WO2021070920A1 - Cyclic peptide - Google Patents

Abstract

A cyclic peptide that has a cyclic structure consisting of 4-30 amino acids or derivative(s) thereof, or a pharmaceutically acceptable salt thereof. In the cyclic structure, the cyclic peptide contains at least carboranylalanine(Cba) and Cys and has a bond to the thiol group of the Cys via the amino group of the Cba. The Cba is linked to the Cys via an amino acid sequence consisting of 2-28 amino acids or derivative(s) thereof to form a structure and the amino acid sequence consisting of 2-28 amino acids or derivative(s) thereof consist of arbitrary amino acids or derivative(s) thereof.

Description

Cyclic peptide

The present invention relates to a cyclic peptide, a method for producing a library, a method for obtaining a cyclic peptide or a pharmaceutically acceptable salt thereof that interacts with a target protein, a human epidermal growth factor receptor (hEGFR) binder, and a boron neutron. It relates to a composition and the like for use in capture therapy (BNCT).

L- ^{Carboranylalanine (L} Cba) is one of the artificial amino acids, and the three-dimensional aromaticity and charge distribution of the carborane side chain are different from those of proteinaceous amino acids in terms of lipophilicity and hydrophilicity. It is known to bring about (Non-Patent Documents 1 to 6). ^L Cba is chemically stable, and a ^{peptide chain containing L} Cba can be easily obtained by solid phase chemical synthesis.

In recent years, as a method for obtaining a compound having a peptide structure, a method performed in vitro (in vitro) has attracted attention.
The cell-free translation system is a system for synthesizing a target peptide or protein in vitro by utilizing a protein synthesis function extracted from cells. The cell-free translation system has a faster synthesis speed and is more convenient than a chemical synthesis method or a production method using a genetically modified organism as a method for synthesizing a protein. Therefore, the cell-free translation system is also used in drug discovery research, and for example, the "Flexible In vitro Translation System (FIT System)" is known (Non-Patent Document 7). The FIT system is a system that creates peptides containing non-proteinogenic amino acids by a cell-free translation system, which enables the construction of a special peptide library having a cyclic structure or the like.
However, no knowledge has been obtained regarding the uptake of ^{L Cba into the ribosome, and it is not known that the peptide into which L} Cba has been introduced can be obtained by a cell-free translation system.

Boron neutron capture therapy (also referred to as Boron Neutron Capture Therapy, BNCT) irradiates a cancer patient who has been administered a boron drug with a neutron beam and uses alpha particles and Li ions generated by the nuclear reaction between boron and neutrons. It is a treatment method that destroys cancer cells. By selectively concentrating a boron drug on cancer cells, it is possible to selectively destroy cancer cells while suppressing damage to normal cells.

Issa, F .; Kassiou, M .; Rendina, L. M. Boron in Drug Discovery: Carboranes as Unique Pharmacophores in Biologically Active Compounds. Chem. Rev. 2011, 111, 5701-5722. Scholz, M .; Hey-Hawkins, E. Carbaboranes as Pharmacophores: Properties, Synthesis, and Application Strategies. Chem. Rev. 2011, 111, 7035-7062. Lesnikowski, Z. J. Challenges and Opportunities for the Application of Boron Clusters in Drug Design. J. Med. Chem., 2016, 59, 7738-7758. Richardson, T. B .; de Gala, S .; Crabtree, R. H .; Siegbahn, P. E. M. Unconventional Hydrogen Bonds: Intermolecular BH ・ ・ ・ HN Interactions. J. Am. Chem. Soc. 1995, 117 , 12875-12876. Fanfrlik, J .; Lepsik, M .; Horinek, D .; Havlas, Z .; Hobza, P. Interaction of Carboranes with Biomolecules: Formation of Dihydrogen Bonds. ChemPhysChem 2006, 7, 1100-1105. Zhang, X .; Dai, H .; Yan, H .; Zou, W .; Cremer, D. BH ・ ・ ・ π Interaction: A New Type of Nonclassical Hydrogen Bonding. J. Am. Chem. Soc. 2016, 138 , 4334-4337. Goto, Y., Katoh, T. & Suga, H. Flexizymes for genetic code reprogramming. Nat Protoc 6, 779-790, (2011).

As described above, the boron drug used for BNCT is required to have the ability to selectively concentrate on cancer cells.
The present invention has been made in view of the above problems, and an object of the present invention is to provide a boron-containing compound capable of selectively forming an interaction with cancer cells.

The present inventors have made intensive studies in order to solve the above problems, a cyclic peptide having a specific structure containing ^L Cba has a structure suitable for the construction of ^L Cba-containing compound library with FIT system , They have found that a cyclic peptide that selectively interacts with cancer cells can be obtained from the library, and have completed the present invention. In addition, the present inventors have found that ^{a cyclic peptide having a specific structure containing L} Cba is suitable for a boron drug used for BNCT because it exists stably in blood and easily reaches a target cancer cell stably. , The present invention has been completed.

（式中、Xは任意のアミノ酸又はその誘導体であり、rは0～10の整数である。）
［６］
　２種以上の環状ペプチドを含むライブラリーの製造方法であって、
　前記環状ペプチドが、
　４～３０のアミノ酸又はその誘導体から構成される環状構造を有し、
　前記環状構造内に、カルボラニルアラニン（Ｃｂａ）及びＣｙｓを少なくとも含み、
　Ｃｂａのアミノ基を介したＣｙｓのチオール基との結合を有すると共に、ＣｂａとＣｙｓとは、２～２８のアミノ酸又はその誘導体から構成されるアミノ酸配列を介して連結した構造を有し、
　２～２８のアミノ酸又はその誘導体から構成されるアミノ酸配列は、任意のアミノ酸又はその誘導体で構成される、
環状ペプチドであり、
　式(1)で表されるペプチド；
　ClAc‐Cba－(Xaa)_n－Cys－(X)_r　　　　　(1)
［式(1)中、
　Xaaは、任意のアミノ酸又はその誘導体であり、
　Xは、任意のアミノ酸又はその誘導体であり、
　nは、２～２８の整数であり、rは０～１０の整数である。］
をコードするmRNAライブラリーを準備する工程と；
　前記mRNAライブラリーを用いて、無細胞翻訳系により前記ペプチドを発現させ、ライブラリーを製造する工程と；
を含む、ライブラリーの製造方法。
［７］
　２種以上の環状ペプチドを含むライブラリーの製造方法であって、
　前記環状ペプチドが、
　４～３０のアミノ酸又はその誘導体から構成される環状構造を有し、
　前記環状構造内に、カルボラニルアラニン（Ｃｂａ）及びＣｙｓを少なくとも含み、
　Ｃｂａのアミノ基を介したＣｙｓのチオール基との結合を有すると共に、ＣｂａとＣｙｓとは、２～２８のアミノ酸又はその誘導体から構成されるアミノ酸配列を介して連結した構造を有し、
　２～２８のアミノ酸又はその誘導体から構成されるアミノ酸配列は、任意のアミノ酸又はその誘導体で構成される、
環状ペプチドであり、
　式(1)で表されるペプチド；
　ClAc‐Cba－(Xaa)_n－Cys－(X)_r　　　　　(1)
［式(1)中、
　Xaaは、任意のアミノ酸又はその誘導体であり、
　Xは、任意のアミノ酸又はその誘導体であり、
　nは、２～２８の整数であり、rは０～１０の整数である。］
をコードするmRNAライブラリーを準備する工程と；
　前記mRNAライブラリーの各mRNAの3’末端にピューロマイシンを結合させ、ピューロマイシン結合mRNAライブラリーを製造する工程と；
　前記ピューロマイシン結合mRNAライブラリーを用いて、無細胞翻訳系により前記ペプチドを発現させ、ペプチド－mRNA複合体ライブラリーを製造する工程と；
を含む、ライブラリーの製造方法。
［８］
　標的タンパク質と相互作用する環状ペプチド又はその医薬的に許容可能な塩を取得する方法であって、
　前記環状ペプチドが、
　４～３０のアミノ酸又はその誘導体から構成される環状構造を有し；
　前記環状構造内に、カルボラニルアラニン（Ｃｂａ）及びＣｙｓを少なくとも含み；
　Ｃｂａのアミノ基を介したＣｙｓのチオール基との結合を有すると共に、ＣｂａとＣｙｓとは、２～２８のアミノ酸又はその誘導体から構成されるアミノ酸配列を介して連結した構造を有し；
　２～２８のアミノ酸又はその誘導体から構成されるアミノ酸配列は、任意のアミノ酸又はその誘導体で構成される、環状ペプチドであり、
　前記方法が、
　式(1)で表されるペプチド；
　ClAc‐Cba－(Xaa)_n－Cys－(X)_r　　　　　(1)
［式(1)中、
　Xaaは、任意のアミノ酸又はその誘導体であり、
　Xは、任意のアミノ酸又はその誘導体であり、
　nは、２～２８の整数であり、rは０～１０の整数である。］
をコードするmRNAライブラリーを準備する工程と；
　前記mRNAライブラリーの各mRNAの3’末端にピューロマイシンを結合させ、ピューロマイシン結合mRNAライブラリーを製造する工程と；
　前記ピューロマイシン結合mRNAライブラリーを用いて、無細胞翻訳系により前記ペプチドを発現させ、ペプチド－mRNA複合体ライブラリーを製造する工程と；
　前記ペプチド－mRNA複合体ライブラリーと、前記標的タンパク質とを接触させて、前記標的タンパク質と相互作用する環状ペプチドを選択する工程と；
を含む、方法。
［９］
　前記標的タンパク質が、がん細胞に発現するタンパク質である、
［８］に記載の方法。
［１０］
　前記標的タンパク質が、ヒト上皮成長因子受容体（ｈＥＧＦＲ）である、
［８］又は［９］に記載の方法。
［１１］
　［１］～［５］のいずれかに記載の環状ペプチド、又はその医薬的に許容可能な塩を含む、ヒト上皮成長因子受容体（ｈＥＧＦＲ）結合剤。
［１２］
　［１］～［５］のいずれかに記載の環状ペプチド、又はその医薬的に許容可能な塩を含む、ホウ素中性子捕捉療法（ＢＮＣＴ）に用いるための組成物。 That is, the present invention is as follows.
[1]
A cyclic peptide having a cyclic structure composed of 4 to 30 amino acids or a derivative thereof, or a pharmaceutically acceptable salt thereof.
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least.
It has a bond with the thiol group of Cys via the amino group of Cba, and has a structure in which the Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof. And
The amino acid sequence composed of the amino acids 2 to 28 or a derivative thereof is composed of any amino acid or a derivative thereof.
Cyclic peptide or pharmaceutically acceptable salt thereof.
[2]
The amino acid sequence composed of the amino acids 2 to 28 or a derivative thereof is a sequence having an interaction with a cell membrane protein expressed in cancer cells.
The cyclic peptide according to [1] or a pharmaceutically acceptable salt thereof.
[2-1]
The amino acid sequence composed of the above 2 to 28 amino acids including Cba and / or Cys or a derivative thereof is a sequence having an interaction with a cell membrane protein expressed in cancer cells.
The cyclic peptide according to [1] or a pharmaceutically acceptable salt thereof.
[3]
The cell membrane protein expressed in the cancer cells is human epidermal growth factor receptor (hEGFR), human EGFR-related substance (HER2-4), epidermal growth factor receptor molecule (EpCAM), transforming growth factor receptor (TGFR), or Any protein selected from transmembrane sugar protein NMB (GPNMB), etc.
The cyclic peptide according to [1] or [2] or a pharmaceutically acceptable salt thereof.
[4]
The amino acid sequence composed of the amino acids 2 to 28 or a derivative thereof comprises any sequence selected from the formulas (I), (II), and (III).
The cyclic peptide according to any one of [1] to [3] or a pharmaceutically acceptable salt thereof.
Equation (I): -Xaa5-Xaa6-Xaa2-Xaa7-Xaa1-Xaa7-Xaa5-Xaa2-Xaa3-Xaa4-H-Xaa2-P-,
Equation (II): -Xaa1-Xaa7-Xaa6-Xaa5-Xaa1-Xaa3-Xaa6-Xaa1-Xaa4-Xaa3-Xaa3-H-Xaa2-Xaa4-,
Equation (III): -Xaa6-Xaa1-Xaa6-HP-Xaa1-Xaa5-Xaa2-Xaa2-Xaa3-Xaa6-Xaa1-
(In the above formula,
Xaa1 is V, L, or I, or a derivative thereof.
Xaa2 is F, Y, or W, or a derivative thereof.
Xaa3 is G or A, or a derivative thereof,
Xaa4 is S or T, or a derivative thereof,
Xaa5 is K or R, or a derivative thereof,
Xaa6 is D or E, or a derivative thereof,
Xaa7 is Q or N, or a derivative thereof. )
[5]
Represented by CbaP5, CbaP14, CbaP16,
The cyclic peptide according to any one of [1] to [4] or a pharmaceutically acceptable salt thereof.

(In the formula, X is any amino acid or derivative thereof, and r is an integer from 0 to 10.)
[6]
A method for producing a library containing two or more cyclic peptides.
The cyclic peptide
It has a cyclic structure composed of 4 to 30 amino acids or derivatives thereof, and has a cyclic structure.
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least.
It has a bond with a thiol group of Cys via an amino group of Cba, and has a structure in which Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof.
An amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is composed of any amino acid or a derivative thereof.
Cyclic peptide
Peptide represented by formula (1);
ClAc－Cba－ (Xaa) _n －Cys－ (X) _r (1)
[In equation (1),
Xaa is any amino acid or derivative thereof
X is any amino acid or derivative thereof
n is an integer of 2 to 28 and r is an integer of 0 to 10. ]
And the process of preparing the mRNA library that encodes;
A step of expressing the peptide by a cell-free translation system using the mRNA library to produce the library;
How to make a library, including.
[7]
A method for producing a library containing two or more cyclic peptides.
The cyclic peptide
It has a cyclic structure composed of 4 to 30 amino acids or derivatives thereof, and has a cyclic structure.
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least.
It has a bond with a thiol group of Cys via an amino group of Cba, and has a structure in which Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof.
An amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is composed of any amino acid or a derivative thereof.
Cyclic peptide
Peptide represented by formula (1);
ClAc－Cba－ (Xaa) _n －Cys－ (X) _r (1)
[In equation (1),
Xaa is any amino acid or derivative thereof
X is any amino acid or derivative thereof
n is an integer of 2 to 28 and r is an integer of 0 to 10. ]
And the process of preparing the mRNA library that encodes;
A step of binding puromycin to the 3'end of each mRNA of the mRNA library to produce a puromycin-bound mRNA library;
A step of expressing the peptide by a cell-free translation system using the puromycin-binding mRNA library to produce a peptide-mRNA complex library;
How to make a library, including.
[8]
A method for obtaining a cyclic peptide that interacts with a target protein or a pharmaceutically acceptable salt thereof.
The cyclic peptide
It has a cyclic structure composed of 4 to 30 amino acids or derivatives thereof;
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least;
It has a bond with the thiol group of Cys via the amino group of Cba, and has a structure in which Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof;
The amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is a cyclic peptide composed of an arbitrary amino acid or a derivative thereof.
The above method
Peptide represented by formula (1);
ClAc－Cba－ (Xaa) _n －Cys－ (X) _r (1)
[In equation (1),
Xaa is any amino acid or derivative thereof
X is any amino acid or derivative thereof
n is an integer of 2 to 28 and r is an integer of 0 to 10. ]
And the process of preparing the mRNA library that encodes;
A step of binding puromycin to the 3'end of each mRNA of the mRNA library to produce a puromycin-bound mRNA library;
A step of expressing the peptide by a cell-free translation system using the puromycin-binding mRNA library to produce a peptide-mRNA complex library;
A step of contacting the peptide-mRNA complex library with the target protein to select a cyclic peptide that interacts with the target protein;
Including methods.
[9]
The target protein is a protein expressed in cancer cells.
The method according to [8].
[10]
The target protein is the human epidermal growth factor receptor (hEGFR).
The method according to [8] or [9].
[11]
A human epidermal growth factor receptor (hEGFR) binder comprising the cyclic peptide according to any one of [1] to [5], or a pharmaceutically acceptable salt thereof.
[12]
A composition for use in boron neutron capture therapy (BNCT), which comprises the cyclic peptide according to any one of [1] to [5], or a pharmaceutically acceptable salt thereof.

The cyclic peptide of the present invention has a structure suitable for constructing a library by the FIT system. Therefore, the cyclic peptide of the present invention is selectively obtained from the library as a peptide that interacts with cancer cells, ^{and since it contains L} Cba and can be stably present in blood, it is a boron drug used for BNCT. It can be said that it is useful as.

A indicates the mRNA and expressed peptide sequences used in this study. The flag indicates the peptide sequence motif DYKDDDK (in the formula, D = aspartic acid, Y = tyrosine, and K = lysine). The expressed peptide is a ^{cyclized Ac-L} Cba-GKKTT C-flag, which is due to cyclization closed by a thioether bond with ^{ClAc-L Cba at the initiation site.} B is the result of tricin-SDS PAGE analysis of the translation product, lane 1 is peptide expression in the Met-deficient FIT system without ^{tRNA fMet} _CAU ; lane 2 is in the Met-deficient FIT system with ^ClAc-L Tyr-tRNA ^fMet _CAU. Peptide expression; Lane 3 shows peptide expression in a Met-deficient FIT system with ^ClAc-L Cba-tRNA ^fMet _CAU. The lower band in each lane is the remaining [ ¹⁴ C] -Asp that was not incorporated into the peptide. C shows the result of MALDI-TOF mass spectrometry of the cyclic peptide containing ^{L Cba.} The calculated mass (Calc.) And the observed mass (Obs.) Of the monovalent charged species [M + H] + are shown in the spectrum, and the asterisk shows the peak corresponding to the potassium type of the peptide. A schematic diagram of the construction of a cyclic peptide library containing ^L Cba and the selection of peptides by the FIT system is shown. 1) The mRNA sequence library is ligated with a puromycin linker (DNA-PEG-CC-Pu) to prepare an mRNA-puromycin library; 2) Translation of a cyclic peptide containing ^{L Cba in an RF1 and methionine-deficient FIT system.} .. The starting AUG codon was ^{assigned to ClAc-L} Cba-tRNA ^fMet _CAU ; 3) Reverse transcription to retrieve cDNA (this step is the (first) post-or (second) preclearance and binding selection. ^{After pre-clearance, selection using the L} Cba-binding cyclic peptide library for human Avi-tagged EGFR immobilized on magnetic beads to remove bead conjugates; 5) final step PCR amplification of cDNA recovered from; 6) In vitro transcription to generate mRNA for the next selection. It is a figure which shows the MALDI-TOF mass spectrum of the cyclic peptide containing ^{selected L} Cba after HPLC purification. The binding kinetics of the cyclic peptide containing ^{L Cba to hEGFR is shown.} Five different concentrations; 10, 25, 50, 100, 250 nM peptides were injected to measure kinetic constants. The binding sensorgram was fitted to the standard 1: 1 binding model. It is a figure which shows the result of the serum stability of the cyclic peptide containing ^{L Cba.} A is the result of serum stability of CbaP5 and CbaP5-1F (P: CbaP5, Q: CbaP5-1F). B is the result of serum stability of CbaP16 and CbaP16-1F (P: CbaP16, Q: CbaP16-1F). The data represent the mean (± standard error) of three or more independent experiments. It is a figure which shows the MALDI-TOF mass spectrum of CbaP5, CbaP14, and CbaP16 labeled with FITC.

Hereinafter, embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and can be variously modified and implemented within the scope of the gist thereof.
In the present specification, the "pharmaceutically acceptable salt" includes, for example, a salt with a pharmaceutically acceptable base or acid.
Non-limiting specific examples of pharmaceutically acceptable salts include addition salts of inorganic acids (hydrochloride, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.) and organic acids (p-toluenesulfonic acid). , Methan sulfonic acid, oxalic acid, p-bromophenyl sulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), inorganic base (ammonium hydroxide or alkali or alkaline earth metal hydroxide) , Carbonate, bicarbonate, etc.), added salts of amino acids, etc.

The present invention is a cyclic peptide having a cyclic structure composed of 4 to 30 amino acids or derivatives thereof, or a pharmaceutically acceptable salt thereof.
Carboranylalanine (Cba) and Cys are contained at least in the cyclic structure of the cyclic peptide of the present invention.
Further, the cyclic peptide of the present invention has a bond with the thiol group of Cys via the amino group of Cba, and the Cba and Cys are amino acids composed of 2 to 28 amino acids or derivatives thereof. It has a structure linked via a sequence.
Further, the amino acid sequence composed of the amino acids 2 to 28 or a derivative thereof is composed of any amino acid or a derivative thereof.

The cyclic peptide of the present invention can be represented by, for example, the formula (A).

In formula (A),
The variable region is a sequence composed of 2 to 28 amino acids or derivatives thereof.
X is an arbitrary amino acid residue or a derivative thereof,
r is an integer of 0 or more, and may be an integer of 0 to 10.

Cba in the present invention is an amino acid represented by the following structure.

The Cba in the present invention may be a D-form, an L-form, or a mixture thereof, but is preferably the L-form ^L- carbolanylalanine (L Cba). .. ^L Cba is represented by the following structure.

The cyclic peptide of the present invention contains Cba and forms a bond with a thiol group of Cys via an amino group of the Cba. Cyclic peptides having this binding form can be synthesized in large quantities by the FIT system, and a cyclic peptide library can be constructed. Although it was generally thought that Cba was not easily incorporated into the ribosome due to its bulky structure, we could apply it to FIT systems by designing mRNAs that introduce Cba into place. We found that it is possible to build a library. By performing a selection targeting a protein expressed on the surface of a cancer cell or the like using the above cyclic peptide library, a cyclic peptide having a high affinity for the protein expressed on the cancer cell can be obtained.
The present inventors have also found that the cyclic peptide containing Cba has high stability in blood. That is, it is considered that the cyclic peptide containing Cba easily stays in the living body and the cyclic peptide can be concentrated in cancer cells.
As described above, the cyclic peptide of the present invention contains a boron atom and can be accumulated in cancer cells, and is therefore useful for boron neutron capture therapy (BNCT).

As used herein, the term "cyclic peptide" means a peptide having at least a cyclic structure formed by four or more amino acids in the molecule. As the molecular structure of the cyclic peptide, in addition to the cyclic structure, it may have a chain structure in which amino acids are linked by peptide bonds, or it may have a structure other than the peptide structure.
As used herein, the term "cyclic structure" means a ring-closed structure formed in a linear peptide by binding two amino acids, Cba and Cys, which are separated by two amino acid residues or more.
As used herein, "separated by two or more amino acid residues" means that at least two amino acids are present between the two amino acids (Cba and Cys).

The ring-closed structure in the cyclic structure is a bond with a thiol group of Cys via an amino group of Cba, and it is preferable that Cba and Cys are formed by a thioether bond. The ring-closed structure referred to here is a cyclic structure formed by binding to a thiol group of Cys via an amino group of Cba in each peptide synthesized by the FIT system, and therefore Cys via an amino group of Cba. The bond with the thiol group is called a ring-closed structure. Specifically, the cyclic peptide of the present invention preferably has a structure represented by the following structure.

Amino acids constituting the cyclic peptide of the present invention include natural amino acids (also simply referred to as “amino acids” in the present specification), artificial amino acid variants and / or derivatives (also referred to as “amino acid derivatives” in the present specification). ) Is also included. Examples of the amino acids constituting the cyclic peptide of the present invention include natural proteinaceous L-amino acids, unnatural amino acids, and chemically synthesized compounds having characteristics known in the art that are characteristic of amino acids. ..
Proteinogenic amino acids are represented by the three-letter notation well known in the art, Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe, Trp, Tyr, and Val. In addition, proteinaceous amino acids are represented by one-letter notation well known in the art, R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M. , F, W, Y, and V.
Non-proteinogenic amino acids mean natural or non-natural amino acids other than proteinogenic amino acids.
Examples of unnatural amino acids include α, α-disubstituted amino acids (α-methylalanine, etc.), N-alkyl amino acids, D-amino acids, β-amino acids, and α-hydroxy acids, which have different main chain structures from the natural type. , Amino acids with different side chain structures (norleucine, homohistidine, etc.), amino acids with excess methylene in the side chain ("homo" amino acids, homophenylalanine, homohistidine, etc.), and carboxylic acids in the side chain Examples thereof include amino acids (such as cysteine acid) in which the functional group is replaced with a sulfonic acid group. Specific examples of unnatural amino acids include the amino acids described in International Publication No. 2015/030014.

As the derivative of the amino acid in the present invention, N-alkyl-α-amino acid, which is an amino acid in which an alkyl group is bonded to an amino group at the α-position, is preferable.

The number of amino acid residues forming the cyclic structure is not particularly limited as long as it is 4 or more, but may be, for example, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more. The number of amino acid residues forming the cyclic structure is not particularly limited as long as it is 30 or less, but may be 25 or less, 20 or less, 17 or less, and 15 or less.
The number of amino acids forming a cyclic structure is usually 5 or more and 30 or less, and the number of amino acids forming a cyclic structure may be 6 or more, 8 or more and 10 or more, and 30 or less within the range of 5 or more and 30 or less. , 25 or less, 20 or less, and 15 or less.
The number of amino acids forming the cyclic structure may be 8 or more and 20 or less, 8 or more and 17 or less, 9 or more and 17 or less, 10 or more and 15 or less, or 10 or more and 13 or less.
The number of amino acids forming the cyclic structure is preferably 9 or more and 25 or less, more preferably 10 or more and 20 or less, and further preferably 10 or more and 15 or less, from the viewpoint of further enhancing the affinity with the target protein.

In the present invention, the cyclic peptide may be modified such as phosphorylation, methylation, acetylation, adenylylation, ADP ribosylation, glycosylation, and addition of polyethylene glycol, and other peptides may be added. And / or may be fused with a protein. The cyclic peptide may also be biotinylated or labeled via a suitable linker.
Further, in the present invention, the cyclic peptide may be a dimer having two cyclic structures in a molecule in which two cyclic peptides having one cyclic structure are bound via a linker structure, and in the molecule. It may have an intramolecular lactam bridge structure forming a lactam structure.
The linker structure connecting the two cyclic peptides is not particularly limited, and a linker having a well-known structure as a linker connecting the peptides in the field of peptide synthesis can be adopted.
The intramolecular lactam bridge structure may be formed by binding the side chains of amino acids constituting the cyclic peptide, for example, the amino group of the side chain of Lys and the carboxyl group of the side chain of Asp or Glu are bonded. By forming a peptide bond, an intramolecular lactam structure is formed, and the cyclic peptide has another ring structure as a bridge structure in the molecule. Instead of Lys, for example, DAP, DAB, and Orn may be bound to Asp or Glu.

In the cyclic peptide of the present invention, the amino acid sequence consisting of 2 to 28 amino acids or derivatives thereof existing between Cba and Cys is arbitrary. The amino acid sequence is appropriately selected according to the target protein. The amino acid sequence consisting of 2 to 28 amino acids or derivatives thereof in the present invention is preferably a sequence capable of interacting with a substance having a physiological action in vivo, and is a sequence capable of interacting with a protein expressed in cancer cells. Is more preferable. Among the amino acid sequences, it is preferable that the sequence interacts with the cell membrane protein expressed in cancer cells. In the present specification, the "protein expressed in cancer cells" and the "cell membrane protein expressed in cancer cells" may be proteins expressed specifically in cancer cells, and are compared to normal cells in cancer cells. It may be a protein that is prominently found in. Further, the protein expressed in the cancer cell may be a protein expressed in the cell or a protein expressed on the cell surface. Furthermore, the protein expressed in cancer cells may be modified with sugar chains or the like.

In the cyclic peptide of the present invention, as described above, the amino acid sequence consisting of 2 to 28 amino acids or a derivative thereof is preferably a sequence capable of interacting with a protein expressed in cancer cells, but "Cba" containing Cba and Cys is preferable. It is preferable that the composition of / 2-28 amino acids or derivatives thereof / Cys ”is a sequence that interacts with the target protein. In other words, an amino acid sequence consisting of 2 to 28 amino acids or a derivative thereof may or may not have an interaction with a target protein, and interacts with Cba and / or Cys. May be demonstrated.
Therefore, in the present invention, when the amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is a sequence having an interaction with a protein expressed in cancer cells, the amino acid sequence is Cba before and after the amino acid sequence. And / or sequences that interact with Cys.

In the present invention, the interaction of an amino acid sequence consisting of 2 to 28 amino acids or a derivative thereof in a cyclic peptide is, for example, a cyclic peptide through a hydrophobic interaction or a hydrophilic interaction with a protein expressed in a cancer cell. Means that can bind to the protein.
It is a preferred embodiment that the peptide moiety composed of the amino acid sequence of 2 to 28 amino acids or a derivative thereof in the cyclic peptide can bind to the target protein as a ligand.
The target protein may be a growth factor receptor protein, a growth factor receptor protein, and a protein in which a ligand can be endogenous to the receptor by binding of the ligand to the growth factor receptor.

Examples of the amino acid sequence composed of 2 to 28 amino acids or derivatives thereof in the cyclic peptide of the present invention include human epidermal growth factor receptor (hEGFR), human EGFR-related substance (HER2-4), and epidermal cell adhesion molecule ( EpCAM), transforming growth factor receptor (TGFR), transmembrane sugar protein NMB (GPNMB) and other sequences that form interactions with proteins.

The amino acid sequence consisting of 2 to 28 amino acids or a derivative thereof preferably contains any sequence selected from the formulas (I), (II), and (III). By including any sequence selected from formulas (I), (II), and (III), the cyclic peptides of the invention can interact with hEGFR.
Equation (I): -Xaa5-Xaa6-Xaa2-Xaa7-Xaa1-Xaa7-Xaa5-Xaa2-Xaa3-Xaa4-H-Xaa2-P-,
Equation (II): -Xaa1-Xaa7-Xaa6-Xaa5-Xaa1-Xaa3-Xaa6-Xaa1-Xaa4-Xaa3-Xaa3-H-Xaa2-Xaa4-,
Equation (III): -Xaa6-Xaa1-Xaa6-HP-Xaa1-Xaa5-Xaa2-Xaa2-Xaa3-Xaa6-Xaa1-

Xaa1 is V, L, or I, or a derivative thereof, preferably V, L, or I, or an N-alkyl amino acid thereof.
Xaa2 is F, Y, or W, or a derivative thereof, preferably F, Y, or W, or an N-alkyl amino acid thereof.
Xaa3 is G or A, or a derivative thereof, preferably G or A, or an N-alkyl amino acid thereof.
Xaa4 is S or T, or a derivative thereof, preferably S or T, or an N-alkyl amino acid thereof.
Xaa5 is K or R, or a derivative thereof, preferably K or R, or an N-alkyl amino acid thereof.
Xaa6 is D or E, or a derivative thereof, preferably D or E, or an N-alkyl amino acid thereof.
Xaa7 is Q or N, or a derivative thereof. It is preferably Q or N, or its N-alkyl amino acid.

The cyclic peptide of the present invention can be obtained by a translational synthesis method using a cell-free translation system.
The cyclic peptide of the present invention can be prepared by preparing a nucleic acid encoding the cyclic peptide of the present invention and translating the nucleic acid with a cell-free translation system. The nucleic acid encoding the cyclic peptide can be appropriately designed by those skilled in the art using the genetic code used in the translation system of the living body, the reprogrammed genetic code, or a combination thereof. The nucleic acid may be DNA or RNA.

According to the method using a cell-free translation system, it is possible to efficiently introduce an unnatural amino acid into a peptide in addition to a natural amino acid by using a tRNA aminoacylated with an unnatural amino acid. For example, by using the artificial aminoacyl-tRNA synthetase flexizyme developed by the present inventors, it is possible to aminoacylate a tRNA having an arbitrary anticodon with an arbitrary natural or non-natural amino acid. Therefore, using this technique, the genetic code composed of triplets of mRNA can be reprogrammed to encode amino acids different from those of the biological translation system (International Publication No. 2008/059823).

In the present specification, the "cell-free translation system" refers to a cell-free translation system, and examples of the cell-free translation system include Escherichia coli extract, wheat germ extract, rabbit erythrocyte extract, insect cell extract and the like. Can be used. In addition, purified ribosomal protein, aminoacyl-tRNA synthetase (aaRS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF) elongation factor (EF), termination factor (RF), and ribosomal regeneration factor ( RRF), as well as a reconstituted cell-free translation system constructed by reconstitution of other factors required for translation may be used.
A system containing RNA polymerase may be used to perform transcription from DNA at the same time. Commercially available cell-free translation systems include RTS-100 (registered trademark) from Roche Diagnostics as a system derived from Escherichia coli, and PURESYSTEM (registered trademark) from PGI as a reconstituted translation system. As a system using wheat germ extract, such as PURE frex from New England Biolabs and PUR Express In Vitro Protein Synthesis Kit from New England BioLabs, those from Zoegene and Self-Free Science can be used.
In addition, as a system using Escherichia coli ribosomes, for example, the techniques described in the following documents are known: HF Kung et al., 1977. The Journal of Biological Chemistry Vol. 252, No. 19, 6889-6894; MC Gonza et al., 1985, Proceeding of National Academy of Sciences of the United States of America Vol. 82, 1648-1652; MY Pavlov and M. Ehrenberg, 1996, Archives of Biochemistry and Biophysics Vol. 328, No. 1, 9 -16; Y. Shimizu et al., 2001, Nature Biotechnology Vol. 19, No. 8, 751-755; H. Ohashi et al., 2007, Biochemical and Biophysical Research Communications Vol. 352, No. 1, 270- 276.
According to the cell-free translation system, the expression product can be obtained in a highly pure form without purification.
The cell-free translation system in the present invention may be used not only for translation but also for transcription by adding a factor necessary for transcription.

As a result of the studies by the present inventors, it has been found that by designing an mRNA that introduces Cba into the N-terminal, a translation synthesis method using a cell-free translation system can be applied, and a library can be constructed.
That is, one of the present inventions is
It has a cyclic structure composed of 4 to 30 amino acids or derivatives thereof;
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least;
It has a bond with the thiol group of Cys via the amino group of Cba, and has a structure in which Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof;
The amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is a method for producing a library containing two or more cyclic peptides composed of an arbitrary amino acid or a derivative thereof:
Peptide represented by formula (1);
ClAc－Cba－ (Xaa) _n －Cys－ (X) _r (1)
[In equation (1),
Xaa is any amino acid or derivative thereof
X is any amino acid or derivative thereof
n is an integer of 2 to 28 and r is an integer of 0 to 10. ]
And the process of preparing the mRNA library that encodes;
A step of expressing the peptide by a cell-free translation system using the mRNA library to produce a peptide library;
It is a manufacturing method of a library including.

Moreover, one of the present inventions is
It has a cyclic structure composed of 4 to 30 amino acids or derivatives thereof;
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least;
It has a bond with the thiol group of Cys via the amino group of Cba, and has a structure in which Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof;
The amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is a method for producing a library containing two or more cyclic peptides composed of an arbitrary amino acid or a derivative thereof:
Peptide represented by formula (1);
ClAc－Cba－ (Xaa) _n －Cys－ (X) _r (1)
[In equation (1),
Xaa is any amino acid or derivative thereof
X is any amino acid or derivative thereof
n is an integer of 2 to 28 and r is an integer of 0 to 10. ]
And the process of preparing the mRNA library that encodes;
A step of binding puromycin to the 3'end of each mRNA of the mRNA library to produce a puromycin-bound mRNA library;
A step of expressing the peptide by a cell-free translation system using the puromycin-binding mRNA library to produce a peptide-mRNA complex library;
It is a manufacturing method of a library including.

Furthermore, one of the present inventions is
A method for obtaining a cyclic peptide that interacts with a target protein or a pharmaceutically acceptable salt thereof. Here, the target protein preferably contains a protein expressed in cancer cells, and more preferably contains a protein of human epidermal growth factor receptor (hEGFR).
The cyclic peptide in the method is
It has a cyclic structure composed of 4 to 30 amino acids or derivatives thereof;
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least;
It has a bond with the thiol group of Cys via the amino group of Cba, and has a structure in which Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof;
The amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is a cyclic peptide composed of an arbitrary amino acid or a derivative thereof.
The method is
Peptide represented by formula (1);
ClAc－Cba－ (Xaa) _n －Cys－ (X) _r (1)
[In equation (1),
Xaa is any amino acid or derivative thereof
X is any amino acid or derivative thereof
n is an integer of 2 to 28 and r is an integer of 0 to 10. ]
And the process of preparing the mRNA library that encodes;
A step of binding puromycin to the 3'end of each mRNA of the mRNA library to produce a puromycin-bound mRNA library;
A step of expressing the peptide by a cell-free translation system using the puromycin-binding mRNA library to produce a peptide-mRNA complex library;
A step of contacting the peptide-mRNA complex library with the target protein to select a cyclic peptide that interacts with the target protein;
including.

The above mRNA library encodes a peptide represented by the formula (1); ClAc-Cba- (Xaa) _n- Cys- (X) _r . For example, the codon of mRNA is AUG- (N1N2N3). It is preferably represented by _n- UGC- (XXX) _r. Here, n and r are synonymous with n and r in the equation (1). AUG and UGC codons are ^{reassigned to ClAc-L} Cba and Cys, respectively. XXX is a codon of any amino acid, and the number of amino acids can be appropriately controlled by setting the STOP codon. Further, XXX is a codon of an arbitrary amino acid, may be a codon represented by N1N2N3, or may be a codon encoding a specific amino acid.
In the present specification, "N1N2N3" means a codon that specifies an arbitrary amino acid, for example, N1, N2 and N3 are independently adenine (A), guanine (G), cytosine (C) and uracil (, respectively). Selected from U). Although one mRNA contains a plurality of N1N2N3, each N1, N2 and N3 are independently selected. Thus, for example, if the mRNA contains -N1N2N3-N1N2N3-, the two N1, N2 and N3, respectively, may be the same or different from each other.

In the present invention, any amino acid is reassigned to N1N2N3. In reassignment, a codon-amino acid relationship that is different from the codon-amino acid relationship in the natural genetic code table can be assigned, or the same relationship can be assigned.
As used herein, the term "natural genetic code table" refers to a table showing amino acids represented by a genetic code consisting of triplets of mRNA in a living body. In the natural genetic code table, N1N2N3 indicates the following amino acids.

The mRNA library contains mRNA containing multiple N1N2N3, for example, multiple N1N2K, multiple N1N2S, multiple N1N2M, multiple N1N2W, multiple N1N2A, multiple N1N2U, multiple N1N2C, and multiple N1N2G. It may include. In the present specification, N1 and N2 are synonymous with the above, K is independently one of uracil (U) and guanine (G), and S is independently of cytosine (C) and guanine (G), respectively. Either, M is either adenine (A) or cytosine (C) independently, and W is independently either adenine (A) or uracil (U), respectively.
Hereinafter, the present invention will be described by taking as an example the case where the mRNA library contains mRNA containing a plurality of N1N2K, that is, the case where the mRNA library contains a plurality of N1N2K codons as N1N2N3. Even when the mRNA library of the above is used, it can be carried out in the same manner as long as the peptides contained in the translated peptide library are prenylated. In the natural genetic code table, N1N2K indicates 20 kinds of amino acids when the right column of the above table is G or U.

Further, in the present specification, for example, Leu may be assigned to UUG according to the natural genetic code table, or amino acids other than Leu may be assigned by suballocating codons. Any amino acid can be assigned to the "N1N2K" codon. "Assigning an amino acid to a codon" means rewriting the genetic code table so that a codon encodes that amino acid. In the present specification, "assigning an amino acid to a codon" and "reassigning a codon" are used interchangeably.
The assignment of amino acids to each codon, which is different from the natural genetic code table, is realized, for example, by codon reallocation using an artificial aminoacyl-tylated RNA-catalyzed Flexizyme. According to flexizyme, a desired amino acid can be bound to a tRNA having an arbitrary anticodon, so that an arbitrary amino acid can be assigned to an arbitrary codon. Flexizyme will be described later. In the present specification, binding an amino acid to a tRNA may mean charging the tRNA with the amino acid, aminoacylating the tRNA, or acylating the tRNA with the amino acid.

In the present invention, a non-proteinogenic amino acid may be assigned to "N1N2K". For example, by using an amino acid having a cyclic structure or an N-alkyl amino acid as a non-protein amino acid, a peptide library having increased resistance to proteolysis, cell membrane permeability, and conformational rigidity can be obtained. Such peptide libraries are useful for screening peptides that target intracellular disease-related molecules or molecules that have protease activity. When mRNA contains 2 or more "N1N2K", all of them may be assigned to non-proteinogenic amino acids, or some of them may be assigned to non-proteinogenic amino acids.

The mRNA library is composed of mRNA encoding _{ClAc-Cba- (Xaa) n-} Cys- (X) _r , which is a peptide represented by the formula (1), and _n in (Xaa) n is usually 2 or more. The upper limit of n is not particularly limited, but is usually 28 or less. Since n is understood to be the number of amino acid residues forming a cyclic structure-2, n is defined as -2, which is exemplified as the number of amino acid residues forming a cyclic structure or is described as a preferable number or range. The number and range may be.

The cyclic peptide of the present invention can form an interaction with a protein expressed in cancer cells. Examples of the protein include human epidermal growth factor receptor (hEGFR). Therefore, one of the present inventions is a hEGFR binder comprising the cyclic peptide of the present invention, or a pharmaceutically acceptable salt thereof. In addition, the cyclic peptide of the present invention can be used for boron neutron capture therapy (BNCT). Therefore, one of the present inventions, one of the present inventions, is a composition for use in BNCT, which comprises the cyclic peptide of the present invention, or a pharmaceutically acceptable salt thereof.
The hEGFR binder or composition of the present invention can be used as a pharmaceutical composition. The administration form of the hEGFR binder or composition of the present invention is not particularly limited, and may be orally or parenterally administered. Examples of parenteral administration include injection administration such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration.
Examples of the administration route of transmucosal administration include nasal administration, eye, lung, vagina, and rectum.
Various modifications may be made to the cyclic peptide in the hEGFR binder or composition from the viewpoint of pharmacokinetics such as metabolism and / or excretion. For example, polyethylene glycol (PEG) and / or a sugar chain can be added to the cyclic peptide to further prolong the residence time in blood and reduce the antigenicity.
In addition, sustained-release bases include biodegradable polymer compounds such as polylactic acid / glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, and nanospheres. These may contain cyclic peptides. When administered transdermally, a weak electric current can be applied to the surface of the skin to allow it to penetrate the stratum corneum (iontophoresis method).

The hEGFR binder or composition may use the cyclic peptide as it is as an active ingredient, or may be formulated by adding a pharmaceutically acceptable additive or the like.
Dosage forms of formulations include, for example, liquids (eg injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, troches. Examples thereof include agents, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.
Formulations include, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjustments. It can be carried out by a conventional method by appropriately using additives such as an agent, a preservative, a wetting agent, a dispersant, and an antioxidant.
The additives used for formulation are not particularly limited, but are, for example, pharmaceutically acceptable organic solvents such as purified water, saline solution, phosphate buffer, dextrose, glycerol, ethanol, and animal and vegetable oils. , Lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, Sodium alginate, water-soluble dextran, sodium carboxymethyl cellulose, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, tragant, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, octyldodecyl myristate, Examples thereof include isopropyl myristate, higher alcohols, stearyl alcohols, stearic acids, and human serum glucose.
Surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile acids such as glycocholic acid, deoxycholic acid, and taurocholic acid; EDTA and salicylic acids, etc. Chelating agents; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamin derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and 1 A nitrogen oxide donor or the like may be used.

The tablet or pill may be a coated tablet or the like coated with a sugar coating, a gastric soluble substance, an enteric substance or the like.
The liquid preparation may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols and the like. As the liquid agent, a wetting agent, an emulsifier, a dispersant, a stabilizer, a dissolving agent, a solubilizing agent, a preservative and the like may be added.

The present invention also provides a method of administering the hEGFR binder or composition of the present invention to a patient in need thereof to treat or prevent a disease in the patient.

The dose of the hEGFR binder or composition of the present invention depends on the symptoms, age, sex, body weight, sensitivity difference, administration method, administration interval, type of preparation, etc. of the patient who needs it. , Can be determined as appropriate.
The patient is a mammal, preferably a human.

<Production Example 1: Preparation of ^ClAc-L Cba-tRNA ^fMet _CAU >
In ^{the preparation of ClAc-L} Cba-tRNA ^fMet _CAU , first, α-N- (2-chloroacetyl) -L-carbolanylalanine cyanomethyl ester (ClAc- ^L Cba-CME) was prepared.

(Preparation of ^ClAc-L Cba-CME)
ClAc- ^L Cba-CME was synthesized according to the following scheme.

^A mixture of L Cba (70 mg, 0.30 mmol), N- (chloroacetoxy) succinimide (72 mg, 0.38 _{mmol) and LVDS 3} (79 mg, 0.94 mmol) in 5 mL of a 50% aqueous solution of 1,4-dioxane at room temperature of 1.5 Stirred for hours. After the reaction, the mixture _{was acidified with KHSO 4} (308 mg, 2.26 mmol). The mixture was then extracted with ethyl acetate (4 times at 10 mL), the combined organic layers were washed 3 times with brine _{, dried over DDL 4} , and concentrated under reduced pressure.
Next, 3 mL of the obtained residue ( ^{containing N-ClAc-L} Cba-OH) was mixed with N, N-diisopropylethylamine (DIPEA; 143 μL, 0.81 mmol) and chloroacetonitrile (1.0 mL), and the reaction mixture was brought to room temperature. Stirred for 12 hours. After the reaction, ethyl acetate (20 mL) was added, and the obtained solution was washed with 1M HCl (5 mL 3 times), saturated LVDS ₃ (5 ml 3 times) and saturated saline (10 ml 3 times), and the organic layer was washed. Was _{dried over DDL 4} and concentrated under reduced pressure.
Finally, the obtained crude product was purified by reverse phase HPLC using 0.1% trifluoroacetic acid (TFA) aqueous solution / 0.1% TFA-containing acetonitrile as the mobile phase to obtain ^ClAc-L Cba-CME as a white solid. (42 mg, yield 40%). Various spectral data of the obtained ^ClAc-L Cba-CME were as follows.

¹ H NMR (300 MHz, CDCl ₃ , δ): 7.13 (d, J = 7.7 Hz, 1H), 4.80 (d, J = 2.1 Hz, 2H), 4.55-4.61 (m, 1H), 4.11 (s, 2H), 3.77 (br s, 1H), 2.75-3.05 (m, 2H), 1.25-3.10 (br m, 10H).
¹³ C NMR (75 MHz, CDCl ₃ , δ): 168.0, 166.4, 113.3, 70.9, 61.6, 52.2, 49.8, 42.1, 38.3.
¹¹ B NMR (96.3 MHz, CDCl ₃ , δ): -1.0, -2.6, -3.9, -5.4, -8.0, -9.7, -11.8.
HRMS (ESI-MS): [ C 9 H 19 B 10 ClN 2 O 3 + TFA-H] - Calculated: 459.1938, Found: 459.1894

(Preparation of ^ClAc-L Cba-tRNA ^fMet _CAU )
The ^{tRNA fMet} _CAU and eFx used to prepare ^ClAc-L Cba-tRNA ^fMet _CAU are Murakami, H .; Ohta, A .; Ashigai, H .; Suga, H. Nat. Methods 2006, 3, 357-359, Goto, Y .; Katoh, T .; Suga, H. Nat. Protoc. 2011, 6, 779-790, Terasaka, N .; Hayashi, G .; Katoh, T .; Suga, H. Nat. Chem. Biol Prepared by run-off in vitro transcription with T7 RNA polymerase according to the method reported in 2014, 10, 555-557.
In 0.17 M HEPES-KOH buffer (pH 7.5), 6 μL of ^{the mixture containing 41.7 μM tRNA fMet} _CAU in the presence of 41.7 μM eFx was heated at 95 ° C. for 2 minutes and then cooled to room temperature for 5 minutes. A 2 μL 3M MgCl ₂ aqueous solution was then added to the mixture and the resulting mixture was allowed to stand at room temperature for 5 minutes and then transferred to ice. After cooling the mixture, ^{2 μL of a 25 mM DMSO solution of ClAc-L} Cba-CME obtained as described above was added and the mixture was incubated on ice for 9 hours. After the reaction, 40 μL of 0.3 M aqueous sodium acetate solution (pH 5.2) and 100 μL of 100% ethanol were added, and the mixture was centrifuged at 13,000 rpm for 15 minutes to pellet aminoacyl-tRNA. The pellet was rinsed twice with 60 μL of 70% EtOH aqueous solution containing 0.1 M sodium acetate aqueous solution (pH 5.2) and once with 40 μL of 70% EtOH aqueous solution to obtain ^{250 pmol of ClAc-L} Cba-tRNA ^fMet _CAU. The reaction volume was scaled up based on the amount of aminoacyl-tRNA required for peptide translation or RaPID selection.

<Examination of introduction ^{of L Cba into peptides>}
Aiming at the construction of a library of cyclic peptides containing ^{L Cba residues, we tested the possibility of L} Cba being incorporated into peptides by initiation or elongation.

[Example 1: Examination of introduction by "start"]
In a methionine-deficient FIT (Flexible In-vitro Translation) system, ClAc- ^L Cba-tRNA ^fMet _CAU was used to reprogram the start codon. Translation was performed according to the method described in Morimoto, J .; Hayashi, Y .; Suga, H. Angew. Chem. Int. Ed. 2012, 51, 3423-3427. Specifically, the reaction by the FIT system ^{was carried out on a 5 μL scale using 0.04 μM mRNA and 100 μM ClAc-L} Cba-tRNA ^fMet _CAU, and incubated at 37 ° C. for 30 minutes to obtain a translation mixture.
Despite the bulkiness of the carborane cluster side chain in ^{L Cba, the initiation factor and ribosome showed resistance to ClAc-L} Cba and expressed the following cyclic peptide peptide (A in FIG. 1).

The expression level of the cyclic peptide shown in FIG. 1A was quantified by Tricine-SDS PAGE analysis (B; lane 3 in FIG. 1), and the expression level was 31% with respect to the Tyr-initiated peptide in comparison with lanes 2 and 3. I found out that there was. In addition, the identity of the peptide was confirmed by MALDI-TOF mass analysis of the translation mixture, and it was found that the desired thioether bond was spontaneously formed and a cyclic peptide skeleton was obtained (see C in FIG. 1).
Despite the bulkiness of the carborane groups, ^L Cba was found to be housed in the ribosomal P site and ribosomal tunnels.

[Comparative example 1: Examination of introduction by "elongation"]
It was also tested whether it was introduced during the extension of ^{L Cba to the nascent peptide chain.} This experiment was performed by preparing ^L Cba-tRNA ^AsnE2 _CAU or ^L Cba-tRNA ^GluE2 _CAU (modified tRNA modified for EF-Tu binding) by eFx (Murakami, H .; Kourouklis, D. Conducted in accordance with Suga, H. Chem. Biol. 2003, 10, 1077-1084, and Katoh, T .; Tajima, K .; Suga, H. Cell Chem. Biol. 2017, 24, 46-54. ). ^L Cba-tRNA ^AsnE2 _CAU and ^L Cba-tRNA ^GluE2 _CAU were used for mRNA translation, respectively.
^{No desired peptide, including L} Cba, was observed under any attempted conditions, and ^L Cba-tRNA ^AsnE2 _CAU had poor binding to EF-Tu, or ^L Cba itself was a carborane group. It was found that the ribosome A site was not tolerated due to its bulkiness.

<Example 2: Library construction and ligand screening for hEGFR>
Ligand screening for hEGFR was performed using the RaPID (Random non-standard Peptides Integrated Discovery) system (see FIG. 2). Yamagishi, Y .; Shoji, I .; Miyagawa, S .; Kawakami, T .; Katoh, T .; Goto, Y .; Suga, H. Chem. Biol. 2011, 18, 1562-1570, and Huang , Y .; Wiedmann, MM; Suga, H. RNA Chem. Rev. 2019, 119, 17, 10360-10391, ligand screening for hEGFR was performed.
The mRNA sequence library consists of AUG- (NNK) _n- UGC- (GGC-AGC) _3- UAG, where (NNK) _n is N and K (N and K are 4 bases and U or G, respectively). , N is a number from 6 to 15), and the starting AUG and extended UGC codons were ^{reassigned to ClAc-L} Cba and Cys, respectively. Specifically, a library was constructed and a ligand screening for hEGFR was performed according to the following procedure. Specifically, the procedure was as follows.

In the first round, using T4 ligase, the mRNA sequence library was first ligated with a puromycin linker (DNA-PEG-CC-Pu, ie 5'-pCTCCCCCCCCCGGTCC-PEG linker-CC-puromycin-3'). did.
The resulting mRNA-puromycin (180 pmol) was then translated using a methionine-deficient FIT system at 37 ° C. for 30 minutes in the presence of ^{150 μL RF1 and 100 μM ClAc-L} Cba-tRNA ^fMet _CAU. In addition, the solution was incubated at room temperature for 12 minutes, 15 μL of EDTA (200 mM, pH 8.0) was added, and the resulting mixture was incubated at 37 ° C. for an additional 30 minutes to allow peptide cyclization to proceed.
Subsequently, the resulting solution was mixed with 165 μL of blocking solution (20 mM phosphate pH 7.4, 275 mM NaCl, 5.4 mM KCl, 0.1% Tween 20 and 0.2% acetyl BSA) and 0.44 mg DynabeadsTM M-280 streptavidin (ThermoFisher Scientific). The bead binder was removed by incubating with the product) at 4 ° C. for 30 minutes 3 times. This process is called pre-cliarance or negative selection. After the pre-clearance step, the collected mixture was incubated at 4 ° C. for 30 minutes in the presence of hEGFR-fixed Dynabeads® M-280 streptavidin (final concentration of hEGFR 200 nM). This process is called positive selection.
The supernatant was then removed and the beads were washed 3 times with 200 μL of selective buffer (1 x PBST: 10 mM phosphate, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20). 40 μL of reverse transcriptase (50 mM Tris-HCl (pH 8.3)), in the presence of 200 units of M-MLV reverse transcriptase RNase H Minus (Promega) and 8 units of RNase inhibitor (Promega). Mixed with 75 mM KCl, 3 mM MgCl ₂ , 10 mM DTT, 0.5 mM dNTP, 2 μM CGS3an13.R39 (5'-TCC GCC CTGCC CTGCC GCTGGC CGC CGCA-3') and reverse transcribed at 42 ° C for 1 hour. The cDNA was heated at 95 ° C. for 5 minutes and 400 μL of PCR mixture (10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl ₂ , 0.25 mM dNTPs, 0.25 μM T7 g10 M. It was eluted with F46 (5'-TAATACGACTCATCATCATAGTTTAAAGTAAGGATAAGATATATA-3'), 0.25 μM CGS3an13.R39).
The isolated cDNA is then quantitatively measured by real-time PCR using Lightcycle 2.0 (Roche), amplified by PCR using Taq DNA polymerase, transcribed in vitro and used for selection in the next round. An mRNA library rich in hEGFR binding sequences was prepared for this purpose.
From the second round of selection, translations were performed on a 5 μL scale and the library was first reverse transcribed by M-MLV prior to pre-clearance and positive selection. Pre-clearance was performed 6 times to remove the bead binder more efficiently. Finally, the observed concentrates appearing in the 4th or 5th round were subjected to further DNA sequencing (deep sequencing) using the MiSeq sequencing system (Ilumina).

Twenty-four candidates shown in Table 2 were selected by deep sequencing data alignment.

<Example 3: Solid phase chemical synthesis of a cyclic peptide containing ^{L Cba>}
In order to confirm the binding ability to hEGFR, the recovery rate of each clone to hEGFR was qualitatively analyzed by the monoclone RaPID display format. We focused on several cloned peptides based on positive recovery (for hEGFR beads) relative to background recovery (for simulated beads) and solid-phase chemosynthesized them. Solid phase chemical synthesis was carried out according to the following procedure.
N-9-fluorenylmethoxycarbonyl- ^L- carbolanylalanine (Fmoc-LCba) used in solid phase chemistry has been reported in reference; de Bruin, G .; Mock, ED; Hoogendoorn, S. .; van den Nieuwendijk, AM; Mazurek, J .; van der Marel, GA; Florea, BI; Overkleeft, HS Chem. Commun. 2016, 52, 4064-4067. Other Fmoc protected amino acids used were obtained from Novabiochem or Watanabe Chemical Industries.
The synthesis of the cyclic peptide selected in Example 2 was performed on a NovaPEG Rink Amide resin on a 25 μmol scale using standard Fmoc solid phase peptide synthesis (SPPS) by a combination of automated and manual approaches.
^{That is, a peptide fragment before L} Cba was synthesized from the C-terminal side using a Syro Wave automatic peptide synthesizer (manufactured by Biotage). Next, a peptide fragment obtained has a free N-terminal α- amino group, incubated for 1 hour at room temperature with a solution (100μmol Fmoc- ^L Cba, DMF solution of 1.2mL containing 100 [mu] mol DIC, and OxymaPure) This allowed coupling with ^Fmoc-L Cba in DMF until a full length peptide was obtained.
^{For L} Cba-binding peptides, the terminal Fmoc groups are incubated in 1 mL of 2% piperazine and 2% DBU in EtOH / NMP (1: 9) at room temperature for 5 minutes to prevent the boron elimination reaction. Removed by. ^{Subsequently, the free N-terminal α-amino group of the L} Cba-binding peptide synthesized on the resin was chloroacetylated by allowing 2 mL of an NMP solution of 0.2M N- (chloroacetoxy) succinimide at room temperature for 1 hour. ..
The resulting peptide-resin was treated with 2 mL of TFA / TIS / water (95: 2.5: 2.5) solution at room temperature for 3 hours. The cleaved peptide was precipitated with diethyl ether and then centrifuged to give pellets. In addition, the pellet was washed with diethyl ether (5 mL x 5). To promote ring closure by thioether binding, the resulting crude peptide was dissolved again in 5 mL DMSO and triethylamine was added to make a basic solution (approximately pH 10), 200 μL of 0.5 M TCEP (Tris (2-carboxy). Incubated at room temperature for 1 hour in the presence of an aqueous ethyl) phosphine solution. Finally, the peptide solution was acidified with TFA and linearly used by reverse phase HPLC (Shimadzu LC-20AP system using Merck Chromolith Prep column) using 0.1% TFA aqueous solution / 0.1% TFA-containing acetonitrile as the mobile phase. Purified under gradient conditions. The purified peptide was confirmed by MALDI-TOF mass and lyophilized in vacuum. The measurement result of MALDI-TOF mass is shown in FIG.
Each cyclic peptide obtained by solid phase chemical synthesis was evaluated by surface plasmon resonance (SPR), and their binding affinity and kinetics for hEGFR were quantitatively measured (see FIG. 4).

<Example 4: Characteristic evaluation of a cyclic peptide having ^{L Cba>}
(Difference in binding activity to hEGFR depending on the presence or absence ^{of L} Cba in the cyclic peptide)
^{To confirm the role of L} Cba residues present in the peptide sequence on binding to hEGFR, three peptides, CbaP5, CbaP14, and CbaP16, were selected and ^{mutant peptides in which L} Cba was replaced with L-phenylalanine were selected. Synthesized. This substitution (mutation) in the mutant peptide reduced its binding to hEGFR. Therefore, the binding activity of peptides having ^L Cba to hEGFR is enhanced by the presence of ^{L Cba, and it is clear that L} Cba is directly involved in the binding to hEGFR or plays an important role. It became.
Table 3 shows the binding activity of CbaP5, CbaP14, and CbaP16, and their mutant peptides. In Table 3, the KD value is a value measured by SPR, and nd means that it could not be determined because the binding was weak.

(Evaluation of peptidase resistance)
The peptidase resistance of the cyclic peptide having a carborane residue was examined.
Peptidase resistance was measured by serum stability measurements using CbaP5 and CbaP16. Peptidase resistance of CbaP5 and CbaP16 was compared to peptidase resistance of CbaP5-1F and CbaP16-1F, respectively.
Serum stability measurements were performed on human serum supernatants at 37 ° C. containing 20 μM cyclic peptide. 50 μL of the reaction mixture was harvested at 0, 1, 3, 8 and 16 hours and quenched by the addition of 100 μL of MeCN containing 0.1% TFA. The resulting mixture was then centrifuged (13000 rpm, 25 ° C., 3 minutes) and filtered to give a supernatant. The recovered supernatant was then analyzed by ultra-high performance liquid chromatography UPLC in the presence of 66.67 μM 4'-hydroxyacetonitrile as standard (UPLC conditions: A: 0.1% TFA-containing H ₂ O; B :. MeCN containing 0.1% TFA; Flow velocity: 0.5 ml / min; Reverse column: ACQUITY UPLC BEH C18 1.7 μm (2.1 × 150 mm column, Waters, USA); B conc. 10% / 2 minutes 75% / 15 minutes). The relative residual rate (%) of each peptide was measured based on the peak area of the UPLC analysis at 280 nm and calculated by the following formula.
Relative survival rate (%) = [(Pt / St) / (P0 / S0)] × 100%
Here, P0 is the peak area of each peptide after incubation for 0 hours, Pt is the peak area of each peptide after incubation at each time point, S0 and St are the peak areas of the standard substance (S0 is 0 hours, St is each). Point in time).
When the degradation of CbaP5 in human serum was monitored by LC-MS, it was shown that the half-life (t1 / 2) was about 6 hours. On the other hand, CabP5-1F _{showed t 1/2} for less than 1 hour, indicating that ^L Cba contributed to serum stability and that the ^{cyclic peptide containing L} Cba was highly resistant to peptidase (see FIG. 5A). Carborane probably disrupted the three-dimensional folding structure of CbaP5 and increased peptidase resistance. Similarly, CbaP16 was more resistant to peptidase than CbaP16-1F (see FIG. 5B).

(Cell test)
^{The binding property of the cyclic peptide containing L} Cba to hEGFR expressed on living cells was evaluated.
For this evaluation, fluorescently active CbaP5, CbaP14, and CbaP16 were synthesized. Each peptide was ^{designed to have β} -alanine (β A) followed by lysine (K), then the K residue was selectively labeled with fluorescein isothiocyanate (FITC). Specifically, β-Ala-Lys (Mmt) groups were coupled to the C-terminus of the peptide for FITC labeling. β-Ala-Lys (Mmt) -containing peptides (12.5 μmol scale) were also synthesized by a combination of automated and manual approaches. After attaching the chloroacetyl group to the N-terminus, the Mmt group was removed by first incubating with 1 mL of solution (1% TFA / 5% TIS / 94% CH ₂ Cl _{2) at room temperature for 30 minutes.} The resulting resin was then equilibrated with 20% DIPEA in NMP and treated with 1 mL of solution in NMP of 0.05M FITC and 0.1M DIPEA for 3 hours at room temperature. Subsequently, the FITC-modified peptide was cleaved, deprotected and cyclized, followed by RP-HPLC purification and vacuum freeze-drying. The analysis results of MALDI-TOF of CbaP5, CbaP14, and CbaP16 labeled with FITC are shown in FIG.
After incubating each peptide with HEK293 cells that stably express hEGFR or HEK293 cells that do not express hEGFR, fluorescence images of each experiment were monitored. Specifically, cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum and high glucose supplemented with penicillin / streptomycin in _{moist air containing 5% CO 2} at 37 ° C. Stable transfection of HEK293 with a lentiviral vector (GenScript, Piscataway, NJ) generated HEK293-mock and HEK293-hEGFR expressing cells with a tetracycline regulatory system. For HEK293-hEGFR cells, stable expression of hEGFR was confirmed by quantitative real-time polymerase chain reaction and Western blotting when the cells were cultured with 500 ng / mL doxycycline.
The cells were then pre-cultured at a density of ^{16 × 10 4} cells / well on a culture cover glass in a 6-well plate for 24 hours. The cells were then gently washed with PBS and FBS-free medium was added. After 30 minutes of incubation with each concentration of FITC-labeled peptide, they were washed twice with PBS to remove FITC-labeled peptide contact medium and medium was added. FITC-labeled peptide-bonded cells were observed using a Nikon fluorescence microscope (Nikon ECLIPSE E600).
From the obtained fluorescence microscope images, it was observed that the control HEK-293 mock cells were not stained, whereas the hEGFR-expressing cells were stained with the FITC-labeled peptide. That is, it was confirmed that the fluorescently labeled cyclic peptide selectively bound to EGFR expressed on living cells.

Claims (12)

A cyclic peptide having a cyclic structure composed of 4 to 30 amino acids or a derivative thereof, or a pharmaceutically acceptable salt thereof.
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least.
It has a bond with the thiol group of Cys via the amino group of Cba, and has a structure in which the Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof. And
The amino acid sequence composed of the amino acids 2 to 28 or a derivative thereof is composed of any amino acid or a derivative thereof.
Cyclic peptide or pharmaceutically acceptable salt thereof. The amino acid sequence composed of the amino acids 2 to 28 or a derivative thereof is a sequence having an interaction with a cell membrane protein expressed in cancer cells.
The cyclic peptide according to claim 1 or a pharmaceutically acceptable salt thereof. The cell membrane protein expressed in the cancer cells is human epidermal growth factor receptor (hEGFR), human EGFR-related substance (HER2-4), epidermal growth factor receptor molecule (EpCAM), transforming growth factor receptor (TGFR), or Any protein selected from the transmembrane sugar protein NMB (GPNMB),
The cyclic peptide according to claim 2 or a pharmaceutically acceptable salt thereof. The amino acid sequence composed of the amino acids 2 to 28 or a derivative thereof comprises any sequence selected from the formulas (I), (II), and (III).
The cyclic peptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.
Equation (I): -Xaa5-Xaa6-Xaa2-Xaa7-Xaa1-Xaa7-Xaa5-Xaa2-Xaa3-Xaa4-H-Xaa2-P-,
Equation (II): -Xaa1-Xaa7-Xaa6-Xaa5-Xaa1-Xaa3-Xaa6-Xaa1-Xaa4-Xaa3-Xaa3-H-Xaa2-Xaa4-,
Equation (III): -Xaa6-Xaa1-Xaa6-HP-Xaa1-Xaa5-Xaa2-Xaa2-Xaa3-Xaa6-Xaa1-
(In the above formula,
Xaa1 is V, L, or I, or a derivative thereof.
Xaa2 is F, Y, or W, or a derivative thereof.
Xaa3 is G or A, or a derivative thereof,
Xaa4 is S or T, or a derivative thereof,
Xaa5 is K or R, or a derivative thereof,
Xaa6 is D or E, or a derivative thereof,
Xaa7 is Q or N, or a derivative thereof. ) Represented by CbaP5, CbaP14, CbaP16,
The cyclic peptide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

(In the formula, X is any amino acid or derivative thereof, and r is an integer from 0 to 10.) A method for producing a library containing two or more cyclic peptides.
The cyclic peptide
It has a cyclic structure composed of 4 to 30 amino acids or derivatives thereof, and has a cyclic structure.
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least.
It has a bond with a thiol group of Cys via an amino group of Cba, and has a structure in which Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof.
An amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is composed of any amino acid or a derivative thereof.
Cyclic peptide
Peptide represented by formula (1);
ClAc－Cba－ (Xaa) _n －Cys－ (X) _r (1)
[In equation (1),
Xaa is any amino acid or derivative thereof
X is any amino acid or derivative thereof
n is an integer of 2 to 28 and r is an integer of 0 to 10. ]
And the process of preparing the mRNA library that encodes;
A step of expressing the peptide by a cell-free translation system using the mRNA library to produce the library;
How to make a library, including. A method for producing a library containing two or more cyclic peptides.
The cyclic peptide
It has a cyclic structure composed of 4 to 30 amino acids or derivatives thereof, and has a cyclic structure.
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least.
It has a bond with a thiol group of Cys via an amino group of Cba, and has a structure in which Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof.
An amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is composed of any amino acid or a derivative thereof.
Cyclic peptide
Peptide represented by formula (1);
ClAc－Cba－ (Xaa) _n －Cys－ (X) _r (1)
[In equation (1),
Xaa is any amino acid or derivative thereof
X is any amino acid or derivative thereof
n is an integer of 2 to 28 and r is an integer of 0 to 10. ]
And the process of preparing the mRNA library that encodes;
A step of binding puromycin to the 3'end of each mRNA of the mRNA library to produce a puromycin-bound mRNA library;
A step of expressing the peptide by a cell-free translation system using the puromycin-binding mRNA library to produce a peptide-mRNA complex library;
How to make a library, including. A method for obtaining a cyclic peptide that interacts with a target protein or a pharmaceutically acceptable salt thereof.
The cyclic peptide
It has a cyclic structure composed of 4 to 30 amino acids or derivatives thereof;
Carboranylalanine (Cba) and Cys are contained in the cyclic structure at least;
It has a bond with the thiol group of Cys via the amino group of Cba, and has a structure in which Cba and Cys are linked via an amino acid sequence composed of 2 to 28 amino acids or derivatives thereof;
The amino acid sequence composed of 2 to 28 amino acids or a derivative thereof is a cyclic peptide composed of an arbitrary amino acid or a derivative thereof.
The above method
Peptide represented by formula (1);
ClAc－Cba－ (Xaa) _n －Cys－ (X) _r (1)
[In equation (1),
Xaa is any amino acid or derivative thereof
X is any amino acid or derivative thereof
n is an integer of 2 to 28 and r is an integer of 0 to 10. ]
And the process of preparing the mRNA library that encodes;
A step of binding puromycin to the 3'end of each mRNA of the mRNA library to produce a puromycin-bound mRNA library;
A step of expressing the peptide by a cell-free translation system using the puromycin-binding mRNA library to produce a peptide-mRNA complex library;
A step of contacting the peptide-mRNA complex library with the target protein to select a cyclic peptide that interacts with the target protein;
Including methods. The target protein is a protein expressed in cancer cells.
The method according to claim 8. The target protein is the human epidermal growth factor receptor (hEGFR).
The method according to claim 8 or 9. A human epidermal growth factor receptor (hEGFR) binder comprising the cyclic peptide according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof. A composition for use in boron neutron capture therapy (BNCT), which comprises the cyclic peptide according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof.

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