Peptide compound

Abstract

To provide a delivery peptide as a DDS carrier capable of efficiently transporting a cargo molecule such as mRNA into a cell.SOLUTION: The peptide compound has a E3 ligase ligand, a basic peptide having basic amino acids, and an endosomal escape moiety. The basic peptide is preferably oligoarginine Rn (n is the number of arginine residues and is a natural number of 6 to 12). ). The E3 ligase ligand is ALAPYIP. The endosomal escape moiety is GFWFG. In order to adjust the physical properties, sarcosine Sarn (n is the number of repetitions of sarcosine and is a natural number of 5 to 30) may be further added. ).SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to peptide compounds, and more particularly to peptide compounds that can efficiently introduce cargo molecules such as mRNA into cells. [Background technology]

[0002] Membrane-permeable peptides, collectively known as cell-penetrating peptides (CPPs), are used as DDS carrier peptides. In recent years, the use of CPPs to deliver mRNA into cells has become increasingly common (Non-Patent Documents 1, 2, 3). This method efficiently delivers mRNA, which is normally not cell membrane permeable, into cells by crosslinking with the CPP via a covalent bond (chemical bond) or by forming a stable complex through non-covalent interactions. Cationic and amphipathic CPPs are used as DDS carrier peptides, and many of them form complexes through electrostatic interactions between the cationic peptide and the anionic mRNA.

[0003] In recent years, the range of applications of ligands for E3 (E3 ubiquitin ligase) has expanded, including not only inhibitors but also applications in a new drug discovery modality called Proteolysis Targeting Chimera (PROTAC) (Non-Patent Documents 4, 5, 6). Peptide ligands for Von Hippel-Lindau (VHL), a type of E3, have a highly hydrophobic structure containing Leu and Ile, and therefore may be useful as hydrophobic sequences for amphipathic peptides consisting of a cationic and hydrophobic region.

[0004] However, there have been no examples of using such sequences as hydrophobic sequences in DDS carrier peptides, nor have there been any findings on their use as mRNA delivery peptides. Because many protein ligand sequences exhibit high hydrophobicity, not only VHL but also other E3 ligand sequences can be applied as lead sequences to improve interaction with the cell membrane.

[0005] On the other hand, if a DDS carrier peptide is taken up into the cell by endocytosis and then cannot escape the endosome, it will not reach the cytoplasm. A common approach is to first take it up into the cell by endocytosis and then transfer it from the endosome to the cytosol (endosomal escape) by some means (Non-Patent Documents 7, 8, and 9). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP-A-6-996620 (page 3, Figure 1) [Patent Document 2] JP-A-7-997730 (page 4, Figure 1) [Non-patent literature]

[0007] [Non-Patent Document 1] M. Oba, Y. Demizu, H. Yamashita, M. Kurihara, M. Tanaka, Bioorg. Med. Chem. 2015, 23, 4911-4918. [Non-patent document 2] H. Yamashita, Y. Demizu, T. Shoda, Y. Sato, M. Oba, M. Tanaka, M. Kurihara, Bioorg. Med. Chem. 2014, 22, 2403-2408. [Non-patent document 3] H. Yamashita, M. Oba, T. Misawa, M. Tanaka, T. Hattori, M. Naito, M. Kurihara, Y. Demizu, ChemBioChem 2016, 17, 137-140. [Non-patent document 4] Lai , AC , and Crews , CM ( 2017 ) Induced protein degradation: an emerging drug discovery paradigm . Nat. Rev. Fr. Drug Discov. 16, 101-1

Direct Environment 5

Outdoor Configuration 6

Direct Environment 7

Outdoor Tools 8

[0008] An object of the present invention is to provide a delivery peptide as a DDS carrier that can efficiently transport cargo molecules into cells. [Means for solving the problem]

[0009] The peptide according to the present invention is characterized in that it comprises an E3 ligase ligand, a basic peptide having basic amino acids, and an endosomal escape moiety, and transports a cargo molecule into cells. [Effects of the Invention]

[0010] The present invention provides a delivery peptide compound that can serve as a DDS carrier, capable of efficiently transporting cargo molecules such as mRNA into cells. Furthermore, the present invention enables the design of various peptide compounds by incorporating an E3 ligase ligand, an endosomal escape moiety, or even sarcosine. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the efficiency of mRNA transfection of peptide R9-VHL and peptide VHL-R9 into MCF-7 cells. [Figure 2] FIG. 1 shows the mRNA transfection efficiency of peptide C6-VHL-R9 and peptide (C6)2-VHL-R9 into Huh-7 cells. [Figure 3] FIG. 1 shows the mRNA transfection efficiency of peptides R9-VHL-Sar5, R9-VHL-Sar10, and R9-VHL-Sar20 into MCF-7 cells. [Figure 4] FIG. 1 shows the mRNA transfection efficiency of peptides VHL-Sar5-R9, VHL-Sar10-R9, and VHL-Sar20-R9 into MCF-7 cells. [Figure 5] FIG. 1 shows the mRNA transfection efficiency of peptides R9-VHL-FED-Sar5, R9-VHL-FED-Sar10, and R9-VHL-FED-Sar20 into MCF-7 cells. [Figure 6] FIG. 1 shows the mRNA transfection efficiency of peptides FED-VHL-R9-Sar5, FED-VHL-R9-Sar10, and FED-VHL-R9-Sar20 into MCF-7 cells. [Figure 7] FIG. 1 shows the mRNA transfection efficiency of peptides FED-VHL-Sar5-R9, FED-VHL-Sar10-R9, and FED-VHL-Sar20-R9 into MCF-7 cells. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, these embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.

[0013] The peptide compound of the present invention is a delivery peptide that can serve as a DDS carrier capable of efficiently transporting cargo molecules into cells, and comprises an E3 ligase ligand, a membrane-permeable basic peptide containing basic amino acids, and an endosomal escape moiety.

[0014] DDS carrier peptides, collectively known as cell-penetrating peptides (CPPs), efficiently deliver mRNA, which is normally impermeable to the cell membrane, into cells by forming a complex with mRNA and a membrane-permeable basic peptide. The present invention is an amphipathic CPP in which a binding peptide sequence for ubiquitin ligase (E3), of which there are hundreds in cells, is introduced as a hydrophobic fragment. However, if mRNA cannot escape the endosome after being taken up into the cell by endocytosis, it cannot effectively reach the cytoplasm. Therefore, the present inventors designed a DDS carrier peptide that contains an endosomal escape moiety in addition to an E3 ligase ligand and a membrane-permeable basic peptide. Based on this concept, the present invention is not limited to any specific sequences for the E3 ligase ligand, the membrane-permeable basic peptide moiety, or the endosomal escape moiety.

[0015] In the membrane-permeable basic peptide having a basic amino acid, the basic amino acid is not particularly limited, and may be, for example, arginine, lysine, tryptophan, or histidine.

[0016] The membrane-permeable basic peptide is, for example, oligoarginine R n where R nIn the formula, n is the number of arginine residues, and is not particularly limited, but is, for example, a natural number of 6 to 12, and is preferably an oligoarginine consisting of 7 to 9 arginine residues, and more preferably an oligoarginine consisting of 8 arginine residues.

[0017] The structure and principle of the membrane-permeable basic peptide are not particularly limited, and examples thereof include, in addition to oligoarginine, Angiopep-5 (RFFYGGSRGKRNNFRTEEY, SEQ ID NO: 1), Antp (RQIKIWFQNRRMKWKK, SEQ ID NO: 2), Bac (YGRKKRRQRRR, SEQ ID NO: 3), BR1 (RAGLQFPVGRLLR, SEQ ID NO: 4), BR2 (RAGLQFPVGRLLRRLLR, SEQ ID NO: 5), Buf IIb[BR3] (RAGLQFPVGRLLRRLLRRLLR, SEQ ID NO: 6), SH-CPPP-2 (KLPVM, SEQ ID NO: 7), CyLoP-1 (CRWRWKCCKK, SEQ ID NO: 8), Cys-Antp (CRQIKIWFQNRRMKWKK, SEQ ID NO: 9), Cys-pVEC (CLLIILRRRIRKQAHAHKS, SEQ ID NO: 10), Cys-SAP (CVRLPPPVRLPPPVRLPPP, SEQ ID NO: 11), Cys-SAPr (CPPPLRVPPPLRVPPPLRV, SEQ ID NO: 12), Cys-TAT (CYGRKKRRQRRR, SEQ ID NO: 13), Cys-TP10 (CAGYLLGKINLKALAALAKKIL, SEQ ID NO: 14), Cys-TP10K (CAGYLLGKINKLKALAALAKKIL, SEQ ID NO: 15), FAK (FAKLAARLYRKALARQLGVAA, SEQ ID NO: 16), H9 (HHHHHHHHH, SEQ ID NO: 17), etc.

[0018] The reason why the membrane-permeable basic peptide contains basic amino acids is that membrane-permeable peptides consisting of an amino acid sequence rich in basic amino acid residues are thought to induce macropinocytosis, a type of endocytosis in which cells take up extracellular substances, and this is thought to enable efficient introduction of mRNA into cells.

[0019] The E3 ligase of the E3 ligase ligand is not particularly limited, and examples thereof include von Hippel-Lindau (VHL), cereblon (CRBN), MDM2, APC, UBR5, SOCS, LNX1, BIRC2, BIRC3, BIRC4, CBX4, CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECTD4, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, and HE. Examples include RC6, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, WWP1, WWP2, or Parkin, and preferably VHL.

[0020] The E3 ubiquitin ligase ligand may be, for example, a von Hippel ligase (VHL) ligand. The VHL ligand may be a peptide fragment of a VHL substrate protein, such as the 7-amino acid peptide fragment ALAPYIP (SEQ ID NO: 18).

[0021] The endosomal escape moiety promotes the escape of mNRAs from the endosome by increasing the osmotic pressure inside the endosome or by destabilizing the endosomal membrane, allowing mNRAs to more efficiently and rapidly translocate to the nucleus or cytoplasm to interact with and act on target genes.

[0022] The endosomal escape moiety is not particularly limited, and examples thereof include peptides, lipid nanoparticles, polyplex nanoparticles, polymer nanospheres, inorganic nanoparticles, cationic lipid-based nanoparticles, cationic polymers, and pH-sensitive polymers.

[0023] When the endosomal escape moiety is a peptide, for example, the peptide fragment GFWFG (SEQ ID NO: 19) consisting of five amino acids can be used. When the endosomal escape moiety is a lipid nanomaterial, for example, lipid, phospholipid, cetyl palmitate, poloxamer 18, Tween 85, tristearinglyceride, Tween 80, etc. can be mentioned. When the endosomal escape moiety is a conjugate nanomaterial, for example, poly(amidoamine) or polyethyleneimine (PEI) can be mentioned.

[0024] The E3 ligase ligand and the endosomal escape moiety can be connected by a linker. The linker is not particularly limited, and examples thereof include 6-aminocaproic acid (Ahx). n Here, n is the number of repetitions of Anx, and is not particularly limited, but is a natural number of, for example, 1 to 6. The linker can be, for example, polyethylene glycol (PEG). n Here, n is the number of repeating PEGs, and is not particularly limited, but is a natural number from 1 to 6, for example.

[0025] Furthermore, the peptide compound of the present invention can be prepared by adding sarcosine (Sar) to adjust the physical properties. n(n is the number of repetitions of sarcosine and is, for example, a natural number from 1 to 30, preferably a natural number from 5 to 30, and more preferably a natural number from 5 to 20).

[0026] For example, Sar5 and Sar 10 , or Sar 20 When the following peptide compounds are provided, each of the peptide compounds can be the following peptide compounds:

[0027] [ka]

[0028] In addition, for example, Sar5 and Sar 10 , or Sar 20 When the amino acid sequence is provided, each of the amino acid sequences can be a peptide compound as shown below.

[0029] [ka]

[0030] In addition, for example, Sar5, Sar 10 , or Sar 20 When sarcosine is used (for example, n and a basic peptide having a basic amino acid attached to its N-terminus), the peptide compounds can be as follows:

[0031] [ka]

[0032] The peptide compounds of the present invention transport cargo molecules into cells. The cargo molecules are not particularly limited, and examples thereof include nucleic acids, proteins, drugs, and nanoparticles.

[0033] The nucleic acid cargo molecule may be a polynucleotide or an oligonucleotide, and may be a DNA or RNA molecule. In the case of DNA, it may be a plasmid DNA, cDNA, genomic DNA, or synthetic DNA. DNA and RNA may be double-stranded or single-stranded. In the case of single-stranded, it may be the coding strand or non-coding strand. The nucleic acid includes DNA derivatives or RNA derivatives, and the derivatives refer to nucleic acids having phosphorothioate bonds or nucleic acids in which the phosphate moiety, sugar moiety, or base moiety of internucleotides has been chemically modified to prevent enzymatic degradation. Nucleic acids also include viruses such as adenoviruses and retroviruses. When the nucleic acid is a vector used in gene therapy, such as plasmid DNA or a virus, it is preferably in a form configured to express the encoded genetic information within the cell when introduced into the cell.

[0034] The method for transporting a cargo molecule into a cell includes the steps of: obtaining a construct by binding the cargo molecule to be transported into the cell with the peptide compound of the present invention; and introducing the construct into a cell. The construct can be administered to a living body (an animal including a human, particularly a mammal including a human) by oral administration, injection, eye drop, nasal drop, pulmonary administration, or absorption through the skin, preferably by injection. [Example]

[0035] (1) Synthesis protocol for each peptide (1-1) Synthetic protocol for R9 The synthesis protocol for R9:5(6)-FAM-βAla-R9-NH2 was as follows.

[0036] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. A 50 μmol-scale peptide was elongated using a Liberty Blue Automatic Synthesizer (CEM) under microwave irradiation. The Liberty Blue Automatic Synthesizer is an automated peptide synthesizer that automatically synthesizes peptides by inputting the target sequence. After the reaction, the mixture was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a nitrogen stream, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (10-90% in 45 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized to obtain R9.

[0037] (1-2) VHL synthesis protocol The synthesis protocol for VHL:5(6)-FAM-βAla-ALAPYIP-NH2 was as follows.

[0038] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. The peptide was elongated on a 50 μmol scale using a Liberty Blue Automatic Synthesizer (CEM) under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a stream of nitrogen, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (20-90% in 40 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized to obtain VHL.

[0039] (1-3) Synthesis protocol of VHL-R9 The synthesis protocol for VHL-R9:5(6)-FAM-βAla-ALAPYIP-R9-NH2 was as follows.

[0040] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. A 50 μmol scale peptide was elongated using a Liberty Blue Automatic Synthesizer (CEM) under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a stream of nitrogen, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (33-39% in 20 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized to obtain VHL-R9.

[0041] (1-4) Synthesis protocol of C6-VHL-R9 The synthesis protocol for C6-VHL-R9:5(6)-FAM-Ahx-ALAPYIP-R9-NH2 was as follows.

[0042] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. A 50 μmol scale peptide was elongated using a Liberty Blue Automatic Synthesizer (CEM) under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a stream of nitrogen, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (25-70% in 40 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized to obtain C6-VHL-R9.

[0043] Synthesis protocol of (1-5)(C6)2-VHL-R9 The synthesis protocol for (C6)2-VHL-R9:5(6)-FAM-(Ahx)2-ALAPYIP-R9-NH2 was as follows.

[0044] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. A 50 μmol scale peptide was elongated using a Liberty Blue Automatic Synthesizer (CEM) under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a stream of nitrogen, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (25-90% in 40 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized to obtain (C6)2-VHL-R9.

[0045] (1-6) Synthesis protocol of VHL-Sar5-R9 The synthesis protocol for VHL-Sar5-R9:5(6)-FAM-βAla-ALAPYIP-Sar5-R9-NH2 was as follows.

[0046] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), 50 μmol of peptides were elongated under microwave irradiation. After the reaction, the mixture was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a stream of nitrogen, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (20-90% in 40 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized to obtain VHL-Sar5-R9.

[0047] (1-7)VHL-Sar 10 Synthesis protocol for -R9 VHL-Sar 10 -R9:5(6)-FAM-βAla-ALAPYIP-Sar 10 The synthesis protocol for -R9-NH2 was as follows.

[0048] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), peptides were elongated on a 50 μmol scale under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a nitrogen stream, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (20-70% in 40 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized and purified to VHL-Sar. 10 -R9 was obtained.

[0049] (1-8)VHL-Sar 20 Synthesis protocol for -R9 VHL-Sar 20 -R9:5(6)-FAM-βAla-ALAPYIP-Sar 20 The synthesis protocol for -R9-NH2 was as follows.

[0050] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), peptides were elongated on a 50 μmol scale under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a nitrogen stream, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (30-50% in 20 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized and purified to VHL-Sar. 20 -R9 was obtained.

[0051] Synthesis protocol of (1-9)EED-VHL-R9-Sar5 The synthesis protocol for EED-VHL-R9-Sar5:5(6)-FAM-βAla-GFWFG-Ahx-ALAPYIP-R9-Sar5-NH2 was as follows.

[0052] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), 50 μmol of peptides were elongated under microwave irradiation. After the reaction, the mixture was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a stream of nitrogen, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (20-70% in 40 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized to obtain EED-VHL-R9-Sar5.

[0053] (1-10)EED-VHL-R9-Sar 10 Synthesis protocol of EED-VHL-R9-Sar 10 :5(6)-FAM-βAla-GFWFG-Ahx-ALAPYIP-R9-Sar 10 The synthesis protocol for -NH2 was as follows.

[0054] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), the peptide was elongated on a 50 μmol scale under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a nitrogen stream, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (20-70% in 40 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized and purified to give EED-VHL-R9-Sar. 10 obtained.

[0055] (1-11)EED-VHL-R9-Sar 20 Synthesis protocol of EED-VHL-R9-Sar 20 :5(6)-FAM-βAla-GFWFG-Ahx-ALAPYIP-R9-Sar 20 The synthesis protocol for -NH2 was as follows.

[0056] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), the peptide was elongated on a 50 μmol scale under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a nitrogen stream, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (20-70% in 40 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized and purified to give EED-VHL-R9-Sar. 20 obtained.

[0057] Synthesis protocol of (1-12)EED-VHL-Sar5-R9 The synthesis protocol for EED-VHL-Sar5-R9:5(6)-FAM-βAla-GFWFG-Ahx-ALAPYIP-Sar5-R9-NH2 was as follows.

[0058] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), 50 μmol of peptides were elongated under microwave irradiation. After the reaction, the mixture was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a stream of nitrogen, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (48-58% in 20 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized to obtain EED-VHL-Sar5-R9.

[0059] (1-13)EED-VHL-Sar 10 Synthesis protocol for -R9 EED-VHL-Sar 10 -R9:5(6)-FAM-βAla-GFWFG-Ahx-ALAPYIP-Sar 10 The synthesis protocol for -R9-NH2 was as follows.

[0060] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), peptides were elongated on a 50 μmol scale under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a nitrogen stream, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (30-60% in 40 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized and purified to EED-VHL-Sar. 10 -R9 was obtained.

[0061] (1-14)EED-VHL-Sar 20 Synthesis protocol for -R9 EED-VHL-Sar 20 -R9:5(6)-FAM-βAla-GFWFG-Ahx-ALAPYIP-Sar 20 The synthesis protocol for -R9-NH2 was as follows.

[0062] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), the peptide was elongated on a 50 μmol scale under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a nitrogen stream, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (40-60% in 30 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized and purified to EED-VHL-Sar. 20 -R9 was obtained.

[0063] Synthesis protocol of (1-15)R9-VHL-EED-Sar5 R9-VHL-EED-Sar 10 The synthesis protocol for 5(6)-FAM-βAla-R9-ALAPYIP-Ahx-GFWFG-Sar5-R9-NH2 was as follows.

[0064] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), 50 μmol of peptides were elongated under microwave irradiation. After the reaction, the mixture was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a stream of nitrogen, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (47–52% in 20 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized to obtain R9-VHL-EED-Sar5.

[0065] (1-16)R9-VHL-EED-Sar 10 Synthesis protocol of R9-VHL-EED-Sar 10 :5(6)-FAM-βAla-R9-ALAPYIP-Ahx-GFWFG-Sar 10 The synthesis protocol for -R9-NH2 was as follows.

[0066] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), the peptide was elongated on a 50 μmol scale under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a nitrogen stream, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (45-50% in 20 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized and purified to give R9-VHL-EED-Sar. 10 obtained.

[0067] (1-17)R9-VHL-EED-Sar 20 Synthesis protocol of R9-VHL-EED-Sar 20 :5(6)-FAM-βAla-R9-ALAPYIP-Ahx-GFWFG-Sar 20 The synthesis protocol for -R9-NH2 was as follows.

[0068] 50 μmol of Protide LL Rink-amide resin (CEM) was suspended in a 1:1 DMF / DCM solution. Using a Biotage Syro I (Biotage) or a Liberty Blue Automatic Synthesizer (CEM), the peptide was elongated on a 50 μmol scale under microwave irradiation. After the reaction, the resin was washed twice with DMF and twice with DCM. The peptide was cleaved from the solid support by adding 4 mL of a 95:2.5:2.5 mixture of TFA / water / TIPS and stirring at room temperature for 2 hours. The TFA was evaporated under a nitrogen stream, and ether was added to the residue. After centrifugation, the supernatant was removed, and the crude peptide was purified by HPLC (43-48% in 20 min MeCN / HO with 0.1% TFA). The purified peptide was lyophilized and purified to give R9-VHL-EED-Sar. 20 obtained.

[0069] (2) mRNA transfection efficiency of each peptide The mRNA introduction efficiency of each peptide was measured by forming a complex with the peptide using luciferase-expressing mRNA and adding it to cells (MCF-7 or Huh-7 cells), and measuring the intracellular luminescence intensity using a luminescence plate reader.

[0070] (2-1) Effect of E3 Ligand Sequence As shown in Figure 1, the R9 peptide, R9-VHL, in which a VHL ligand peptide was introduced at the N-terminus, increased the efficiency of mRNA transduction into MCF-7 cells. Furthermore, the R9 peptide, VHL-R9, in which a VHL ligand peptide was introduced at the C-terminus, showed mRNA transduction efficiency into MCF-7 cells comparable to that of R9-VHL.

[0071] [ka]

[0072] (2-2) Effect of the linker aminohexanoic acid (Ahx) As shown in Figure 2, the introduction of one or two aminohexanoic acid (Ahx) moieties, which are linkers that connect fluorescent substances, into the peptide VHL-R9 increased the efficiency of mRNA transfection into Huh-7 cells.

[0073] [ka]

[0074] (2-3) Effect of sarcosine introduced into the N-terminus As shown in Figure 3, peptides R9-VHL-Sar were prepared by introducing sarcosine of various lengths into the N-terminus of the R9-VHL peptide. n showed similar mRNA transfection efficiencies into MCF-7 cells.

[0075] [ka]

[0076] (2-4) Effect of sarcosine introduced into the center of the sequence As shown in Figure 4, peptides VHL-Sar, in which sarcosine of various lengths was introduced into the center of the VHL-R9 peptide sequence, were n -R9 showed similar mRNA transfection efficiencies into MCF-7 cells.

[0077] [ka]

[0078] (2-5) Effect of sarcosine introduced into the center of the sequence Examples of the present application As shown in Figure 5, peptide R9-VHL-Sar n The peptide R9-VHL-EED-Sar has the endosomal escape sequence EED in the center of the sequence. n showed improved efficiency of mRNA transfection into MCF-7 cells.

[0079] [ka]

[0080] (2-6) Effect of introducing the endosomal escape sequence EED into the C-terminus Examples of the present application As shown in Figure 6, the peptide VHL-R9-Sar n The peptide EED-VHL-R9-Sar was derived by introducing the endosomal escape sequence EED at the C-terminus of VHL. n showed a significant improvement in the efficiency of mRNA transfection into MCF-7 cells.

[0081] [ka]

[0082] [ka]

[0083] (2-7) Effect of introducing the endosomal escape sequence EED into the C-terminus Examples of the present application As shown in Figure 7, the peptide VHL-Sar n -R9 peptide EED-VHL-Sar, which is an endosomal escape sequence EED at the C-terminus of R9 n -R9 showed a significant improvement in the efficiency of mRNA transfection into MCF-7 cells.

[0084] [ka] [Industrial Applicability]

[0085] It can be used in the pharmaceutical and drug discovery fields.

Claims

1. A peptide compound that transports a cargo molecule into a cell, comprising an E3 ligase ligand, a basic peptide having a basic amino acid, and an endosomal escape moiety.

2. 2. The peptide compound according to claim 1, wherein the basic amino acid is arginine, lysine, or histidine.

3. The basic peptide is oligoarginine R ｎ (n is the number of arginine residues and is a natural number from 6 to 12).

4. The peptide compound according to claim 1, wherein the E3 ligase of the E3 ligase ligand is VHL.

5. The peptide compound according to claim 1, wherein the E3 ligase ligand is ALAPYIP.

6. 2. The peptide compound according to claim 1, wherein the endosomal escape moiety is at least one selected from the group consisting of peptides, lipid nanoparticles, polymer nanoparticles, polymer nanospheres, inorganic nanoparticles, cationic lipid-based nanoparticles, cationic polymers, and pH-sensitive polymers.

7. The peptide compound of claim 1, wherein the endosomal escape moiety is GFWFG.

8. The peptide compound according to claim 1, characterized in that it has a linker connecting the E3 ligase ligand and the endosomal escape moiety.

9. Furthermore, sarcosine Sar ｎ (n is the number of sarcosine repeats and is a natural number from 5 to 30).

10. The sarcosine Sar ｎ 10. The peptide compound according to claim 9, wherein the E3 ligase ligand is connected to the C-terminus of the peptide and the basic peptide having the basic amino acid is connected to the N-terminus of the peptide.

11. The peptide compound of claim 1 , wherein the cargo molecule is a nucleic acid, a protein, a drug, or a nanoparticle.

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