JP2021107327A - Method for improving pharmacokinetics of medium molecules with bioactivity, and method for manufacturing medium molecule library using improvement of pharmacokinetics - Google Patents

Abstract

To provide methods for enabling binding to an intracellular target by imparting membrane permeability to a medium molecular compound.SOLUTION: The method comprises fusing a peptide sequence binding to a protein that is present in the target cell and has the activity of drawing out medium molecules from a lipid bilayer membrane with medium molecules having a molecular weight of 500 to 5000.SELECTED DRAWING: Figure 4

Description

The present invention relates to a method for allowing a medium molecule having a molecular weight of 500 to 5000 to permeate the membrane. More specifically, the medium molecule binds to a protein existing in a target cell and having an activity of extracting the medium molecule from the lipid bilayer membrane. The present invention relates to a method for allowing the medium molecule to permeate the membrane by fusing the membrane permeation-promoting peptide sequence, and a cyclic peptide library that has acquired the membrane permeation ability by the method.

Organic compounds having a molecular weight of up to about 500 have been developed as many pharmaceuticals because of their low production cost, oral administration, and low immunotoxicity. However, this small molecule drug has low specificity, and it has become increasingly difficult to develop in recent years due to the problem of side effects due to off-targeting. On the other hand, high molecular weight drugs derived from antibodies and proteins having a molecular weight of more than 10,000 are highly specific and have few side effects, so that sales are increasing, but they are expensive, cannot be orally administered, and show immunotoxicity. There is a problem that there is a possibility.

So-called medium-molecular-weight drugs such as peptides, cyclic peptides, and natural products with a molecular weight of about 500 to 5000 have the advantages of low-molecular-weight drugs that are inexpensive and have low immunotoxicity, and that they are highly specific to target molecules and have few side effects. It is expected that it has the advantages of high molecular weight drugs and has a wide range of applications. However, when a medium-molecular-weight drug targets an intracellular molecule, a method of permeating the cell membrane to reach the desired intracellular target molecule has not been established. It is also necessary to improve stability such as protease resistance in order to show high bioavailability by oral administration.

Cyclosporin A (CsA), which is one of such medium-molecular-weight drugs, is an immunosuppressive drug that targets intracellular calcineurin, such as interleukin-2 (IL-2) by T lymphocytes and interferon-γ. It specifically and reversibly suppresses the transcription of cytokines and suppresses cytokine production and release. This action dephosphorylates the activated T cell nuclear factor (NFAT) that controls IL-2 production, and inhibits intracellular signal transduction by calcineurin, an intracellular calcium-dependent dephosphorylating enzyme that activates T cells. It is known that As shown in FIG. 1, CsA is an 11-residue cyclic peptide containing a non-proteinogenic special amino acid, and 7 of the amino groups forming a peptide bond are N-methylated. As a result, cell membrane permeability and resistance to proteases have been acquired.

Many of the peptide sequences known to have membrane permeation activity have a characteristic of being rich in basic linear residues, and this sequence should be added to a peptide having the desired physiological activity. There is a report of trying to achieve both membrane permeation activity and physiological activity (see, for example, Patent Documents 1 to 5). However, such a cell-penetrating peptide does not impart membrane-penetrating activity to any physiologically active substance, and its membrane-penetrating mechanism is not clear.

Attempts have also been made to improve the membrane permeability of cyclic peptides by imparting a specific structure to the medium molecule. For example, the invention described in Patent Document 6 improves the membrane permeability and / or metabolic stability of a cyclic peptide by introducing a cyclopropane amino acid unit having a specific structure into the ring of the cyclic peptide. Further, in the invention described in Patent Document 7, at least two amino acids of the cyclic peptide that are not adjacent to each other have a hydrophobic side chain, and in a hydrophilic environment, the hydrophobic side chains are inside the ring of the cyclic peptide. By interacting with each other, peptides that can penetrate the cell membrane and reach the inside of the cell are obtained.

WO2011 / 126010 WO2016 / 136708 WO2016 / 136707 WO2013 / 061818 WO2010 / 134537 JP-A-2017-43615 JP-A-2015-42159

Wu X, Stockdill JL, Wang P, Danishefsky SJ., Total synthesis of cyclosporine: access to N-methylated peptides via isonitrile coupling reactions. J Am Chem Soc. 2010 Mar 31; 132 (12): 4098-100.

Medium-molecular-weight compounds are expected as new drug discovery materials that inhibit intracellular protein-protein interactions, because of their strong bioactivity. However, since most of them do not exhibit membrane permeability, a method for obtaining a medium-molecular-weight compound capable of acting on a desired intracellular target has not yet been established. An object of the present invention is to impart membrane permeability to a medium molecular weight compound and enable binding to an intracellular target.

The present inventors have focused on the common feature of medium molecules exhibiting membrane permeability that they exhibit the ability to bind to peptidyl prolyl isomerase (PPI), which is one of the proteins abundantly present in cells. .. Then, the membrane-permeable fusion thus obtained by fusing a peptide sequence that binds to a protein that exists in a cell represented by PPI and has an activity of extracting a medium molecule from a lipid bilayer with a medium molecule. The present invention has been completed by finding that the transferability into cells of the body is enhanced.

In one embodiment of the present invention, the medium molecule membrane permeation method is a peptide that binds to a protein present in a target cell in a medium molecule having a molecular weight of 500 to 5000 and having an activity of extracting the medium molecule from the lipid bilayer membrane. It is characterized by a step of fusing the sequences to obtain a membrane-permeable fusion and contacting the membrane-permeable fusion with a cell.
The protein that exists in the target cell and has the activity of extracting medium molecules from the lipid bilayer is one of the proteins that are abundant in the cell and is weakly localized in the lipid bilayer, and is in the lipid bilayer. It is a presentation protein that has the activity of binding to medium molecules. This protein is preferably a protein having peptidyl prolyl isomerase activity, more preferably peptidyl prolyl isomerase A (PPIA), as long as it has an activity of extracting medium molecules from the lipid bilayer membrane. Not limited to these. Then, the peptide sequence that binds to the protein having peptidyl prolyl isomerase activity is the amino acid residue sequence represented by the following formula (I):
Leu-Leu-Val-Bmt-Abu ... (I)
(In the formula, Bmt indicates (4R) -4-[(E) -2-butenyl] -4-methyl-L-threonine; Abu indicates L-2-aminobutyric acid, forming a peptide bond. The amino group may be N-alkylated) or a homologous amino acid residue sequence thereof.

Further, in the method for producing a membrane-permeable fusion in another embodiment, a medium molecule having a molecular weight of 500 to 5000 is prepared, and the medium molecule has an activity of extracting the medium molecule from the lipid bilayer membrane existing in the target cell. It is characterized by fusing a peptide sequence that binds to a protein having.

The membrane-permeable cyclic peptide of the present invention in still another embodiment has the following formula (II):
Cyclo [(X) _n- Leu-Leu-Val-Bmt-Abu] ... (II)
(In the formula, n Xs are independent of each other, natural or non-natural amino acids or derivatives thereof, n is an integer of 5 to 50, and Bmt is (4R) -4-[(E). ) -2-Butenyl] -4-methyl-L-threonine; Abu indicates L-2-aminobutyric acid, and the amino group forming the peptide bond may be N-alkylated.)
It is characterized by consisting of an amino acid sequence represented by or a homologous amino acid residue thereof. However, the above cyclic peptide excludes cyclosporin A.

Yet another embodiment of the present invention is a method for screening a membrane-permeable cyclic peptide having a binding ability to a target molecule, in which n X-random amino acids represented by _{(X) n in the above formula (II) are used.} It is characterized by including a step of constructing a cyclic peptide library into which the above is introduced, a step of contacting the cyclic peptide library with a target molecule, and a step of selecting a membrane-permeable cyclic peptide bound to the target molecule.

According to the method of the present invention, various medium molecules can be permeated into a membrane by fusing a peptide sequence that binds to a protein existing in a target cell and having an activity of extracting a medium molecule from a lipid bilayer membrane. In addition, by using a cyclic peptide library composed of membrane-permeable fusions, which are various fusion peptides, a medium molecule having membrane permeability and binding ability to a desired intracellular target can be selected. Is possible.

FIG. 1 shows the structure of cyclosporin A (CsA). FIG. 2 shows the three-dimensional structure of a complex of calcineurin (Cn), cyclosporin A (CsA) and PPIA. FIG. 3A shows the three-dimensional structure of the Cn, FK506 and FKBP complexes. FIG. 3B shows the three-dimensional structure of the rapamycin target protein (mTOR), rapamycin (Rap) and FKBP complex. FIG. 4 is a schematic diagram showing how the medium molecule embedded in the cell membrane is pulled out by the PPI and migrates into the cell. FIG. 5A is a schematic view showing how PPIA pulls out CsA contained in the DPC micelle. FIG. 5B is the result of NMR analysis of the binding between CsA and PPIA contained in the DPC micelle. FIG. 6A shows the chemical shift change of each residue due to the binding between CsA and PPIA added in the state of being encapsulated in the DPC micelle. FIG. 6B shows the mapping onto the PPIA / CsA complex (in the absence of micelles) structure. FIG. 7 shows the results of NMR analysis of the binding between DPC micelles containing no CsA and PPIA. FIG. 8 shows the results of a liposome addition experiment for PPIA. FIG. 9 shows the results of an experiment in which CsA-containing liposomes were added to PPIA. FIG. 10 is a graph showing changes in signal intensity between CsA-bound and non-bound PPIA when CsA-containing liposomes are added to PPIA.

Next, a preferred embodiment of the present invention will be described with reference to the drawings. It should be noted that the embodiments described below do not limit the invention according to the claims, and all of the elements and combinations thereof described in the embodiments are indispensable for the means for solving the present invention. Is not always the case.

[Membrane permeation method for medium molecules]
The method according to this embodiment is a medium molecule membrane permeation method. In the present specification, the "medium molecule" is a peptide or macrolide compound having a molecular weight of about 500 to 5000, which is neither a small molecule (organic compound having a molecular weight of about 500) nor a polymer (protein having a molecular weight of 10,000 or more). , Nucleic acid and natural products or derivatives thereof. Preferably, it is a peptide having a molecular weight of about 500 to 2000, that is, a linear or cyclic peptide having about 5 to 20 amino acid residues. As the peptide, a cyclic peptide is preferable, and the details thereof will be described later. The macrolide compound is a macrocycle lactone, and is a general term for compounds having 12 or more ring members. For example, FK506 and rapamycin can be mentioned. Nucleic acids include DNA and RNA, and include, for example, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short-chain hairpin RNA (SHRNA), nucleic acid aptamers, and the like.
By using such a medium molecule as a drug, it is expected to develop a drug that is inexpensive, has low immunogenicity, and has high specificity. Then, in the medium molecule, a peptide sequence (sometimes referred to as "membrane permeation promoting peptide sequence") that binds to a protein that is abundant in the target cell and has an activity of extracting the medium molecule from the lipid bilayer membrane is used. By fusing, it is possible to realize a medium-molecular-weight drug that can permeate the cell membrane and reach the target molecule in the details.

As used herein, the term "target cell" means a cell into which a medium molecule is introduced by the method of the present embodiment, and is a eukaryotic cell, a prokaryotic cell, an animal cell, a plant cell, a fungal cell, an archaeal cell, or a eubacteria. Includes cells and the like. Cells include eukaryotic cells such as yeast cells, plant cells and animal cells. Specific examples of cells include mammalian cells. In addition, cells include any cells that are beneficial or desirable by introducing medium molecules. Such cells may include certain cells that cause disease or adverse health conditions. For example, tumor cells and immune cells, and by introducing medium molecules into these cells, cell proliferation can be suppressed or the enhancement of immune response can be suppressed. Alternatively, medium molecules can be introduced into harmful bacterial cells to inhibit their growth or kill them. In this way, therapeutic treatment is provided by the methods described herein. The type of cell is not particularly limited, and various tissues (eg, liver, kidney, pancreas, lung, spleen, heart, blood, muscle, bone, brain, stomach, small intestine, large intestine, skin, etc. It is possible to introduce a fusion in which a medium molecule and a membrane permeation-promoting peptide sequence are fused into cells in (adipose tissue, etc.). As used herein, the term "membrane" means a plasma membrane such as a cell or a lipid bilayer membrane.

In the present specification, the “protein existing in the target cell and having an activity of extracting a medium molecule from the lipid bilayer membrane” is not particularly limited, but is abundantly present in the target cell and the lipid bilayer membrane. Proteins with activity to extract medium molecules from, such as proteins with peptidyl prolyl isomerase activity, i.e. a group of enzymes or thyroid hormone receptors that catalyze cis-trans isomerization of proline residues in protein molecules. Examples include intracellular receptors. Peptide bonds between amino acids are generally much more stable (low energy state) in the trans form than in the cis form, and this state is achieved naturally. However, due to the unique structure of the proline residue (to be exact, imino acid rather than amino acid), the N-side peptide bond exists relatively stably as a cis form. These need to be determined for accurate folding of the protein. However, since the activation energy required for cis-trans isomerization of this bond is relatively high at about 20 kcal / mol, this bond is difficult to isomerize naturally, and folding requires catalyzing the isomerization of proline residues. There is. Prolyl isomerase works here and is therefore one of the chaperones. About 6000 kinds of PPIs from prokaryotes to eukaryotes have been confirmed, and they are present in a large amount in cells. For example, peptidyl prolyl isomerase A (also called PPIA or cyclophilin A) is the 22nd most abundant protein in mammalian cells and is presumed to be abundant in cells at μM level concentrations (presumably). Schwanhausser B et al., Global quantification of mammalian gene expression control., Nature. 2011 May 19; 473 (7347): 337-42). PPIs are classified into three subfamilies: the cyclophilin family, the FKBP (FK506 binding protein) family, and the parvulin family, based on their amino acid sequence homology. In addition, some intracellular receptors such as thyroid hormone receptors can be used for the same purpose because hormones having a molecular weight of more than 500 can be extracted from the cell membrane and transferred into the cytoplasm or nucleus.

Cyclophilin A is known to be involved in AIDS virus HIV-1 infection, and cyclophilin D is known to be involved in myocardial infarction and cerebral infarction. It has been reported that FKBP12 functions in the signal transduction system of cell proliferation. Cyclophilin A and FKBP12 are also direct receptors for immunosuppressants, and they are also called immunophilins. On the other hand, Pin1 of the parbrin subfamily has high specificity for the phosphorylated Ser / ThrPro sequence and functions in various aspects such as transcription, apoptosis, and regulation of amyloid β production. It is considered that all of these PPIs regulate activation / deactivation by catalyzing the cis-trans isomerization reaction in the cell to change the structure around the proline peptide bond of the substrate protein. There is. However, the catalytic mechanism of the cis-trans isomerization reaction by PPI is still unclear. Prolyl isomerases may include subunits or modules of different function, eg, modules exhibiting catalytic activity and modules exhibiting chaperones or binding activity.

Therefore, the medium molecule obtained by the method of the present embodiment is a peptide sequence (membrane permeation promoting peptide sequence) that exists in the target cell and binds to a protein (for example, PPI) having an activity of extracting the medium molecule from the lipid bilayer membrane. It is considered that the protein is extracted from the cell membrane by PPI or the like and presented to the target protein in the cell by fusing with. As used herein, "binding" includes forming a complex of two molecules in a non-covalent bond. "Fusion" means that two molecules are covalently bonded into one molecule. The fusion of the medium molecule with the membrane permeation-promoting peptide sequence may form one or more covalent bonds. Further, it is preferable that the medium molecule and the membrane permeation-promoting peptide sequence are fused by two or more covalent bonds to form a ring. In a preferred embodiment, peptidyl prolyl isomerase A (PPIA or cyclophilin A) can be used as the protein having the peptidyl prolyl isomerase activity. In a more preferred embodiment, the peptide sequence of this embodiment can use the binding site of cyclosporin A (CsA) and PPIA (cyclophilin A).

FIG. 2 schematically shows the three-dimensional structure of a complex (Cn / CsA / PPIA complex) of calcineurin (Cn), cyclosporin A (CsA), and PPIA, which is a model of the present invention. .. Cn consists of two subunits, in which CnA represents a catalytic subunit and CnB represents a calcium binding subunit. In the Cn / CsA / PPIA complex, most of the contact surfaces of the three are borne by CsA, and PPIA only presents CsA to Cn. Furthermore, the three-dimensional structure of CsA is fixed by interacting with PPIA, and the binding ability to Cn is acquired on the surface opposite to the binding site with PPIA.

As another model example, FIG. 3 shows the binding mode between FKBP and FK506 (A) or rapamycin (B). FKBP, a type of PPI, has the interesting feature of binding to macrolide compounds such as FK506 and rapamycin (Rap). Since the target protein in the cell of FK506 and Rap is different from that of Cn or mTOR, even medium molecules introduced into the cell by the same PPI are different because they have different structures other than the binding site with PPI. It shows that it can bind to the target protein.

From this, the estimation mechanism in which CsA existing in the cell membrane is extracted by PPI and transferred into the cell is schematically shown in FIG. CsA, which has high hydrophobicity, has a high affinity for the cell membrane, and is considered to be easily transferred to the membrane when absorbed in the body (arrow A in FIG. 4). However, since it has low solubility in an aqueous solution, it cannot move from the membrane into the cell as it is. At this time, PPIA existing in the cell assists membrane permeation. PPIA is weakly bound to the membrane as shown later (arrow B in FIG. 4). The PPIA bound to the membrane efficiently forms a complex with the PPI because the CsA binding surface is directed toward the membrane, and is transferred from the cell membrane into the cytoplasm (arrow C in FIG. 4). This complex is presented in the cytoplasm to the target protein calcineurin, as shown in FIG. At this time, either PPI, which is abundant in the cell, directly acts on CsA existing in the membrane to extract it, or CsA slightly distributed in the cell from the membrane is captured and transferred from the membrane to the cell. good.

The present invention is an intracellular antigen-presenting protein that concentrates and presents medium molecules, such as PPIA, which are abundant in cells and other proteins capable of extracting medium molecules in the membrane. By utilizing this, the membrane permeability of medium molecules is enhanced by a mechanism similar to that in which CsA obtains membrane permeability by PPIA. As will be demonstrated in the examples described later, since CsA is transferred from the membrane fraction to the soluble fraction by the addition of PPIA, it is surely predicted that the CsA will permeate the membrane and migrate into the cytoplasm by the method of the present invention. can.

In one embodiment of the invention, the membrane permeation-promoting peptide sequence consists of a peptide of 5 or more residues, including a natural or non-natural amino acid having no side chain charge or a derivative thereof. Amino acids having no side chain charge include natural amino acids such as glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan. Among these, hydrophobic non-polar amino acids such as glycine, alanine, valine, leucine, isoleucine, proline and phenylalanine are preferable.

For example, the amino acid residue represented by the following formula (I) (SEQ ID NO: 1):
Leu-Leu-Val-Bmt-Abu ... (I)
(In the formula, Bmt indicates (4R) -4-[(E) -2-butenyl] -4-methyl-L-threonine; Abu indicates L-2-aminobutyric acid, forming a peptide bond. The amino group may be N-alkylated). The amino acid residue represented by the above formula (I) contains five amino acid residues in which CsA is presumed to bind to PPIA: MeLeu-MeLeu-MeVal-MeBmt-Abu (SEQ ID NO: 3), but binds to PPIA. As long as it is capable, it may be substituted with homologous amino acid residues as found in other cyclosporin families. As used herein, "homologous amino acid residues" are amino acid residues that share similar characteristics or properties, such as residues that are similar in polarity, charge, size, aroma and / or hydrophobicity. To say. For example, Bmt may be MeBmt, MeLeu, Desoxy-MeBmt or methylaminooctanoic acid, and Abu may be L-Ala, L-Thr, L-Val, L-norvaline, L-Ala. , D-Ala and other optical isomers are also included. In addition, in this specification, the notation such as MeBmt and MeLeu indicates N-methylBmt and N-methylLeu.

Further, since the amino acid sequence represented by the above formula (I) is highly hydrophobic, it is considered that the medium molecule containing this sequence easily migrates to the cell membrane only by mixing with the cell. In particular, N-alkylation of the peptide backbone has been reported as a method for increasing the membrane permeability of peptides and their resistance to proteolysis (eg, Yan T, Feringa BL, Barta K, Direct N-alkylation of unprotected). amino acids with alcohols. Sci Adv. 2017 Dec 8; 3 (12): See eaao6494 etc.). Among the N-alkylations, N-methylation is preferable.

The method for fusing the medium molecule and the membrane permeation-promoting peptide sequence is not particularly limited, but as a method generally applicable to all medium molecules, the medium molecule and the membrane permeation-promoting peptide sequence prepared separately are used. There is a method of fusing by a well-known and usual chemical reaction means. For example, various homobifunctional and / or heterobifunctional cross-linking agents reagents such as bis (sulfosuccinimidyl) sverate, bis (diazobenzidine), dimethyl adipimide acid, dimethyl pimerimite, dimethyl suberimide, sverin. Disuccinimidyl acidate, glutaaldehyde, m-maleimide benzoyl-N-hydroxysuccinimide, sulfo-m-maleimide benzoyl-N-hydroxysuccinimide, sulfosuccinimidyl 4- (N-maleimidemethyl) cyclohexane-1-carboxylate, sulfosucci Nimidyl 4- (p-maleimide-phenyl) butyrate and (1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide can be used. An appropriate linker between the medium molecule and the membrane permeation promoting peptide sequence. May be inserted.

Further, in a different embodiment, a DNA encoding a medium molecule composed of a peptide and a DNA encoding a membrane permeation-promoting peptide sequence or a variant thereof are ligated in a form capable of functioning in-frame, and the fusion DNA is expressed as an expression vector. The vector can be genetically engineered to be produced as a recombinant peptide by introducing the vector into a suitable host cell and expressing it. At this time, elements such as promoters and terminators may be linked in a functional manner. When a linker composed of only genetically encoded amino acids is arranged, the above-mentioned linker peptide is placed between the DNA encoding the medium molecule and the DNA encoding the membrane permeation-promoting peptide sequence or a variant thereof. DNA encoding the above may be interposed.

More specifically, when the medium molecule is a peptide or a derivative thereof, a known peptide synthesis method can be used for fusion with the membrane permeation-promoting peptide sequence, the details of which will be described later. When the medium molecule is a macrolide compound, a known chemical synthesis method similar to the peptide synthesis method can be used. For example, a convergent synthesis method has been reported in which multiple parts called building blocks are created in advance and combined to synthesize the desired macrolide compound (A platform for the discovery of new macrolide antibiotics, Nature volume 533, pages). 338-345 (19 May 2016)). A functional group that reacts with the above-mentioned homobifunctional and / or heterobifunctional cross-linking reagent may be introduced into these building blocks.

On the other hand, as a method for covalently binding a nucleic acid and a peptide, a method using an enzyme called terminal deoxynucleotidyl transferase (hereinafter abbreviated as TdT) has also been reported (see, for example, Japanese Patent Application Laid-Open No. 2004-331574). This enzyme is an enzyme capable of incorporating triphosphate at the 3'end of DNA. This enzyme can be used to introduce a modified nucleic acid into the 3'end and covalently attach it to a peptide. For example, as a modified nucleic acid, a nucleotide in which a maleimide group or a thiol group is bound to a nucleobase via a linker can react with a thiol group present in the peptide to form a covalent bond. Further, the thiol group in the modified nucleic acid can react with a maleimide group introduced into the peptide or a compound having a disulfide bond or a methyl halide group to form a covalent bond. Furthermore, as a method of performing without a protecting group in an efficient and mild aqueous medium, a method of chemically selectively fusing a peptide and a nucleic acid (oligonucleotide) using an oxime or thiazolidine formation reaction has also been reported ( Forget D, Boturyn D, Defrancq E, Lhomme J, Dumy P., Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation. Chemistry. 2001, 7 (18): 3976-84.). For the oxime formation reaction, _{a peptide containing an oxyamine group (-ONH 2} ), a nucleic acid containing an aldehyde group (-CHO), or a reaction product vice versa is used. The thiazolidine formation reaction is a reaction for forming a thiazolidine ring using a peptide containing a cysteine residue (-CH (NH ₂ ) CH ₂ SH) and a nucleic acid containing an aldehyde group (-CHO).

In addition, there are a method of binding a peptide and a nucleic acid on a ribosome using puromycin, a method of conjugating a photosensitive cross-linking reagent to the end of the nucleic acid, and a method of binding by affinity between streptavidin and biotin. In addition, although it is not strictly a covalent fusion, peptide nucleic acids (PNAs) and ordinary nucleic acids are annealed to form non-covalent but rigid double strands, or nucleic acids are attached to the ends of proteins. It can also be attached to form a double chain.
Hereinafter, a membrane-permeable cyclic peptide will be described as a typical example of such a fusion of a medium molecule and a membrane-permeability-promoting peptide.

[Membrane-permeable cyclic peptide]
In the present specification, the “membrane-permeable cyclic peptide” refers to the following formula (II):
Cyclo [(X) _n- Leu-Leu-Val-Bmt-Abu] ... (II)
(In the formula, n Xs are independent of each other, natural or non-natural amino acids or derivatives thereof, n is an integer of 5 to 50, and Bmt is (4R) -4-[(E). ) -2-Butenyl] -4-methyl-L-threonine; Abu indicates L-2-aminobutyric acid, and the amino group forming the peptide bond may be N-alkylated.)
It consists of an amino acid sequence represented by or a homologous amino acid residue thereof. However, the membrane-permeable cyclic peptide of the present embodiment excludes cyclosporin A.

In the membrane-permeable cyclic peptide represented by the above formula (II), the _{sequence represented by (X) n} corresponds to the medium molecule in the present invention, and is represented by Leu-Leu-Val-Bmt-Abu (SEQ ID NO: 1). The sequence represented is the membrane permeation-promoting peptide sequence in the present invention. Therefore, the membrane-permeable cyclic peptide represented by the formula (II) is a peptide containing a cyclic structure consisting of 8 or more amino acids as a whole. From the viewpoint of stabilizing the cyclic structure, it is preferably composed of 9 or 10 or more amino acids. The upper limit of the number of amino acid residues as a whole is preferably 25 or less, more preferably 13 or less, from the viewpoint of stabilizing the cyclic structure. Therefore, a cyclic peptide consisting of 11 amino acid residues is most preferred. In the case of a cyclic peptide consisting of 11 amino acid residues, about 47,000 kinds (6 to the 6th power) of hydrophobic non-polar natural amino acids (Ile, Leu, Val, Ala, Gly, Phe) excluding Pro alone. If both L-form and D-form are included, there is a diversity of about 3 million species (12 to the 6th power). And, of course, the addition of special amino acids and derivatives of amino acids can provide a wider variety (infinite) variety.

The cyclic structure consists of two amino acids, a disulfide bond, a peptide bond, an alkyl bond, an alkenyl bond, an ester bond, a thioester bond, an ether bond, a thioether bond, a phosphonate ether bond, an azo bond, a CSC bond, and a CN. It is formed by binding by -C bond, C = NC bond, amide bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, amine bond, thioamide bond, etc., but the type of bond is limited to these. Not done. Cyclization of the peptide may stabilize the structure of the peptide and increase its affinity for the target.

The cyclization is not limited to the bond between the N-terminal and C-terminal amino acids of the peptide, and may be due to the bond between the terminal amino acid and the amino acid other than the terminal, or the bond between the amino acids other than the terminal. In a cyclic peptide, when one of the amino acids bound for ring formation is a terminal amino acid and the other is a non-terminal amino acid, the cyclic peptide has a structure in which a linear peptide is attached to a cyclic structure like a tail. In the present specification, such a structure may be referred to as a "lasso type".

As used herein, the term "amino acid or derivative thereof" is used in its broadest sense and includes artificial amino acid variants and derivatives in addition to natural amino acids. Amino acids may also be indicated in the conventional one-letter or three-letter notation. In the present specification, examples of amino acids or derivatives thereof include natural proteinaceous L-amino acids; unnatural amino acids; chemically synthesized compounds having characteristics known in the art that are characteristic of amino acids. Examples of unnatural amino acids are α, α-disubstituted amino acids (such as α-methylalanine), N-alkyl-α-amino acids, D-amino acids, β-amino acids, and α-, which have different main chain structures from the natural type. Hydroxy acid, amino acids with different side chain structures (norleucine, homohistidine, etc.), amino acids with extra methylene in the side chain ("homo" amino acids, homophenylalanine, homohistidine, etc.), extra in the side chain Amino acids with halogens (F, Cl, Br, I), extra amines (N) and amino groups on the side chains, amino acids with extra oxins (O) and carboxy groups on the side chains, extra sulfone on the side chains ( Examples thereof include, but are not limited to, S), amino acids having a thio group, and amino acids (such as cysteine acid) in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group.

Amino acids include proteinogenic amino acids and non-proteinogenic amino acids. As used herein, the term "proteinogenic amino acid" refers to the amino acids that make up a protein (Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Ile, Leu, Met, Phe. , Trp, Tyr, and Val).
As used herein, the term "non-proteinogenic amino acid" means a natural or non-natural amino acid other than a proteinogenic amino acid.

[Peptide production method]
The method for preparing the peptide according to the present invention is not particularly limited. The peptide or cyclic peptide according to the present invention is known to include a liquid phase method, a solid phase method, a chemical synthesis method such as a hybrid method combining a liquid phase method and a solid phase method, a gene recombination method, and translation synthesis by a cell-free translation system. It can be prepared by the method of the above or a method similar thereto.

1. 1. Synthesis by solid phase method The peptide or cyclic peptide according to the present invention can be prepared by solid phase synthesis in addition to the method shown in Scheme 1 described later, but is not limited thereto.
In the solid phase method, for example, the hydroxyl group of a resin having a hydroxyl group is subjected to an esterification reaction with the carboxy group of the first amino acid (usually the C-terminal amino acid of the target peptide) in which the α-amino group is protected by a protecting group. .. As the esterification catalyst, known dehydration condensing agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used.

Next, the protective group of the α-amino group of the first amino acid is eliminated, and the second amino acid in which all functional groups other than the carboxy group of the main chain are protected is added to activate the carboxy group. , The first and second amino acids are bound. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid in which all functional groups other than the carboxy group of the main chain are protected is added to activate the carboxy group to activate the second and second amino acids. Bind the third amino acid. This is repeated to deprotect all functional groups once the peptide of the desired length has been synthesized.

The protecting group for the α-amino group includes a benzyloxycarbonyl (Cbz or Z) group, a tert-butoxycarbonyl (Boc) group, a fluorenylmethoxycarbonyl (Fmoc) group, a benzyl group, an allyl group, and an allyloxycarbonyl (Allloc) group. ) Group etc. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine.

For protection of the α-carboxy group, methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester and the like can be used. As other functional groups of amino acids, the hydroxy group of serine or threonine can be protected with a benzyl group or tert-butyl group, and the hydroxy group of tyrosine is protected with a 2-bromobenzyloxycarbonyl dilute or tert-butyl group. The amino group of the lysine side chain and the carboxy group of glutamic acid and aspartic acid can be protected in the same manner as the α-amino group and the α-carboxy group.

Activation of the carboxy group can be carried out using a condensing agent. Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC or WSC), and (1H-benzotriazole-1-yloxy) tris. Examples thereof include (dimethylamino) phosphonium hexafluorophosphate (BOP), 1- [bis (dimethylamino) methyl] -1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), and the like. Cleavage of the peptide chain from the resin can be performed by treating with an acid such as TFA or hydrogen fluoride (HF).

2. Cyclicization method The linear peptide biologically or chemically synthesized by the above-mentioned method can be cyclized by various methods in addition to the method shown in Scheme 1 described later. For example, there is a technique for forming a disulfide bond via a cysteine residue introduced by chemical modification or genetic engineering modification (see US Pat. No. 4033940 and US Pat. No. 4,102,877). Another technique produces intracellular linear precursors, which are then added with exogenous agents such as proteases and nucleophiles to chemically cyclize these linear precursors (eg,). , Julio A. Camarero and Tom W. Muir, J. Am. Chem. Soc., 1999, 121 (23), pp 5597-5598). In yet another technique, genetic engineering modifications are used to produce cyclized peptides in cells. For example, the trans-splicing ability of the split intein is used to cyclize the precursor peptide that intervenes between the two parts of the split intein. Here, an intein (sometimes referred to as a protein intron) is a part of a protein molecule that is automatically excised and the remaining part (extein) is recombined by a peptide bond (“protein splicing”). Say something.

Alternatively, an unnatural amino acid or cross-linking agent molecule is introduced to produce a cyclic peptide through chemical modification. For example, a technique is known in which (S') -α- (2'-pentenyl) alanines are crosslinked with each other using an olefin metathesis reaction to cyclize the peptide in the molecule (see Japanese Patent Application Laid-Open No. 2008-501623). ).

_{Furthermore, a method utilizing the S N} 2 reaction of a chloroacetyl group and a thiol group of cysteine (Goto, Y., et al., Reprogramming the translation initiation for the synthesis of physiologically stable cyclic peptides. ACS Chem. Biol. 2008; 3 (2): 120-9) is more stable under intracellular reduction conditions than the disulfide bonds between cysteine residues found in natural peptides and proteins. In click chemistry utilizing the Huisgen reaction between an azide group and an alkynyl group, for example, by allowing a monovalent copper ion to act on azidohomoalanine and propargylglycine, two functional groups react to form a triazole ring. However, since these two functional groups do not react with any proteinaceous amino acid under these conditions, a cyclic structure can be selectively formed at the desired position (Sako, Y., et al.,, Ribosomal synthesis of bicyclic peptides via two functional inter-side-chain reactions. J. Am. Chem. Soc. 2008; 130 (23): 7232-4).

[Cyclic peptide library and screening method]
The present invention also provides the above-mentioned library of cyclic peptides and a screening method using the same. As used herein, the term "cyclic peptide library" refers to the following formula (II):
Cyclo [(X) _n- Leu-Leu-Val-Bmt-Abu] ... (II)
(In the formula, n Xs are independent of each other, natural or non-natural amino acids or derivatives thereof, n is an integer of 5 to 50, and Bmt is (4R) -4-[(E). ) -2-Butenyl] -4-methyl-L-threonine; Abu indicates L-2-aminobutyric acid, and the amino group forming the peptide bond may be N-alkylated.)
In the cyclic peptide represented by, an amino acid residue which is a binding site with PPI, preferably a binding site with PPIA: A random amino acid sequence is provided in a portion other than MeLeu-MeLeu-MeVal-MeBmt-Abu (SEQ ID NO: 3). An aggregate of peptides containing a plurality of cyclic peptides. As for the amino acid residues other than the binding site with PPI, all amino acids may have a random sequence, or some sequences may have a random sequence, but at least 40,000 kinds of cyclic peptide libraries are preferable. Preferably contains 2 million or more cyclic peptides.

As a method other than the method for preparing the peptide library described in Example 3, for example, there is also a method called a split mix method based on the peptide solid phase synthesis method. In this method, resin beads on which different amino acids (for example, 20 types) are immobilized are prepared and mixed together. Then, the resin beads are equally divided (for example, 20 equal parts), and 20 different kinds of amino acids are coupled to each. By repeating the operation of mixing the individually reacted resin beads together again, dividing them into equal parts, and coupling different amino acids, the different peptide sequences are finally immobilized on each resin bead. The completed peptide library is completed.

On the other hand, by using a translation system, which is a polypeptide synthesis system using ribosomes, peptides having different sequences can be synthesized simply by changing the mRNA sequence. Since it is easy to prepare mRNA having a random sequence, it is possible to easily construct a highly diverse peptide library by using a translation system.

The screening method using the cyclic peptide library includes a step of contacting and incubating the cyclic peptide library with the target molecule. In the present specification, the target molecule is not particularly limited and may be a small molecule compound, a high molecular compound, a nucleic acid, a peptide, a protein, a sugar, a lipid or the like, but is typically a protein. In particular, the cyclic peptide according to the present invention is highly stable in vivo and has excellent cell membrane permeability, and therefore it is preferable to target intracellular proteins. Since the cyclic peptide according to the present invention is also excellent in protease resistance, screening for a target molecule having protease activity can also be performed.

The target molecule can be immobilized on a solid phase carrier, for example, and contacted with a cyclic peptide library. In the present specification, the "solid phase carrier" is not particularly limited as long as it is a carrier capable of immobilizing a target molecule, and is made of glass, metal, resin or the like microtiter plate, substrate, beads, nitrocellulose membrane, nylon. Membranes, PVDF membranes and the like can be mentioned. The target molecule can be immobilized on these solid phase carriers by a known method.
The target molecule and the library are brought into contact with each other in an appropriately selected buffer solution, and the pH, temperature, time, etc. are adjusted to interact with each other.

The screening method according to the present invention then comprises the step of selecting a cyclic peptide bound to the target molecule. For binding to the target molecule, for example, the peptide is detectableally labeled by a known method, and after the above incubation, the surface of the solid phase carrier is washed with a buffer solution, and the compound bound to the target molecule is subjected to mass spectrometry or mass spectrometry. It can be done by identification using the NMR method. Detectable labels include peroxidase, an enzyme such as alkaline ^{phosphatase, 125 I, 131 I, 35} S, 3 radioactive isotopes such H, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, near Examples thereof include fluorescent substances such as infrared fluorescent materials, luminescent substances such as luciferase, luciferin and equolin, and nanoparticles such as gold colloids and quantum dots. In the case of an enzyme, a substrate of the enzyme may be added to develop color and detect it. It is also possible to bind biotin to a peptide and bind avidin or streptavidin labeled with an enzyme or the like for detection.

In the above step, not only the presence or absence or degree of binding is detected and measured, but also the enhancement or inhibition of the activity of the target molecule is measured, and the cyclic peptide having such enhancing activity or inhibitory activity is subjected to mass spectrometry or NMR method. It can also be used for identification. By such a method, a cyclic peptide having physiological activity and useful as a medicine can be obtained.

The cyclic peptide that binds to the target molecule obtained by screening the cyclic peptide library according to the present invention may be further modified and optimized by a known method or a method similar thereto.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

[Example 1] Interaction analysis between PPIA and micelle-mixed CsA Cyclosporin A (CsA; see FIG. 1 and SEQ ID NO: 2) exhibits high hydrophobicity due to its structure, and is therefore considered to easily transfer to a membrane. However, its solubility in an aqueous solution is very low, and it is originally difficult to escape from the membrane by itself and bind to the intracellular target (Cn). Therefore, the present inventors consider that the intracellularly abundant PPI extracts CsA from the membrane, thereby preventing CsA from being trapped in the cell membrane and realizing efficient intracellular translocation of CsA. rice field. Therefore, in order to verify this, first, a sample in which CsA was incorporated into a DPC (dodecylphosphocholine) micelle was prepared (Fig. 5A, upper left), ^{and the stable isotope-labeled PPIA with 15} N (Fig. 5A, upper right) was used. It was analyzed whether or not CsA binds to PPIA when added (Fig. 5A, bottom).

Figure ^{5B, 15} N stable isotope labeled PPIA (200 [mu] M) shows a ¹ H ¹⁵ N HSQC measurement results of the spectrum before and after the addition of 20mMDPC containing therein the CsA of 200 [mu] M with respect to (before addition: black, added After: Gray). ^{In the 1} H ¹⁵ N HSQC spectrum, each signal corresponds to one amino acid residue, and the position of the signal in the spectrum changes when the chemical shift changes due to a change in the surrounding chemical environment such as binding. Therefore, in this case, the residue of PPIA whose signal position has changed depending on the presence or absence of DPC containing CsA can be identified as a CsA binding site. At this time, only the labeled molecule gives a signal, so this time, only the signal of PPIA labeled with a stable isotope at ^{15 N was observed.} FIG. 6 (A) shows a chemical shift change of each residue due to the binding of CsA and PPIA added in a state of being encapsulated in a DPC micelle (those with a large change of 0.8 ppm or more are uniformly plotted at 0.8 ppm). 6 (B) shows the mapping onto the PPIA / CsA complex (in the absence of micelles) structure. Furthermore, FIG. 7 shows ¹ H ¹⁵ N HSQC spectra before and after the addition of 20 mM DPC containing no CsA to ¹⁵ N stable isotope-labeled PPIA (200 μM) (before addition: black, after addition: gray). ) Is shown.

According to FIG. 5, since the signal of PPIA bound to CsA was detected in the presence of DPC, it was clarified that PPIA can extract and bind CsA from the DPC micelle. Moreover, since the chemical shift change associated with the binding was consistent with the PPIA / CsA complex structure determined in the absence of micelles, the binding mode of CsA to PPIA is the same regardless of the presence or absence of DPC micelles. It was shown (Fig. 6 (A)). ^Furthermore, when comparing the ¹ H ¹⁵ N HSQC spectra before and after the addition of 20mM of DPC without the CsA against stable isotope-labeled PPIA (200 [mu] M), and chemical shift changes only DPC residue with ¹⁵ N Most of the residues were also involved in the binding of CsA (Fig. 7, signal surrounded by a dotted line). Therefore, it was strongly suggested that PPIA efficiently extracts membrane-permeable medium molecules such as CsA from the membrane by directing the CsA binding surface toward the membrane (center of FIG. 4, left of FIG. 7). From the center to the right).

[Example 2] Preparation of CsA-containing lipid bilayer membrane (liposome) and NMR measurement In the case of an experiment using DPC micelles, considering the molecular weights of CsA (1.2 kDa) and DPC (0.35 kDa), the inside of the DPC micelle Since the weight ratio of CsA: DMPC is 3:97 and CsA accounts for 3%, it is considered that the environment is likely to be pulled out by PPI. Therefore, we examined the extraction of CsA by PPIA on the same lipid bilayer membrane (liposome) as the biological membrane. 60 mg of DMPC (dipalmitoylphosphatidylcholine) dissolved in chloroform was dried under a nitrogen stream and then placed under vacuum for 2 hours to prepare a completely dried lipid film film, to which 1.0 mL of NMR buffer was added. Liposomes were prepared by vortexing. The prepared liposomes were further homogenized by ultrasonic treatment. The final concentration of DMPC in this liposome solution is 89 mM. CsA was incorporated into the lipid bilayer membrane by adding CsA to a final concentration of 200 μM and performing vortexing. The final CsA: DMPC molar ratio is 1: 445 and the CsA: DMPC weight ratio is 0.4: 99.6, which creates an environment closer to that of a biological membrane, and the CsA is thin, making it difficult to pull out PPIA. become. First, DMPC liposomes containing no CsA were added to PPIA (200 μM) stably isotope-labeled with ^{15 N, and the 1} H ¹⁵ N HSQC spectra were compared before and after, and chemical shift changes were observed in some residues. (Fig. 8). Residues that have undergone a chemical shift change are residues that are also involved in CsA binding, and PPIA can release a membrane-permeable medium molecule such as CsA from the membrane by directing the CsA binding surface toward the membrane in advance (center of FIG. 4). Efficient pulling was further supported. Next, when ^{CsA-containing liposomes were added to 13} C stable isotope-labeled PPIA and the signal change of PPIA was observed, it was observed that CsA was withdrawn from DMPC liposomes in a time-dependent manner (Fig.). 9 and 10). Therefore, it is considered that PPI is not only an intracellular presentation protein responsible for presenting medium molecules to a target, but also improves membrane permeability by efficiently extracting medium molecules in the membrane. ..

[Example 3] Preparation of peptide library CsA is a cyclic peptide of 11 residues, but the motif that binds to PPIA is 5 residues of MeLeu-MeLeu-MeVal-MeBmt-Abu (Fig. 1 and SEQ ID NO: 3). Therefore, while retaining this peptide sequence, a group of 11-residue cyclic peptides in which the sequences of other sites of CsA were randomly modified was created and made into a library.

The library was prepared by a synthesis scheme of Non-Patent Document 1 (Wu et al, Total Synthesis of Cyclospholine: Access to N-methylated Peptide via Isonitrile Coupling Reactions, JACS, 2010, 132, 4098-), which discusses the total synthesis method of CsA. Was partially modified. According to this document, CsA can be roughly divided into three blocks and totally synthesized (Scheme 1). In Scheme 1 below, in each block, the site where the side chain structure can be modified is shown in black, and the site which is necessary for the binding activity with PPIA and cannot be changed is shown in gray. Block A is immutable. Block B can modify the side chain of a site corresponding to 1 amino acid and block C can modify the side chain corresponding to a maximum of 5 amino acids.

[Scheme 1] Total synthesis scheme of CsA

Block A is a known compound known to be obtained by the method of Aebi et al., Which is a partial modification of the method of Evans et al. (Evans DA, Weber AE. J Am Chem Soc. 1986; 108: 6757-6761.). Aebi JD, Dhaon MK, Rich DH. J Org Chem. 1987; 52: 2881-2886).

Block B was totally synthesized according to Non-Patent Document 1 by the following scheme, and at that time, the side chain (R ₁ ) of B3 can be freely modified within a range where a side reaction does not occur. From this, even if R ₁ is limited to the side chains of Gly, Ala, Val, Leu, Ile, and Ph, which have low reactivity, the D-form and L-form can have 6 × 2 = 12 variations in total. Is possible.

[Synthesis scheme of block B]

Block C is totally synthesized in Schemes described below by non-patent document 1, this time, C1, C2, C3, C4, C6 side-chain (respectively _{R 2,} R ₃ of, R _{4, R} 5, R ₆ ) can be freely modified within the range where side reactions do not occur. From this, even if it is limited to the side chains of Gly, Ala, Val, Leu, Ile, and Ph, which have low reactivity, the D-form and L-form are combined to have (6 × 2) ⁵ = 12 ⁵ kinds of variations. It is possible. Thus the synthetic scheme shown here in conjunction with _{R 1} it is possible to create up to ¹² 6 = 2,985,984 different libraries.

[Synthesis scheme of block C]

The peptide whose membrane permeability has been enhanced by the method of the present invention, particularly the cyclic peptide obtained by screening from the cyclic peptide library of the present invention, is expected to be developed as a medium-molecular-weight drug capable of binding to a desired target molecule in cells. , Useful in the pharmaceutical industry.

Claims (13)

It is a method of permeating medium molecules into the membrane.
The medium molecule has a molecular weight of 500 to 5000 and has a molecular weight of 500 to 5000.
The method comprises a step of fusing the medium molecule with a peptide sequence that binds to a protein existing in the target cell and having an activity of extracting the medium molecule from the lipid bilayer membrane to obtain a membrane-permeable fusion. A method comprising contacting a membrane permeable fusion with a cell. The method according to claim 1, wherein the peptide sequence comprises a peptide having 5 or more residues, which comprises a natural or non-natural amino acid having no charge on the side chain or a derivative thereof. The method according to claim 1 or 2, wherein the medium molecule contains a peptide, a macrolide compound, a nucleic acid or a derivative thereof. The method according to any one of claims 1 to 3, wherein the target cell is a prokaryotic cell, an animal cell or a plant cell, and the medium molecule is introduced into these cells. The method according to any one of claims 1 to 4, wherein the protein present in the target cell and having an activity of extracting a medium molecule from a lipid bilayer membrane is a protein having a peptidyl prolyl isomerase activity. The method according to claim 5, wherein the protein having peptidyl prolyl isomerase activity is peptidyl prolyl isomerase A (cyclophilin A). The peptide sequence is the amino acid sequence represented by the following formula (I):
Leu-Leu-Val-Bmt-Abu ... (I)
(In the formula, Bmt indicates (4R) -4-[(E) -2-butenyl] -4-methyl-L-threonine; Abu indicates L-2-aminobutyric acid, forming a peptide bond. The method according to any one of claims 1 to 6, wherein the amino group may be N-alkylated) or a homologous amino acid residue thereof. A medium molecule having a molecular weight of 500 to 5000 is prepared, and the medium molecule is fused with a peptide sequence that binds to a protein existing in the target cell and having an activity of extracting the medium molecule from the lipid bilayer membrane. A method for producing a membrane-permeable fusion. The method according to claim 8, wherein the protein present in the target cell and having an activity of extracting a medium molecule from the lipid bilayer membrane is a protein having a peptidyl prolyl isomerase activity. The method according to claim 9, wherein the protein having peptidyl prolyl isomerase activity is peptidyl prolyl isomerase A (cyclophilin A). The following formula (II):
Cyclo [(X) _n- Leu-Leu-Val-Bmt-Abu] ... (II)
(In the formula, n Xs are independent of each other, natural or non-natural amino acids or derivatives thereof, n is an integer of 5 to 50, and Bmt is (4R) -4-[(E). ) -2-Butenyl] -4-methyl-L-threonine; Abu indicates L-2-aminobutyric acid, and the amino group forming the peptide bond may be N-alkylated.)
A membrane-permeable cyclic peptide consisting of the amino acid sequence represented by (excluding cyclosporin A). A cyclic peptide library containing more than 2 million types of membrane-permeable cyclic peptides according to claim 11. The step of constructing a cyclic peptide library in which a random amino acid is introduced into n Xs represented by (X) _{n according to claim 11.}
The step of contacting the cyclic peptide library with the target molecule,
A step of selecting a membrane-permeable cyclic peptide bound to the target molecule, and
A method for screening a membrane-permeable cyclic peptide having the ability to bind to a target molecule.

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