Glycosylated bicyclic stapled peptides and their applications

Abstract

The present invention belongs to the field of polypeptide drugs, and specifically relates to a glycosylated bicyclic stapled peptide that can improve the anti-drug-resistant bacteria activity, improve the serum stability, and has good enzymatic hydrolysis stability on the basis of a template polypeptide, and its application. The present invention modifies the amino acid sequence of the template polypeptide SLP-0: Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH2 to obtain a glycosylated bicyclic stapled peptide. Compared with the template polypeptide SLP-0, its antibacterial effect is significantly improved, especially the antibacterial performance against Klebsiella pneumoniae (KP) is increased by 31 times compared with the template peptide SLP-0. Among them, SLP-51 has significant stability against trypsin, chymotrypsin and serum.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide drugs, and particularly relates to a glycosylated bicyclic stapled peptide which can improve the anti-drug-resistant bacteria activity, improve the serum stability and has good enzymatic stability on the basis of a template polypeptide, and an application thereof. Background Art

[0002] The abuse 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 drug-resistant strains, such as Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, etc., can form a biofilm on their own surface. Bacteria encapsulated by the biofilm are 10 to 1000 times more tolerant to conventional antibiotics than other bacteria, resulting in certain limitations of clinically available antibiotics in combating drug-resistant bacteria. In recent years, the development of antibacterial drugs has been slow, and some marketed antibiotic drugs have shown drug-resistant bacteria in a short time. Therefore, 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. Patients with low immune function are particularly susceptible to Klebsiella pneumoniae infection. Therefore, Klebsiella pneumoniae infection has become one of the main causes of nosocomial infections. Klebsiella can cause various forms of infection, including pneumonia, blood infection, and wound and postoperative infection. At the same time, it is also the main force of drug-resistant bacteria, and antibiotics are often ineffective in treatment. Therefore, solving 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 various pathways, such as bacterial membrane lysis, oxidative damage, inhibition of biofilm formation, etc. They do not involve the binding of specific proteins and are not easily resistant to drugs. They can fight against bacterial infections ineffective against traditional antibiotics. Therefore, AMPs have the potential to be a new generation of antibiotics for treating drug-resistant bacterial infectious diseases. However, most AMPs are linear polypeptides, which are easily degraded by proteases in vivo and have unstable structures, greatly limiting their clinical applications. Previous studies have made some progress in improving the structural stability of polypeptides by chemically modifying AMPs, such as changing the backbone, replacing D-type and non-natural amino acids, etc. However, in the past few decades, the structural stability of AMPs has still been an important factor hindering the clinical application of such drugs. Therefore, there is an urgent need 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 developed an antibacterial peptide SAAP-148 (SLP-0) based on human LL-37. It contains 24 residues and has an α-helical structure. Compared with the model peptide LL-37, it exhibits higher resistance to drug-resistant bacteria in vitro and in vivo. Although SLP-0 has high resistance to drug-resistant bacteria, as a linear polypeptide, it is easily enzymatically degraded 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. In the present invention, Rink amide MBHA amino resin is used as a solid-phase carrier, and based on the amino acid sequence of the template polypeptide SLP-0: Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH2, modifications are made. On the basis of retaining key amino acid residues, S5 is used to replace the original amino acids at the i,i+4 amino acid positions, and a total of 13 stapled peptides (SLP-1 - SLP-13) are synthesized. The screened polypeptides have improved antibacterial activity and can produce inhibitory positive regulation on harmful drug-resistant bacteria. However, the antibacterial activity of SLP-1 - SLP-13 stapled peptides against drug-resistant bacteria is limited, and high concentrations are required to inhibit Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae, which hinders their drug development. Moreover, SLP-1 - SLP-13 has good antibacterial effects on some bacteria among Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii, but most hospital infections are mixed infections of multi-drug-resistant bacteria, and being effective only against some bacteria means that it needs to be used in combination with other anti-drug-resistant bacteria drugs, affecting its scope of use. Summary of the Invention

[0006] The object 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, this glycosylated bicyclic stapled peptide has improved antibacterial activity, improved serum stability, good enzymatic stability, better comprehensive performance, and is more conducive to drug formation.

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

[0008] To achieve the above first object, the technical solution adopted by the present invention is:

[0009] A stapled peptide, which is selected from one of the following:

[0010] a) Using Ac-LKRVWKRVFKLLKRYWRQLKKPVR-NH2 as a peptide chain template, wherein amino acid residues 1L and 5W are replaced by K, 8V, 12L, 14R, and 18Q are replaced by S5 and cyclized, and then a glycosyl Glc is connected to the C-terminal Arg.

[0011] b) Using Ac-LKRVWKRVCHLLKRYWRQLKKPVR-NH2 as the peptide chain template, where amino acid residues 1L and 5W are replaced by K, 8V, 12L, 14R, and 18Q are replaced by S5 and cyclized, and then the glycosyl Lac is linked to the C-terminal Arg;

[0012] c) Using Ac-LKRVWKRVCHLLKRYWRQLKKPVR-NH2 as the peptide chain template, where amino acid residues 1L and 5W are replaced by K, 8V, 12L, 14R, and 18Q are replaced by S5 and cyclized, and then the glycosyl Mal is linked to the C-terminal Arg;

[0013] The sequences of the modified glycosylated bicyclic stapled peptides in the present invention are as follows in the table:

[0014] Table 1 Sequences of the glycosylated bicyclic stapled peptides and template polypeptides in the present invention

[0015]

[0016] To achieve the second above-mentioned purpose, the technical solution adopted by the present invention is:

[0017] The above-mentioned glycosylated bicyclic stapled peptide is used in the preparation of antibacterial drugs.

[0018] Preferably, the bacterium is Streptococcus pneumoniae (SP).

[0019] Preferably, the bacterium is Staphylococcus aureus (MRSA).

[0020] Preferably, the bacterium is Pseudomonas aeruginosa (PA).

[0021] Preferably, the bacterium is Klebsiella pneumoniae (KP).

[0022] In the present invention, the abbreviations involved are explained as follows:

[0023] Fmoc: 9-fluorenylmethoxycarbonyl;

[0024] DCM: Dichloromethane

[0025] DCE: 1,2-Dichloroethane

[0026] DMF: N,N-Dimethylformamide

[0027] Oxyme: Ethyl 2-oximinoacetate

[0028] DIC: N,N-Diisopropylcarbodiimide

[0029] S5: (2R)-2-N-Fmoc-amino-2-methyl-6-heptenoic acid

[0030] EDT: 1,2-Ethanedithiol

[0031] TFA: Trifluoroacetic acid

[0032] Grubbs Ⅰ: Benzylidene bis(tricyclohexylphosphine) ruthenium dichloride

[0033] MS: Mass spectrometry

[0034] HR-Q-TOF-MS: High resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometry

[0035] Glc: N-acetylglucosamine

[0036] Lac: Lactose

[0037] Mal: Maltotriose

[0038] MRSA: Methicillin-resistant Staphylococcus aureus

[0039] KP: Klebsiella pneumoniae

[0040] Streptococcus pneumoniae: Streptococcus pneumoniae

[0041] P. aeruginosa: Pseudomonas aeruginosa

[0042] The advantages of the present invention are as follows:

[0043] 1. By structurally modifying SAAP-148 (SLP-0), a new series of glycosylated bicyclic stapled peptides are obtained in the present invention. The antibacterial experiment results show that, compared with the template polypeptide, the antibacterial properties of the three glycosylated stapled peptides obtained in the present invention are significantly improved. The three stapled peptides all 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 properties of the three stapled peptides against Staphylococcus aureus (MRSA) are improved by about 5 times compared with the template peptide SLP-0, and the antibacterial properties against Klebsiella pneumoniae (KP) are improved by 31 times compared with the template peptide SLP-0; the antibacterial properties of the stapled peptide SLP-50 against Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) are improved by 1 time compared with the template peptide SLP-0; the antibacterial properties of the stapled peptide SLP-51 against Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) are improved by 2 times compared with the template peptide SLP-0; the antibacterial properties of the stapled peptide SLP-52 against Streptococcus pneumoniae (SP) are improved by 1 time compared with the template peptide SLP-0, and the antibacterial properties against Pseudomonas aeruginosa (PA) are improved by 2 times compared with the template peptide SLP-0. Among the three stapled peptides, the glycosylated bicyclic stapled peptide SLP-51 shows the most prominent anti-drug-resistant bacteria effect and has potential application value in the treatment of clinical drug-resistant bacteria infections and other related diseases.

[0044] 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 trypsin treatment. This indicates that the stability of the modified glycosylated bicyclic stapled peptide SLP-51 against trypsin has been greatly improved. SLP-51 was not degraded under chymotrypsin exposure and remained 100% present, indicating that the modified glycosylated bicyclic stapled polypeptide SLP-51 no longer has chymotrypsin cleavage sites in the polypeptide sequence, and its stability against chymotrypsin has been greatly enhanced.

[0045] 3. The glycosylated stapled peptide SLP-51 with the best activity and enzyme stability and the linear peptide SLP-0 were selected for the evaluation of serum stability. It was found that SLP-0 was completely degraded at 24 hours, while 11.36% of the peptides of the glycosylated bicyclic stapled peptide remained intact at 72 hours, showing very good serum stability. The experiment shows that the stability of the glycosylated bicyclic stapled peptide SLP-51 is significantly improved compared to the template peptide SLP-0. Brief Description of the Drawings

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments and comparative examples. It should be understood that the following shows the result comparison and method verification of the embodiments and comparative examples of the present invention.

[0047] Figure 1 It is the synthetic route diagram of the sugar intermediate of the present invention.

[0048] Figure 2 It is the synthetic route diagram of the glycosylated bicyclic stapled peptide of the present invention

[0049] Figure 3 It is the schematic diagram of the amino acid sequence of SLP-50 and its characterization map. SLP-50 was purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes). SLP-50 was obtained. Among them, A is the amino acid sequence of SLP-50, B is the HPLC map of SLP-50, analysis 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.

[0050] Figure 4Schematic diagram of the SLP-51 amino acid sequence and its characterization map. SLP-51 was purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes). SLP-51 was obtained. Among them, A is the amino acid sequence of SLP-51, B is the HPLC map 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.

[0051] Figure 5 Schematic diagram of the SLP-52 amino acid sequence and its characterization map. SLP-52 was purified by HPLC (purification conditions: 10%-55% CH3CN (0.1% TFA) in H2O (0.1% TFA), on a Welch C18 column for 55 minutes). SLP-52 was obtained. Among them, A is the amino acid sequence of SLP-52, B is the HPLC map 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.

[0052] Figure 6 Trypsin degradation kinetic curves of SLP-0 and SLP-51.

[0053] Figure 7 Chymotrypsin degradation kinetic curves of SLP-0 and SLP-51.

[0054] Figure 8 Comparison chart of serum stability between template peptide SLP-0 and glycosylated stapled peptide SLP-51. Detailed implementation manners

[0055] The following will further illustrate the present application in conjunction with the accompanying drawings and specific implementation manners, 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 not to limit the scope of the present invention, that is, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0056] Therefore, the following detailed description of some embodiments of the present invention provided is not intended to limit the scope of the claimed invention, but is merely selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present invention.

[0057] The present invention designs and synthesizes 3 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 as shown in the attached Figure 3-5 shown.

[0058] The sources of the experimental materials involved in the examples of the present invention are as follows:

[0059] Fmoc-amino acids and Rink amide MBHA amino resin were purchased from Nankai Synthesis Co., Ltd.; NMP, DIC, Oxyme, TFA, and acetonitrile (chromatographically pure) were purchased from the Exploration Platform; DMF, anhydrous ether, DCM, DCE, piperidine, and phenol were all of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai

[0060] Example 1 Preparation of Glycosylated Bicyclic Stapled Peptide Based on SAAP-148 (SLP-0)

[0061] 1. Synthesis of Bicyclic Stapled Peptide

[0062] (1) Swelling

[0063] Take 400 mg of amino resin (loading capacity is 0.30 mmol·g -1 ) and add it to the solid-phase synthesis reaction tube. Soak the resin in DCM for 30 min to fully swell the resin, and drain it for later use.

[0064] (2) Deprotection

[0065] Add 7 ml of 20% piperidine-DMF solution to the resin, and shake it at 35 °C for 5 min × 2 to remove the Fmoc protecting group on the resin. Wash the resin with DMF, DCM, and DMF 3 times each in turn.

[0066] (3) Amino Acid Condensation Reaction

[0067] Mix the first amino acid (1 mmol) in the sequence, Oxyme (142 mg, 1 mmol), and DIC (200 μL) in 7 ml of DMF, add it to the resin, and shake it at 60 °C for 20 min (the reaction time for the amino acid after S5 is 1 h, and the reaction is repeated once). Wash the resin with DMF, DCM, and DMF 3 times each in turn.

[0068] (4) Blocking

[0069] After the reaction of the first amino acid, acetylation blocking is carried out. Use a 10 ml solution with a volume ratio of N,N-diisopropylethylamine (DIPEA): acetic anhydride: DMF = 1:1:8 and shake it in a shaker at 37 °C for 5 + 10 minutes.

[0070] (5)Synthetic polypeptide amino acid sequence

[0071] Repeat the procedures in steps (2) and (3). According to the polypeptide sequence, dissolve Fmoc amino acid (1 mmol), Oxyme (142 mg), and DIC (200 μl) in 7 ml of DMF in sequence, then add them to the resin, and react with shaking at 60 °C for 30 min. Repeat the process of removing Fmoc protection → condensation → removing Fmoc protection until all amino acids are condensed completely.

[0072] (6)The first cyclization of the polypeptide

[0073] After condensing to the first group of special unnatural amino acid S5, perform the first cyclization of the polypeptide. Use a DCE solution of the first-generation Grubbs reagent to react at 37 °C for 2 h to carry out olefin metathesis reaction (RCM) to cyclize the two special unnatural amino acids.

[0074] (7)The second cyclization of the polypeptide

[0075] Repeat step (5) to continue synthesizing the peptide chain according to the amino acid sequence until the second group of unnatural amino acid S5 residues are assembled completely; use a DCE solution of the first-generation Grubbs reagent to react at 37 °C for 2 h to carry out olefin metathesis reaction (RCM) to cyclize the two special unnatural amino acids.

[0076] (8)Acetylation

[0077] After all the amino acids in the sequence are synthesized, use a DMF solution containing 20% piperidine to remove the Fmoc protecting group of the last amino acid, and then carry out acetylation blocking. Use a 10 mL solution with a volume ratio of DIPEA: acetic anhydride: DMF = 1:1:8 to shake in a shaker at 37 °C for 5 + 10 minutes. Wash the resin with DMF, DCM, and DMF in sequence.

[0078] (9)Cleavage

[0079] After synthesizing the polypeptide, use 20 mL of K reagent (volume ratio of trifluoroacetic acid: water: 1,2-ethanedithiol: anisole: phenol = 82.5: 5: 2.5: 5: 5) to cleave the polypeptide on the resin, and cleave at 37 °C for 3 h. Blow with nitrogen, place the reaction solution in a centrifuge tube, and use nitrogen to bubble and concentrate it to about 5 - 7 mL.

[0080] (10)Precipitation

[0081] Add 20 mL of ice-cold diethyl ether for precipitation, centrifuge at 3500 r / min for 3 min, repeat three times to obtain the precipitate, discard the supernatant, and air-dry to obtain the crude polypeptide.

[0082] 2. Synthesis of the sugar donor (see the roadmap in Figure 1 )

[0083] (1) Synthesis of the crude chlorosugar-based product 2

[0084] Taking the synthesis of monosaccharide glycosylation as an example for illustration. Using β-D-glucosamine 1 as the starting material, it first reacts with acetyl chloride for 48 h to obtain the crude chloroglycoside product 2, which is directly used in the next reaction without purification.

[0085] (2) Synthesis of the isothiocyanate sugar-based compound 3

[0086] Compound 2, 0.02 mmol of tetrabutylammonium iodide, 0.04 mmol of potassium thiocyanate, and a certain amount of molecular sieve are added to a round-bottom flask. Under the protection of N2, the mixture is refluxed in acetonitrile for 3 h to introduce an isothiocyanate group at the anomeric position of the sugar. After filtration and concentration, the crude product of isothiocyanate sugar-based 3 is obtained.

[0087] (3) Synthesis of the final product sugar intermediate

[0088] 5.2 mmol of compound 3, 5.2 mmol of Pbf-NH2, and 5.0 mmol of potassium tert-butoxide are reacted in a round-bottom flask containing tetrahydrofuran. The reaction solution is adjusted to pH = 7, the reaction solution is removed, and the crude product is dried. Then the crude product is 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 processes of other sugar intermediates (5, 6) are the same as that of intermediate 4.

[0089] 3. Synthesis of the glycosylated bicyclic stapled peptide

[0090] Before the cleavage step in the above bicyclic stapled peptide process, 3 eq of the sugar intermediate and 3 eq of silver nitrate are weighed and dissolved in 6 mL of anhydrous DMF. Ultrasonic treatment is used to completely dissolve it. Finally, 10 eq of triethylamine solution is added. After mixing evenly, it is quickly poured into a peptide synthesis tube and reacted with shaking at 35 °C overnight. This process needs to be protected from light throughout. In this reaction, under the catalysis of silver nitrate, the guanidination reaction between the polypeptide and the sugar intermediate occurs on the solid phase to obtain the glycopeptide. Then the crude polypeptide is obtained by cleavage using the K reagent.

[0091] 4. Removal of the acetyl group of the sugar donor

[0092] First, dissolve the crude polypeptide in 15 mL of anhydrous methanol. Then, dissolve 150 μL of sodium methoxide in 1.5 mL of anhydrous methanol. Slowly add the prepared methanol / sodium methoxide solution to the methanol solution of the polypeptide and stir for 30 min to remove the acetyl group on the glycosyl donor under the alkaline condition of methanol / sodium methoxide. Then, use acetic acid for acid-base neutralization to adjust the pH value to 7.0. Centrifuge the reaction solution, take the supernatant, and perform rotary evaporation using a rotary evaporator to finally obtain a dry crude polypeptide product.

[0093] 5. Purification of the stapled peptide sample

[0094] Dissolve the crude polypeptide in a mixed solvent of acetonitrile and water, and purify it by reverse-phase preparative RP-HPLC to obtain a purified stapled peptide pure product. The separation conditions are as follows:

[0095] Instrument: Shimadzu LC-20A reverse-phase high-performance liquid chromatograph;

[0096] Chromatographic column: Ultimate XB-C18, 21.2×250 mm, 5 μm;

[0097] Mobile phase: Mobile phase A is an 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;

[0098] Steps and parameters: Elute with 90% B for 3 min, and elute with 90% B to 50% B for 40 min; the flow rate is 8 ml / min, the injection volume is 3 ml, and the detection wavelengths are 214 nm and 254 nm.

[0099] Identification and structural analysis of the product of Example 2

[0100] Identify the product obtained in Step 5 of Example 1 by reverse-phase HPLC and perform structural analysis by HR-Q-TOF-MS. The chromatographic mobile phase is acetonitrile and water. Mobile phase A is an 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. Gradient elution (0 - 2 min, mobile phase B: 90%; 3 - 25 min, mobile phase B: 90% - 10%); flow rate 1.0 mL·min -1 ; The detection wavelengths are 214 nm and 254 nm, and the injection volume is 24 μl. It is determined that the retention time is consistent with that of the crude product main peak, and the purity of the stapled peptide prepared by this method > 95%. The mass spectrometry analysis results are as Figures 3-5 shown. After analysis, the structure of the obtained stapled peptide is shown in Table 1.

[0101] Example 3 Antibacterial experiment of the glycosylated bicyclic stapled peptide of the present invention

[0102] In vitro anti-drug-resistant bacteria test: Prepare solid LB medium, plate it after autoclaving, and prepare LB liquid medium for standby in a 4°C refrigerator. Spread the bacterial solution on the solid LB medium and incubate it upside down in a 37°C incubator overnight; pick a monoclonal colony, add it to 3 mL of liquid LB medium, and culture it at 37°C and 220 rpm in a constant temperature shaker for 6 h to grow the bacteria to the logarithmic phase; take 1 mL of the bacterial solution, centrifuge it at 4000 rpm for 5 min, discard the supernatant, add PBS, and adjust the concentration of the bacterial solution to 2×106 CFU / mL by OD value. Add antibacterial peptides with different concentrations to a 96-well plate, and at the same time add the bacterial solution to the 96-well plate, incubate at 37°C for 8 h, detect it with an ELISA reader at 595 nm, repeat three times, and statistically analyze the MIC value.

[0103] The results are shown in Table 2.

[0104] Table 2 Glycosylated bicyclic stapled peptide sequences and antibacterial activities of the present invention

[0105]

[0106] Table 2 shows that the antibacterial performance of the three glycosylated stapled peptides obtained in the present invention is significantly improved compared with the template polypeptide. The three glycosylated stapled peptides obtained in the present invention show good in vitro antibacterial performance 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) is about 5 times higher than that of the template peptide SLP-0, and the antibacterial performance against Klebsiella pneumoniae (KP) is 31 times higher than that of the template peptide SLP-0; the antibacterial performance of the stapled peptide SLP-50 against Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) is 1 time higher than that of the template peptide SLP-0; the antibacterial performance of the stapled peptide SLP-51 against Streptococcus pneumoniae (SP) and Pseudomonas aeruginosa (PA) is 2 times higher than that of the template peptide SLP-0; the antibacterial performance of the stapled peptide SLP-52 against Streptococcus pneumoniae (SP) is 1 time higher than that of the template peptide SLP-0, and the antibacterial performance against Pseudomonas aeruginosa (PA) is 2 times higher than that of the template peptide SLP-0. Among the three stapled peptides, the glycosylated bicyclic stapled peptide SLP-51 has the most prominent anti-drug-resistant bacteria effect.

[0107] The above examples show that the present invention successfully prepares glycosylated bicyclic stapled peptides based on SAAP-148 (SLP-0). Through in vitro antibacterial experiments, it is proved that the synthesized glycosylated bicyclic stapled peptide SLP-51 can significantly inhibit the growth and reproduction of pathogenic drug-resistant bacteria, and has the application prospect of developing into a new antibacterial drug.

[0108] Example 4 Enzymatic hydrolysis and serum stability experiments of the stapled peptides of the present invention

[0109] 1. Determination method and results of trypsinolytic stability

[0110] (1)Preparation of buffer solution: Weigh 11.1 mg of anhydrous calcium chloride and dissolve it in PBS solution (50 mM, pH = 7.4). After complete dissolution, the final concentration is 2 mM.

[0111] (2)Preparation of trypsin solution: Weigh a small amount of trypsin and dissolve it in the prepared buffer solution to make the final concentration 0.01 ng / μL.

[0112] (3)Preparation of polypeptide solution: Weigh 1 mg of template peptide SLP-0 and the most active stapled peptide derivative in each round of modification respectively, and add them into the buffer solution to make the final concentration 1 mM.

[0113] (4)Reaction and sampling: Place 1950 μL of trypsin solution in a 5 mL centrifuge tube. Then, take 50 μL of the prepared 1 mM polypeptide solution and add it to the centrifuge tube containing trypsin solution for trypsin degradation experiment. At time points of 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, etc. during the experiment, take 60 μL of the reaction mixture solution and add 20 μL of concentrated hydrochloric acid (1M) to quench and terminate the enzymatic hydrolysis reaction.

[0114] (5)Result analysis: Take 50 μL for HPLC analysis. Determine the residual amount of the polypeptide by integrating the peak area of the polypeptide, calculate the percentage of the peak area at each moment to the peak area at 0 moment and draw a curve, then the analysis chart of the chymotrypsin hydrolysis stability of the polypeptide can be obtained.

[0115] Trypsin mainly cleaves the peptide bond at the carboxyl terminus of arginine or lysine, and then can specifically shear the peptide. The experimental results show that 26.65% of the stapled peptide of the modified glycosylated bicyclic stapled peptide SLP-51 remains intact after 3 h of trypsin treatment. This indicates that the stability of the modified glycosylated bicyclic stapled peptide SLP-51 against trypsin is greatly improved.

[0116] Table 3 Trypsin stability data of SLP-0

[0117]

[0118] Table 4 Trypsin stability data of SLP-51

[0119] 2. Determination method and results of chymotrypsinolytic stability

[0120] (1) Preparation of buffer solution: Weigh 11.1 mg of anhydrous calcium chloride and dissolve it in PBS solution (50 mM, pH = 7.4). Dissolve it thoroughly to make its final concentration 2 mM.

[0121] (2) Preparation of chymotrypsin solution: Weigh a trace amount of chymotrypsin and dissolve it in the prepared buffer solution to make its final concentration 0.01 ng / μL.

[0122] (3) Preparation of polypeptide solution: Weigh 1 mg of template peptide SLP-0 and the most active stapled peptide derivative from each round of modification respectively, and add them to the buffer solution to make its final concentration 1 mM.

[0123] (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 polypeptide solution and add it to the centrifuge tube containing chymotrypsin solution for trypsin degradation experiment. Take 60 μL of the reaction mixture at time points such as 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min during the experiment, and add 20 μL of concentrated hydrochloric acid (1M) to quench and terminate the enzymatic hydrolysis reaction.

[0124] (5) Result analysis: Take 50 μL for HPLC analysis. Determine the residual amount of the polypeptide by integrating the peak area of the polypeptide, calculate the percentage of the peak area at each moment to the peak area at 0 moment and draw a curve, then the analysis chart of the chymotrypsin hydrolysis stability of the polypeptide can be obtained.

[0125] The principle of chymotrypsin degrading peptides 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 the exposure of chymotrypsin and remained 100% present. The experimental results show that after the modification of the glycosylated bicyclic stapled polypeptide SLP-51, there is no cleavage site of chymotrypsin in the polypeptide sequence, and its stability to chymotrypsin is greatly improved.

[0126] Table 5 Chymotrypsin hydrolysis data of SLP-0

[0127]

[0128] Table 6 Chymotrypsin hydrolysis data of SLP-51

[0129] 3. Serum stability determination method and its results

[0130] (1) Preparation of polypeptide solution: First, dissolve the polypeptide powder in water to prepare a solution with a concentration of 2 mg / mL.

[0131] (2) Preparation of reaction solution: Mix serum and polypeptide solution in a ratio of 4:1.

[0132] (3) Reaction sampling: Place the mixture in an incubator at 37 °C. Set the sampling intervals as 0 h / 12 h / 24 h / 36 h / 48 h / 60 h / 72 h. Take 50 μL of the mixture each time, and then add 50 μL of acetonitrile to terminate the reaction.

[0133] (4) Result analysis: Centrifuge the mixed solution at 10000 rpm for 10 min and take the supernatant. Perform HPLC analysis on 50 μL of the supernatant at a wavelength of 220 nm. Determine the residual amount of the polypeptide by integrating the peak area of the polypeptide, calculate the percentage of the peak area at each moment to the peak area at 0 h, and draw a curve to obtain the analysis chart of the serum stability of the polypeptide.

[0134] Select the glycosylated stapled peptide SLP-51 with the best activity and good enzyme stability and the linear peptide SLP-0 for the evaluation of serum stability. It is found that SLP-0 is completely degraded at 24 h, while 11.36% of the peptide of the glycosylated bicyclic stapled peptide remains intact at 72 h, showing very good serum stability. The experiment shows that the glycosylated bicyclic stapled peptide SLP-51 has a significantly improved stability compared to the template peptide SLP-0.

[0135] Table 7 Serum stability data of SLP-0

[0136]

[0137] Table 8 Serum stability data of SLP-51

[0138] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A glycosylated bicyclic stapled peptide, characterized in that: The stapled 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; Wherein, the S5 is (2R)-2-N-fluorenylmethoxycarbonylamino-2-methyl-6-heptenoic acid; The 8V, 12L, 14R, and 18Q are replaced by S5 and cyclized, and the 8V and 12L are replaced by S5 and cyclized, and the 14R and 18Q are replaced by S5 and cyclized.

2. The use of the glycosylated bicyclic stapled peptide according to claim 1 in the preparation of antibacterial drugs, characterized in that: The bacteria is Streptococcus pneumoniae.

3. The use of the glycosylated bicyclic stapled peptide according to claim 1 in the preparation of antibacterial drugs, characterized in that: The bacteria is Staphylococcus aureus.

4. The use of the glycosylated bicyclic stapled peptide according to claim 1 in the preparation of antibacterial drugs, characterized in that: The bacterium is Pseudomonas aeruginosa.

5. Use of the glycosylated bicyclic stapled peptide according to claim 1 in the preparation of antibacterial drugs, characterized in that: The bacteria is Klebsiella pneumoniae.

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