WO2020195302A1

WO2020195302A1 - Linker molecule for producing cell membrane-permeable ring-shaped peptide and use thereof

Info

Publication number: WO2020195302A1
Application number: PCT/JP2020/005760
Authority: WO
Inventors: 慶士高津; 敏裕鹿倉; 優佳石場; 達也馬渡; 博文前田; 寛士北; 北野　光昭
Original assignee: 株式会社カネカ
Priority date: 2019-03-28
Filing date: 2020-02-14
Publication date: 2020-10-01
Also published as: JPWO2020195302A1

Abstract

Provided is a linker molecule for producing a cell membrane-permeable ring-shaped peptide, the linker molecule having a cell membrane permeability-imparting group; and a ring-forming group which forms a peptide into a ring shape.

Description

Linker molecule for producing cell-penetrating cyclic peptide and its use

The present invention presents a linker molecule for producing a cell-penetrating cyclic peptide, a peptide complex into which the linker molecule has been introduced and a method for producing the same, a peptide library containing the peptide complex, and screening of a functional peptide using the peptide library. Regarding the method.

In recent years, therapeutic agents for specific diseases and molecules having a high affinity for target molecules have been selected from a peptide (polypeptide) library having various amino acid sequences.

As a method for producing the peptide library, for example, as an in vitro translation system method, a ribosome display method (RD method) using a ribosome display complex containing a peptide chain, an mRNA molecule, and a ribosome is known (for example, a patent). Reference 1). The RD method, if they have in vitro translation system and mRNA, only mixing them, in which very good useful which can be produced 10 ¹² or more peptide library in a few minutes.

On the other hand, attention is also focused on the development of pharmaceuticals in which polypeptides such as antibodies are chemically modified with functional molecules to expand their functions. For example, cyclization of a peptide improves its stability in vivo, cyclization stabilizes its structure, which improves its affinity and selectivity for a target compound, and resistance to degrading enzymes. It is also known that cell membrane permeability may be expressed.

In the development of pharmaceuticals, it is also important to be able to efficiently take in various active ingredients such as proteins and nucleic acids into cells.

Techniques for incorporating the active ingredient into the cell include, for example, a method of fusing the amino acid sequences of a cell membrane penetrating peptide containing a large amount of basic amino acids, and a dendrimer which is a dendrimer polymer having a regularly branched structure from the center. The method to be used (see, for example, Patent Document 2) and the like are known.

Therefore, it is considered that a molecule that imparts cell membrane penetrating performance to a peptide having a cyclic structure can be a valuable drug discovery seed, and a peptide library containing such a peptide is a very useful means for the search.

So far, as a technique for easily and easily cyclizing (modifying) a peptide chain in the ribosome complex, RNA is bound to a peptide translated from the RNA by modifying it with a modifying reagent at a specific time. It is known that the adhesive function of ribosomes can be maintained without losing the original function (see, for example, Patent Document 3).

Further, as a technique for cyclizing a peptide and imparting cell membrane permeability, an example in which these are performed stepwise has been reported (see, for example, Non-Patent Document 1).

However, a technique capable of simultaneously cyclizing a peptide and imparting cell membrane permeability has not yet been developed, and the current situation is that rapid development thereof is strongly required.

Japanese Unexamined Patent Publication No. 2008-271903 U.S. Pat. No. 7,862,807 International Publication No. 2017/213158

An object of the present invention is to solve the above-mentioned problems in the past and to achieve the following objects. That is, in the present invention, a linker molecule for producing a cell-penetrating cyclic peptide capable of simultaneously performing cyclization of the peptide and imparting cell membrane permeability to the peptide, a peptide complex into which the linker molecule has been introduced, and production thereof It is an object of the present invention to provide a method, a peptide library containing the peptide complex, and a method for screening a functional peptide using the peptide library.

As a result of diligent studies to solve the above problems, the present inventors unexpectedly synthesize a molecule in which a cell membrane permeability-imparting group and a cyclic group that cyclizes a peptide coexist in the same molecule. It was found that the peptide can be cyclized and cell membrane permeability can be imparted to the peptide at the same time by using this molecule.

The present invention is based on the above-mentioned findings of the present inventors, and the means for solving the above-mentioned problems are as follows. That is,
<1> A linker molecule for producing a cell-penetrating cyclic peptide, which comprises a cell membrane-penetrating cyclic peptide-imparting group and a cyclic group that cyclizes a peptide.
<2> A peptide complex comprising a peptide and the linker molecule according to <1> introduced into the peptide.
<3> A peptide library comprising the peptide complex according to <2> above.
<4> A method for producing a peptide complex, which comprises the step of introducing the linker molecule according to <1> into the peptide.
<5> A method for screening a functional peptide, which comprises a step of screening a functional peptide using the peptide library according to the above <3>.

According to the present invention, a cell-penetrating cyclic peptide capable of solving the above-mentioned problems in the past, achieving the above-mentioned object, and simultaneously performing cyclization of the peptide and impartation of cell membrane permeability to the peptide. It is possible to provide a linker molecule for production, a peptide complex into which the linker molecule has been introduced and a method for producing the same, a peptide library containing the peptide complex, and a method for screening a functional peptide using the peptide library.

FIG. 1 is a diagram showing the results of preparative HPLC of peptide P in Example 2. FIG. 2 is a diagram showing the results of preparative HPLC of the cyclic peptide (peptide complex G3-DCX-P) in Example 2. FIG. 3 is a schematic diagram of the structure of the template DNA used to prepare the ribosome display complex in Example 3. FIG. 4 is a diagram showing the measurement results of MALDI-TOF MS in Example 4. FIG. 5 is a diagram showing the measurement results of MALDI-TOF MS in Example 5. FIG. 6 is a diagram showing the measurement results of MALDI-TOF MS in Example 6.

(Linker molecule for producing cell-penetrating cyclic peptide)
The linker molecule for producing a cell-penetrating cyclic peptide of the present invention (hereinafter, may be referred to as “linker molecule”) has at least a cell membrane-penetrating cyclic peptide-imparting group and a cyclic group that cyclizes the peptide, and is necessary. It also has other configurations such as linking groups, depending on the situation.

The linker molecule can be represented by the following general formula (1).
ABC ・・・ General formula (1)
In the general formula (1), "A" represents a cyclic group, "B" represents a linking group or a single bond, and "C" represents a cell membrane permeability-imparting group.

<Cyclic group>
The cyclic group contributes to the cyclization of the peptide by reacting with a reactive amino acid residue in the peptide described later.
The reactive amino acid residue is an amino acid residue that reacts with the cyclic group, may be an amino acid residue that directly reacts with the cyclic group, or is modified so as to react with the cyclic group. It may be an amino acid residue.
The cyclic group is not particularly limited and may be appropriately selected depending on the intended purpose, but an electron-withdrawing group is preferable.

-Electronic suction group-
The electron-withdrawing group is not particularly limited and may be appropriately selected depending on the intended purpose, but it preferably has a halogen.

The type of halogen is not particularly limited and can be appropriately selected according to the purpose.

The number of halogens is not particularly limited and may be appropriately selected depending on the purpose, but 2 or more is preferable.

The electron-withdrawing group preferably has two or more chlorine atoms, more preferably benzyl chloride, which may have a substituent, and particularly preferably a 3,5-bis (chloromethyl) benzyl group.

Cyclization of a peptide by the cyclic group may be carried out by reacting the cyclic group with at least one group selected from the group consisting of a thiol group, an amino group and a hydroxy group contained in the peptide. preferable.

<Cell membrane permeability-imparting group>
The cell membrane permeability-imparting group is a group that imparts cell membrane permeability to the peptide.
The cell membrane permeability-imparting group preferably has a basic functional group.

The mode of the cell membrane permeability-imparting group is not particularly limited and may be appropriately selected depending on the intended purpose, but a mode having a dendritic structure is preferable.
The number of dendritic structural units in the cell membrane permeability-imparting group is not particularly limited and may be appropriately selected depending on the intended purpose, and may be one or two or more.
The number of branches per dendritic structural unit is not particularly limited and may be appropriately selected depending on the intended purpose, but 3 or more is preferable.
The structure of the branch of the dendritic structure is not particularly limited and may be appropriately selected depending on the intended purpose.

-Basic functional group-
The basic functional group is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a guanidino group, an amino group and an imidazole group. Among these, it is preferable to have a guanidino group. The basic functional group may be used alone or in combination of two or more.
The number of the basic functional groups is not particularly limited and may be appropriately selected depending on the intended purpose, but 2 or more is preferable.
The basic functional group preferably has two or more guanidino groups.
The position of the basic functional group in the linker molecule is not particularly limited and may be appropriately selected depending on the intended purpose, but it is preferably located at the end of the branch of the dendritic structure.

Specific examples of the cell membrane permeability-imparting group having a dendritic structure include those represented by the following general formula (C).

The general formula (C) represents a cell membrane permeability-imparting group having a dendritic structure having three branches, and "Y" in the formula represents a basic functional group.
The basic functional group may be formed at the ends of all branches, or may be formed at the ends of some branches.
Further, the cell membrane permeability-imparting group may have a plurality of dendritic structures represented by the general formula (C), and in that case, the number of branches may be, for example, 6 or 9.

<Other configurations>
The other constitution of the linker molecule is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected depending on the intended purpose. Examples thereof include a linking group.

<< Linking group >>
The linking group is a group that links the cell membrane permeability-imparting group and the cyclic group.
The structure of the linking group is not particularly limited and may be appropriately selected depending on the intended purpose.

Specific examples of the linker molecule include compounds represented by the following structural formulas. The linker molecule represented by the following structural formula has a 3,5-bis (chloromethyl) benzyl group as a cyclic group and three guanidino groups at the end of the dendritic structure as a cell membrane permeability-imparting group. This is an example in which the cyclic group and the cell membrane permeability-imparting group are linked via a linking group.

<Manufacturing method of linker molecule>
The method for producing the linker molecule is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a cell membrane permeability-imparting group precursor forming step and a linking group are added to the cell membrane permeability-imparting group precursor. The step of introducing the linking group to be introduced, the step of introducing the cyclic group precursor into the linking group, and the cell membrane penetrating group and the cyclic group from the cell membrane penetrating group precursor and the cyclic group precursor, respectively. Examples include a step of forming and a method including.
Each of the above steps can be carried out by appropriately selecting a known chemical synthesis technique.
For example, the cell membrane permeability-imparting group precursor forming step can be performed by the method described in US Pat. No. 7,862,807 and the like.

The method for confirming whether or not the obtained linker molecule has a desired structure is not particularly limited, and a known analysis method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance spectroscopy can be selected. , Carbon 13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, infrared spectroscopy and other analytical methods.

According to the linker molecule of the present invention, cyclization of the peptide and impartation of cell membrane permeability to the peptide can be performed in one step, so that these can be easily achieved in a short time.

(Peptide complex)
The peptide complex of the present invention comprises at least the peptide and the linker molecule of the present invention introduced into the peptide, and further comprises other configurations as required.

<Peptide>
The peptide is not particularly limited as long as it is cyclized by introducing the linker molecule, and can be appropriately selected depending on the intended purpose.
The amino acid residue (hereinafter, may be referred to as “reactive amino acid residue”) used for introducing the linker molecule is not particularly limited and may be appropriately selected depending on the intended purpose. For example, cysteine. Residues, lysine residues, serine residues, threonine residues and the like can be mentioned. The reactive amino acid residue may be used alone or in combination of two or more.
The number of the reactive amino acid residues in the peptide is preferably 2 or more in terms of cyclizing the peptide. The upper limit of the number of the reactive amino acid residues in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose. However, as the number of reaction sites increases, the number and position of the linker molecules that bind to the peptide increase. It is preferably 10 or less because it may not be stable and it may be difficult to compare the characteristics of peptides derived from the amino acid sequence.
In addition, for example, when the cysteine residue in the peptide is involved in the stabilization of the higher-order structure of the peptide by disulfide bond, the reactive amino acid residue may be separately introduced into the peptide. preferable.

The position of the reactive amino acid residue in the peptide is not particularly limited and can be appropriately selected depending on the intended purpose.

For example, as the peptide, an mRNA molecule, a peptide chain which is a translation thereof (hereinafter, may be referred to as “polypeptide chain”), and a ribosome display complex containing ribosome (hereinafter, referred to as “RD complex”). When (may be used) is used, for example, it is a portion protruding from the exit tunnel (exit tunnel) of the ribosome, and specifically, the position (N) from the second N-terminal to the 30th C-terminal. It is preferable to set it between (including the second position from the terminal and the 30th position from the C terminal) in that the modification reaction by the linker molecule can be less likely to be sterically inhibited by the ribosome.
As the position from the C-terminal, the 50th position from the C-terminal is preferable, and the 100th position is more preferable.
Further, when the position of the reactive amino acid residue is counted from the N-terminal side, the position can be appropriately set according to the chain length of the peptide, and is, for example, the 2nd to 1,000th position from the N-terminal. , The 2nd to 100th positions from the N-terminal are preferable, and the 2nd to 50th positions from the N-terminal are more preferable.

The method for producing the RD complex is not particularly limited, and a known method can be appropriately selected. Examples thereof include the method described in International Publication No. 2017/213158. It can also be manufactured using a commercially available kit.

The amino acid sequence of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, but one containing a random sequence at a specific position is preferable so as to be useful as a peptide library. From such a random sequence, a useful amino acid sequence can be specified according to a predetermined purpose.

The position of the random sequence in the peptide is not particularly limited and may be appropriately selected depending on the intended purpose. For example, when the RD complex is used as in the position of the reactive amino acid residue, the position is not particularly limited. It is preferably between the 2nd position from the N-terminal to the 30th position from the C-terminal (including the 2nd position from the N-terminal and the 30th position from the C-terminal). That is, the reactive amino acid residue is preferably contained in a random sequence. Therefore, the preferred position of the random sequence can be set from the same range as the preferred position of the reactive amino acid residue.

The number of the random sequences in the peptide may be one or two or more. The upper limit of the number of the random sequences is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 or less.
The number of amino acid residues per random sequence is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 1 or more and 30 or less.
The longer one random sequence and the larger the number of random sequences, the greater the diversity of the peptide library.

The peptide may further contain a sequence for purifying a polypeptide chain such as a FLAG® sequence or a polyHis sequence, a sequence selectively cleaved by a protease or the like, a spacer sequence, or the like.

The number of amino acid residues of the peptide is not particularly limited and may be appropriately selected depending on the intended purpose, and may be, for example, 10 or more and 5,000 or less.
The lower limit of the number of amino acid residues of the peptide is preferably 150 or more, more preferably 200 or more. The upper limit of the number of amino acid residues of the peptide is preferably 800 or less, more preferably 600 or less, and particularly preferably 500 or less. The lower limit value and the upper limit value can be appropriately combined and selected.

The method for synthesizing the peptide is not particularly limited, and a known method can be appropriately selected.

<Linker molecule>
The linker molecule is the above-mentioned linker molecule for producing a cell-penetrating cyclic peptide of the present invention.
The linker molecule is introduced into the peptide by reacting the cyclic group in the linker molecule with the reactive amino acid residue in the peptide. During the reaction, the structure of the cyclic group changes.

<Other configurations>
The other constitution of the peptide complex is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected depending on the intended purpose. For example, a luminescent substance such as a fluorescent substance, a dye, a radioactive substance, etc. Examples thereof include drugs, toxins, nucleic acids, amino acids, sugars, lipids, and various polymers. These may be used alone or in combination of two or more.
The fluorescent substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include fluorescent dyes such as fluorescein, rhodamine, coumarin, pyrene and cyanine.
The other constitution can be attached to the above-mentioned peptide, for example, directly or via a linking group or the like.

(Method for producing peptide complex)
The method for producing a peptide complex of the present invention includes at least a linker molecule introduction step of introducing the linker molecule of the present invention into the peptide, and further includes other steps as necessary.

<Linker molecule introduction process>
The linker molecule introduction step is a step of introducing the linker molecule of the present invention into a peptide (hereinafter, may be referred to as "binding", "inserting", or "linking").
By the linker molecule introduction step, the cyclization of the peptide and the impartation of cell membrane permeability to the peptide can be performed at the same time.
In the linker molecule introduction step, the linker molecule may be introduced into at least one peptide in the reaction product, but it is preferable that the linker molecule is introduced into all the peptides.

-peptide-
The peptide is the same as that described in the item <Peptide> of the above (peptide complex). The peptide may be in the form of a peptide library.

-Introduction-
The method of introduction is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method of reacting the linker molecule with the peptide in the presence of a reducing agent.
The reducing agent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include tris (2-carboxyethyl) phosphine hydrochloride.
The conditions such as the temperature and time of the reaction are not particularly limited and may be appropriately selected depending on the intended purpose.

<Other processes>
The other steps are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose. Examples thereof include a peptide complex purification step using preparative HPLC.

The method for confirming whether or not the obtained peptide complex has a desired structure is not particularly limited, and a known analysis method can be appropriately selected. For example, mass spectrometry and proton nuclear magnetic resonance spectroscopy can be selected. Analytical methods such as method, carbon-13 nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, and infrared spectroscopy can be mentioned.

(Peptide library)
The peptide library of the present invention contains at least the peptide complex of the present invention, and further contains other configurations as required.
The peptide library may consist only of the peptide complex of the present invention, or may contain a peptide that has not been cyclized.

The peptide library can be produced in the same manner as described above (method for producing a peptide complex).

(Screening method for functional peptides)
The method for screening a functional peptide of the present invention includes at least a step of screening a functional peptide using the peptide library of the present invention, and further includes other steps as necessary.
The screening method is not particularly limited as long as the peptide library of the present invention is used, and a known method can be appropriately selected. For example, a desired target substance and the peptide library are mixed, a bound peptide complex (for example, RD complex) is selected, RNA is dissociated from the RD complex, and DNA is prepared from the RNA. Examples thereof include a method of screening for a functional peptide having an affinity for the target substance by repeating the step of transcribing to mRNA and preparing an RD complex library again after amplification.

The present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited to these Examples and the like.

(Preparation Example 1: Peptide Synthesis)
A peptide having the following sequence was synthesized on a link amide resin (0.2 mmol / g) by a solid-phase synthesis method using microwaves.
FITC-Ahx-Cys-Gly-Ser-Gly-Leu-Ala-Ser-Pro-Asn-Gly-Tyr-Cys-NH ₂
[In the above sequence, "FITC" represents fluorescein isothiocyanate and "Ahx" represents 6-aminohexanoic acid. ]

The resin on which the peptide was formed was used as trifluoroacetic acid (TFA) / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (92.5 / 2.5 / 2.5 / 2.5). The peptide was excised from the resin by immersing it in (volume ratio)) for 3 hours.
The obtained peptide was purified by reverse phase HPLC and freeze-dried to obtain a peptide having the above sequence (hereinafter, may be referred to as "P". See the structural formula below).
The identification data of the peptide P by electrospray ionization mass spectrometry (ESI-MS) were as follows.
ESI-MS C ₇₂ H ₉₂ N ₁₆ O ₂₂ O ₃ Calculated value (M + 2H +) 815.295, measured value 814.67

(Example 1: Synthesis of linker molecule G3-DCX for producing cell membrane penetrating cyclic peptide)
<Synthesis of compound C1>
Compound C1 represented by the following structural formula was synthesized by the same method as described in US Pat. No. 7,862,807.
In addition, "Boc" in the structural formula represents "tert-butoxycarbonyl group".

The compound C3 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, a methylene chloride solution (15 mL) of compound C1 (500 mg, 0.42 mmol) was cooled to 0 ° C., and compound C2 (manufactured by Tokyo Kasei Kogyo Co., Ltd., product number A2293) (117.5 mg, 0.504 mmol) was cooled. ), 1-Hydroxybenzotriazole (HOBT) (85.1 mg, 0.630 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC / HCl) (120 mg, 0.630 mmol). The mixture was stirred at 25 ° C. for 17 hours. H ₂ O (20 mL) is added to this, and extraction is performed with methylene chloride (30 mL 3 times), and the organic layer is 1N hydrochloric acid (20 mL twice), saturated sodium bicarbonate water (20 mL twice) and water (20 mL). Washed twice). The compound C3 was obtained as a white solid by drying with Na ₂ SO ₄ , filtering and concentrating, and purifying the obtained residue by silica gel chromatography (methanol (MeOH) / CH ₂ Cl ₂ = 1/10) (methanol (MeOH) / CH ₂ Cl ₂ = 1/10). 533 mg, 0.379 mmol, 90% yield).
The identification data of the compound C3 by 1H NMR was as follows.
1H NMR (CDCl ₃ ): δ 11.4 (s, 3H), 8.58 (t, ³ J _HH = 6.0 Hz, 3 H), 7.69 (t, ³ J _HH = 5.0 Hz, 3 H) , 6.84 (s, 1H), 3.89 (s, 2H), 3.71-3.65 (m, 22H), 3.56-3.53 (m, 6H), 3.42-3 .38 (m, 8H), 2.43 (t, ³ J _HH = 6.0 Hz, 6H), 1.49 (s, 27H), 1.48 (s, 27H)

<Synthesis of compound DBXA>
Sodium hydride (132 mg, 3.02 mmol) was suspended in tetrahydrofuran (THF) (6 mL) and cooled to 0 ° C. 2-Propyne-1-ol (0.165 mL, 2.80 mmol) was added thereto, and the mixture was stirred at 0 ° C. for 30 minutes. 1,3,5-Tris (bromomethyl) benzene (1.00 g, 2.80 mmol) was added, and the mixture was stirred at 25 ° C. for 20 hours. Ethyl acetate (100 mL) was added and then washed with water (100 mL) and saturated brine (100 mL), and the organic layer was washed with magnesium sulfate. The residue obtained by filtration and concentration was purified by preparative thin layer chromatography (PTLC) (methylene chloride / hexane = 1/2), and the compound DBXA (416.6 mg, 1.25 mmol) represented by the following structural formula was obtained. , Yield 45%) was obtained as a pale yellow oil.
The identification data of the compound DBXA by 1H NMR were as follows.
1H NMR (CDCl ₃ ): δ 7.29 (s, 1H), 7.26 (s, 2H), 4.53 (s, 2H), 4.40 (s, 4H), 4.15 ( ⁴ J) _HH = 2.5Hz, 2H), 2.43 ( ⁴ J _HH = 2.0Hz, 1H)

The compound C4 represented by the above structural formula was synthesized by the above reaction formula.
Specifically, nitrogen gas bubbling was performed on a solution consisting of compound C3 (533 mg, 0.379 mmol), compound DBXA (188 mg, 0.568 mmol) and THF (40 mL) to create a nitrogen atmosphere. An aqueous solution of copper sulfate (200 mM; 1.89 mL, 0.379 mmol) and an aqueous solution of sodium ascorbate (100 mM; 7.58 mL, 0.758 mmol) were added thereto, and the mixture was stirred at 25 ° C. for 3 hours. Water is added to the reaction solution, extraction is performed with methylene chloride (20 mL, 3 times), the organic layer is dried with Na ₂ SO ₄ , filtered and concentrated, and the obtained residue is purified by silica gel chromatography (MeOH / CH ₂ Cl). _{By 2} = 1/20), compound C4 was obtained as a white solid (497 mg, 0.286 mmol, yield 76%).
The identification data of the compound C4 by 1H NMR was as follows.
1H NMR (CDCl ₃ ): δ 8.58 (t, ³ J _HH = 5.5 Hz, 3 H), 7.77 (s, 1 H), 7.74 (t, ³ J _HH = 5.0 Hz, 3 H) , 7.34 (s, 1H), 7.32 (s, 2H), 6.82 (s, 1H), 4.69 (s, 2H), 4.58 (s, 2H), 4.56 ( t, ³ J _HH = 5.0 Hz, 2H), 4.47 (s, 4H), 3.89 (t, ³ J _HH = 5.0 Hz, 2H), 3.87 (s, 2H), 3. 69 (t, ³ J _HH = 6.0 Hz, 6H), 3.66 (s, 6H), 3.61 (s, 8H), 3.56-3.52 (m, 6H), 3.41- 3.38 (m, 6H), 2.42 (t, ³ J _HH = 6.0 Hz, 6H), 1.49 (s, 27H), 1.48 (s, 27H)

The linker molecule G3-DCX for producing a cell-penetrating cyclic peptide represented by the above structural formula was synthesized by the above reaction formula.
Specifically, compound C4 (497 mg, 0.286 mmol) and a 4N dioxane hydrochloride solution (30 mL) were mixed and stirred at 25 ° C. for 46 hours. After the reaction, a white solid was precipitated. The supernatant was removed to obtain a crude product of the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide. Purification by preparative HPLC (high performance liquid chromatography) was obtained to obtain the linker molecule G3-DCX for producing a cell-penetrating cyclic peptide as a white solid (116 mg, 0.110 mmol, yield 54%).
The identification data of the compound G3-DCX by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) were as follows.
MALDI-TOF MS C ₂₄ H ₅₁ N ₁₄ O ₈ Calculated value (M + H +) 1047.502, measured value 1047.953

(Example 2: Synthesis of peptide complex G3-DCX-P)

The peptide complex G3-DCX-P represented by the above structural formula was synthesized by the above reaction formula.
Specifically, the peptide P (4.8 mg, 3.0 μmol) synthesized in Preparation Example 1 was dissolved in 50 mM ammonium bicarbonate buffer (1.0 mL), and tris (2-carboxyethyl) phosphine (TCEP) (TCEP). 0.94 mg (3.3 μmol) was added, and the mixture was stirred at 25 ° C. for 1 hour. A solution consisting of the linker molecule G3-DCX (4.7 mg, 4.5 μmol) for producing a cell-penetrating cyclic peptide synthesized in Example 1 and water (500 μL) was added thereto, and the mixture was stirred at 25 ° C. for 20 hours. The reaction solution was purified by preparative HPLC to obtain a cyclic peptide (peptide complex G3-DCX-P) (1.01 mg, 0.388 μmol, yield 13%).
The analysis conditions of the preparative HPLC are as follows. The analysis chart of the peptide P synthesized in Preparation Example 1 is shown in FIG. 1, and the analysis chart of the cyclic peptide (peptide complex G3-DCX-P) is shown in FIG.
[Analysis conditions]
-Column: SUPELCO C18 4.7 mm x 250 mm
・ Detection wavelength: 220 nm
-Flow flow rate: 1.0 mL / min-Solvent: A = 0.1% TFA-containing aqueous solution, B = 0.1% TFA-containing acetonitrile solution-Gradient: Solvent B concentration (%) = 0% → 100% (0 minutes) → 20 minutes)
The identification data of the peptide complex G3-DCX-P by MALDI-TOF MS was as follows.
MALDI-TOF MS C ₁₁₄ H ₁₆₃ N ₃₂ O ₃₃ O ₃ Calculated value (M + H +) 2604.12., Measured value 2603.575

(Test Example 1: Evaluation of cell membrane permeability)
HeLa cells (Human cervix adenocarcinoma cell) in a cell culture medium containing 2 μM of the peptide synthesized in Preparation Example 1 or the peptide complex synthesized in Example 2 under the conditions of 5% (v / v) CO ₂ , 37 ° C. Was cultured for 2 hours. For culturing HeLa cells, FluroBrite D-MEM (manufactured by Thermo Fisher) (10% (v / v) FCS (fetal bovine serum) added), 10% (v / v) GlutaMax (manufactured by Thermo Fisher) was added).
Then, after washing the cell surface with D-PBS (-) (addition of heparin (20 units / mL)), the cells were collected, and D-PBS (-) (0.5% (v / v) BSA (bovine serum albumin)) was collected. ), 200 mM EDTA (ethylenediaminetetraacetic acid), 0.2% (v / v) propidium iodide (manufactured by Sigma-Aldrich) was suspended), and the fluorescence intensity was determined using a flow cytometer (BD FACS Maria III). It was measured. At the time of flow cytometry analysis, the mode of fluorescence intensity was calculated for the living cell population excluding the dead cells positive for propidium iodide. The results are shown in Table 1.

As shown in Table 1, the peptide synthesized in Preparation Example 1 hardly permeated the membrane after 2 hours of culturing, whereas the peptide complex synthesized in Example 2 permeated the membrane. From this, it was demonstrated that the cell membrane permeability was acquired at the same time as the cyclization of the peptide by introducing the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide into the peptide.

(Example 3: Preparation of a peptide library containing a peptide complex having a peptide cyclized with the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide)
<(1) Preparation of RNA library>
In this section (1), a random base sequence (NNK) by the NNK method ₁₀ [In the formula, N indicates A, U, G or C, K indicates G or U, and NNK corresponds to all codons. ], A method for producing an RNA library having a diversity of 10 ¹² or more composed of RNA containing [] will be described.

A template DNA (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2) was used to prepare this RNA library. Specifically, a reaction solution having the composition shown in Table 2-1 was used, and a 5'fragment was prepared using the plasmid as a template DNA in the PCR cycle of Table 2-2. In Table 2-1, 5FragF_150409 is a forward primer (SEQ ID NO: 4) and Ma5frag_withoutHis_R150310 is a reverse primer (SEQ ID NO: 5).

Next, using the reaction solution having the composition shown in Table 2-3, a 3'fragment of the template DNA was prepared by the PCR cycle in Table 2-4. In Table 2-3, A1MaNNK10_withoutHis_150531 is a forward primer (SEQ ID NO: 6) and 3FragR_150409 is a reverse primer (SEQ ID NO: 7).

Next, using the reaction solution having the composition shown in Table 2-5, overlap extension PCR was performed in the PCR cycle of Table 2-6, and the above 5'fragment and 3'fragment were ligated to obtain the total length. Amplification was performed to obtain template DNA. In Table 2-5, X ~ Z indicates adjusted by adding of _{H 2} O to total volume 60μL in using 5 'fragment and 3' fragment of 1 × ^{10 12,} the reaction solution. Further, 5FFnew_150409 is a forward primer (SEQ ID NO: 8), and 3F-Rnew_150409 is a reverse primer (SEQ ID NO: 9).

The obtained the DNA as a template, using a reaction solution having the composition shown in Table 2-7, by reacting for 2 hours at 37 ° C., RNA library containing ^{10 12} or more mRNA having the nucleotide sequence of SEQ ID NO: 3 Got As shown in FIG. 3, the mRNA contained in this library has FLAG® site, cysteine, random sequence, cysteine, TEV protease site, and spacer sequence in order from the 5'side, and has a stop codon. Absent.

<(2) Preparation of ribosome display (RD) complex library>
The RD complex was prepared using the above RNA library using a reconstituted cell-free protein synthesis kit (“PURE flex®” manufactured by Gene Frontier).
This RD complex reaction solution was mixed with anti-FLAG (registered trademark) M2 antibody-bound agarose beads (manufactured by Sigma-Aldrich, 20 μL) and stirred at 4 ° C. for 60 minutes. Anti-FLAG M2 antibody-bound agarose beads to which an RD complex having a FLAG sequence was selectively bound to the peptide moiety were collected.

<(3) Preparation of ribosome display complex library into which the linker molecule G3-DCX for producing cell membrane penetrating cyclic peptide is introduced>
After diluting the agarose beads recovered in (2) above to 80 μL, 10 mM tris (2-carboxyethyl) phosphine hydrochloride (4 μL) (final concentration 0.5 mM) and 200 mM G3-DCX (Example 1) were used as reducing agents. A linker molecule for producing a cell membrane-permeable cyclic peptide synthesized in (0.8 μL) (final concentration 2 mM) was added, and the reaction was carried out overnight at 4 ° C. After the cyclization reaction, the FLAG peptide was added to elute the RD complex from the agarose beads, and a ribosomal display complex library into which the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide was introduced was obtained.

(Example 4: Preparation of RD complex containing a peptide complex having a peptide cyclized with the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide)
<Preparation of RNA>
Except that the random sequence in the mRNA prepared in Example 3 (in FIG. 3, the portion of "(NNK) ₁₀ " located between the cysteine residues (C)) was used as the base sequence encoding the amino acid sequence "LYRSLPAWRYL". , MRNA was prepared in the same manner as in Example 3.

<Preparation of RD complex>
An RD complex was prepared in the same manner as in Example 3 except that the mRNA prepared in <Preparation of RNA> was used.

<Preparation of ribosome display complex introduced with linker molecule G3-DCX for cell membrane penetrating cyclic peptide production>
An RD complex into which the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide was introduced was obtained in the same manner as in Example 3 except that the RD complex prepared in <Preparation of RD complex> was used.

Agarose beads are separated and removed from the reaction solution containing the RD complex, and a phosphate buffered saline (pH 7.5, 100 μL) containing no Mg ²⁺ is added to dissociate the RD complex, and the obtained peptide is TEV. It was cleaved with protease to give a peptide fragment.
Further, a peptide fragment was obtained in the same manner for the RD complex in which the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide was not introduced.
The results of measuring the molecular weight of the obtained peptide by MALDI-TOF MS are shown in FIG. In FIG. 4, the upper row shows the measurement results of the peptide fragment obtained when the linker molecule G3-DCX for producing a cell-penetrating cyclic peptide was not introduced, and the lower row shows the linker molecule G3- for producing a cell-penetrating cyclic peptide. The measurement result of the peptide fragment obtained when DCX was introduced is shown. As shown in FIG. 4, it was confirmed that a cyclic peptide (black arrow in FIG. 4) can be obtained by introducing the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide. The white arrows in FIG. 4 indicate peptides that are not cyclized.

(Example 5: Preparation of RD complex containing a peptide complex having a peptide cyclized with the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide)
In the same manner as in Example 4, mRNA, RD complex, and linker molecule G3-DCX for producing a cell-penetrating cyclic peptide were introduced, except that the amino acid sequence “LYRSLPAWRYL” in Example 4 was changed to “PLFPWPSLWHR”. A ribosome display complex was prepared.
Further, in the same manner as in Example 4, a peptide fragment was obtained from the obtained RD complex, and its molecular weight was measured by MALDI-TOF MS. The results are shown in FIG.
In FIG. 5, the upper row shows the measurement results of the peptide fragment obtained when the linker molecule G3-DCX for producing a cell-penetrating cyclic peptide was not introduced, and the lower row shows the linker molecule G3- for producing a cell-penetrating cyclic peptide. The measurement result of the peptide fragment obtained when DCX was introduced is shown. As shown in FIG. 5, it was confirmed that a cyclic peptide (black arrow in FIG. 5) can be obtained by introducing the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide. The white arrows in FIG. 5 indicate peptides that are not cyclized.

(Example 6: Preparation of RD complex containing a peptide complex having a peptide cyclized with the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide)
The mRNA, RD complex, and linker molecule G3-DCX for producing a cell-penetrating cyclic peptide were introduced in the same manner as in Example 4, except that the amino acid sequence “LYRSLPAWRYL” in Example 4 was changed to “AGRWNVLWRTYMH”. A ribosome display complex was prepared.
Further, in the same manner as in Example 4, a peptide fragment was obtained from the obtained RD complex, and its molecular weight was measured by MALDI-TOF MS. The results are shown in FIG.
In FIG. 6, the upper row shows the measurement results of the peptide fragment obtained when the linker molecule G3-DCX for producing a cell-penetrating cyclic peptide was not introduced, and the lower row shows the linker molecule G3- for producing a cell-penetrating cyclic peptide. The measurement result of the peptide fragment obtained when DCX was introduced is shown. As shown in FIG. 6, it was confirmed that a cyclic peptide (black arrow in FIG. 6) can be obtained by introducing the linker molecule G3-DCX for producing a cell membrane penetrating cyclic peptide. The white arrows in FIG. 6 indicate peptides that are not cyclized.

As shown in Examples 4 to 6, it was confirmed that the cyclization reaction proceeds even when the length of the peptide and the amino acids constituting the peptide are different.

Examples of aspects of the present invention include the following.
<1> A linker molecule for producing a cell-penetrating cyclic peptide, which comprises a cell membrane-penetrating cyclic peptide-imparting group and a cyclic group that cyclizes a peptide.
<2> The linker molecule according to <1>, wherein the cell membrane permeability-imparting group has a basic functional group.
<3> The linker molecule according to <2>, wherein the basic functional group has a guanidino group.
<4> The linker molecule according to <3>, wherein the basic functional group has two or more guanidino groups.
<5> The above-mentioned <2> to <4>, wherein the cell membrane permeability-imparting group has a dendritic structure and the basic functional group is introduced into the end of a branch of the dendritic structure. It is a linker molecule.
<6> The cyclic group is the linker molecule according to any one of <1> to <5>, which is an electron-withdrawing group.
<7> The linker molecule according to <6>, wherein the electron-withdrawing group has a halogen.
<8> The linker molecule according to <7>, wherein the electron-withdrawing group has two or more halogens.
<9> The linker molecule according to <8>, wherein the electron-withdrawing group has two or more chlorine atoms.
<10> The linker molecule according to <9>, wherein the electron-withdrawing group is a benzyl chloride which may have a substituent.
<11> The linker molecule according to <10>, wherein the electron-withdrawing group is a 3,5-bis (chloromethyl) benzyl group.
<12> The cyclization of the peptide by the cyclizing group is carried out by the reaction of the cyclizing group with at least one group selected from the group consisting of a thiol group, an amino group and a hydroxy group contained in the peptide. The linker molecule according to any one of <1> to <11>.
<13> A linker molecule for producing a cell-penetrating cyclic peptide, which is represented by the following structural formula.

<14> A peptide complex comprising a peptide and the linker molecule according to any one of <1> to <13> introduced into the peptide.
<15> A peptide library comprising the peptide complex according to <14>.
<16> A method for producing a peptide complex, which comprises the step of introducing the linker molecule according to any one of <1> to <13> into the peptide.
<17> The method for producing a peptide complex according to <16>, wherein the peptide is a peptide contained in a peptide library.
<18> A method for screening a functional peptide, which comprises a step of screening a functional peptide using the peptide library according to <15>.

Claims

A linker molecule for producing a cell-penetrating cyclic peptide, which comprises a cell membrane-permeable cyclic peptide-imparting group and a cyclic group that cyclizes the peptide.
The linker molecule according to claim 1, wherein the cell membrane permeability-imparting group has a basic functional group.
The linker molecule according to claim 2, wherein the basic functional group has a guanidino group.
The linker molecule according to claim 3, wherein the basic functional group has two or more guanidino groups.
The linker molecule according to any one of claims 2 to 4, wherein the cell membrane permeability-imparting group has a dendritic structure, and the basic functional group is introduced at the end of a branch of the dendritic structure.
The linker molecule according to any one of claims 1 to 5, wherein the cyclic group is an electron-withdrawing group.
The linker molecule according to claim 6, wherein the electron-withdrawing group has a halogen.
The linker molecule according to claim 7, wherein the electron-withdrawing group has two or more halogens.
The linker molecule according to claim 8, wherein the electron-withdrawing group has two or more chlorine atoms.
The linker molecule according to claim 9, wherein the electron-withdrawing group is a benzyl chloride which may have a substituent.
The linker molecule according to claim 10, wherein the electron-withdrawing group is a 3,5-bis (chloromethyl) benzyl group.
Claim that the cyclization of a peptide by the cyclizing group is carried out by the reaction of the cyclizing group with at least one group selected from the group consisting of a thiol group, an amino group and a hydroxy group contained in the peptide. The linker molecule according to any one of 1 to 11.
A peptide complex comprising a peptide and the linker molecule according to any one of claims 1 to 12 introduced into the peptide.
A peptide library comprising the peptide complex according to claim 13.
A method for producing a peptide complex, which comprises the step of introducing the linker molecule according to any one of claims 1 to 12 into a peptide.
The method for producing a peptide complex according to claim 15, wherein the peptide is a peptide contained in a peptide library.
A method for screening a functional peptide, which comprises a step of screening a functional peptide using the peptide library according to claim 14.