JP2007223958A - Cell death-inducing peptide, map(multiple antigenic peptide)-converted cell death-inducing peptide, cell death inducer and anticancer agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide effective for cell death induction, etc., and to provide a cell death inducer and an anticancer agent each comprising the above peptide as active ingredient. <P>SOLUTION: The cell death-inducing peptide is composed of consecutive 4-12 amino acids selected from the domain corresponding to amino acid No.39 to 281 in the amino acid sequence shown in the sequence No.1 of the Sequence Table. This peptide meets the following requirements(a) and (b): (a) Containing no 3 or more consecutive amino acids in SNLHL and (b) The average measurement of viable cell counts after a cell death induction test by cell culture for 48 h on a chip immobilized with the peptide is 80% or less of that after the same test on a control chip non-immobilized with the peptide. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell death inducing peptide, a MAP-activated cell death inducing peptide, a cell death inducing agent, and an anticancer agent, and more specifically, an amino acid in the extracellular domain of a tumor necrosis factor-related apoptosis-inducing ligand TRAIL A cell death-inducing peptide invented from or derived from a sequence, a MAP-modified cell death-inducing peptide obtained by MAP-forming this cell death-inducing peptide, and these cell death-inducing peptides and / or MAP The present invention relates to a cell death inducing agent and an anticancer agent comprising a modified cell death inducing peptide as an active ingredient.

  TRAIL is a cytokine belonging to the TNF family, and is a type 2 membrane-permeable protein consisting of 281 amino acids. This TRAIL induces apoptosis in cancer cells via a receptor (death receptor), for example, as reported in Non-Patent Document 1 below. Moreover, TRAIL has little effect on normal cells.

Wilev et al., Immunity, 3: 673-682 (1995), Pitti etal., J. Biol. Chem., 271: 12697 Therefore, TRAIL can be used for cancer treatment without side effects. As exemplified in the patent literature, research related to cancer treatment, immune diseases, and the like is underway.

Garber, K., Arbor, A., Nat. Biotech., 23, 409-411 (2005) However, the binding mechanism of TRAIL to the receptor has not been elucidated yet, and its stable mass production process is also It is still undeveloped.

  On the other hand, it is generally known that a small molecule such as a peptide has higher bioabsorbability than a protein itself, and chemical modification with a targeting molecule or other drug molecule is simple. In addition, peptides have various physiological activities in vivo while being small molecules compared to proteins. In addition to many biological hormones and cytokines, peptides having many functions such as antibacterial peptides and cell adhesion peptides have been reported so far. In addition, the organic synthetic chemistry of peptides has been greatly developed and can be stably supplied as a drug.

  Conventionally, there are many research examples of cell functional peptides such as antibacterial activity, promotion of cell adhesion, apoptosis, and other protein activity inhibitors. However, there are very few reported examples of peptides having short cytotoxicity and cytotoxicity, even if the following Non-Patent Document 3 and Non-Patent Document 4 are mentioned. Furthermore, even if it is a peptide which has such activity, there is no report which retains activity in the state by which the peptide terminal was fix | immobilized.

Buckley CD, et al., RGD peptides induce apoptosis by direct caspase-3 activation., "Nature", (UK), 1999, Vol. 397 (6719) p.534-9 Terui Y, et al., NH2-terminal pentapeptide ofendothelial interleukin 8 is responsible for the induction ofapoptosis in leukemic cells and has an antitumor effect in vivo., "Cancer Res.", 1999, Vol.59 (22), p.5651 -Five

  Therefore, the present invention provides a cell death-inducing peptide and a MAP-activated cell death-inducing peptide obtained by multimerizing such a peptide, and further provides a cell death inducing agent and an anticancer agent containing these peptides as active ingredients. This is a technical problem to be solved.

  The inventor of the present application has constructed a peptide library that partially covers the amino acid sequence of the extracellular domain that is the site of interaction with the receptor in TRAIL. And about these peptides, the cell death induction peptide was screened through the assay of the cell death inducibility of leukemia cells, and several effective cell death induction peptides were found. The present invention has been completed based on these findings.

(First invention)
The structure of the first invention of the present application for solving the above problem is a peptide comprising 4 to 12 consecutive amino acids selected from the region of amino acid numbers 39 to 281 in the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing. These are cell death-inducing peptides corresponding to the following (a) and (b).
(A) It does not contain 3 or more consecutive amino acids in SNLHL.
(B) The average value of the number of living cells after the cell death induction assay by 48-hour cell culture on the chip on which the peptide is immobilized is the living cell after the same assay on the peptide non-immobilized chip as a control. It is 80% or less of the number measurement average value.

  The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is the amino acid sequence of TRAIL, and the region of amino acid numbers 39 to 281 is the extracellular domain.

  In the cell death inducing peptide of the first invention, the average value of the number of living cells in the cell death induction assay on the peptide-immobilized chip is higher than that in the same assay on the peptide non-immobilized chip as a control. It shows a significant cell death inducing property of 80% or less. Therefore, it is considered that it can be sufficiently used as an active ingredient of a cell death inducer or an anticancer agent as a peptide drug. These cell death-inducing peptides are also useful as new drug lead compounds.

  Further, as described above, TRAIL is known to have little effect on normal cells. Therefore, the cell death-inducing peptide of the first invention comprising the partial amino acid sequence has almost no effect on normal cells. It is thought that it does not reach

(Second invention)
The configuration of the second invention of the present application for solving the above problem is a cell death-inducing peptide in which the average number of living cells measured for the peptide according to the first invention is 70% or less of the average number of living cells measured for control. is there.

  As the cell death-inducing peptide of the present invention, a considerable number of peptides with particularly high cell death-inducing activity are found, in which the average value of the live cell count of the peptide is 70% or less of the average value of the live cell count of the control. ing. These cell death-inducing peptides are particularly effective. This percentage is more preferably 65% or less.

(Third invention)
The configuration of the third invention of the present application for solving the above problem is a cell death-inducing peptide having any one of the following amino acid sequences (1) to (14).
(1) YSKSGIAC
(2) KSGIACFL
(3) GIACLFLKE
(4) ACFLKEDD
(5) KEDSYSYWD
(6) SPCWQVKW
(7) CWQVKWQL
(8) QVKWQLRQ
(9) KWQLRRQLV
(10) LRNGELVI
(11) NGELVIHE
(12) KENTKNDK
(13) NTKNDKQM
(14) KNDKQMVQ
The amino acid sequence of the cell death-inducing peptide according to any one of (1) to (14) defined in the third invention is found from the region of amino acid numbers 39 to 281 in the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing. It has been done. As described in the Examples below, these cell death-inducing peptides have been specifically confirmed to have cell death-inducing activity corresponding to (b) of the first invention.

(Fourth invention)
The structure of the fourth invention of the present application for solving the above-mentioned problem is that any one or two amino acids in the amino acid sequence of (1) to (14) according to the third invention are substituted, deleted, deleted, Alternatively, the average value of the number of viable cells after the cell death induction assay by cell culture for 48 hours on the chip comprising the added amino acid sequence and immobilizing the peptide is on the peptide non-immobilized chip as a control. The cell death-inducing peptide, which is 80% or less of the average value of the number of living cells after the same test in FIG.

  The cell death-inducing peptide according to the above-mentioned third invention as generally seen as “sequence fluctuation” in the amino acid sequence of a protein or peptide having an arbitrary function or in the base sequence of a gene or the like having an arbitrary function There are also a large number of cell death-inducing peptide groups consisting of amino acid sequences in which any one or two amino acids in the amino acid sequence are substituted, deleted, or added, and functionally equivalent.

(Fifth invention)
The composition of the fifth invention of the present application for solving the above-mentioned problems consists of 4 to 12 amino acids containing one or more C, and an aromatic amino acid is bonded to the carboxyl terminal side of the C. It is a cell death inducing peptide.

  The amino acid sequence of the extracellular domain of TRAIL contains a total of 3 C (cysteine: Cys), all of which are components of the typical effective cell death-inducing peptide described in the third invention. It has become. Moreover, an aromatic amino acid (F: Phe or W: Trp) is bound to the carboxyl terminal side of the three Cs in any case. Therefore, although it has not been confirmed experimentally yet, in a cell death inducing peptide, one of the important elements of cell death inducing is to contain a cysteine residue and an aromatic amino acid bonded to the carboxyl terminal side. It is more reasonable to think that there is.

(Sixth invention)
The structure of the sixth invention of the present application for solving the above problem is that, in the cell death inducing peptide according to the fifth invention, one or two amino acids of any kind are added to the carboxyl terminal side of the aromatic amino acid. Is a cell death-inducing peptide to which K is bound.

  With respect to the fifth invention described above, with respect to the aromatic amino acid bonded to the carboxyl terminal side of the C, the carboxyl terminal side is K in any case via one or two amino acids of any kind. (Lysine) is bound. Therefore, although not confirmed experimentally yet, in the cell death-inducing peptide, via a cysteine residue, an aromatic amino acid bonded to the carboxyl terminal side, and one or two amino acids of any kind It is particularly reasonable to consider that an amino acid sequence part to which lysine binds is one of the important elements for inducing cell death.

(Seventh invention)
The structure of the seventh invention of the present application for solving the above problem is that the cell death-inducing peptide according to any one of the first to sixth inventions is in a solubilized state, or in its amino group or carboxyl group. It is a cell death-inducing peptide that is in an immobilized state.

  The cell death-inducing peptide according to any one of the first to sixth inventions described above is effective even in a solubilized state (free state) or in a state where the N-terminal side or C-terminal side is immobilized. Show cell death-inducing activity. Immobilization refers to chemical bonding to an arbitrary base compound or material or immobilization in a state equivalent thereto. In view of the effectiveness of the immobilized cell death-inducing peptide, the invention of the MAP-activated cell death-inducing peptide described below is established.

(Eighth invention)
The configuration of the eighth invention of the present application for solving the above-described problem is that MAP-mediated cell death is obtained by MAP-izing one or more of the cell death-inducing peptides according to any of the first to sixth inventions. Derived peptide.

  Here, “MAP” refers to “Multiple Antigenic Peptide”, which is obtained by chemically bonding a plurality of units of cell death-inducing peptide to each other. In the MAP-mediated cell death-inducing peptide, a plurality of the same type of cell death-inducing peptides may be bound, or a plurality of two or more different cell death-inducing peptides may be bound.

  In the MAP-mediated cell death-inducing peptide, the cell death-inducing peptide acts on cells at a high density, so that the cell death-inducing activity can be further improved as confirmed in the Examples.

  Examples of the form of MAP include, for example, binding a carboxyl group of a cell death-inducing peptide to each of a plurality of amino groups in a basic amino acid, or a group of amino acids of a cell death-inducing peptide to each of a plurality of carboxyl groups in an acidic amino acid. Can be exemplified by the MAP-ized form in which the cell death-inducing peptide constitutes each branch. Since one branch part can be constituted by one basic amino acid or acidic amino acid, if these are combined in two stages in series, a two-stage branch part can be constituted, resulting in a total of four The cell death-inducing peptide can be branched into a MAP.

  In addition, according to the method disclosed in Non-Patent Document 5 below, a cell death-inducing peptide is incorporated into a liposome after being bound to a lipid such as palmitic acid, and more lipophilic (cell affinity) MAP as an anticancer agent or the like. It can also be a cell death-inducing peptide.

FEBS Lett. 2003; 540 (1-3): 133-40.Liposome entrapment and immunogeni studies of a synthetic lipophilicmultiple antigenic peptide bearing VP1 and VP3 domains of thehepatitis A virus: a robust method for vacccine design.Haro I, et al. Furthermore, since the cell death-inducing peptide immobilized on an arbitrary base material also maintains cell death-inducing activity, for example, a large number of cell death-inducing peptides can be immobilized on a chip substrate, or a minute base on the order of mm to μm. A MAP-activated cell death-inducing peptide in which a large number of cell death-inducing peptides are immobilized on material particles (for example, viscosity mineral particles) can also be used. It can also be set as the MAP-ized cell death induction peptide of the form which fix | immobilized many cell death induction peptides with respect to the said functional group of the organic polymer material provided with a functional group.

(9th invention)
The structure of the ninth invention of the present application for solving the above-mentioned problems is the active ingredient of the cell death-inducing peptide according to any one of the first to seventh inventions and / or the MAP cell death-inducing peptide according to the eighth invention. And a cell death inducer.

(10th invention)
The configuration of the tenth invention of the present application for solving the above-mentioned problem is the active ingredient of the cell death-inducing peptide according to any one of the first to seventh inventions and / or the MAP cell death-inducing peptide according to the eighth invention. It is an anticancer agent.

  The present invention provides a cell death-inducing peptide that is effective against cancer cells such as blood cell cancer cells. Since these cell death-inducing peptides exhibit effective cell death-inducing activity not only in a solubilized state but also in an immobilized state, they can be applied to various functional materials and have a wide range of applications. The present invention also provides a MAP-activated cell death-inducing peptide that is even more effective. These cell death-inducing peptides and MAP-activated cell death-inducing peptides can be used as active ingredients of cell death inducing agents and anticancer agents.

  Next, embodiments of the present invention will be described including the best mode.

[Screening of cell death-inducing peptides]
The present inventor used a peptide chip in high-throughput combinatorial chemistry as a technique for identifying a peptide having cell death-inducing activity. This method is useful for screening a peptide having a novel physiological activity, and enables cell culture on a chip.

  As a cell death inducing peptide screening target, a cell death induction site was searched for the amino acid sequence of the extracellular domain (region of amino acid numbers 39 to 281) in TRAIL shown in SEQ ID NO: 1 in the Sequence Listing. In the search, a library of peptides consisting of 8 amino acids was constructed so as to cover the entire region of the extracellular domain. Each peptide constituting the library has two amino acids at both ends so that two amino acids overlap each other with the peptide covering the amino acid sequence adjacent to each other in the sequence (ie, two residues). Set by shifting each group sequentially).

  As a result, it has been found that peptides having the amino acid sequences listed in (1) to (14) of the third invention show high cell death-inducing activity against cancer cells. These cell death-inducing peptides exhibited high cell death-inducing activity in the state of being immobilized on the chip and in the solubilized state. In the state of being immobilized as a MAP-activated cell death-inducing peptide, the density of peptides that act simultaneously is high, so that particularly strong cell death-inducing activity is exhibited.

  The peptides having the amino acid sequences listed in the above (1) to (14) all have an average value of the number of living cells after the cell death induction assay by cell culture for 48 hours on a chip on which the peptide is immobilized. It is 80% or less of the average value of the number of living cells after the same test on a peptide non-immobilized chip as a control.

The above-mentioned cell death-inducing peptide is set so that two amino acids at the end of each other overlap each other, and there are many cell death-inducing peptides that commonly include a specific number of amino acids. Has been issued. Therefore, it is a peptide consisting of 4 to 12 consecutive amino acids selected from the region of amino acid numbers 39 to 281 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and corresponds to the following (a) and (b) A cell death-inducing peptide can also be proposed.
(A) It does not contain 3 or more consecutive amino acids in SNLHL.
(B) The average value of the number of living cells after the cell death induction assay by 48-hour cell culture on the chip on which the peptide is immobilized is the living cell after the same assay on the peptide non-immobilized chip as a control. It is 80% or less of the number measurement average value.

  The reason for the condition that “it does not contain 3 or more consecutive amino acids in SNLHL” is that these cell death-inducing peptides are already disclosed in Japanese Patent Application Laid-Open No. 2005-306762 (Japanese Patent Application No. 2004-124338). It is because it has been.

  Among the cell death-inducing peptide group according to the first invention described above, as a particularly preferable one, the above-mentioned cell death-inducing peptide has an average value of viable cell counts of 70% or less of the control viable cell count measurement average value Can be mentioned.

  Furthermore, a chip comprising an amino acid sequence in which any one or two amino acids are substituted, deleted, or added in any one of the amino acid sequences of (1) to (14), and the peptide is immobilized The average value of the number of living cells after the cell death induction test by 48 hours of cell culture is 80% or less of the average value of the number of living cells after the same test on the peptide non-immobilized chip as a control. A cell death-inducing peptide group can also be proposed.

  Generally speaking, the “amino acid substitution” described above has little effect on the activity of the substituted polypeptide, and the total charge, hydrophobicity / hydrophilicity, and / or stericity of the substituted amino acid. So-called conservative substitutions that substantially preserve the general size can be preferably exemplified.

  Furthermore, the inventor of the present application paid attention to the role of cysteine (C) in the TRAIL-derived cell death-inducing peptide as described in the fifth or sixth invention. That is, a peptide consisting of 4 to 12 amino acids containing one or more cysteines is likely to be a potent cell death-inducing peptide. Such a peptide in which an aromatic amino acid is bonded to the carboxyl terminal side of cysteine is likely to be a more potent cell death-inducing peptide. Moreover, when lysine (K) is bonded to the carboxyl terminal side of the aromatic amino acid via one or two amino acids of any kind, it is highly likely that it is a particularly potent cell death-inducing peptide. .

  The cell death-inducing peptide of the present invention can be produced by various known peptide synthesis methods such as a solid phase synthesis method and a liquid phase synthesis method. When the peptide or amino acid that can constitute a part of the peptide is condensed with the remaining part and the product has a protecting group, the protecting peptide is eliminated to produce the target peptide. Any known automatic synthesizer may be used for peptide synthesis.

  Specifically, for example, using the Fmoc method, it can be synthesized by solid phase synthesis in which amino acids are bonded to the amino acid derivative fixed on the resin one by one from the carboxyl end. In the Fmoc method, a peptide derivative in which the α-amino group is protected by an Fmoc group that can be removed with a base (for example, piperidine) is used. After removing this protecting group, an amino acid derivative (for example, an ester with HOBt) that has been washed with N, N-dimethylformamide (DMF) or the like, dried, and activated with a subsequent condensing agent (for example, HOBt). ). Next, after washing with DMF or the like, deprotection for the next cycle is performed. By repeating the above cycle, a synthetic peptide having a desired sequence can be obtained.

  The peptide thus obtained is bound to the resin at the terminal position by a linker. The side chain of the peptide has a protecting group. Next, a free peptide can be obtained by cleaving the bond with this resin. For example, trifluoroacetic acid treatment may be performed under reducing conditions to deprotect the side chain protecting group and cleave from the resin. The released protecting group can be removed by washing the peptide after deprotection and cleavage treatment several times with diethyl ether or the like. In addition, in order to obtain a peptide immobilized on a membrane, a peptide is synthesized as described in the following examples and used without performing a cleavage treatment.

  The cell death-inducing peptide of the present invention can also be produced by culturing a transformant introduced with a DNA encoding the peptide. For example, the peptide of the present invention can be expressed by incorporating DNA encoding the peptide of the present invention under the control of a promoter in an appropriate expression vector and transfecting the vector into a host cell. The peptide thus expressed may be purified and isolated for use. For the purification of peptides, known separation / purification methods can be combined appropriately. Separation and purification methods include salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, isoelectric focusing Can be illustrated.

  The obtained cell death-inducing peptide can be dialyzed and freeze-dried to obtain a dry powder as long as the cell death-inducing activity is not impaired. In addition, the cell death-inducing peptide of the present invention has cell death-inducing activity even when the C-terminal is immobilized, and thus can be bound (modified) to various compounds having a functional group, and immobilized on a carrier. It may be made. Examples of the carrier include liposomes utilizing lipids, polyvalent peptide polyethylene glycol utilizing carbohydrate dendrimers, and immobilization on inorganic base materials.

  In addition, since the cell death-inducing peptide of the present invention has cell death-inducing activity even when the C-terminal (carboxyl group) or N-terminal (amino group) is immobilized, it can be combined with polyethylene glycol (PEG) to give PEG It can also be a hybrid. For this method, see Non-Patent Document 6 below.

Gene Ther. 2000; 7 (11): 1183-92 Protective copolymers for nonviral gene vectors: synthesis, vector characterization and application in gene delivery. Finsinger D et al. Based on a small core molecule consisting of a dendrimer of lysine branched radially. A huge polymer having a three-dimensional conformation can be produced by binding a series of peptides thereon. The cell recognition function is improved by designing such a polymer. Therefore, the cell-killing effect when administered to cells (particularly to cancer cells) can be enhanced.

  Next, the cell death inducer and anticancer agent according to the present invention will be described. These cell death-inducing agents and anticancer agents are any of the above-mentioned cell death-inducing peptides (one or more of them) and / or any of the above-mentioned MAP-activated cell death-inducing peptides (one or more of them) Is the active ingredient. Only the active ingredient may be used alone, or it may be immobilized or mixed in a pharmacologically acceptable carrier.

  Those skilled in the art can easily prepare these drugs using methods known in the art. These cell death inducers and anticancer agents can be safely administered orally or parenterally (for example, topical, rectal, intravenous administration, etc.).

  The cell death inducer and anticancer agent of the present invention may be a membrane using a synthetic polymer, a hydrogel using a natural polymer, or a peptide alone immobilized on an inorganic carrier such as hydroxyapatite. It is good also as a formulation containing the fixed peptide. In recent years, since it is known that a peptide forms a gel by self-organizing itself, the peptide itself may be used as a material. In these embodiments, the present invention may be applied directly to cells, tissues, etc. that are desired to induce cell death.

  Examples of the pharmaceutically acceptable carrier that may be used in the production of the cell death inducer and anticancer agent of the present invention include various organic polymers or inorganic carrier substances that are commonly used as pharmaceutical materials, such as solvents in liquid formulations, Examples include solubilizers and suspending agents, or excipients, lubricants, binders and disintegrants in solid preparations. Furthermore, additives such as colorants, sweeteners, adsorbents, wetting agents and the like can be included as necessary.

  Examples of the excipient include starch, corn starch, crystalline cellulose, and light anhydrous silicic acid.

  Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Examples of the binder include crystalline cellulose, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, gelatin, methylcellulose, sodium carboxymethylcellulose and the like.

  Examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl starch sodium, L-hydroxypropyl cellulose, and the like.

  Examples of the solvent include propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.

  Examples of the solubilizer include polyethylene glycol, propylene glycol, trisaminomethane, cholesterol, triethanolamine and the like.

  Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, carboxy Examples include hydrophilic polymers such as sodium methylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

  In the cell death inducing agent and anticancer agent of the present invention, the active ingredient (cell death inducing peptide and / or MAP-activated cell death inducing peptide) is usually about 0.1 to 100% by weight, preferably about 10%, based on the whole preparation. ˜99.9 wt%, more preferably about 20 to 90 wt%. The content of components other than the active ingredient and its carrier varies depending on the form of the preparation, but is usually about 10 to 99.9% by weight, preferably about 20 to 90% by weight, based on the whole preparation.

  Next, examples of the present invention will be described. The technical scope of the present invention is not limited by the examples.

[Assumptions of Examples]
Peptide chips can be produced with a very high density and an exhaustive chip, and currently 8000 spots / membranes are possible. It is expected to be useful as a high-throughput combinatorial chemistry for screening peptides with novel physiological activities.

  In this example, a peptide chip was used as a method for searching for a novel peptide having cell death inducing activity (particularly, an antitumor function), and cell death inducing activity against tumor cells was screened. As the screening target, the amino acid sequence of the extracellular domain of TRAIL known as an apoptosis-inducing factor (region of amino acid numbers 39 to 281 in SEQ ID NO: 1 in the sequence listing) was used.

[Example 1]
TRAIL covers the entire extracellular domain, and the amino acids are sequentially shifted by 2 residues as described above, and a total of 119 peptides each consisting of 8 amino acids are solid-phased by the aforementioned Fmoc method. Synthesized. Peptide numbers (serial numbers as peptide samples) were assigned to these peptides in order from the youngest amino acid number in SEQ ID NO: 1 in the sequence listing. That is, Peptide No. 1 is a peptide having an amino acid sequence corresponding to amino acid numbers 39 to 46, Peptide No. 2 is a peptide having an amino acid sequence corresponding to amino acid numbers 41 to 48, and so on. A peptide spot synthesized on a cellulose membrane was cut into a diameter of 6 mm and placed on the bottom of a 96-well plate.

Jurkat cells (human T lymphocyte culture cells) were cultured thereon using serum-free medium RPMI (Gibco). The cell concentration was adjusted to 2.0 × 10 ⁵ cells / mL, and 100 μL was added to 1 well of the plate. The cells were cultured for 48 hours on each chip under conditions of 37 ° C. and 5% CO ₂ .

  Thereafter, the cells were stained with a fluorescent reagent, and the state of living cells and cell death after culture were measured using a fluorescence plate reader Fluoroskan Ascent manufactured by Labsystems. For the fluorescent reagent, calcein AM (molecular probes) (measurement: excitation 485 nm, fluorescence 538 nm) was used for the number of living cells, and Cytotoxone (Promega) (measurement: excitation 560 nm, fluorescence 590 nm) was used for cell death.

  The peptide chip was prepared as follows on a cellulose membrane coated with β-alanine using an automatic peptide synthesizer ASP 222 (manufactured by Intavis). That is, 1.5 μL of Fmoc amino acid solution (concentration: 0.25 mol / L) is spotted on the amino group of β-alanine on the membrane, and the carboxyl group of the first amino group is bound to the membrane. After the washing operation, the next amino acid reacts as Fmoc amino acid and is immobilized. Thereafter, amino acids were sequentially combined to synthesize a peptide having the target sequence. The C-terminal part of the synthesized peptide is fixed in a state of being bound to the membrane.

  Regarding the process of the above example, FIG. 1 for explaining the production of the peptide chip and FIG. 2 for explaining the method of the example are presented for reference.

[Results of Example 1]
In FIG. 3, the analysis result of the growth inhibitory activity by a peptide chip, ie, cell death induction activity, is shown. The horizontal axis of Fig. 3 shows Peptide No. The vertical axis in FIG. 3 is the specific fluorescence intensity of each peptide number when the measured fluorescence intensity value in the control (measurement example performed without immobilizing the peptide on the chip) is 1. Actually, nine measurements were performed for each Peptide No., and the average value was shown in FIG. Also, the broken lines inserted above and below in FIG. 3 are described as one index for effect determination.

  Among the Peptide Nos., The Peptide No. related to the example showing the relatively excellent measured value, its amino acid sequence, the average value of the measurement and the error range are listed in a bar graph form in FIG. Peptide No. related to the example showing the measured values, the amino acid sequence thereof, the average value of the measurement, and the error range are shown numerically in FIG.

  The inventor of the present application actually measured the number of living cells after the cell death induction assay by 48 hours cell culture on a chip on which the specific fluorescence intensity was 0.80 or less, that is, the chip on which the peptide was immobilized. However, it is considered that a peptide that is 80% or less of the average value of the number of living cells after the same test on a peptide non-immobilized chip as a control is practical as a cell death-inducing peptide.

  From the consideration of Example 1, the following three points were reasonably estimated as described above with respect to the fifth to seventh inventions.

  1) Possibility that a peptide consisting of 4 to 12 amino acids containing one or more C and having an aromatic amino acid bonded to the carboxyl terminal side of the C can be effectively used as a cell death-inducing peptide.

  2) In the cell death-inducing peptide of 1) above, a peptide in which K is bonded to the carboxyl terminal side of the aromatic amino acid via one or two amino acids of any kind is particularly a cell death-inducing peptide. Possibility to use effectively.

[Example 2]
Since Example 1 is an activity measurement for a cell death-inducing peptide in an immobilized state, the cell death induction in the solubilized state of several cell death-inducing peptides whose effects were confirmed in Example 1 was then performed. Activity was assayed.

Briefly, Example 2 was performed as follows. A soluble peptide was added at a concentration of 2 mM to RPMI medium (with serum) containing 2.0 × 10 ⁵ cells / mL of Jurkat cells, and cultured in an incubator at 37 ° C. and 5% CO ₂ for 48 hours. Then, cells 24 hours and 48 hours after the start of the culture were stained with the fluorescent dye calcein AM, and the number of viable cells was calculated from the fluorescence intensity.

  The results of Example 2 are illustrated in FIG. FIG. 6 (a) is an example performed on a peptide chip having a sequence with reduced specific fluorescence intensity, and shows measurement results for three types of cell death-inducing peptides displaying amino acid sequences. As described in the right end part of FIG. 6A, “no peptide” means a control without addition of peptide. Further, “1 mM, 2 mM, 0.1 mM” means an example in which soluble peptides are added to the indicated concentrations in the RPMI medium in the manner described in Example 2 above. “TRAIL” indicates an example of addition of a cell death-inducing peptide having the amino acid sequence shown at the bottom of the three diagrams shown in FIG. 6 (a), and the addition concentration is 2 mM, 1 mM, and 0.1 mM. . Further, in the three diagrams shown in FIG. 6A, the horizontal axis represents the elapsed time after the start of the culture, and the vertical axis represents the number of surviving (proliferated) cells.

  Next, FIG. 6 (b) is an example performed for a peptide (REGPQRV) in which no cell death was observed, and the way of viewing the figure is the same as FIG. 6 (a), but FIG. 6 (b) “TRAIL” is a tumor necrosis factor-related apoptosis-inducing ligand.

  FIG. 6 (a) shows that the cell death-inducing peptide of the present invention exhibits cell death-inducing activity in a concentration-dependent manner even in a solubilized state.

Example 3
Using four types of peptides shown in FIG. 7, a soluble peptide was added at a concentration of 2 mM to RPMI medium (with serum) containing 1.0 × 10 ⁵ cells / mL of Jurkat cells, and 37 ° C., 5% CO ₂ . After culturing under conditions for 24 hours, Cytotoxone was added and incubated for 10 minutes. Next, the reaction stop solution was added and stirred for 10 seconds, and then fluorescence at 590 nm was measured with excitation at 560 nm, and LDH released from dead cells was measured. “No peptide” is an example in which no peptide was added to the Jurkat medium in the above.

  From the results of FIG. 7, it can be seen that cell growth is not actively suppressed by the peptide, but cell death is positively induced.

Example 4
A MAP-mediated cell death-inducing peptide (MAP branched by the side chain of Lys residue) shown in FIG. 8 (b) was synthesized. The amino acid sequence of the 4-unit cell death-inducing peptide used for MAP is also shown in FIG. 8 (b). On the other hand, a linear multimerization of the same cell death-inducing peptide was also synthesized.

  Using these, the same test as in Example 2 was performed, and the results shown in FIG. 8A were obtained. The view of FIG. 8A is the same as that of FIG. In FIG. 8A, “MAP (0.5 mM)” means an example in which 0.5 mM of the branched MAP is added. “Linear” means the peptide multimerized in the above linear form. “TRAIL” indicates the result of addition to a concentration of 100 ng / mL.

  As can be seen from FIG. 8, “TRAIL” and “MAP (0.5 mM)” show almost the same strong cell death inducing activity. When considering the molar concentration of both, it can be seen that branched MAP has substantially enhanced cell death-inducing activity compared to linear cell death-inducing peptide.

[Evaluation of the whole example]
From the results of the above Examples, it was revealed that the cell death-inducing peptide and MAP-activated cell death-inducing peptide of the present invention have effective cell death-inducing activity against cancer cells. Therefore, there is a possibility of application to peptide medicine that kills cancer cells, and the discovery of further useful cell death-inducing peptides using the screening method established by this study is expected.

According to the present invention, there are provided a cell death inducing peptide and a MAP-activated cell death inducing peptide having effective cell death inducing activity against cancer cells, and a cell death inducing agent and an anticancer agent containing these as active ingredients.

[Sequence Listing]
SEQUENCE LISTING
<110> National University Corporation Nagoya University
<120> Cell death-inducing peptide, MAP-mediated cell death-inducing peptide, cell death-inducing agent and anticancer agent
<130> POK-05-028
<160> 17

<210> 1
<211> 281
<212> PRT
<213> Homo Sapiens
<400> 1
Met Ala Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys 16
Val Leu Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val Ala 32
Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 48
Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 64
Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val 80
Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 96
Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 112
Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly 128
Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu 144
Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser Ser Arg Ser Gly 160
His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val Ile 176
His Glu Lys Gly Phe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 192
Gln Glu Glu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 208
Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 224
Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr 240
Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile 256
Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala 272
Ser Phe Phe Gly Ala Phe Leu Val Gly 281

<210> 2
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 2
Tyr Ser Lys Ser Gly Ile Ala Cys 8

<210> 3
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 3
Lys Ser Gly Ile Ala Cys Phe Leu 8

<210> 4
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 4
Gly Ile Ala Cys Phe Leu Lys Glu 8

<210> 5
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 5
Ala Cys Phe Leu Lys Glu Asp Asp 8

<210> 6
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 6
Lys Glu Asp Asp Ser Tyr Trp Asp 8

<210> 7
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 7
Ser Pro Cys Trp Gln Val Lys Trp 8

<210> 8
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 8
Cys Trp Gln Val Lys Trp Gln Leu 8

<210> 9
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 9
Gln Val Lys Trp Gln Leu Arg Gln 8

<210> 10
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 10
Lys Trp Gln Leu Arg Gln Leu Val 8

<210> 11
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 11
Leu Arg Asn Gly Glu Leu Val Ile 8

<210> 12
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 12
Asn Gly Glu Leu Val Ile His Glu 8

<210> 13
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 13
Lys Glu Asn Thr Lys Asn Asp Lys 8

<210> 14
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 14
Asn Thr Lys Asn Asp Lys Gln Met 8

<210> 15
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 15
Lys Asn Asp Lys Gln Met Val Gln 8

<210> 16
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 16
Ser Ala Arg Asn Ser Cys Trp Ser 8

<210> 17
<211> 8
<212> PRT
<213> Homo Sapiens
<400> 17
Arg Asn Ser Cys Trp Ser Lys Asp 8

It is a figure explaining preparation of the peptide chip in Example 1. FIG.

2 is a diagram for explaining a process method in Embodiment 1. FIG.

It is a figure which shows the analysis result of the cell death induction activity in Example 1.

It is a figure which shows the cell death induction activity measured value of the peptide in Example 1 in a bar graph format.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which enumerates the peptide which shows the especially outstanding effect in Example 1.

It is a figure which shows the cell death induction activity of the peptide of the solubilized state in Example 2. FIG.

It is a figure which shows the evidence of the cell death induction in Example 3.

It is a figure which shows the effect of the MAP-ized cell death induction peptide in Example 4.

Claims (10)

A peptide consisting of 4 to 12 consecutive amino acids selected from the region of amino acid numbers 39 to 281 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and corresponding to the following (a) and (b) A characteristic cell death-inducing peptide.
(A) It does not contain 3 or more consecutive amino acids in SNLHL.
(B) The average value of the number of living cells after the cell death induction assay by 48-hour cell culture on the chip on which the peptide is immobilized is the living cell after the same assay on the peptide non-immobilized chip as a control. It is 80% or less of the number measurement average value. 2. The cell death-inducing peptide according to claim 1, wherein the average number of living cells measured for the peptide is 70% or less of the average number of living cells measured for control. A cell death-inducing peptide having the amino acid sequence of any one of (1) to (14) below.
(1) YSKSGIAC
(2) KSGIACFL
(3) GIACLFLKE
(4) ACFLKEDD
(5) KEDSYSYWD
(6) SPCWQVKW
(7) CWQVKWQL
(8) QVKWQLRQ
(9) KWQLRRQLV
(10) LRNGELVI
(11) NGELVIHE
(12) KENTKNDK
(13) NTKNDKQM
(14) KNDKQMVQ On a chip comprising an amino acid sequence in which any one or two amino acids are substituted, deleted, or added in any one of the amino acid sequences of (1) to (14) and having the peptide immobilized thereon The average value of the number of living cells after the cell death induction test in 48 hours of cell culture was 80% or less of the average number of living cells after the same test on a peptide non-immobilized chip as a control. The cell death-inducing peptide according to claim 3. A cell death-inducing peptide comprising 4 to 12 amino acids containing 1 or more C, and an aromatic amino acid bound to the carboxyl terminal side of the C. 6. The cell according to claim 5, wherein in the cell death-inducing peptide, K is bound to the carboxyl terminal side of the aromatic amino acid via one or two amino acids of any kind. Death-inducing peptide. The cell according to any one of claims 1 to 6, wherein the cell death-inducing peptide is in a solubilized state or in a state of being immobilized at its amino group or carboxyl group. Death-inducing peptide. A MAP-mediated cell death-inducing peptide, wherein one or more of the cell death-inducing peptides according to any one of claims 1 to 6 are MAP (Multiple Antigenic Peptide). A cell death inducer comprising the cell death-inducing peptide according to any one of claims 1 to 7 and / or the MAP-activated cell death-inducing peptide according to claim 8 as an active ingredient. An anticancer agent comprising the cell death-inducing peptide according to any one of claims 1 to 7 and / or the MAP-mediated cell death-inducing peptide according to claim 8 as an active ingredient.

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