JP2021048838A - Peptides - Google Patents

Abstract

To provide novel proteasome inhibitors.SOLUTION: Provided is a proteasome inhibiting peptide comprising an amino acid sequence corresponding to the 1st to 7th, 2nd to 7th, 3rd to 7th, 1st to 6th, 2nd to 6th, or 3rd to 6th amino acids of Ser-Xaa1-Leu-Xaa2-His-Trp-Trp (SEQ ID NO:1) where Xaa1 and Xaa2 represent any amino acid residue.SELECTED DRAWING: None

Description

The present invention relates to peptides.

The proteasome can degrade a specific protein at a specific time by recognizing and degrading the ubiquitin modification added to the protein as a marker of degradation. This ubiquitin-proteasome system is the major pathway that negatively regulates the intracellular abundance of proteins. The ubiquitin-proteasome system functions centrally in a wide range of biological phenomena, including cell cycle, gene expression, signal transduction, and immune response.

Proteasome inhibitors are positioned as anticancer agents and are used in the treatment of hematological malignancies.

Bortezomib was the first clinically applied proteasome inhibitor and has become the standard of care in multidrug therapy for multiple myeloma.

Bortezomib reversibly inhibits chymotrypsin-like activity in the proteasome. However, after a certain period of treatment, it is often made resistant by mutation of β subunit (Non-Patent Document 1). The second generation carfilzomib is an irreversible proteasome inhibitor. Carfilzomib exhibits higher selectivity for chymotrypsin-like activity than bortezomib (Non-Patent Document 2). Third-generation ixazomib can now be administered orally. Examples of those in the development stage include irreversible inhibitor marizomib (Non-Patent Document 3), orally administrable delanzomib (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 pharmacologicallogic profile competitive with bortezomib. Blood, 111, pp. 2665-2675

Multidrug therapy with proteasome inhibitors greatly improved the life expectancy of patients with multiple myeloma. However, on the other hand, the problem of resistance of cancer cells to proteasome inhibitors has not been overcome. Therefore, it is necessary to overcome the resistance formation or to develop a new proteasome inhibitor having a mechanism different from the inhibition mechanism of the proteasome in which the resistance mechanism has been acquired.

An object of the present invention is to provide a composition having a proteasome inhibitory activity having an inhibitory mechanism different from the conventional one in preparation for the resistance of the existing proteasome inhibitor.

As a result of diligent research, the present inventor has found a peptide having proteasome inhibitory activity having an inhibitory mechanism different from the conventional one.
The present invention has been completed based on the findings and includes the following aspects.
Item 1. From the 1st to 7th, 2nd to 7th, 3rd to 7th, 1st to 6th, 2nd to 6th, or 3rd to the following amino acid sequences Peptide containing the sixth sequence and having an action of inhibiting the proteolytic enzyme activity of the proteasome 20S core: Ser-Xaa ¹ -Leu-Xaa ² -His-Trp-Trp (SEQ ID NO: 1); where Xaa ¹ And Xaa ² indicate any amino acid residue.
Item 2. Item 2. The peptide according to Item 1, ^{further comprising Xaa 3} , which is an arbitrary amino acid residue, at the C-terminal of the amino acid sequence represented by SEQ ID NO: 1.
Item 3. ^{Further, the amino acid sequence Met-Xaa 4-} Xaa ^5- Pro- (SEQ ID NO: 5) is provided at the N-terminal of the amino acid sequence represented by SEQ ID NO: 1, ^{and Xaa 4} and Xaa ⁵ have arbitrary amino acid residues. Item 2. The peptide according to Item 1 or 2.
Item 4. A polynucleotide comprising a base sequence encoding the peptide according to any one of Items 1 to 3.
Item 5. A vector containing 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. Item 6. The composition according to Item 6, wherein the composition is a pharmaceutical composition.
Item 8. Item 2. The composition according to Item 7, wherein the pharmaceutical composition is used for the prevention and / or treatment of malignant tumors.
Item 9. A method for protecting a target peptide from degradation by the proteasome 20S core, which comprises binding the peptide according to any one of Items 1 to 3 to the target peptide.

According to the present invention, it is possible to provide a peptide that inhibits the proteolytic enzyme activity of the proteasome 20S core.

The analysis result of the inhibition mode of PI-Rank1 peptide is shown. (A) A Lineweaver-Burk plot of PI-Rank 1 peptide at each concentration is shown. (B) The Dixon plot of the PI-Rank 1 peptide at each concentration is shown. The Ki values for chymotrypsin-like activity and caspase-like activity of PI-Rank1 are shown. It shows an inhibitory effect on PI-Rank 1 (6-17) and PI-Rank 1 (1-12) with the N-terminal or C-terminal deleted. _{(A) IC 50} for CT-L activity of proteasome 20S of PI-Rank 1 (1-17), PI-Rank 1 (6-17) and PI-Rank 1 (1-12) is shown. (B) The concentration-dependent inhibition curve of each peptide for CT-L activity is shown. It shows a change in _{the 20S enzyme inhibitory activity (50% inhibitory concentration (IC 50} )) of the PI-Rank1 peptide analog in which amino acids are gradually deleted from the N-terminal side or the C-terminal side. The change in _{the 20S enzyme inhibitory activity (50% inhibitory concentration (IC 50} )) of the PI-Rank 1 peptide analog in which each amino acid is replaced with alanine is shown. (A) The inhibitory activity shown in FIG. 3 is shown graphically. (B) The inhibitory activity shown in FIG. 4 is shown graphically. Shows the inhibitory effect of PI-Rank 1 (5-12) on the 26S proteasome. (A) Shows the connectivity between the 20S core and PI-Rank 1 (5-12). (B) Shows the binding property between 20S core or 26S proteasome and PI-Rank 1 (5-12). (A) Among the subunits constituting the 20S core, the analysis results by two-dimensional electrophoresis for identifying the site that binds to PI-Rank 1 (5-12) are shown. (B) A schematic view of the three-dimensional structures of the α subunit and the β subunit as viewed from the side is shown. (C) A cross-sectional view of the α subunit at the α1 subunit site is shown. (A) A schematic diagram of the binding mode of Rpt3 and β1 subunit is shown. (B) The cross-sectional view of (A) is shown. (C) The three-dimensional structure in the Rpt3 binding pocket of the α1 subunit and the binding pocket of the three amino acid residues on the C-terminal side of Rpt3 is shown. (D) A comparison of the C-terminal amino acid sequences of Rpt3 and PI-Rank1 (5-12) is shown. In (D), "acid" indicates an acidic amino acid, "medium" indicates a neutral amino acid, "salt" indicates a basic amino acid, and "good" indicates an aromatic amino acid. In addition, (parent) indicates a hydrophilic amino acid, and (sparse) indicates a hydrophobic amino acid. The effect of the C-terminal peptide of Rpt3 and PI-Rank 1 (5-12) on 20S enzyme activity is shown. (A) Shows changes over time in substrate decomposition. The dotted line shows the CT-L activity in the absence of PI-Rank1 (5-12), and the solid line shows the CT-L activity in the absence of PI-Rank1 (5-12). (B) Shows the 20S enzyme activity promoting action of the C-terminal peptide of Rpt3 in the presence and absence of PI-Rank1 (5-12). It shows the effect of cell membrane permeation type PI-Rank 1 (5-12). (A) A list of compounds used in the experiment is shown. [AC] indicates acetylation. (B) The amount of ATP (cell survival rate) 24 hours after the addition of each compound to the multiple myeloma cell line is shown. (C) The amount of ATP (cell survival rate) 48 hours after the addition of each compound to the multiple myeloma cell line is shown. Purified thioredoxin fusion human α-synuclein recombinant protein (represented by “αSYN (Trx fusion)”), and purified thioredoxin fusion human α-synuclein-PI- with the PI-Rank1 (1-12) amino acid sequence added to its C-terminus. The degradation rate of the Rank 1 (1-12) adduct (represented by "αSYN + Rank 1 (Trx fusion)") by the human 20S proteasome is shown. (A) shows the result of SDS-PAGE. In (A), "M" indicates a molecular weight marker. “-” Indicates a negative control without the addition of human purified 20S proteasome. “1h” or “4h” indicates the reaction time (hours) after the addition of the human purified 20S proteasome. (B) shows the quantitative result obtained by image analysis of the concentration of the protein band of (A). Recombinant poly of thioredoxin fusion human α-synuclein-PI-Rank1 (1-12) adduct (represented by "αSYN + Rank1 (Trx fusion)") and wild-type human α-synuclein that does not contain additional sequences such as tags. The degradation rate of the peptide (represented by "αSYN recombinant") by the 20S proteasome is shown. (A) shows the result of SDS-PAGE. In (A), "M" indicates a molecular weight marker. “-” Indicates a negative control without the addition of human purified 20S proteasome. “1h” or “4h” indicates the reaction time (hours) after the addition of the human purified 20S proteasome. In addition, (B) shows the quantitative results obtained by image analysis of the concentration of the protein band of (A).

1. 1. Proteasome Inhibiting Peptide An embodiment of the present invention relates to a peptide having an action of inhibiting the proteolytic enzyme activity of the proteasome 20S core (hereinafter, also referred to as “proteasome inhibitory peptide”). The 26S proteasome consists of a complex of 20S core, which exists inside the cell and has protease activity, and 19S associated at both ends thereof. Further, it is known that some proteasome 20S cores exhibit proteolytic enzyme activity without forming a complex with 19S.

The structure of the proteasome-inhibiting peptide is shown below, and the amino acid sequence is described from the N-terminal side to the C-terminal side.
The description and physical characteristics of the amino acid sequence shall be in accordance with Table 1 below.

Amino acids include both natural and artificial amino acids.

In the present specification, the "residue" of various amino acids is a structural unit of an amino acid constituting a peptide, and the hydrogen atom is removed from the amino acid for the amino group of the main chain, and / or the carboxyl group of the main chain. Represents a group with -OH removed.

Also, as used herein, "having" may include both the concepts of "including" and "consisting of".

Some embodiments of the proteasome-inhibiting peptide, as the main chain, have the following amino acid sequences, 1st to 7th, 2nd to 7th, 3rd to 7th, 1st to 6th, ^{Ser-Xaa 1} -Leu-Xaa ² -His-Trp-Trp (SEQ ID NO: 1) containing the 2nd to 6th or 3rd to 6th sequences ^{(where Xaa 1} and Xaa ² are (Indicating any amino acid residue).

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, ^{both Xaa 1} and Xaa ² are Arg residues. Most preferably, the first embodiment of the proteasome inhibitory peptide comprises, as the main chain, the amino acid sequence represented by Ser-Arg-Leu-Arg-His-Trp-Trp (SEQ ID NO: 2).

^{The second embodiment of the proteasome inhibitory peptide has, as the main chain, Xaa 3} which is an arbitrary amino acid residue on the C-terminal side of the amino acid sequence represented by SEQ ID NO: 1. That is, the second embodiment of the proteasome inhibitory peptide has an amino acid sequence represented by ^{Ser-Xaa 1} -Leu-Xaa ^2- His-Trp-Trp-Xaa ^{3 (SEQ ID NO: 3) as a 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. A second embodiment of the most preferred proteasome inhibitory peptide has an amino acid sequence represented by Ser-Arg-Leu-Arg-His-Trp-Trp-Arg (SEQ ID NO: 4) as the backbone.

^{In the third embodiment of the proteasome-inhibiting peptide, the amino acid sequence Met-Xaa 4-} Xaa ^5- Pro- (SEQ ID NO: 5) is located 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 inhibitory peptide, as the main chain, is Met-Xaa ⁴ -Xaa ⁵ -Pro-Ser-Xaa ¹ -Leu-Xaa ² -His-Trp-Trp (SEQ ID NO: 6) or Met- It ^{has an amino acid sequence represented by Xaa 4} -Xaa ⁵ -Pro-Ser-Xaa ¹ -Leu- Xaa ² -His-Trp-Trp-Xaa ³ (SEQ ID NO: 7). Here, Xaa ⁴ and Xaa ⁵ are arbitrary amino acid residues. Preferably ^{, either Xaa 4} or Xaa ⁵ 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 Ala or Arg residues. A third embodiment of the most preferred proteasome inhibitory peptide, as the backbone, is Met-Ala-Arg-Pro-Ser-Arg-Leu-Arg-His-Trp-Trp (SEQ ID NO: 8) or Met-Ala-Arg- It has an amino acid sequence represented by Pro-Ser-Arg-Leu-Arg-His-Trp-Trp-Arg (SEQ ID NO: 9).

The main chain of the proteasome inhibitory peptide is preferably about 5 to 150 amino acid residues in total length. 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-inhibiting peptide can be 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, it can be synthesized by a tert-butoxycarbonyl group (Boc) solid-phase synthesis method, a 9-fluorenylmethyloxycarbonyl group (Fmoc) solid-phase synthesis method, or the like. When the total length of the main chain of the proteasome-inhibiting peptide exceeds 100 amino acid residues, it may be synthesized as a recombinant peptide using Escherichia coli, insect cells, mammalian cells, yeast, silk moth and the like.

Proteasome inhibition peptide, C-terminal, carboxyl group (-COOH), a carboxylate (-COO ^-), or those which are ester (-COOR), etc. may also be included.

The proteasome inhibitory peptide may have a modification in addition to the main chain. Modifications here are not limited unless they inhibit the proteolytic enzyme activity of the proteasome 20S core of the proteasome inhibitory peptide. The modification may be present at least one selected from the N-terminus, C-terminus of the backbone and at least one side chain of the amino acid residues constituting the backbone.

Examples of the N-terminal modification include modifications generally used as the N-terminal modification of a peptide. For example, a modification of the N-terminal, azide modification, C _1-6 acylation modifications such as C _1-6 alkanoyl (acetylation modification, formylation modification, etc.), succinylated modified, Boc modification, Fmoc modification, biotinylation modification , Myristinization modification, palmitoylation modification, stearoylation modification, fluorescent dye modification (TAMRA label, FITC label, Rhodamine label, FAM label, etc.), pyroglutamylation modification, dansylation modification, methylation modification and the like.

As the C-terminal modification, modifications generally used as the C-terminal modification of peptides can be raised. For example, examples of the C-terminal modification include amidation modification, biotination modification, methyl esterification modification, aldehydeation modification, N-hydroxyesterification modification, pNA labeling, MCA labeling, β-naphthylamide modification and the like.

Side chains of amino acid residues constituting the main chain can be modified by polyethylene glycolization, phosphorylation, acetylation, methylation, fluorescence, biotination, sugar or sugar chain, lipid, etc. Can be mentioned. However, it is preferable not to modify the side chain at least on the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. More preferably, it is not applied to the amino acid sequences represented by SEQ ID NOs: 3 to 9.

The proteasome inhibitory peptide may contain a basic peptide in order to enhance the cell membrane permeability of the proteasome inhibitory peptide. A basic peptide is a peptide that is rich in basic amino acid residues. As the basic amino acid residue, an arginine residue is preferable. The basic peptide is preferably about 5 to 25 amino acid residues. The ratio of basic amino acid residues contained in the basic peptide is at least about 25%, preferably about 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80. It is about% or more and about 90% or more. For example, as basic peptides, arginine peptides consisting of 5 to 8 consecutive arginine residues, 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), P22 N- (14-30) and the like. The basic peptide is a linker moiety of several amino acid residues (preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues) continuous with the N-terminal or C-terminal of the main chain of the proteasome-inhibiting peptide. Can be concatenated including.

Hereinafter, it has (1) an amino acid sequence of the main chain of a proteasome-inhibiting peptide, a peptide having a basic polypeptide sequence, and (2) an amino acid sequence of the main chain of a proteasome-inhibiting peptide, a basic polypeptide sequence, and a linker sequence. The peptide is called 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-inhibiting activity of the proteasome-inhibiting peptide is evaluated by whether or not it inhibits the proteasome 20S core proteolytic activity, preferably the chymotrypsin-like activity or caspase-like activity of the proteasome 20S, more preferably the chymotrypsin-like activity. be able to.

The chymotrypsin-like activity, caspase-like activity, and trypsin-like activity of the proteasome 20S can be measured by a known method. The activity of these proteolytic enzymes can be evaluated by whether or not a specific peptide contained in the substrate is cleaved.

As a substrate used for evaluation of chymotrypsin-like activity, for example, Suc-LLVY-AMC (Suc represents a succinyl group, LLVY represents a peptide chain, and AMC represents 7-amide-4-methylcoumarin) should be used. Can be done. As a substrate used for evaluation of trypsin-like activity, for example, Boc-LRR-AMC (Boc represents a tert-butoxycarbonyl group, LRR represents a peptide chain, and AMC represents 7-amide-4-methylcoumarin). Can be used. As a substrate used for evaluation of caspase-like activity, for example, Z-LLE-AMC (Z represents a benzyloxycarbonyl group, LLE represents a peptide chain, and AMC represents 7-amide-4-methylcoumarin). For example, about 100 ng of human proteasome 20S core (Enzo ENZ Life Sciences, Inc.) is added with about 50 mM substrate and proteasome inhibitory peptide to about 100 μL of enzyme reaction buffer. As the enzyme reaction buffer used for measuring chymotrypsin-like activity or caspase-like activity, for example, an enzyme reaction buffer containing HEPES (pH 8.0) of about 25 mM, EDTA of about 0.5 mM and SDS of about 0.03% should be used. Can be done. As the enzyme reaction buffer used for measuring the trypsin-like activity, for example, an enzyme reaction buffer containing HEPES (pH 8.5) of about 25 mM and EDTA of about 0.5 mM can be used. The proteolytic enzyme activity can be measured at about 30 ° C. at 10-minute intervals for about 1 hour 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.

2. A polynucleotide encoding a proteasome-inhibiting peptide and a vector containing the polynucleotide An embodiment of the present invention is a polynucleotide encoding an amino acid sequence constituting the main chain of a proteasome-inhibiting peptide, or an amino acid sequence of a cell-permeable proteasome-inhibiting peptide. (Hereinafter, both are simply referred to as "polynucleotides"). The polynucleotide can be DNA or RNA. The polynucleotide is preferably DNA. The amino acid sequence constituting the main chain of the proteasome-inhibiting peptide or the amino acid sequence of the cell-penetrating proteasome-inhibiting peptide can be converted into a polynucleotide sequence according to a general codon table.

In addition, the polynucleotide may be inserted into the vector so that it can be expressed in the host cell. The vector into which the polynucleotide is inserted can be selected depending on the host cell. For example, when a polynucleotide is expressed in mammalian cells including humans, a plasmid vector containing a promoter capable of expressing the polynucleotide, a multicloning site, a poly A signal, etc., an adenovirus vector, a retrovirus vector, a lentiviral vector, etc. Can be used. When expressing a polynucleotide in Escherichia coli, a plasmid vector containing a promoter capable of expressing the polynucleotide, a multicloning site, or the like can be used.

3. 3. Composition and Pharmaceutical Composition The composition according to the present invention contains the proteasome-inhibiting peptide, the polynucleotide, or a vector containing the polynucleotide as an active ingredient. The composition according to the present invention can be prepared by combining the active ingredient with an appropriate carrier or additive. The carriers and additives used in the preparation of the pharmaceutical composition include various substances commonly used in ordinary drugs depending on the dosage form of the pharmaceutical composition, such as excipients, binders, disintegrants, and lubricants. , Colorants, flavoring agents, odorants, surfactants and the like can be exemplified.

As the carrier, a transfection reagent containing a polymer, a lipid, magnetism, or the like can be used.

The dosage form when the composition is orally administered is not particularly limited, but is limited to tablets, powders, granules, capsules (including hard capsules and soft capsules), liquids, pills, and suspensions. Examples thereof include agents and emulsions. When the pharmaceutical composition is administered parenterally, examples thereof include injections, infusions, suppositories, nasal drops, and transpulmonary administrations.

When the composition is an oral solid composition such as tablets, powders, granules, rounds, capsules, etc., as a carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate , Kaolin, crystalline cellulose, silicic acid, methyl cellulose, glycerin, sodium alginate, gum arabic, etc .; simple syrup, pudo sugar solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, carboxymethyl cellulose, cellac , Methyl cellulose, ethyl cellulose, water, ethanol, potassium phosphate, etc .; dried starch, sodium alginate, canten powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid Disintegrants such as monoglyceride, starch, lactose; disintegrators such as sucrose, stearic acid, cacao butter, hydrogenated oil; absorption enhancers such as sodium lauryl sulfate; moisturizers such as glycerin, starch; starch, lactose, kaolin, Excipients such as bentonite and colloidal silicic acid; purified starch, stearate, boric acid powder, lubricants such as polyethylene glycol and the like can be used. Further, the tablet may be a tablet coated with a normal skin, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, a multi-layer tablet, or the like.

When the composition is an oral solid composition of pills, as a carrier, for example, excipients such as glucose, lactose, starch, cacao butter, hardened vegetable oil, kaolin, talc; gum arabic powder, Binders such as talc powder and gelatin; disintegrants such as laminarin and canten can be used.

When the composition is an oral solid composition of a capsule, the capsule is prepared by mixing the active ingredient with various carriers exemplified above and filling it in a hard capsule, a soft capsule, or the like. Will be done.

When the preparation is a liquid preparation, it may be an aqueous or oily suspension, a solution, a syrup, or an elixir preparation, and is prepared according to a conventional method using ordinary additives.

In the preparation when the composition is an injection, as a carrier, for example, a diluent such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like. Acidity regulators such as sodium citrate, sodium acetate, sodium phosphate; buffers such as dipotassium phosphate, trisodium phosphate, sodium hydrogen phosphate, sodium citrate; sodium pyrosulfite, EDTA, thioglycolic acid, Stabilizers such as thiolactic acid; sugars such as mannitol, inositol, maltose, sucrose, and lactose can be used as molding agents when freeze-dried. In this case, a sufficient amount of glucose or glycerin to prepare an isotonic solution may be contained in the preparation, or a usual solubilizing agent, soothing agent, local anesthetic, etc. may be added. good. These carriers can be added to produce subcutaneous, intramuscular, and intravenous injections by conventional methods.

When the preparation is a drip infusion, the administration compound can be prepared by dissolving it 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 dose of the pharmaceutical composition according to the present embodiment is not particularly limited as long as the effects of the present invention are exhibited, and may be appropriately set depending on the dosage form, the age, sex, degree of medical condition, etc. of the patient.

For example, when a pharmaceutical composition containing a proteasome-inhibiting peptide is systemically administered, it is administered so as to be 0.01 to 1,000 mg / day per 1 kg of adult body weight in terms of the weight of the main chain of the proteasome-inhibiting peptide. can do. When the pharmaceutical composition containing the proteasome-inhibiting peptide is locally administered, it can be administered so as to be 0.01 to 100 mg per ^{1 cm 2 of} the target tissue in terms of the weight of the main chain of the proteasome-inhibiting peptide. ..

The polypeptide or the pharmaceutical composition containing the vector containing the polypeptide is converted into a polynucleotide encoding the amino acid sequence of the main chain of the proteasome inhibitory peptide, and in the case of systemic administration, 0.1 to 1 per kg of adult body weight. It can be administered at a dose of 000 mg / day.

When locally administered, the polypeptide or the pharmaceutical composition containing the vector containing the polypeptide is converted into a polynucleotide encoding the amino acid sequence of the main chain of the proteasome-inhibiting peptide, and is 0 per ^{1 cm 2 of the target tissue.} It can be administered at a dose of 0.01 to 100 mg / day. The vector can be linearized if desired. The polynucleotide, or 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 malignancies. Specifically, respiratory malignancies originating from the trachea, bronchi, lungs, etc; Gastrointestinal malignancies originating from parts, etc .; liver cancer; pancreatic cancer; urinary system malignant tumors originating from the bladder, urinary tract, or kidney; female reproductive system malignant tumors originating from the ovary, oviduct, uterus, etc .; breast cancer: Prostatic cancer; Skin cancer; Endocrine malignancies such as hypothalamus, pituitary gland, thyroid gland, parathyroid gland, adrenal gland; Central nervous system malignancies; Solid tumors such as malignant tumors originating from bone and soft tissue, and myeloplastic 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 total myelogenous leukemia Leukemia, acute meganuclear leukemia, red leukemia, eosinophilia, chronic eosinophilia, chronic neutrophil leukemia, adult T-cell leukemia, hairy cell leukemia, plasmacytoid leukemia, multiple myelomas, Hematopoietic malignancies such as malignant lymphoma; Hematopoietic tumors such as lymphoid malignancies can be mentioned. More preferably, it is a hematopoietic tumor. The most preferred hematopoietic tumor is multiple myeloma.

The pharmaceutical composition may be used as an anticancer agent. Antineoplastic agents include alkylating agents, anti-metaplastic agents, antitumor antibiotics, microvascular inhibitors, hormones or hormone-like agents, platinum preparations, topoisomerase inhibitors, cytokines, antibody agents, radioimmunotherapy agents, molecules. It can be appropriately selected from target drugs, non-specific immunoactive drugs, and other anticancer drugs according to the target tumor.

Although not limited here, examples of alkylating agents include cyclophosphamide, iphosphamide, busulfan, melfaran, bendamstin hydrochloride, nimustin hydrochloride, lanimustin, dagalvazine, procarbazine hydrochloride temozolomid, etc .; as metabolic antagonists. , Metotrexate, Pemetrexed Sodium, Fluorouracil, Doxyflulysine, Capecitabin, Tegafur, Citarabin, Citarabin ocphosphate hydrate, Enocitabin, Gemcitabine hydrochloride, Mercaptopurine hydrate, Fludarabin phosphate ester, Nerarabin , Holinate calcium, hydroxycarbamide, L-asparaginase, azacitidine, etc .; Examples of antitumor antibiotics include doxorubicin hydrochloride, daunorbisin hydrochloride, pyrarbisin, epirubicin hydrochloride, idarbisin hydrochloride, acralbisin hydrochloride, amurubicin hydrochloride, mitoxane. Tron hydrochloride, mitomycin C, actinomycin D, bleomycin, pepromycin sulfate, dinostatin styramer, etc .; Examples of microvascular inhibitors include vincrystin sulfate, vinblastin sulfate, bindesin sulfate, binorelbin tartrate, paclitaxel, docetaxel water. Japanese products, elibrin mesylate, etc .; as hormones or hormone-like drugs (hormone agents), anastrozole, exemethan, retrozol, tamoxyphencitrate, tremiphencitrate, flubestland, flutamide, bicartamide, medroxy Progesterone acetate, estramustin phosphate sodium hydrate, goselelin acetate, leuprorelin acetate, etc .; as platinum preparations, cisplatin, milliplatin hydrate, carboplatin, nedaplatin, oxaliplatin, etc .; as topoisomerase inhibitors Is a topoisomerase I inhibitor such as irinotecan hydrochloride hydrate and nogitecan hydrochloride, and a topoisomerase II inhibitor such as etoposide and sobzoxane; as cytokines, interferon gamma-1a, teseloykin, cellmoloikin, etc.; as antibody drugs. , Trustuzumab, rituximab, gemtuzumab ozogamicin, bebashizumab, setuximab, panitumumab, alemtuzumab, etc .; Lutezomib, calfilzomib, ixazomib citrate, gefitinib, imatinib mesylate, erlotinib hydrochloride, sorafenib tosilate, sunitinib malate, salidamide, nilotinib hydrochloride hydrate, dasatinib hydrate, lapatinib tosilate hydrate Substances, Eberolimus, Lenalidemid hydrate, Dexametazone, Temsirolimus, Bolinostat, Tretinoin, Tamivarotene, etc .; Non-specific immunoactive agents include OK-432, Dried BCG, Kawatake polysaccharide preparation, Lentinan, Ubenimex, etc. Examples thereof include acegraton, porphimer sodium, taraporfin sodium, ethanol, arsenic trioxide and the like. Although not limited, a metabolic antagonist can be preferably mentioned from the viewpoint of action and effect, and a molecular target drug and an antibody drug can be preferably mentioned from the viewpoint of having few side effects.

Molecular-targeted drugs include bortezomib, carfilzomib, ixazomib citrate, lenalidomide, dexamethasone, cyclophosphamide, imatinib mesylate, nilotinib hydrochloride hydrate, and dasatinib water when targeting hematopoietic tumors. Japanese products are preferable.

As the antibody drug, trastuzumab and panitumumab are preferable, and trastuzumab is particularly preferable.

As a treatment method for multiple myeloma, chemotherapy in combination with bortezomib and other drugs is generally performed. For example, chemotherapy includes VCD therapy (bortezomib, cyclophosphamide, dexamethasone), VRD therapy (bortezomib, lenalidomide, dexamethasone), BD therapy (bortezomib, dexamethasone) and the like. As a method for treating multiple myeloma, the proteasome inhibitory peptide of the present invention can be used instead of bortezomib.

The mode of the combined use (combination) of the pharmaceutical composition of the present invention and the anticancer agent may be any mode of use that exhibits the effect of the present invention, and is not particularly limited. For example, the pharmaceutical composition may be administered in parallel with the administration of the anticancer drug, or the pharmaceutical composition may be administered prior to the administration of the anticancer drug or after the administration of the anticancer drug. May be good. In this case, the pharmaceutical composition and the anticancer drug may be administered alternately. Further, after the administration of the anticancer drug, the pharmaceutical composition may be co-administered from the middle of the administration of the anticancer drug according to the degree of shrinkage of the tumor tissue, or conversely, after the administration of the pharmaceutical composition. , An anticancer drug may be administered in combination from the middle of the process.

In addition, the pharmaceutical composition of the present invention may be administered once, continuously, or intermittently as 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 the anticancer drug, 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 on the day of the start of administration. Up to, a method of administering once a day every day can be exemplified.

The pharmaceutical composition of the present invention is prepared as a pharmaceutical composition of various forms (dosage forms) according to its administration route (administration method), and is administered to a target tumor patient in combination with an anticancer agent. To. The tumor patient is a patient who has not undergone antitumor treatment such as chemotherapy, radiation therapy and / or excision surgery (surgical therapy), and who is at any stage during and after treatment. There may be.

The route of administration (administration method) of the pharmaceutical composition is not particularly limited, and oral administration; parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, transmucosal administration, transdermal administration, and rectal administration. Can be mentioned. Oral administration and intravenous administration are preferable, and oral administration is more preferable.

The dose of the pharmaceutical composition of the present invention depends on the type and location of the tumor to be treated, the stage (progression) of the tumor, the pathological condition of the patient, the age, the sex, the weight, the type of the anticancer drug to be used in combination, and the like. Therefore, it can fluctuate and can be appropriately set according to these factors.
When the pharmaceutical composition and the anticancer drug are used in combination, the anticancer drug can be administered according to the general administration method and dose of the drug.

4. Methods for Protecting Target Peptides Certain embodiments of the present invention relate to methods for protecting target peptides.
The main chain of the proteasome-inhibiting peptide according to the first, second and third embodiments can inhibit the proteolytic enzyme activity of the proteasome 20S core. Therefore, by adding the main chain of the proteasome-inhibiting peptide according to the first, second and third embodiments to the other peptide, it is possible to suppress the degradation of the other peptide by the proteasome.

That is, in this embodiment, the target peptide is protected from degradation by the proteasome 20S core by binding the main chain of the proteasome-inhibiting peptide according to the first, second and third embodiments to a target peptide other than the proteasome-inhibiting peptide. Including doing.

Methods of binding the target peptide to the backbone of the proteasome inhibitory peptide are known. For example, the peptide may be synthesized by directly or indirectly linking the main chain of the proteasome-inhibiting peptide to the N-terminal or C-terminal of the target peptide. Further, for example, a polypeptide encoding the main chain of a proteasome-inhibiting peptide is directly or indirectly linked to the 5'-terminal side or 3'-terminal side of the polynucleotide encoding the target polypeptide, and the peptide is synthesized from this polynucleotide. It can be obtained by doing.

5. Complex of anti-cancer agent and proteasome-inhibiting peptide The proteasome-inhibiting peptide of the present invention may also include a proteasome-inhibiting peptide complexed with an anti-cancer agent. The complex formation of the anticancer drug and the proteasome-inhibiting peptide can be carried out by utilizing the N-terminal amino group or the C-terminal carboxyl group of the proteasome-inhibiting peptide. For example, the carboxyl group contained in the main component of the anticancer drug is previously mixed with carbodiimide and N-hydroxysuccinimide to form an active ester type. By mixing this active ester type anticancer agent with a peptide and forming an amide bond with the N-terminal amino group of the peptide, the anticancer agent and the proteasome-inhibiting peptide can be formed into a complex.

In addition, the N-terminal amino group of the proteasome-inhibiting peptide is first protected by acetylation or the like, and the carboxyl group on the C-terminal side of the proteasome-inhibiting peptide is converted into an active ester type by mixing carbodiimide and N-hydroxysuccinimide. , An active ester-type proteasome-inhibiting peptide can be reacted with an anticancer agent having an amino group to form an amide bond.

The reaction for forming the amide bond is known. For example, the reaction can be carried out in the presence of a dehydration condensing agent and, if necessary, a catalyst. Carbodiimide compounds such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide); imidazole compounds such as N, N-carbonyldiimidazole); triazine compounds Phosphonium-based compounds; uronium-based compounds and the like. It is preferably a carbodiimide-based compound, and is particularly preferable. A catalyst can also be used with the dehydration condensing agent. The specific catalyst is not limited. For example, 1-hydroxybenzotriazole), N-hydroxysuccinimide (HOSu) 1-hydroxy-7-azabenzotriazole (HOAt) and the like can be mentioned. HOBt is preferred.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not construed as being limited to Examples.

1. Search for 20S core-binding peptide
The cDNA display method was used to search for peptides that bind to the proteasome 20S core.

1-1. Method (1) Preparation of IVV 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) A DNA library was created as a template for in vitro virus (IVV) containing. This DNA library was ^{transcribed from IVV template mRNA using RiboMAX TM} Large Scale RNA Production Systems (Promega Co., Ltd.), and the IVV template mRNA was purified by RNeasy Mini Kit (Qiagen Co., Ltd.). 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 to translate a peptide derived from IVV template mRNA, and IVV in which the peptide and IVV template mRNA were linked was prepared. Furthermore, in order to stabilize IVV, the mRNA in IVV was reverse transcribed to prepare a double-stranded second complex of peptide, puromycin and mRNA-cDNA.

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

(3) Peptide identification Three steps of peptide translation, IVV selection, and amplification of cDNA in the second complex were set as one cycle, and five cycles were repeated to obtain a pool of cDNA in each cycle. The cDNA pool of each cycle was sequence-analyzed by a next-generation sequencer to identify the peptide sequence.

1-2. Sequence analysis results In the analysis by the next-generation sequencer, an average of 1.5 million reads were obtained in each cycle including the peripheral sequences of the IVV diversity region (the region encoding the amino acid sequence of 8 random residues). When the second and subsequent read sequences are sequentially added to the read sequences obtained in the first cycle, the duplicated read sequences increase as the cycle progresses from the second cycle onward, and about 400,000 reads are performed in the fifth cycle. Convergence to a specific sequence was observed. The most frequently occurring read sequence accounted for 5.2% of the approximately 1.5 million reads analyzed in the 5th cycle.

Of the 17-residue amino acid peptide sequences that can be translated from the sequenced cDNA sequence, the most frequently filed sequence is MARPSRLRHWWRLRRRV (SEQ ID NO: 10) from the N-terminus to the C-terminus (hereinafter, "PI-Rank 1"). It was called). The conversion from the cDNA sequence to the amino acid sequence followed the general peptide notation.

2. Enzyme inhibitory effect of 20S core-binding peptide 1. It was examined whether or not the peptide identified in (1) can inhibit the proteolytic enzyme activity of the 20S proteasome.

2-1. Method (1) Purification of Peptides Each peptide was synthesized by a contractor (Cosmo Bio Co., Ltd.) by a standard solid-phase synthesis method, and a crude product cleaved from a carrier resin was purchased. The crude peptide was purified and used according to the following procedure.

Reverse-phase column (COSMOSIL Packed Column 5C18-AR-II 4.6 ID x 150 mm) in which crude peptide was dissolved in 5% CH ₃ CN / 0.05% TFA / H _{2 O and connected to HPLC (HITACHI ELITE LaChrom L-2000 series).} ) (Buffer A: 0.05% TFA / H ₂ O Buffer B: 0.05% trifluoroacetic acid (TFA) / CH ₃ CN, detection wavelength: 220 nm, temperature: 40 ° C, flow velocity: 1 mL / min, CH ₃ CN concentration change: 5% -80%). The recovered fraction was dried under reduced pressure and then stored at -20 ° C.

In the peptides purified by the above method, the N-terminal of each peptide becomes a hydrogen atom and the C-terminal becomes a hydroxyl group. In addition, each peptide will be abbreviated as PI-Rank1 below.

(2) Measurement of enzyme activity The proteasome enzyme activity at the time of addition of the inhibitor was evaluated by measuring the decomposition rate of the fluorescent substrate peptide. Suc-LLVY-AMC was used for chymotrypsin-like activity, and Z-LLE-AMC was used as a fluorescent substrate peptide for caspase-like activity (PGPH-like activity) (Suc is a succinyl group, Z is a benzyloxycarbonyl group, and Boc is a butoxycarbonyl. Group, AMC stands for 7-amide-4-methylcoumarin). 100 ng human proteasome 20S core (Enzo ENZ Life Sciences, Inc.) 50 mM each fluorescent substrate peptide, 100 μL of inhibitory peptide at each concentration in an enzyme reaction buffer (measurement of chymotrypsin-like activity, PGPH-like activity: 25 mM HEPES pH 8.0 , 0.5 mM EDTA, 0.03% SDS, measurement of trypsin-like activity: 25 mM HEPES pH8.5, 0.5 mM EDTA). The fluorescence change due to free AMC at an excitation wavelength of 380 nm and a fluorescence wavelength of 460 nm was measured at 30 ° C. for 1 hour at 10-minute intervals using FusionTMα-FP (Parkin Lumer Japan Co., Ltd.).

2-2. Results The inhibitory effect is shown as an _{IC 50 value.} PI-Rank1 the IC ₅₀ values for CT-L activity was 1.46MyuM. Further, IC ₅₀ values of PGPH activity of PI-Rank1 is 0.65 [mu] M, were more inhibitory effect on PGPH activity.

Next, in order to clarify the Ki value of the inhibitory activity against the 20S core and the mode of inhibition, PI-Rank1 was used to change the substrate concentration and the peptide concentration with respect to the CT-L activity to change the reaction rate. Was analyzed by the Lineweaver-Burk plot and the Dixon plot. The result is shown in FIG. FIG. 1 (A) is a Lineweaver-Burk plot for determining the mode of inhibition, and FIG. 1 (B) is a Dixon plot for determining the Ki value. The regression lines of the Lineweaver-Burk plot all intersected on the negative side of the X-axis. This suggests that the PI-Rank1 peptide has a non-antagonistic inhibitory effect on the enzymatic activity of the 20S core. The Ki values for chymotrypsin-like activity and caspase-like activity of PI-Rank1 are shown in FIG. The second complex (IVV) used in the cDNA display method consists of a 168-base-long cDNA, a puromycin linker, and a translated 10-odd-residue peptide. Due to the size of this molecule, it is unlikely that it will get inside the 20S core. Therefore, if it binds to the outer surface of the 20S core and shows an inhibitory effect, it is considered to be a rational result to show a non-antagonistic formula.

3. 3. Structural activity correlation of PI-Rank1 Next, in order to clarify the site of the 17-residue amino acid sequence of PI-Rank1 that has an inhibitory effect on the enzymatic activity of the 20S core, the amino acid sequence of PI-Rank1 was deleted. Alternatively, PI-Rank1 analogs introduced with one amino acid substitution were prepared, and the inhibitory effect of these analogs on the enzymatic activity of the 20S core was evaluated.

3-1. Method PI-Rank1 (6-17) and PI-Rank1 (1-12) were prepared by deleting 5 residues each of the N-terminal or C-terminal region of PI-Rank1 shown in FIG. In addition, a short-chain analog was prepared in which a maximum of 6 amino acid residues were deleted by 1 amino acid residue from the N-terminal or C-terminal side of PI-Rank 1 (1-12) shown in FIG. Further, as shown in FIG. 5, an alanine-substituted analog was prepared by substituting alanine for each amino acid residue other than alanine "A" among the 12 amino acid residues of PI-Rank 1 (1-12). The production of each analog is described in 2-1. According to (1). The chymotrypsin-like (CT-L) activity of the 20S core is described in 2-1. _{The IC 50} value was determined by measuring according to (2).

3-2. Results Figure 3 (A), showing an IC ₅₀ value of PI-Rank1 (6-17) and PI-Rank1 (1-12). PI-Rank1 (1-12) was confirmed to have the same inhibitory effect on CT-L activity as PI-Rank1 (1-17) (IC ₅₀ = 0.93 ± 0.30 μM), but PI-Rank1 (6-17) IC ₅₀ was higher than 10 μM, and the inhibitory effect disappeared. Moreover, as shown in FIG. 3 (B), the inhibitory effect of PI-Rank1 (1-12) was concentration-dependent as in PI-Rank1 (1-17). From this, it was clarified that PI-Rank 1 (1-12) has a site of action on CT-L activity. It was considered consistent with this that the puromycin linker was bound to the C-terminal side at the selection stage by the cDNA display method, and a region responsible for the inhibitory action was found on the N-terminal side.

Next, using the short-chain analog of PI-Rank1 (1-12) and the alanine-substituted analog, the site of action responsible for the inhibitory action was identified.

Figure 4 shows an IC ₅₀ value for the short-chain analogs of CT-L activity of PI-Rank1 (1-12). In addition, FIG. 6 (A) shows a graph _{of IC 50} values for CT-L activity of short-chain analogs. In the short-chain analog deleted from the N-terminus, the activity was lost when 2 residues were deleted, and conversely, the activity was restored when 3 and 4 residues were deleted. PI-Rank 1 (5-12) with 4 residues deleted _{showed an IC 50} value of 0.82 μM. However, further deletion significantly attenuated the inhibitory activity. When the sequence is deleted from the C-terminal side, the IC ₅₀ value increases and the inhibitory activity decreases, except for the peptide PI-Rank 1 (1-11), which lacks only one residue, showing _{an IC 50 value of 0.72 μM.} , More strongly inhibited CT-L activity. From the above results, it is the region of 8 residues from serine at 5th residue to arginine at 12th residue that has an inhibitory effect on CT-L activity in PI-Rank1 (1-12). The shortest peptide necessary and sufficient for inhibitory activity was considered to be PI-Rank 1 (5-11).

FIG. 5 shows _{IC 50} values for CT-L activity of alanine-substituted analogs. In addition, FIG. 6 (B) shows a graph _{of IC 50} values for CT-L activity of alanine-substituted analogs. Substitution of 1st methionine, 4th proline, 5th serine, 7th leucine, 9th histidine, and 10th and 11th tryptophan with alanine greatly attenuated the inhibitory effect. Substitution of the 3rd, 6th, 8th, and 12th arginines with alanine did not show a significant change in activity.

4.26 Enzyme inhibitory effect of PI-Rank1 on the S proteasome
The following experiments were conducted to investigate how PI-Rank1 affects the function of the 26S proteasome.

4-1. Method
As the 26S proteasome activity, chymotrypsin-like activity was measured by measuring the degradation rate of Suc-LLVY-MCA. Purified 26S proteasome (0.1 μg, Enzo Life Sciences, Inc.) in 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) Fluorescent substrate peptide (50 μM) was added in 100 μL of dithiothreitol in the presence of various inhibitory compound concentrations (0.1-10 μM). The fluorescence of released 7-amino-4-methylcoumarin (AMC) (λ _ex = 380 nm, λ _em = 480 nm) in the reaction mixture was monitored at 37 ° C. for 1 hour. Was used. Define IC ₅₀ values as the concentration of compound required for 50% inhibition of proteasome activity was determined for each compound from the respective inhibition curve.

4-2. Results As shown in FIG. 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 means that PI-Rank1 (5-12) cannot bind to the 20S core when the 20S core and 19S complex bind, or even if PI-Rank1 (5-12) binds, it controls the enzyme activity of the 20S core. It is possible that the necessary structural changes did not occur.

5. Confirmation of binding between 20S core and PI-Rank1 Next, it was confirmed by the affinity label method that PI-Rank1 actually binds to the 20S core.

5-1. Method (1) Generation of photoreactive peptide In order to introduce an allyl azide group that forms a covalent bond with surrounding molecules by nitrene that is activated by ultraviolet rays into PI-Rank 1 (5-12), an NHS ester of a modifying group Was reacted with the N-terminal amino group of PI-Rank 1 (5-12). Specifically, 50 nmol PI-Rank 1 (5-12) and 50 nmole Sulfo-SBED (Thermo SCIENTIFIC) were reacted in 10 μL DMF containing 1% TEA for 48 to 72 hours. After the reaction, it was purified by reverse phase HPLC and its molecular weight was confirmed by mass spectrometry. Since a biotin group is also introduced into this Sulfo-SBED, the photoreaction of Sulfo-SBED is performed by binding the Sulfo-SBED-labeled PI-Rank1 (5-12) to the target protein and then irradiating with ultraviolet rays. The group is crosslinked to the target protein. Therefore, by detecting biotin, the target protein bound to Sulfo-SBED-labeled PI-Rank 1 (5-12) can be detected by Western blotting or the like.

Mass spectrometry was performed by the following method. _{The sample was dissolved in 50% CH 3} CN / water (MS measurement solvent) containing 0.1% TFA. As a matrix, α-CHCA saturated with MS measurement solvent was prepared, mixed 1: 1 and measured with a MALDI-TOF mass spectrometer (SpiralTOF JMS-S3000, JEOL Ltd.). By mass spectrometry, molecules matching the target substance were confirmed in the fraction with a retention time of 23 minutes. Two mass peaks, 1833.6 and 1860.4, were detected in the mass spectrum. Since the theoretical amount of the target photoreactive peptide is 1859.23, 1860.4 is the hydrogen ion adduct, and 1833.6 is the same as the molecular weight when phenyl azide is changed to dehydroazebin and hydrogen ions are added at the same time. Hereinafter, 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 lysed in DMSO. 10 μL Buffer (25 mM HEPES, 0.5 mM EDTA, 0.03% SDS, pH 8.0) with a total of 10 ng so that the final concentration is 10 μM for SBED-PI-Rank1 (5-12) and 1 ng / μL for the 20S core. ), And allowed to undergo a light-shielding reaction at room temperature for 30 minutes. When confirming the specific binding, uncrosslinked PI-Rank 1 (5-12) was added as a competing peptide in a 10-fold amount (final concentration 100 μM). After the reaction, it was irradiated with a UV lamp at 365 nm and a height of 5 cm for 30 minutes. At that time, the sample was ice-cooled. Samples after UV irradiation were subjected to SDS-PAGE electrophoresis using 16.5% acrylamide gel (26 mA constant current 1 hour per gel). 3 filter papers soaked in solution A (0.3 M tris, 20% methanol, 0.05% SDS) on a positive electrode, 1 filter paper soaked in solution B (25 mM tris, 20% methanol, 0.05% SDS), PVDF A semi-dry blotting device (Co., Ltd.), which stacks two filter papers soaked in a film, gel, and C solution (25 mM Tris, 40 mM 6-aminohexanoic acid, 20% methanol, 0.05% SDS) in this order, sets a negative electrode, and sets a negative electrode. Blotting (25V 1mA / cm2 30 minutes) with Biocraft BE-331). After that, it was attached to Blocking One-P (Nacalai Tesque) and blocked while vibrating for 1 hour. After washing with TBS-T (50 mM Tris, 138 mM NaCl, 2.7 mM KCl, 0.1% Tween 20), the cells were reacted with streptavidin-HRP (GE Healthcare) antibody for 1 hour. Finally, biotin was detected on LAS4000 using the ECL reagent (AmershamTM ECLTM Prime Western Blotting).

5-2. Results Although not shown, a protein band was confirmed in the silver-stained image after SDS-PAGE near 25 kDa, which is the molecular weight band of subunits. When a biotin-labeled band was detected after Western blotting, as shown in FIG. 8 (A), SBED-PI-Rank1 (5-12) was added, and a band was seen around 25 kDa only when UV irradiation was performed. (Dotted square frame). Furthermore, a comparative study was conducted on the UV irradiation time. The irradiation time was changed to 0, 15, and 30 minutes. At the same time, in order to confirm the labeling inhibition due to competition, a sample supplemented with a 10-fold amount of PI-Rank 1 (5-12) was prepared in the same manner. As a result, it was confirmed that the band after 30 minutes of UV irradiation was best labeled, and at the same time, the band was thinned by excessive addition of PI-Rank 1 (5-12). From the above results, it is considered that SBED-PI-Rank1 (5-12) specifically labeled the subunit of the 20S core.

Similarly, it was confirmed whether the 26S proteasome was labeled with SBED-PI-Rank1 (5-12). After reacting with the samples of 26S proteasome and 20S core only, UV irradiation was performed for 30 minutes, and the results were confirmed by Western blotting. As a result, as shown in FIG. 8 (B), the labeled band was observed only in the case of the 20S core, and the labeled band could not be confirmed in the 26S proteasome. From the above results, it was considered that PI-Rank1 (5-12) did not bind to the 26S proteasome. 4. In, PI-Rank1 (5-12) did not inhibit the CT-L activity of the 26S proteasome, which was considered to be due to the fact that PI-Rank1 (5-12) did not bind to the 26S proteasome. In addition, PI-Rank1 (5-12) was considered to have a high possibility of acting on the bonding surface of the 20S core and the 19S complex.

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

6-1. Method (1) Two-dimensional electrophoresis The above 5-1. SBED-PI-Rank1 (5-12) and the 20S core were crosslinked according to the method described in (2). The total volume was 60 μl and used as an analysis sample. 60 μL of analytical sample was mixed with 1D sample Buffer (60 mM Tris-HCl, pH8.8, 5M urea, 1M thiourea, 5 mM EDTA, 1% CHAPS, 1% NP-40, 65 mM DTT). 6 μL of 1M iodoacetamide was added and reacted for 10 minutes. It was applied to agar gel (mini size (75 mm) pH range 3-10, AM 310) and layered with 2M urea. The electrophoresis was performed using an isoelectric focusing device (ATTA discRun AE-6541) (upper electrode solution: 0.2 M NaOH, lower electrode solution: 10 mM phosphoric acid, 300 V, 210 minutes). After electrophoresis, the gel was removed from the column and immobilized (2.5% trichloroacetic acid, 3 minutes). Soaked in SDS equilibration solution (50 mM Tris-hydrochloric acid, 2% SDS, 0.001% BPB, 10 minutes) for second-dimensional electrophoresis. 1% agarose was adhered to a 2D 16.5% acrylamide gel and subjected to SDS-PAGE electrophoresis. After that, CBB staining Western blotting was performed.

(2) Identification of 20S core subunits by LC-MS
The two-dimensional electrophoresed sample was stained with CBB (fixed solution: 20% methanol, 7.5% acetic acid stain: 0.25% CBB, 40% methanol, 7.5% acetic acid decolorizing solution 2.5% methanol, 7.5% acetic acid). All the bands visible by CBB staining were cut out. It was decolorized with 50% decolorizing solution (50% acetonitrile, 25 mM ammonium bicarbonate, 2 times), 25% decolorizing solution (25% acetonitrile, 25 mM ammonium bicarbonate, 2 times), 100% acetonitrile, and then centrifuged. The reducing solution was put therein and reacted at 56 ° C. for 300 minutes to remove the excess solution. The alkylated solution was added, and the reaction was performed at 37 ° C. for 30 minutes in the dark. After washing with 100 mM ammonium hydrogen carbonate, the mixture was dehydrated with acetonitrile for 10 minutes and dried by centrifugation. The trypsin solution was placed in the dried sample and allowed to stand for 45 minutes. The non-sucked solution was discarded, and a solution of 10 mM Tris-hydrochloric acid (pH 8.8) was added until the gel was completely immersed, and the mixture was reacted at 37 ° C. for 4 to 16 hours. The reaction product was recovered using a solution of 50% acetonitrile and 0.1% formic acid. Therefore, MilliQ water of 0.1% formic acid was added, and the acetonitrile concentration was reduced to 5% or less by repeating centrifugation. Finally, it was analyzed by LC-MS and analyzed.

6-2. result
Two-dimensional electrophoresis was performed to identify the labeled sites of SBED-PI-Rank1 (5-12) of the 14 subunits (7 types) that make up the 20S core. As a result, the three labeled spots shown in FIG. 9 (A) were observed. Two of these were found to be α1 and β4 subunits by mass spectrometric protein identification. The remaining one could not be identified, but it is highly possible that it is β5 or β6 from the same analysis example. These β subunits are considered to be in the vicinity of the CT-L active site as shown in Fig. 9 (B), and when SBED-PI-Rank1 (5-12) is degraded inside the 20S core, the labeling reaction occurs. Is thought to have occurred. On the other hand, the detected α1 subunit is located at the bonding surface of the 20S core and the 19S complex, as shown in FIG. 9C, and is a subunit involved in the opening and closing of the substrate uptake gate.

7. Interaction of Rpt3 with C-terminal peptide
The α1 subunit of the 20S core is known to interact with the C-terminal peptide of Rpt3, which is a constituent subunit of the 19S complex (Fig. 10 (A)). On the binding surface of the 19S complex of the α1 subunit, there is a groove on which the peptide acts (Fig. 10 (B)), and the C-terminal peptide of Rpt3 binds to this groove to open and close the substrate uptake gate of the 20S core. It is controlled (Fig. 10 (C)). Therefore, when the C-terminal 8 residues of PI-Rank1 (5-12) and Rpt3 were compared, it seemed that the hydrophilic and hydrophobic positions of the amino acid sequences were the same (Fig. 10 (D)). Since it was considered possible that PI-Rank1 (5-12) binds to the Rpt3 interaction site of the α1 subunit, the peptide (Rpt3-C) at the C-terminal 8 residue of Rpt3 shown in FIG. 10 (D) was synthesized. Then, the effect of 20S core on the enzyme activity was verified.

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

7-2. Results Figure 11 (A) shows the effect of Rpt3-C on CT-L activity in the absence / presence of PI-Rank 1 (5-12). FIG. 11 (A) is a diagram showing the activity by a polygonal line cluff, and the dotted line shows the activity in the absence of PI-Rank 1 (5-12). The solid line shows the activity in the presence of PI-Rank 1 (5-12). FIG. 11B is a bar graph showing the activity. Black bars indicate activity in the absence of PI-Rank 1 (5-12). White bars indicate activity in the presence of PI-Rank 1 (5-12). When Rpt3-C was added and the CT-L activity of the 20S core was measured in the absence of PI-Rank1 (5-12), no inhibitory effect was shown on the 20S core, and conversely, it depended on the concentration of Rpt3-C. CT-L activity was enhanced. In addition, in the presence of PI-Rank1 (5-12), the enhancing effect of CT-L activity by Rpt3-C was suppressed. From these experiments, it was clarified that Rpt3-C positively regulates the activity of the 20S core, and PI-Rank1 (5-12) negatively regulates the activity of the Rpt3-C. In previous studies, SDS was added when measuring the enzyme reaction to promote the opening of the substrate uptake gate of the 20S core. In this experiment as well, SDS was added at the time of measurement. When Rpt3-C was measured under the condition that SDS was not added, Rpt3-C further enhanced the enzymatic activity of the 20S core, suggesting that it 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. Method To confirm cytotoxic activity, multiple myeloma cell line RPMI8226 cells were used as cultured cells. RPMI8226 cells were maintained using RPMI1640 medium supplemented with 10% FBS, 100 U / mL Penicillin-Streptomycin.

After seeding 1 × 10 ³ cells in a 96-well plate, after culturing for 12 hours, the medium was changed, and the four test agents shown in FIG. 12 (A) were used in FIGS. 12 (B) and 12 (C), respectively. ) Was added at the concentration shown in). Since PI-Rank1 (5-11) has no cell membrane permeability, a polyarginine tag consisting of eight arginine "R" was bound to the C-terminal side to impart cell membrane permeability ("PI-Rank1 (5-12)"). ) R7G "). As negative controls, PI-Rank1 (5-12), which has no cell membrane permeability, and only the polyarginine tag (consisting of "RRRRRRRRG" (SEQ ID NO: 36) and represented by "R8G") were used. Bortezomib is an existing proteasome inhibitor approved for the treatment of multiple myeloma. The cells were collected 24 hours and 48 hours after the administration of the test drug, and the amount of intracellular ATP was measured with a plate reader using an ATP measurement reagent for cells (Toyo Beanet Co., Ltd.).

8-2. Results As shown in Fig. 12 (B), 24 hours after administration of the test drug, a decrease in ATP concentration was observed as compared with Bortezomib when 1 μM of PI-Rank1 (5-12) R7G was administered. In addition, even 48 hours after administration of the test drug, a decrease in ATP concentration similar to that of Bortezomib was observed when 1 μM of PI-Rank1 (5-12) R7G was administered. Decreased ATP levels mean less live cells.

From the above results, it was shown that PI-Rank1 (5-12) has cytotoxic activity. In addition, unmodified PI-Rank 1 (5-12) had no cell membrane permeability and showed no cytotoxicity. From this, it was considered that cell-specific introduction is possible by controlling the cell membrane permeability of PI-Rank 1 (5-12) in combination with the drug derivation system.

As described above, the proteasome-inhibiting peptide of the present invention was found to have tumor toxicity and was shown to have activity as an anticancer agent. Therefore, it has been shown that it may be used for tumors that have acquired resistance to existing proteasome inhibitors having other inhibitory mechanisms.

9. Protection of Target Peptide by Proteasome Inhibiting Peptide Next, it was examined whether the target peptide could be protected from degradation by the proteasome 20S core by binding the proteasome inhibitory peptide to the target peptide.

9-1. Expression of recombinant protein Human α-synuclein (amino acid sequence 1-140) polypeptide and human α-synuclein (MARPSRLRHWWR: SEQ ID NO: 9) with the amino acid sequence of PI-Rank1 (1-12) added to its C-terminal (Amino acid sequence 1-140) Amino acid sequence 1-140) Polynucleotides in which the recognition cleavage sequences of the restriction enzymes Hind III and EcoR I were added to the DNA sequence encoding the polypeptide were chemically synthesized. These synthetic polynucleotides were inserted at the cleavage sites of the corresponding restriction enzymes in the multicloning site of the pET32a plasmid. These two plasmids E. coli using by transforming and the N-terminal side of each peptide was expressed thioredoxin and hexahistidine (His ₆₎ fusion sequence recombinant protein. Thioredoxin-fused human α-synuclein and a thioredoxin-fused human α-synuclein-PI-Rank1 (1-12) -added protein having a PI-Rank1 (1-12) amino acid sequence (MARPSRLRHWWR: SEQ ID NO: 9) added to its C-terminal. ^{, Transducted into Origami TM} 2 (DE3) Competent Cells (Novagen) for massive expression. After transformation according to a general protocol, colonies were isolated by culturing in LB medium (containing 100 μg / ml ampicillin). Inoculate the colonies into 4 ml of LB liquid medium (100 μg / ml ampicillin), incubate until the OD600 value reaches 0.6 to 0.9, add isopropyl-β-thiogalactopyranoside to 1 mM, and add 2 more. The cells were cultured with shaking for hours. The culture broth 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 Buffer solution (100 mM phosphate pH 8.0 buffer, 300 mM NaCl, 0.2 mg / ml lysoteam, 0.2 mg / ml benzamidine, 0.5 mM phenylmethanesulfonylfloride) for 1 g of frozen E. coli pellets 5 After adding ml and sonicating 10 times on ice for 30 seconds, the mixture was frozen and thawed at -80 ° C. It was further 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.

Ni-NTA agarose (Qiagen) with a bed volume of 200 μl was placed on a chromatography column (0.8 × 4 cm), and 200 μl of an equilibration buffer solution containing 10 mM imidazole added to the above buffer solution was added for washing and equilibration. 500 μl of the above Escherichia coli crushed solution was added, and the mixture was inverted and mixed at 4 ° C. for 60 minutes on a rotator. The pass-through fraction was collected, washed twice with 500 μl of 10 mM imidazole, and eluted 6 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) and the above-mentioned purified thioredoxin fusion human α-synuclein (4 pmol equivalent, 0.15 μg), or PI at the C-terminal thereof -Thioredoxin fusion human α-synuclein with the addition of the Rank1 (1-12) amino acid sequence-PI-Rank1 (1-12) adduct (equivalent to 50 pmol, 1.8 μg) in 10 μL of 20 mM HEPES (pH 7.5) buffer. , 37 ° C. for a certain period of time. The reaction was stopped by mixing with an equal volume of 2 × SDS buffer. It was heated at 98 ° C. for 3 minutes and analyzed by SDS-PAGE electrophoresis. In addition, as a comparative study, a similar experiment was conducted using a commercially available purified human α-synuclein recombinant protein (Cosmo Bio Co., Ltd., equivalent to 50 pmol, 0.73 μg).

9-4. Results Purified thioredoxin fusion human α-synuclein recombinant protein (represented by “αSYN (Trx fusion)” in the figure) or purified thioredoxin fusion human α-with the PI-Rank1 (1-12) amino acid sequence added to its C-terminus. The time course of addition of human 20S proteasome to each of the synuclein-PI-Rank1 (1-12) adduct (represented by "αSYN + Rank1 (Trx fusion)" in the figure) was observed by SDS-PAGE. The result is shown in FIG. 13 (A). In FIG. 13 (A), “M” indicates a molecular weight marker. “-” Indicates a negative control without the addition of human purified 20S proteasome. “1h” or “4h” indicates the reaction time (hours) after the addition of the human purified 20S proteasome. Further, FIG. 13 (B) shows the quantitative results obtained by image analysis of the concentration of the protein band of FIG. 13 (A). Each purified recombinant protein was observed as a molecular weight band of 35 kDa. Multiple bands around 20-30 kDa are derived from the human 20S proteasome. With the addition of the human 20S proteasome, the purified thioredoxin-fused human α-synuclein recombinant protein was gradually degraded by about 28% in 1 hour and about 72% in 4 hours. On the other hand, the thioredoxin-fused human α-synuclein-PI-Rank1 (1-12) adduct having the PI-Rank1 (1-12) amino acid sequence added to the C-terminal was about 23% in 1 hour and in 4 hours. Only about 31.5% was decomposed.

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

From the above results, it was shown that by adding the proteasome-inhibiting peptide sequence to the target polypeptide, the degradation reaction of the target peptide was suppressed depending on the proteasome-inhibiting peptide.

Claims (9)

Of the following amino acid sequence,
1st to 7th,
2nd to 7th,
3rd to 7th,
1st to 6th,
A peptide containing the 2nd to 6th or 3rd to 6th sequences and having an action of inhibiting the proteolytic enzyme activity of the proteasome 20S core:
Ser-Xaa ¹ -Leu-Xaa ² -His-Trp-Trp (SEQ ID NO: 1);
Here, Xaa ¹ and Xaa ² indicate arbitrary amino acid residues. The peptide according to claim 1, ^{further comprising Xaa 3} , which is an arbitrary amino acid residue, at the C-terminal of the amino acid sequence represented by SEQ ID NO: 1. ^{Further, the amino acid sequence Met-Xaa 4-} Xaa ^5- Pro- (SEQ ID NO: 5) is provided at the N-terminal of the amino acid sequence represented by SEQ ID NO: 1, ^{and Xaa 4} and Xaa ⁵ have arbitrary amino acid residues. The peptide according to claim 1 or 2 shown. A polynucleotide comprising a base sequence encoding the peptide according to any one of claims 1 to 3. A vector comprising expressively the polynucleotide according to claim 4. A composition comprising the peptide according to any one of claims 1 to 3, the polynucleotide according to claim 4, or the vector according to claim 5. The composition according to claim 6, wherein the composition is a pharmaceutical composition. The composition according to claim 7, wherein the pharmaceutical composition is used for the prevention and / or treatment of malignant tumors. The target peptide comprises binding the peptide according to any one of claims 1 to 3.
A method of protecting a target peptide from degradation by the proteasome 20S core.

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