JP7572710B2 - peptide - Google Patents

Description

Patent Law Article 30, Paragraph 2 Application Publication 2018 Master's Thesis Public Review Session "Abstract Collection" Issued February 8, 2019

Patent Law Article 30, Paragraph 2 applies Meeting name and location 2018 Master's Thesis Public Examination Nagahama Institute of Bio-Sciences (1266 Tamura-cho, Nagahama, Shiga Prefecture) Date February 13, 2019

The present invention relates to a peptide.

The proteasome recognizes and degrades ubiquitin modifications that are added to proteins as a marker for degradation, enabling it to degrade specific proteins at specific times. This ubiquitin-proteasome system is a major pathway that negatively regulates the abundance of proteins in cells. The ubiquitin-proteasome system plays a central role in a wide range of biological phenomena, including the cell cycle, gene expression, signal transduction, and immune responses.

Proteasome inhibitors are positioned as anticancer drugs and are used to treat hematological malignancies.

Bortezomib was the first proteasome inhibitor to be used clinically and has become the standard treatment for multiple myeloma in combination therapy.

Bortezomib reversibly inhibits the chymotrypsin-like activity of the proteasome. However, after a certain period of treatment, resistance often develops due to mutations in the β subunit (Non-Patent Document 1). The second generation, carfilzomib, is an irreversible proteasome inhibitor. Carfilzomib shows higher selectivity for chymotrypsin-like activity than bortezomib (Non-Patent Document 2). The third generation, ixazomib, can be administered orally. Other inhibitors in the development stage include marizomib, an irreversible inhibitor (Non-Patent Document 3), and delanzomib, which can be administered orally (Non-Patent Document 4).

E. Suzuki, et al (2011): Molecular Mechanisms of Bortezomib Resistant Adenocarcinoma Cells. PLoS One, 6: e27996, doi: 10.1371/journal.pone.0027996. S.D. Demo, et al (2007): Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome. Cancer Res., 67, pp. 6383-6391 D. Chauhan, T. Hideshima, and K.C. Anderson (2006): A novel proteasome inhibitor NPI-0052 as an anticancer therapy. Br. J. Cancer, 95, pp. 961-965 R. Piva, B. Ruggeri, et al, (2008): CEP-18770: a novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib. Blood, 111, pp. 2665-2675

Combination therapy using proteasome inhibitors has significantly improved the life expectancy of multiple myeloma patients. However, the problem of cancer cells developing resistance to proteasome inhibitors has not been overcome. Therefore, it is necessary to either overcome resistance or develop new proteasome inhibitors that have a different mechanism of inhibition from the proteasome that has developed resistance.

The objective of the present invention is to provide a composition having proteasome inhibitory activity with an inhibitory mechanism different from conventional ones, in preparation for the development of resistance to the existing proteasome inhibitors.

As a result of extensive research, the present inventors have discovered a peptide having proteasome inhibitory activity with an inhibitory mechanism different from conventional mechanisms.
The present invention has been completed based on this finding, and includes the following aspects.
Item 1. A peptide having an effect of inhibiting the protease activity of the proteasome 20S core, comprising the 1st to 7th, the 2nd to 7th, the 3rd to 7th, the 1st to 6th, the 2nd to 6th, or the 3rd to 6th amino acid residues of the following amino acid sequence: Ser- ^Xaa1 -Leu- ^Xaa2 -His-Trp-Trp (SEQ ID NO: ^{1 )} ;
Item 2. The peptide according to Item 1, further comprising any amino acid residue, ^Xaa3 , at the C-terminus of the amino acid sequence represented by SEQ ID NO:1.
Item 3. The peptide according to Item 1 or 2, further comprising an amino acid sequence Met- ^Xaa4 - ^Xaa5 -Pro- (SEQ ID NO: 5) at the N-terminus of the amino acid sequence represented by SEQ ID NO: 1, wherein ^Xaa4 and ^Xaa5 represent any amino acid residue.
Item 4. A polynucleotide comprising a base sequence encoding the peptide according to any one of Items 1 to 3.
Item 5. A vector comprising the polynucleotide according to Item 4 in an expressible manner.
Item 6. A composition comprising the peptide according to any one of items 1 to 3, the polynucleotide according to item 4, or the vector according to item 5.
Item 7. The composition according to Item 6, which is a pharmaceutical composition.
Item 8. The composition according to Item 7, wherein the pharmaceutical composition is used for the prevention and/or treatment of a malignant tumor.
Item 9. A method for protecting a target peptide from degradation by the proteasome 20S core, comprising binding the peptide according to any one of items 1 to 3 to the target peptide.

The present invention provides a peptide that inhibits the protease activity of the proteasome 20S core.

The results of analyzing the inhibition pattern of the PI-Rank1 peptide are shown. (A) Lineweaver-Burk plots of the PI-Rank1 peptide at each concentration. (B) Dixon plots of the PI-Rank1 peptide at each concentration. The Ki values of PI-Rank1 for chymotrypsin-like activity and caspase-like activity are shown. The inhibitory effect of N-terminally or C-terminally deleted PI-Rank1(6-17) and PI-Rank1(1-12) is shown. (A) The _IC50 values of PI-Rank1(1-17), PI-Rank1(6-17) and PI-Rank1(1-12) against the CT-L activity of the proteasome 20S are shown. (B) The concentration-dependent inhibition curves of each peptide against CT-L activity are shown. The graph shows the change in 20S enzyme inhibitory activity (50% inhibitory concentration (IC ₅₀ )) of PI-Rank1 peptide analogs in which amino acids were deleted stepwise from the N-terminus or C-terminus. The graph shows changes in 20S enzyme inhibitory activity (50% inhibitory concentration (IC ₅₀ )) of PI-Rank1 peptide analogs in which each amino acid was replaced with alanine. (A) A graph showing the inhibitory activity shown in Figure 3. (B) A graph showing the inhibitory activity shown in Figure 4. 1 shows the inhibitory effect of PI-Rank1(5-12) on the 26S proteasome. (A) Binding of 20S core to PI-Rank1(5-12) (B) Binding of 20S core or 26S proteasome to PI-Rank1(5-12). (A) The results of two-dimensional electrophoresis analysis to identify the site that binds to PI-Rank1(5-12) among the subunits that make up the 20S core. (B) A schematic diagram of the three-dimensional structures of the α subunit and β subunit from the side. (C) A cross-sectional view of the α subunit at the α1 subunit site. (A) A schematic diagram of the binding mode between Rpt3 and the β1 subunit. (B) A cross-sectional view of (A). (C) The Rpt3-binding pocket of the α1 subunit and the three-amino acid residue binding pocket on the C-terminus of Rpt3 are shown. (D) A comparison of the C-terminal amino acid sequences of Rpt3 and PI-Rank1(5-12). In (D), "acid" indicates acidic amino acids, "neutral" indicates neutral amino acids, "salt" indicates basic amino acids, and "aromatic" indicates aromatic amino acids. Additionally, (parent) indicates hydrophilic amino acids, and (phobic) indicates hydrophobic amino acids. The effect of the C-terminal peptide of Rpt3 and PI-Rank1(5-12) on 20S enzyme activity is shown. (A) Changes in substrate degradation over time are shown. The dotted line indicates CT-L activity in the absence of PI-Rank1(5-12), and the solid line indicates CT-L activity in the absence of PI-Rank1(5-12). (B) The promotion of 20S enzyme activity by the C-terminal peptide of Rpt3 in the presence and absence of PI-Rank1(5-12) is shown. The effect of cell membrane permeable PI-Rank1(5-12) is shown. (A) List of compounds used in the experiment. [AC] indicates acetylation. (B) ATP amount (cell viability) 24 hours after addition of each compound to multiple myeloma cell line is shown. (C) ATP amount (cell viability) 48 hours after addition of each compound to multiple myeloma cell line is shown. The figures show the degradation rates by human 20S proteasome of purified thioredoxin-fused human α-synuclein recombinant protein (referred to as "αSYN (Trx fusion)") and its C-terminally-added purified thioredoxin-fused human α-synuclein-PI-Rank1(1-12) adduct (referred to as "αSYN+Rank1 (Trx fusion)"). (A) shows the results of SDS-PAGE. In (A), "M" indicates the molecular weight marker. "-" indicates the negative control to which human purified 20S proteasome was not added. "1h" or "4h" indicates the reaction time (hours) after addition of human purified 20S proteasome. (B) shows the quantitative results obtained by image analysis of the concentration of the protein bands in (A). The degradation rates of thioredoxin-fused human α-synuclein-PI-Rank1(1-12) adduct (represented as "αSYN+Rank1 (Trx fusion)") and wild-type human α-synuclein recombinant polypeptide without additional sequences such as tags (represented as "αSYN recombinant") by the 20S proteasome are shown. (A) shows the results of SDS-PAGE. In (A), "M" indicates the molecular weight marker. "-" indicates the negative control to which human purified 20S proteasome was not added. "1h" or "4h" indicates the reaction time (hours) after the addition of human purified 20S proteasome. (B) shows the quantitative results obtained by image analysis of the concentration of the protein bands in (A).

1. Proteasome Inhibitory Peptides One embodiment of the present invention relates to a peptide (hereinafter also referred to as a "proteasome inhibitor peptide") that has the effect of inhibiting the protease activity of the proteasome 20S core. The 26S proteasome is present in cells and consists of a complex of a 20S core having protease activity and 19S associated with both ends of the 26S core. In addition, it is known that some proteasome 20S cores exhibit protease activity without forming a complex with 19S.

The structures of the proteasome inhibitory peptides are shown below, with the amino acid sequences being written from the N-terminus to the C-terminus.
The amino acid sequence and physical properties are as shown in Table 1 below.

Amino acids include both natural and artificial amino acids.

In this specification, the "residue" of each amino acid is a structural unit of an amino acid that constitutes a peptide, and refers to a group obtained by removing a hydrogen atom from the amino group in the main chain and/or removing an -OH from the carboxyl group in the main chain of the amino acid.

In addition, in this specification, "having" can include both the concepts of "including" and "consisting of."

One embodiment of a proteasome inhibitor peptide relates to Ser-Xaa1-Leu-Xaa2-His-Trp-Trp (SEQ ID NO: ¹ ) (wherein Xaa1 and Xaa2 represent any amino acid residue) having as its backbone the 1st to 7th, the 2nd to 7th, the ^3rd to 7th, the 1st to 6th, the ^2nd to ^6th , or the 3rd to 6th amino acid residues of the following amino acid sequence:

Preferably, in the proteasome inhibitory peptide, Xaa ¹ and Xaa ² are the same or different hydrophilic amino acid residues. More preferably, Xaa ¹ and Xaa ² are the same or different basic amino acid residues, and even more preferably, Xaa ¹ and Xaa ² are both Arg residues. Most preferably, the first embodiment of the proteasome inhibitory peptide comprises an amino acid sequence represented by Ser-Arg-Leu-Arg-His-Trp-Trp (SEQ ID NO: 2) as the backbone.

The second embodiment of the proteasome inhibitor peptide has an arbitrary amino acid residue Xaa ³ at the C-terminal side of the amino acid sequence represented by SEQ ID NO: 1 as the main chain. That is, the second embodiment of the proteasome inhibitor peptide has an amino acid sequence represented by Ser-Xaa ¹ -Leu-Xaa ² -His-Trp-Trp-Xaa ³ (SEQ ID NO: 3) as the main chain. Xaa ³ is preferably a hydrophilic amino acid residue, more preferably a basic amino acid residue, and even more preferably an Arg residue. The most preferred second embodiment of the proteasome inhibitor peptide has an amino acid sequence represented by Ser-Arg-Leu-Arg-His-Trp-Trp-Arg (SEQ ID NO: 4) as the main chain.

The third embodiment of the proteasome inhibitor peptide has an amino acid sequence Met- ^Xaa4 - ^Xaa5- Pro- (SEQ ID NO: 5) on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 as the main chain. That is, the third embodiment of the proteasome inhibitor peptide has an amino acid sequence represented by Met- ^Xaa4 - ^Xaa5 -Pro-Ser- ^Xaa1 -Leu- ^Xaa2 -His-Trp-Trp (SEQ ID NO: 6) or Met- ^Xaa4 - ^Xaa5 -Pro-Ser-Xaa1 ^- Leu- ^Xaa2 -His-Trp-Trp- ^Xaa3 (SEQ ID NO: 7) as the main chain. Here, ^Xaa4 and ^Xaa5 are any amino acid residues. Preferably, either one of ^Xaa4 and ^Xaa5 may be a hydrophobic amino acid residue. More preferably, Xaa ⁴ is a hydrophobic amino acid residue and Xaa ⁵ is a hydrophilic amino acid residue. Most preferably, Xaa ⁴ and Xaa ⁵ are the same or different and are Ala or Arg residues. The most preferred third embodiment of the proteasome inhibitor peptide has an amino acid sequence represented by Met-Ala-Arg-Pro-Ser-Arg-Leu-Arg-His-Trp-Trp (SEQ ID NO: 8) or Met-Ala-Arg-Pro-Ser-Arg-Leu-Arg-His-Trp-Trp-Arg (SEQ ID NO: 9) as the backbone.

The main chain of the proteasome inhibitory peptide preferably has a total length of about 5 to 150 amino acid residues. The upper limit of the length of the proteasome inhibitory peptide is more preferably about 7 amino acid residues. The upper limit of the length of the proteasome inhibitory peptide is more preferably selected from about 100 amino acid residues, about 50 amino acid residues, about 20 amino acid residues, about 15 amino acids, or about 12 amino acids.

Methods for synthesizing peptides are known. For example, peptides can be synthesized by tert-butoxycarbonyl (Boc) solid-phase synthesis or 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis. When the total length of the main chain of the proteasome inhibitor peptide exceeds 100 amino acid residues, the peptide may be synthesized as a recombinant peptide using Escherichia coli, insect cells, mammalian cells, yeast, silkworms, or the like.

Proteasome inhibitory peptides may also include those in which the C-terminus is a carboxyl group (-COOH), a carboxylate ( ^-COO- ), or an ester (-COOR).

The proteasome inhibitor peptide may have modifications in addition to the main chain. There are no limitations on the modifications, so long as they do not inhibit the protease activity of the proteasome 20S core of the proteasome inhibitor peptide. The modifications may be present at at least one site selected from the N-terminus, C-terminus, and at least one side chain of an amino acid residue constituting the main chain.

Examples of N-terminal modifications include modifications generally used as N-terminal modifications of peptides, such as azido modification, _C1-6 acylation modification such as _C1-6 alkanoyl (acetylation modification, formylation modification, etc.), succinylation modification, Boc modification, Fmoc modification, biotinylation modification, myristination modification, palmitoylation modification, stearoylation modification, fluorescent dye modification (TAMRA label, FITC label, rhodamine label, FAM label, etc.), pyroglutamylation modification, dansylation modification, methylation modification, etc.

C-terminal modifications include those generally used as C-terminal modifications of peptides. For example, C-terminal modifications include amidation modification, biotinylation modification, methyl esterification modification, aldehyde modification, N-hydroxyesterification modification, pNA labeling, MCA labeling, β-naphthyl amidation modification, etc.

Examples of modifications of the side chains of the amino acid residues constituting the main chain include polyethylene glycol modification, phosphorylation modification, acetylation modification, methylation modification, fluorescent modification, biotinylation modification, modification with sugars or sugar chains, lipid modification, etc. However, it is preferable that side chain modifications are not performed at least on the amino acid sequences represented by SEQ ID NO: 1 or SEQ ID NO: 2. It is more preferable that side chain modifications are not performed on the amino acid sequences represented by SEQ ID NO: 3 to SEQ ID NO: 9.

The proteasome inhibitory peptide may contain a basic peptide to enhance the cell membrane permeability of the proteasome inhibitory peptide. The basic peptide is a peptide that is rich in basic amino acid residues. The basic amino acid residue is preferably an arginine residue. The basic peptide preferably contains about 5 to 25 amino acid residues. The proportion of basic amino acid residues contained in the basic peptide is at least about 25%, and is preferably about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more. For example, basic peptides include arginine peptides consisting of 5 to 8 consecutive arginine residues, such as HIV-1 Tat-(48-60), HIV-1 Rev-(34-50), FHV Coat-(35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), CCMV Gag-(7-25), and P22 N-(14-30). The basic peptide can be linked to the N-terminus or C-terminus of the main chain of the proteasome inhibitor peptide either consecutively or via a linker portion of several amino acid residues (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues).

Hereinafter, (1) a peptide having an amino acid sequence of the main chain of a proteasome inhibitory peptide and a basic polypeptide sequence, and (2) a peptide having an amino acid sequence of the main chain of a proteasome inhibitory peptide, a basic polypeptide sequence, and a linker sequence are referred to as a cell membrane-permeable proteasome inhibitory peptide.

The proteasome 20S core has proteolytic enzyme activity such as chymotrypsin-like activity, caspase-like activity, and trypsin-like activity. Therefore, the proteasome inhibitory activity of a proteasome inhibitor peptide can be evaluated based on whether it inhibits the proteolytic activity of the proteasome 20S core, preferably the chymotrypsin-like activity or caspase-like activity of the proteasome 20S, and more preferably the chymotrypsin-like activity.

The chymotrypsin-like activity, caspase-like activity, and trypsin-like activity of the proteasome 20S can be measured by known methods. These proteolytic enzyme activities can be evaluated based on whether or not they cleave a specific peptide contained in a substrate.

For example, Suc-LLVY-AMC (Suc represents a succinyl group, LLVY represents a peptide chain, and AMC represents 7-amido-4-methylcoumarin) can be used as a substrate for evaluating chymotrypsin-like activity. For example, Boc-LRR-AMC (Boc represents a tert-butoxycarbonyl group, LRR represents a peptide chain, and AMC represents 7-amido-4-methylcoumarin) can be used as a substrate for evaluating trypsin-like activity. For example, Z-LLE-AMC (Z represents a benzyloxycarbonyl group, LLE represents a peptide chain, and AMC represents 7-amido-4-methylcoumarin) can be used as a substrate for evaluating caspase-like activity. For example, about 100 ng of human proteasome 20S core (Enzo ENZ Life Sciences, Inc.) is added to about 100 μL of enzyme reaction buffer with about 50 mM substrate and proteasome inhibitor peptide. For example, an enzyme reaction buffer containing about 25 mM HEPES (pH 8.0), about 0.5 mM EDTA, and about 0.03% SDS can be used as the enzyme reaction buffer used to measure chymotrypsin-like activity or caspase-like activity. For example, an enzyme reaction buffer containing about 25 mM HEPES (pH 8.5) and about 0.5 mM EDTA can be used as the enzyme reaction buffer used to measure trypsin-like activity. Protease activity can be measured by monitoring the fluorescence change due to free AMC at an excitation wavelength of 380 nm and a fluorescence wavelength of 460 nm with a fluorometer or the like at 10-minute intervals at about 30°C for about 1 hour.

2. Polynucleotides encoding proteasome inhibitor peptides and vectors containing said polynucleotides Some embodiments of the present invention relate to polynucleotides encoding the amino acid sequence constituting the backbone of a proteasome inhibitor peptide, or polynucleotides encoding the amino acid sequence of a cell-permeable proteasome inhibitor peptide (hereinafter, both are simply referred to as "polynucleotides"). The polynucleotides may be DNA or RNA. The polynucleotides are preferably DNA. The conversion of the amino acid sequence constituting the backbone of a proteasome inhibitor peptide or the amino acid sequence of a cell-permeable proteasome inhibitor peptide to a polynucleotide sequence can be performed according to a general codon table.

The polynucleotide may be inserted into a vector so that it can be expressed in a host cell. The vector into which the polynucleotide is inserted can be selected depending on the host cell. For example, when expressing a polynucleotide in a mammalian cell, including a human cell, a plasmid vector, an adenovirus vector, a retrovirus vector, a lentivirus vector, or the like, which includes a promoter capable of expressing a polynucleotide, a multicloning site, a polyA signal, or the like, can be used. When expressing a polynucleotide in E. coli, a plasmid vector which includes a promoter capable of expressing a polynucleotide, a multicloning site, or the like, can be used.

3. Composition and pharmaceutical composition The composition of the present invention contains the proteasome inhibitor peptide, the polynucleotide, or a vector containing the polynucleotide as an active ingredient. The composition of the present invention can be prepared by combining the active ingredient with a suitable carrier or additive. Examples of carriers and additives used in the preparation of the pharmaceutical composition include various types of carriers and additives commonly used in ordinary drugs depending on the dosage form of the pharmaceutical composition, such as excipients, binders, disintegrants, lubricants, colorants, flavorings, odorants, surfactants, etc.

The carrier may be a transfection reagent containing a polymer, lipid, magnet, etc.

When the composition is administered orally, the dosage form is not particularly limited, and examples thereof include tablets, powders, granules, capsules (including hard capsules and soft capsules), liquids, pills, suspensions, and emulsions. When the pharmaceutical composition is administered parenterally, examples thereof include injections, drips, suppositories, nasal drops, and pulmonary preparations.

When the composition is prepared as a solid oral composition such as a tablet, powder, granule, pill, or capsule, the carrier may be, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, methylcellulose, glycerin, sodium alginate, or gum arabic; simple syrup, glucose liquid, starch liquid, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, carboxymethylcellulose, shellac, methylcellulose, ethylcellulose, water, ethanol, or potassium phosphate. binders; disintegrants such as dried starch, sodium alginate, powdered agar, powdered laminaran, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearic acid, starch, and lactose; disintegration inhibitors such as sucrose, stearic acid, cocoa butter, and hydrogenated oil; absorption promoters such as sodium lauryl sulfate; moisturizers such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid; lubricants such as purified talc, stearates, powdered boric acid, and polyethylene glycol. Furthermore, tablets can be made into tablets with a conventional coating, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets, if necessary.

When the composition is prepared as a solid oral composition in the form of a pill, the following carriers can be used: excipients such as glucose, lactose, starch, cacao butter, hardened vegetable oil, kaolin, talc, etc.; binders such as powdered gum arabic, powdered tragacanth, gelatin, etc.; disintegrants such as laminaran, agar, etc.

When the composition is prepared as an oral solid composition in the form of a capsule, the capsule is prepared by mixing the active ingredient with the various carriers listed above and filling the mixture into a hard or soft capsule.

When the preparation is a liquid, it may be an aqueous or oily suspension, solution, syrup, or elixir, and is prepared in the usual manner using conventional additives.

When the composition is prepared as an injection, carriers that can be used include diluents such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc.; pH adjusters such as sodium citrate, sodium acetate, sodium phosphate, etc.; buffers such as dipotassium phosphate, trisodium phosphate, sodium hydrogen phosphate, sodium citrate, etc.; stabilizers such as sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, etc.; and sugars such as mannitol, inositol, maltose, sucrose, lactose, etc., as molding agents for lyophilization. In this case, glucose or glycerin may be contained in the preparation in an amount sufficient to adjust the solution to an isotonicity, and conventional dissolving agents, pain-relieving agents, local anesthetics, etc. may also be added. By adding these carriers, subcutaneous, intramuscular, and intravenous injections can be prepared by conventional methods.

When the preparation is a drip infusion, it can be prepared by dissolving the compound to be administered in an isotonic electrolyte infusion preparation based on physiological saline, Ringer's solution or the like.
The composition can be used as a pharmaceutical composition.

The dosage of the pharmaceutical composition of this embodiment is not particularly limited as long as the effects of the present invention are achieved, and can be set appropriately depending on the dosage form, the patient's age, sex, and severity of the condition, etc.

For example, when a pharmaceutical composition containing a proteasome inhibitory peptide is administered systemically, it can be administered at 0.01 to 1,000 mg/kg of body weight for an adult, calculated as the weight of the main chain of the proteasome inhibitory peptide, per day. When a pharmaceutical composition containing a proteasome inhibitory peptide is administered locally, it can be administered at 0.01 to 100 mg/ ^cm2 of target tissue, calculated as the weight of the main chain of the proteasome inhibitory peptide.

The pharmaceutical composition containing the polypeptide or the vector containing the polypeptide can be administered at 0.1 to 1,000 mg/kg of body weight per day in adult humans when administered systemically, calculated as a polynucleotide encoding the amino acid sequence of the main chain of the proteasome inhibitor peptide.

When the pharmaceutical composition containing the polypeptide or the vector containing the polypeptide is administered locally, it can be administered at 0.01 to 100 mg/day per 1 ^cm2 of target tissue, calculated as a polynucleotide encoding the amino acid sequence of the main chain of the proteasome inhibitor peptide. The vector can be linearized as necessary. The polynucleotide or the vector containing the polynucleotide can be injected into the target tissue using a syringe or catheter. In this case, a nucleic acid delivery reagent such as liposome may be used in combination.

The pharmaceutical composition can be used for the prevention and/or treatment of malignant tumors. Malignant tumors include both non-epithelial and epithelial malignant tumors. Specifically, the following tumors can be used: respiratory system malignant tumors arising from the trachea, bronchi, or lungs; digestive system malignant tumors arising from the nasopharynx, esophagus, stomach, duodenum, jejunum, ileum, cecum, appendix, ascending colon, transverse colon, sigmoid colon, rectum, or anus; liver cancer; pancreatic cancer; urinary system malignant tumors arising from the bladder, ureter, or kidney; female reproductive system malignant tumors arising from the ovaries, fallopian tubes, and uterus; breast cancer; prostate cancer; skin cancer; endocrine system malignant tumors such as the hypothalamus, pituitary gland, thyroid gland, parathyroid gland, and adrenal gland; central nervous system malignant tumors; malignant tumors arising from bone and soft tissues. Examples of the tumor include solid tumors such as tumors, and hematopoietic tumors such as myelodysplastic syndrome, acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelomonocytic leukemia, chronic myelomonocytic leukemia, acute monocytic leukemia, chronic monocytic leukemia, acute panmyelogenous leukemia, acute megakaryocytic leukemia, erythroleukemia, eosinophilic leukemia, chronic eosinophilic leukemia, chronic neutrophilic leukemia, adult T-cell leukemia, hairy cell leukemia, plasma cell leukemia, multiple myeloma, and malignant lymphoma; and hematopoietic tumors such as lymphatic tumors. More preferably, the tumor is a hematopoietic tumor. The most preferred hematopoietic tumor is multiple myeloma.

The pharmaceutical composition may be used as an anticancer drug. The anticancer drug may be appropriately selected from alkylating agents, metabolic antagonists, antitumor antibiotics, microvascular inhibitors, hormones or hormone analogues, platinum preparations, topoisomerase inhibitors, cytokines, antibody drugs, radioimmunotherapy drugs, molecular targeted drugs, nonspecific immunoactivators, and other anticancer drugs according to the target tumor.

Examples of alkylating agents include, but are not limited to, cyclophosphamide, ifosfamide, busulfan, melphalan, bendamustine hydrochloride, nimustine hydrochloride, ranimustine, dagalbazine, procarbazine hydrochloride, temozolomide, etc.; examples of metabolic antagonists include methotrexate, pemetrexed sodium, fluorouracil, doxifluridine, capecitabine, tegafur, cytarabine, cytarabine ocfosfate hydrate, enocitabine, gemcitabine hydrochloride, mercaptopurine hydrate, fludarabine phosphate, nelarabine, pentostatin, cladribine, calcium levofolinate, calcium folinate, hydroxycarbamide, L-asparaginase, azacitidine, etc.; examples of antitumor antibiotics include Substances include doxorubicin hydrochloride, daunorubicin hydrochloride, pirarubicin, epirubicin hydrochloride, idarubicin hydrochloride, aclarubicin hydrochloride, amrubicin hydrochloride, mitoxantrone hydrochloride, mitomycin C, actinomycin D, bleomycin, peplomycin sulfate, zinostatin styramer, etc.; microvascular inhibitors include vincristine sulfate, vinblastine sulfate, vindesine sulfate, vinorelbine tartrate, paclitaxel, docetaxel hydrate, eribulin mesylate, etc.; hormones or hormone-like drugs (hormonal drugs) include anastrozole, exemestane, letrozole, tamoxifen citrate, toremifene citrate, fulvestrant, flutamide, bicalutamide, medicamentosyltransferase ... roxyprogesterone acetate, estramustine phosphate sodium hydrate, goserelin acetate, leuprorelin acetate, etc.; platinum preparations such as cisplatin, miriplatin hydrate, carboplatin, nedaplatin, oxaliplatin, etc.; topoisomerase inhibitors such as topoisomerase I inhibitors such as irinotecan hydrochloride hydrate and nogitecan hydrochloride, and topoisomerase II inhibitors such as etoposide and sobuzoxane; cytokines such as interferon gamma-1a, teceleukin, celmoleukin, etc.; antibody drugs such as trastuzumab, rituximab, gemtuzumab ozogamicin, bevacizumab, cetuximab, panitumumab, alemtuzumab, etc.; radioimmunotherapy drugs such as , ibritumomab, tiuxetan combination drugs, etc.; molecular targeted drugs include bortezomib, carfilzomib, ixazomib citrate ester, gefitinib, imatinib mesylate, erlotinib hydrochloride, sorafenib tosylate, sunitinib malate, thalidomide, nilotinib hydrochloride hydrate, dasatinib hydrate, lapatinib tosylate hydrate, everolimus, lenalidomide hydrate, dexamethasone, temsirolimus, vorinostat, tretinoin, and tamibarotene, etc.; non-specific immunoactive drugs include OK-432, dried BCG, Kawaratake polysaccharide preparations, lentinan, ubenimex, etc.; and other examples include aceglatone, porfimer sodium, talaporfin sodium, ethanol, arsenic trioxide, etc. Although not limited thereto, metabolic antagonists are preferred in terms of their effectiveness, and molecular targeted drugs and antibody drugs are preferred in terms of fewer side effects.

As molecular targeted drugs, when targeting hematopoietic tumors, bortezomib, carfilzomib, ixazomib citrate, lenalidomide, dexamethasone, cyclophosphamide, imatinib mesylate, nilotinib hydrochloride hydrate, and dasatinib hydrate are preferred.

As an antibody drug, trastuzumab and panitumumab are preferred, and trastuzumab is particularly preferred.

As a method for treating multiple myeloma, three-drug or two-drug combination chemotherapy combining bortezomib with other drugs is generally performed. Examples of chemotherapy include VCD therapy (bortezomib, cyclophosphamide, dexamethasone), VRD therapy (bortezomib, lenalidomide, dexamethasone), and BD therapy (bortezomib, dexamethasone). As a method for treating multiple myeloma, the proteasome inhibitor peptide of the present invention can be used instead of bortezomib.

The mode of combination of the pharmaceutical composition of the present invention with an anticancer agent is not particularly limited as long as it is a mode of use that produces the effects of the present invention. For example, the pharmaceutical composition may be administered in parallel with the administration of the anticancer agent at the same time, or the pharmaceutical composition may be administered prior to or after the administration of the anticancer agent. In this case, the pharmaceutical composition and the anticancer agent may be administered alternately. Furthermore, after the administration of the anticancer agent, the pharmaceutical composition may be administered in combination with the anticancer agent halfway through the administration of the anticancer agent depending on the degree of shrinkage of the tumor tissue, or conversely, the anticancer agent may be administered in combination with the pharmaceutical composition halfway through the administration of the anticancer agent.

In addition, the pharmaceutical composition of the present invention may be administered in a single dose, continuously, or intermittently, so long as it is used in combination with an anticancer drug. For example, in the case where the pharmaceutical composition of the present invention is administered prior to the administration of an anticancer drug, an example of the method is administration once a day from 7 days before the start of administration of the anticancer drug to the day of the start of administration, or from 3 days before the start of administration to the day of the start of administration.

The pharmaceutical composition of the present invention is prepared in various forms (dosage forms) depending on the administration route (administration method) and administered to the target tumor patient in combination with an anticancer drug. The tumor patient may be a patient before undergoing antitumor treatment such as chemotherapy, radiation therapy and/or resection surgery (surgical therapy), or may be a patient at any stage during or after treatment.

The route of administration (method of administration) of the pharmaceutical composition is not particularly limited, and examples thereof include oral administration; and parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, transmucosal administration, transdermal administration, and rectal administration. Oral administration and intravenous administration are preferred, and oral administration is more preferred.

The dosage of the pharmaceutical composition of the present invention may vary depending on the type and location of the tumor to be treated, the stage (degree of progression) of the tumor, the patient's condition, age, sex, weight, type of anticancer drug used in combination, etc., and can be appropriately set depending on these factors.
When a pharmaceutical composition is used in combination with an anticancer agent, the anticancer agent can be administered in accordance with the general administration method and dosage for that agent.

4. Methods for protecting a target peptide Certain embodiments of the present invention relate to methods for protecting a target peptide.
The main chains of the proteasome inhibitor peptides according to the first, second and third embodiments can inhibit the protease activity of the proteasome 20S core, and therefore, by adding the main chains of the proteasome inhibitor peptides according to the first, second and third embodiments to other peptides, the degradation of the other peptides by the proteasome can be suppressed.

That is, this embodiment involves protecting a target peptide from degradation by the proteasome 20S core by binding the main chain of the proteasome inhibitor peptide according to the first, second, and third embodiments to a target peptide other than the proteasome inhibitor peptide.

Methods for binding a target peptide to the main chain of a proteasome inhibitor peptide are known. For example, a peptide may be synthesized by directly or indirectly linking the main chain of a proteasome inhibitor peptide to the N-terminus or C-terminus of a target peptide. Alternatively, for example, a target polypeptide can be obtained by directly or indirectly linking a polypeptide encoding the main chain of a proteasome inhibitor peptide to the 5'-terminus or 3'-terminus of a polynucleotide encoding the target polypeptide, and synthesizing a peptide from this polynucleotide.

5. Complex of Anticancer Agent and Proteasome Inhibitory Peptide The proteasome inhibitor peptide of the present invention may also include a proteasome inhibitor peptide complexed with an anticancer agent. The complex of an anticancer agent and a proteasome inhibitor peptide can be formed by utilizing the amino group at the N-terminus or the carboxyl group at the C-terminus of the proteasome inhibitor peptide. For example, the carboxyl group contained in the main component of the anticancer agent is converted into an active ester type in advance by mixing a carbodiimide and N-hydroxysuccinimide. This active ester type anticancer agent is mixed with a peptide, and an amide bond is formed with the N-terminal amino group of the peptide, thereby forming a complex between the anticancer agent and the proteasome inhibitor peptide.

Also, the amino group at the N-terminus of the proteasome inhibitor peptide can first be protected by acetylation or the like, and the carboxyl group at the C-terminus of the proteasome inhibitor peptide can be converted to an active ester type by mixing carbodiimide and N-hydroxysuccinimide, and the active ester type proteasome inhibitor peptide can be reacted with an anticancer drug having an amino group to form an amide bond.

The above-mentioned amide bond forming reaction is known. For example, the above-mentioned reaction can be carried out in the presence of a dehydration condensation agent and, if necessary, a catalyst. Examples of the catalyst include carbodiimide-based compounds such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; imidazole-based compounds such as N,N-carbonyldiimidazole; triazine-based compounds; phosphonium-based compounds; and uronium-based compounds. Carbodiimide-based compounds are preferred, and among these, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxy-7-azabenzotriazole, 1-hydroxy-7-azabenzotriazole, and ...

The present invention will be described in more detail below with reference to examples, but the present invention should not be construed as being limited to these examples.

1. Search for 20S core-binding peptides
Using the cDNA display method, we screened for peptides that bind to the proteasome 20S core.

1-1. Method (1) Preparation of IVV A DNA library was prepared as a template for an in vitro virus (IVV) containing DNA encoding a random 8-residue amino acid sequence (represented by the base sequence NNKNNKNNKNNKNNKNNKNNKNNK, where N represents A, T, G, or C, and K represents T or G). This DNA library was used to transcribe the IVV template mRNA using RiboMAX ^TM Large Scale RNA Production Systems (Promega Corporation), and the IVV template mRNA was purified using RNeasy Mini Kit (Qiagen, Inc.). Puromycin was bound to the purified IVV template mRNA via a linker to prepare a first complex of IVV template mRNA and puromycin. The prepared complex was added to a cell-free translation system using a reticulocyte extract, and a peptide derived from the IVV template mRNA was translated to prepare an IVV in which the peptide was linked to the IVV template mRNA. Furthermore, to stabilize the IVV, the mRNA in the IVV was reverse transcribed to prepare a second complex of the peptide, puromycin, and double-stranded mRNA-cDNA.

(2) Selection of IVV and amplification of nucleic acid in IVV In the selection process, selection was performed based on affinity to the target molecule, human 20S core. For this, a magnetic carrier with 20S core immobilized was used. 200 μg of magnetic carrier FG beads for affinity purification (Tamagawa Seiki Co., Ltd.) was washed three times with N,N-dimethylformamide (DMF). 250 μL of activation solution (1 M N-hydroxyimide (Peptide Institute Co., Ltd.), 0.5 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, solvent DMF) was added and reacted at room temperature for 2 hours with shaking. After the reaction, the supernatant was removed and the mixture was washed five times with 100 μl of DMF. After washing, 10 μg of human 20S proteasome (Enzo Life Sciences) diluted in 25 mM HEPES (pH 7.0) was added and reacted at 4°C for 30 minutes with shaking. The supernatant was removed, and the protein-immobilized magnetic carrier was washed five times with 200 μl of washing and storage buffer (10 mM HEPES buffer (pH 7.9), 50 mM KCl, 1 mM EDTA, 10% glycerol) and stored at 4°C until use. 100 μl of the dispersion of the 20S proteasome-immobilized carrier (200 ng of carrier) was washed twice with 150 μl of selection separation buffer (100 mM NaCl, 0.1% Tween-20, PBS(-)). The selection separation buffer and IVV solution were added to the proteasome-immobilized magnetic carrier from which the buffer had been removed to make 100 μl, and the reaction was carried out at 4°C with rotational mixing. After the reaction, the carrier was washed with 150 μl of selection separation buffer and 150 μl of 1× washing buffer (250 mM NaCl, 0.1% Tween-20, PBS(-)). The washing buffer was removed, and 35 μl of 0.1 M KOH was added and reacted at 37°C for 10 minutes. The supernatant (elution fraction) was collected and neutralized with 15 μl of 1 M Tris-HCl (pH 7.5). Then, the cDNA of the second complex bound to the target molecule was amplified by PCR (94°C 15 sec → 60°C 5 sec → 72°C 30 sec, 15 to 25 cycles).

(3) Identification of peptides The three steps of peptide translation, IVV selection, and amplification of cDNA in the second complex were counted as one cycle, and five cycles were repeated to obtain a cDNA pool at each cycle. The cDNA pools from each cycle were sequenced by a next-generation sequencer to identify the peptide sequences.

1-2. Sequence analysis results In the analysis using a next-generation sequencer, an average of 1.5 million reads were obtained in each cycle, including the surrounding sequences of the IVV diversity region (the region encoding a random 8-residue amino acid sequence). When the read sequences from the second cycle onwards were added sequentially to the read sequences obtained in the first cycle, the number of overlapping read sequences increased with the progress of the cycles from the second cycle onwards, and by the fifth cycle, convergence to a specific sequence of about 400,000 reads was observed. The most frequently occurring read sequence accounted for 5.2% of the approximately 1.5 million reads analyzed in the fifth cycle.

The most frequently applied sequence among the peptide sequences consisting of 17 amino acid residues that can be translated from the sequenced cDNA sequence was MARPSRLRHWWRLRRRV (SEQ ID NO: 10) (hereafter referred to as "PI-Rank 1") from the N-terminus to the C-terminus. The conversion from the cDNA sequence to the amino acid sequence followed the general peptide notation method.

2. Enzyme Inhibitory Effect of 20S Core Binding Peptides We examined whether the peptides identified in 1 above can inhibit the proteolytic enzyme activity of the 20S proteasome.

2-1. Method (1) Peptide purification Each peptide was synthesized by a contractor (Cosmo Bio Co., Ltd.) using a standard solid-phase synthesis method, and the crude product cleaved from the support resin was purchased. The crude peptide was purified by the following procedure before use.

The crude peptide was dissolved in 5% _CH3CN /0.05% TFA/ _H2O and purified using a reversed-phase column (COSMOSIL Packed Column 5C18-AR-II 4.6ID x 150mm) connected to an HPLC (HITACHI ELITE LaChrom L-2000 series) (Buffer A: 0.05% TFA/ _H2O , Buffer B: 0.05% trifluoroacetic acid (TFA)/ _CH3CN , detection wavelength: 220nm, temperature: 40℃, flow rate: 1mL/min, _CH3CN concentration change: 5%-80%). The collected fraction was dried under reduced pressure and stored at -20℃.

The peptides purified by the above method have a hydrogen atom at the N-terminus and a hydroxyl group at the C-terminus. In the following, each peptide will be abbreviated as PI-Rank1.

(2) Enzyme activity measurement Proteasome enzyme activity when inhibitors were added was evaluated by measuring the decomposition rate of fluorescent substrate peptides. Suc-LLVY-AMC was used as the fluorescent substrate peptide for chymotrypsin-like activity, and Z-LLE-AMC was used for caspase-like activity (PGPH-like activity) (Suc stands for succinyl group, Z stands for benzyloxycarbonyl group, Boc stands for butoxycarbonyl group, and AMC stands for 7-amido-4-methylcoumarin). 100ng of human proteasome 20S core (Enzo ENZ Life Sciences, Inc.), 50mM of each fluorescent substrate peptide, and each concentration of inhibitor peptide were reacted in 100μL of enzyme reaction buffer (Chymotrypsin-like activity, PGPH-like activity measurement: 25mM HEPES pH8.0, 0.5mM EDTA, 0.03% SDS, trypsin-like activity measurement: 25mM HEPES pH8.5, 0.5mM EDTA). The fluorescence change due to free AMC was measured at 30℃ at 10-minute intervals for 1 hour using FusionTMα-FP (PerkinLumer Japan Co., Ltd.) at an excitation wavelength of 380nm and an emission wavelength of 460nm.

2-2. Results The inhibitory effect is shown as _IC50 value. The _IC50 value of PI-Rank1 against CT-L activity was 1.46 μM. The _IC50 value of PI-Rank1 against PGPH activity was 0.65 μM, indicating a high inhibitory effect against PGPH activity.

Next, to clarify the Ki value and inhibition mode of the inhibitory activity against the 20S core, the change in reaction rate was analyzed by Lineweaver-Burk plot and Dixon plot when the substrate concentration and peptide concentration were changed against CT-L activity using PI-Rank1. The results are shown in Figure 1. Figure 1 (A) is a Lineweaver-Burk plot for determining the inhibition mode, and (B) is a Dixon plot for determining the Ki value. The regression lines of the Lineweaver-Burk plots all intersected on the negative side of the X-axis. This suggests that the PI-Rank1 peptide exhibits a non-competitive inhibitory effect on the enzyme activity of the 20S core. The Ki values of PI-Rank1 for chymotrypsin-like activity and caspase-like activity are shown in Figure 2. The second complex (IVV) used in the cDNA display method consists of a 168-base cDNA, a puromycin linker, and a translated peptide of 10 or so residues. Due to the large molecular size, it is unlikely that it can enter the inside of the 20S core. Therefore, if the inhibitory effect was due to binding to the outer surface of the 20S core, the non-antagonistic behavior was considered to be a reasonable result.

3. Structure-activity relationship of PI-Rank1 Next, to clarify the site within the 17-residue amino acid sequence of PI-Rank1 that is responsible for the inhibitory effect on the enzymatic activity of the 20S core, we created PI-Rank1 analogs by introducing deletions or single amino acid substitutions into the amino acid sequence of PI-Rank1 and evaluated the inhibitory effect of these analogs on the enzymatic activity of the 20S core.

3-1. Methods As shown in Figure 3, PI-Rank1(6-17) and PI-Rank1(1-12) were prepared by deleting 5 residues each from the N-terminus or C-terminus of PI-Rank1. In addition, short-chain analogs were prepared by deleting up to 6 amino acid residues, one each from the N-terminus or C-terminus of PI-Rank1(1-12) shown in Figure 4. Furthermore, as shown in Figure 5, alanine-substituted analogs were prepared by substituting one amino acid residue each from the 12 amino acid residues of PI-Rank1(1-12) with alanine, except for alanine "A". Each analog was prepared according to 2-1. (1) above. The chymotrypsin-like (CT-L) activity of the 20S core was measured according to 2-1. (2) above, and the _IC50 value was calculated.

3-2. Results Figure 3 (A) shows the _IC50 values of PI-Rank1(6-17) and PI-Rank1(1-12). PI-Rank1(1-12) had the same inhibitory effect on CT-L activity as PI-Rank1(1-17) ( _IC50 = 0.93 ± 0.30 μM), but PI-Rank1(6-17) had an _IC50 higher than 10 μM and lost its inhibitory effect. As shown in Figure 3 (B), the inhibitory effect of PI-Rank1(1-12) was concentration-dependent, as was PI-Rank1(1-17). This demonstrated that PI-Rank1(1-12) has an active site on CT-L activity. In the selection stage of the cDNA display method, a puromycin linker was bound to the C-terminus, and the fact that a region responsible for the inhibitory effect was found on the N-terminus was considered to be consistent with this.

Next, we used short-chain and alanine-substituted analogs of PI-Rank1(1-12) to identify the site of action responsible for the inhibitory effect.

Figure 4 shows the _IC50 values of short analogs of PI-Rank1(1-12) for CT-L activity. Figure 6(A) shows a graph of _IC50 values of short analogs for CT-L activity. In the short analogs deleted from the N-terminus, deletion of two residues abolished activity, whereas deletion of three or four residues restored activity. PI-Rank1(5-12), which deleted four residues, showed an _IC50 value of 0.82 μM. However, further deletions significantly weakened the inhibitory activity. Deletion of sequences from the C-terminus increased the _IC50 value and reduced the inhibitory activity, with the exception of peptide PI-Rank1(1-11), which deleted only one residue, showing an _IC50 value of 0.72 μM and more potently inhibiting CT-L activity. These results suggest that the region of eight residues from the fifth serine residue to the twelfth arginine residue in PI-Rank1(1-12) that exerts an inhibitory effect on CT-L activity is the region of eight residues in PI-Rank1(1-12), and that the shortest peptide necessary and sufficient for the inhibitory activity is PI-Rank1(5-11).

Figure 5 shows the _IC50 values of alanine-substituted analogs for CT-L activity. Figure 6(B) shows a graph of the _IC50 values of alanine-substituted analogs for CT-L activity. When the 1st methionine, 4th proline, 5th serine, 7th leucine, 9th histidine, and 10th and 11th tryptophans were substituted with alanine, the inhibitory effect was greatly weakened. When the 3rd, 6th, 8th, and 12th arginines were substituted with alanine, no significant change in activity was observed.

4. Enzyme inhibitory effect of PI-Rank1 on the 26S proteasome
To examine how PI-Rank1 affects the function of the 26S proteasome, the following experiment was carried out.

4-1. Method
Chymotrypsin-like activity was measured by measuring the rate of degradation of Suc-LLVY-MCA as the 26S proteasome activity. Purified 26S proteasome (0.1 μg, Enzo Life Sciences, Inc.) was added to 100 μL of test buffer (50 mM HEPES [pH 7.5], 40 mM KCl, 5 mM MgCl ₂ , 50 μg bovine serum albumin, 0.5 mM ATP, 1 mM dithiothreitol) in the presence of various inhibitor compound concentrations (0.1–10 μM) and fluorescent substrate peptide (50 μM). The fluorescence of released 7-amino-4-methylcoumarin (AMC) (λ _ex = 380 nm, λ _em = 480 nm) in the reaction mixture was monitored for 1 h at 37°C. IC ₅₀ values were defined as the compound concentration required for 50% inhibition of proteasome activity and were determined for each compound from the respective inhibition curves.

4-2. Results As shown in Figure 7, PI-Rank1(5-12) was added up to 10 μM, but no inhibitory effect was observed on the CT-L activity of the 26S proteasome. This may be because PI-Rank1(5-12) is unable to bind to the 20S core when the 20S core and 19S complex bind, or because the structural change required for controlling the enzyme activity of the 20S core does not occur even if PI-Rank1(5-12) binds.

5. Confirmation of binding between 20S core and PI-Rank1 Next, the actual binding of PI-Rank1 to the 20S core was confirmed by affinity labeling.

5-1. Method (1) Generation of photoreactive peptides To introduce an aryl azide group, which forms a covalent bond with surrounding molecules by the nitrene generated by activation with ultraviolet light, into PI-Rank1(5-12), the NHS ester of the modifying group was reacted with the N-terminal amino group of PI-Rank1(5-12). Specifically, 50 nmol of PI-Rank1(5-12) and 50 nmol of Sulfo-SBED (Thermo SCIENTIFIC) were reacted in 10 μL of DMF containing 1% TEA for 48 to 72 hours. After the reaction, the product was purified by reversed-phase HPLC and the molecular weight was confirmed by mass spectrometry. Since a biotin group was also introduced into this Sulfo-SBED, the photoreactive group of Sulfo-SBED was crosslinked to the target protein by irradiating it with ultraviolet light after binding Sulfo-SBED-labeled PI-Rank1(5-12) to the target protein. Therefore, by detecting biotin, the target protein bound to Sulfo-SBED-labeled PI-Rank1(5-12) can be detected by Western blotting or the like.

Mass spectrometry was performed as follows. The sample was dissolved in 50% CH ₃ CN/water (MS measurement solvent) containing 0.1% TFA. The matrix was prepared by saturating α-CHCA in the MS measurement solvent, and the mixture was mixed 1:1 and measured with a MALDI-TOF mass spectrometer (SpiralTOF JMS-S3000, JEOL Ltd.). Mass spectrometry confirmed that the target molecule was found in the fraction with a retention time of 23 minutes. Two mass peaks were detected in the mass spectrum: 1833.6 and 1860.4. Since the theoretical amount of the target photoreactive peptide is 1859.23, 1860.4 corresponds to the molecular weight of the hydrogen ion adduct, and 1833.6 corresponds to the molecular weight when phenyl azide is converted to dehydroazepin and hydrogen ions are added at the same time. Hereafter, the obtained photoreactive peptide is referred to as SBED-PI-Rank1(5-12).

(2) Reaction of SBED-PI-Rank1(5-12) with 20S core or 26S proteasome SBED-PI-Rank1(5-12) was dissolved in DMSO. A total of 10 ng was added to 10 μL of buffer (25 mM HEPES, 0.5 mM EDTA, 0.03% SDS, pH 8.0) so that the final concentration of SBED-PI-Rank1(5-12) was 10 μM and that of 20S core was 1 ng/μL, and the reaction was carried out at room temperature for 30 minutes in the dark. When confirming specific binding, 10 times the amount of uncrosslinked PI-Rank1(5-12) (final concentration 100 μM) was added as a competing peptide. After the reaction, the sample was irradiated with a UV lamp at 365 nm and a height of 5 cm for 30 minutes. The sample was cooled on ice. The samples after UV irradiation were subjected to SDS-PAGE electrophoresis using 16.5% acrylamide gel (constant current of 26 mA per gel for 1 hour). Three pieces of filter paper soaked in solution A (0.3 M Tris, 20% methanol, 0.05% SDS), one piece of filter paper soaked in solution B (25 mM Tris, 20% methanol, 0.05% SDS), PVDF membrane, gel, and two pieces of filter paper soaked in solution C (25 mM Tris, 40 mM 6-aminohexanoic acid, 20% methanol, 0.05% SDS) were stacked on top of the positive electrode, and the negative electrode was set and blotted (25V 1mA/cm2 30 minutes) using a semi-dry blotting device (Biocraft BE-331). The gel was then placed in Blocking One-P (Nacalai Tesque) and blocked with shaking for 1 hour. After washing with TBS-T (50 mM Tris, 138 mM NaCl, 2.7 mM KCl, 0.1% Tween 20), the sections were reacted with streptavidin-HRP (GE Healthcare) antibody for 1 hour. Finally, biotin was detected using ECL reagent (AmershamTM ECLTM Prime Western Blotting) on the LAS4000.

5-2. Results Although not shown, a protein band was confirmed at around 25 kDa, which is the molecular weight band of the subunit, in the silver stained image after SDS-PAGE. After Western blotting, biotin-labeled bands were detected, and as shown in Figure 8 (A), a band was observed at around 25 kDa only when SBED-PI-Rank1(5-12) was added and UV irradiation was performed (dotted square frame). Furthermore, a comparison was made of UV irradiation times. The irradiation time was changed to 0, 15, and 30 minutes. At the same time, to confirm the inhibition of labeling due to competition, a sample was prepared in the same manner with 10 times the amount of PI-Rank1(5-12). As a result, it was confirmed that the band was most well labeled after 30 minutes of UV irradiation, and at the same time, the band became fainter when excessive PI-Rank1(5-12) was added. From the above results, it is considered that SBED-PI-Rank1(5-12) specifically labeled the 20S core subunit.

Similarly, we confirmed whether the 26S proteasome was labeled by SBED-PI-Rank1(5-12). After reacting with the 26S proteasome and the 20S core only samples, we irradiated them with UV for 30 minutes and confirmed by Western blotting. As a result, as shown in Figure 8 (B), a labeled band was observed only in the case of the 20S core only, and no labeled band was observed in the case of the 26S proteasome. From these results, it was considered that PI-Rank1(5-12) does not bind to the 26S proteasome. In the above 4., the fact that PI-Rank1(5-12) did not inhibit the CT-L activity of the 26S proteasome was considered to be due to the fact that PI-Rank1(5-12) does not bind to the 26S proteasome. In addition, it was considered that PI-Rank1(5-12) is likely to act on the binding surface between the 20S core and the 19S complex.

6. Identification of the 20S core subunit to which PI-Rank1 binds Next, we performed an experiment to identify which 20S core subunit PI-Rank1 binds to.

6-1. Method (1) Two-dimensional electrophoresis According to the method described in 5-1. (2) above, SBED-PI-Rank1(5-12) was crosslinked with 20S core. The total volume was 60 μl and used as the analysis sample. 60 μl of the analysis sample was mixed with the first dimension sample buffer (60 mM Tris-HCl, pH 8.8, 5 M urea, 1 M thiourea, 5 mM EDTA, 1% CHAPS, 1% NP-40, 65 mM DTT), 6 μl of 1 M iodoacetamide was added, and the reaction was allowed to proceed for 10 minutes. The gel was applied to an agar gel (mini size (75 mm), pH range 3-10, AM 310) and overlaid with 2 M urea. Electrophoresis was performed using an isoelectric focusing apparatus (ATTA discRun AE-6541) (upper electrode solution: 0.2 M NaOH, lower electrode solution: 10 mM phosphoric acid, 300 V, 210 min). After electrophoresis, the gel was removed from the column and immobilized (2.5% trichloroacetic acid, 3 min). For second-dimensional electrophoresis, it was immersed in SDS equilibration solution (50 mM Tris-HCl, 2% SDS, 0.001% BPB, 10 min). 1% agarose was attached to the second-dimensional 16.5% acrylamide gel and SDS-PAGE electrophoresis was performed. CBB staining and western blotting were then performed.

(2) Identification of 20S core subunits by LC-MS
The samples subjected to 2D electrophoresis were stained with CBB (fixing solution: 20% methanol, 7.5% acetic acid, staining solution: 0.25% CBB, 40% methanol, 7.5% acetic acid, destaining solution: 2.5% methanol, 7.5% acetic acid). All bands visible in CBB staining were excised. After destaining with 50% destaining solution (50% acetonitrile, 25 mM ammonium bicarbonate, twice), 25% destaining solution (25% acetonitrile, 25 mM ammonium bicarbonate, twice), and 100% acetonitrile, the samples were centrifuged and dried. The reducing solution was added and reacted at 56°C for 300 minutes, and the remaining solution was removed. The alkylation solution was added and reacted at 37°C for 30 minutes in the dark. After washing with 100 mM ammonium bicarbonate, the samples were dehydrated with acetonitrile for 10 minutes and centrifuged and dried. Trypsin solution was added to the dried samples and left to stand for 45 minutes. The solution that was not absorbed was discarded, and 10 mM Tris-HCl (pH 8.8) solution was added until the gel was completely immersed, and the reaction was carried out at 37°C for 4 to 16 hours. The reaction product was recovered using a 50% acetonitrile, 0.1% formic acid solution. The acetonitrile concentration was then reduced to less than 5% by adding MilliQ water with 0.1% formic acid and repeatedly centrifuging and drying. Finally, the gel was analyzed by LC-MS.

6-2. Results
To identify the labeling sites of SBED-PI-Rank1(5-12) in the 14 (7 types) subunits that make up the 20S core, two-dimensional electrophoresis was performed. As a result, three labeled spots were observed, as shown in Figure 9 (A). Two of these were found to be α1 and β4 subunits by protein identification using mass spectrometry. The remaining one could not be identified, but similar analysis showed that it is likely to be β5 or β6. These β subunits are thought to be in the vicinity of the CT-L active site, as shown in Figure 9 (B), and it is thought that the labeling reaction occurred when SBED-PI-Rank1(5-12) was degraded inside the 20S core. On the other hand, the detected α1 subunit is located at the binding surface between the 20S core and the 19S complex, as shown in Figure 9 (C), and is a subunit involved in opening and closing the substrate import gate.

7. Interaction with the C-terminal peptide of Rpt3
It is known that the α1 subunit of the 20S core interacts with the C-terminal peptide of Rpt3, a subunit of the 19S complex (Fig. 10(A)). The binding surface of the α1 subunit to the 19S complex has a groove where the peptide acts (Fig. 10(B)), and the binding of the C-terminal peptide of Rpt3 to this groove controls the opening and closing of the substrate uptake gate of the 20S core (Fig. 10(C)). Comparing the C-terminal 8 residues of PI-Rank1(5-12) and Rpt3, the hydrophilic and hydrophobic positions of the amino acid sequences appeared to match (Fig. 10(D)). Since it was thought that PI-Rank1(5-12) might bind to the Rpt3 interaction site of the α1 subunit, we synthesized a peptide of the C-terminal 8 residues of Rpt3 (Rpt3-C) shown in Fig. 10(D) and examined its effect on the enzyme activity of the 20S core.

7-1. Methods The CT-L activity of the 20S core in the presence of Rpt3-C was measured according to the method described in 2-1. (2) above. In addition, the CT-L activity of the 20S core in the presence of Rpt3-C and PI-Rank1(5-12) was measured.

7-2. Results Figure 11 (A) shows the effect of Rpt3-C on CT-L activity in the absence/presence of PI-Rank1(5-12). Figure 11 (A) shows the activity as a line graph, and the dotted line shows the activity in the absence of PI-Rank1(5-12). The solid line shows the activity in the presence of PI-Rank1(5-12). Figure 11 (B) shows the activity as a bar graph. The black bar shows the activity in the absence of PI-Rank1(5-12). The white bar shows the activity in the presence of PI-Rank1(5-12). When Rpt3-C was added in the absence of PI-Rank1(5-12) and the CT-L activity of the 20S core was measured, no inhibitory effect was observed on the 20S core, and conversely, the CT-L activity was enhanced in a concentration-dependent manner by Rpt3-C. Furthermore, in the presence of PI-Rank1(5-12), the enhancement of CT-L activity by Rpt3-C was suppressed. These experiments revealed that Rpt3-C positively regulates the activity of the 20S core, and PI-Rank1(5-12) negatively regulates the activity of Rpt3-C. In previous studies, SDS was added during enzyme reaction measurements to promote the opening of the substrate uptake gate of the 20S core. In this experiment, SDS was also added during the measurements. When Rpt3-C was measured under conditions without the addition of SDS, it further enhanced the enzyme activity of the 20S core, suggesting the possibility that Rpt3-C may promote the opening of the substrate uptake gate of the 20S core.

8. Cytotoxic activity of PI-Rank1(5-12) Next, we examined whether PI-Rank1(5-12) has cytotoxic activity.

8-1. Methods To confirm cytotoxic activity, multiple myeloma cell line RPMI8226 cells were used as cultured cells. RPMI8226 cells were maintained in RPMI1640 medium supplemented with 10% FBS and 100 U/mL penicillin-streptomycin.

After seeding 1×10 ³ cells on a 96-well plate, the cells were cultured for 12 hours, and the medium was changed. The four test drugs shown in FIG. 12(A) were added at the concentrations shown in FIG. 12(B) and FIG. 12(C). PI-Rank1(5-11) does not have cell membrane permeability, so a polyarginine tag consisting of eight arginines "R" was attached to the C-terminus to impart cell membrane permeability (referred to as "PI-Rank1(5-12)R7G"). As negative controls, PI-Rank1(5-12) without cell membrane permeability and only a polyarginine tag (consisting of "RRRRRRRRG" (SEQ ID NO: 36) and represented as "R8G") were used. Bortezomib is an existing proteasome inhibitor and has been approved as a treatment for multiple myeloma. After 24 and 48 hours from the administration of the test drug, the cells were harvested and the amount of intracellular ATP was measured using a plate reader with a cell ATP measurement reagent (Toyo B-Net Co., Ltd.).

8-2. Results As shown in Figure 12 (B), 24 hours after administration of the test drug, when PI-Rank1(5-12)R7G was administered at 1 μM, the ATP concentration was reduced more than when Bortezomib was administered. Even 48 hours after administration of the test drug, when PI-Rank1(5-12)R7G was administered at 1 μM, the ATP concentration was reduced to the same extent as when Bortezomib was administered. The reduced ATP concentration means that there are fewer live cells.

These results indicate that PI-Rank1(5-12) has cytotoxic activity. Furthermore, unmodified PI-Rank1(5-12) is not cell membrane permeable and did not show cytotoxicity. This suggests that cell-specific delivery is possible by using a drug delivery system in combination to control the cell membrane permeability of PI-Rank1(5-12).

As described above, the proteasome inhibitor peptide of the present invention was found to have tumor toxicity and was shown to have activity as an anticancer drug. Therefore, it was shown that it may be possible to use it against tumors that have acquired resistance to existing proteasome inhibitors that have other inhibitory mechanisms.

9. Protection of target peptide by proteasome inhibitor peptide Next, it was examined whether a target peptide can be protected from degradation by the proteasome 20S core by binding the proteasome inhibitor peptide to the target peptide.

9-1. Expression of recombinant proteins Polynucleotides were chemically synthesized by adding the recognition and cleavage sequences of the restriction enzymes Hind III and EcoR I to the DNA sequence encoding the human α-synuclein (amino acid sequence 1-140) polypeptide and the human α-synuclein (amino acid sequence 1-140) polypeptide with the amino acid sequence of PI-Rank1 (1-12) (MARPSRLRHWWR: SEQ ID NO: 9) added to its C-terminus. These synthetic polynucleotides were inserted into the corresponding restriction enzyme cleavage sites in the multicloning site of the pET32a plasmid. By transforming E. coli with these two types of plasmids, recombinant proteins in which thioredoxin and hexahistidine (His ₆ ) sequences were fused to the N-terminus of each peptide were expressed. Thioredoxin-fused human α-synuclein and thioredoxin-fused human α-synuclein-PI-Rank1(1-12) protein with the PI-Rank1(1-12) amino acid sequence (MARPSRLRHWWR: SEQ ID NO: 9) added to its C-terminus were transduced into Origami ^TM 2(DE3) Competent Cells (Novagen) for mass expression. After transformation according to a general protocol, colonies were isolated by culturing in LB medium (containing 100 μg/ml ampicillin). The colonies were inoculated into 4 ml of LB liquid medium (100 μg/ml ampicillin) and cultured until the OD600 value reached 0.6-0.9, isopropyl-β-thiogalactopyranoside was added to 1 mM, and the cells were further cultured with shaking for 2 hours. The culture was transferred to a 50 ml centrifuge tube and centrifuged at 4,400 rpm for 10 minutes to collect the cells.

9-2. Purification of recombinant protein 5 ml of buffer (100 mM phosphate pH 8.0 buffer, 300 mM NaCl, 0.2 mg/ml lysozyme, 0.2 mg/ml benzamidine, 0.5 mM phenylmethanesulfonyl fluoride) was added to 1 g of frozen E. coli pellet, and the pellet was sonicated 10 times for 30 seconds on ice, then frozen and thawed at -80°C. The pellet was then centrifuged at 15,000 rpm for 30 minutes at 4°C. The supernatant was used as a sample and purified using Ni-NTA agarose as described below.

A bed volume of 200 μl of Ni-NTA agarose (Qiagen) was placed in a chromatography column (0.8 × 4 cm), and 200 μl of equilibration buffer containing 10 mM imidazole added to the above buffer was added for washing and equilibration. 500 μl of the above E. coli lysate was added, and the column was mixed by inversion on a rotator for 60 minutes at 4°C. The flow-through fraction was collected, washed twice with 500 μl of 10 mM imidazole, and eluted six times with 500 μl of 40 mM imidazole. The recombinant protein contained in this eluate was used as a purified sample in the next experiment.

9-3. Degradation experiment of purified recombinant protein Human purified 20S proteasome (Enzo Life Sciences, Inc., 1 pmol equivalent, 1 μg) was reacted with the above-mentioned purified thioredoxin-fused human α-synuclein (4 pmol equivalent, 0.15 μg), or its thioredoxin-fused human α-synuclein-PI-Rank1(1-12) adduct (50 pmol equivalent, 1.8 μg) with the PI-Rank1(1-12) amino acid sequence added to its C-terminus in 10 μL of 20 mM HEPES (pH 7.5) buffer at 37°C for a certain period of time. The reaction was stopped by mixing with an equal amount of 2×SDS buffer. The mixture was heated at 98°C for 3 minutes and analyzed by SDS-PAGE electrophoresis. For comparison, a similar experiment was performed using commercially available purified human α-synuclein recombinant protein (Cosmo Bio Co., Ltd., 50 pmol equivalent, 0.73 μg).

9-4. Results The time course of changes in the concentration of purified thioredoxin-fused human α-synuclein recombinant protein (represented as "αSYN (Trx fusion)" in the figure) or purified thioredoxin-fused human α-synuclein-PI-Rank1(1-12) adduct (represented as "αSYN+Rank1 (Trx fusion)" in the figure), in which the PI-Rank1(1-12) amino acid sequence has been added to its C-terminus, was observed by SDS-PAGE. The results are shown in Figure 13(A). In Figure 13(A), "M" indicates the molecular weight marker. "-" indicates the negative control to which no human purified 20S proteasome was added. "1h" or "4h" indicates the reaction time (hours) after the addition of human purified 20S proteasome. Figure 13(B) shows the quantitative results obtained by image analysis of the concentration of the protein bands in Figure 13(A). Each purified recombinant protein was observed as a 35 kDa molecular weight band. Multiple bands around 20-30 kDa were derived from human 20S proteasomes. Upon addition of human 20S proteasomes, the purified thioredoxin-fused human α-synuclein recombinant protein was gradually degraded, with approximately 28% degraded in 1 hour and approximately 72% degraded in 4 hours. In contrast, the thioredoxin-fused human α-synuclein-PI-Rank1(1-12) adduct, which had the PI-Rank1(1-12) amino acid sequence added to the C-terminus, was only degraded by approximately 23% in 1 hour and approximately 31.5% in 4 hours.

Furthermore, the degradation rates of thioredoxin-fused human α-synuclein-PI-Rank1(1-12) adduct (represented as "αSYN+Rank1 (Trx fusion)" in the figure) and wild-type human α-synuclein recombinant polypeptide without additional sequences such as tags (represented as "αSYN recombinant" in the figure) by the 20S proteasome were compared. The results are shown in Figure 14(A). In Figure 14(A), "M" indicates the molecular weight marker. "-" indicates the negative control without the addition of human purified 20S proteasome. "1h" or "4h" indicates the reaction time (hours) after the addition of human purified 20S proteasome. Figure 14(B) shows the quantitative results obtained by image analysis of the concentration of the protein bands in Figure 14(A). A protein band was observed at 15 kDa for wild-type α-synuclein without the PI-Rank1 sequence. The recombinant human α-synuclein polypeptide was degraded by about 64% in 4 hours, whereas the thioredoxin-fused human α-synuclein-PI-Rank1(1-12) adduct, which had the PI-Rank1(1-12) amino acid sequence added to the C-terminus, was degraded by only about 34% in 4 hours.

These results show that adding a proteasome inhibitor peptide sequence to a target polypeptide inhibits the degradation of the target peptide in a proteasome inhibitor peptide-dependent manner.

Claims (9)

A peptide having the effect of inhibiting the proteolytic enzyme activity of the proteasome 20S core, comprising a sequence represented by SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33 . A polynucleotide comprising a base sequence encoding the peptide of claim 1 . A vector comprising the polynucleotide of claim 2 in an expressible manner. A composition comprising the peptide of claim 1 , the polynucleotide of claim 2 , or the vector of claim 3 . The composition of claim 4 , wherein the composition is a pharmaceutical composition. The composition according to claim 5 , wherein the pharmaceutical composition is used for the prevention and/or treatment of malignant tumors. The method comprises binding the peptide of claim 1 to a target peptide.
A method for protecting a target peptide from degradation by the proteasome 20S core. A peptide having cytotoxic activity comprising the sequence represented by SEQ ID NO:15. The peptide according to claim 1 or 8, further comprising a basic peptide attached to the N-terminus or C-terminus.

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