KR100517055B1 - Peptides with increased hydrophobicity by substituting one or more amino acids of Pep-27 peptide and pharmaceutical compositions containing thereof - Google Patents

Abstract

The present invention provides a peptide comprising an increased hydrophobic region by replacing a hydrophilic region of Pep ²⁷ , a peptide known to be involved in apoptosis in Streptococcus pneumoniae , with hydrophobic other amino acids, and a pharmaceutical composition comprising the same. In particular, the present invention relates to a synthetic peptide set forth in SEQ ID NO: 2 prepared by substituting a hydrophilic portion of the Pep ²⁷ peptide described in SEQ ID NO: 1 with another hydrophobic amino acid, and a pharmaceutical composition for anticancer comprising the same. Since the synthetic peptide of the present invention has no cytotoxicity and shows excellent anticancer activity, it may be usefully used as a safe anticancer agent for human body.

Description

Peptides with increased hydrophobicity by substituting one or more amino acids of PeJ-27 peptide and pharmaceutical compositions containing ©} Peptides with increased hydrophobicity by substituting one or more amino acids

The present invention provides a peptide comprising an increased hydrophobic region by replacing a hydrophilic region of Pep ²⁷ , a peptide known to be involved in apoptosis in Streptococcus pneumoniae , with hydrophobic other amino acids, and a pharmaceutical composition comprising the same. In particular, the present invention relates to a synthetic peptide set forth in SEQ ID NO: 2 prepared by substituting a hydrophilic portion of the Pep ²⁷ peptide described in SEQ ID NO: 1 with another hydrophobic amino acid, and a pharmaceutical composition for anticancer comprising the same.

In general, one of the obstacles to successful cancer treatment is the acquisition of multi-drug resistance (MDR) mechanism of tumor cells. This multidrug resistance is mediated by overexpression of human MDR1 or MRP genes, which are the respective genes encoding P-glycoprotein and multidrug resistance related protein (MRP). When the P-glycoprotein or MRP protein is overexpressed, the drug accumulation in the cell is reduced, thereby allowing cancer cells to have a multidrug resistant phenotype. To date many hydrophobic peptides are known as substrates of P-glycoproteins. Therefore, it is possible to develop chemotherapy sensitizers using hydrophobic peptides (Sharom, FJ, et al., Biochem. J. , 1998 , 333, 621-630).

Meanwhile, the dolastatin 10 peptide isolated from Dolabella auricularia , a type of mollusk, has anticancer activity, and P- resistance to the peptide in rat and human leukemia cells. Glycoproteins are known to be involved (Toppmeyer, DL, Biochem. Pharmacol. , 1994 , 48, 609-612). In addition, two hydrophobic peptides, reversin 121 and 205, have recently been reported to increase drug accumulation in drug-resistant cells to drug-sensitive cells in response to P-glycoproteins (Sharom, FJ, et al., Biochem. Pharmacol. , 1999 , 58 (4): 571-586).

Accordingly, the present inventors have tried to develop a new peptide showing effective anticancer activity in cancer cells with general or drug resistance, synthesized a novel peptide with increased hydrophobic region by substituting the hydrophilic region of Pep ²⁷ peptide with other amino acids, The present invention was completed by identifying the antibiotics, anticancer activity and cytotoxicity (hemolytic activity) of the synthetic peptides synthesized as described above and revealing that the synthetic peptides can be useful as antibiotics and anticancer agents.

It is an object of the present invention to provide a peptide and derivatives thereof which have a hydrophobic region, a cytotoxicity and excellent anticancer activity by substituting the hydrophilic portion of the Pep ²⁷ peptide described in SEQ ID NO: 1 with other amino acids.

In order to achieve the above object, the present invention provides a peptide and derivative thereof having an increased hydrophobic region by substituting the hydrophilic portion of the Pep ²⁷ peptide described in SEQ ID NO: 1 with other hydrophobic amino acids.

The present invention also provides a pharmaceutical composition for anticancer containing the peptide and derivatives thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides peptides and derivatives thereof having an increased hydrophobic region by substituting hydrophilic sites of the Pep ²⁷ peptide set forth in SEQ ID NO: 1 with other hydrophobic amino acids.

Peptides and derivatives thereof of the present invention are hydrophobic to the hydrophilic sites of Pep ²⁷ described in SEQ ID NO: 1 , a peptide known to initiate apoptosis through signaling through the component system VncR / S in pneumococci. It is a synthetic peptide having an affinity structure by replacing with an amino acid to increase the hydrophobic region.

Peptides of the present invention can be prepared using a Merrifield liquid phase solidification method using a Fmoc amino group protective container (Merrifield, RB, J. Am. Chem. Soc. , 1963 , 85, 2149). The peptide of the present invention and derivatives thereof may be any synthetic peptide having increased hydrophobic region by substituting one or more amino acids of the hydrophilic portion of the Pep ²⁷ peptide as set forth in SEQ ID NO: 1 with other hydrophobic amino acids, SEQ ID NO: 1 The 11th amino acid serine and the 13th amino acid glutamine in the hydrophilic region of the Pep ²⁷ peptide described in Fig. ³ are hydrophobic amino acids selected from the group consisting of tryptophan, alanine, isoleucine, methionine, phenylalanine, proline, valine and leucine. It is preferable to prepare by substitution, and more preferably, the peptides described in SEQ ID NO: 2 prepared by substituting tryptophan and derivatives thereof (see Fig. 1 ).

The present inventors have isolated and purified the peptide of the present invention and confirmed the purity, the purity was more than 95%, the molecular weight obtained by using the Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry is calculated from the amino acid sequence Since it is consistent with the peptide having the exact amino acid sequence described in SEQ ID NO: 2 it was confirmed that the synthesis.

The present invention also provides a pharmaceutical composition for anticancer containing the peptide and derivatives thereof as an active ingredient.

The peptides and derivatives thereof of the present invention can be effectively used for any kind of cancer cells, in particular for breast cancer, gastric cancer and lymphoma.

In order to confirm whether the peptide of the present invention and its derivatives can be used as an anticancer agent, the present inventors measured the anticancer activity of the peptide synthesized in the present invention. Anticancer activity was observed by measuring 50% inhibitory concentration (hereinafter abbreviated as "IC ₅₀ ").

In order to confirm whether the synthetic peptide of the present invention exhibits anticancer activity against cancer cells, anticancer activity was measured in human acute myeloid leukemia cell line, promyeloid leukemia cell line, breast cancer cell line, gastric adenocarcinoma cell line and T-cell leukemia cell line. , It was confirmed that the synthetic peptide of the present invention described in SEQ ID NO: 2 shows a strong anticancer activity compared to the Pep ²⁷ peptide used in the comparison group (see Table 1 ).

In addition, the present inventors have identified acute myeloid leukemia cell lines sensitive to drugs, cell lines overexpressing P-glycoprotein, and multi-drug-resistant proteins to determine whether the peptides of the present invention exhibit anticancer activity against drug resistant cancer cells. As a result of measuring the anticancer activity in the overexpressing cell line, it was confirmed that the synthetic peptide of the present invention described in SEQ ID NO: 2 showed excellent anticancer activity compared to the Pep ²⁷ peptide used in the comparison group (see Table 2 ).

In addition, the present inventors treated the synthetic peptides of the present invention with human leukemia cell line, and observed the intracellular distribution and site of action of the peptides over time. As a result, the peptides of the present invention diffused into the cells over time. It was confirmed that (see Fig. 2 ).

In addition, after treating the peptides of the present invention in human leukemia cell line, the cell death and membrane integrity were observed through flow cytometry, and it was confirmed that the cell death was caused without significant damage to the cell membrane (see FIG. 3 ).

In addition, after treating the peptide of the present invention in human leukemia cell line, and observed the change of the enzyme activity according to the treatment time and concentration of the peptide, it was confirmed that the activity of phosphatase increases with increasing treatment time and concentration. (See FIG. 4 ).

From the above results, it was confirmed that the synthetic peptide described in SEQ ID NO: 2 of the present invention can be usefully used as an anticancer agent because it shows an excellent anticancer effect.

Peptides and derivatives thereof of the present invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

That is, the peptides and derivatives thereof of the present invention may be administered in various parenteral formulations, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. It is prepared. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the peptides and derivatives thereof of the present invention can be used in combination with various carriers which are accepted as medicaments such as physiological saline or organic solvents, and carbo such as glucose, sucrose or dextran to increase stability or absorption. Antioxidants such as hydrates, ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

The effective dose of the peptide of the present invention and its derivatives is 0.1-2 mg / kg, preferably 0.5-1 mg / kg, and can be administered 1-3 times a day.

In the pharmaceutical composition of the present invention, the total effective amount of the peptide of the present invention and its derivatives may be administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. Multiple doses may be administered by a fractionated treatment protocol with long term administration. Since the concentration of the peptide and its derivatives is determined in consideration of various factors such as the age and health status of the patient as well as the route of administration and the number of treatments of the drug, the general knowledge in this field is considered in view of this point. Those of skill in the art will be able to determine appropriate effective dosages for particular uses of the peptides and derivatives thereof as pharmaceutical compositions.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Synthesis and Separation of Peptides

In order to synthesize an anti-cancer peptide having an amphiphilic structure with an increased hydrophobic region, the present inventors substituted the hydrophilic region of the Pep ²⁷ peptide described in SEQ ID NO: 1 with a hydrophobic amino acid. Specifically, the peptide described in SEQ ID NO: 2, respectively substituted with tryptophan for the 11th amino acid serine as the 13th amino acid of glutamine Pep ²⁷ peptides described in SEQ ID NO: 1 was prepared (Fig. 1). For the synthesis of peptides, Merrifield's liquid phase solidification method using Fmoc (9-fluorenylmethoxycarbonyl) as an amino group protective container was used (Merrifield, RB, J. Am. Chem. Soc. , 1963 , 85, 2149). In the present invention, the peptide having the carboxyl terminus of -NH ₂ form was used as the starting material of the link amide (Rink Amide) MBHA-resin (Resin), the peptide of the carboxyl terminus of the -OH form is Fmoc-amino acid-Wang Resin ( SynPep Corporation) was used as starting material. The extension of the peptide chain by the coupling of Fmoc-amino acids was by N-hydroxybenzo triazole (HOBt) -dicyclo-hexycarbodiimide (DCC) method. First, after coupling Fmoc-amino acid at the amino terminal of each peptide, remove the Fmoc group with 20% piperidine / NMP (N-methyl pyrolidone) solution and wash it several times with NMP and DCM (dichoromethane). Dry with nitrogen gas. To this was added a solution of trifluoroacetic acid (TFA) -phenol-thioanisole-H ₂ O-triisopropylsilane (85: 5: 5: 2.5: 2.5, vol / vol) and 2 The reaction was carried out for 3 hours to remove the protecting group and to separate the peptide from the resin, and then the peptide was precipitated with diethylether. Crude peptides obtained by the above method were purified in reverse phase (RP) -HPLC column (Delta Pak, C ₁₈ 300Å, 15) in acetonitrile gradient containing 0.05% TFA. , 19.0 mm x 30 cm, Waters). The synthesized peptide was hydrolyzed at 110 ° C. with 6 N-HCl, the residue was concentrated under reduced pressure, dissolved in 0.02 N-HCl, and the amino acid composition was measured by an amino acid analyzer (Hitachi 8500 A). As a result of confirming the purity of the peptide thus prepared, it showed a purity of 95%, and the molecular weight obtained using MALDI mass spectrometry (Hill, et al., Rapid Commun. Mass Spectrometry , 1991 , 5, 395) was calculated from the amino acid sequence. Since it is consistent with the obtained molecular weight, it was confirmed that the peptide having the correct amino acid sequence described in SEQ ID NO: 2 .

Experimental Example 1 Antitumor Activity of Synthetic Peptides

<1-1> Growth Inhibitory Concentration (IC ₅₀ ) Measure

In order to measure the anticancer activity of the synthetic peptide of the present invention prepared in Example 1 , the present inventors MTT analysis of human lung cancer cell line Calu-6 and gastric cancer cell line SNU 601, and T cell lymphoma Jurkat cell line Was performed. First, 90 μl of each cell line at a concentration of 2 × 10 ⁵ cells / ml was dispensed into 96-well plates. At this time, only the culture solution was used as a negative control. The plate was shaken well and incubated in a CO ₂ incubator for 3 days, and the formazan produced by the mitochondrial enzyme of living cells was well dissolved with 100 μl of 0.04 N HCl-isopropanol solution and absorbance at 540 nm using an ELISA reader. Measured. The anti-cancer activity of the peptide of the present invention was shown as a percentage obtained by dividing the absorbance value of the wells treated with the synthetic peptide by diluting 70 μM to the maximum concentration by the absorbance value containing the control cells not treated with the synthetic peptide ( Table 1 ).

Peptide 50% growth inhibition concentration (IC ₅₀ ) AML-2 HL-60 Jurkat SNU-601 MCF-7 Pep ²⁷ 70 < 70 < 70 < 70 < 70 < Synthetic peptide of SEQ ID NO: 2 29 20 23 25 10

As a result, as shown in Table 1 , the synthetic peptide of the present invention was confirmed that the anti-cancer activity of each cell line was 3 to 7 times stronger than that of Pep ²⁷ used as a comparative group for all the cell lines tested.

<1-2> Cytotoxicity Measurement of Cancer Cells with Drug Resistance

In order to determine whether the synthetic peptide of the present invention exhibits anticancer activity against cancer cells with drug resistance, we have also used AML-2, acute myeloid leukemia cell line, drug-sensitive AML-2 cell line, P-glycoprotein overexpression resistant AML- MTT analysis was performed on AML-2 / D100, which is a 2 blast cell line, and AML-2 / DX100, which is a protein overexpression resistant AML-2 blast line related to multidrug resistance. First, 90 μl of AML-2 / WT, AML-2 / D100, and AML-2 / DX100 cell lines at a concentration of 2 × 10 ⁵ cells / ml were dispensed into 96-well plates. At this time, only the culture solution was used as a negative control. The plate was shaken well and incubated overnight in a CO ₂ incubator, followed by treatment with 20 μl of the synthetic peptides of the present invention (17.5 μM, 35 μM and 70 μM) and incubated at 37 ° C. for 3 days. 100 [mu] l of MTT (3- [4,5-dimethyl-2-thiazolyl] -2,5-diphenyl-2H-tetrazolium bromide) -formazan produced by mitochondrial enzymes in living cells with 100 μl of 0.04 N HCl-isopropanol solution It was thawed well and the absorbance was measured at 540 nm using an ELISA reader. The absorbance values of the wells treated with the synthetic peptides were divided by the absorbance values containing control cells not treated with the synthetic peptides to show the anticancer activity of the synthetic peptides of the present invention ( Table 2 ).

Peptide 70 μM 35 μM 17.5 μM AML-2 / WT AML-2 / D100 AML-2 / DX100 AML-2 / WT AML-2 / D100 AML-2 / DX100 AML-2 / WT AML-2 / D100 AML-2 / DX100 Pep ²⁷ 89.7 68.95 100 93 84.35 100 93.4 98.3 98.7 Synthetic peptide of SEQ ID NO: 2 0 0 0 30.4 49.9 100 83.2 91.95 100

As a result, as shown in Table 2 , it was confirmed that the synthetic peptide of the present invention showed stronger anti-cancer activity than Pep ²⁷ peptide for all the cell lines tested. The synthetic peptide of the present invention did not show anticancer activity at a concentration of 17.5 μM, but showed a rapid anticancer activity as the concentration was increased, showing a strong anticancer activity completely inhibiting the growth of cancer cells at a concentration of 70 μM.

<1-3> Identification of intracellular distribution and site of action of peptides over time

The present inventors treated the Jurkat cell line, which is a human T-cell leukemia cell line, with the peptide described in SEQ ID NO: 2 of the present invention, and then examined the confocal laser scanning microscope in order to check the cell integrity of the cell line over time. Scanning microscopy) was used to perform the following experiments. Specifically, the Jurkat cell line was grown on a cover glass and then treated with a peptide of the present invention to which fluoresceinisothio cyanate (FITC) was added overnight. After treatment for 30 minutes, 60 minutes, and 120 minutes, the solution was washed with phosphate buffer, fixed for 15 minutes using 3.7% formaldehyde, and mounted on a glass slide. The distribution of labeled peptides was visualized using a Leica TCS 4D (Leica Lastertech. GmbH, Heidelberg, Germany) connected to a Leica DAS uplight microscope.

As a result, it could be confirmed that the peptide described in SEQ ID NO: 2 of the present invention was diffused and distributed in the whole intracellular plasma membrane over time, which indicates that the peptide of the present invention penetrates the cell membrane and exhibits anticancer activity. ( FIG. 2 ).

<1-4> Apoptosis Apoptosis and Membrane Integrity Measurement

The inventors performed flow cytometry experiments to determine the effect of the peptide described in SEQ ID NO: 2 of the present invention on apoptosis and membrane integrity of Jurkat cells, a human leukemia cell line. Specifically, 12.5 μM and 62.5 μM of the synthetic peptides of the present invention were treated with Jurkat cells at a concentration of 1 × 10 ⁵ cells / ml and mixed for 4 hours. As a control, etoposide (etopside), which is an anticancer therapy, and melittin, which has strong anticancer activity, were used. The mixture was washed three times with phosphate buffer solution and then resuspended in 400 μl of binding buffer (10 mM Hepes / NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl ₂ ) and 10 μl of Annexin (Annexin). V-FITC (MedSystems Diagnostics GmbH, Australia) was added and incubated at room temperature for 10 minutes, and then exposed to 1 μM of propidium iodide (PI). Flow cytometry was performed using FACScan (Becton Dickinson, San Jose, Calif.).

As a result, apoptosis apoptosis was induced in the positive control etoposide 100 μM, but cell membrane damage was small. On the other hand, apoptotic cell death was small at 12.5 μM of melittin, but the membrane damage was severe. Treatment of Jurkat cells with 12.5 μM of the peptide set forth in SEQ ID NO: 2 of the present invention resulted in apoptosis apoptosis without significant damage of the cell membrane to the extent similar to 100 μM of etoposide, and apoptosis without significant damage of the cell membrane even at high concentration of 62.5 μM. Apoptosis resulted remarkably ( FIG. 3 ). From the above results, it can be seen that the anticancer action of SEQ ID NO: 2 of the present invention was shown through apoptosis apoptosis mechanism rather than damage to the cell membrane, unlike meritin, and the effect of etoposide apoptosis apoptosis was about 9 times stronger. there was.

<1-5> Confirmation of phosphatase activity by time and concentration

The present inventors treated the peptide described in SEQ ID NO: 2 to the Jurkat cell line, a human leukemia cell line, and observed changes in phosphatase activity according to treatment time and concentration of the peptide. Specifically, Jurkat cells treated with peptides were centrifuged and washed with tris-buffered saline (pH 7.4). The cells were sonicated in storage buffer (25 mM Tris-Hcl, pH 7.5, 10 mM β-mercaptoethanol, 2 mM EDTA, 0.1 mM phenylmethylsufonyl fluoride) and loaded onto Sephadex G-25 columns. The sample received here was analyzed by Serine / Threonine Phosphate Assay Kit (Ser / Thr Phosphatase Assay kit, Promega, USA) and the absorbance was measured at a wavelength of 620 nm.

As a result, it was confirmed that the activity of serine / threonine phosphatase (Ser / Thr Phosphatase) increased as the treatment time and concentration of the peptide described in SEQ ID NO: 2 of the present invention increased ( FIG. 4 ).

Experimental Example 2 Parenteral Administration Acute Toxicity in Rats

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended parenterally at a dose of 1 g / kg / 15 ml by suspending the peptide of SEQ ID NO: 2 of the present invention in a 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, the antibiotic peptide of the present invention tested did not show a toxicity change in rats up to 5 mg / kg and the parenteral administration minimum dose (LD ₅₀ ) was determined to be a safe substance of 10 mg / kg or more.

From the above results, the synthetic peptide described in SEQ ID NO: 2 of the present invention exhibits excellent anti-cancer activity not only for general cancer cells but also for cancer cells that are sensitive or drug-resistant to drugs, and thus can be usefully used as a safe anticancer drug for humans. Can be.

1 is a schematic diagram of an analogue peptide derived from Pep ²⁷ peptide of the present invention,

Figure 2 shows the results of observing the distribution pattern of peptides over time with a laser scanning microscope (left) and a phase contrast microscope (right) after treatment with a synthetic leukemia cell line (Jurkat) of the present invention. It is a photograph,

A: peptide 30 minutes treatment group,

B: peptide 60 minutes treatment group,

C: 120 minutes treatment group peptide

3 is a photograph showing the results of observing the effect of the synthetic peptide of the present invention on apoptosis and membrane integrity state by flow cytometry,

Left: Bistaining (Annexin V-FITC [FL1-H] and PI [FL2-H]),

Center: Annexin V-FITC Dyeing,

Right: PI dyed,

A: control group, B: etoposide treatment group,

C: melittin treatment group, D: peptide treatment group as set forth in SEQ ID NO: 2

Figure 4 is a graph showing the change in phosphatase activity with time and concentration after the peptide of the present invention treated with human leukemia cell line (Jurkat).

A: 5 to 60 minutes of peptide

B: Treating Peptides from 12.5 μM to 62.5 μM

<110> Chosun University <120> Novel peptides with increased hydrophobicity by substituting one or more amino acids of Pep27 peptide and pharmaceutical compositions containing <130> 2p-01-23B <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> PRT <213> Streptococcus pneumoniae <400> 1 Met Arg Lys Glu Phe His Asn Val Leu Ser Ser Gly Gln Leu Leu Ala 1 5 10 15 Asp Lys Arg Pro Ala Arg Asp Tyr Asn Arg Lys 20 25 <210> 2 <211> 27 <212> PRT <213> Streptococcus pneumoniae <400> 2 Met Arg Lys Glu Phe His Asn Val Leu Ser Trp Gly Trp Leu Leu Ala 1 5 10 15 Asp Lys Arg Pro Ala Arg Asp Tyr Asn Arg Lys 20 25

Claims (5)

The 11 th amino acid serine and the 13 th amino acid glutamine of the Pep ²⁷ peptide described in SEQ ID NO: 1 were substituted with an amino acid selected from the group consisting of tryptophan, alanine, isoleucine, methionine, phenylalanine, proline, valine and leucine Peptides with increased hydrophobic regions. delete The peptide of claim 1, wherein the peptide is set forth in SEQ ID NO: 2 . An anticancer pharmaceutical composition comprising the peptide of claim 1 or 3 as an active ingredient. The pharmaceutical composition of claim 4, wherein the cancer is selected from the group consisting of breast cancer, gastric cancer, and lymphoma.