Peptide compound

Abstract

To provide a means capable of inducing decomposition of a new protein which can be comprehensively and generally used for all cells.SOLUTION: A peptide compound comprising a target-binding moiety, a cell membrane-penetrating peptide moiety, and a lysosome-inducing peptide moiety.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to peptide compounds. [Background technology]

[0002] Compounds that specifically induce the degradation of specific proteins include proteolysis targeting chimeras (PROTACs) and lysosome targeting chimeras (LYTACs). PROTACs are chimeric compounds that primarily induce the degradation of intracellular proteins, while LYTACs are a collective term for molecules that induce the degradation of plasma membrane proteins or extracellular proteins. LYTACs induce protein degradation by forming a molecular complex consisting of a dimer of a ligand that binds to receptors (IGF2R, ASGPR) that travel between the lysosome, an intracellular organelle responsible for protein degradation, and a molecule that binds to the target protein. Using these complexes, it is possible to induce the degradation of specific plasma membrane proteins or extracellular proteins. Recently, various receptors that target proteins to lysosomes have been identified, and research into the development of LYTACs using molecules that selectively bind to these receptors has been active.

[0003] For example, Patent Document 1 discloses a nucleic acid chimera having a nucleic acid aptamer element that specifically binds to a lysosomal targeting receptor and an aptamer element that targets a protein to be degraded. Patent Document 2 also discloses a bifunctional molecule that includes a first portion that specifically binds to a cell surface molecule or an extracellular molecule and a second portion that specifically binds to a lysosomal targeting molecule. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2022 / 095853 [Patent Document 2] US Patent Application Publication No. 2022 / 0023434 Summary of the Invention [Problem to be solved by the invention]

[0005] Existing LYTACs, such as those described in Patent Documents 1 and 2, require the chimerization and conjugation of a ligand molecule that binds to a receptor (shuttling receptor) that travels between the cell membrane surface and lysosomes and a binding ligand for the protein targeted for degradation at an appropriate distance. Furthermore, it is known that LYTAC activity varies significantly depending on the expression level of the shuttling receptor, making it difficult to develop LYTACs that can target all cells. Furthermore, many of the known ligands for shuttling receptors are sugar ligands, which poses the challenge of complex organic synthesis.

[0006] Therefore, an object of the present invention is to provide a new means for inducing protein degradation that can be used comprehensively and universally in all cells. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems, and as a result have found that the above problems can be solved by a peptide compound comprising a target-binding portion, a cell membrane-penetrating peptide portion, and a lysosome-targeting peptide portion, thereby completing the present invention. [Effects of the Invention]

[0008] The present invention provides a new means for inducing protein degradation that can be used comprehensively and universally in all cells. [Brief explanation of the drawings]

[0009] [Figure 1]1 is an electrophoretic photograph showing the results of evaluation of the concentration-dependent decomposition activity of a peptide compound targeting epidermal growth factor receptor (EGFR) protein in Test Example 1. [Figure 2] 1 shows electrophoresis photographs showing the results of evaluating the concentration-dependent degradation activity of peptide compounds targeting PD-L1 protein in Test Example 2. [Figure 3] 1 is an electrophoretic photograph showing the results of evaluation of the concentration-dependent decomposition activity of a peptide compound targeting HER2 protein in Test Example 3. [Figure 4] 1 is an electrophoretic photograph showing the results of evaluation of the time-dependent decomposition activity of a peptide compound targeting HER2 protein in Test Example 4. [Figure 5] 1 is an electrophoretic photograph showing the results of evaluation of the time-dependent decomposition activity of a peptide compound targeting EGFR protein in Test Example 5. [Figure 6] 10 shows fluorescence micrographs showing the results of an analysis of changes in the localization of HER2 protein by immunostaining in Test Example 6. [Figure 7] 1 is an electrophoresis photograph showing the results of evaluation of HER2 protein degradation activity under lysosomal inhibition conditions with chloroquine in Test Example 7. [Figure 8] 1 shows electrophoretic photographs showing the results of evaluation of EGFR proteolytic activity under lysosomal inhibition conditions with chloroquine in Test Example 8. [Figure 9] 1 is an electrophoretic photograph showing the results of evaluation of the concentration-dependent decomposition activity of a peptide compound targeting EGFR protein in Test Example 9. [Figure 10] 1 is an electrophoretic photograph showing the results of evaluation of the concentration-dependent decomposition activity of a peptide compound targeting HER2 protein in Test Example 10. [Figure 11] 1 is an electrophoretic photograph showing the results of evaluation of the concentration-dependent target protein degradation activity of a peptide compound targeting integrin αv in Test Example 11. [Figure 12]1 is an electrophoretic photograph showing the results of examining the amino acid sequence of the lysosome-inducing peptide portion of the peptide compound in Test Example 12. [Figure 13] 1 shows fluorescence micrographs showing the evaluation results of FITC-modified streptavidin (extracellular protein model) uptake activity in Test Example 13. DETAILED DESCRIPTION OF THE INVENTION

[0010] An embodiment of the present invention will be described below, but the present invention is not limited to the following embodiment.

[0011] In this specification, the range "X to Y" means "X or more and Y or less." Unless otherwise specified, operations and measurements of physical properties are performed at room temperature (20 to 25°C) and a relative humidity of 40 to 50% RH.

[0012] In the present invention, the term "amino acid residue" refers to a portion of a peptide or protein molecule that corresponds to one unit of an amino acid constituting the peptide or protein. More specifically, it refers to a divalent group derived from an α-amino acid, as represented by the following formula:

[0013] [ka]

[0014] However, the above R 0 is the side chain of the amino acid, for example, a hydrogen atom for Gly and a methyl group for Ala.

[0015] The "amino acid residue" is derived from a natural or unnatural α-amino acid, and when optically active forms are possible, it may be either the L-form or the D-form, with the L-form being preferred.

[0016] More specifically, examples of "amino acid residues" include Arg, Lys, Asp, Asn, Glu, Gln, His, Pro, Tyr, Trp, Ser, Thr, Gly, Ala, Met, Cys, Phe, Leu, Val, and Ile, and analogs thereof. The analogues may be, for example, derivatives in which the side chains of the 20 amino acid residues are substituted with any substituent, and examples thereof include halogenated derivatives of the 20 amino acid residues (e.g., 3-chloroalanine), 2-aminobutyric acid, norleucine, norvaline, isovaline, 2-aminoisobutyric acid, homophenylalanine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, 2-hydroxyglycine, homoserine, hydroxylysine, hydroxyproline, 3,4-didehydroproline, homoproline, homocysteine, homomethionine, aspartic acid esters (e.g., aspartic acid methyl ester, aspartic acid ethyl ester, aspartic acid propyl ester, aspartic acid cyclohexyl ester), and the like. Examples of the amino acid residues include, but are not limited to, amino acid residues derived from amino acids such as glutamic acid esters (glutamic acid cyclohexyl ester, glutamic acid ethyl ester, glutamic acid propyl ester, glutamic acid methyl ester, glutamic acid benzyl ester, etc.), glutamic acid esters (glutamic acid cyclohexyl ester, glutamic acid ethyl ester, glutamic acid propyl ester, glutamic acid methyl ester, glutamic acid benzyl ester, etc.), formyltryptophan, 2-cyclopentylglycine, 2-cyclohexylglycine, 2-phenylglycine, 2-pyridylalanine, 3-cyclopentylalanine, 3-cyclohexylalanine, 3-pyridylalanine, 3-pyrazolylalanine, 3-furanylalanine, 3-thienylalanine, methoxyphenylalanine, 3-naphthylalanine, and 4-pyridylalanine. Furthermore, for those having an asymmetric carbon in the side chain, such as Ile and Thr, which exist as diastereomers, the natural form (e.g., (2R * ,3R * )-2-amino-3-methylpentanoic acid, and (2R * ,3S * )-2-amino-3-hydroxybutanoic acid) and unnatural forms (e.g., (2R * ,3S *)-2-amino-3-methylpentanoic acid, and (2R * ,3R * )-2-amino-3-hydroxybutanoic acid) can be used without distinction. That is, "Ile" means (2R * ,3R * )-2-amino-3-methylpentanoic acid and (2R * ,3S * )-2-amino-3-methylpentanoic acid, and "Thr" is used to mean both (2R * ,3S * )-2-amino-3-hydroxybutanoic acid and (2R * ,3R * )-2-amino-3-hydroxybutanoic acid. Preferably, the natural diastereomer (i.e., Ile, (2R * ,3R * )-2-amino-3-methylpentanoic acid, if Thr, (2R * ,3S * )-2-amino-3-hydroxybutanoic acid) is used.

[0017] Unless otherwise specified, the amino acid sequences described herein are conventionally written from the N-terminus (amino terminus) to the C-terminus (carboxyl terminus).

[0018] As used herein, the term "pharmaceutically acceptable salt" refers to a metal salt, ammonium salt, organic acid salt, inorganic acid salt, or salt with an organic or inorganic base that does not produce undesirable physiological effects after administration to a patient or subject. More specifically, examples include, but are not limited to, sodium salt, potassium salt, calcium salt, magnesium salt, barium salt, aluminum salt, zinc salt, ammonium salt, methylamine salt, ethylamine salt, aniline salt, dimethylamine salt, diethylamine salt, pyrrolidine salt, piperidine salt, morpholine salt, piperazine salt, trimethylamine salt, triethylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, trifluoroacetate, phthalate, fumarate, oxalate, tartrate, maleate, citrate, succinate, malate, methanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0019] The structure of the N-terminus of the peptide according to the present invention is not particularly limited, and may be, for example, a hydrogen atom (ie, unmodified), or a structure into which a modifying group has been introduced by a conventionally known method. Examples of N-terminal modifying groups include alkyl groups having 1 to 20 carbon atoms, alkenyl groups having 1 to 20 carbon atoms, alkynyl groups having 1 to 20 carbon atoms, aromatic hydrocarbon groups having 6 to 20 carbon atoms, heterocyclic groups, sulfonyl groups, carboxyl groups, glyoxyl groups, and formyl groups; polyethylene glycol groups (PEGylated), polyoxyethylene glycol groups, and polypropylene glycol groups; protecting groups such as tert-butoxycarbonyl groups (Boc groups), benzyloxycarbonyl groups (Z groups), and fluorenylmethoxycarbonyl groups (Fmoc groups); cycloalkyloxycarbonyl groups such as cyclopentyloxycarbonyl groups, cyclohexyloxycarbonyl groups, adamantyloxycarbonyl groups, norbornyloxycarbonyl groups, and isobornyloxycarbonyl groups; protecting groups derived from amino acids such as pyroglutamic acid and morotanoic acid; carbamate protecting groups; and protecting groups derived from sulfonic acids such as benzenesulfonic acid or phosphoric acid.

[0020] The structure of the C-terminus of the peptide according to the present invention is also not particularly limited, and may be a structure modified with a protecting group commonly used for protecting carboxylic acids. More specifically, the C-terminus of the peptide according to the present invention may be, for example, a carboxyl group (-COOH), a carboxylate (-COO - ), amide (-CONH2), alkylamide (-CONHR 31 , -CONR 31 R 32 ), ester (-COOR 31 ), acyloxyalkyl (-R 33 -OCOR 31 ), a phthalidyl group optionally substituted with an alkyl or alkoxy group having 1 to 4 carbon atoms (for example, a phthalidyl group, a dimethylphthalidyl group, or a dimethoxyphthalidyl group), or a (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl group. Of these, the C-terminus of the peptide is preferably an amide. In the above alkylamide, ester, and acyloxyalkyl groups, R 31 and R 32 are each independently an alkyl group having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an amyl group, an isoamyl group, a tert-amyl group, a hexyl group, or a cyclohexyl group; an aryl group having 6 to 10 carbon atoms, such as a phenyl group or a naphthyl group; an aralkyl group having 7 to 18 carbon atoms, such as a benzyl group, a phenethyl group, or a benzhydryl group; a sugar, such as glucose; or a polyethylene glycol group optionally modified with an alkyl group having 1 to 6 carbon atoms (e.g., a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an amyl group, an isoamyl group, a tert-amyl group, or a hexyl group). 33 is an alkylene group having 1 to 4 carbon atoms such as a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, an isobutylene group, an s-butylene group, or a t-butylene group.

[0021] It is known in the art that each amino acid residue can be substituted with an amino acid residue having similar properties based on differences in its side chain (conservative substitution). For example, the aliphatic hydrophobic amino acids Val, Leu, Ile, 2-aminobutyric acid (Abu), norleucine (Nle), norvaline (Nva), and isovaline (Iva) can be substituted for each other. Gly, Ala, and 2-aminoisobutyric acid (Aib), whose side chains are hydrogen atoms or methyl groups, can be substituted for each other. The neutral polar amino acids Asn and Gln can be substituted for each other. 2,4-diaminobutanoic acid (Dbu) and ornithine (Orn) can be substituted for each other. Pro and homoproline (homoPro) can be substituted for each other.

[0022] <Peptide compounds> One aspect of the present invention is a peptide compound comprising a target-binding portion, a cell membrane-penetrating peptide portion, and a lysosome-targeting peptide portion.

[0023] In the peptide compound according to the present invention, the target is a compound that is degraded in the lysosome. In one embodiment, the target according to the present invention is a protein. The target protein is not particularly limited, and may be a cell membrane protein or an extracellular protein.

[0024] The peptide compounds of the present invention contain a cell membrane-penetrating peptide moiety and a lysosome-targeting peptide moiety as substitutes for shuttling receptors used to deliver compounds to intracellular lysosomes. When presented intracellularly, the lysosome-targeting peptide moiety is recognized by AP-2 protein, a component protein of clathrin-dependent endocytosis. Therefore, it is believed that the peptide compounds of the present invention are taken up into cells without the involvement of shuttling receptors and targeted to lysosomes. Therefore, the peptide compounds of the present invention can induce target degradation without being affected by the increase or decrease in activity due to the presence or absence of shuttling receptor expression.

[0025] The peptide compounds of the present invention can theoretically be used as a comprehensive and versatile method for inducing target degradation in all cells. As shown in Test Examples 9 to 11 in the Examples, the peptide compounds of the present invention can induce target degradation even in the case of low-molecular-weight peptides with 25 amino acid residues. The peptide compounds of the present invention can be synthesized inexpensively and stably, and can also be easily chemically modified using amino acid side chains, making them advantageous over existing LYTACs and the like. Furthermore, the peptide compounds of the present invention do not require cross-linking between the shuttling receptor and the target (e.g., a protein), making molecular design extremely simple, and are believed to comprehensively solve the problems associated with previous methods.

[0026] The target-binding moiety of the present invention includes a compound capable of binding to a target. The compound capable of binding to a target can be appropriately selected depending on the target to be degraded. In one embodiment, the target-binding moiety of the present invention includes an antibody, a protein-binding peptide, or a low-molecular-weight compound that binds to a protein. Conventional antibodies and protein-binding peptides can be used as the antibody and protein-binding peptide. Examples of protein-binding peptides include RGD peptides such as a peptide consisting of the amino acid sequence represented by SEQ ID NO: 34 (EGFR-binding peptide), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 35 (HER2-binding peptide), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 36 (see M. Alipour et al., J Biomed Mater Res. 2020;108:839-850). As used herein, a low-molecular-weight compound that binds to a protein refers to a compound with a molecular weight of 1000 or less (excluding protein-binding peptides with a molecular weight of 1000 or less). Conventionally known compounds can be used as low molecular weight compounds that bind to proteins, such as osimertinib, gefitinib, genistein, cilengitide, BMS-1, BMS-202, CWHM-12, bexotegrast, and EMD527040.

[0027] For example, when the target is epidermal growth factor receptor (EGFR) protein, the target-binding moiety can be cetuximab (antibody) or a peptide (protein-binding peptide) consisting of the amino acid sequence represented by LARLLT (SEQ ID NO: 34). For example, when the target is PD-L1 protein, the target-binding moiety can be atezolizumab (antibody). For example, when the target is HER2 protein, the target-binding moiety can be trastuzumab (antibody) or a peptide (protein-binding peptide) consisting of the amino acid sequence represented by KCCYSL (SEQ ID NO: 35). For example, when the target is integrin αv, the target-binding moiety can be RGDTFI (SEQ ID NO: 36).

[0028] The cell membrane-permeable peptide portion according to the present invention has the function of passing through the cell membrane and being transported into the cell. The cell membrane-permeable peptide portion includes a cell membrane-permeable peptide. The cell membrane-permeable peptide is not particularly limited, and any conventionally known cell membrane-permeable peptide can be used. Examples of cell membrane-permeable peptides are shown in Table 1.

[0029] [Table 1]

[0030] In one embodiment, the cell membrane-permeable peptide portion of the present invention preferably comprises an amino acid sequence represented by any one of SEQ ID NOs: 19 to 33, more preferably comprises an amino acid sequence represented by any one of SEQ ID NOs: 19 to 22, and even more preferably consists of an amino acid sequence represented by any one of SEQ ID NOs: 19 to 22.

[0031] The lysosome-targeting peptide portion of the present invention has the function of targeting the peptide compound of the present invention to lysosomes. The lysosome-targeting peptide portion contains a lysosomal sorting signal sequence.

[0032] In one embodiment, the lysosomotropic peptide moiety of the present invention is X 1 X 2 X 3 X 4 The lysosome-inducing peptide portion contains 6 to 19 amino acid residues. 1 X 2 X 3 X 4 In the amino acid sequence represented by 1 is a Tyr or Ala residue, and X 2 and X 3 are each independently any amino acid residue, and X 4 is an amino acid residue selected from the group consisting of Leu, Ile, Val, Phe, Tyr, Trp, Met, Cys, Gly and Ala. 1 X 2 X 3 X 4 The amino acid sequence represented by the formula (I) preferably does not include the amino acid sequence represented by YSKV (SEQ ID NO: 97).

[0033] The lysosome-inducing peptide portion preferably has 7 to 15 amino acid residues, and more preferably has 8 to 11 amino acid residues.

[0034] X 1 X 2 X 3 X 4 The amino acid sequence represented by 1 is a Tyr residue and X 2 is an amino acid residue selected from the group consisting of Gln, Glu, Asp, Asn, Met, His, Lys, Arg, Thr, Ser, Val, and Ala; and X 3 is an amino acid residue selected from the group consisting of Arg, Lys, His, Thr, Ser, Gln, Asp, Glu, Asn, Val, Leu, Ile, Ala, Gly, and Pro; and X 4 is an amino acid residue selected from the group consisting of Leu, Ile, Val, Phe, and Met. 1 X 2 X3 X 4 More preferably, the amino acid sequence represented by SEQ ID NOs: 37 to 63 is included.

[0035] The amino acid sequences represented by SEQ ID NOs: 37 to 63 are shown in Table 2 below.

[0036] [Table 2]

[0037] In one embodiment, the lysosome-inducing peptide portion comprises a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 10 to 18, or comprises a peptide consisting of an amino acid sequence that has 95% or more sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS: 10 to 18 and has the function of targeting to lysosomes, or comprises a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 10 to 18, or comprises a peptide consisting of an amino acid sequence that has 95% or more sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS: 10 to 18 and has the function of targeting to lysosomes. Herein, sequence identity can be determined using an analysis program such as BLAST. When BLAST is used, the program's default parameters are used.

[0038] In a preferred embodiment, the lysosome-inducing peptide portion comprises a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 10 and 13 to 18, or consists of a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 10 and 13 to 18.

[0039] The amino acid sequences represented by SEQ ID NOs: 10 to 18 are shown in Table 3 below.

[0040] [Table 3]

[0041] In one embodiment, the lysosome-inducing peptide portion comprises a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 64 to 96, or a peptide consisting of amino acids with 95% or more sequence identity to the amino acid sequence represented by any one of SEQ ID NOs: 64 to 96 and having the function of inducing to lysosomes, or a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 64 to 96, or a peptide consisting of an amino acid sequence with 95% or more sequence identity to the amino acid sequence represented by any one of SEQ ID NOs: 64 to 96 and having the function of inducing to lysosomes.

[0042] In one embodiment, the lysosomotropic peptide portion does not comprise the amino acid sequence set forth in SEQ ID NO:98.

[0043] The amino acid sequences represented by SEQ ID NOs: 64 to 96 and 98 are shown in Table 4 below.

[0044] [Table 4]

[0045] In the peptide compound of the present invention, the order in which the target-binding portion, the cell membrane-penetrating peptide portion, and the lysosome-targeting peptide portion are bound is not particularly limited. The peptide compound of the present invention can have the following six configurations: Configuration 1: (target-binding portion)-(cell membrane-penetrating peptide portion)-(lysosome-targeting peptide portion); Configuration 2: (target-binding portion)-(lysosome-targeting peptide portion)-(cell membrane-penetrating peptide portion); Configuration 3: (cell membrane penetrating peptide portion)-(target binding portion)-(lysosome-targeting peptide portion); Configuration 4: (cell membrane-penetrating peptide portion)-(lysosome-targeting peptide portion)-(target-binding portion); Configuration 5: (lysosome-targeting peptide portion)-(target-binding portion)-(cell membrane-penetrating peptide portion); and Configuration 6: (lysosome-targeting peptide portion)-(cell membrane-penetrating peptide portion)-(target-binding portion).

[0046] The peptide compound according to the present invention preferably has Configuration 1 or Configuration 6, more preferably Configuration 1.

[0047] In the peptide compounds of the present invention, each moiety may be linked via a linker, directly, or a mixture thereof. For example, when the target-binding moiety, cell membrane-penetrating peptide moiety, and lysosome-targeting peptide moiety are peptides, the target-binding moiety, cell membrane-penetrating peptide moiety, and lysosome-targeting peptide moiety may be linked directly without a linker, or may be linked via a linker. In this case, the peptide compounds of the present invention may be in the form of a single peptide. For example, in Configuration 1, when the target-binding moiety comprises an antibody and the cell membrane-penetrating peptide moiety and lysosome-targeting peptide moiety comprise peptides, the target-binding moiety and the cell membrane-penetrating peptide moiety may be linked via a linker, and the cell membrane-penetrating peptide moiety and the lysosome-targeting peptide moiety may be linked directly without a linker, or may be linked via a linker. In this case, the peptide compounds of the present invention may be in the form of an antibody-peptide complex.

[0048] In the peptide compound according to the present invention, when each moiety is bonded via a linker, the linker is not particularly limited and any conventionally known linker can be used. Examples of linkers include a substituted or unsubstituted alkylene group having 1 to 20 carbon atoms, a substituted or unsubstituted alkenylene group having 2 to 20 carbon atoms, a substituted or unsubstituted alkynylene group having 2 to 20 carbon atoms, a substituted or unsubstituted cycloalkylene group having 3 to 20 carbon atoms, a substituted or unsubstituted cycloalkenylene group having 3 to 20 carbon atoms, a substituted or unsubstituted arylene group having 6 to 20 carbon atoms, a substituted or unsubstituted heteroarylene group having 3 to 20 carbon atoms, -NH-, -O-, -S-, -C(=O)-NH-, -NH-C(=O)-, -O-, -C(=O)-O-, -OC(=O)-, -S-, -C(=O)-, a polyoxyalkylene group, an amino acid residue, a peptide chain, polyethylene glycol, and combinations thereof.

[0049] The peptide compound according to the present invention can be produced using a conventionally known method so that the target-binding portion, the cell membrane-penetrating peptide portion, and the lysosome-inducing peptide portion have the above-mentioned configurations 1 to 6.

[0050] The peptides included in the peptide compounds of the present invention (also referred to herein simply as "peptides of the present invention") can be produced by conventionally known methods, including chemical synthesis and recombinant technology. To prepare the peptide moiety by chemical synthesis, each amino acid can be produced by a method commonly used in peptide chemistry, such as the methods described in "The Peptides," Vol. 1 (Schroder and Luhke, Academic Press, New York, USA (1966)) and "Fundamentals and Experiments of Peptide Synthesis" (Izumiya Nobuo et al., Maruzen Co., Ltd., 1985). Production can be performed by either a liquid-phase method or a solid-phase method. Furthermore, either a column method or a batch method can be used.

[0051] The peptides of the present invention may also be produced by recombinant technology using animal cells, insect cells, microorganisms, or the like, for example, by the techniques described in Current Protocols in Molecular Biology, Chapter 16 below. After being produced in cultured cells or microorganisms, the peptides can be purified by conventionally known methods. Methods for purifying and isolating peptides are known to those skilled in the art and can be carried out by the techniques described, for example, in Current Protocols in Molecular Biology, Chapter 16 (Ausubel et al., John Wiley and Sons, 2006).

[0052] Condensation methods for forming peptide bonds include the azide method, acid halide method, acid anhydride method, carbodiimide method, carbodiimide-additive method, active ester method, carbonylimidazole method, oxidation-reduction method, enzymatic method, and methods using Woodward reagent K, HATU reagent, Bop reagent, etc. Of the above-mentioned methods for condensation reactions using solid phase methods, the acid anhydride method, carbodiimide method, and active ester method are the main methods.

[0053] Furthermore, when extending a peptide chain using the solid-phase method, the C-terminal amino acid is bound to a support such as a resin that is insoluble in the organic solvent used. Such resins may include those into which functional groups have been introduced for the purpose of binding the amino acid to the resin, or those into which a spacer has been inserted between the resin and the functional group, depending on the purpose. More specific examples include halomethyl resins such as chloromethyl resin, oxymethyl resin, 4-(oxymethyl)-phenylacetamidomethyl resin, 4-(oxymethyl)-phenoxymethyl resin, and Rink amide resin. Prior to these condensation reactions, carboxyl groups, amino groups, hydroxyl groups, amidino groups, and the like that do not participate in the condensation reaction can be protected by commonly known means. Conversely, carboxyl groups and amino groups that directly participate in the condensation reaction can also be activated.

[0054] Protective groups used to protect functional groups not involved in the condensation reaction can be those commonly used in organic chemistry, such as those described in "Protective Groups in Organic Synthesis" (Greene, John Wiley & Sons, Inc., 1981). More specifically, protective groups for carboxyl groups include commonly known protective groups such as various methyl esters, ethyl esters, benzyl esters, p-nitrobenzyl esters, t-butyl esters, and cyclohexyl esters. Protective groups for amino groups include benzyloxycarbonyl, t-butoxycarbonyl, isobornyloxycarbonyl, and 9-fluorenylmethoxycarbonyl (Fmoc) groups.

[0055] Examples of activated carboxyl groups include acid anhydrides corresponding to the carboxyl groups, azides, and activated esters with pentafluorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, etc. Examples of activated amino groups include phosphoric acid amides corresponding to the amino groups.

[0056] Condensation reactions during peptide synthesis are usually carried out in a solvent. Examples of such solvents include chloroform, dichloromethane, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, N-methylpyrrolidone, water, methanol, and mixtures thereof. The reaction temperature for the condensation reaction is usually within the range of -30°C to 50°C.

[0057] Furthermore, the type of protecting group elimination reaction in the peptide production process can be selected depending on the type of protecting group used, as long as it can eliminate the protecting group without affecting the peptide bond. Examples of such reactions include acid treatment with hydrogen chloride, hydrogen bromide, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof; alkali treatment with sodium hydroxide, potassium hydroxide, hydrazine, diethylamine, piperidine, or the like; sodium treatment in liquid ammonia; reduction with palladium on carbon; and silylation treatment with trimethylsilyl triflate, trimethylsilyl bromide, or the like. In the deprotection reaction using the above acid or silylating agent treatment, it is preferable to add a cation scavenger such as anisole, phenol, cresol, thioanisole, or ethanedithiol, from the viewpoint of efficient deprotection.

[0058] The cleavage of peptides synthesized by the solid-phase method from the solid phase can also be performed according to commonly known methods. For example, the cleavage method can include treatment with the above-mentioned acid or silylating agent. After the completion of the above series of reactions, the peptides produced in this manner can be subjected to commonly known separation and purification procedures. For example, the peptides can be obtained with higher purity by extraction, distribution, reprecipitation, recrystallization, solid-phase extraction, column chromatography, etc.

[0059] The N- and C-terminal modifications of the peptide of the present invention can be carried out by conventionally known methods. When synthesizing a peptide by solid-phase synthesis, N-terminal modification can be achieved by deprotecting the last amino acid residue and then introducing a desired functional group (linker). Furthermore, C-terminal modification can be achieved by solid-phase synthesis using, for example, Rink amide resin, a resin for synthesizing amides, to obtain an amide form of the peptide.

[0060] The peptide of the present invention may be isolated or purified. "Isolated or purified" means that a procedure for removing components other than the target component has been performed. The purity of the isolated or purified peptide compound of the present invention is usually 50% or more (e.g., 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%).

[0061] When the target-binding moiety of the peptide compound according to the present invention is an antibody, the method for linking the antibody to the peptide is not particularly limited, and a conventionally known method such as a click reaction can be used. For example, a method can be used in which an antibody into which dibenzocyclooctyne (DBCO) has been introduced is linked to a peptide into which an azide group has been introduced by a click reaction.

[0062] In the peptide compound according to the present invention, when the target-binding moiety is a low-molecular-weight compound that binds to a protein, the method for binding the low-molecular-weight compound to the peptide is not particularly limited, and any conventionally known reaction can be used, such as an amide bond formation reaction, an ester bond formation reaction, a disulfide bond formation reaction, or a covalent bond formation reaction by click chemistry.

[0063] <Pharmaceutical Composition> One embodiment of the present invention is a pharmaceutical composition comprising the above-described peptide compound according to the present invention or a pharmaceutically acceptable salt thereof.

[0064] The pharmaceutical composition according to this embodiment may be composed of one or more peptide compounds according to the present invention or pharmaceutically acceptable salts thereof, or may be composed of one or more peptide compounds according to the present invention or one or more pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier.

[0065] Pharmaceutically acceptable carriers include, but are not limited to, excipients such as lactose, sucrose, mannitol, starch, corn starch, crystalline cellulose, and light anhydrous silicic acid; lubricants such as silica, talc, calcium stearate, and magnesium stearate; binders such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, polyvinylpyrrolidone, crystalline cellulose, dextrin, and gelatin; antioxidants such as ascorbic acid, sodium sulfite, sodium bisulfite, and tocopherol; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as borate, bicarbonate, Tris-HCl, citrate, phosphate, and other organic acids; water for injection, physiological saline, ethanol, propanediol, and the like. Examples of suitable surfactants include solvents such as alcohol, ethylene glycol, propylene glycol, macrogol, olive oil, and corn oil; surfactants or humectants such as Pluronic®, polyethylene glycol, sorbitan fatty acid esters, polysorbates, Triton®, lecithin, cholesterol, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; isotonicity agents such as sodium chloride, potassium chloride, glycerin, glucose, sorbitol, and mannitol; preservatives such as benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, and chlorhexidine; complexing agents; amino acids; antimicrobial agents; colorants; flavoring agents and diluents; emulsifiers; salt-forming counterions such as sodium; delivery vehicles; and diluents (Remington's Pharmaceutical Sciences, 18th ed., edited by A.R. Gennaro, Mack Publishing Company, 1990).

[0066] Examples of diseases to which the pharmaceutical composition according to this embodiment can be applied include cancer, Alzheimer's disease, Parkinson's disease, metabolic disorder-related diseases (e.g., MASH and MAFLD), and hereditary muscular atrophy diseases (e.g., muscular dystrophy).

[0067] The pharmaceutical composition of this embodiment can be used in any mammal or bird, preferably a mammal or bird suffering from cancer. Mammals include primates such as humans, monkeys, gorillas, chimpanzees, and orangutans, as well as non-human mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, camels, and goats. Birds include chickens, quails, and pigeons.

[0068] The pharmaceutical composition according to the present embodiment can be provided as an oral agent, an external agent, an injection, an inhalant, a nasal drop, an eye drop, or the like, and can be made into a desired dosage form such as a tablet, a liquid, an injection, an ointment, a cream, a lotion, an aerosol, or a suppository depending on the method of use.

[0069] The content of the peptide compound according to the present invention in the pharmaceutical composition can be 0.01 to 100% by mass based on the total mass of the pharmaceutical composition.

[0070] <Proteolysis inducers and methods for inducing proteolysis> One embodiment of the present invention is a protein degradation inducer comprising the peptide compound of the present invention or a pharmaceutically acceptable salt thereof. Another embodiment of the present invention is a method for inducing protein degradation, comprising contacting a peptide compound of the present invention or a pharmaceutically acceptable salt thereof with at least one of a membrane protein and an extracellular protein of a eukaryotic cell in the presence of the eukaryotic cell. The protein inducer and method for inducing protein degradation according to this embodiment can be used to induce target (e.g., protein) degradation in vivo or in vitro. For example, when used in vitro for testing, research, or the like, the degradation of a desired membrane protein or extracellular protein can be induced by adding the peptide compound of the present invention to a solution containing eukaryotic cells having the desired membrane protein, or by adding the peptide compound of the present invention to a solution containing eukaryotic cells and the desired extracellular protein. For example, when used in vivo for the purpose of treating a disease, the degradation of the target protein can be induced by selecting an administration route, administration method, etc., as appropriate for the disease being treated.

[0071] The protein inducer according to this embodiment may be composed of one or more peptide compounds according to the present invention or pharmaceutically acceptable salts thereof, or may be composed of one or more peptide compounds according to the present invention or one or more pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier.

[0072] The pharmaceutically acceptable carriers are as described above.

[0073] The content of the peptide compound according to the present invention in the proteolysis inducer may be 0.01 to 100% by mass based on the total mass of the proteolysis inducer.

[0074] As described above, the peptide compound according to the present invention can induce protein degradation without being affected by whether or not the shuttling receptor is expressed, and therefore, there are no particular limitations on the eukaryotic cells used in the method for inducing protein degradation according to this embodiment.

[0075] <Embodiment> The following describes an embodiment of the present invention. [1] A peptide compound comprising a target-binding portion, a cell membrane-penetrating peptide portion, and a lysosome-targeting peptide portion. [2] The peptide compound according to [1] above, wherein the target is a protein. [3] The peptide compound according to [2] above, wherein the target-binding moiety comprises an antibody, a protein-binding peptide, or a small molecule compound that binds to a protein. [4] The lysosome-inducing peptide portion is X 1 X 2 X 3 X 4 The amino acid sequence represented by X 1 is a Tyr or Ala residue, X 2 and X 3 are each independently any amino acid residue, X 4 is an amino acid residue selected from the group consisting of Leu, Ile, Val, Phe, Tyr, Trp, Met, Cys, Gly and Ala, The peptide compound according to any one of [1] to [3] above, wherein the lysosome-inducible peptide moiety has 6 to 19 amino acid residues. [5] X 1 is a Tyr residue, X 2 is an amino acid residue selected from the group consisting of Gln, Glu, Asp, Asn, Met, His, Lys, Arg, Thr, Ser, Val, and Ala; X 3is an amino acid residue selected from the group consisting of Arg, Lys, His, Thr, Ser, Gln, Asp, Glu, Asn, Val, Leu, Ile, Ala, Gly, and Pro; X 4 is an amino acid residue selected from the group consisting of Leu, Ile, Val, Phe, Met, and Ala. [6] X 1 X 2 X 3 X 4 The peptide compound according to [4] or [5] above, wherein the amino acid sequence represented by the formula (I) consists of an amino acid sequence represented by any one of SEQ ID NOs: 37 to 63. [7] The peptide compound according to any one of [1] to [6] above, wherein the lysosome-inducing peptide portion comprises a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 10 to 18. [8] The peptide compound according to any one of [1] to [7] above, wherein the cell membrane-permeable peptide portion comprises an amino acid sequence represented by any one of SEQ ID NOs: 19 to 33. [9] A pharmaceutical composition comprising the peptide compound or a pharmaceutically acceptable salt thereof according to any one of [1] to [8] above.

[10] A proteolysis inducer comprising the peptide compound according to any one of the above [1] to [8] or a pharmaceutically acceptable salt thereof. [Example]

[0076] The present invention will be described below using specific examples, but the present invention is not limited to these examples.

[0077] Peptide synthesis The peptides listed in Table 5 were manually synthesized by Fmoc solid-phase synthesis, and the C-terminus was prepared in the amide form.

[0078] First, the resin (4-(2,4-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin, rink amide resin) was placed in a PD-10 column (Cytiva), and dimethylformamide (DMF) containing 20% ​​(v / v) piperidine was added. The column was then shaken for 3 hours to deprotect the Fmoc group of the resin. The resin was then washed with DMF, and Fmoc-protected amino acids and 1-hydroxybenzotriazole (HOBt) were added to the DMF. Condensation was then carried out using diisopropylcarbodiimide with stirring at room temperature for 1 hour. Subsequently, DMF containing 20% ​​piperidine was added to deprotect the Fmoc group of the amino acid. The peptide was then elongated by repeating the condensation of each amino acid and deprotection of the Fmoc group of the amino acid. Finally, the column was dried by washing with methanol and diethyl ether.

[0079] Next, a mixture of trifluoroacetic acid (TFA): m-cresol: 1,2-ethanedithiol: thioanisole: Milli-Q water (80:5:5:5:5 v / v / v / v / v) was added to the resin-containing column and incubated at room temperature for 3 hours to simultaneously remove the peptide side chain protecting groups and desorb the resin. The resin was removed through a filter, and diethyl ether was added to the filtrate. The precipitate was centrifuged at 3000 rpm for 10 minutes. The resulting precipitate was washed twice with diethyl ether. The precipitate was dried at room temperature and dissolved in an appropriate amount of aqueous acetic acid containing 0.1% TFA to obtain a crude peptide solution. The resulting crude peptide solution was purified by reverse-phase HPLC using a mobile phase of Milli-Q water containing 0.1% TFA and acetonitrile containing 0.1% TFA. The purified peptide solution was lyophilized to obtain a white, fluffy powder (peptide).

[0080] The purity of the peptide was confirmed using analytical HPLC (Shimadzu Corporation), and its molecular weight was confirmed by mass spectrometry (HRMS).

[0081] [Table 5]

[0082] In the peptides listed in Table 5, double underlining indicates the target-binding portion, bolding indicates the cell membrane-penetrating peptide portion, and single underlining indicates the lysosome-targeting peptide portion.

[0083] Preparation of dibenzocyclooctyne (DBCO)-modified antibodies A solution containing DBCO-modified antibodies was prepared by reacting 2 equivalents of DBCO-sulfo-NHS (CAS No.: 1400191-52-7) with various antibodies (trastuzumab, cetuximab, and atezolizumab; 5 mg / mL) in PBS overnight at 4°C. After the reaction was completed, unreacted DBCO-sulfo-NHS was removed from the solution using an ultrafiltration membrane (10,000 Da MWCO) to prepare the DBCO-modified antibodies.

[0084] Preparation of DBCO-modified antibody-peptide conjugates (peptide compounds) The DBCO-modified antibody was added to serum-free cell culture medium DMEM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.; hereinafter simply referred to as "serum-free medium") to a final concentration of 100 nM. Subsequently, a peptide (50 mM DMSO solution) consisting of the amino acid sequence represented by SEQ ID NO: 7, 8, or 9 (Table 6) was added to a final DMSO concentration of 0.1% or less, and the mixture was incubated at 37°C for 1 hour to prepare peptide compounds 1 to 5. The target-binding moiety, cell membrane-penetrating peptide moiety, and lysosome-targeting peptide moiety contained in peptide compounds 1 to 5 are shown in Table 7. The synthesized peptide compounds were confirmed by separation by SDS-PAGE followed by CBB staining.

[0085] [Table 6]

[0086] The peptide consisting of the amino acid sequence shown in SEQ ID NO: 7, 8, or 9 is a peptide having an azide group at the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, 5, or 6. The azide modification of the N-terminus was carried out by condensing azidoacetic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) with the N-terminal amino group in the final step of Fmoc solid-phase synthesis.

[0087] [Table 7]

[0088] Test Example 1: Evaluation of concentration-dependent degradation activity of peptide compounds targeting epidermal growth factor receptor (EGFR) protein A549 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 1 (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, or 25 μM), DBCO-modified cetuximab (100 nM), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, or 25 μM), or DBCO-modified cetuximab (100 nM) was added and incubated for 24 hours at 37°C. After incubation, whole cells were lysed in 1x SDS sample buffer and heated at 100°C for 10 minutes to prepare samples for SDS-PAGE. SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the samples were transferred to a PVDF membrane using a BioRad Transturbo Blot. Blocking was performed for 1 hour with 5% TBS-T solution. Antibody binding was performed (37°C, 1 hour) using a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-EGFR antibody, Cell Signaling Technology) or a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-GAPDH antibody, Santa Cruz Biotechnology). After washing with 5% TBS-T solution, the secondary antibody solution diluted 1000-fold with 5% TBS-T solution was treated in the same manner. After washing again with 5% TBS-T solution, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 1. In Figure 1, "NT" indicates the results for a sample containing no peptide compound 1, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, or DBCO-modified cetuximab, and "peptide of SEQ ID NO: 7" indicates the amino acid sequence represented by SEQ ID NO: 7.

[0089] As shown in FIG. 1, it can be seen that peptide compound 1 degraded the EGFR protein.

[0090] Test Example 2: Evaluation of concentration-dependent degradation activity of peptide compounds targeting PD-L1 protein MDA-MB-231 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 2 (1 μM, 5 μM, 10 μM, or 25 μM), DBCO-modified atezolizumab (100 nM), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 (1 μM, 5 μM, 10 μM, or 25 μM), or DBCO-modified atezolizumab (100 nM) was added and incubated for 24 hours at 37°C. After incubation, whole cells were lysed in 1× SDS sample buffer and heated at 100°C for 10 minutes to prepare SDS-PAGE samples. SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the gel was transferred to a PVDF membrane using a BioRad Transturbo blot. Blocking was performed with 5% TBS-T solution for 1 hour. Antibody binding was performed with a primary antibody solution (anti-PD-L1 antibody, Proteintech Japan, Inc.) diluted 1:1000 in 5% TBS-T solution or a primary antibody solution (anti-GAPDH antibody, Santa Cruz Biotechnology, Inc.) diluted 1:1000 in 5% TBS-T solution (37°C, 1 hour). After washing with 5% TBS-T solution, secondary antibody solution (diluted 1:1000 in 5% TBS-T) was treated in the same manner. After washing again with 5% TBS-T solution, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 2. In Figure 2, "NT" indicates the results for a sample containing no peptide compound 2, the peptide consisting of the amino acid sequence represented by SEQ ID NO:7, or DBCO-modified atezolizumab, and "peptide of SEQ ID NO:7" indicates the amino acid sequence represented by SEQ ID NO:7.

[0091] As shown in Figure 2, it can be seen that peptide compound 2 degraded the PD-L1 protein on the cell surface.

[0092] Test Example 3: Evaluation of concentration-dependent degradation activity of peptide compounds targeting HER2 protein SKBR3 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 3 (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, or 25 μM), DBCO-modified trastuzumab (100 nM), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, or 25 μM), or DBCO-modified trastuzumab (100 nM) was added and incubated for 24 hours at 37°C. After incubation, whole cells were lysed in 1x SDS sample buffer and heated at 100°C for 10 minutes to prepare samples for SDS-PAGE. SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the samples were transferred to a PVDF membrane using a BioRad Transturbo Blot. Blocking was performed for 1 hour with 5% TBS-T solution. Antibody binding was performed (37°C, 1 hour) using a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-HER2 antibody, Cell Signaling Technology) or a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-GAPDH antibody, Santa Cruz Biotechnology). After washing with 5% TBS-T solution, the secondary antibody solution diluted 1000-fold with 5% TBS-T solution was treated in the same manner. After washing again with 5% TBS-T solution, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 3. In Figure 3, "NT" indicates the results for a sample containing no peptide compound 3, no peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, or no DBCO-modified trastuzumab, and "peptide of SEQ ID NO: 7" indicates the amino acid sequence represented by SEQ ID NO: 7.

[0093] As shown in FIG. 3, it can be seen that peptide compound 3 degraded the HER2 protein on the cell surface.

[0094] Test Example 4: Evaluation of time-dependent degradation activity of peptide compounds targeting HER2 protein SKBR3 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 3 (25 μM) and DBCO-modified trastuzumab (100 nM) were added and incubated at 37°C for 1, 3, 6, or 24 hours. After incubation, whole cells were lysed in 1x SDS sample buffer and heated at 100°C for 10 minutes to prepare SDS-PAGE samples. Furthermore, peptide compound 3 (25 μM) was added and the same procedure as above was repeated without incubation to prepare SDS-PAGE samples. SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the samples were transferred to a PVDF membrane using a BioRad Transturbo blot. Blocking was performed for 1 hour with 5% TBS-T solution. Antibody binding was performed (37°C, 1 hour) using a primary antibody solution diluted 1:1000 in 5% TBS-T (anti-HER2 antibody, Cell Signaling Technology) or a primary antibody solution diluted 1:1000 in 5% TBS-T (anti-ACTB antibody, Fujifilm Wako Pure Chemical Industries, Ltd.). After washing with 5% TBS-T, the secondary antibody solution diluted 1:1000 in 5% TBS-T was treated in the same manner. After washing again with 5% TBS-T, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 4.

[0095] As shown in FIG. 4, it can be seen that peptide compound 3 exhibited time-dependent HER2 proteolytic activity.

[0096] Test Example 5: Evaluation of time-dependent degradation activity of peptide compounds targeting EGFR protein Cultured A549 cells were seeded in a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 1 (25 μM) and DBCO-modified cetuximab (100 nM) were added and incubated at 37°C for 1, 3, 6, or 24 hours. After incubation, whole cells were lysed in 1x SDS sample buffer and heated at 100°C for 10 minutes to prepare SDS-PAGE samples. Furthermore, peptide compound 1 (25 μM) was added and the same procedure as above was repeated without incubation to prepare SDS-PAGE samples. SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the samples were transferred to a PVDF membrane using a BioRad Transturbo blot. Blocking was performed for 1 hour with 5% TBS-T solution. Antibody binding was performed (37°C, 1 hour) using a primary antibody solution diluted 1:1000 in 5% TBS-T (anti-EGFR antibody, Cell Signaling Technology) or a primary antibody solution diluted 1:1000 in 5% TBS-T (anti-ACTB antibody, Fujifilm Wako Pure Chemical Industries, Ltd.). After washing with 5% TBS-T, the secondary antibody solution diluted 1:1000 in 5% TBS-T was treated in the same manner. After washing again with 5% TBS-T, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 5.

[0097] As shown in FIG. 5, it can be seen that peptide compound 1 exhibited time-dependent EGFR proteolytic activity.

[0098] Test Example 6: Analysis of changes in HER2 protein localization by immunostaining SKBR3 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 3 (10 μM), DBCO-modified trastuzumab (100 nM), a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 (10 μM), or DBCO-modified trastuzumab (100 nM) was added and incubated for 24 hours at 37°C. After incubation, the cells were washed three times with phosphate-buffered saline (PBS) and then fixed by incubating in 4% PFA / PBS for 15 minutes at room temperature. Blocking was then performed by adding 3% BSA / PBS and incubating at room temperature for 1 hour. Fluorescently modified anti-HER2 antibodies were then added and incubated at room temperature for 1 hour. After washing with PBS, nuclear staining was performed with DAPI dissolved in PBS for 15 minutes at room temperature. After washing three times with PBS, fluorescence imaging was performed directly using a Keyence Corporation BZ-X800 fluorescence microscope. The results are shown in Figure 6. In Figure 6, "No Treatment" shows the results for a sample to which none of peptide compound 3, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 7, and DBCO-modified trastuzumab was added, and "peptide of SEQ ID NO: 7" shows the amino acid sequence represented by SEQ ID NO: 7. In Figure 6, the HER2 protein is shown in green, and the nucleus is shown in blue.

[0099] As shown in Figure 6, it can be seen that the HER2 protein on the cell surface was degraded by peptide compound 3. On the other hand, no degradation of the HER2 protein was observed with the peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 or DBCO-modified trastuzumab.

[0100] Test Example 7: Evaluation of HER2 protein degradation activity under lysosomal inhibition conditions with chloroquine SKBR3 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37° C. After washing once with serum-free medium, the cells were treated with serum-free medium containing chloroquine at the final concentrations shown in Table 8 (chloroquine-containing medium; 7-4 and 7-5), or with serum-free medium alone without chloroquine (chloroquine-free medium; 7-1 to 7-3). After incubation at 37°C for 1 hour, peptide compound 3 and DBCO-modified trastuzumab were added to chloroquine-containing medium to the final concentrations shown in Table 8 (7-4 and 7-5), or chloroquine-free medium without peptide compound 3 or DBCO-modified trastuzumab (7-1), chloroquine-free medium with DBCO-modified trastuzumab (7-2), or chloroquine-free medium with peptide compound 3 and DBCO-modified trastuzumab (7-3) to the final concentrations shown in Table 8, and the mixture was incubated at 37°C for 24 hours. After incubation, whole cells were lysed in 1x SDS sample buffer and heated at 100°C for 10 minutes to prepare SDS-PAGE samples (Samples 7-1 to 7-5). SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the samples were transferred to a PVDF membrane using a BioRad Transturbo blot. Blocking was performed for 1 hour with 5% TBS-T solution. Antibody binding was performed (37°C, 1 hour) using a primary antibody solution diluted 1:1000 in 5% TBS-T solution (anti-HER2 antibody, Cell Signaling Technology) or a primary antibody solution diluted 1:1000 in 5% TBS-T solution (anti-GAPDH antibody, Santa Cruz Biotechnology). After washing with 5% TBS-T solution, the secondary antibody solution diluted 1:1000 in 5% TBS-T solution was treated in the same manner. After washing again with 5% TBS-T solution, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 7.

[0101] [Table 8]

[0102] Lysosomal proteolytic activity is known to be inhibited by chloroquine treatment (Mauthe, M. et al., Autophagy, 14, 1435-1455 (2018)). As shown in Figure 7, the addition of chloroquine inhibited the degradation of HER2 protein, whereas the addition of peptide compound 3 without chloroquine resulted in the degradation of HER2 protein. This indicates that HER2 protein was directed to the lysosome for degradation.

[0103] Test Example 8: Evaluation of EGFR proteolytic activity under lysosomal inhibition conditions with chloroquine A549 cells were seeded onto 96-well cell culture plates and cultured overnight at 37°C. After washing once with serum-free medium, the cells were treated with serum-free medium at the final concentrations listed in Table 9 (chloroquine-containing medium; 8-4 and 8-5), or with serum-free medium alone without chloroquine (chloroquine-free medium; 8-1 to 8-3). After 1 hour of incubation at 37°C, peptide compound 1 and DBCO-modified cetuximab were added to chloroquine-containing medium at the final concentrations listed in Table 9 (8-4 and 8-5), or chloroquine-free medium without peptide compound 1 and DBCO-modified cetuximab (8-1), chloroquine-free medium with DBCO-modified cetuximab (8-2), or chloroquine-free medium with peptide compound 3 and DBCO-modified cetuximab (8-3), and then incubated at 37°C for 24 hours. After incubation, whole cells were lysed in 1x SDS sample buffer and heated at 100°C for 10 minutes to prepare SDS-PAGE samples (Samples 8-1 to 8-5). SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the gel was transferred to a PVDF membrane using a BioRad Transturbo blot. Blocking was performed for 1 hour with 5% TBS-T solution. Antibody binding was performed (37°C for 1 hour) using a primary antibody solution diluted 1:1000 in 5% TBS-T (anti-EGFR antibody, Cell Signaling Technology) or a primary antibody solution diluted 1:1000 in 5% TBS-T (anti-GAPDH antibody, Santa Cruz Biotechnology). After washing with 5% TBS-T, secondary antibody solution diluted 1:1000 in 5% TBS-T was treated in the same manner. After washing again with 5% TBS-T solution, detection was carried out using a LuminoGraph manufactured by ATTO Corporation. The results are shown in Figure 8.

[0104] [Table 9]

[0105] 8, the addition of chloroquine inhibited the degradation of EGFR protein, whereas the addition of peptide compound 1 without the addition of chloroquine resulted in the degradation of EGFR protein, indicating that the EGFR protein was directed to lysosomes for degradation.

[0106] Test Example 9: Evaluation of concentration-dependent degradation activity of peptide compounds targeting EGFR protein As peptide compound 6, a peptide consisting of the amino acid sequence shown in SEQ ID NO:2 was used.

[0107] Cultured A549 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 6 (10 μM, 25 μM, or 50 μM) was added and incubated for 24 hours at 37°C. After incubation, whole cells were lysed in 1× SDS sample buffer and heated at 100°C for 10 minutes to prepare SDS-PAGE samples. Additionally, SDS-PAGE samples were prepared by the same procedure as above after incubation without peptide compound 6. SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the gel was transferred to a PVDF membrane using a BioRad Transturbo blot. Blocking was performed for 1 hour with 5% TBS-T solution. Antibody binding was performed (37°C, 1 hour) using a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-EGFR antibody, Cell Signaling Technology) or a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-ACTB antibody, Fujifilm Wako Pure Chemical Industries, Ltd.). After washing with 5% TBS-T solution, secondary antibody solution diluted 1000-fold with 5% TBS-T solution was treated in the same manner. After washing again with 5% TBS-T solution, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 9.

[0108] As shown in FIG. 9, it can be seen that peptide compound 6, whose target-binding moiety (EGFR protein-binding moiety) is a peptide, can degrade EGFR protein in a concentration-dependent manner.

[0109] Test Example 10: Evaluation of concentration-dependent degradation activity of peptide compounds targeting HER2 protein As peptide compound 7, a peptide consisting of the amino acid sequence shown in SEQ ID NO:3 was used.

[0110] SKBR3 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 7 (10 μM, 25 μM, or 50 μM) was added and incubated for 24 hours at 37°C. After incubation, whole cells were lysed in 1× SDS sample buffer and heated at 100°C for 10 minutes to prepare SDS-PAGE samples. Additionally, SDS-PAGE samples were prepared by the same procedure as above after incubation without peptide compound 7. SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the samples were transferred to a PVDF membrane using a BioRad Transturbo blot. Blocking was performed for 1 hour with 5% TBS-T solution. Antibody binding was performed (37°C, 1 hour) using a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-HER2 antibody, Cell Signaling Technology) or a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-ACTB antibody, Fujifilm Wako Pure Chemical Industries, Ltd.). After washing with 5% TBS-T solution, secondary antibody solution diluted 1000-fold with 5% TBS-T solution was treated in the same manner. After washing again with 5% TBS-T solution, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 10.

[0111] As shown in FIG. 10, it can be seen that peptide compound 7, whose target-binding moiety (HER2 protein-binding moiety) is a peptide, can degrade HER2 protein in a concentration-dependent manner.

[0112] Test Example 11: Evaluation of concentration-dependent degradation activity of target protein of peptide compounds targeting integrin αv As peptide compound 8, a peptide consisting of the amino acid sequence shown in SEQ ID NO:4 was used.

[0113] HeLa cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 8 (5 μM, 10 μM, 25 μM, or 50 μM) was added and incubated for 24 hours at 37°C. After incubation, whole cells were lysed in 1× SDS sample buffer and heated at 100°C for 10 minutes to prepare SDS-PAGE samples. SDS-PAGE samples were also prepared by incubation without peptide compound 8 and the same procedure as above. SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the gel was transferred to a PVDF membrane using a BioRad Transturbo blot. Blocking was performed for 1 hour with 5% TBS-T solution. Antibody binding was performed (37°C, 1 hour) using a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-integrin αv antibody, Cell Signaling Technology) or a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-GAPDH antibody, Santa Cruz Biotechnology). After washing with 5% TBS-T solution, secondary antibody solution diluted 1000-fold with 5% TBS-T solution was treated in the same manner. After washing again with 5% TBS-T solution, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 11.

[0114] As shown in FIG. 11, it can be seen that peptide compound 8, whose target-binding moiety (integrin αv-binding moiety) is a peptide, can degrade integrin αv in a concentration-dependent manner.

[0115] Test Example 12: Examination of the amino acid sequence of the lysosome-inducing peptide portion of peptide compounds SKBR3 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 3 (50 μM) and DBCO-modified Herceptin (100 nM), peptide compound 4 (50 μM) and DBCO-modified Herceptin (100 nM), peptide compound 5 (50 μM) and DBCO-modified Herceptin (100 nM), or DBCO-modified Herceptin (100 nM) was added and incubated for 24 hours at 37°C. After incubation, whole cells were lysed in 1x SDS sample buffer and heated at 100°C for 10 minutes to prepare samples for SDS-PAGE. SDS-PAGE was performed using a 5-20% gradient gel at a constant voltage of 20 mA. After electrophoresis, the gel was transferred to a PVDF membrane using a BioRad Transturbo blot. Blocking was performed with 5% TBS-T solution for 1 hour. Antibody binding was performed (37°C, 1 hour) using a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-HER2 antibody, Cell Signaling Technology) or a primary antibody solution diluted 1000-fold with 5% TBS-T solution (anti-GAPDH antibody, Santa Cruz Biotechnology). After washing with 5% TBS-T solution, secondary antibody solution diluted 1000-fold with 5% TBS-T solution was treated in the same manner. After washing again with 5% TBS-T solution, detection was performed using a LuminoGraph (ATTO Corporation). The results are shown in Figure 12. In Figure 12, "No Treatment" indicates the results for samples to which neither peptide compounds 3 to 5 nor DBCO-modified Herceptin was added.

[0116] As shown in Figure 12, peptide compound 4, in which the tyrosine residue in "YQRL" (YXXΦ peptide) contained in peptide compound 3 was replaced with an alanine residue, and peptide compound 5, in which the leucine residue was replaced with an alanine residue, exhibited HER2 proteolytic activity. Comparing peptide compounds 3 to 5 revealed that the amino acids "Y" and "Φ" in the YXXΦ peptide are important for HER2 proteolytic activity.

[0117] Test Example 13: Evaluation of FITC-modified streptavidin (extracellular protein model) uptake activity As peptide compound 9, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 with biotin added to the N-terminus was used.

[0118] SKBR3 cells were seeded onto a 96-well cell culture plate and cultured overnight at 37°C. After washing once with serum-free medium, peptide compound 9 (10 μM) and FITC-modified streptavidin (500 nM) or FITC-modified streptavidin (500 nM) were added and incubated overnight at 37°C. After incubation, the plate was washed three times with phosphate-buffered saline (PBS). Nuclear staining was performed with DAPI dissolved in PBS for 15 minutes at room temperature. After washing once more with PBS, fluorescent imaging was performed directly using a Keyence BZ-X800 fluorescence microscope. The results are shown in Figure 13. In Figure 13, "No Treatment" indicates the results for a sample to which neither peptide compound 9 nor FITC-modified streptavidin was added. In Figure 13, FITC-modified streptavidin is shown in green, and nuclei are shown in blue.

[0119] As shown in FIG. 13, it is clear that peptide compound 9 can induce the uptake of an extracellular protein (streptavidin) into cells and its degradation.

Claims

1. A peptide compound comprising a target-binding portion, a cell membrane-penetrating peptide portion, and a lysosome-targeting peptide portion.

2. The peptide compound of claim 1 , wherein the target is a protein.

3. The peptide compound of claim 2 , wherein the target-binding moiety comprises an antibody, a protein-binding peptide, or a small molecule compound that binds to a protein.

4. The lysosome-inducing peptide portion is X １ X ２ X ３ X ４ The amino acid sequence represented by X １ is a Tyr or Ala residue, X ２ and X ３ are each independently any amino acid residue, X ４ is an amino acid residue selected from the group consisting of Leu, He, Val, Phe, Tyr, Trp, Met, Cys, Gly and Ala; The peptide compound according to claim 1, wherein the lysosome-inducing peptide portion has 6 to 19 amino acid residues.

5. The X １ is a Tyr residue, The X ２ is an amino acid residue selected from the group consisting of Gln, Glu, Asp, Asn, Met, His, Lys, Arg, Thr, Ser, Val, and Ala; The X ３ is an amino acid residue selected from the group consisting of Arg, Lys, His, Thr, Ser, Gln, Asp, Glu, Asn, Val, Leu, Ile, Ala, Gly, and Pro; The X ４ is an amino acid residue selected from the group consisting of Leu, Ile, Val, Phe, Met, and Ala.

6. The X １ X ２ X ３ X ４ The peptide compound according to claim 4, wherein the amino acid sequence represented by the formula (I) consists of an amino acid sequence represented by any one of SEQ ID NOs: 37 to 63.

7. The peptide compound according to claim 1, wherein the lysosome-inducing peptide portion comprises a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 10 to 18.

8. The peptide compound according to claim 1, wherein the cell membrane-permeable peptide portion comprises an amino acid sequence represented by any one of SEQ ID NOs: 19 to 33.

9. A pharmaceutical composition comprising the peptide compound of claim 1 or a pharmaceutically acceptable salt thereof.

10. A protein degradation inducer comprising the peptide compound of claim 1 or a pharmaceutically acceptable salt thereof.

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