Bee venom peptide derived alicyclic peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to melittin-derived alicyclic peptide, which is prepared by replacing 2-site isoleucine and 26-site glutamine in a melittin peptide chain with 2, 4-diaminobutyric acid (Dab), coupling butyric acid, hexanoic acid, octanoic acid or decanoic acid on 1-site glycine (Gly) at a nitrogen terminal, and performing dehydration condensation and cyclization on the 2-site Dab and the 26-site Dab. Compared with a parent melittin, the melittin-derived alicyclic peptide disclosed by the invention has a better inhibition effect on pet-derived gram positive and negative germs, has lower hemolysis and cytotoxicity, shows better stability in in-vitro pepsin and trypsin solutions, and can be used for preparing the melittin-derived alicyclic peptide. The compound can be used for preparing novel anti-bacterial infection medicines, feed additives, in-vitro sprays and the like for pets, and has a very wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biochemistry, and particularly relates to a melittin-derived aliphatic cyclic peptide as well as a preparation method and application thereof. BACKGROUND

[0002] With the development of social economy and the continuous improvement of people's living standards, pets play an increasingly important role in contemporary families and become an indispensable emotional partner for many families. However, while enjoying the spiritual comfort and emotional companionship brought by pets, their potential health risks cannot be ignored. The living habits and activity characteristics of pets make them extremely susceptible to contact and carry a variety of pathogenic microorganisms, including dozens of zoonotic pathogenic bacteria. Because of the frequent close contact between humans and pets, the risk of pathogen transmission through direct contact is greatly increased. More worrying is that when pets have bacterial infections, a large amount of antibiotics are often used for treatment, which not only affects the treatment effect, but also accelerates the generation of bacterial drug resistance, thereby causing a series of more serious public health and safety problems, posing a potential threat to human health and the ecological environment.

[0003] Antimicrobial peptides (AMPs) are a class of small molecule active peptides widely existing in nature, which are an important part of the innate immune system of organisms and play a key role in host defense system. They not only can effectively resist the invasion of foreign pathogenic microorganisms, but also show significant antibacterial activity against traditional antibiotic-resistant strains and are less likely to induce bacterial drug resistance. Among the many antimicrobial peptides, melittin exhibits broad-spectrum and high-efficiency antibacterial activity and has attracted much attention. However, although natural melittin has excellent antibacterial properties, it still faces many challenges in practical application. On the one hand, melittin is easily degraded by proteases in physiological environment and has poor stability; on the other hand, its strong hemolytic activity seriously limits the feasibility of clinical application. Therefore, it is of great significance to develop melittin derivatives with high antibacterial activity, good in-vivo and in-vitro stability, and low hemolytic activity through structural modification and modification strategies. SUMMARY

[0004] The application aims to provide a melittin-derived aliphatic cyclic peptide to solve the problems of instability of natural melittin in vivo and in vitro and strong hemolytic activity.

[0005] In order to achieve the above-mentioned purpose, the scheme of the application is as follows: a melittin-derived aliphatic cyclic peptide, the structure of the melittin-derived aliphatic cyclic peptide is as follows:

[0006] butyric acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab]

[0007] or, hexanoic acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab];

[0008] or, octanoic acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab];

[0009] or, decylic acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab].

[0010] The working principle and beneficial effects of the present scheme are that: the inventors have found through experiments that the melittin derivative aliphatic cyclic peptide obtained by replacing the 2nd isoleucine (Ile) and the 26th glutamine (Gln) in the peptide chain of melittin with 2,4-diaminobutyric acid (Dab), coupling butyric acid, hexanoic acid, octanoic acid or decylic acid on the 1st glycine (Gly) at the nitrogen end, and then dehydrating and condensing the 2nd Dab and the 26th Dab to form a ring has significantly higher pepsin stability, trypsin stability and antibacterial property than melittin, and its hemolytic property is lower than that of melittin. Among them, the melittin derivative aliphatic cyclic peptide coupled with octanoic acid has the most optimal stability and antibacterial effect, and its MIC value is 2-16 μg / mL.

[0011] The present application also provides a preparation method of the above-mentioned melittin derivative aliphatic cyclic peptide, comprising the following steps:

[0012] 1) Replacing the 2nd Ile and the 26th Gln at the carbon end of melittin with 2,4-diaminobutyric acid to obtain melittin derivative I, and the structure of the melittin derivative I is as follows: G-Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab;

[0013] 2) Coupling butyric acid, hexanoic acid, octanoic acid or decylic acid with the 1st Gly at the nitrogen end of the melittin derivative I to obtain melittin derivative II;

[0014] 3) Dehydrating and condensing the 2nd Dab and the 26th Dab of the melittin derivative II to form a ring to obtain the melittin derivative aliphatic cyclic peptide.

[0015] The scheme takes Rink-MBHA Resin as a starting material, adopts the Fmoc solid-phase synthesis method, introduces Dab at the corresponding positions of the natural melittin peptide segment according to the designed replacement site, further couples butyric acid, hexanoic acid, octanoic acid or decanoic acid on the Gly at the 1st position, and synthesizes the melittin derivative aliphatic cyclic peptide through the dehydration condensation of the Dabs at the 2nd and 26th positions, which has the advantages of mild reaction condition, few side reactions, high yield, and the Fmoc group itself has characteristic ultraviolet absorption, which is easy to monitor and control the reaction.

[0016] Optionally, the 2,4-diaminobutyric acid is D-type and / or L-type 2,4-diaminobutyric acid.

[0017] The application also provides application of the above-mentioned melittin derivative aliphatic cyclic peptide in preparation of a broad-spectrum pet antibacterial drug or antibacterial composition.

[0018] Optionally, the bacteria targeted by the broad-spectrum pet antibacterial drug or antibacterial composition include gram-positive bacteria and gram-negative bacteria.

[0019] Optionally, the bacteria include Escherichia coli, Pasteurella canis, Haemophilus felis, Campylobacter coli, Streptococcus canis and Staphylococcus pseudintermedius.

[0020] The application also provides application of the above-mentioned melittin derivative aliphatic cyclic peptide in preparation of a pet feed additive or pet feed additive composition.

[0021] The application also provides application of the above-mentioned melittin derivative aliphatic cyclic peptide in preparation of a pet eye drop.

[0022] The application also provides application of the above-mentioned melittin derivative aliphatic cyclic peptide in preparation of a pet external antibacterial spray. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a high-performance liquid chromatogram for detecting MEL-Dab in the embodiment 1 of the application;

[0024] Figure 2HPLC detection chart of cyclo-MEL-Dab in Example 2 of the present application;

[0025] Figure 3 HPLC detection chart of butyric acid-cyclo-MEL-Dab in Example 3 of the present application;

[0026] Figure 4 HPLC detection chart of hexanoic acid-cyclo-MEL-Dab in Example 4 of the present application;

[0027] Figure 5 HPLC detection chart of octanoic acid-cyclo-MEL-Dab in Example 5 of the present application;

[0028] Figure 6 HPLC detection chart of decylic acid-cyclo-MEL-Dab in Example 6 of the present application;

[0029] Figure 7 HPLC detection chart of MEL in Comparative Example 1 of the present application;

[0030] Figure 8 Hemolytic evaluation result chart of melittin-derived aliphatic cyclic peptide in Experimental Example 2 of the present application;

[0031] Figure 9 Cytotoxicity evaluation result chart of melittin-derived aliphatic cyclic peptide in Experimental Example 3 of the present application;

[0032] Figure 10 Pepsin solution stability evaluation result chart of melittin-derived aliphatic cyclic peptide in Experimental Example 4 of the present application;

[0033] Figure 11 Trypsin solution stability evaluation result chart of melittin-derived aliphatic cyclic peptide in Experimental Example 5 of the present application. DETAILED DESCRIPTION

[0034] The following will be further described in detail through specific embodiments. If not specifically indicated, the technical means used in the embodiments are the conventional means well known to those skilled in the art, and the raw materials used are all commercially available goods.

[0035] The experimental materials used in the following embodiments: the amino acids required for synthesis of melittin and its derived aliphatic cyclic peptides, trypsin, pepsin, and Triton X-100 enzyme were purchased from Sigma Aldrich (Shanghai) Trading Co., Ltd. Other conventional reagents were imported in small quantities or of domestic analytical purity. The sources of the antibacterial test strains are shown in Table 1.

[0036] Table 1 Strains and their sources

[0037] Strains Source Escherichia coli CVCC3034 Bought from CVCC Pasteurella canis CAU0082 Bought from CVCC Haemophilus felis BNCC364403 Bought from North Na Biological Campylobacter coli CICC23925 Bought from CICC Streptococcus canis ATCC43496 Bought from ATCC Staphylococcus pseudintermedius ATCC49444 Bought from ATCC

[0038] Synthesis of Melittin Derivative I (MEL-Dab)

[0039] Weigh 2.0 g of 2-chloro-trityl chloride resin (degree of substitution 0.4 mmol / g, the content of effective chlorine on the resin is 2.0 x 0.4 = 0.8 mmol), and add to the reaction column containing N,N-dimethylformamide (DMF) and stir for 1 hour to swell the resin completely. Remove the DMF, and add a DMF solution containing Fmoc-Gln-OH (2 x 0.8 mmol) and diisopropylethylamine (DIEA) (4 x 0.8 mmol) to the resin, and shake the reaction at room temperature for 2 hours. Remove the reaction solution, and wash the resin with DMF 3 times, then add a mixed solution containing excess methanol and DIEA (4 x 0.8 mmol) in DMF to the resin and continue stirring for 1 hour to block the active chlorine groups on the resin that are not involved in the amino acid coupling reaction. Wash the resin with DMF 3 times, add 15 mL of 20% piperidine / DMF solution (V / V) to the resin, and shake the reaction at room temperature for 10 minutes, repeat 2 times to remove the Fmoc protecting group on the terminal amino group. Wash the resin with DMF 4 more times. Dissolve the amino acid (3 x 0.8 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (4 x 0.8 mmol), 1-hydroxybenzotriazole (HOBt) (4 x 0.8 mmol), and DIEA (6 x 0.8 mmol) in DMF solution, activate the carboxyl group, and add to the resin, and shake the reaction at room temperature for 1.5 hours. After the reaction is complete, wash the resin with DMF 3 times, and use a ninhydrin solution in methanol (10 mg / mL) to verify that the amino group is not condensed completely. If the solution is blue or red, it indicates that the condensation is not complete, and the amino acid and activating agent need to be added again to repeat the condensation step; if the solution is bright yellow or unchanged, it indicates that the condensation is complete, the Fmoc protecting group is removed, and the designed Gly-Dab-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Dab (i.e., G-Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab) amino acid sequence continues to extend the peptide chain. The synthesized derivative is purified by preparative RP-HPLC to obtain the pure peptide MEL-Dab (i.e., melittin derivative I), and the RP-HPLC analysis spectrum of MEL-Dab is shown in Figure 1. Figure 1

[0040] Synthesis of Melittin Derivative Cyclopeptide (cyclo-MEL-Dab) ​

[0041] Weigh 2.0 g of 2-chloro-trityl chloride resin (degree of substitution 0.4 mmol / g, the content of effective chlorine on the resin is 2.0 x 0.4 = 0.8 mmol), and add to the reaction column containing N,N-dimethylformamide (DMF), and stir for 1 hour to swell the resin completely. Remove the DMF, and add a DMF solution containing Fmoc-Gln-OH (2 x 0.8 mmol) and diisopropylethylamine (DIEA) (4 x 0.8 mmol) to the resin, and oscillate the reaction at room temperature for 2 hours. Remove the reaction solution, and wash the resin with DMF 3 times, then add a mixed solution containing excess methanol and (4 x 0.8 mmol) DIEA in DMF, and continue to stir for 1 hour to block the active chlorine groups on the resin that are not involved in the amino acid coupling reaction. Wash the resin with DMF 3 times, add 15 mL of 20% piperidine / DMF solution (V / V) to the resin, and oscillate the reaction at room temperature for 10 minutes, and repeat 2 times to remove the Fmoc protecting group on the terminal amino group. Wash the resin with DMF 4 more times. Dissolve the amino acid (3 x 0.8 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (4 x 0.8 mmol), 1-hydroxybenzotriazole (HOBt) (4 x 0.8 mmol), and DIEA (6 x 0.8 mmol) in a DMF solution, activate the carboxyl group, and add to the resin, and oscillate the reaction at room temperature for 1.5 hours. After the reaction is complete, wash the resin with DMF 3 times, and use a ninhydrin solution in methanol (10 mg / mL) to verify that the amino group is not condensed completely. If the solution is blue or red, the condensation is not complete, and the amino acid and activating agent need to be added again to repeat the condensation step; if the solution is bright yellow or unchanged, the condensation is complete, the Fmoc protecting group is removed, and the designed Gly-Dab-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Dab amino acid sequence is used to continue to extend the peptide chain. The condensing agent HBTU is used to activate the carboxyl group of the synthesized peptide chain, and then DIEA is added to adjust the pH to 8-9, the linear peptide chain is dissolved in DMF, and is slowly added to a dilute system under vigorous stirring, the reaction temperature is 0-25 °C, and the time is 2-24 h. Acetic acid is added to terminate the reaction, and the dehydrated condensation and cyclization of the Dab at position 2 and the Dab at position 26 are completed to obtain the cyclic peptide. The synthesized cyclic peptide is purified by preparative RP-HPLC to obtain the pure cyclic peptide cyclo-MEL-Dab, and the RP-HPLC analysis spectrum of cyclo-MEL-Dab is as follows: Figure 2As shown, the structure of cyclo-MEL-Dab is: Gly-cyclo[Dab-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Dab] (i.e., G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab]).

[0042] Example 3 Synthesis of Melittin-Derived Aliphatic Cyclic Peptide (butyric acid-cyclo-MEL-Dab)

[0043] Take 2.0 g 2-chloro-trityl chloride resin (degree of substitution 0.4 mmol / g, the content of effective chlorine on the resin is 2.0 x 0.4 = 0.8 mmol), add to the reaction column containing N,N-dimethylformamide (DMF), and stir for 1 hour to fully swell the resin. Remove the DMF, add a DMF solution containing Fmoc-Gln-OH (2 x 0.8 mmol) and diisopropylethylamine (DIEA) (4 x 0.8 mmol) to the resin, and oscillate the reaction at room temperature for 2 hours. Remove the reaction solution, wash the resin with DMF 3 times, then add a mixed solution containing excess methanol and (4 x 0.8 mmol) DIEA in DMF, and continue stirring for 1 hour to close the active chlorine groups in the resin that are not involved in the amino acid coupling reaction. Wash the resin with DMF 3 times, add 15 mL of 20% piperidine / DMF solution (V / V) to the resin, oscillate the reaction at room temperature for 10 minutes, and repeat 2 times to remove the Fmoc protecting group on the terminal amino group. Wash the resin with DMF 4 more times. Dissolve the amino acid (3 x 0.8 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (4 x 0.8 mmol), 1-hydroxybenzotriazole (HOBt) (4 x 0.8 mmol), and DIEA (6 x 0.8 mmol) in a DMF solution, activate the carboxyl group, and add to the resin, oscillate the reaction at room temperature for 1.5 hours. After the reaction is complete, wash the resin with DMF 3 times, and use a ninhydrin methanol solution (10 mg / mL) to verify whether the amino group is completely condensed. If the solution is blue or red, it indicates that the condensation is not complete, and the amino acid and activating agent need to be added again to repeat the condensation step; if the solution is bright yellow or unchanged, it indicates that the condensation is complete, the Fmoc protecting group is removed, and the designed Gly-Dab-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Dab (i.e., G-Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab) amino acid sequence continues to extend the peptide chain, and finally butyric acid is coupled to the Gly of the peptide chain as the last amino acid. The synthesized lipopeptide chain uses the condensing agent HBTU to activate the carboxyl group, and then adds DIEA to adjust the pH to 8-9. The linear peptide chain is dissolved in DMF, slowly added to a vigorously stirred dilution system, the reaction temperature is 0-25°C, and the time is 2-24 h. Add acetic acid to terminate the reaction, complete the dehydration condensation of 2-position Dab and 26-position Dab, complete the dehydration condensation of 2-position Dab and 26-position Dab, and obtain the alicyclic peptide. The synthesized alicyclic peptide is purified by preparative RP-HPLC to obtain the pure alicyclic peptide butyric acid-cyclo-MEL-Dab.The RP-HPLC analysis chart of butyric acid-cyclo-MEL-Dab is shown in Figure 1. Figure 3 As shown in Figure 1, the structure of butyric acid-cyclo-MEL-Dab is: butyric acid-Gly-cyclo[Dab-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Dab] (i.e. butyric acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab]).

[0044] Example 4 Synthesis of melittin-derived aliphatic cyclic peptide (hexanoic acid-cyclo-MEL-Dab)

[0045] The difference between this example and Example 3 is that hexanoic acid is used instead of butyric acid in Example 3, and hexanoic acid-cyclo-MEL-Dab is synthesized. The RP-HPLC analysis chart of hexanoic acid-cyclo-MEL-Dab is shown in Figure 2. Figure 4 As shown in Figure 2, the structure of hexanoic acid-cyclo-MEL-Dab is: hexanoic acid-Gly-cyclo[Dab-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Dab] (i.e. hexanoic acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab]).

[0046] Example 5 Synthesis of melittin-derived aliphatic cyclic peptide (octanoic acid-cyclo-MEL-Dab)

[0047] The difference between this example and Example 3 is that octanoic acid is used instead of butyric acid in Example 3, and octanoic acid-cyclo-MEL-Dab is synthesized. The RP-HPLC analysis chart of octanoic acid-cyclo-MEL-Dab is shown in Figure 3. Figure 5The structure of octanoic acid-cyclo-MEL-Dab is shown as follows: octanoic acid-Gly-cyclo[Dab-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Dab] (i.e. octanoic acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab]).

[0048] Example 6 Synthesis of decylic acid-cyclo-MEL-Dab

[0049] The difference between this example and Example 3 is that this example uses decylic acid instead of butyric acid in Example 3, and decylic acid-cyclo-MEL-Dab is synthesized. The RP-HPLC analysis spectrum of decylic acid-cyclo-MEL-Dab is shown in Figure 6. Figure 6 The structure of decylic acid-cyclo-MEL-Dab is shown as follows: decylic acid-Gly-cyclo[Dab-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Dab] (i.e. decylic acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab]).

[0050] Comparative Example 1 Synthesis of melittin (MEL)

[0051] Example 1-6 and Comparative Example 1 are shown in Table 2. Figure 7

[0052] Example 1-6 and Comparative Example 1 are shown in Table 2.

[0053] Example 1-6 and Comparative Example 1 are shown in Table 2.

[0054]

[0055] Example 1-6 and Comparative Example 1 are shown in Table 2. Figure 1-Figure 7 ​It can be seen that the products obtained in Examples 1-6 and Comparative Example 1, after purification, all have a purity >90%.

[0056] Example 1: Evaluation of the antibacterial properties of bee venom peptide-derived lipocyclic peptides

[0057] Antimicrobial assays were performed using a broth-based microdilution method, in accordance with the CLSI Guidelines for Antimicrobial Susceptibility Testing. *Escherichia coli* CVCC 3034, *Pasteurella canis* CAU0082, *Haemophilus felis* BNCC 364403, *Campylobacter coli* CICC 23925, *Streptococcus canis* ATCC 43496, and *Staphylococcus pseudintermedius* ATCC 49444 were cultured in broth and grown to the logarithmic growth phase. The bacterial suspensions were then diluted with broth to a concentration of 10. 5 ~10 6 CFU / mL. Meliostepin and its derivative lipocyclic peptide were dissolved in sterile broth to obtain a stock solution with a concentration of 512 μg / mL. Test concentrations were prepared by two-fold dilution. 50 μL of each of the continuously two-fold diluted meliostepin and its derivative lipocyclic peptide solutions, along with 50 μL of indicator bacterial dilution, were added to sterile 96-well plates, with three replicates for each concentration. The plates were incubated at 37°C for 18 h. Solution clarity was observed visually; if indicator bacteria were present, the solution would become turbid. The concentration of meliostepin and its derivative lipocyclic peptide corresponding to the clear well at the boundary between turbidity and clarity was the minimum inhibitory concentration (MIC). Sterile MH broth was used as a negative control, and polymyxin and vancomycin were used as positive controls. The results are shown in Table 3.

[0058] Table 3. MIC values ​​of meliostepin and meliostepin-derived lipocyclic peptides

[0059]

[0060] As shown in Table 3, vancomycin has a significant antibacterial effect against Gram-positive bacteria (MIC value of 1-2 μg / mL), but its antibacterial effect against Gram-negative bacteria is not obvious (MIC value of 64-512 μg / mL); colistin sulfate has the opposite antibacterial effect, with a significant antibacterial effect against Gram-negative bacteria (MIC value of 1-8 μg / mL). Neither of them has broad-spectrum antibacterial activity.

[0061] The lipocyclic peptide derived from melittin exhibits broad-spectrum antibacterial activity. Its antibacterial effect against Gram-negative bacteria such as Escherichia coli, Pasteurella canis, Haemophilus felis, and Campylobacter coli (MIC values ​​of 2–32 μg / mL) is superior to that of the parent melittin (MIC values ​​of 32–64 μg / mL). Its antibacterial effect against Gram-positive bacteria such as Streptococcus canis and Staphylococcus pseudintermedius (MIC values ​​of 8–32 μg / mL) is significantly superior to that of the parent melittin (MIC values ​​of 32–128 μg / mL). This indicates that the bee venom peptide-derived lipocyclic peptide in this invention significantly enhances the antibacterial effect of the polypeptide, and has good antibacterial effects against both Gram-positive and Gram-negative bacteria. In particular, octanoic acid-cyclo-MEL-Dab shows the best broad-spectrum antibacterial activity (MIC value of 2-16 μg / mL), making it suitable for preparing novel antibacterial drugs for pets and possessing very broad application prospects.

[0062] Experimental Example 2: Evaluation of the hemolytic activity of bee venom peptide-derived lipocyclic peptides

[0063] Rabbit erythrocytes were isolated from rabbit blood and washed 4–5 times with phosphate-buffered saline (PBS, pH 7.4). The washed erythrocytes were dispersed in PBS to prepare a 0.25% (v / v) erythrocyte suspension. Meliostein and its derivative lipocyclic peptide were dissolved in PBS and diluted to a stock solution of 512 μg / mL. The stock solution was then diluted 2-fold with PBS to obtain the working solution. Equal volumes of erythrocyte suspension and different concentrations of meliostein and its derivative lipocyclic peptide working solutions were added to 96-well plates. Triton X-100 solution (1%, v / v 20 μL) was used as a positive control, and the erythrocyte PBS solution served as a negative control. The 96-well plates were incubated at 37°C for 1 hour, then centrifuged at 3000 rpm at 4°C for 10 minutes. The supernatant was carefully transferred to new wells, and the absorbance was measured at 490 nm. The hemolysis rate was calculated using the following formula:

[0064]

[0065] All experiments were repeated three times, and the results were as follows: Figure 8 As shown.

[0066] Bee venom peptides themselves exhibit high hemolytic activity; the hemolytic activity of MEL obtained in Comparative Example 1 was 99.3%, which is unfavorable for the clinical application of bee venom peptides. Cyclation of bee venom peptides alters the peptide chain conformation, making it difficult for bee venom peptide-derived cyclic peptides to bind to the lipid membrane of erythrocytes, thereby reducing hemolytic activity. Experimental results showed that within a concentration range of 1–512 μg / mL, the hemolytic activity of the bee venom peptide parent (MEL) ranged from 11.41% to 99.3%, while the hemolytic activity of bee venom peptide-derived lipocyclic peptides was 2.68%–36.2%, hexanoic acid-cyclo-MEL-Dab was 0%–4.66%, octanoic acid-cyclo-MEL-Dab was 0%–1.69%, and decylic acid-cyclo-MEL-Dab was 0%–1.86%. This indicates that bee venom peptide-derived lipocyclic peptides have lower hemolytic activity and broad application prospects.

[0067] Experimental Example 3: Evaluation of the cytotoxicity of bee venom peptide-derived lipocyclic peptides

[0068] The cytotoxicity of melittin and its derivative lipocyclic peptides to eukaryotic cells was determined using the Cell Proliferation and Toxicity Assay (CCK-8). The specific steps were as follows: Resuscitated mouse mononuclear macrophages (RAW 264.7) were transferred to 5 mL of DMEM containing 10% fetal bovine serum and cultured overnight at 37°C with 5% CO2. After discarding the culture medium, 2 mL of 0.2% trypsin was added for 2 min of digestion, followed by the addition of 3 mL of cell culture medium to form a single-cell suspension. This suspension was then added to 96-well plates at 50 μL per well. After cell adhesion, 50 μL of melittin and its derivative lipocyclic peptide solutions with the same final MIC concentration gradient were added to wells 1–10. After 6 h of incubation, 10 μL of CCK-8 solution was added, and the cells were incubated for another 1–4 h. The OD value was measured at 450 nm using a microplate reader, and cell viability was calculated. The results are shown below. Figure 9 As shown.

[0069] Meliostepin itself exhibits high cytotoxicity; the MEL obtained in Comparative Example 1 showed a high cytotoxicity of 99.12%, which is unfavorable for the clinical application of meliostepin. Experimental results showed that within a concentration range of 1–512 μg / mL, the cytotoxicity of meliostepin-derived lipocyclic peptides on mouse monocytes and macrophages ranged from 10.08% to 99.12%. In contrast, the cytotoxicity of meliostepin-derived lipocyclic peptides, such as butyric acid-cyclo-MEL-Dab, was 3.35–33.98%, hexanoic acid-cyclo-MEL-Dab from 0–8.31%, octanoic acid-cyclo-MEL-Dab from 0–3.09%, and decylic acid-cyclo-MEL-Dab from 0–4.76%. This indicates that meliostepin-derived lipocyclic peptides possess lower cytotoxicity and have broad application prospects.

[0070] Example 4: Stability evaluation of melitoxic peptide-derived lipocyclic peptides in pepsin solution

[0071] The stability of melittin and its derived lipocyclic peptides in pepsin solution was determined by high performance liquid chromatography (HPLC). According to the Pharmacopoeia of the People's Republic of China (2020 edition), 16.4 mL of dilute hydrochloric acid was mixed with approximately 800 mL of water and 10 g of pepsin, then transferred to a 1000 mL volumetric flask and diluted to the mark with ultrapure water to obtain artificial gastric fluid. The melittin and its derived lipocyclic peptides were dissolved and diluted with the artificial gastric fluid to a concentration of 200 μg / mL, and placed in a 37℃ water bath. Samples were taken at 0, 0.5, 1, 2, 3, 4, 5, and 6 h. After inactivating the protease by bathing the samples in an 85℃ water bath for 5 min, they were centrifuged at 10000 rpm and 4℃ for 15 min. The peak areas of melittin and its derived lipocyclic peptides at different time points were detected, and the degradation rate was calculated. The results are shown below. Figure 10 As shown.

[0072] from Figure 10 The study revealed that melitoxic peptides and their derived lipocyclic peptides exhibited degradation rates ranging from 43.70% to 96.99% after incubation in pepsin solution for 0, 0.5, 1, 2, 3, 4, 5, and 6 hours. In comparison, butyric acid-cyclo-MEL-Dab showed degradation rates ranging from 0% to 2.82%, hexanoic acid-cyclo-MEL-Dab from 0% to 1.20%, octanoic acid-cyclo-MEL-Dab from 0% to 0.82%, and decylic acid-cyclo-MEL-Dab from 0% to 1.01%. This indicates that cyclization and esterification can improve the pepsin degradation stability of melitoxic peptides.

[0073] Example 5: Stability evaluation of melitoxic peptide-derived lipocyclic peptides in trypsin solution

[0074] The stability of melittin and its derived lipocyclic peptides in trypsin solution was determined by high performance liquid chromatography (HPLC). According to the Pharmacopoeia of the People's Republic of China (2020 edition), 250 mL of 0.2 mol / L potassium dihydrogen phosphate solution was dissolved in 118 mL of 0.2 mol / L sodium hydroxide solution. Separately, 10 g of trypsin was dissolved in water. The two solutions were mixed and transferred to a 1000 mL volumetric flask, then diluted to the mark with water to obtain the trypsin solution. The melittin and its derived lipocyclic peptides were dissolved and diluted with trypsin solution to a concentration of 200 μg / mL. The solutions were then placed in a 37℃ water bath, and samples were taken at 0, 0.5, 1, 2, 3, 4, 5, and 6 h. After inactivating the protease by incubating the samples in an 85℃ water bath for 5 min, they were centrifuged at 10000 rpm and 4℃ for 15 min. The peak areas of the melittin and its derived lipocyclic peptides at different time points were detected, and the degradation rate was calculated. The results are shown below. Figure 11 As shown.

[0075] from Figure 11 The results show that after incubation in protease solution for 0, 0.5, 1, 2, 3, 4, 5, and 6 hours, the degradation rate of meliostepin ranged from 77.03% to 98.78%. In comparison, the degradation rates of butyric acid-cyclo-MEL-Dab ranged from 0% to 3.90%; hexanoic acid-cyclo-MEL-Dab from 0% to 2.49%; octanoic acid-cyclo-MEL-Dab from 0% to 2.69%; and decylic acid-cyclo-MEL-Dab from 0% to 2.83%. This indicates that cyclization and esterification can improve the trypsin degradation stability of meliostepin.

[0076] In summary, cyclization and fatty acid coupling can significantly improve the activity and structural stability of meliostepin. Derivative lipocyclic peptides of meliostepin have more advantages than meliostepin in various applications for pets and have a broader application prospect.

[0077] The above are merely embodiments of the present invention. The invention is not limited to the fields covered by these embodiments. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can improve and implement this solution based on the guidance provided in this application and their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness or practicality of the invention. The scope of protection claimed in this application should be determined by the content of its claims. The specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A melittin-derived aliphatic cyclic peptide, characterized in that: The structure of the melittin-derived lipocyclic peptide is as follows: butyric acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab]; or, hexanoic acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab]; or, octanoic acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab]; or, decylic acid-G-cyclo[Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab].

2. The method for preparing bee venom peptide-derived lipocyclic peptides as described in claim 1, characterized in that: The method comprises the following steps: 1) replacing Ile at the 2nd position of the N-terminal and Gln at the 26th position of the C-terminal of melittin with 2,4-diaminobutyric acid to obtain melittin derivative I, the structure of which is as follows: G-Dab-GAVLKVLTTGLPALISWIKRKRQ-Dab; 2) coupling butyric acid, hexanoic acid, octanoic acid or decylic acid with Gly at the 1st position of the N-terminal of melittin derivative I to obtain melittin derivative II; 3) dehydrating and condensing Dab at the 2nd position and Dab at the 26th position of melittin derivative II to obtain a melittin-derived lipocyclic peptide.

3. The method for preparing bee venom peptide-derived lipocyclic peptides according to claim 2, characterized in that: The 2,4-diaminobutyric acid is D-type and / or L-type 2,4-diaminobutyric acid.

4. Use of the melittin-derived lipocyclic peptide of claim 1 in the preparation of a broad-spectrum pet antibacterial drug or antibacterial composition.

5. Use according to claim 4, characterized in that: The bacteria targeted by the broad-spectrum pet antibacterial drug or antibacterial composition include gram-positive bacteria and gram-negative bacteria.

6. Use according to claim 5, characterized in that: The bacteria include Escherichia coli, Pasteurella canis, Haemophilus felis, Campylobacter coli, Streptococcus canis and Staphylococcus pseudintermedius.

7. Use of the melittin-derived lipocyclic peptide of claim 1 in the preparation of a pet feed additive or pet feed additive composition.

8. Use of the melittin-derived lipocyclic peptide of claim 1 in the preparation of a pet eye drop.

9. Use of the melittin-derived lipocyclic peptide of claim 1 in the preparation of a pet external use antibacterial spray.

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