KR100465230B1 - Novel polypeptide having anticancer and antimicrobial activity, and anticancer and antimicrobial composition comprising the same - Google Patents

Abstract

The present invention provides a polypeptide, a derivative thereof, and an antibiotic and an anticancer composition, wherein the N-terminal ten amino acids have an amino acid sequence of SEQ ID NO: 1 and have a total amino acid length of 20 to 24, and a polypeptide or a derivative thereof as an active ingredient. In the present invention, the polypeptide of the present invention exhibits excellent antibiotic and anticancer activity against various microorganisms and cancer cell lines, and particularly, the same anticancer efficacy without being resistant to MDR (multi-drug resistance) cancer having resistance to existing anticancer agents. This can overcome the problems caused by MDR. In addition, when used in the human body does not show a hemolytic action, even when broken down in the cell has fewer side effects.

Description

Novel polypeptide having anticancer and antimicrobial activity and anticancer and antibiotic composition containing the same {NOVEL POLYPEPTIDE HAVING ANTICANCER AND ANTIMICROBIAL ACTIVITY, AND ANTICANCER AND ANTIMICROBIAL COMPOSITION COMPRISING THE SAME}

The present invention provides an anticancer and antibiotic composition in which the N-terminal ten amino acids have an amino acid sequence represented by SEQ ID NO: 1 and a polypeptide having a total amino acid length of 20 to 24, a derivative thereof, and a polypeptide or a derivative thereof as an active ingredient. It is about.

Most organisms produce and release antimicrobial polypeptides into mucous membranes or body fluids to inhibit the growth of pathogenic microorganisms from the outside. Representative examples of such polypeptides include cecropins, attackcins, lectins, hemolins, etc., found in the chrysalis of Hyalophora cecropia by Haberman et al. (Boman, HC and Hultmark, D., 1987, Annu. Rev. Microbiol., 41: 103-126) and defensins found in mucosal cells of vertebrates (Lehrer, RI, Ganz, T. and Seisted , ME, 1991, Cell 64, 229-230, and magainins (US Pat. No. 4,810,777) found in Xenopus laevis are known.

However, most of the above-described anti-biogenic peptides have low biological activity and very limited microorganisms of interest, or when used in the human body, there is a problem of causing hemolysis.

On the other hand, the incidence of various cancers such as gastric cancer, lung cancer, liver cancer, breast cancer, rectal and colorectal cancer is rapidly increasing due to various factors such as deepening environmental pollution and westernization of diet. Various chemicals have been used in chemotherapy to treat various types of cancer, but these chemicals are highly toxic, affecting not only cancer cells but also normal cells, causing severe side effects such as hair loss, weight loss, vomiting and diarrhea. In addition, the chemicals used in chemotherapy often only work against certain types of cancer. In particular, when one anticancer drug is continuously administered, the limitations of the existing anticancer drugs have become a big problem due to MDR having resistance to various anticancer drugs not related to this.

Accordingly, efforts to develop a treatment that has less side effects than the chemotherapy and is not affected by MDR have continued, and as a result, biotherapy using a biological agent has emerged. However, until now, only monoclonal antibodies and interferons combined with lysine or other toxins have been used as biological agents for the treatment of cancer.

Accordingly, the present inventors have steadily tried to search for novel peptides that exhibit excellent anticancer and antibiotic effects with little side effects and can overcome the MDR problem. As a result, the present inventors have found that specific polypeptides satisfy such conditions. .

An object of the present invention is to provide a novel antibiotic and anticancer polypeptide having excellent antimicrobial and anticancer activity and broad spectrum of application and harmless to human body.

Another object of the present invention is to provide an antibiotic and anticancer composition containing the polypeptide as an active ingredient.

In accordance with the above object, the present invention provides a polypeptide and a derivative thereof having 10 amino acids in N-terminal sequence having the amino acid sequence of SEQ ID NO: 1 and having a total length of 20 to 24 amino acids.

According to the above another object, the present invention provides an anticancer and antibiotic composition containing the polypeptide or a derivative thereof as an active ingredient.

Polypeptides or derivatives thereof of the present invention exhibit excellent anti-cancer and anti-life properties even when used alone or in admixture with each other.

As used herein, anti-cancer and anti-liver means growth inhibitory or killing activity against bacteria, viruses, protozoa, cancer cells, spores and other microorganisms.

Preferred polypeptides of the present invention are those in which the N-terminal 10 amino acids of SEQ ID NO: 1 and the C-terminal 6 amino acids have the sequence of SEQ ID NO: 2, in particular the amino acids of SEQ ID NOs: 3-5 Most preferred are polypeptides having a sequence. In addition, all derivatives of the polypeptide of the present invention, except for SEQ ID NO: 1, have one or more amino acids substituted or deleted from other amino acid sequences and exhibit antibiotic or anticancer activity, and are included in the scope of the present invention. Examples of the derivative of the polypeptide include those having the amino acid sequence of SEQ ID NO: 6.

The polypeptide and its derivatives may be synthesized chemically, or may be produced by genetic engineering methods using the genes encoding them according to known methods.

The anticancer and antibiotic compositions of the present invention exhibit excellent anticancer and antimicrobial properties against various microorganisms, such as bacteria, viruses, spores, protozoa, cancer cells, etc. In case of less side effects, it is very useful as anticancer and antibiotic.

On the other hand, the pharmaceutical formulation can be prepared according to a conventional method using the composition of the present invention. In the preparation of the formulations, it is preferable to enclose the composition prepared by mixing or diluting the active ingredient with the carrier, excipient and diluent, etc. in a capsule, sachet or other container-type carrier or the like. Such formulations include, for example, tablets, pills, granules, powders, sasei, elecils, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders, ointments, patching agents and the like. It may be in the form.

Examples of suitable carriers, excipients and diluents include water, saline, lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, methylhydroxy Benzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. In addition, the compositions of the present invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The anticancer and antibiotic compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. For humans, typical daily dosages of the anticancer and antibiotic compositions of the invention may range from 10 to 50 mg / kg body weight, preferably from 20 to 25 mg / kg body weight, once or as active peptides. Can be administered in several divided doses. However, it should be understood that the actual dosage of the anticancer and antibiotic compositions of the present invention should be determined in light of several related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease, and therefore, The dosage does not limit the scope of the invention in any way.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, and the present invention is not limited to these.

Example 1 : Synthesis and Purification of Polypeptides

Polypeptides having an amino acid sequence of SEQ ID NOs: 3 to 6 (named Polypeptides 1 to 4) were subjected to a solid-phase peptide synthesis method (Merrifield, RB, 1963, J. Am Chem. Soc., 85: 2149). ), And then purified using HPLC. In the HPLC analysis, a column of C18 Vydac 218TP101522 (22 × 250 mm) reverse phase HPLC column was used, and acetonitrile was flowed as an eluent at a flow rate of 10 ml / min. Mass spectra of the purified polypeptides were measured by a mass spectrometer (Matrix-associated laser desorption ionization (MALDI-I); PerSeptive Biosystem, Votager Biospectrometry Workstation) to confirm that they had a purity of 95% or more.

Test Example 1 : Investigation of antibiotic activity of polypeptide

Streptococcus epidermidis (ATCC 12228), Micrococcus luteus (ATCC 4698), Escherichia coli (ATCC 9637) to quantitatively determine the antibiotics of polypeptides 1-4. , Minimal inhibitory concentration (MIC) for Klebsiella pneumoniae (ATCC 10031), Salmonella typhimurium (ATCC 14028) and Proteus mirabilis (ATCC 25933) ) Was measured by the following method.

A single colony of the bacteria cultured on LB medium was collected and inoculated into 5 ml of LB liquid medium, followed by shaking culture at 37 ° C. to 2 × 10 ⁶ colony forming units (CFU) / ml, which was then prepared as a strain inoculum. . 50 μl of the strain was inoculated into a 96-well plate, and any one of peptides 1 to 4 was treated with 50 μl of a peptide solution diluted with LB to a final concentration of 1.56 μg / ml to 100 μg / ml (final volume 100 Μl) After incubation at 37 ° C for 12 hours, absorbance at 600 nm was measured using a scanning multiwell spectrophotometer (Molecular Devices (Versa Mas)) to determine the growth of the strain and the minimum growth inhibition concentration for each bacteria. Was calculated using Maggainin 2 (Magainin 2; Zasloff, M. et al., 1988, Proc. Natl. Acad. Sci. USA , 85, 910-913), which had been reported to have antibiotics as a comparative group . The same experiment was repeated. The results are shown in Table 1.

microbe Minimum growth inhibitory concentration (MIC, μg / ml) Peptide 1 Peptide 2 Peptide 3 Peptide 4 Marghein 2 Staphylococcus epidermidis (ATCC 12228) 50 25 12.5 50 7 Microcaucus Luteus (ATCC 4698) 6.25 6.25 6.25 6.25 - E. coli (ATCC 9637) 25 12.5 12.5 100 100 Klebsiella pneumoniae (ATCC 10031) 50 25 25 50 100 Salmonella typhimurium (ATCC 14028) > 100 100 100 > 100 70 Proteus Mirabilis (ATCC 25933) > 100 > 100 > 100 > 100 > 100

Test Example 2 : Hemolysis test

Polypeptides 1 and 4 prepared in Example 1 were diluted 10-fold with a 10-fold dilution of erythrocytes with phosphate buffered saline (0.8% sodium chloride, 0.02% potassium chloride, 0.144% Na ₂ HPO ₄ ; pH was adjusted to 7.4 with hydrochloric acid). Mixed at concentrations of μg / ml and 100 μg / ml and then incubated at 37 ° C. for 10 minutes. The culture solution was centrifuged at 10,000 rpm for 10 minutes at room temperature using an ultracentrifuge (VISION VS-1500), and the absorbance of the supernatant was measured at 350 nm. The results are shown in Table 2. The absorbance of 350 nm of the supernatant caused by hemolysis in phosphate buffered saline with a final concentration of Triton X-100 of 0.1% (v / v) was considered to be 100% and the relative absorbance to this value was calculated. Hemolysis hardly occurred at the peptide concentration of. As a control material, bee melittin (Sigma Chemicals Co.), a natural polypeptide known to have anti-bioactivity, was used.

Concentration (µg / ml) Peptide 1 Peptide 2 Peptide 3 Peptide 4 Melittin 10 0.00 0.00 0.00 0.00 2.9 100 0.69 0.13 0.02 0.06 100

Test Example 3 : Investigation of anticancer activity of polypeptide

In order to investigate anticancer activity, 50% growth inhibitory concentration (IC ₅₀ ) was measured by MTT assay (Carmichael, J. et al. , Cancer Res. , 47 : 936-942, 1987) as follows.

In 96-well plates, 293 cell lines (human kidney cancer cell line; ATCC CRL-1573), A549 cell line (human lung cancer cell line; ATCC CCL-185), MCF-7 cell line (human mammary cancer cell line; ATCC HTB-22) and MCF-7 MDR cell line, MCF-7-DOX cell line (Dr. Sung-Jin Kim, NCI, NIH USA), 90 μl of each 5 × 10 ³ cells (293 cell line DMEM medium containing 10% FBS (GIBCO BRL 12800-017) , A549 cell line, MCF-7 cell line and its MDR variant, MCF-7-DOX cell line, were inoculated with RPMI 1640 medium (GIBCO BRL 31800-022) containing 10% FBS and then at 37 ° C., 5% CO ₂ conditions. The cells were incubated for 1 day, and any one of peptides 1 to 4 was treated with a peptide solution diluted in each medium to a final concentration of 0.8 μg / ml to 100 μg / ml (final volume 100 μl). for 4 days [4,5-dimethyl thiazol-2-yl] after incubation MTT solution for measuring viable cells (in phosphate buffer solution 3 under 5% CO ₂ conditions, 2,5-di Carbonyl thiazol imidazolium bromide is 2 mg / ㎖ solution dissolved at a concentration) was reacted with handle 50㎕ and 37 ℃, 5% CO ₂ for 4 hours. Remove the medium and MTT solution, DMSO (dimethyl sulfoxide) in 200㎕ The crystals formed were dissolved and absorbance was measured at 570 nm using a scanning multiwell spectrophotometer (Molecular Devices (Versa Max)).

The anticancer efficacy was investigated by calculating IC ₅₀ from the obtained absorbance data, and the result shown in Table 3 was obtained.

Cell line IC ₅₀ (μg / ml) Peptide 1 Peptide 2 Peptide 3 Peptide 4 MCF-7 18 15 25 50 293 20 18 20 65 A549 14 22 25 60 MCF-7-DOX 49 57 32 70

The anticancer and antibiotic composition of the present invention exhibits excellent anticancer and antimicrobial activity against various microorganisms and cancer cell lines, and in particular, exhibits anticancer efficacy without being resistant to MDR (multi-drug resistance) cancer having resistance to existing anticancer agents. In addition, when used in the human body does not show a hemolytic action, even when broken down in the cell is very useful as antibiotics and anticancer drugs because side effects are small.

<110> Peptipharm Inc. <120> Novel polypeptide having anticancer and antimicrobial activity, and anticancer and antimicrobial composition comprising the same <130> FPD / 200202-0038 <150> KR 10-2001-0008894 <151> 2001-02-22 <160> 6 <170 > KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> N-terminal amino acids of synthetic anticancer and antimicrobial polypeptide <400> 1 Phe Leu Lys Leu Leu Lys Lys Leu Ala Ala 1 5 10 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> C-terminal amino acids of synthetic anticancer and antimicrobial polypeptide <400> 2 Lys Ile Ser Tyr Lys Cys 1 5 <210> 3 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic anticancer and antimicrobial polypeptide <400> 3 Phe Leu Lys Leu Leu Lys Lys Leu Ala Ala Lys Phe Leu Pro Thr Ile 1 5 10 15 Ile Cys Lys Ile Ser Tyr Lys Cys 20 <210> 4 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic anticancer and antimicrobial polypeptide <400> 4 Phe Leu Lys Leu Leu Lys Lys Leu Ala Ala Lys Phe Leu Pro Ile Ile 1 5 10 15 Cys Lys Ile Ser Tyr Lys Cys 20 <210> 5 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic anticancer and antimicrobial polypeptide <400 > 5 Phe Leu Lys Leu Leu Lys Lys Leu Ala Ala Lys Phe Leu Pro Ile Ile 1 5 10 15 Lys Ile Ser Tyr Lys Cys 20 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> < 223> Synthetic anticancer and antimicrobial polypeptide <400> 6 Phe Leu Lys Leu Leu Lys Lys Leu Ala Ala Leu Pro Ile Ile Lys Lys 1 5 10 15 Ile Lys Leu Gly 20

Claims (10)

A polypeptide that exhibits anticancer and antibiotic activity and has an amino acid sequence of any one of SEQ ID NOs: 3-6. delete delete delete delete delete An anticancer and antibiotic composition comprising the polypeptide of claim 1 as an active ingredient. The method of claim 7, wherein A composition characterized by having anticancer and antibiotic activity against microorganisms including bacteria, viruses, protozoa, cancer cells and spores. The method of claim 8, Bacteria include Streptococcus epidermidis , Micrococcus luteus , Escherichia coli , Klebsiella pneumoniae , Salmonella typhimurium Or Proteus mirabilis . The method of claim 7, wherein Composition for use in the treatment of mammary gland cancer, kidney cancer, prostate cancer, liver cancer, leukemia, gastric cancer and lung cancer.