Glycosylated bicyclic stapled peptides and uses thereof

Abstract

The invention belongs to the field of polypeptide drugs, and particularly relates to a glycosylated double-ring stapled peptide which can improve drug-resistant bacterium resistance activity and serum stability on the basis of template polypeptide and is good in enzymolysis stability and application. The amino acid sequence of the template polypeptide SLP-0: Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH2 is modified and transformed to obtain the glycosylated double-ring stapled peptide, compared with the template polypeptide SLP-0, the antibacterial effect of the glycosylated double-ring stapled peptide is remarkably improved, and particularly, the antibacterial performance of the glycosylated double-ring stapled peptide on klebsiella pneumoniae (KP) is improved by 31 times compared with that of the template peptide SLP-0. Wherein the SLP-51 has remarkable stability on trypsin, chymotrypsin and serum.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide drugs, and specifically relates to a glycosylated bicyclic stapled peptide and its application which can improve the activity against drug-resistant bacteria, improve serum stability and have good enzymatic stability based on a template polypeptide. Background Art

[0002] The misuse of antibiotics has led to the emergence of drug-resistant strains, and infectious diseases caused by drug-resistant strains have become a serious threat to global public health. Clinically resistant strains, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, can form a biofilm on their own surface. The tolerance of bacteria wrapped in biofilms to conventional antibiotics is 10 to 1000 times that of other bacteria, resulting in certain limitations of clinically available antibiotics in combating drug-resistant bacteria. In recent years, the development of antimicrobial drugs has been slow, and some marketed antibiotics have developed drug-resistant bacteria in a relatively short period of time. For this reason, there is an urgent need to find new therapeutic drugs to deal with infectious diseases caused by drug-resistant bacteria.

[0003] Klebsiella pneumoniae is a facultative anaerobic Gram-negative rod-shaped bacterium. Immunocompromised patients are particularly susceptible to Klebsiella pneumoniae infection, so Klebsiella pneumoniae infection has become one of the main causes of hospital-acquired infections. Klebsiella pneumoniae can cause a variety of infections, including pneumonia, bloodstream infections, and wound and postoperative infections. At the same time, it is the main force of drug-resistant bacteria, and antibiotics are often powerless in treatment. Therefore, solving the problem of drug-resistant Klebsiella pneumoniae has become an urgent problem to be solved.

[0004] Antimicrobial peptides (AMPs) are a class of host defense peptides that can kill bacteria through a variety of pathways, such as biofilm lysis, oxidative damage, and inhibition of biofilm formation. They do not involve binding to specific proteins, are not prone to drug resistance, and can fight bacterial infections that are ineffective with traditional antibiotics. Therefore, AMPs have the potential to be a new generation of antibiotics for the treatment of drug-resistant bacterial infections. However, most AMPs are linear peptides that are easily degraded by proteases in the body and have unstable structures, which greatly limits their clinical application. Previous researchers have improved the structural stability of peptides and made certain progress by chemically modifying AMPs, such as skeleton changes, D-type, and non-natural amino acid substitutions. However, in the past few decades, the structural stability of AMPs is still an important factor that hinders the clinical application of this type of drug. Therefore, it is urgent to find more effective strategies to modify AMPs to better solve the problem of drug-resistant bacterial infections.

[0005] LL-37 is one of the main human AMPs and plays an important role in defending against local and systemic infections. In 2018, the Nibbering group modified the antimicrobial peptide SAAP-148 (SLP-0) based on human LL-37. It contains 24 residues and has an alpha-helical structure. Compared with the model peptide LL-37, it exhibits a higher ability to resist drug-resistant bacteria in vivo and in vitro. Although SLP-0 has a high ability to resist drug-resistant bacteria, it is a straight-chain polypeptide that is easily enzymatically hydrolyzed and has an unstable structure, which is not conducive to its development as a drug. Patent CN116655766A discloses a stapled peptide and its preparation method and application. The present invention uses Rink amide MBHA amino resin was used as a solid phase carrier to modify the amino acid sequence of the template polypeptide SLP‑0:Ac‑LKRVWKRVFKLLKRYWRQLKKPVR‑NH2. On the basis of retaining the key amino acid residues, S5 was used to replace the original amino acid at the i,i+4 amino acid position, and a total of 13 stapled peptides (SLP-1—SLP-13) were synthesized. The screened peptides have improved antibacterial activity and can produce inhibitory positive regulation on harmful resistant bacteria; however, the anti-resistant bacteria activity of SLP-1-SLP-13 stapled peptides is limited, and the inhibition of Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae requires a higher concentration to be achieved, which hinders its drug development; and SLP-1-SLP-13 has a good antibacterial effect on some bacteria in Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii, while most nosocomial infections are mixed infections with multidrug-resistant bacteria. If it is only effective against some bacteria, it means that it must be used in combination with other anti-resistant bacteria drugs, which affects its scope of use. Summary of the invention

[0006] The purpose of the present invention is to provide a glycosylated bicyclic stapled peptide in view of the deficiencies in the prior art. Compared with the template polypeptide, the glycosylated bicyclic stapled peptide has improved antibacterial activity, serum stability, and enzymatic stability, and has better comprehensive performance and is more conducive to drug development.

[0007] Another object of the present invention is to provide the use of the glycosylated bicyclic stapled peptide.

[0008] To achieve the above first purpose, the technical solution adopted by the present invention is: A stapler peptide, wherein the stapler peptide is selected from one of the following: a) Ac- LKRVWKRVFKLLKRYWRQLKKPVR-NH2 was used as a peptide chain template, wherein amino acid residues 1L and 5W were replaced by K, 8V, 12L, 14R, and 18Q were replaced by S5 and cyclized, and then the sugar group Glc was connected to the C-terminal Arg; b) Ac- LKRVWKRVFKLLKRYWRQLKKPVR -NH2 was used as a peptide chain template, wherein amino acid residues 1L and 5W were replaced by K, 8V, 12L, 14R, and 18Q were replaced by S5 and cyclized, and then the sugar group Lac was connected to the C-terminal Arg; c) Ac- LKRVWKRVFKLLKRYWRQLKKPVR -NH2 was used as a peptide chain template, wherein amino acid residues 1L and 5W were replaced by K, 8V, 12L, 14R, and 18Q were replaced by S5 and cyclized, and then the sugar group Mal was connected to its C-terminal Arg; The sequence of the modified glycosylated bicyclic stapled peptide in the present invention is as follows: Table 1 Sequences of glycosylated bicyclic stapled peptides and template polypeptides in the present invention To achieve the above second purpose, the technical solution adopted by the present invention is: The use of the above-mentioned glycosylated bicyclic stapled peptide in the preparation of antibacterial drugs.

[0009] Preferably, the bacteria is Streptococcus pneumoniae (SP).

[0010] Preferably, the bacteria is Staphylococcus aureus (MRSA).

[0011] Preferably, the bacteria is Pseudomonas aeruginosa (PA).

[0012] Preferably, the bacteria is Klebsiella pneumoniae (KP).

[0013] In the present invention, the abbreviations involved are explained as follows: Fmoc: fluorenylmethoxycarbonyl; DCM: dichloromethane DCE: 1,2-dichloroethane DMF: N,N-dimethylformamide Oxyme: Ethyl 2-oxime cyanoacetate DIC: N,N-diisopropylcarbodiimide S5: (2R)-2-N-Fluorenylmethoxycarbonylamino-2-methyl-6-heptenoic acid EDT: 1,2-ethanedithiol TFA: trifluoroacetic acid Grubbs Ⅰ: phenylmethylenebis(tricyclohexylphosphine)ruthenium dichloride MS: Mass spectrometry HR-Q-TOF-MS: high-resolution matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.

[0014] Glc: acetylglucosamine Lac: Lactose Mal: Maltotriose MRSA: Staphylococcus aureus KP: Klebsiella pneumoniae Streptococcus pneumoniae: Streptococcus pneumoniae P. aeruginosa: Pseudomonas aeruginosa The advantages of the present invention are: 1. The present invention obtains a new series of glycosylated bicyclic stapled peptides by structural modification of SAAP-148 (SLP-0). The antibacterial experimental results show that the three glycosylated stapled peptides obtained by the present invention have significantly improved antibacterial properties compared with the template polypeptide. The three stapled peptides show good in vitro antibacterial properties against four common drug-resistant bacteria, namely Staphylococcus aureus (MRSA), Klebsiella pneumoniae (KP), Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA). Among them, the antibacterial performance of the three stapled peptides against Staphylococcus aureus (MRSA) was improved by about 5 times compared with the template peptide SLP-0, and the antibacterial performance against Klebsiella pneumoniae (KP) was improved by 31 times compared with the template peptide SLP-0; the antibacterial performance of the stapled peptide SLP-50 against Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) was improved by 1 times compared with the template peptide SLP-0; the antibacterial performance of the stapled peptide SLP-51 against Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) was improved by 2 times compared with the template peptide SLP-0; the antibacterial performance of the stapled peptide SLP-52 against Streptococcus pneumoniae (SP) was improved by 1 times compared with the template peptide SLP-0, and the antibacterial performance against Pseudomonas aeruginosa (PA) was improved by 2 times compared with the template peptide SLP-0. Among the three stapled peptides, the glycosylated bicyclic stapled peptide SLP-51 has the most outstanding anti-resistant bacteria effect. It has potential application value in the treatment of clinical drug-resistant bacterial infections and other related diseases.

[0015] 2. The results of the enzymatic hydrolysis experiment showed that 26.65% of the stapled peptides of SLP-51 remained intact after 3 hours of trypsinization, which indicates that the stability of the modified glycosylated bicyclic stapled peptide SLP-51 to trypsin is greatly improved. SLP-51 was not degraded under the exposure of chymotrypsin and maintained 100% existence, indicating that the modified glycosylated bicyclic stapled peptide SLP-51 no longer has a chymotrypsin cleavage site in the polypeptide sequence, and its stability to chymotrypsin is greatly improved.

[0016] 3. The glycosylated stapled peptide SLP-51 with the best activity and enzyme stability and the linear peptide SLP-0 were selected for serum stability evaluation. It was found that SLP-0 was completely degraded at 24 hours, while 11.36% of the glycosylated bicyclic stapled peptide remained intact at 72 hours, showing very good serum stability. The experiment showed that the glycosylated bicyclic stapled peptide SLP-51 had significantly improved stability compared with the template peptide SLP-0. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the illustrations required in the embodiments and comparative examples. It should be understood that the following shows the comparison of results and method verification presentation of the embodiments of the present invention and comparative examples.

[0018] Figure 1 The figure is a synthetic route diagram of the sugar intermediate of the present invention.

[0019] Figure 2 The synthetic route of the glycosylated bicyclic stapled peptide of the present invention is Figure 3 The figure is a schematic diagram of the amino acid sequence of SLP-50 and its characterization spectrum. SLP-50 was purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA), 55 minutes on a Welch C18 column). SLP-50 was obtained. A is the amino acid sequence of SLP-50, B is the HPLC spectrum of SLP-50, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm). C is the mass spectrum of SLP-50.

[0020] Figure 4 The figure is a schematic diagram of the amino acid sequence of SLP-51 and its characterization spectrum. SLP-51 was purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA), 55 minutes on a Welch C18 column). SLP-51 was obtained. A is the amino acid sequence of SLP-51, B is the HPLC spectrum of SLP-51, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm). C is the mass spectrum of SLP-51.

[0021] Figure 5The figure is a schematic diagram of the amino acid sequence of SLP-52 and its characterization spectrum. SLP-52 was purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA), 55 minutes on a Welch C18 column). SLP-52 was obtained. A is the amino acid sequence of SLP-52, B is the HPLC spectrum of SLP-52, analytical column: Welch C18, gradient: 10%-90% CH3CN (0.1% TFA) in H2O (0.1% TFA), 25 minutes (λ=214 nm). C is the mass spectrum of SLP-52.

[0022] Figure 6 This is the kinetic curve of trypsin degradation of SLP-0 and SLP-51.

[0023] Figure 7 This is the kinetic curve of chymotrypsin degradation of SLP-0 and SLP-51.

[0024] Figure 8 Comparison of serum stability of template peptide SLP-0 and glycosylated stapled peptide SLP-51. DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with the accompanying drawings and specific implementation methods so that those skilled in the art can better understand the present application. However, these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. That is, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0026] Therefore, the following detailed description of some embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but is only a selected embodiment of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0027] The present invention designs and synthesizes three glycosylated bicyclic stapled peptides according to the amino acid sequence of the template polypeptide SLP-0: Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH2. The structure of the glycosylated bicyclic stapled peptide is shown in the attached manual. Figure 3-5 shown.

[0028] The sources of the experimental materials involved in the embodiments of the present invention are as follows: Fmoc-amino acid and Rink amide MBHA amino resin were purchased from Nankai Synthesis Co., Ltd.; NMP, DIC, Oxyme, TFA, and acetonitrile (chromatographic grade) were purchased from the exploration platform; DMF, anhydrous ether, DCM, DCE, piperidine, and phenol were all analytically pure and purchased from Sinopharm Chemical Reagent Shanghai Co., Ltd. Example 1 Preparation of glycosylated bicyclic stapled peptide based on SAAP-148 (SLP-0) 1. Synthesis of bicyclic stapled peptides (1) Swelling Take 400 mg of amino resin (loaded with 0.30 mmol g -1 ) was added into the solid phase synthesis reaction tube, soaked in DCM for 30 min to allow the resin to fully swell, and then drained for later use.

[0029] (2) Deprotection Add 7 ml of 20% piperidine-DMF solution to the resin, shake at 35°C for 5 min × 2 to remove the Fmoc protecting group on the resin, and wash the resin with DMF, DCM, and DMF three times each.

[0030] (3) Amino acid condensation reaction The first amino acid in the sequence (1 mmol), Oxyme (142 mg, 1 mmol) and DIC (200 μL) were mixed in 7 ml of DMF, added to the resin and shaken at 60 °C for 20 min (one amino acid after S5 was reacted for 1 h, and the reaction was repeated once), and the resin was washed 3 times with DMF, DCM and DMF respectively.

[0031] (4) Closed After the first amino acid reaction, acetylation blocking was performed using 10 ml of a solution with a volume ratio of N,N-diisopropylethylamine (DIPEA): acetic anhydride: DMF = 1:1:8 and shaking at 37 °C in a shaker for 5+10 minutes.

[0032] (5) Synthetic peptide amino acid sequence Repeat steps (2) and (3). Dissolve Fmoc amino acid (1 mmol), Oxyme (142 mg) and DIC (200 μl) in 7 ml DMF according to the peptide sequence, then add them to the resin and shake the reaction at 60 °C for 30 min. Repeat the process of deprotection → condensation → deprotection until all amino acids are condensed.

[0033] (6) The first cyclization of the peptide After condensation to the first group of special non-natural amino acids S5, the first cyclization of the peptide was carried out, and the two special non-natural amino acids were cyclized by olefin metathesis reaction (RCM) using a DCE solution of the first-generation Grubbs reagent at 37 °C for 2 h.

[0034] (7) Second cyclization of peptide Repeat step (5) to continue synthesizing the peptide chain according to the amino acid sequence until the second group of non-natural amino acid S5 residues are assembled; use the first-generation Grubbs reagent DCE solution at 37 ° C for 2 h to carry out olefin metathesis reaction (RCM) to cyclize the two special non-natural amino acids.

[0035] (8) Acetylation After the synthesis of all amino acids in the sequence was completed, the Fmoc protecting group of the last amino acid was removed using a DMF solution containing 20% ​​piperidine, followed by acetylation blocking, and 10 mL of a solution with a volume ratio of DIPEA: acetic anhydride: DMF = 1:1:8 was shaken in a shaker at 37 °C for 5+10 minutes. The resin was washed with DMF, DCM, and DMF in sequence.

[0036] (9) Cutting After the peptide was synthesized, 20 mL of K reagent (volume ratio of trifluoroacetic acid: water: 1, 2-ethanedithiol: thioanisole: phenol = 82.5: 5: 2.5: 5: 5) was used to cut the peptide on the resin at 37 °C for 3 h. Nitrogen was blown, and the reaction solution was placed in a centrifuge tube and concentrated by bubbling with nitrogen to about 5-7 mL.

[0037] (10) Sedimentation Add 20 mL of ice ether for precipitation, centrifuge at 3500 r / min for 3 min, repeat three times to obtain a precipitate, discard the supernatant, and air dry to obtain a crude polypeptide.

[0038] 2. Synthesis of sugar donors (see the roadmap) Figure 1 ) (1) Synthesis of crude chloroglycoside product 2 The synthesis of monosaccharide glycosylation is used as an example. β-D-acetylglucosamine 1 is used as the starting material and first reacted with acetyl chloride for 48 h to obtain the crude chloroglycoside product 2, which is directly used in the next step without purification.

[0039] (2) Synthesis of isothiocyanate sugar compound 3 Compound 2, 0.02 mmol of tetrabutylammonium iodide, 0.04 mmol of potassium thiocyanate and a certain amount of molecular sieves were added to a round-bottom flask. Under N2 protection conditions, the reaction was refluxed in acetonitrile for 3 h to introduce an isothiocyanate group at the end of the sugar. The crude product of isothiocyanate glycosyl 3 was obtained by filtration and concentration.

[0040] (3) Synthesis of final product sugar intermediates 5.2 mmol of compound 3 was reacted with 5.2 mmol of Pbf-NH2 and 5.0 mmol of potassium tert-butoxide in a round-bottom flask containing tetrahydrofuran. The reaction solution was adjusted to pH=7, the reaction solution was removed and the crude product was dried. The crude product was then reacted with 25 mmol of anhydrous potassium carbonate and 10 mmol of iodoethane under anhydrous conditions for 16 h to obtain the final product 4 sugar intermediate. The synthesis process of other sugar intermediates (5, 6) was the same as that of intermediate 4.

[0041] 3. Synthesis of glycosylated bicyclic stapled peptides Before the cleavage step of the above-mentioned bicyclic stapled peptide process, 3 eq of sugar intermediate and 3 eq of silver nitrate were weighed, dissolved in 6 mL of anhydrous DMF, and completely dissolved by ultrasound. Finally, 10 eq of triethylamine solution was added, mixed evenly, and quickly poured into the peptide synthesis tube. The reaction was shaken at 35 °C overnight. This process needs to be protected from light throughout. In this reaction, the peptide and the sugar intermediate were guanidinated on the solid phase under the catalysis of silver nitrate to obtain a glycopeptide. Then, the crude peptide was cut using K reagent.

[0042] 4. Removal of acetyl group from glycosyl donor First, dissolve the crude polypeptide in 15 mL of anhydrous methanol, then take 150 μL of sodium methoxide and dissolve it in 1.5 mL of anhydrous methanol, slowly add the prepared methanol / sodium methoxide solution to the polypeptide methanol solution and stir for 30 min to remove the acetyl group on the glycosyl donor under the alkaline conditions of methanol / sodium methoxide. Then use acetic acid for acid-base neutralization and adjust the pH value to 7.0. Centrifuge the reaction solution, take the supernatant and use a rotary evaporator to perform rotary evaporation to finally obtain a dry polypeptide crude product.

[0043] 5. Purification of stapled peptide samples The crude peptide was dissolved in a mixed solvent of acetonitrile and water and purified by reverse phase preparative RP-HPLC to obtain the purified stapled peptide pure product. The separation conditions were as follows: Instrument: Shimadzu LC-20A reversed-phase high performance liquid chromatograph; Chromatographic column: UltimateXB-C18, 21.2×250mm, 5μm; Mobile phase: Mobile phase A is acetonitrile solution with a volume fraction of 0.1% TFA, and mobile phase B is an aqueous solution with a volume fraction of 0.1% TFA; Steps and parameters: 90% B elution for 3 min, 90% B~50% B elution for 40 min; flow rate of 8 ml / min, injection volume of 3 ml, detection wavelengths of 214 nm and 254 nm.

[0044] Example 2 Identification and structural analysis of the product The product obtained in step 5 of Example 1 was identified by reverse phase HPLC and structurally analyzed by HR-Q-TOF-MS. The chromatographic mobile phases were acetonitrile and water. Mobile phase A was an acetonitrile solution with a volume fraction of 0.1% TFA, and mobile phase B was an aqueous solution with a volume fraction of 0.1% TFA. The gradient elution (0 to 2 min, mobile phase B: 90%; 3-25 min, mobile phase B: 90% to 10%) was performed; the flow rate was 1.0 mL·min -1 ; Detection wavelengths were 214 nm and 254 nm, and the injection volume was 24 μl. The peak time was consistent with that of the crude product, and the purity of the stapled peptide prepared by this method was >95%. The results of mass spectrometry analysis were as follows Figure 3-Figure 5 After analysis, the obtained stapled peptide structure is shown in Table 1.

[0045] Example 3 Antibacterial Experiment of Glycosylated Bicyclic Stapled Peptide of the Present Invention In vitro anti-resistant bacteria test: Prepare solid LB medium, sterilize by high pressure, plate and prepare LB liquid medium, and keep in a refrigerator at 4℃ for later use. Spread the bacterial solution on solid LB medium and invert and culture overnight in a 37℃ incubator; take a single clone, add it to 3 mL of liquid LB medium, and culture it in a constant temperature shaker at 37℃, 220 rpm for 6 h to allow the bacteria to grow to the logarithmic phase; take 1 mL of bacterial solution, centrifuge at 4000 rpm for 5 min, discard the supernatant, add PBS, and adjust the bacterial solution concentration to 2×106CFU / mL by OD value. Add different concentrations of antimicrobial peptides to a 96-well plate, and add the bacterial solution to a 96-well plate at the same time, culture at 37℃ for 8 h, and detect it at 595 nm with an enzyme reader. Repeat three times, and statistically analyze the MIC value.

[0046] The results are shown in Table 2.

[0047] Table 2 Glycosylated bicyclic stapled peptide sequences and antibacterial activity of the present invention The results in Table 2 show that the antibacterial properties of the three glycosylated stapled peptides obtained in the present invention are significantly improved compared with the template polypeptide. The antibacterial properties of the three glycosylated stapled peptides obtained in the present invention are significantly improved compared with the template polypeptide. The three stapled peptides showed good in vitro antibacterial properties against four common drug-resistant bacteria, namely Staphylococcus aureus (MRSA), Klebsiella pneumoniae (KP), Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA). Among them, the antibacterial performance of the three stapled peptides against Staphylococcus aureus (MRSA) was improved by about 5 times compared with the template peptide SLP-0, and the antibacterial performance against Klebsiella pneumoniae (KP) was improved by 31 times compared with the template peptide SLP-0; the antibacterial performance of the stapled peptide SLP-50 against Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) was improved by 1 times compared with the template peptide SLP-0; the antibacterial performance of the stapled peptide SLP-51 against Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) was improved by 2 times compared with the template peptide SLP-0; the antibacterial performance of the stapled peptide SLP-52 against Streptococcus pneumoniae (SP) was improved by 1 times compared with the template peptide SLP-0, and the antibacterial performance against Pseudomonas aeruginosa (PA) was improved by 2 times compared with the template peptide SLP-0. Among the three stapled peptides, the glycosylated bicyclic stapled peptide SLP-51 had the most outstanding anti-resistant bacteria effect.

[0048] The above examples show that the present invention successfully prepared a glycosylated bicyclic stapled peptide based on SAAP-148 (SLP-0). Through in vitro antibacterial experiments, it was proved that the synthesized glycosylated bicyclic stapled peptide SLP-51 can significantly inhibit the growth and reproduction of pathogenic and drug-resistant bacteria, and has the application prospect of being developed into a new type of antibacterial drug.

[0049] Example 4 Enzymatic hydrolysis and serum stability test of the stapled peptide of the present invention 1. Method and results of trypsin hydrolysis stability test (1) Preparation of buffer solution: Weigh 11.1 mg of anhydrous calcium chloride and dissolve it in PBS solution (50 mM, pH = 7.4) until it is fully dissolved to a final concentration of 2 mM.

[0050] (2) Preparation of trypsin solution: Weigh a small amount of trypsin and dissolve it in the buffer solution prepared above to a final concentration of 0.01 ng / μL.

[0051] (3) Preparation of peptide solution: Weigh 1 mg of the template peptide SLP-0 and the stapled peptide derivative with the best activity in each round of modification, respectively, and add them to the buffer solution to make the final solubility of 1 mM.

[0052] (4) Reaction and sampling: Place 1950 μL of trypsin solution in a 5 mL centrifuge tube, and then take 50 μL of the prepared 1 mM peptide solution and add it to the centrifuge tube containing the trypsin solution to perform the trypsin degradation experiment. Take 60 μL of the reaction mixture at 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min and other time points during the experiment, and add 20 μL of concentrated hydrochloric acid (1M) to quench and terminate the enzymatic reaction.

[0053] (5) Result analysis: Take 50 μL for HPLC analysis. Determine the residual amount of the peptide by integrating the peak area of ​​the peptide. Calculate the percentage of the peak area at each time point to the peak area at time 0 and draw a curve to obtain an analysis chart of the peptide's anti-chymotrypsin hydrolysis stability.

[0054] The main cleavage site of trypsin is the peptide bond at the carboxyl terminal of arginine or lysine, and then it can specifically cleave the peptide. The experimental results showed that 26.65% of the modified glycosylated bicyclic stapled peptide SLP-51 remained intact after 3 hours of trypsin. This shows that the stability of the modified glycosylated bicyclic stapled peptide SLP-51 to trypsin is greatly improved.

[0055] Table 3 SLP-0 trypsin stability data Table 4 SLP-51 trypsin stability data 2. Chymotrypsin hydrolysis stability determination method and results (1) Preparation of buffer solution: Weigh 11.1 mg of anhydrous calcium chloride and dissolve it in PBS solution (50 mM, pH = 7.4) until it is fully dissolved to a final concentration of 2 mM.

[0056] (2) Preparation of chymotrypsin solution: Weigh a small amount of chymotrypsin and dissolve it in the buffer solution prepared above to a final concentration of 0.01 ng / μL. (3) Preparation of peptide solution: Weigh 1 mg of the template peptide SLP-0 and the stapled peptide derivative with the best activity in each round of modification, respectively, and add them to the buffer solution to make the final solubility of 1 mM.

[0057] (4) Reaction and sampling: Place 1950 μL of chymotrypsin solution in a 5 mL centrifuge tube, and then take 50 μL of the prepared 1 mM peptide solution and add it to the centrifuge tube containing the chymotrypsin solution to perform the trypsin degradation experiment. Take 60 μL of the reaction mixture at 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min and other time points during the experiment, and add 20 μL of concentrated hydrochloric acid (1M) to quench and terminate the enzymatic reaction.

[0058] (5) Result analysis: Take 50 μL for HPLC analysis. Determine the residual amount of the peptide by integrating the peak area of ​​the peptide. Calculate the percentage of the peak area at each time point to the peak area at time 0 and draw a curve to obtain an analysis chart of the peptide's anti-chymotrypsin hydrolysis stability.

[0059] The principle of peptide degradation by chymotrypsin is that the main cleavage points of chymotrypsin are tryptophan, tyrosine, phenylalanine and threonine. The glycosylated bicyclic stapled peptide SLP-51 was not degraded under chymotrypsin exposure and maintained 100% existence. The experimental results show that the modified glycosylated bicyclic stapled peptide SLP-51 no longer has chymotrypsin cleavage sites in the polypeptide sequence, and its stability to chymotrypsin is greatly improved.

[0060] Table 5 SLP-0 chymotrypsin hydrolysis data Table 6 SLP-51 chymotrypsin hydrolysis data 3. Serum stability determination method and results (1) Preparation of peptide solution: First, dissolve the peptide powder in water to prepare a solution with a concentration of 2 mg / mL.

[0061] (2) Preparation of reaction solution: Mix serum and polypeptide solution in a ratio of 4:1.

[0062] (3) Reaction sampling: The mixed solution was placed in a 37°C incubator for incubation. The sampling interval was set to 0 h / 12 ​​h / 24 h / 36 h / 48 h / 60 h / 72 h. 50 μL of the mixed solution was taken each time, and then 50 μL of acetonitrile was added to terminate the reaction.

[0063] (4) Result analysis: The mixed solution was centrifuged at 10,000 rpm for 10 min and the supernatant was collected. 50 μL of the supernatant was subjected to HPLC analysis at a wavelength of 220 nm. The residual amount of the peptide was determined by integrating the peak area of ​​the peptide, and the percentage of the peak area at each time to the peak area at time 0 was calculated and a curve was drawn to obtain an analysis chart of the stability of the peptide serum.

[0064] The glycosylated stapled peptide SLP-51 with the best activity and enzyme stability and the linear peptide SLP-0 were selected for serum stability evaluation. It was found that SLP-0 was completely degraded at 24 hours, while 11.36% of the glycosylated bicyclic stapled peptide remained intact at 72 hours, showing very good serum stability. The experiment showed that the glycosylated bicyclic stapled peptide SLP-51 had significantly improved stability compared to the template peptide SLP-0.

[0065] Table 7 SLP-0 serum stability data Table 8 SLP-51 serum stability data The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be regarded as the scope of protection of the present invention.

Claims

1. A glycosylated bicyclic stapled peptide, characterized in that: The stapled peptide is: a) Ac- LKRVWKRVFKLLKRYWRQLKKPVR-NH2 was used as a peptide chain template, wherein amino acid residues 1L and 5W were replaced by K, 8V, 12L, 14R, and 18Q were replaced by S5 and cyclized, and then the sugar group Glc was connected to the C-terminal Arg; b) Ac- LKRVWKRVFKLLKRYWRQLKKPVR -NH2 was used as a peptide chain template, wherein amino acid residues 1L and 5W were replaced by K, 8V, 12L, 14R, and 18Q were replaced by S5 and cyclized, and then the sugar group Lac was connected to the C-terminal Arg; c) Using Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH2 as the peptide chain template, amino acid residues 1L and 5W were replaced by K, 8V, 12L, 14R, and 18Q were replaced by S5 and cyclized, and then the sugar group Mal was connected to its C-terminal Arg.

2. Use of the glycosylated bicyclic stapled peptide according to claim 1 in the preparation of antibacterial drugs.

3. The use of the glycosylated bicyclic stapled peptide according to claim 2 in the preparation of antibacterial drugs, characterized in that: The bacteria is Streptococcus pneumoniae.

4. The use of the glycosylated bicyclic stapled peptide according to claim 2 in the preparation of antibacterial drugs, characterized in that: The bacteria is Staphylococcus aureus.

5. The use of the glycosylated bicyclic stapled peptide according to claim 2 in the preparation of antibacterial drugs, characterized in that: The bacterium is Pseudomonas aeruginosa.

6. Use of the glycosylated bicyclic stapled peptide according to claim 2 in the preparation of antibacterial drugs, characterized in that: The bacteria is Klebsiella pneumoniae.

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