Chain molecule and pseudocyclic peptide molecule using same

Abstract

The present invention addresses the problem of providing a peptide cyclization technology that makes it possible to obtain a molecule that can be used as a cyclic peptide. The problem is solved by this chain molecule that comprises, in order of arrangement, a PNA chain (a) comprising a base sequence (a), a peptide chain capable of forming a cyclic peptide, and a PNA chain (b) comprising a base sequence (b).

Description

Chain-like molecules and pseudo-cyclic peptide molecules using the same

[0001] This invention relates to cyclic peptide molecules, etc.

[0002] Cyclic peptides are peptides in which the peptide chain is closed in a ring within the molecule, and they may have different properties from linear peptides. For example, cyclization has been reported to improve stability (improved protease resistance), enhance affinity and / or specificity to target molecules such as proteins, and confer functions such as membrane permeability (Non-Patent Literature 1). However, cyclization of peptides often requires cost and time to avoid side reactions as much as possible.

[0003] Peptide nucleic acid (PNA) is an artificial nucleic acid reported by Nielsen et al. in 1991. Due to its similar structure to DNA, PNA forms a double helix with complementary DNA via Watson-Crick base pairs. While the double helix of DNA, which contains phosphate groups, becomes unstable due to electrostatic repulsion of negative charges, PNA has an electrostatically neutral backbone, thus forming a more stable double helix with DNA. Although the double helix of PNA with DNA is stable, it also possesses sequence recognition capabilities equivalent to or better than DNA, and can recognize single-base mismatches.

[0004] K. Matsui, et al., Chem. Eur. J. 2017, 23, 3034-3041.

[0005] The object of this invention is to provide a peptide cyclization technology that can obtain molecules that can be used as cyclic peptides.

[0006] In view of the above problems, the inventors diligently conducted research and found that the above problems can be solved by using a chain-like molecule comprising a PNA chain a consisting of base sequence a, a cyclic peptide-forming peptide chain, and a PNA chain b consisting of base sequence b, arranged in this order. Based on this finding, the inventors furthered their research and completed the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. A chain-like molecule comprising a PNA chain a consisting of base sequence a, a cyclic peptide-forming peptide chain, and a PNA chain b consisting of base sequence b, arranged in this order.

[0008] Item 2. The chain-like molecule according to Item 1, wherein the cyclic peptide is a cell membrane-permeable cyclic peptide.

[0009] Item 3. The chain-like molecule according to item 1 or 2, wherein the peptide chain does not contain a protease recognition sequence.

[0010] Item 4. A chain-like molecule according to any one of items 1 to 3, wherein the number of bases in base sequence a is 3 or more and the number of bases in base sequence b is 3 or more.

[0011] Item 5. A chain-like molecule according to any one of items 1 to 4, wherein the number of amino acid residues in the peptide chain is 4 to 50.

[0012] Item 6. A chain-like molecule according to any one of items 1 to 5, wherein a polynucleotide comprising a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b, forms a double chain based on the complementary relationship between the base sequence a and the complementary sequence a' and the complementary relationship between the base sequence b and the complementary sequence b', thereby forming a cyclic structure for the peptide chain and a pseudo-cyclic peptide molecule, or (2) the PNA chain a and the PNA chain b form a double chain based on the complementary relationship between the base sequence a and the base sequence b, thereby forming a cyclic structure for the peptide chain and a pseudo-cyclic peptide.

[0013] Item 7. A combination of a chain-like molecule as described in Item 1 and a polynucleotide comprising a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b, wherein a cyclic peptide-forming peptide chain is optionally positioned between the two chains a' and b'.

[0014] Item 8. A pseudo-cyclic peptide molecule in which the peptide chain of the chain-like molecule described in any of Items 1 to 7 forms a cyclic structure.

[0015] Item 9. (1) A pseudo-cyclic peptide molecule according to Item 8, comprising a chain-like molecule according to any one of Items 1 to 6 and a polynucleotide comprising a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b, wherein a cyclic peptide-forming peptide chain is optionally positioned between the two chains a' and b', and the polynucleotide is double-stranded based on the complementary relationship between the base sequence a and the complementary sequence a' and the complementary relationship between the base sequence b and the complementary sequence b', thereby forming a cyclic structure for the peptide chain, or (2) The PNA chain a and the PNA chain b are double-stranded based on the complementary relationship between the base sequence a and the base sequence b, thereby forming a cyclic structure for the peptide chain.

[0016] Item 10. The pseudocyclic peptide molecule according to item 9, wherein it is embodiment (1) and the number of bases between the polynucleotide chain a' and the polynucleotide chain b' is 0 to 3.

[0017] Item 11. The pseudocyclic peptide molecule according to item 9 or 10, wherein both the polynucleotide chain a' and the polynucleotide chain b' are PNA chains.

[0018] Item 12. A pseudocyclic peptide molecule according to any one of items 8 to 11, which is permeable to the cell membrane.

[0019] Item 13. A method for producing a pseudo-cyclic peptide molecule, comprising forming a cyclic structure on the peptide chain of a chain-like molecule described in any of Items 1 to 6.

[0020] Item 14. (1) A method for producing a peptide chain according to any one of claims 1 to 6, comprising a chain-like molecule and a polynucleotide comprising a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b, wherein a cyclic peptide-forming peptide chain is optionally positioned between the two chains a' and b', wherein the two chains are double-stranded based on the complementary relationship between the base sequence a and the complementary sequence a' and the complementary relationship between the base sequence b and the complementary sequence b', thereby forming a cyclic structure of the peptide chain, or (2) A method for producing a peptide chain according to claim 13, wherein the PNA chain a and the PNA chain b are double-stranded based on the complementary relationship between the base sequence a and the base sequence b, thereby forming a cyclic structure of the peptide chain.

[0021] Item 15. A compound library comprising multiple compounds, each containing at least one selected from the group consisting of a chain-like molecule described in any of Items 1 to 6, a combination described in Item 7, and a pseudocyclic peptide molecule described in any of Items 8 to 12.

[0022] Item 16. A method for evaluating the activity of a pseudocyclic peptide molecule, comprising contacting a target molecule and / or cell with at least one selected from the group consisting of a chain molecule described in any of Items 1 to 6, a combination described in Item 7, and a pseudocyclic peptide molecule described in any of Items 8 to 12.

[0023] According to the present invention, a peptide cyclization technology can be provided that can obtain molecules usable as cyclic peptides. Specifically, the present invention can provide chain-like molecules used in the technology, the obtained pseudocyclic peptide molecules, a method for evaluating the activity of the pseudocyclic peptide molecules, and a compound library containing the pseudocyclic peptide molecules.

[0024] This shows the sequence design concept for Test Example 1. This shows a fluorescence microscope image (after 24 hours of incubation) showing the results of Test Example 2. This shows a fluorescence microscope image (after 3 hours of incubation) showing the results of Test Example 2. This shows a schematic diagram of a pseudo-cyclic peptide molecule obtained by forming a double helix based on the complementary relationship of antiparallel (left side in the figure) or parallel (right side in the figure) between the PNA chains on both sides of the peptide chain of the chain-like molecule. This shows a schematic diagram of the case in which multiple cyclic structures are formed on the chain-like molecule side of the present invention. This shows a schematic diagram of the case in which cyclic structures are formed on both the chain-like molecule side and the polynucleotide side of the present invention.

[0025] The upper and lower limits set forth herein may be combined in any way.

[0026] In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0027] In this specification, the bases constituting nucleic acids include not only typical bases (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.) in natural nucleic acids such as RNA and DNA, but also other bases such as hypoxanthine (I), modified bases, etc. Examples of modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, etc. These nucleic acid bases may further have one or more substituents.

[0028] 1. Linear molecule In one aspect, the present invention relates to a linear molecule (which may also be referred to as "the linear molecule of the present invention" in this specification) that includes a PNA strand a consisting of a base sequence a, a cyclic peptide-forming peptide strand, and a PNA strand b consisting of a base sequence b, and these are arranged in this order.

[0029] The PNA strand is single-stranded, and the monomers constituting the strand have a carboxy group, an amino group, and a nucleobase, and the monomers are linked by amide bonds, and are not particularly limited as long as they are polymers (artificial nucleic acids) capable of binding complementarily to other nucleic acids. The monomers may include stereoisomers and optical isomers, but these are not particularly limited. As the monomers, for example, those described in known literature (for example, Chem Biol Drug Des 2017; 89: 16-37) can be adopted. Examples of the monomers include monomers in which a group containing an amino group, a group containing a carboxy group, and a group containing a nucleobase are linked to a nitrogen atom. More specifically, as the monomer, the general formula (1):

[0030]

[0031] [In the formula: R 1 represents a hydrogen atom, an alkyl group, an amino acid residue, or a water-soluble polymer group. R 2 represents a hydrogen atom, an alkyl group, an amino acid residue, or a water-soluble polymer group. R 3 represents a hydrogen atom, an alkyl group, an amino acid residue, or a water-soluble polymer group. R 4 represents a hydrogen atom, an alkyl group, an amino acid residue, or a water-soluble polymer group. Base represents a nucleobase.]. The compounds represented thereby are exemplified.

[0032] R 1 、R 2 、R 3 、and R 4 The alkyl groups represented by include both linear or branched (preferably linear) ones. The number of carbon atoms of the alkyl group is not particularly limited, and is, for example, 1 to 4, preferably 1 to 2, more preferably 1. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, and the like.

[0033] R 1 、R 2 、R 3 、and R 4The amino acid-derived group shown is not particularly limited, and any group derived from various natural or unnatural amino acids (for example, a group consisting of an amino acid with one hydrogen atom or functional group removed) can be used without restriction. Examples include groups derived from amino acids such as lysine, glutamine, and alanine.

[0034] The water-soluble polymer is not particularly limited, and for example, polyalkylene glycols such as polyethylene glycol can be used.

[0035] The nucleic acid base indicated by Base can be any base that makes up a nucleic acid without any particular restrictions.

[0036] In this invention, R 1 , R 2 , R 3 , and R 4 It is particularly preferable that all of them are hydrogen atoms.

[0037] Base sequence a and base sequence b are arbitrary base sequences. The number of bases in each of base sequence a and base sequence b is not particularly limited, as long as PNA chain a / PNA chain b can complementarily bind to each other or to other nucleic acids. The number of bases is, for example, 3 or more, preferably 4 or more, more preferably 5 or more, and even more preferably 6 or more. The upper limit of the number of bases is not particularly limited, and is, for example, 100, 50, 30, 20, 10, or 8.

[0038] The peptide chain is cyclic peptide-forming, that is, it has an amino acid sequence capable of forming a cyclic peptide, and is not particularly limited to that extent. In this specification, a cyclic peptide is one that has or is intended to exert physiological activity such as target molecule binding or cell membrane permeability. Furthermore, for this reason, the peptide chain is not intended to be cleaved midway, and therefore preferably does not contain a protease recognition sequence.

[0039] The number of amino acid residues in the peptide chain is not particularly limited as long as it can form a cyclic peptide. The number of residues is, for example, 4 to 100, preferably 4 to 50, more preferably 4 to 30, even more preferably 4 to 20, even more preferably 5 to 12, and most preferably 5 to 8.

[0040] In the chain-like molecule of the present invention, PNA chain a, peptide chain, and PNA chain b are arranged in this order. That is, the chain-like molecule of the present invention has a structure consisting of (N-terminal side) PNA chain a - peptide chain - PNA chain b (C-terminal side).

[0041] The PNA chain a and the peptide chain, and the peptide chain and PNA chain b, may be directly linked, or other PNA chains / other peptide chains may be positioned between them. In a particularly preferred embodiment of the present invention, they are directly linked.

[0042] The number of bases in the other PNA chain and the number of amino acid residues in the other peptide chain between PNA chain a and the peptide chain, and between the peptide chain and PNA chain b, are preferably as few as possible, for example, 0 to 5, preferably 0 to 3, more preferably 0 to 2, even more preferably 0 to 1, and particularly preferably 0 (direct linkage).

[0043] Furthermore, the N-terminal side of PNA chain a and the C-terminal side of PNA chain b may or may not have other PNA chains / other peptide chains. From the viewpoint of improving water solubility, it is preferable that polar amino acid residues (e.g., 1-3, 1-2, or 1) are located at the N-terminal side of PNA chain a and / or the C-terminal side of PNA chain b. Examples of polar amino acids include basic amino acids such as arginine, lysine, and histidine; acidic amino acids such as aspartic acid and glutamic acid; and neutral amino acids such as serine, threonine, cysteine, asparagine, glutamine, and tyrosine.

[0044] The number of bases in the other PNA chain and the number of amino acid residues in the other peptide chain at the N-terminal end of PNA chain a and the C-terminal end of PNA chain b are, for example, 0 to 5, preferably 0 to 3, more preferably 1 to 2, and even more preferably 1.

[0045] The chain-like molecule of the present invention may contain only one unit comprising a PNA chain a, a cyclic peptide-forming peptide chain, and a PNA chain b arranged in this order, or it may contain multiple units (for example, two or more, two to five, two to three, or two).

[0046] The chain-like molecules of the present invention may be chemically modified.

[0047] The chain molecule of the present invention has a carboxyl group (-COOH) at the C-terminus, and a carboxylate (-COO) at the carboxylate (-COOH) － It may be any of the following: amide (-CONH2) or ester (-COOR).

[0048] Here, R in esters can be C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3－8 Cycloalkyl groups; for example, phenyl, α-naphthyl, etc. 6－12 Aryl group; for example, phenyl-C such as benzyl and phenethyl. 1－2 Alkyl groups; such as α-naphthylmethyl and α-naphthyl-C 1－2 C such as alkyl groups 7－14 Aralkyl groups and pivaloyloxymethyl groups are used.

[0049] In the chain-like molecule of the present invention, carboxyl groups (or carboxylates) other than the C-terminus may be amidated or esterified. In this case, the ester can be, for example, the C-terminus ester described above.

[0050] Furthermore, the chain-like molecule of the present invention has a protecting group (for example, a formyl group, an acetyl group, etc.) at the N-terminus amino group. 1－6 C such as Alkanoyl 1－6 Protected by an acyl group, etc., the N-terminal glutamine residue which can be cleaved and produced in vivo has been pyroglutamine-oxidized, or the substituent on the side chain of an amino acid within the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) is a suitable protecting group (e.g., formyl group, acetyl group, etc.) 1－6 C such as alkanoyl groups 1－6 This also includes those protected by acyl groups, etc.

[0051] The chain-like molecules of the present invention may have modified substances such as fluorescent substances attached to them.

[0052] The chain-like molecules of the present invention can be produced by solid-phase synthesis in accordance with or in accordance with conventional methods.

[0053] 2. Pseudocyclic peptide molecules The present invention relates, in one embodiment, to a pseudocyclic peptide molecule (which may also be referred to herein as "the pseudocyclic peptide molecule of the present invention") in which the peptide chain of the chain-like molecule of the present invention forms a cyclic structure.

[0054] In the chain-like molecule of the present invention, a peptide chain is positioned between PNA chain a and PNA chain b. Therefore, the peptide chain can be bent by complementary chain formation using PNA chain a and PNA chain b, thereby forming a cyclic structure. As shown in the examples described later, the obtained cyclic structure has the properties of a cyclic peptide and can be used as a pseudo-cyclic peptide molecule.

[0055] Specific examples of the manner in which a cyclic structure is formed include: (1) a polynucleotide (the polynucleotide of the present invention) comprising a chain-like molecule of the present invention and a polynucleotide chain a' consisting of a polynucleotide chain a' comprising a polynucleotide chain b' consisting of a polynucleotide chain b' comprising a polynucleotide chain b' comprising a polynucleotide chain b' comprising a polynucleotide chain b' consisting of

[0056] Therefore, the chain-like molecule of the present invention can be: (1) a polynucleotide comprising a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b, which forms a double helix based on the complementary relationship between the base sequence a and the complementary sequence a' and the complementary relationship between the base sequence b and the complementary sequence b', thereby forming a cyclic structure for the peptide chain and a pseudo-cyclic peptide molecule; or (2) a PNA chain a and a PNA chain b form a double helix based on the complementary relationship between the base sequence a and the base sequence b, thereby forming a cyclic structure for the peptide chain and a pseudo-cyclic peptide.

[0057] In the case of embodiment (1), the present invention relates in one embodiment to a combination of the chain molecule of the present invention and a polynucleotide comprising a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b (the combination of the present invention). In one embodiment, the combination may be a composition comprising the chain molecule of the present invention and the polynucleotide, and in another embodiment, it may be a kit in which the chain molecule of the present invention and the polynucleotide are housed in separate containers. The composition and kit can be used to produce the pseudocyclic peptide molecule of the present invention. Furthermore, the pseudocyclic peptide molecule of the present invention may or may not be formed in the composition.

[0058] An overview of embodiment (1) is shown in Figure 1.

[0059] The complementary sequence a' is complementary to base sequence a. Similarly, the complementary sequence b' is complementary to base sequence b. Complementarity indicates that the bases are complementary to each other (e.g., A and T, A and U, G and C, etc.). The complementary sequences a' / b' may contain non-complementary bases not only when they are completely complementary to the corresponding base sequences a / b (A and T, A and U, or G and C), but also insofar as they can hybridize under stringent conditions. The number of non-complementary bases in the complementary sequences a' / b' relative to base sequences a / b is, for example, 30% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 10% or less, relative to 100% of the base number of the complementary sequences a' / b', or for example, 3 or less, preferably 2 or less, and more preferably 1 or less.

[0060] In this specification, "stringent conditions" means salt concentration and / or temperature conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. For example, this may be conditions in which an amplified reaction solution containing 5 to 15 mM each of KCl, MgSO4, and / or (NH4)2SO4 is incubated at 55 to 70°C.

[0061] By making the distance between polynucleotide chain a' and polynucleotide chain b' shorter than the distance between PNA chain a and PNA chain b, the chain-like molecule of the present invention and the polynucleotide of the present invention can form a cyclic structure when double-stranded based on the complementary relationship between base sequence a and complementary sequence a' and the complementary relationship between base sequence b and complementary sequence b'. The number of bases between polynucleotide chain a' and polynucleotide chain b' is, for example, 0 to 5, preferably 0 to 4, more preferably 0 to 3, even more preferably 0 to 2, even more preferably 0 to 1, and particularly preferably 0.

[0062] Complementary relationships are usually of the antiparallel type, meaning that the two strands can form complementary base pairs in opposite directions. An example of an antiparallel type is shown below.

[0063]

[0064] Complementary relationships are of the parallel type, meaning that the two strands can form complementary base pairs in the same orientation. An example of a parallel type is shown below.

[0065]

[0066] In this specification, the complementary relationship is preferably antiparallel. This allows for stronger complementary base pair formation and the creation of a stable pseudocyclic peptide molecule.

[0067] When utilizing an antiparallel complementary relationship, the polynucleotide of the present invention is arranged from the N-terminus (or 5' terminus) in the order of a polynucleotide chain b' consisting of complementary sequence b' and a polynucleotide chain a' consisting of complementary sequence a'. When utilizing a parallel complementary relationship, the polynucleotide of the present invention is arranged from the N-terminus (or 5' terminus) in the order of a polynucleotide chain a' consisting of complementary sequence a' and a polynucleotide chain b' consisting of complementary sequence b'.

[0068] If the chain-like molecule of the present invention contains multiple units in which a PNA chain a, a cyclic peptide-forming peptide chain, and a PNA chain b are arranged in that order, then correspondingly, the polynucleotide of the present invention may also contain multiple units containing polynucleotide chains a' and b'. In this case, as shown in Figure 5, multiple cyclic structures can be formed on the chain-like molecule side of the present invention.

[0069] In the pseudocyclic peptide molecule of the present invention, the double chain formed from the chain-like molecule of the present invention and the polynucleotide of the present invention may further be bonded to other polynucleotide chains (e.g., by base pairing) to form a multi-chain (e.g., triple-chain, quadruple-chain).

[0070] The polynucleotide of the present invention may be a polynucleotide comprising a polynucleotide chain a' and a polynucleotide chain b', with a cyclic peptide-forming peptide chain positioned between the two chains. In this case, as shown in Figure 6, a cyclic structure can be formed on both the chain-like molecule side and the polynucleotide side of the present invention.

[0071] In this specification, "polynucleotide" refers to a molecule in which monomers containing nucleic acid bases are linked together in a chain, and which can form a double helix through complementary base pairing. Monomers include not only those having the backbone of natural nucleic acids, but also artificial nucleic acids.

[0072] The polynucleotides of the present invention can be PNA, but also, for example, DNA, RNA, etc. Furthermore, the invention includes not only DNA and RNA, but also those that have been subjected to known chemical modifications, as exemplified below. In order to prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residue of each nucleotide can be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. In addition, the hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may be replaced with -OR (where R represents, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at position 5 of the pyrimidine base, or by substituting the carbonyl group at position 2 with a thiocarbonyl group. Furthermore, examples include, but are not limited to, polynucleotides in which the phosphate or hydroxyl portion is modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group. The polynucleotides of the present invention also include BNA (Bridged Nucleic Acid), LNA (Locked Nucleic Acid), and the like.

[0073] The polynucleotide of the present invention is preferably PNA. This allows for stronger complementary base pair formation and the creation of a stable pseudocyclic peptide molecule.

[0074] The method for forming a double helix between the chain molecule of the present invention and the polynucleotide of the present invention is not particularly limited, and the two can be brought into contact under conditions that allow complementary base pair formation to occur. The molar ratio of the chain molecule of the present invention to the polynucleotide of the present invention (moles of the chain molecule of the present invention / moles of the polynucleotide of the present invention) is, for example, 0.5 to 2.0, preferably 0.7 to 1.5, more preferably 0.8 to 1.3, and even more preferably 0.9 to 1.1. The two are usually brought into contact in a cell culture medium, buffer solution, etc. Upon contact, double helix formation can be rapidly achieved, for example, within 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute.

[0075] An overview of embodiment (2) is shown in Figure 4. The case where the complementary relationship is antiparallel is shown on the left side of Figure 4, and the case where it is parallel is shown on the right side of Figure 4. Double-strand formation in embodiment (2) can also be carried out according to or similar to embodiment (1).

[0076] The pseudo-cyclic peptide molecule of the present invention can exhibit the properties of a cyclic peptide, as well as physiological activities such as target molecule binding and cell membrane permeability.

[0077] 3. Compound Library In one embodiment, the present invention relates to a compound library (which may be referred to herein as "the compound library of the present invention") comprising a plurality of compounds, each comprising at least one selected from the group consisting of the chain molecules of the present invention, the combinations of the present invention, and the pseudocyclic peptide molecules of the present invention.

[0078] The activity of a pseudocyclic peptide molecule can be evaluated by contacting at least one selected from the group consisting of the chain molecule of the present invention, the combination of the present invention, and the pseudocyclic peptide molecule of the present invention with a target molecule and / or cell. Therefore, by using a compound library containing this, pseudocyclic peptide molecules having the desired activity can be screened.

[0079] The total number of compound types included in the compound library of the present invention is, for example, 5 or more, preferably 10 or more, more preferably 20 or more, even more preferably 50 or more, and even more preferably 100 or more. The upper limit of this number of types is not particularly limited, but for example, 20,000, 10,000, 5,000, 2,000, 1,000, and 500.

[0080] The compound library of the present invention preferably comprises multiple chain molecules of the present invention, multiple combinations of the present invention, and / or multiple pseudocyclic peptide molecules of the present invention, more preferably multiple pseudocyclic peptide molecules of the present invention. The number of types of chain molecules, multiple combinations of the present invention, and pseudocyclic peptide molecules of the present invention contained in the compound library of the present invention is, for example, 5 or more, preferably 10 or more, more preferably 20 or more, even more preferably 50 or more, and even more preferably 100 or more. The upper limit of the number of types is not particularly limited, but for example, 20,000, 10,000, 5,000, 2,000, 1,000, and 500. The ratio of the number of types to 100% of the total number of types of compounds is, for example, 10% or more, preferably 30% or more, more preferably 50% or more, even more preferably 70% or more, even more preferably 90% or more, even more preferably 95% or more, and particularly preferably 99% or more.

[0081] In the compound library of the present invention, the listed compounds are usually housed in containers. The type of container is not particularly limited as long as it is suitable for use in a compound library, and examples include multi-well plates (e.g., 24-well plates, 96-well plates, 384-well plates, 1536-well plates, etc.) and microtubes. The manner of housing is not particularly limited, and all compounds may be housed separately without being mixed with other compounds (for example, one compound housed in one well of a multi-well plate, and one compound housed in one microtube), or multiple compounds (e.g., 2 to 100, 2 to 50, 2 to 20, 2 to 10, 2 to 5, 2 to 3) may be housed in a mixed state (for example, multiple compounds housed in one well of a multi-well plate, and multiple compounds housed in one microtube).

[0082] Furthermore, the compound library of the present invention may also be in the form of a compound array in which the listed compounds are immobilized on a solid phase. The solid phase is not particularly limited as long as it can immobilize the listed compounds. Examples of solid phases include substrates such as glass, polydimethylsiloxane, polystyrene, and gold. The shape of the substrate can be any substrate reported to date, such as a flat plate, a series of pillars, or a series of wells. Functional groups such as amino groups and carboxyl groups are introduced onto these substrates by coating the surface with a silane coupling agent or polysaccharide in the case of glass, by coating the surface with a polysaccharide or modifying the surface with plasma in the case of polydimethylsiloxane, or by modifying the surface with a thiol derivative in the case of gold. Immobilization can then be achieved by using a crosslinking agent, activating the terminal carboxyl group, introducing a formyl group or maleimide group, and then reacting the peptide terminal as a cysteine ​​residue.

[0083] In the compound library of the present invention, the amounts of each compound are, for example, 1 μg or more, 10 μg or more, 100 μg or more, and 1 mg or more. The upper limit of these amounts is not particularly limited and is, for example, 1 g, 100 mg, and 10 mg.

[0084] In the compound library of the present invention, the listed compounds may exist alone, or they may exist together with other components, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc. (for example, in a mixed state).

[0085] The compound library of the present invention can be provided as a kit, either by itself, or by combining the compound library of the present invention with other compound libraries, or, if necessary, by adding them together with instruments, reagents, etc., that can be used for compound screening.

[0086] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0087] The PNA synthesized in the example is R in the general formula (1) described above.1 , R 2 , R 3 , and R 4 It is PNA, which consists entirely of hydrogen atoms.

[0088] Example 1. Figure 1 shows the concept of designing the synthetic sequence of chain-like molecules. Based on this concept, two types of chain molecules (PP1 and PP1G) were synthesized by Fmoc solid-phase synthesis and purified by HPLC. These consisted of a peptide chain (amino acid sequence: YLALL (SEQ ID NO: 1) or YLALLG (SEQ ID NO: 2)) and PNA chains positioned on both sides of it (PNA chain a1 (nucleotide sequence a1: N-terminus - gcatgt) and PNA chain b1 (nucleotide sequence b1: N-terminus - cgtatc)), and a PNA chain (complementary chain (nucleotide sequence: gatacgacatgc (SEQ ID NO: 3)) consisting of a PNA chain b1' (nucleotide sequence b1': N-terminus - gatacg) with a complementary sequence b1' for nucleotide sequence b1, and a PNA chain a1' (nucleotide sequence a1': N-terminus - acatgc) with a complementary sequence a1. MS analysis confirmed that the target PNA had been synthesized.

[0089]

[0090] Test Example 2. Intracellular uptake test. D-MEM medium was used as the cell culture medium. HeLa cells (chamber slide, 0.8 cm) 2 To each well, 1. complementary chain only, 2. PP1 + complementary chain, and 3. PP1G + complementary chain were added. The final concentration of each complementary chain and chain molecule in the culture medium was 5 μM. After addition, the cells were incubated for 3 or 24 hours under conditions of 37°C, 90% humidity, and 5% CO2. After incubation, the cells were washed with PBS(-) and the cells in the colorless culture medium were observed under a fluorescence microscope.

[0091] Fluorescence microscope images are shown in Figure 2 (after 24 hours of incubation) and Figure 3 (after 3 hours of incubation). Peptides composed of YLALL do not exhibit membrane permeability in their linear form, but only become permeable when cyclic. It has also been reported that the curvature of the peptide chain affects membrane permeability (Non-Patent Literature 1). This study reflects this, demonstrating that the peptide cyclic structure formed by the combination of PP1 and PP1G with complementary chains is a pseudo-cyclic peptide that reflects the properties (cell membrane permeability) of cyclic peptides composed of YLALL. From these results, it was found that by forming a double chain based on a complementary relationship using a chain-like molecule with a structure similar to that synthesized in Test Example 1, a pseudo-cyclic peptide molecule possessing the properties of a cyclic peptide (cell membrane permeability, target binding, etc.) can be obtained. Furthermore, these results suggest that a pseudo-cyclic peptide molecule can also be obtained by forming a double chain (Figure 4) using a chain-like molecule with a structure similar to that synthesized in Test Example 1, based on an anti-parallel or parallel complementary relationship between the PNA chains on both sides of the peptide chain.

Claims

1. A chain-like molecule comprising a PNA chain a consisting of base sequence a, a cyclic peptide-forming peptide chain, and a PNA chain b consisting of base sequence b, arranged in this order.

2. The chain-like molecule according to claim 1, wherein the cyclic peptide is a cell membrane-permeable cyclic peptide.

3. The chain-like molecule according to claim 1, wherein the peptide chain does not contain a protease recognition sequence.

4. The chain-like molecule according to claim 1, wherein the number of bases in base sequence a is 3 or more and the number of bases in base sequence b is 3 or more.

5. The chain-like molecule according to claim 1, wherein the number of amino acid residues in the peptide chain is 4 to 50.

6. (1) A polynucleotide comprising a polynucleotide a' consisting of a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b, and a double-stranded polynucleotide formed based on the complementary relationship between the base sequence a and the complementary sequence a' and the complementary relationship between the base sequence b and the complementary sequence b', thereby forming a cyclic structure of the peptide chain and a pseudo-cyclic peptide molecule, or (2) The PNA chain a and the PNA chain b form a double-stranded polynucleotide based on the complementary relationship between the base sequence a and the base sequence b, thereby forming a cyclic structure of the peptide chain and a pseudo-cyclic peptide, according to claim 1.

7. A combination of the chain-like molecule described in claim 1 and a polynucleotide comprising a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b, wherein a cyclic peptide-forming peptide chain is optionally positioned between the two chains a' and b'.

8. A pseudo-cyclic peptide molecule in which the peptide chain of the chain-like molecule according to any one of claims 1 to 7 forms a cyclic structure.

9. (1) A chain-like molecule according to any one of claims 1 to 6, and a polynucleotide comprising a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b, wherein a cyclic peptide-forming peptide chain is optionally positioned between the two chains a' and b', and the polynucleotide is double-stranded based on the complementary relationship between the base sequence a and the complementary sequence a' and the complementary relationship between the base sequence b and the complementary sequence b', thereby forming a cyclic structure for the peptide chain, or (2) The PNA chain a and the PNA chain b are double-stranded based on the complementary relationship between the base sequence a and the base sequence b, thereby forming a cyclic structure for the peptide chain, according to claim 8.

10. The pseudocyclic peptide molecule according to claim 9, wherein it is the embodiment (1) and the number of bases between the polynucleotide chain a' and the polynucleotide chain b' is 0 to 3.

11. The pseudocyclic peptide molecule according to claim 10, wherein both the polynucleotide chain a' and the polynucleotide chain b' are PNA chains.

12. A pseudocyclic peptide molecule according to claim 8, which has cell membrane permeability.

13. A method for producing a pseudo-cyclic peptide molecule, comprising forming a cyclic structure on the peptide chain of a chain-like molecule according to any one of claims 1 to 6.

14. (1) A method for producing a peptide chain according to claim 13, comprising a chain-like molecule according to any one of claims 1 to 6 and a polynucleotide comprising a polynucleotide chain a' consisting of a complementary sequence a' to the base sequence a and a polynucleotide chain b' consisting of a complementary sequence b' to the base sequence b, wherein a cyclic peptide-forming peptide chain is optionally positioned between the two chains a' and b', wherein the two chains are double-stranded based on the complementary relationship between the base sequence a and the complementary sequence a' and the complementary relationship between the base sequence b and the complementary sequence b', thereby forming a cyclic structure of the peptide chain; or (2) A method for producing a peptide chain according to claim 13, wherein the PNA chain a and the PNA chain b are double-stranded based on the complementary relationship between the base sequence a and the base sequence b, thereby forming a cyclic structure of the peptide chain.

15. A compound library comprising a plurality of compounds, each containing at least one selected from the group consisting of a chain-like molecule according to any one of claims 1 to 6, a combination according to claim 7, and a pseudo-cyclic peptide molecule according to claim 8.

16. A method for evaluating the activity of a pseudocyclic peptide molecule, comprising contacting a target molecule and / or cell with at least one selected from the group consisting of a chain-like molecule according to any one of claims 1 to 6, a combination according to claim 7, and a pseudocyclic peptide molecule according to claim 8.

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