Novel alpha-helical amphipathic cell-permeable peptide and its applications

Abstract

The present invention relates to novel alpha-helical amphiphilic cell-penetrating peptides and their uses. The cell-penetrating peptides of the present invention have nanoparticle self-assembly properties and can penetrate eukaryotic cells at nanomolar concentrations, making them useful for delivering drugs that are difficult to penetrate into cells.

Description

[Technical Field]

[0001] This invention relates to a novel alpha-helical amphipathic cell-permeable peptide and its applications. The cell-permeable peptide of this invention has the property of nanoparticle self-assembly and can be permeated into eukaryotic cells at nanomolar concentrations, making it useful for delivering drugs and other substances that are difficult to permeate into cells. [Background technology]

[0002] Cell-penetrating peptides (CPPs) are used to deliver regulatory substances, particularly biological drugs, that are difficult to transfer into cells. In this process, the regulatory substance can either be covalently linked to the CPP or form a complex with the cell-penetrating peptide. Using CPPs, regulatory substances that would otherwise only be able to be delivered intracellularly at extremely high concentrations can be delivered at low concentrations.

[0003] Since the 1980s, when Tat peptide was reported as the first CPP, dozens of CPPs have been developed, revealing that they can be used to deliver small molecular weight regulatory substances or fluorescent substances into cells. However, there are very few examples of CPPs actually being used to deliver disease-causing agents into cells and improve therapeutic effects. This is because the cell permeability of known CPPs appears at relatively high concentrations of micromoles or more, which is a very high concentration compared to therapeutic agents that produce nanomolar effects in the body (or cells). In other words, when delivering therapeutic agents into cells at concentrations of micromoles or more, the toxicity of therapeutic agents, which usually have nanomolar binding forces, can become severe. Moreover, CPPs themselves can be toxic, so there have been many problems in commercially applying cell-permeable peptides of micromolar or higher concentrations in vivo.

[0004] The inventor has confirmed in previous studies that if a peptide containing a covalent linkage site at a specific amino acid position is produced as a dimer to maximize the alpha helix, the cell permeability is significantly improved and it can be transmitted into cells even at nanomolar concentrations (Patent Document 1). In particular, when leucine was replaced with cysteine at the i and i+7 positions on the hydrophobic surface of the amphiphilic alpha helix peptide (LK-1) composed of Leu and Lys (LK-2), a dimeric peptide (LK-3) that forms two disulfide bonds under the oxidation conditions of cysteine was produced, and it was revealed that LK-3 has the characteristic of having cell permeability even at a concentration of several nanomolars. However, LK-3 is a dimeric bundle peptide in which the monomer is composed of a total of 16 amino acids, which is not suitable for commercial use because the production process is slow and the cytotoxicity is not satisfactory.

[0005] There is a need for a CPP that still exhibits excellent cell permeability while minimizing cytotoxicity.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention aims to provide a cell-penetrating peptide that can be commercially and practically used in consideration of cell permeability, simplification of the production process, substance properties such as the solubility of the dimeric peptide in water, and cytotoxicity.

[0008] The present invention also aims to provide a platform technology that can transmit a biologically active substance, such as a therapeutic drug, into cells using the cell-penetrating peptide.

Means for Solving the Problems

[0010] <Chemical Formula 1> X1X2X3X4X5X6X7X8X9X 10 X1, X4, X5, and X8 are each independently hydrophobic amino acids, X3, X6, X7, and X 10 are each independently hydrophilic amino acids, X2 and X9 are each independently amino acids that form a bond such that each unit peptide can be linked to each other at at least one of the positions of X2 and X9 to form a dimeric peptide.

[0011] The present invention also provides a pharmaceutical composition containing the dimeric peptide and a biologically active substance.

Effects of the Invention

[0012] The alpha-helical amphiphilic peptide according to the present invention exhibits high cell permeability due to the characteristics of nanoparticle self-assembly. Along with this, the peptide of the present invention can not only effectively transmit various biologically active substances into cells, but also minimize the cytotoxicity after cell permeation, and thus can be usefully used in the fields of disease prevention or treatment.

[0013] In addition, the dimeric peptide of the present invention is useful also from an economic aspect because the formation rate from the unit is rapid and is suitable for commercial use.

Brief Description of the Drawings

[0014] [Figure 1A] It is a diagram showing the formation rate of a dimeric cell-penetrating peptide (measured by the change in the concentration of the unit) and the rate constant k value due to the change in the length of the unit. [Figure 1B] It is a diagram showing the formation rate of a dimeric cell-penetrating peptide (measured by the change in the concentration of the unit) and the rate constant k value due to the change in the length of the unit. [Figure 2A]This figure shows the formation rates of various dimeric cell-permeable peptides (measured by changes in unit concentration). In each graph, the monomer concentration (%) at 24 hours is lowest for FK10, LK10, FR10, IR10, LR10, IK10, NpgR10, LH10, ​​VK10, FH10, and NpgK10 (however, the graphs for FR10, IR10, and LR10 almost overlap after 8 hours). [Figure 2B] This figure shows the schematic structure of a dimeric peptide that exhibits the characteristics of nanoparticle self-assembly. [Figure 3A] This figure shows a schematic structure of the deformation of the unit interval in a dimer: A) 16-mer(LK-3) dimer. [Figure 3B] This figure illustrates the deformation of the unit interval in a dimer: B) 10-mer dimer. [Figure 4A] This figure shows the cell permeability of the dimeric peptides of the present invention. A) Percentage of cells that sense fluorescence (%). At a peptide concentration of approximately 15 nM, the cell percentage (%) is lowest for R9 (control group), followed by diFK10, diLK10, diIK10, diFR10, diLR10, diIR10, diNpgK10, diNpgR10, and diChaK10. [Figure 4B] This figure shows the cell permeability of the dimeric peptide of the present invention. B) Comparison of average fluorescence intensity at the same concentration (62.5 nM). B) shows the relative fluorescence intensity when the fluorescence intensity of diFH10 is set to 1. [Figure 4C] This figure shows the cell permeability of the dimeric peptide of the present invention. C) Comparison of average fluorescence intensity at the same concentration (62.5 nM). [Figure 4D] This figure shows the cell permeability of the dimeric peptide of the present invention. D) Comparison of average fluorescence intensity at the same concentration (62.5 nM). [Figure 4E] This figure shows the cell permeability of the dimeric peptide of the present invention. E) Comparison of average fluorescence intensity at the same concentration (62.5 nM). [Figure 5A] This figure shows the cytotoxicity of the dimeric peptide of the present invention (measured by cell viability). [Figure 5B]This figure shows the cytotoxicity of the dimeric peptide of the present invention (measured by cell viability). [Figure 6A] This figure shows the correlation between the cell permeability and cytotoxicity of the dimeric peptide of the present invention. [Figure 6B] This figure shows the nanoparticle self-assembly tendency of the dimer peptides of the present invention as a state change in temperature-dependent retention time using reversed-phase chromatography (at the highest temperature (Temp) in each graph, the ΔRt values ​​are lowest in the following order: AcLR10mono, diFR10, diLR10, LK03, helix A, diLK10 (almost overlapping with helix A), diNpgR10, and diNpgK10). [Figure 6C] This figure shows the correlation between the tendency of the dimeric peptide of the present invention to self-assemble nanoparticles and cytotoxicity. [Figure 7] This figure shows the synthesis of parallel or antiparallel diLK10 dimers and the cell permeability of each dimer. [Figure 8] This figure shows the results obtained by confirming the degree of intracellular transduction of cyclosporine using non-covalent bonding with cell-permeable peptides, using fluorescently labeled cyclosporine. [Figure 9] This figure shows the intracellular transfer of antibodies using a cell-permeable peptide conjugated with streptavidin and biotin. [Modes for carrying out the invention]

[0015] Alpha-helix amphipathic cell-permeable peptide

[0016] The present invention provides a cell-permeable peptide in dimer form containing a 10-mer peptide unit consisting of a total of 10 amino acids. The 10-mer unit peptide can contain hydrophobic amino acids and hydrophilic amino acids in an appropriate ratio, for example, 1:1, more specifically, 4 of each, and each unit can be linked to one or two others through bonding to form a dimer peptide.

[0017] The inventors aimed to provide an optimal cell-permeable peptide by considering factors such as the rate of dimer formation, cell permeability, cytotoxicity, and the correlation between the characteristics of nanoparticle self-assembly and cell permeability. Specifically, they confirmed that a unit peptide consisting of 10 amino acids is most suitable in terms of the rate of dimer formation, and found that when this is made as a dimer bundle, it has optimal cell permeability. In particular, the dimer peptide composed of 10 amino acids has excellent cell permeability at the nanomolar level, and while it is superior, it has lower cytotoxicity compared to conventionally known 16-amino acid dimer peptides, making it suitable for use in vivo. Furthermore, since the short 10-mer cell-permeable peptide permeates into cells even faster than existing 16-mer peptides, it also has the advantage of being able to deliver regulatory substances into cells more quickly.

[0018] The present invention provides a dimerized peptide containing a peptide represented by the following chemical formula 1 as a unit.

[0019] <Chemical formula 1> X1X2X3X4X5X6X7X8X9X 10 X1, X4, X5, and X8 are each independently hydrophobic amino acids. X3, X6, X7 and X 10 These are each hydrophilic amino acids, X2 and X9 are amino acids that independently form a bond so that each unit peptide can be linked to each other at at least one of the positions of X2 and X9 to form a dimer peptide. X1 is an N-terminus, X 10 This is the C-terminus.

[0020] The dimeric peptide may be an alpha-helical amphiphilic peptide in homodimer or heterodimer form. For example, when additional fluorescent substances, biotin, or lipids are linked, it may be used in heterodimer form.

[0021] The term "peptide" as used in this application refers to an amino acid polymer, which can include not only natural amino acids but also unnatural amino acids as constituent elements.

[0022] In one embodiment, the amino acids constituting the peptide are not limited in type as long as they exhibit amphiphilicity while maintaining an alpha-helical structure, and hydrophobic or hydrophilic amino acids known to those skilled in the art can be appropriately selected and used. Through previous research, the inventors have shown that peptides with increased alpha-helical degree have increased intracellular permeability, and the alpha-helical structure is removed within the cell, thereby reducing the toxicity of the peptide (Patent Document 1).

[0023] In one specific example, the hydrophobic amino acid may be selected from the group consisting of leucine (L), isoleucine (I), phenylalanine (F), valine (V), norvaline (norV), tryptophan (W), pentylglycine (pg), neopentylglycine (Npg), and cyclohexylalanine (Cha). Accordingly, X1, X4, X5, and X8 may each be independently selected from the group consisting of leucine (L), isoleucine (I), phenylalanine (F), valine (V), norvaline (norV), tryptophan (W), pentylglycine (pg), neopentylglycine (Npg), and cyclohexylalanine (Cha).

[0024] In further specific examples, the hydrophilic amino acid may be selected from the group consisting of lysine (K), arginine (R), homoarginine (hR), noarginine (norR), histidine (H), ornithine (O), diaminobutanoic acid (Dab), and diaminopropanoic acid (Dap). Accordingly, X3, X6, X7 and X 10Each of these may be a hydrophilic amino acid independently selected from the group consisting of lysine (K), arginine (R), homoarginine (hR), noarginine (norR), histidine (H), ornithine (O), diaminobutanoic acid (Dab), and diaminopropanoic acid (Dap).

[0025] The dimeric peptide of the present invention is formed by the bonding of unit peptides to each other, where the bonding site can be one or both of the X2 and X9 positions. Accordingly, X2 and X9 can each be independently selected from any amino acids that form a bond so that each unit peptide is linked to each other at at least one of the X2 and X9 positions to form a dimeric peptide. For example, X2 and X9 are amino acids independently selected from the group consisting of cysteine ​​(C), homocysteine ​​(Hcy), penicillamine (Pen), selenocysteine ​​(Sec, U), and leucine (L), provided that X2 and X9 are not both leucine (L).

[0026] At this time, the bonds formed between each unit peptide may include any form of bond that links the peptides together to exhibit the properties desired by the present invention, and may include, for example, covalent bonds. The covalent bond is not particularly limited as long as it is a form of covalent bond that can improve the alpha helix degree without inhibiting the function of the peptide, but may be one or more bonds selected from the group consisting of disulfide bonds, diselenide bonds, ester bonds, maleimide bonds, thioester bonds, thioether bonds, and click reaction bonds between cysteine ​​molecules. More specifically, the covalent bond may be one or more bonds selected from the group consisting of disulfide bonds between cysteine ​​(C), homocysteine ​​(Hcy), or penicillamine (Pen), diselenide bonds, ester bonds between selenocysteine ​​(See, U), maleimide bonds utilizing thiol functional groups that can participate in the reaction, thioester bonds, thioether bonds, and click reaction bonds if the non-natural amino acids have triple bonds or azide groups that can induce a click reaction.

[0027] When a disulfide bond is formed between two units of a dimeric peptide, if cysteine ​​(C) is used as one of the units, there are four atoms (CSSC) between the two unit peptide skeletons. However, if homocysteine ​​(Hcy) is used instead of cysteine ​​(C), the bond distance between the two unit peptide skeletons can be adjusted to five atoms (CCSSC or CSSCC) or six atoms (CCSSCC). As an example, a dimer formed by the oxidative linkage of one cysteine ​​and one homocysteine ​​can be used. Furthermore, selenocysteine ​​(Sec, U) or penicillamine (Pen) can be used instead of cysteine ​​to form diselenides, hybrids with cysteine ​​or penicillamine, or esters with disulfide bonds.

[0028] Each dimer peptide can be linked in a parallel direction while all the monomer peptides maintain the direction from the N-terminus to the C-terminus, or one of the two peptides can be linked from the N-terminus to the C-terminus and the other from the C-terminus to the N-terminus, that is, in an anti-parallel direction. In one specific example, each monomer peptide is linked in an anti-parallel direction to each other.

[0029] The N-terminus of the dimer peptide can be lipidated with a fatty acid. Specifically, the dimer peptide can have a C6-C 16 A fatty acid may be bound. Through such a fatty acid binding, the cell permeability can be further improved.

[0030] In one specific embodiment, the present invention provides a dimer peptide containing, as a monomer, a peptide represented by the following Chemical Formula 1.

[0031] <Chemical Formula 1> X1X2X3X4X5X6X7X8X9X 10 X1, X4, X5, or X8 is independently leucine (L), isoleucine (I), phenylalanine (F), valine (V), neopentylglycine (Npg), or cyclohexylalanine (Cha), X3, X6, X7 and X 10 are independently lysine (K), arginine (R), homoarginine (hR), norarginine (norR), histidine (H), ornithine (O), diaminobutyric acid (Dab), or diaminopropanoic acid (Dap), X2 and X9 are independently cysteine (C), homocysteine (Hcy), penicillamine (Pen), selenocysteine (Sec, U) or leucine (L), provided that X2 and X9 are not leucine (L) at the same time.

[0032] In particular, as hydrophobic amino acids, X1, X4, X5, or X8 may each be independently phenylalanine (F), isoleucine (I), leucine (I), or neopentylglycine (Npg). As hydrophilic amino acids, X3, X6, X7, and X 10 These can each be independently arginine (R) or lysine (K).

[0033] However, the unit peptide constituting the dimeric peptide is chemical formula 1, where X1, X4, X5, or X8 are independently leucine (L), cyclohexylalanine (Cha), or phenylalanine (F), X2 and X9 are cysteine ​​(C), and X3, X6, X7, and X 10 If all are arginine(R), then the invention may be excluded from the scope of this invention. That is, in one embodiment, in chemical formula 1, X2 and X9 are cysteine(C), and X3, X6, X7 and X 10 If all are arginine (R), then X1, X4, X5, or X8 are not leucine (L), cyclohexylalanine (Cha), or phenylalanine (F), respectively.

[0034] The present invention also provides the unit peptide itself as described above. In one embodiment, the unit peptide of the present invention is represented by the following chemical formula 1.

[0035] <Chemical formula 1> X1X2X3X4X5X6X7X8X9X 10 X1~X 10 This is as defined above.

[0036] In yet another embodiment, the unit peptide of the present invention is a peptide represented by chemical formula 1 or a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 8-46. The term "unit peptide" as used in this application refers to a peptide that forms the basis for producing a dimeric peptide and can be used interchangeably with "monomer peptide".

[0037] In a more specific embodiment, the dimeric peptide of the present invention may include a unit peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 8-26. In yet another example, the dimeric peptide of the present invention may include a unit peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 8-26. In yet another example, the dimeric peptide of the present invention may include a unit peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 8-26. In this case, the unit peptide is formed of 10 amino acids, which are linked at one or more positions among X2 and / or X9 via the aforementioned covalent bonds to form the dimeric peptide.

[0038] The dimerized peptide according to the present invention can ensure cell permeability even at low concentrations in the nanomolar range. Conventional cell-permeable peptides required concentrations of at least micromolar to deliver biologically active substances into cells, but the cell-permeable dimerized peptide according to the present invention can ensure the desired cell permeability even at nanomolar concentrations, and it has been confirmed that it can be usefully used to effectively deliver biologically active substances.

[0039] Thus, by using cell-permeable dimerized peptides at very low concentrations, particularly tens of nanomoles or less, biologically active substances can exert their desired effects even at much lower concentrations than conventional methods. Low concentrations of cell-permeable peptides and low concentrations of biologically active substances can be sufficient conditions to minimize cytotoxicity.

[0040] One of the most important considerations when using cell-permeable peptides is the balance between cell permeability and toxicity. Since cell-permeable peptides themselves possess cell-permeability, they can cause toxicity once they enter cells. In other words, cell permeability and toxicity can be related. Therefore, it is important to develop peptides that have high cell permeability while minimizing toxicity. In the case of the dimeric peptide according to the present invention, it exhibits cell permeability even at concentrations of a few nanomoles to several hundred nanomoles, and has the characteristic that the ratio of cell permeability to the concentration at which cytotoxicity occurs is 1 / 10 or less. If physiologically active substances can be delivered into cells at low concentrations, cytotoxicity caused by cell-permeable peptides can be minimized.

[0041] The cell-permeable dimeric peptide according to the present invention can become a unit peptide when it penetrates into the cytoplasm, which is a reducing condition, as the covalent bond between the dimers breaks. If the covalent bond is continuously maintained within the cell and the peptide exists in the dimeric peptide form, the dimeric peptide generally has excellent binding ability to DNA or RNA, so cytotoxicity may be significant. However, the peptide unit whose covalent bond has broken in the cytoplasm has a rapidly decreasing alpha helix degree and very low chemical stability, and can be easily hydrolyzed by many proteolytic enzymes within the cell.

[0042] Through this, the cell-permeable dimeric peptide according to the present invention not only exhibits excellent cell transduction ability, but also enables the acquisition of the desired effect even when using low concentrations of biologically active substances, and minimizes cytotoxicity by being degraded within cells.

[0043] In a specific embodiment, the cytotoxicity (IC) of the dimeric peptide of the present invention at 62.5 nM is described. 50 -1 The ratio of cell permeability to cellular permeability is higher than that of conventionally known peptides (e.g., LK-3 dimers), and can be, for example, 130,000 or more.

[0044] In yet another specific embodiment, the cytotoxicity (IC) of the dimer peptide of the present invention at 62.5 nM is described. 50-1 The ratio of the nanoparticle self-assembly properties (ΔΔRt) to the ) is higher than that of conventionally known peptides (e.g., LK-3 dimers), and can be, for example, 8 or higher.

[0045] Intracellular signaling compositions

[0046] The present invention provides a platform technology that can deliver biologically active substances, such as therapeutic drugs, into cells using the cell-permeable peptide.

[0047] Since the dimeric peptide according to the present invention exhibits cell permeability, it can be used to effectively utilize biologically active substances that were previously difficult to deliver into cells and therefore could not exhibit effective therapeutic effects.

[0048] Accordingly, the present invention provides a pharmaceutical composition comprising the aforementioned dimeric peptide and a biologically active substance.

[0049] The aforementioned biologically active substance is a type of cargo that binds to a cell membrane permeable domain and is transmitted into the cell, thereby regulating the biological activity or function that controls all physiological phenomena in the body. This includes, but is not limited to, DNA, RNA, siRNA, aptamers, proteins, antibodies, small molecule compounds, or cytotoxic compounds.

[0050] A substance or other carrier that modulates biological activity or function can be additionally attached to the dimeric peptide according to the present invention, in which case the dimeric peptide and the substance or other carrier that modulates biological activity or function can form a complex structure. The substance or carrier can be linked to the peptide of the present invention, for example, through non-covalent or covalent bonds. The non-covalent bond may be one or more selected from the group consisting of hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, π-π interactions, and cation-π interactions. The covalent bond may be a degradable or non-degradable bond, and the degradable bond may be a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzymatically degradable bond, and the non-degradable bond may be an amide bond or a phosphate bond, but is not limited thereto.

[0051] In the case of the cytotoxic compounds, they may be linked to the dimeric peptide by electrostatic bonds or non-covalent bonds such as host-guest bonds, and may be, but are not limited to, doxorubicin, methotrexate, paclitaxel, cisplatin, bleomycin, taxol, berberine, or urcumin. In the case of proteins or antibodies, they may include any form of drug that specifically binds to a particular intracellular target and may be introduced into the peptide in a form fused to the N-terminus or C-terminus.

[0052] In some cases, this new substance can be used to kill drug-resistant cancer cells (e.g., MCF7 or MDA-MB-231) by linking methotrexate (MTX) to a dimeric peptide. Hydrophobic physiologically active low-molecular-weight molecules (such as taxol, berberine, and curcumin) can be attached to the peptide to increase the concentration of intracellular signaling. In one specific example, when MTX was covalently bonded to the dimeric peptide according to this invention, the efficacy of MTX in MTX-resistant MDA-MB-231 cells was increased by more than 20 times. In some cases, cell-permeable peptides that allow action at concentrations up to 40 times lower can also be provided.

[0053] Furthermore, antibodies, proteins that are part of antibodies, and protein drugs can be linked to dimeric peptides and delivered into cells, and oligonucleotide drugs (siRNA, asDNA, DNA, aptamer) can be linked to dimeric peptides and delivered into cells. Alternatively, cell-permeable peptides can be linked to biologically active substances (e.g., antibodies) via non-covalent bonds such as biotin-streptavidin and delivered into cells.

[0054] The compositions according to the present invention may be manufactured by further comprising one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers must be compatible with the active ingredients of the present invention and can be used by mixing saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Diluents, dispersants, surfactants, binders, and lubricants may be added to further formulate the compositions into injectable dosage forms such as aqueous solutions, suspensions, and emulsions. In particular, it is preferable to provide the compositions in lyophilized dosage forms. Methods commonly known in the art to which the present invention belongs may be used to manufacture the lyophilized dosage forms, and stabilizers for lyophilization may be added. Furthermore, the compositions may be preferably formulated according to each disease or component by appropriate methods in the art.

[0055] The present invention will be described in more detail below through examples. It will be obvious to those ordinary in the art that these examples are merely illustrative and should not be construed as limiting the scope of the present invention. [Examples]

[0056] Example 1. Comparison of unit and dimer peptide synthesis and dimer formation rates.

[0057] Each unit peptide shown in Table 1 was prepared, and dimers were formed by oxidation in air.

[0058] [Table 1]

[0059] First, unit peptides were synthesized using solid-phase peptide synthesis. Specifically, they were synthesized using a peptide microwave synthesizer (CEM) with standard fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide. Link amide MBHA resin (0.59 mmol / g loading, 50 mg, 29.5 μmol) was used in Discover SPS. The resin was deprotected with 20% piperidine in DMF. Coupling reactions were carried out using amino acids in each sequence, PyBOP, and N-diisopropylethylamine (DIPEA). Fluorescent labeling was performed by reacting 5-TAMRA with the N-terminus of the synthesized peptide in the presence of HOBt, HCTU, and DIPEA. The synthesized peptides were separated from the resin at room temperature for 2 hours using a cleavage cocktail (using 940 μL of trifluoroacetic acid, 25 μL of 1,2-ethanedithiol, 25 μL of water, and 10 μL of triisopropylsilane). The separated peptides were precipitated with n-hexane and diethyl ether (v / v=1 / 1) and purified by reverse-phase chromatography-HPLC. Purification was performed using an HPLC system (Agilent HPLC 1100 series) equipped with a Zorbax C18 column (3.5 μm, 4.6 × 150 mm).

[0060] HPLC conditions: Buffer A (water containing 0.1% v / v TFA) and Buffer B (acetonitrile containing 0.1% v / v TFA), flow rate 1 mL / min; 5 minutes, followed by a linear gradient of 70% B over 25 minutes, reaching 100% B over 10 minutes, and then maintained at this level for 10 minutes.

[0061] The manufactured units were oxidized under air oxidation conditions (air oxidation in a 0.1 M NH4HCO3 aqueous solution) to produce dimers.

[0062] At this time, it was assumed that the rate of dimer formation was the same as the rate at which the unit was consumed, and the rate of dimer formation was expressed as the reciprocal of the rate at which the unit was consumed. Each experiment was carried out under the same unit concentration conditions (air oxidation with a unit concentration of 1 mM in a 0.1 M NH4HCO3 aqueous solution). The results are shown in Figures 1A and 1B.

[0063] Surprisingly, the fastest dimerization occurred from a unit peptide consisting of 10 amino acids (LK10), with over 90% of the reaction completed within 8 hours and nearly 100% completed within 24 hours. In contrast, dimerization from unit peptides consisting of 16 or 18 amino acids (LK16 or LK18) required several days for 100% reaction completion. With 14 units (LK14), the reaction proceeded relatively quickly but was slower than with LK10. We also observed that the reaction proceeded more slowly than with LK10 with the shorter 8-unit unit (LK8). With 12 amino acid units (LK12), the reaction rate was the fastest, but crystals formed quickly, and we observed that the units produced a variety of oligopeptides (trimers, tetramers, etc.) rather than dimers. In the case of LK10, the dimerization rate was faster compared to the 16-unit peptide (LK-2) that is already known.

[0064] When comparing the first-order reaction rate constants k, it was confirmed that LK10 showed the highest value (Figure 1B).

[0065] From a commercial standpoint, it is crucial that CPPs can rapidly form amphiphilic dimers. Therefore, a peptide composed of 10 amino acids was found to be the optimal length in terms of dimer production rate (formation rate).

[0066] Example 2. Comparison of 10-mer unit and dimer peptide synthesis and dimer formation rates.

[0067] Each unit peptide in Table 2 was prepared using the same method as in Example 1, and dimers were formed under oxidation conditions.

[0068] [Table 2]

[0069] The dimer formation rates of unit peptides number 8-17 were measured and compared with those of LK10, and the results are shown in Figure 2A. Dimers were rapidly formed from the 10-mer units, and in particular, FK10, LK10, FR10, IR10, LR10, and IK10 showed dimer formation rates almost as fast as LK10, with the reaction almost 100% completed within 24 hours, and NpgR10 also showed a particularly fast rate (Figure 2A).

[0070] Based on the above, we confirmed that when the unit is composed of 10 amino acids, and particularly when Leu, Ile, Val, Phe, or Npg are located on the hydrophobic surface and Lys, Arg, or His are located on the hydrophilic surface, the formation of the dimeric peptide proceeds rapidly. Peptides satisfying this condition are shown in Helical Wheel image in Figure 2B.

[0071] Example 3. Confirmation of changes in dimer formation ability due to changes in the amino acid sequence of the unit body.

[0072] The peptide of the present invention is an alpha-helical amphiphilic peptide in which the hydrophilic and hydrophobic residue regions of the peptide are separated when it forms an alpha helix. To confirm the effect of this amphiphilicity on dimer formation ability, the amino acid sequence of the unit was partially modified to reduce the amphiphilicity and then checked whether a dimer of this unit was formed.

[0073] For this purpose, LR10 (Ac-LCRLLRRLCR-NH2) is a unit in which amphiphilicity is reduced by changing the positions of leucine at the 8th position, which is the hydrophobic surface, and arginine at the 10th position, which is the hydrophilic surface. 8,10exch (Ac-LCRLLRRRCL-NH2) was prepared, and its dimerization ability was confirmed under the same oxidation conditions. As a result, LR10 8,10exchNo dimers were formed; instead, a kink was mainly formed within the unit via a disulfide bond between the 2nd and 9th cysteine ​​molecules. These results suggest that the amphiphilicity of the peptide's alpha-helical structure is a major factor in dimerization.

[0074] Additionally, we prepared RL10 (Ac-RCLRRLLRCL-NH2), which maintains the amphiphilicity of the peptide but changes its orientation, and similarly confirmed its dimerization ability. RL10 under oxidative conditions is LR10 8,10exch Similarly, a kink was partially formed, along with the formation of an insoluble white precipitate. This indicates that not only the amphiphilicity of the peptide but also its directionality significantly influences its dimerization ability.

[0075] From the above, it can be seen that in order for the alpha-helic amphiphilic peptide units of the present invention to efficiently form dimers, the amphiphilicity and directionality of the units must be maintained.

[0076] Example 4. Production of units and dimer peptides with altered inter-dimer bundle peptide spacings.

[0077] Two disulfide bonds connect unit peptides to form a dimer. By replacing the cysteine ​​(Cys) that forms the disulfide bond with homocysteine ​​(Hcy), which has one more carbon chain length, unit peptides are created, and then using these to produce dimer peptides, a new dimer with an increased distance between the two units can be obtained. For example, in the case of LK-3+1, LK-2 (SEQ ID NO:1) and LK-2 C5Hcy / C12Hcy (SEQ ID NO:48) can be obtained by mixing it in a 1:1 mole ratio and separating the heteromorphic dimer obtained under oxidative conditions in air.

[0078] The peptides shown in Table 2 were prepared using the same method as in Example 1.

[0079] [Table 3]

[0080] To aid understanding, Figures 3A and 3B illustrate the process of adjusting the spacing between unit molecules. Depending on the degree of homocysteine ​​(Hcy) use, the distance between unit peptide molecules was divided into four atoms (CSSC; using only Cys), five atoms (CCSSC or CSSCC), or six atoms (CCSSCC).

[0081] Example 5. Confirmation of the cell permeability of the dimeric peptide.

[0082] During the production of CPP in dimer form as shown in Tables 1-3, one unit was produced with 5-TAMRA fluorescence attached to the N-terminus instead of Acetyl. Acetyl units and 5-TAMRA fluorescent units were mixed in a 1:1 mole ratio and produced in dimer form under air oxidation conditions to create dimers with only one fluorescence label per molecule, and then their cell permeability was confirmed. Using the fluorescently labeled peptide, human-derived HeLa cells (5 × 10⁻¹⁶) were tested. 4 The cells (per well) were processed, and the ratio of fluorescent cells to the total number of cells was obtained by flow cytometry.

[0083] The results of examining the cell permeability at different concentrations of each dimeric peptide are shown in Figures 4A-E.

[0084] The dimeric peptide according to the present invention showed excellent cell permeability at the tens of nanomolar level (Figure 4A). Figures 4B to 4D show the results of treating cells with each dimer at the same concentration (62.5 nM), measuring the fluorescence intensity of the cells with a flow cytometer, and comparing the relative fluorescence intensities.

[0085] Figure 4E shows the results of confirming the cell permeability of dimerized peptides due to changes in the spacing between dimers. In the case of diLR10, the length between units was increased by one carbon (i.e., the length of 5 elements). C9HcyThe best results were obtained with diLK10. A similar result was observed with diL*R10 (i.e., diNpgR10), and it was found that both cases with 4 and 5 atoms between the units performed equally well. From this, it was confirmed that the cell permeability was best when there were 5 atoms between the two units forming the dimer.

[0086] Example 6. Confirmation of cytotoxicity of dimeric peptides

[0087] Cytotoxicity in HeLa cells was measured in 96 wells (1 × 10⁻¹⁶ wells). 4 The cells were treated in a 24-hour cell / well and confirmed using the WST-1 assay method.

[0088] Specifically, HeLa cells were cultured in DMEM at 37°C under 5% CO2 conditions. After culturing the cells individually in cell culture plates, they were separated with Trypsin and then transferred to a 96-well plate at a rate of 1 x 10⁶ cells per well. 4 Cells were seeded. After 24 hours, peptides were added to the media and cultured again at 37°C and 5% CO2 for 24 hours. 10 μL of WST-1 reagent was added to each well and reacted at 37°C and 5% CO2 for 30 minutes, after which UV absorbance was measured at 450 / 700 nm using a 96-well plate reader.

[0089] The results are shown in Table 4 and Figures 5A and 5B.

[0090] Compared to the existing 16-mer CPP, LK-3, the dimeric peptide formed from the 10-mer CPP was found to exhibit significantly lower cytotoxicity even at high concentrations. 50 Even when comparing the values, the 10-mer CPP IC 50The IC50 ratio increased by more than double compared to LK-4, confirming a corresponding decrease in toxicity (see Ratio in Table 4). Furthermore, in the case of diLH10, ​​no toxicity was observed even at 40 μM (data not shown). In particular, the cytotoxicity of diLR10 was confirmed to decrease at the highest value, and when compared to the existing 16-mer dimer LK-3, the IC50 ratio was approximately 5 times lower. 50 An increase in the value was observed, and it was confirmed that the value was approximately 20 μM.

[0091] [Table 4]

[0092] We examined the cytotoxicity based on the spacing between dimers, and found that cytotoxicity was lowest when the length was 5 atoms (CCSSC) (Figure 5B).

[0093] Example 7. Correlation between cytotoxicity of dimeric peptides and cell permeability / nanoparticle self-assembly properties.

[0094] 7.1 Correlation between cytotoxicity and cell permeability of dimeric peptides

[0095] The correlation between the cell permeability and cytotoxicity of the dimeric peptides of the present invention was confirmed based on these two criteria. The cytotoxicity (IC) of each peptide was determined at 62.5 nM using the methods described in Examples 5 and 6. 50 -1 After confirming the cell permeability, this was shown in a graph.

[0096] The results are shown in Figure 6A.

[0097] Compared to LK-3, a conventional 16-mer CPP dimeric peptide, the dimeric peptides diNpgK10, diLR10, diFR10, diIR10, and diNpgR10 of the present invention exhibited lower toxicity relative to their cell permeability. In other words, the cytotoxicity (IC) compared to LK-3 was lower. 50 -1 ) as the ratio of cell permeability (i.e., cell permeability / cytotoxic IC) 50 -1The value was approximately 130,000, but all of the dimeric peptides of the present invention showed higher values. This indicates that the dimeric peptides of the present invention are more suitable for use as cell permeable peptides than conventional LK-3 because they have an appropriate balance between cell permeability and cytotoxicity.

[0098] 7.2 Correlation between the cytotoxicity of dimeric peptides and the self-assembly properties of nanoparticles

[0099] First, to confirm whether the dimeric peptide of the present invention exhibits self-association properties through peptide-peptide interactions, we conducted experiments that could quantify the self-association properties (Ref. JBC (2003) 278, 22918). This involved observing the retention time (Rt) on a C18 reverse-phase HPLC column under varying temperatures to understand the tendency towards self-association. The results are shown in Figure 6B. Subsequently, through separate experiments, we confirmed that stronger self-association properties were associated with stronger cell permeability (results not shown).

[0100] Furthermore, the correlation between the tendency for self-assembly of each dimeric peptide and its cytotoxicity was confirmed. The cytotoxicity (IC) of each peptide was determined at 62.5 nM using the method described in Example 6. 50 -1 After confirming the cytotoxicity at 62.5 nM (IC) 50 -1 The ratio of the properties of nanoparticle self-assembly (ΔΔRt) to (i.e., the properties of nanoparticle self-assembly (ΔΔRt) / cytotoxicity IC) 50 -1 ) was confirmed. The results are shown in Figure 6C.

[0101] We were able to confirm that the tendency for self-assembly of each dimer peptide correlates with cytotoxicity. In particular, compared to LK-3, a conventional 16-mer CPP dimer peptide, the dimer peptides diFR10, di-LR10, diLK10, diNpgR10, and diNpgK10 of the present invention were found to exhibit less cytotoxicity relative to their self-assembly tendency. That is, the cytotoxicity (IC) compared to LK-3 was found to be less.50 -1 The ratio of nanoparticle self-assembly properties to ) was approximately 8, but all of the dimeric peptides of the present invention showed higher values.

[0102] The results in Figures 6A and 6C suggest that the cell permeability of the dimeric peptide of the present invention is determined by the nanoparticle self-assembly properties, and in particular, that the dimeric peptide of the present invention is optimized for use in vivo due to its relatively low cytotoxicity.

[0103] Example 8. Production of parallel or antiparallel dimeric peptides and confirmation of their cell permeability.

[0104] As shown in Table 5, parallel or antiparallel dimers were produced using cysteine ​​amino acids containing different protecting groups.

[0105] [Table 5]

[0106] The results of confirming the cell permeability of the dimer in question are shown in Figure 7.

[0107] The cell permeability of air-oxidized dimer was similar to that of antiparallel dimer, but showed a completely different trend from that of parallel dimer. This indicates that diLK10, which is naturally produced under air-oxidation conditions, possesses antiparallel alignment.

[0108] Example 9. Confirmation of drug delivery effect of methotrexate-conjugated peptide (drug delivery by covalent bond)

[0109] To confirm the drug delivery effect of the peptide, a peptide in which methotrexate (MTX) was bound to the N-terminus of one of the dimer units was synthesized using a previously known method (Kim et al. Biomacromolecules 2016).

[0110] Table 6 shows the IC of MTX in three cell lines. 50 The values ​​are shown, but the IC is high in MDA-MB-231 cells. 50 The value indicates that MTX is resistant to these cells.

[0111] [Table 6]

[0112] Accordingly, the cytotoxicity of MDA-MB-231 cells resistant to MTX was confirmed using the WST-1 assay method described in Example 5. MDA-MB-231 cells were placed in a 96-well plate in a 2.5 × 10⁶ solution. 3 Cells were coated at a density of cells / well, and treated with MTX-conjugated peptides for 48 hours starting the following day. The results are shown in Table 7.

[0113] [Table 7]

[0114] As shown in Table 7, the IC of MTX-bound peptides 50 The level was at the tens of nanomolar level. The peptide itself IC 50 Considering that the levels are at the micromolar level, we confirmed that the peptide according to the present invention can efficiently deliver MTX into cells with low toxicity at the nanomolar level.

[0115] Example 10. Confirmation of drug delivery effect of cyclosporine and peptide complex (drug delivery by non-covalent linkage)

[0116] We attempted to confirm that complexes formed by simply mixing peptides and drugs have a drug delivery effect.

[0117] A hydrophobic drug, cyclosporine (CsA), and a peptide were mixed and incubated at room temperature for 20 minutes to form a complex with the hydrophobic drug. Then, Jurkat cells (1.0 × 10⁻¹⁰) were used. 4 Cells (per well) were treated for 4.5 hours. The amount of IL-2 secreted extracellularly was measured by ELISA to determine whether the anti-inflammatory effect of CsA was enhanced by the cell-permeable peptide diLR10 by the relative decrease in IL-2. 50 I confirmed it through the values.

[0118] As a result, as shown in Table 8, we confirmed that using diLR10 resulted in a 21% reduction.

[0119] [Table 8]

[0120] For peptides with diverse lysine and arginine compositions, we confirmed intracellular transduction via non-covalent bonding with fluorescent CsA using CsA. As a result, as shown in Figure 8, diLR10 R3K It was confirmed that CsA has the most superior intracellular signal transduction capacity.

[0121] Example 11. Confirmation of drug (siRNA and antibody) delivery effect using streptavidin (drug delivery by analogous covalent linkage)

[0122] In addition to the non-covalent intracellular delivery of hydrophobic drugs via dimeric cell-permeable peptides, we also investigated whether drug delivery is possible via analogous covalent bonds. Specifically, we investigated whether a bond with a similar strength to a covalent bond (Kd=10) is possible. -15 We investigated the intracellular signaling method using streptavidin and biotin (which possess M) and confirmed whether poorly permeable drugs such as siRNA and antibodies could be transmitted using a biotin-bound cell-permeable peptide.

[0123] The results are shown in Figure 9. When a cell-permeable peptide conjugated with 25 nM streptavidin and 50 nM biotin was used, it was confirmed that antibodies in concentrations of 5–100 μg / mL (30–600 nM) were delivered into the cells (Figure 9: Flow cytometry analysis results).

[0124] Next, we used an antibody against BCl2 to confirm whether the intracellularly transmitted antibody could successfully label the intracellular target. AF488-conjugated BCl2 antibody was transmitted using EZ-Link™ Sulfo-NHS-LC-LC-Biotin after biotin binding, and mitochondria were labeled using mitotracker red. The results showed that many regions of green fluorescence from AF488 were located at the same positions as red fluorescence from mitotracker (results not shown).

Claims

1. A dimeric peptide containing the peptide represented by the chemical formula 1 below as its unit. <Chemical formula 1> X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １ , X ４ , X ５ and X ８ Each of these is independently a hydrophobic amino acid selected from the group consisting of leucine (L), isoleucine (I), phenylalanine (F), valine (V), norvaline (norV), tryptophan (W), pentylglycine (pg), neopentylglycine (Npg), and cyclohexylalanine (Cha). X ３ , X ６ , X ７ and X １０ Each of these is independently a hydrophilic amino acid selected from the group consisting of lysine (K), arginine (R), homoarginine (hR), noarginine (norR), histidine (H), ornithine (O), diaminobutanoic acid (Dab), and diaminopropanoic acid (Dap). X ２ and X ９ Each of these is an amino acid independently selected from the group consisting of cysteine ​​(C) and homocysteine ​​(Hcy), and each unit peptide is X ２ and X ９ These are amino acids that form a covalent bond so that they can be linked to each other at both positions to form a dimeric peptide. X １ It is the N-terminus, X １０ It is the C-terminus, Each unit peptide is linked to the others in an anti-parallel direction.

2. The dimerized peptide according to claim 1, wherein the covalent bond is cysteine ​​(C), homocysteine ​​(Hcy), or a disulfide bond between cysteine ​​(C) and homocysteine ​​(Hcy).

3. X １ C at position ６ ~C １２ The dimeric peptide according to claim 1, wherein a fatty acid is bonded to it.

4. X １ , X ４ , X ５ , or X ８ These are, independently, leucine (L), isoleucine (I), phenylalanine (F), valine (V), neopentylglycine (Npg), or cyclohexylalanine (Cha), X ３ , X ６ , X ７ and X １０ These are independently lysine (K), arginine (R), homoarginine (hR), noarginine (norR), histidine (H), ornithine (O), diaminobutanoic acid (Dab), or diaminopropanoic acid (Dap), X ２ and X ９ The dimeric peptide according to claim 1, wherein each is independently cysteine ​​(C).

5. The dimer peptide according to claim 1, wherein the dimer peptide is an alpha-helical amphiphilic peptide in homodimer or heterodimer form.

6. The dimeric peptide according to claim 1, which exhibits cell permeability at nanomolar concentration.

7. Cytotoxicity (IC) of the aforementioned dimer peptide at 62.5 nM ５０ －１ The dimeric peptide according to claim 1, wherein the ratio of cell permeability to ) is 130,000 or more.

8. Cytotoxicity (IC) of the aforementioned dimer peptide at 62.5 nM ５０ －１ The dimeric peptide according to claim 1, wherein the ratio of the nanoparticle self-assembly property (ΔΔRt) to ) is 8 or more.

9. The dimer peptide according to claim 1, wherein the peptide represented by chemical formula 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 8 to 46.

10. A peptide represented by the chemical formula 1 below. <Chemical formula 1> X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １ ~X １０ These are as defined independently in claim 1 or claim 4.

11. A pharmaceutical composition comprising a dimeric peptide and a biologically active substance as described in any one of claims 1 to 9.

12. The pharmaceutical composition according to claim 11, characterized in that the biologically active substance is DNA, RNA, siRNA, aptamer, protein, antibody, or low molecular weight compound.

13. The pharmaceutical composition according to claim 11, wherein the dimeric peptide and the biologically active substance are linked by a covalent bond or exist in the form of a complex by a non-covalent bond.

14. The pharmaceutical composition according to claim 11, wherein the dimeric peptide is conjugated with biotin and streptavidin.

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