JP2005053855A - Peptide and medicine for induction of cell death - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicine useful for the induction of cell death, more specifically, an anticancer agent obtained by applying a peptide having cell death-inducing activities. <P>SOLUTION: The peptide has a sequence having 3-8 amino acid and selected from the group consisting of CNN, CNNL, CNNLP, CNNLPLSH, NNL, NNLP, NNLPL, NNLPL, INNLP, VNNLP, CNCLP, TNNLP, CTNLP, CNTLP, NNN and CNNXP (wherein, X is an amino acid I, V, L, F, C, M, A, G, T, S, W, Y, P, H, D, F, N, O, K or R). <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cell death-inducing peptide. More particularly, it relates to peptides derived from the extracellular domain sequence of Fas ligand (FasL).

  FasL is known to be a protein that binds to one of its receptors, Fas antigen, and induces apoptosis in cells.

  FasL is known to induce cell death not only in normal cells but also in tumor cells. Therefore, FasL can be used as an antitumor agent. However, since FasL is a large molecule, it is difficult to use for treatment such as cancer treatment.

  Usually smaller molecules are preferred for use in therapy. For this reason, smaller molecules having cell death inducing activity are desired.

  On the other hand, peptides have various physiological activities in vivo while being small molecules compared to proteins such as FasL. 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.

To date, there have been many previous examples of cell functional peptides such as antibacterial activity, promotion of cell adhesion, apoptosis and other protein activity inhibitors. However, there are very few reports of peptides that are short peptides and have cytotoxicity (Non-patent Documents 1 and 2). Furthermore, there has been no report of peptides having such activity that retain activity even when the terminal of the peptide is immobilized.
Buckley CD, et al., RGD peptides induce apoptosis by direct caspase-3 activation. “Nature” (UK), 1999, 397 (6719), p.534-9 Terui Y, et al., NH2-terminal pentapeptide of endothelial interleukin 8 is responsible for the induction of apoptosis in leukemic cells and has an antitumor effect in vivo., “Cancer Res.”, 1999, 59 (22), p. .5651-5

  An object of this invention is to provide the pharmaceutical effective for cell death induction. More specifically, an object of the present invention is to provide an anticancer agent to which a peptide having cell death inducing activity is applied.

  The present inventors 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 screening target, synthetic peptides based on the amino acid sequence of Fas ligand (FasL), an apoptosis inducer, were used to search for peptides that induce cell death. As a result, it was found that 5 residues of the extracellular domain sequence CNNLP of Fas ligand have high cell death-inducing activity against hematopoietic cancer cells. In addition, we confirmed the cell killing effect of CNNLP analogues in the experimental system we established for screening CNNLP peptides, and as shown below, they showed high activity in CNCLP, TNNLP, CNNCP, CNNTP, CNNNP, NNLPL, etc. Higher activities were also shown in CNNL, NNL and NNLP, which are some of them. Further, when the peptide was immobilized, a higher cell death inducing activity was shown. In the immobilized state, it is meaningful that the density of peptides acting simultaneously is high, and it is considered that high cell death-inducing activity was exhibited.

  Based on the above findings, a cell death inducer comprising the peptide as an active ingredient has been completed.

  That is, the present invention is a peptide having a sequence selected from the group consisting of CNN, CNNL, CNNLP, CNNLPLSH, NNL, NNLP, NNLPL, NNLPL, INNLP, VNNLP, CNCLP, TNNLP, CTNLP, CNTLP, NNN and CNNXP Provide: where X is amino acid I, V, L, F, C, M, A, G, T, S, W, Y, P, H, D, F, N, O, K or R Represent.

  Moreover, the pharmaceutical containing the said peptide is provided.

  Furthermore, the said pharmaceutical which is the said cell death inducer or an anticancer agent is provided.

  Furthermore, the use of the above peptide for producing a cell death inducer or an anticancer agent is provided.

  Here, the peptide is a peptide in which two or more amino acids are bound by peptide bonds. The peptide of the present invention is a peptide in which 3 to 5 amino acids are bound. The peptide of the present invention may be variously modified and immobilized on a carrier as long as it has a cell death-inducing activity.

  The cell death inducing activity is an activity that can induce cell death by administering a peptide, a carrier or a cell death inducing agent.

Peptide The peptide of the present invention can be produced by the following method. For example, it can be produced by a peptide synthesis method. It can also be produced by culturing a transformant containing DNA encoding the peptide of the present invention.

  When producing by a peptide synthesis method, it can be produced according to a known peptide synthesis method. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.

  For example, the peptide or amino acid that can constitute the peptide is condensed with the remaining part, and when the product has a protecting group, the protecting peptide can be eliminated to produce the desired peptide. Automated synthesis groups may be used for the synthesis of such peptides (Figure 1).

  Specifically, for example, using the Fmoc method, it can be synthesized by solid-phase synthesis in which amino acids are bound to the amino acid derivatives immobilized on the resin one by one from the carboxyl end (Fig. 2 is immobilized on the membrane). Schematic diagram of the production of the prepared peptide chip is shown). As such a resin, a commercially available resin for peptide synthesis may be used.

  The Fmoc method uses a peptide derivative in which the α-amino group is protected with an Fmoc group that can be removed with a base (eg, piperidine). After removing this protecting group, it is washed with N, N-dimethylformamide (DMF) or the like and dried. Subsequently, the amino acid derivative (for example, ester with HOBt) which activated the carboxyl group with the following condensing agent (for example, HOBt) is condensed. 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 end 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, the peptide may be synthesized as described in the following examples and used without performing a cleavage treatment.

  On the other hand, it can also be produced by culturing a transformant containing DNA encoding the peptide of the present invention. 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 cell. The peptide expressed in this way may be purified and isolated for use.

  The obtained peptide can also be purified as necessary. In order to purify the peptide, known separation and 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 For example, electrophoresis.

  As long as the cell death-inducing activity is not impaired, the peptide thus obtained can be dialyzed and freeze-dried to obtain a dry powder.

  In addition, the 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. In particular, as shown in the following examples, the peptide of the present invention has improved cell death-inducing activity when immobilized. Therefore, the peptide of the present invention may be immobilized on a carrier. Examples of the carrier include liposomes using lipids and immobilization on polyvalent peptide polyethylene glycol using carbohydrate dendrimers.

  The peptide of the present invention is bound to a lipid (for example, palmitic acid and the like) and incorporated into a liposome (FEBS Lett. 2003; 540 (1-3): 133-40. Liposome entrapment and immunogeni studies of a synthetic lipophilic multiple antigenic peptide bearing VP1 and VP3 domains of the hepatitis A virus: a robust method for vacccine design. (See Haro I, et al.), as a medicine described below, especially for anti-cancer drugs with higher cell affinity be able to.

  In addition, since the peptide of the present invention has cell death-inducing activity even when the C-terminus is immobilized, it can be made into a PEG hybrid by binding to polyethylene glycol (PEG) (Gene Ther. 2000; 7 (11): 1183-92 Protective copolymers for nonviral gene vectors: synthesis, vector characterization and application in gene delivery. (See Finsinger D et al.), Which can also be applied as pharmaceuticals described below.

  In addition, since the peptide of the present invention has cell death-inducing activity even when the C-terminus is immobilized, various molecular designs are possible. For example, it can be a multivalent peptide. By using a small core molecule composed of a radially branched lysine dendrimer as a basic structure and binding a series of peptides thereon, a huge polymer having a three-dimensional conformation can be produced. The cell recognition function is improved by designing the peptide of the present invention into such a polymer. Therefore, the cell killing effect can be enhanced when administered to cells (especially to cancer cells as an anticancer agent) as a medicine described below.

Drug The drug of the present invention may be the above peptide alone, or may be immobilized or mixed with a pharmacologically acceptable carrier. Such medicaments can be safely administered orally or parenterally (for example, topical, rectal, intravenous administration, etc.).

  In particular, as shown in the following examples, the medicament of the present invention is composed of a membrane using a synthetic polymer, a hydrogel using a natural polymer, a peptide alone immobilized on an inorganic carrier such as hydroxyapatite. It may be a preparation containing a peptide immobilized on a carrier as described above. In the case of this embodiment, the present invention may be applied directly to cells, tissues, etc. that want to induce cell death.

  Examples of the pharmaceutically acceptable carrier that may be used for the production of the medicament of the present invention include various organic polymers or inorganic carrier substances that are commonly used as pharmaceutical materials. Examples include solvents, dissolution aids, 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 inducer of the present invention, the peptide of the present invention is usually about 0.1 to 100% by weight, preferably about 10 to 99.9% by weight, more preferably about 20 to 90% by weight based on the whole preparation. %. In the cell death inducer of the present invention, the content of components other than the peptide or carrier of the present invention varies depending on the form of the preparation, but is usually about 10 to 99.9% by weight, preferably about It is about 20 to 90% by weight.

  The CNNLP peptide of the present invention is advantageous in that it is more effective in an immobilized state, and is very useful in that it can be applied to various functional materials.

  The peptide of the present invention is preferably CNNLP.

  The cell death-inducing peptide of the present invention can be applied to pharmaceuticals as a cancer therapeutic agent. In particular, it can be used as a cell death inducer of blood cell cancer cells.

  Peptide chips can be used to produce very high-density and comprehensive chips (currently possible with 8000 spot / membrane), and are expected to be useful for screening peptides with novel bioactivity as high-throughput combinatorial chemistry. . 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 Fas ligand known as an apoptosis-inducing factor was used.

〔experimental method〕
Peptides solid-phase synthesized by the Fmoc method were spotted on a cellulose membrane, cut into a diameter of 6 mm, and placed on the bottom of a 96-well plate. Using serum-free medium DMEM-F-12 (Gibco), cell K562 (human chronic myelogenous leukemia cell line) was adjusted to a cell concentration of 1 × 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 (37 ° C., 5% CO ₂ ). Cells were stained with a fluorescent reagent, and the state of living cells and cell death after culture were measured using a fluorescent plate reader Fluoroskan Ascent (Labsystems). As the fluorescent reagent, calceinAM (molecular probes) (measurement: excitation 485 nm, fluorescence 538 nm) was used for the number of living cells, and Cytotox one (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). Spot 0.9 μl of Fmoc amino acid solution (concentration 0.25 mol / l) on the amino group of β-alanine on the membrane, and bind the carboxyl group of the first amino group to the membrane. After the washing operation, the next amino acid reacts as Fmoc amino acid and is immobilized. A peptide having the target sequence was synthesized by sequentially combining amino acids (FIG. 2). The C-terminal part of the synthesized peptide is fixed in a state of being bound to the membrane.

〔Experimental result〕
The peptide chip was searched using a membrane in which the amino acid sequence of Fas ligand was comprehensively spotted.

  FIG. 3 shows the analysis result of the growth inhibitory activity by the peptide chip, that is, the cell death inducing activity. A: Color bars related to growth inhibition, B: Growth inhibitory activity of comprehensive sequence peptides of FasL, C: Growth inhibitory activity of peptides comprehensively prepared by substituting one base with CNNLP as a reference, D: Check for sequence specificity (The reverse peptide of PLNNC has no activity and NNN is also active).

  From the above results, it can be seen that CNN to NNLPL have a specific growth inhibitory effect (FIG. 3B). The amino acid 5-residue CNNLP was found to have particularly high growth inhibitory activity against K562 cells.

  In a similar experimental system, CNNLP analogs were confirmed to have cell death-inducing activity. CNCLP, TNNLP, CNNCP, CNNTP, CNNNP, NNLPL, etc. also showed high activity (Fig. 3C), some of which are CNNL, NNL NNLP also showed high activity (FIG. 3D).

  When experiments were performed under the same conditions using Jurkat (acute Tcell leukemia) and A3 (subclone of jurkat cell line) other than K562, the CNNLP peptide immobilized on the chip had the same effect as K562. (Fig. 4).

In addition, the effect of non-immobilized powder synthetic peptide CNNLP (BEX Co., Ltd.) on cells was examined. 100 μl of cell K562 (human chronic myelogenous leukemia cell line) adjusted to a cell concentration of 1 × 10 ⁵ cell / ml was added to 1 well of the plate, 10 μl of the adjusted peptide solution was added and cultured for 48 hours. After culturing, the state of living cells was measured with a fluorescence plate reader, and a growth inhibitory effect of about 50% was observed at a peptide concentration of 2 mM (FIG. 5).

  From the above results, it was revealed that the peptide of the present invention has a cell death inhibitory activity and thus has a cell growth inhibitory effect.

  Such peptides have the potential for application to peptide medicine that kills cancer cells, and the discovery of further useful peptides using the screening method established by this study is expected.

The figure which shows a peptide chip automatic synthesis group. The figure which shows the peptide chip preparation scheme. The figure which shows the analysis result by a peptide chip. The figure which shows the effect with respect to the cell of a CNNLP peptide. The figure which shows the effect with respect to the cell of a powder peptide.

Claims (6)

  Peptides having a sequence selected from the group consisting of CNN, CNNL, CNNLP, CNNLPLSH, NNL, NNLP, NNLPL, NNLPL, INNLP, VNNLP, CNCLP, TNNLP, CTNLP, CNTLP, NNN and CNNXP, where X is Amino acids I, V, L, F, C, M, A, G, T, S, W, Y, P, H, D, F, N, O, K or R are represented.   A peptide having a sequence selected from the group consisting of CNN, CNNL, CNNLP, CNNLPLSH, NNL, NNLP, NNLPL, NNLPL, CNCLP, TNNLP, CNNCP, CNNTP, CNNNP and NNN.   A medicament comprising the peptide according to claim 1 or 2.   4. The medicament according to claim 3, which is a cell death inducer.   4. The medicament according to claim 3, which is an anticancer agent.   Use of the peptide according to claim 1 or 2 for producing a cell death inducer or an anticancer agent.

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