Multifunctional cyclic peptide and application thereof

Abstract

The invention discloses a multifunctional cyclic peptide, and the structure of the multifunctional cyclic peptide is an end-to-end cyclization structure of tryptophan, leucine, lysine, arginine, tryptophan, leucine, arginine, lysine, leucine, tyrosine, lysine, phenylalanine, glycine and glycine. The multifunctional cyclic peptide has the advantages of the cyclic peptide, can be used as a novel bioactive molecule integrating antibacterial, anti-inflammatory and osteogenesis / tooth promoting functions, can meet various clinical requirements, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of novel bioactive polypeptide molecules, and specifically refers to a multifunctional cyclic peptide and its application. Background Art

[0002] Infectious tissue defects are characterized by bacterial infection, local immune homeostasis disorder, and limited tissue regeneration ability. Usually, the treatment or research measures can only improve one aspect of infection, inflammation, or tissue repair promotion. To improve multiple aspects, multiple treatments or drugs need to be used in combination, which increases the complexity, cost, and side effects.

[0003] For example, anti-infection relies on the use of antibiotics. With the increasingly serious global problem of antibiotic resistance, the development of new antibacterial drugs has become an urgent task. At the same time, the demand for anti-inflammatory and tissue repair promotion functions in the medical field is also increasing, especially in the treatment of chronic inflammatory diseases and hard tissue repair.

[0004] A multifunctional peptide refers to a peptide molecule with multiple biological activities that can simultaneously participate in the regulation of multiple physiological processes. For example:

[0005] Some peptide molecules not only have antibacterial effects but also can regulate the immune system, promote tissue repair, etc.

[0006] In recent years, the application of multifunctional peptides in drug research and development has gradually increased, especially in the fields of anti-tumor, anti-infection, and rare disease treatment.

[0007] However, traditional linear peptides have poor stability in vivo and are easily enzymatically degraded, which limits their clinical applications.

[0008] Cyclic peptides are cyclic structures formed by amino acids through peptide bonds, and have the characteristics of high stability, strong permeability, and good specificity. Compared with linear peptides, cyclic peptides are more stable in vivo, are not easily enzymatically degraded, and can better bind to targets. In recent years, significant progress has been made in the research of cyclic peptides in the fields of antibacterial, anti-inflammatory, and tissue repair promotion. However, there is little research on cyclic peptides that integrate multiple functions. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the above technical defects and provide a multifunctional cyclic peptide with multiple biological activities, which provides an innovative solution for the treatment of related diseases and its application.

[0010] To solve the above technical problem, the technical solution provided by the present invention is: a multifunctional cyclic peptide, and the structure of the multifunctional cyclic peptide is shown in formula (I):

[0011]

[0012] It is the head-to-tail cyclized structure of tryptophan-leucine-lysine-arginine-tryptophan-leucine-arginine-lysine-leucine-tyrosine-lysine-phenylalanine-glycine-glycine.

[0013] On the other hand, the present invention discloses a pharmaceutical composition, the structural fragment is selected from a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0014] Preferably, it further contains a pharmaceutically acceptable diluent, excipient or carrier.

[0015] On the other hand, the present invention also discloses the application of the multifunctional cyclic peptide or a pharmaceutically acceptable salt thereof in drugs for promoting hard tissue repair.

[0016] Preferably, it includes the application in promoting the osteogenic and odontoblastic differentiation of bone marrow mesenchymal stem cells and dental pulp stem cells.

[0017] On the other hand, the present invention also discloses the application of the multifunctional cyclic peptide or a pharmaceutically acceptable salt thereof in drugs for anti-infection of oral pathogenic bacteria.

[0018] Preferably, the oral pathogenic bacteria include Staphylococcus aureus, drug-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Porphyromonas gingivalis, Candida albicans.

[0019] On the other hand, the present invention also discloses the application of the multifunctional cyclic peptide or a pharmaceutically acceptable salt thereof in anti-inflammatory drugs.

[0020] Preferably, it is applied in inhibiting macrophage inflammation caused by lipopolysaccharide and down-regulating the secretion of pro-inflammatory factors.

[0021] The advantages of the present invention compared with the prior art are as follows: The multifunctional cyclic peptide disclosed by the present invention is more stable in vivo, not easily enzymatically degraded, and can better bind to the target. While having the advantages of cyclic peptides, as a novel bioactive molecule integrating antibacterial, anti-inflammatory and osteogenic / dental functions, it can meet various clinical needs and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the molecular structure of Cyclo-WG14.

[0023] Figure 2 It is a schematic diagram of the hemolytic toxicity data of the multifunctional cyclic peptide Cyclo-WG14.

[0024] Figure 3 It is a schematic diagram of anti-inflammatory data.

[0025] Figure 4 It is a schematic diagram of the application for promoting hard tissue repair. DETAILED DESCRIPTION OF THE INVENTION

[0026] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. The same components are denoted by the same reference numerals.

[0027] It should be noted that the multifunctional cyclic peptide provided by the present invention is hereinafter referred to as Cyclo-WG14.

[0028] Combined with the attached Figures 1-4 , its molecular formula and structure Figure 1 as shown

[0029] Example 1:

[0030] Cyclo-WG14 antibacterial experiment. In the present invention, various clinically and orally common pathogenic bacteria such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Porphyromonas gingivalis, Candida albicans are used, and the MIC / MBC against different bacteria is detected by the microplate dilution method for drug susceptibility test.

[0031] 1 Pick the preserved S. aureus, E. coli, MRSA, P. aeruginosa single colonies and resuscitate them aerobically in an LB liquid medium in a 37 °C constant temperature incubator. Resuscitate the E. faecalis and C. albicans colonies aerobically in a 10 mL BHI liquid medium in a constant temperature incubator. Resuscitate the P. gingivalis single colony in a BHI medium containing hemin and vitamin K in a 37 °C constant temperature anaerobic incubator (80% N 2 , 10% H 2 , 10% CO 2 ).

[0032] 2 Dilute the mid-logarithmic growth phase bacterial solution with the corresponding medium to 1×10 6 CFU / mL for standby.

[0033] 3 Add the cyclic peptide to the 96-well plate by the two-fold dilution method, 10 μL per well. Add 90 μL of the corresponding medium with a bacterial concentration of 1×10 6 CFU / mL to make the final concentration of the antibacterial peptide 2 - 128 μg / mL. Sterile water is used as a negative control.

[0034] Four 96-well plates were placed in a 37 °C constant-temperature anaerobic incubator (80% N 2 , 10% H 2 , 10% CO 2 ) or in a constant-temperature aerobic incubator for 24 h.

[0035] 5 The MIC is the minimum inhibitory concentration of the clear wells in the plate.

[0036] 6 50 μL of the bacterial liquid in the clear wells was aspirated and spread on the corresponding culture medium agar plate and cultured for 48 h. The MBC is the minimum bactericidal concentration at which no colonies grow on the plate.

[0037] As shown in Table 1, Cyclo-WG14 has good antibacterial ability and has good antibacterial ability against a variety of pathogenic bacteria, including drug-resistant bacteria and bacteria with natural resistance to antibacterial drugs.

[0038]

[0039] Table 1: Antibacterial ability of Cyclo-WG14

[0040] Example 2:

[0041] Hemolytic toxicity of the multifunctional cyclic peptide Cyclo-WG14:

[0042] ① Defibrinated sheep blood was centrifuged at 1000 rpm for 10 min, the supernatant was discarded, washed with PBS, and centrifuged repeatedly until the supernatant was clear to obtain red blood cells. The red blood cells were resuspended with ten volumes of PBS to obtain a red blood cell suspension.

[0043] ② The cyclic peptide was added to an EP tube by a two-fold dilution method, 100 μL per tube. 900 μL of the red blood cell suspension was added to each well to make the final concentration of the antibacterial peptide 8 - 128 μg / mL. Another 100 μL of 1% Triton X-100 was used as a positive control, and 100 μL of PBS was used as a negative control. Incubate at 37 °C for 1 h. After centrifugation at 1000 rpm for 10 min, the supernatant was taken to measure OD540. Hemolysis rate % = (OD540 treatment group - OD540 negative control group) / (OD540 Triton X-100 - OD540 negative control group) × 100%.

[0044] Figure 2 The multifunctional cyclic peptide Cyclo-WG14 shown has low hemolytic toxicity and obvious toxicity appears at 128 μg / mL, and this concentration has covered its highest antibacterial concentration, indicating that it will not cause obvious toxicity to the body when exerting its antibacterial effect;

[0045] Figure 2 shows the hemolytic toxicity of the multifunctional cyclic peptide Cyclo-WG14. Different lowercase letters represent significant differences, P

[0046] <0.05.

[0047] Example 3:

[0048] Anti-inflammatory ability of the multifunctional cyclic peptide Cyclo-WG14:

[0049] ① RAW 264.7 macrophages were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The medium was changed every 3 days.

[0050] ② To establish an inflammatory cell model, RAW 264.7 cells were pretreated with Escherichia coli lipopolysaccharide for 24 hours and then co-cultured with LPS or different concentrations of the polypeptide for 24 hours.

[0051] ③ The supernatant was collected, and an ELISA kit was used to detect the levels of pro-inflammatory cytokines such as IL-1β and TNF-α.

[0052] As Figure 3 shown, Cyclo-WG14 can significantly inhibit macrophage inflammation caused by lipopolysaccharide, downregulate the secretion of pro-inflammatory factors, and has anti-inflammatory activity;

[0053] Figure 3 showed the inhibitory effect of the multifunctional cyclic peptide Cyclo-WG14 on the release of pro-inflammatory cytokines from inflammatory macrophages. Different lowercase letters represent significant differences, P < 0.05.

[0054] Example 4:

[0055] Hard tissue repair ability of the multifunctional cyclic peptide Cyclo-WG14:

[0056] (1) Bone marrow mesenchymal stem cells and dental pulp stem cells were seeded in 12-well plates and cultured in DMEM / F-12 medium containing double antibodies and 10% fetal bovine serum in a CO 2 incubator (5% CO 2 , 95% air, 100% humidity, 37 °C) at a constant temperature.

[0057] (2) The adherent cells in culture were daily replaced with fresh medium containing 32 μg / mL of the cyclic peptide.; The negative control group used medium without the cyclic peptide.

[0058] (3) After 7 days of cell culture, alkaline phosphatase staining solution was used for qualitative observation of alkaline phosphatase, and after 14 days of cell culture, alizarin red staining solution was used for qualitative observation of mineralized nodules.

[0059] As Figure 4As shown, the ALP staining and alizarin red staining of bone marrow mesenchymal stem cells and dental pulp stem cells under the treatment of the multifunctional cyclic peptide Cyclo-WG14 were significantly stronger than those of the control group, indicating its potential to promote hard tissue repair.

[0060] The content not described in detail in this specification belongs to the prior art well known to those skilled in the art.

[0061] The above description of the present invention and its embodiments is not restrictive. What is shown in the drawings is only one of the embodiments of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural modes and embodiments without creative efforts without departing from the purpose of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. A multifunctional cyclic peptide, characterized in that: The structure of the multifunctional cyclic peptide is shown in formula (I): It is a head-to-tail cyclized structure of tryptophan-leucine-lysine-arginine-tryptophan-leucine-arginine-lysine-leucine-tyrosine-lysine-phenylalanine-glycine-glycine.

2. A pharmaceutical composition, characterized in that: The structural fragment is selected from a compound of formula (I) or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to any one of claim 2, characterized in that: It also contains a pharmaceutically acceptable diluent, excipient or carrier.

4. Use of the multifunctional cyclic peptide or a pharmaceutically acceptable salt thereof according to claim 1 in a drug for promoting hard tissue repair.

5. Use of the multifunctional cyclic peptide or a pharmaceutically acceptable salt thereof according to claim 1 in an anti-infection drug for oral pathogens.

6. Use of the multifunctional cyclic peptide or a pharmaceutically acceptable salt thereof according to claim 1 in anti-inflammatory drugs.

7. The use according to claim 5, characterized in that: The oral pathogens include Staphylococcus aureus, drug-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Porphyromonas gingivalis, and Candida albicans.

8. The use according to claim 6, characterized in that: Its application in inhibiting macrophage inflammation caused by lipopolysaccharide and downregulating the secretion of pro-inflammatory factors.

9. The use according to claim 4, characterized in that: Including the application in promoting the osteogenic and odontogenic differentiation of bone marrow mesenchymal stem cells and dental pulp stem cells.

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