A cationic cyclic peptide and its application

Abstract

This invention discloses a cationic cyclic bound polypeptide and its applications, belonging to the field of biomedical technology. The amino terminus of this cationic cyclic bound polypeptide is acetylated, and the carboxyl terminus is amidated. This invention develops a novel class of cationic cyclic bound polypeptides through a multi-modification strategy involving the introduction of non-natural amino acids into the polypeptide sequence, a bound polypeptide structure, and amino-terminal acetylation. Activity testing shows that the cationic cyclic bound polypeptide provided by this invention exhibits high serum stability and good broad-spectrum antibacterial and antitumor activity, possessing potential pharmaceutical value. It can be used to develop drugs for treating bacterial infections caused by Gram-positive and / or Gram-negative bacteria, and also for the treatment of various tumor diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cationic cyclic peptide and its applications. Background Technology

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are a class of short-chain, cationic, or amphiphilic molecules that constitute key components of the innate immune system in many organisms. In addition to their antimicrobial functions, many antimicrobial peptides exhibit selective cytotoxicity against cancer cells. This selectivity is largely attributed to the unique membrane composition of malignant tumor cells, whose outer membranes are often rich in negatively charged phospholipids (such as phosphatidylserine, PS), a feature rarely seen in healthy cells. The cationic nature of antimicrobial peptides promotes electrostatic interactions with these anionic membrane surfaces, leading to membrane disruption, pore formation, and rapid lytic cell death. Moreover, the therapeutic potential of some oncolytic antimicrobial peptides extends beyond direct membrane lysis; they can also trigger immunogenic cell death (ICD), a process characterized by the release of damage-associated molecular patterns (DAMPs), including ATP, calreticulin, and HMGB1. These signals can promote dendritic cell maturation and activate tumor-specific T-cell responses, thereby bridging innate and adaptive immunity and potentially achieving durable antitumor effects. A prime example highlighting this translational potential is the synthetic peptide LTX-315, which is currently in clinical trials.

[0003] Despite the promising prospects of antimicrobial peptides, the clinical translation of antimicrobial peptides and their analogues is still hindered by several inherent pharmaceutical limitations, the most prominent being their susceptibility to rapid protease degradation, poor pharmacokinetic properties, and potential off-target membrane toxicity. To overcome these obstacles, various peptide modification strategies have been developed and applied. Among them, peptide bonding strategies have been shown to effectively enhance resistance to protease degradation, membrane permeability, and bioactivity. However, current modification strategies for developing peptides based on cationic antimicrobial peptides remain very limited and require further development. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a cationic cyclic chelated polypeptide and its applications. The cationic cyclic chelated polypeptide provided by the present invention exhibits significant broad-spectrum antibacterial and antitumor activity, while also possessing high resistance to serum protease degradation, and can be applied to the development of antibacterial and antitumor drugs for humans or animals.

[0005] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A cationic cyclic steric polypeptide is provided, wherein the amino terminus of the cationic cyclic steric polypeptide is acetylated and the carboxyl terminus is amidated, and its structural formula is as follows:

[0006] Where Ac represents acetyl; Pep1, Pep2, and Pep3 are polypeptide sequences; K m This indicates that the (S)-2,6-diamino-2-methylhexanoic acid residue (i.e., α-methyl-substituted lysine) in the polypeptide sequence has a cyclic cross-linked structure between the side chain amino groups of the two α-methyl-substituted lysine residues through 1,2-dimethylenephenyl groups.

[0007] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the cationic cyclic peptide consists of 21 amino acid residues.

[0008] Furthermore, the amino acid sequence of the cationic cyclic peptide is: XKFFKKLKKAVKK m GFKK m FAKV, where the first X is (S)-2,3-diaminopropionic acid, and the 13th and 17th positions are both α-methyl-substituted lysine residues, with the side chain amino groups cross-linked by 1,2-dimethylenebenzene. This cationic cyclic peptide is acetylated at the amino terminus and amidated at the carboxyl terminus. This cationic cyclic peptide is designated MeLS-01, and its specific structural formula is as follows: .

[0009] Furthermore, the amino acid sequence of the cationic cyclic peptide is: ZKFFKKLKKAVKK m GFKK m FAKV, where the first Z is (S)-2,4-diaminobutyric acid, and the 13th and 17th positions are both α-methyl-substituted lysine residues. Furthermore, the side chain amino groups are cross-linked via 1,2-dimethylenebenzene. This cationic cyclic peptide is acetylated at the amino terminus and amidated at the carboxyl terminus. This cationic cyclic peptide is designated MeLS-02, and its specific structural formula is as follows: .

[0010] Furthermore, the amino acid sequence of the cationic cyclic peptide is: OKFFKKLKKAVKK m GFKK m FAKV, where the first O is ornithine, and the 13th and 17th positions are both α-methyl-substituted lysine residues, with the side chain amino groups cross-linked by 1,2-dimethylenebenzene. This cationic cyclic peptide is acetylated at the amino terminus and amidated at the carboxyl terminus. This cationic cyclic peptide is designated MeLS-03, and its specific structural formula is as follows: .

[0011] Furthermore, the amino acid sequence of the cationic cyclic stapled polypeptide is: KKFFKKLKKAVKK m GFKK m FAKV, where the first K is lysine, the 13th and 17th positions are two α-methyl substituted lysine residues, and the side-chain amino groups are crosslinked by 1,2-dimethylenebenzene. The amino terminus of the cationic cyclic stapled polypeptide is acetylated and the carboxyl terminus is amidated; this cationic cyclic stapled polypeptide is denoted as MeLS-04, and the specific structural formula is as follows: .

[0012] A pharmaceutical composition comprising the above-mentioned cationic cyclic stapled polypeptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0013] Use of the above-mentioned cationic cyclic stapled polypeptide or pharmaceutical composition in the preparation of an antibacterial infection drug.

[0014] Furthermore, the bacterium is a bacterium.

[0015] Use of the above-mentioned cationic cyclic stapled polypeptide or pharmaceutical composition in the preparation of an anti-tumor drug.

[0016] Furthermore, the tumors include but are not limited to breast cancer, colon cancer, hepatoblastoma, malignant glioma, osteosarcoma, lung cancer, malignant melanoma, and cervical cancer.

[0017] The present invention has the following beneficial effects: By introducing non-natural amino acids, polypeptide stapling structures, and amino-terminal acetylation multiple modification strategies in the polypeptide sequence, the present invention has developed a novel class of cationic cyclic stapled polypeptides. Through activity testing, the cationic cyclic stapled polypeptides provided by the present invention have high serum stability, good broad-spectrum antibacterial activity, and anti-tumor activity, have potential medicinal value, can be used to develop drugs for treating bacterial infections caused by Gram-positive and / or Gram-negative bacteria, and can also be used to develop drugs for treating various tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a preparation flow chart of 4 cationic cyclic stapled polypeptides.

[0019] Figure 2 It is the stability analysis of the cationic antibacterial peptide MeLS-01 in 25% serum; where 1 is linear peptide 1, 2 is cyclic stapled polypeptide 2, and 3 is cyclic stapled polypeptide 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0021] Example 1: Preparation of cationic cyclic peptide MeLS-01 A schematic diagram of the preparation process of the cationic cyclic peptide MeLS-01 is shown below. Figure 1 The specific preparation process includes the following steps: (1) Pretreatment of Rink-AM resin: Weigh Rink-AM resin (0.35 mmol / g, 1 g, 0.35 mmol, 1 eq) into a 25 mL glass solid-phase tube. Then, add 15 mL of DCM into the glass solid-phase tube, let stand for 15 minutes to allow the resin to fully swell, and finally remove DCM by vacuum filtration. (2) Removal of Fmoc protecting groups: Add 15 mL of 20% (v / v) piperidine / DMF to the solid-phase tube, seal the tube, and shake it on a shaker for 15 min. Repeat the deprotection process twice. Finally, wash the resin three times with MeOH, DCM, and DMF, 10 mL each time. (3) Preparation of linear peptide MeLS-01-I: The linear peptide MeLS-01-I was synthesized using a standard solid-phase synthesis method. The following peptides were used: Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, (S)-2,6-diamino-2-methylhexanoic acid (i.e., α-methyl-substituted lysine), Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, (S)-2,6-diamino-2-methylhexanoic acid (i.e., α-methyl-substituted lysine), Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, and Ac-Dap(Boc)-OH were sequentially coupled onto Rink-AM solid-phase resin. The coupling conditions were as follows: amino acid (1.05 mmol, 3 eq), coupling reagents HBTU (1.05 mmol, 3 eq), HOBT (1.05 mmol, 3 eq), and DIEA (2.10 mmol, 6 eq). The deprotection condition for Fmoc was 20% piperidine / DMF (v / v). After each amino acid coupling reaction and deprotection step, the resin was washed three times each with MeOH, DCM, and DMF to obtain the linear peptide cationic cyclic peptide MeLS-01-I. (4) Preparation of cationic cyclic peptide MeLS-01-II by N-alkylation reaction: MeLS-01-I was swollen in DCM for 10 minutes, the DCM was removed, THF (10 mL) and TBAH (2.1 mmol, 6 eq) were added and shaken for 10 min, then alkylation reagent 1,2-dibromomethylbenzene (2.1 mmol, 6 eq) was added, and the mixture was shaken on a shaker for 4 h. The mixture was then washed 3 times each with MeOH, DCM and DMF to obtain MeLS-01-II. (5) Preparation of cationic cyclic peptide MeLS-01-III by removing o-Ns protecting groups: MeLS-01-II was swollen in DCM for 10 minutes. After removing the DCM, DMF (10 mL) was added, followed by 2-mercaptoethanol (1.75 mmol, 5 eq) and DBU (3.5 mmol, 10 eq). After shaking on a shaker for 10 h, the mixture was washed three times each with MeOH, DCM, and DMF to obtain MeLS-01-III. (6) Preparation of cationic cyclic peptide MeLS-01 by cleavage of peptides on solid-phase resin: MeLS-01-III was placed in a cleavage reagent (TFA:TES:H2O, 95:2.5:2.5, 15 mL) and cleaved overnight. The filtrate was collected, TFA was removed by rotary evaporation to obtain a yellow oily crude product, which was precipitated by cold diethyl ether, centrifuged, the diethyl ether was discarded, the product was dissolved in water, lyophilized, and semi-preparatively purified to obtain MeLS-01 with a purity >95%. HRMS m / z calcd for C135H219N33O22 [M+H]+ 2656.7138, found 2656.7131.

[0022] Example 2: Preparation of cationic cyclic peptide MeLS-02 A schematic diagram of the preparation process of the cationic cyclic peptide MeLS-02 is shown below. Figure 1 Its preparation method is the same as that of MeLS-01, except that the first coupled amino acid is different, which is Ac-Dab(Boc)-OH. The rest of the process is the same. MeLS-02 was obtained by semi-preparative separation and purification with a purity >95%. HRMS m / z calcd for C136H221N33O22 [M+H]+ 2670.7295, found 2670.7298.

[0023] Example 3: Preparation of cationic cyclic peptide MeLS-03 A schematic diagram of the preparation process of the cationic cyclic peptide MeLS-03 is shown below. Figure 1 Its preparation method is the same as that of MeLS-01, except that the first coupled amino acid is different, which is Ac-Orn(Boc)-OH. The rest of the process is the same. MeLS-03 was obtained by semi-preparative separation and purification with a purity >95%. HRMS m / z calcd for C137H223N33O22 [M+H]+ 2684.7451, found 2684.7460.

[0024] Example 4: Preparation of cationic cyclic peptide MeLS-04 A schematic diagram of the preparation process of the cationic cyclic peptide MeLS-04 is shown below. Figure 1 Its preparation method is the same as that of MeLS-01, except that the first coupled amino acid is different, which is Ac-Lys(Boc)-OH. The rest of the process is the same. MeLS-04 was obtained by semi-preparative separation and purification with a purity >95%. HRMS m / z calcd for C138H225N33O22 [M+H]+ 2698.7608, found 2698.7614.

[0025] Experimental Example 1: Serum Stability Test The peptide (1.28 mg / mL) was incubated with 25% human serum (v / v) at 37 °C. At different time points (0, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h), 60 μL of the mixture was taken and 120 μL of a solution containing 12% trichloroacetic acid (water:acetonitrile = 1:3) was added to precipitate the protein. The precipitate was then incubated at 4 °C for 30 min. The precipitated protein was separated from the solution by centrifugation at 12000 rpm for 10 min. 60 μL of the supernatant was used for HPLC quantitative analysis of the peptide concentration change after co-incubation with serum. The results are shown in Table 1. Figure 2 As shown.

[0026] Table 1. Serum stability of cationic cyclic peptides

[0027] Linear peptide 1, cyclic peptide 2, and cyclic peptide 3 are all peptides based on MeLS-01 in different ways. The amino acid sequence of linear peptide 1 is: KFFKKLKKAVKKGFKKFAKV; cyclic peptide 2 does not contain α-methyl-substituted lysines or N-terminal acetylation, and its specific amino acid sequence is: KFFKKLKKAVKKGFKKFAKV, where the side chain amino groups of the lysine residues at positions 12 and 16 are cross-linked by 1,2-dimethylbenzene; cyclic peptide 3 contains α-methyl-substituted lysines but does not contain N-terminal acetylation, and its specific amino acid sequence is: KFFKKLKKAVKK m GFKK m FAKV, wherein the side chain amino groups of the α-methyl-substituted lysine residues at positions 12 and 16 are cross-linked by 1,2-dimethylbenzene.

[0028] From Table 1 and Figure 2 It is evident that the cationic cyclic peptides provided by this invention have a half-life >48 h in 25% human serum, significantly longer than their corresponding linear peptide 1, their corresponding cyclic peptide 2 (which does not contain α-methyl substituted lysine and does not contain N-terminal acetylation), and their corresponding cyclic peptide 3 (which contains α-methyl substituted lysine but does not contain N-terminal acetylation). Therefore, it can be concluded that the cationic cyclic peptides provided by this invention, through the simultaneous introduction of α-methyl substituted lysine ligation and N-terminal acetylation, greatly enhance the serum stability of the peptides, giving them potential for clinical drug development.

[0029] Experimental Example 2: Determination of Antibacterial Activity Take a certain amount of TSB liquid medium (tryptic soy broth) into a petri dish, and dilute the bacteria cultured to the logarithmic phase to 1×10⁻⁶. 5CFU / mL. Add 180 μL of bacterial culture and 20 μL of the test peptide solution (prepared as an initial concentration of 1.28 mg / mL aqueous solution) to each well in the first row of a 96-well plate. Each sample was tested in triplicate. TSB medium was used as a negative control. Subsequent 2-fold dilutions were performed to decrease the final concentration proportionally. The plates were incubated at 37 ℃ for 16–20 h. The minimum inhibitory concentration (MIC) of the cyclic antimicrobial peptide was determined by observing the clarity and transparency of the solution in each well and the absorbance value of each well at 490 nm using a multi-mode microplate reader. The results are shown in Table 2.

[0030] Table 2 Antibacterial activity of cationic cyclic peptides

[0031] A: MIC 2-4 ug / mL; B: MIC 4-8 ug / mL; C: MIC 8-16 ug / mL.

[0032] The bacterial strains used in this experiment included four Gram-positive bacteria: LM (Listeria), SA (Staphylococcus aureus), MRSA (methicillin-resistant Staphylococcus aureus), and EF (Enterococcus faecium); and five Gram-negative bacteria: EC (Escherichia coli), AB (Acinetobacter baumannii), PA (Pseudomonas aeruginosa), MDR-EC (Multidrug-resistant Escherichia coli), and MDR-PA (Multidrug-resistant Pseudomonas aeruginosa). The multidrug-resistant strains were all derived from clinical isolates and were resistant to multiple traditional antibiotics, including carbapenems.

[0033] As shown in Table 2, the cationic cyclic peptide provided by the present invention has broad-spectrum and highly efficient antibacterial activity, including good antibacterial activity against multiple drug-resistant strains, and is expected to be used as a broad-spectrum antibacterial drug in the clinical treatment of bacterial infections.

[0034] Experimental Example 3: Assay for Antitumor Activity (1) Dilute the tumor cells to 5×10 4 Cells were cultured at a density of 100 μL / mL and then transferred to 96-well plates, 100 μL per well. The 96-well plates were incubated overnight at 37 °C in a 5% CO2 cell culture incubator until the cells adhered.

[0035] (2) Co-incubation of peptides with cells: The 320 μM peptide was diluted with DMEM medium (containing 10% FBS) to six concentrations: 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, and 32 μM. The medium in the 96-well plates was discarded, and different concentrations of peptide solution were added to the 96-well plates, with each concentration repeated in 5 wells, and 100 μL added to each well. The medium without peptide was used as a negative control, and the medium without peptide was used as a blank control. Finally, the 96-well plates were placed in a 5% CO2 cell culture incubator and cultured at 37 °C for 24 h.

[0036] (3) MTT staining and absorbance detection: Discard the culture medium and peptides from the 96-well plate. Add 100 μL of 500 μg / mL MTT solution to each well and incubate the 96-well plate at 37 °C for 4 h in a 5% CO2 cell culture incubator. Remove the 96-well plate, discard the supernatant, and add 100 μL of DMSO to each well. Place the 96-well plate on a shaker and gently shake for 15 minutes to dissolve the formazan. Finally, detect the absorbance at 490 nm using a multi-mode microplate reader. Repeat the above experiment 3 times.

[0037] The in vitro antitumor activity of the cationic cyclic peptide of the present invention against nine tumor cell lines is shown in Table 3. The tumor cell lines used include: MCF-7 (breast cancer), HCT116 (colon cancer), HepG2 (hepatoblastoma), U87 (malignant glioma), SJSA-1 (osteosarcoma), 4T1 (mouse breast cancer), A549 (non-small cell lung cancer), A375 (malignant melanoma), and HeLa (cervical cancer).

[0038] Table 3. Antiproliferative activity of cationic cyclic peptides

[0039] As shown in Table 3, the cationic cyclic peptides provided by this invention have good anti-tumor cell proliferation activity against a variety of tumor cell lines and are expected to be used as anti-tumor drugs in the clinical treatment of cancer.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cationic cyclic chelated polypeptide, characterized in that, The amino terminus of the cationic cyclic peptide is acetylated, and the carboxyl terminus is amidated. Its structural formula is as follows: wherein Ac is acetyl; Pep1, Pep2 and Pep3 are polypeptide sequences; K m represents an alpha-methyl substituted lysine, and the side chain amino groups of the two alpha-methyl substituted lysine residues form a cyclic cross-linking structure via a 1,2-dimethylene phenyl group.

2. The cationic cyclic steric polypeptide according to claim 1, characterized in that, The cationic cyclic peptide consists of 21 amino acid residues.

3. The cationic cyclic skewered polypeptide according to claim 1 or 2, characterized in that, The amino acid sequence of the cationic cyclic stapled polypeptide is: XKFFKKLKKAVKK m GFKK m FAKV, wherein the first X is (S)-2,3-diaminopropionic acid.

4. The cationic cyclic peptide according to claim 1 or 2, characterized in that, The amino acid sequence of the cationic cyclic peptide is: ZKFFKKLKKAVKK m GFKK m FAKV, where the first Z is (S)-2,4-diaminobutyric acid.

5. The cationic cyclic skewered polypeptide according to claim 1 or 2, characterized in that, The amino acid sequence of the cationic cyclic peptide is: OKFFKKLKKAVKK m GFKK m FAKV, where the first O is ornithine.

6. The cationic cyclic skewing polypeptide according to claim 1 or 2, characterized in that, The amino acid sequence of the cationic cyclic peptide is: KKFFKKLKKAVKK m GFKK m FAKV, where the first K is lysine.

7. A pharmaceutical composition, characterized in that, Includes the cationic cyclic skewer polypeptide of any one of claims 1-6 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier thereof.

8. The use of the cationic cyclic peptide according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of antibacterial or antitumor drugs.

9. The application according to claim 8, characterized in that, Bacteria are bacteria.

10. The application according to claim 8, characterized in that, The tumors include breast cancer, colon cancer, hepatoblastoma, malignant glioma, osteosarcoma, lung cancer, malignant melanoma, and cervical cancer.

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