WO2014198096A1 - Small polypeptide zy13 and applications thereof - Google Patents

Abstract

Disclosed are applications of a small polypeptide, ZY31, in preparing an acne treatment, anti-infection, and anti-tumor medicament. The polypeptide comprises 15 amino acid residues, has a molecular weight of 2228.77 Da and an isoelectric point of 12.05, and is of an amino acid sequence as represented by SEQ ID:1. The polypeptide has a small molecular weight, is convenient to synthesize, provides a significant bacteriostatic effect, provides significant therapeutic effect for acne, also is capable of inhibiting tumor cell growth, and provides cytotoxicity against tumor cells.

Description

[Name of invention established by ISA according to Rule 37.2] Small molecule peptide ZY13 and its application Technical field

The invention belongs to the field of biomedical technology, and particularly relates to the application of the small molecule polypeptide ZY13 in the preparation of a medicament for treating acne, anti-infection and anti-tumor.

Background technique

Acne is a chronic inflammation of the hair follicle sebaceous glands. It is a common inflammatory skin disease in adolescence. It occurs in the cheeks, forehead, cheeks and nasolabial folds, followed by the chest, back and shoulders. Level, sebaceous gland secretion, P. acnes proliferation, keratinization abnormalities of hair follicle sebaceous gland ducts and inflammation. Adolescent skin surface lipid composition has changed, and male hormones increase, causing hyperkeratosis of sebaceous hair follicles. Epithelial cells that are detached from the hair follicle wall are mixed with sebum, embolized in the hair follicle mouth to form acne, and then acted as propionic acid bacteria. In addition to bacterial infection, it causes inflammation, produces abscesses, and is extremely painful after suffering.

Tumor is a new organism in which a certain cell of a local tissue loses its normal regulation at the genetic level due to various carcinogenic factors, resulting in its clonal abnormal proliferation. According to the size of the tumor and its growth characteristics, it is divided into benign tumors and malignant tumors. Malignant tumors grow rapidly, often infiltrating into surrounding tissues during growth, with few envelopes on the surface, and often have systemic metastases. At present, the incidence of malignant tumors has been increasing year by year, and it is also among the top causes of death of various diseases, and thus it is a disease that is mainly controlled.

The antibacterial peptide is a biologically active small molecule polypeptide induced by the living body, and has a molecular weight of about 1000 to 7000 and consists of 10 to 60 amino acid residues. Antimicrobial peptides are high-efficiency, spectral antibacterial peptide molecules that are rapidly produced by organisms when they are invaded by microorganisms to participate in the body's immune response. Antibacterial peptides are widely used as effective defense molecules in the body. At present, thousands of antimicrobial peptides have been identified from microorganisms, plants, insects, arthropods, amphibians, mammals and even humans. Antibacterial peptides have complex antibacterial mechanisms, but most of them believe that the mechanism involves the cationic and hydrophobic properties of antimicrobial peptides and the action of negatively charged microbial membranes. When the antimicrobial peptides and bacterial cell membranes are in contact, the membrane permeability changes. Or forming a transmembrane pore on the bacterial cell membrane, which eventually causes the bacterial contents to leak out and die. Therefore, antibacterial peptides are much faster than traditional antibiotics in killing bacteria, and unlike antibiotics, which inhibit the growth of bacteria at low concentrations, the effects of antimicrobial peptides on bacteria are almost fatal. Studies have shown that antibacterial peptides are less likely to induce drug-resistant strains than traditional antibiotics; antibacterial spectrum light has effects on bacteria, fungi, viruses, protozoa and cancer cells. Three family antibacterial peptides have been found in poultry, including cathelicidin, liver-expressed MICROstatic peptide(LEAP),and --defensin. These antibacterial peptides are crucial for poultry to resist bacterial and viral diseases, and mutations or deletions associated with these genes will have a significant impact on the ability of poultry to be resistant to microbial infections; in addition to spectral antibacterial activity, these antimicrobial peptides also Highly effective antifungal, antiviral, antiprotozoal and/or antitumor activities, such as Bat5 and Imc7 can kill Leptospira, and have good killing against Candida albicans, Cryptococcus and membrane viruses and parasites. Some antibacterial peptides have obvious lethal effect on herpesvirus, influenza virus and HIV enveloped virus; in addition, some antibacterial peptides also have various other regulatory functions, such as Cathelicidin, which promotes wound healing and tissue damage. Repair, chemotactic, pro-angiogenic and anti-parasitic functions have important biological activities in regulating animal immunity.

technical problem

The object of the present invention is to overcome the shortcomings of the prior art, providing a small molecule polypeptide ZY13;

Another object of the present invention is to provide a small molecule polypeptide ZY13 for use in the preparation of a medicament for treating acne, anti-infective and anti-tumor drugs.

Technical solution

The object of the present invention is achieved by the following technical scheme: small molecule polypeptide ZY13, which contains 15 amino acid residues and has a molecular weight of 2228.77. Da, the isoelectric point is 12.05, and its amino acid sequence is SEQ. ID: 1 is: valine-lysine-arginine-tryptophan-lysine-lysine-tryptophan-arginine-tryptophan-lysine-color Amino-lysine-lysine-tryptophan-valine.

The use of the small molecule polypeptide ZY13 of the invention for preparing acne, anti-infective and anti-tumor drugs.

Beneficial effect

The beneficial effects of the invention are: the polypeptide ZY13 of the invention is artificially synthesized, has the advantages of small molecular weight, convenient artificial synthesis, obvious antibacterial effect, and has significant therapeutic effect on acne; the polypeptide can also inhibit the growth of tumor cells, Tumor cells have a killing effect; small molecule polypeptide ZY13 can be used in the preparation of anti-infective, anti-tumor and acne treatment drugs.

DRAWINGS

Figure 1 is a schematic diagram of the therapeutic effect of the small molecule polypeptide ZY13 on the mouse ear acne model.

Figure 2 is a schematic diagram showing the therapeutic effect of small molecule polypeptide ZY13 on inflammation of mouse ear acne model.

Figure 3 is a schematic representation of the activity of the small molecule polypeptide ZY13 against lung cancer cell A549.

Figure 4 is a schematic representation of the activity of the small molecule polypeptide ZY13 against breast cancer cell MDA-435.

Figure 5 is a schematic representation of the activity of the small molecule polypeptide ZY13 against melanoma cell A357.

Figure 6 is a schematic representation of the activity of the small molecule polypeptide ZY13 against hepatoma cells HepG2.

Figure 7 is a schematic representation of the activity of the small molecule polypeptide ZY13 against gastric cancer cell SGC7901.

Figure 8 Comparison of the minimum inhibitory concentration of small molecule polypeptide ZY13 against Escherichia coli, Candida albicans, Staphylococcus aureus and Bacillus subtilis.

Figure 9 Comparison of the minimum inhibitory concentration of small molecule polypeptide ZY13 against acne-associated Staphylococcus aureus, hemolytic staphylococcus, Staphylococcus epidermidis, and Staphylococcus aureus.

Embodiments of the invention

The present invention will be further described below in conjunction with the accompanying drawings, and the scope of protection of the present invention is not limited to the following:

Example 1: Preparation of Small Molecule Peptide ZY13

1. 0.2 g of the resin was weighed and placed in a dry and clean reaction tube, and an appropriate amount of N,N-dimethylamide (DMF) was added thereto for activation for 30 minutes. Weigh 1mmol of the first amino acid residue, 150mg of 4-dimethylaminopyridine (DMAP) was added to the reaction tube, DMF was used as a solvent for 3h, and after the reaction was completed, it was washed 3-6 times with DMF, and appropriate pyridine and acetic anhydride were added. , volume ratio is 1:1, reaction 30 Min, DMF is washed 3-6 times after the reaction is completed. Then elute the amino acid protecting group Fmoc with piperidine, elute twice, each time for 15 min, then wash 4 times with DMF, and wash twice with methanol;

2. Weigh 2mmol of the second amino acid, 3mmol of HBTU in the reaction tube, and add DIEA. 0.5ml, reaction for 40min, wash 3-6 times with DMF, add the piperidine solution twice to elute the amino acid protecting group Fmoc, each time 10min, then wash 4 times with DMF, wash twice with methanol;

3. Repeat the second step until the last amino acid residue;

4. After the last amino acid reaction is completed, it is cleaved with trifluoroacetic acid for 2 hours, and the reaction is filtered with suction to obtain a solution of the polypeptide in trifluoroacetic acid, which is precipitated with diethyl ether, centrifuged, and washed with diethyl ether for 3-5 times to obtain a white solid, which is desalted by HPLC. A sample of the polypeptide is obtained.

Example 2: Small molecule polypeptide ZY13 antibacterial experiment

1. Prepared Escherichia coli, Candida albicans, Staphylococcus aureus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus aureus, and cultured at 37 ° C for 18 h, ready for use;

2. Preparing a solution of small molecule polypeptide ZY13 with a concentration of 0.8-20 μg/ml, sterilizing and drying a circular qualitative filter with a diameter of 5-7 mm, and immersing it in the above-mentioned different concentration of small molecule polypeptide ZY13 solution;

3. Prepare high-column broth agar medium, sterilize, and set aside;

4. Dissolve high column broth agar medium, cool to 50 ° C, add E. coli, Candida albicans, Staphylococcus aureus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus aureus 1ml, light Shake well, pour into a sterile plate, shake gently to spread the medium evenly on the plate;

5. The sterilized tweezers were used to place the filter neatly and orderly in a cooled dish, covered with a terracotta cover, and marked, placed in a 37 ° C incubator for 24 h;

6. The size of the inhibition zone was measured with a caliper, and the intensity of action of different concentrations of the small molecule polypeptide ZW13 on different bacteria was compared. The results are shown in Fig. 8 and Fig. 9.

In Figure 8, the minimum inhibitory concentration of the small molecule polypeptide ZY13 against Escherichia coli, Candida albicans, Staphylococcus aureus and Bacillus subtilis is 18.75, respectively. Gg/ml, 1.17 μg/ml, 2.34 μg/ml, 1.17 μg/ml.

In Figure 9, the minimum inhibitory concentration of small molecule peptide ZY13 against acne-related Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus aureus was 2.34 μg/ml, 4.7 μg/ml, 4.7 μg/ Ml, 1.17 μg/ml.

The experimental results show that the small molecule polypeptide ZY13 has significant bacteriostatic action.

Example 3: Therapeutic effect of small molecule polypeptide ZY13 on mouse ear acne model

1. Culture P. acnes ATCC11817 with brain heart infusion medium, incubate to log phase, wash twice with normal saline, and resuspend physiological saline to 5×10 ⁸ CFU/ml;

2. Male mice weighing 18g, Kunming, were randomly divided into 3 groups, 6 in each group, respectively, treatment group, positive control group and negative control group. Each intraperitoneal injection of 120μl, 1% sodium pentobarbital Anesthesia was performed, and then 20 μl/mu of the resuspended bacterial solution was injected into the left ear of the mouse;

3. The small molecule polypeptide ZY13 and clindamycin were separately prepared into 2 mg/ml ointment by using polyethylene glycol and glycerin, wherein the ratio by weight of polyethylene glycol to glycerin was 5:1, and the surface of the left ear skin of the mouse was rubbed. The treatment group was coated with small molecule polypeptide ZY13, the positive control group was coated with clindamycin, the negative control group was rubbed with polyethylene glycol, once every 8 hours, a total of 3 times, and the mice were sacrificed after 24 hours;

4. The left ear of the mouse was disinfected with a clean alcohol cotton ball, the left ear was cut and cut, transferred to a homogenizer for full homogenization, and 1 ear was added with 1 ml of physiological saline homogenate;

5. Dilute the homogenate to 1000 times, take 50 μl of the diluted solution and apply the brain heart infusion medium plate, anaerobic culture for 72 h at 37 ° C, and count the colonies. The results are shown in Fig. 1. The number of colonies of P. acnes after treatment with 2 mg/ml of small molecule polypeptide ZY13 was 2.4×10 ⁵ , and the number of colonies of P. acnes after treatment with 2 mg/ml of clindamycin was 5.6. ×10 ⁵ , the number of colonies of P. acnes after treatment with polyethylene glycol was 9.44×10 ⁵ , which indicated that ZY13 was significantly better than clindamycin in treating ear decompression in mice.

Example 4: Therapeutic effect of small molecule polypeptide ZY13 on inflammation of mouse ear acne model

1. Culture P. acnes ATCC6919 with brain heart infusion medium, incubate to log phase, wash twice with physiological saline, and resuspend physiological saline to 5×10 ⁸ CFU/ml;

 2. Male mice weighing 18g, Kunming, were randomly divided into 3 groups, 9 in each group, respectively, treatment group, positive control group, negative control group, each intraperitoneal injection of 50μl ketamine for anesthesia, then in mice The left ear was intradermally injected with 20 μl of bacteria solution after resuspending;

 3. The small molecule polypeptide ZY13 and clindamycin were separately prepared into 2 mg/ml ointment with polyethylene glycol, wherein the ratio of polyethylene glycol to glycerin was 5:1, and the surface of the left ear skin of the mouse was rubbed. The treatment group The small molecule polypeptide ZY13 was rubbed, the positive control group was coated with clindamycin, the negative control group was rubbed with polyethylene glycol, and once every 8 hours, a total of 3 times were administered, and the mice were sacrificed 24 hours later;

 4. Disinfect the left ear of the mouse with a clean alcohol cotton ball and measure the thickness of the mouse ear. The results are shown in Figure 2. The thickness of the mouse ears before treatment is 0.20. Mm, given 2mg/ml The thickness of the mouse ear was 0.26 mm after 1 day of treatment with the small molecule polypeptide ZY13. The thickness of the mouse ear was 0.33 mm after 1 day of treatment with 2 mg/ml clindamycin, and the thickness of the mouse ear was 0.42 after 1 day of treatment with polyethylene glycol. Mm, indicating that ZY13 anti-inflammatory effect is significantly better than clindamycin.

Example 5: Activity of small molecule polypeptide ZY13 against lung cancer tumor cell A549

1. Collect a logarithmic lung cancer tumor cell A549, make a cell suspension, adjust the concentration to 5×10 ⁶ to 10×10 ⁶ /ml, add 100 μl per well, and plate the cells to adjust the density to 1000-10000. Holes, edge holes filled with sterile PBS;

2. Incubate with 5% CO ₂ at 37 °C until the cell monolayer is covered with 96-well flat-bottomed bottom. After the cells are attached for 2-12 hours, add the small molecule peptide ZY13 at a concentration of 100 μg/ml and 200 μg/ml. 10 μg/ml and 5 μg/ml paclitaxel, 5% CO ₂ , 37 ° C for 16 to 48 h, observed under an inverted microscope;

3. Add 20 μl, 5 mg/ml MTT solution to each well, continue to culture for 4 h, and aspirate the culture medium in the well;

4. 150 μl of dimethyl sulfoxide was added to each well, and shaken at a low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance of each well was measured at the OD490nm of the enzyme-linked immunosorbent detector, and the zero-reducing well and the control well were set at the same time. 3: 200μg/ml ZY13 acts on lung cancer cells A549, the OD value is 0.3, 100μg/ml ZY13 acts on lung cancer cells A549, the OD value is 0.5; 10μg/ml paclitaxel acts on cancer cells, lung cancer cells, A549, OD The value of 0.5, 10μg/ml paclitaxel on cancer cell lung cancer cell A549 after OD value of 0.7, indicating that ZY13 has a significant inhibitory effect on lung cancer tumor cell A549.

Example 6: Activity of small molecule polypeptide ZY13 against breast cancer cell MDA-435

1. Collect the cell suspension of MDA-435 in log phase breast cancer cells, adjust the concentration to 5×10 ⁶ ～10×10 ⁶ /ml, add 100 μl per well, and plate the cells to adjust the density to 1000~ 10,000 wells, the edge holes are filled with sterile PBS;

2. Incubate with 5% CO ₂ at 37 °C until the cell monolayer is covered with a 96-well flat-bottomed well. After the cells are attached for 2-12 hours, add the small molecule peptide ZY13 at a concentration of 50-300 μg/ml. Physiological saline, 5% CO ₂ , 37 ° C for 16 ~ 48h, observed under an inverted microscope;

 3. Add 20 μl, 5 mg/ml MTT solution to each well, continue to culture for 4 h, and aspirate the culture medium in the well;

4. 150 μl of dimethyl sulfoxide was added to each well, and shaken at a low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance of each well was measured at the OD490nm of the enzyme-linked immunosorbent detector, and the zero-reducing well and the control well were set at the same time. 4 shows: 300 The OD value of μg/ml ZY13 in breast cancer cells after MDA-435 is 0.17, and the OD value of MZ-435 after treatment with 200 μg/ml of ZY13 is 0.20, and the OD of MZ-435 after treatment with 100 μg/ml of ZY13 The value of 0.61, 50μg/ml of ZY13-treated breast cancer cells after MDA-435 was 0.7, indicating that ZY13 inhibited MDA-435 in a concentration-dependent manner.

Example 7: Small molecule polypeptide ZY13 anti-melanoma cell A357 activity experiment

1. Collect log phase melanoma cells A357, make cell suspension, adjust the concentration to 5×10 ⁶ ～10×10 ⁶ /ml, add 100μl per well, and plate the cells to adjust the density to 1000～10000 Holes, edge holes filled with sterile PBS;

2. Incubate with 5% CO ₂ at 37 °C until the cell monolayer is covered with a 96-well flat-bottomed well. After the cells are attached for 2-12 hours, add the small molecule peptide ZY13 at a concentration of 100 μg/ml and 200 μg/ml. Incubate with the same volume of normal saline, 5% CO ₂ , 37 ° C for 16 to 48 h, observe under an inverted microscope;

 3. Add 20 μl, 5 mg/ml MTT solution to each well, continue to culture for 4 h, and aspirate the culture medium in the well;

 4. 150 μl of dimethyl sulfoxide was added to each well, and shaken at a low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance of each well was measured at the OD490nm of the enzyme-linked immunosorbent detector, and the zero-reducing well and the control well were set at the same time. 5 shows that the OD value of 100 μg/ml of ZY13 acting melanoma cell A357 is 1.0, the OD value of ZY13 after 200 μg/ml of melanoma cells is 0.72, and the OD value of 1.0 is normal saline after treatment of melanoma cells. It indicated that 200μg/ml of ZY13 had a significant inhibitory effect on melanoma.

Example 8: Small molecule polypeptide ZY13 anti-hepatocarcinoma cell HepG2 activity experiment

1. Collect the hepatocellular carcinoma cell HepG2, make the cell suspension, adjust the concentration to 5×10 ⁶ ～10×10 ⁶ /ml, add 100 μl per well, and plate the cells to adjust the density to 1000-10000 The edge holes are filled with sterile PBS;

2. Incubate with 5% CO ₂ at 37 °C until the cell monolayer is covered with a 96-well flat-bottomed well. After the cells are attached for 2-12 hours, add the small molecule peptide ZY13 at a concentration of 100 μg/ml and 200 μg/ml. Incubate with the same volume of normal saline, 5% CO ₂ , 37 ° C for 16 to 48 h, observe under an inverted microscope;

 3. Add 20 μl, 5 mg/ml MTT solution to each well, continue to culture for 4 h, and aspirate the culture medium in the well;

 4. 150 μl of dimethyl sulfoxide was added to each well, and shaken at a low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance of each well was measured at the OD490nm of the enzyme-linked immunosorbent detector, and the zero-reducing well and the control well were set at the same time. 6 shows that the OD value of HepG2 after treatment with 100 μg/ml of ZY13 is 0.89, and the OD value of HepG2 after treatment with 200 μg/ml of ZY13 is 0.6, and the OD value of HepG2 after treatment with physiological saline is 1.0, indicating: 200 μg/ Ml and 100 μg/ml of ZY13 have a significant inhibitory effect on HepG2 cells.

Example 9: Activity of small molecule polypeptide ZY13 against gastric cancer cell SGC7901

1. Collect log phase gastric cancer cells SGC7901, make cell suspension, adjust the concentration to 5×10 ⁶ ～10×10 ⁶ /ml, add 100 μl per well, and plate the cells to adjust the density to 1000-10000 wells. The edge holes are filled with sterile PBS;

2. Incubate with 5% CO ₂ at 37 °C until the cell monolayer is covered with a 96-well flat-bottomed well. After the cells are attached for 2-12 hours, add the small molecule peptide ZY13 at a concentration of 50-300 μg/ml. Physiological saline, 5% CO ₂ , incubated at 37 ° C for 16 to 48 h, observed under an inverted microscope;

 3. Add 20 μl, 5 mg/ml MTT solution to each well, continue to culture for 4 h, and aspirate the culture medium in the well;

 4. 150 μl of dimethyl sulfoxide was added to each well, and shaken at a low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance of each well was measured at the OD490nm of the enzyme-linked immunosorbent detector, and the zero-reducing well and the control well were set at the same time. 7: 50μg/ml ZY13 effect gastric cancer cell SGC7901 OD value 0.65, 100μg/ml ZY13 effect gastric cancer cell SGC7901 OD value 0.57, 200μg/ml ZY13 effect gastric cancer cell SGC7901 OD value 0.2,300μg The OD value of /ml of ZY13 after gastric cancer cell SGC7901 was 0.09, indicating: ZY13 has a significant inhibitory effect on gastric cancer cell line SGC7901.

Claims (4)

1.  Small molecule polypeptide ZY13, which is characterized in that it contains 15 amino acid residues, has a molecular weight of 2228.77 Da, and has an isoelectric point of 12.05.

   The acid sequence is shown in SEQ ID: 1.
2. Use of the small molecule polypeptide ZY13 according to claim 1 for the preparation of an anti-infective medicament.
3. Use of the small molecule polypeptide ZY13 according to claim 1 for the preparation of a medicament for treating acne.
4. Use of the small molecule polypeptide ZY13 of claim 1 for the preparation of an antitumor drug.

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