method according to any one of claims 5 to 8, wherein the fusion protein has an amino acid sequence of 4 to 10 consecutive lysine residues at the N-terminus or C-terminus of the fusion protein or inside the tag protein. The method according to any one of claims 5 to 9, wherein the target protein is a signal transduction protein. the protein of interest is an endogenous protein,
The method according to any one of claims 5 to 10, wherein the cell is a cell in which a gene fragment encoding the tag protein has been knocked in to a gene locus encoding the target protein. A kit for controlling the localization of a tagged protein in a cell, comprising the localization agent according to claim 4. A cell expressing a fusion protein of a target protein and the tag protein;
The kit according to claim 12 , further comprising: an expression vector for a fusion protein of the target protein and the tag protein; or a vector for producing a fusion protein of the target protein and the tag protein. The kit according to claim 12 or 13, further comprising a ligand for the tag protein. A compound represented by the following formula (1):

[In formula (1), Z represents a group represented by the following formula (2), L represents a divalent hydrocarbon group having 1 to 50 carbon atoms which may be interrupted by substitution of -C- with -O-, -CO-, -NH- or a group represented by the following formula (3), R ¹ represents a group that functions as a ligand for a protein or a group in which a photodegradable protecting group is further bonded to the group that functions as a ligand for a protein, and in the following formula (2), R ² represents a saturated or unsaturated linear hydrocarbon group having 9 or more carbon atoms.

］ The compound according to claim 15, wherein the combination of the protein and the group functioning as a ligand for the protein is a combination of Escherichia coli dihydrofolate reductase (eDHFR) protein and a group represented by the following formula (4), a combination of FK506 binding protein and a group represented by the following formula (5), a combination of carbonic anhydrase I protein or carbonic anhydrase II protein and a group represented by the following formula (6), a combination of SNAP-tag (registered trademark) protein and a group represented by the following formula (7) or (8), a combination of HaloTag (registered trademark) protein and a group represented by the following formula (9), or a combination of photoactive yellow protein and a group represented by the following formula (10).

The compound according to claim 15 or 16, wherein the photodegradable protecting group is a group represented by any one of the following formulas (11) to (19):

An agent for localizing a protein to a cell membrane or Golgi apparatus, comprising the compound according to any one of claims 15 to 17. A method for controlling intracellular protein localization, comprising:
19. The localization agent of claim 18, wherein the R ¹ a step (a) of introducing a localization agent, the group of the localization agent which functions as a ligand for the protein, such that the group which functions as a ligand of the localization agent binds to the protein and the protein is localized to a cell membrane or a Golgi apparatus. The method further comprises, after the step (a), a step (b) of introducing a ligand for the protein into the cell,
The method according to claim 19, wherein in step (b), the group functioning as a ligand competes with the ligand, and the ligand displaces the group functioning as a ligand in at least a portion of the protein localized in the cell membrane or the Golgi apparatus, resulting in the protein being translocated from the cell membrane or the Golgi apparatus. A method for controlling intracellular protein localization, comprising:
19. The localization agent of claim 18, wherein the R ¹ a step (a1) of introducing a localization agent which is a group having a photolabile protecting group further bonded to a group which functions as a ligand for the protein;
and step (a2) of irradiating the cell with light after step (a1), such that the photolabile protecting group is cleaved from the localization agent, the group of the localization agent that functions as a ligand is bound to the protein, and the protein is localized to the cell membrane or Golgi apparatus. The method further comprises, after the step (a2), a step (b) of introducing a ligand for the protein into the cell,
The method according to claim 21, wherein in step (b), the group functioning as a ligand competes with the ligand, and the ligand displaces the group functioning as a ligand in at least a portion of the protein localized in the cell membrane or the Golgi apparatus, resulting in the protein being translocated from the cell membrane or the Golgi apparatus. The method of any one of claims 19 to 22, wherein the protein is a signal transduction protein. A kit for controlling intracellular protein localization, comprising the localization agent according to claim 18. The kit of claim 24, further comprising a ligand for the protein.

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