Trypsin inhibitory peptide with antibacterial activity and application thereof

Abstract

The invention discloses a trypsin inhibitory peptide with antibacterial activity and application of the trypsin inhibitory peptide. Drug-resistant bacteria cause millions of infections in the world every year, and development of novel antibacterial agents becomes an important task. The invention discloses a bifunctional polypeptide with strong trypsin inhibitory activity and antibacterial activity. The peptide is also a novel derivative (BBI) of Borman-Burkholderia trypsin inhibitor polypeptide; the derivative peptide not only has strong bactericidal ability to two drug-resistant strains of Escherichia coli and methicillin-resistant staphylococcus, but also has excellent performance in safety evaluation of erythrocyte dissolution activity and anti-proliferative activity to human epithelial cuticle forming cells. Besides, the antibacterial activity of the novel bifunctional peptide is not obviously interfered under the conditions of salt ions, serum and trypsin, and the novel bifunctional peptide shows certain resistance to the salt ions, the serum and the trypsin. Therefore, the peptides have clinical development potential as novel clinical antibacterial preparations.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a trypsin inhibitory peptide, specifically a trypsin inhibitory peptide with antibacterial activity, structural modification ideas for such peptides, and applications of these structurally derived peptides. Background Technology

[0002] In recent years, antibiotic resistance (AMR) has increasingly become a serious threat to human health. The overuse of antibiotics, such as poor prescription adherence and the release of antibiotics before complete metabolism, has led to the emergence of antibiotic-resistant superbugs, which cause serious problems in human medicine, veterinary medicine, and agriculture. The rapid evolution of drug-resistant microorganisms and the formation of antibiotic resistance have greatly limited the future application of traditional antibiotics. According to the latest survey by the Centers for Disease Control and Prevention (CDC), millions of patients worldwide are infected with drug-resistant bacteria each year, and it is projected that by 2050, the number of deaths related to drug resistance will reach tens of millions. Therefore, the development of new and effective antimicrobial drugs has become an urgent priority.

[0003] In recent years, there has been extensive research on the multifunctional components derived from amphibian skin secretions. Amphibian skin secretions have long been considered a unique source of bioactive compounds. Among them, antimicrobial peptides (AMPs), with their antibacterial activity, are widely regarded as the most promising candidates for novel antibiotics. However, due to their low selectivity for cell membranes, some AMPs exhibit non-selective cytotoxicity and hemolytic activity; furthermore, because AMPs are rich in lysine and arginine, they are readily degraded by trypsin or chymotrypsin. Summary of the Invention

[0004] This invention discloses an antibacterial trypsin inhibitory peptide, with the parent peptide being OTI-1872. This peptide exhibits strong inhibitory activity against trypsin and possesses certain antibacterial activity. Further structural modification of the parent peptide optimized its antibacterial activity while maintaining its trypsin inhibitory activity, yielding derived peptides OTI-2369, OTI-2483, and OTI-2380. These derived peptides all demonstrated enhanced antibacterial activity and strong trypsin inhibitory activity similar to that of the parent peptide. These derived peptides also exhibited excellent safety profiles, with low hemolytic activity and low inhibitory activity against HaCaT cell proliferation. Surprisingly, the antibacterial activity of these derived peptides remained largely unchanged after 1 hour of trypsin treatment. In summary, these bifunctional peptides possess novel antibacterial properties and safety profiles, and their value warrants further investigation. They may become strong candidates for novel clinical antibacterial drugs in the future.

[0005] The present invention adopts the following technical solution.

[0006] A trypsin inhibitory peptide with antibacterial activity, wherein the trypsin inhibitory peptide with antibacterial activity comprises one or more of a parent peptide with the amino acid sequence SEQ ID NO: 1 and a derivative peptide of the parent peptide.

[0007] Furthermore, the amino acid sequence of the derived peptide of the parent peptide is SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0008] Preferably, the terminal end of the parent peptide or its derivative peptide of the present invention is an amino group.

[0009] This invention discloses nucleotides encoding the aforementioned parent peptide or derived peptide.

[0010] This invention discloses a structural modification strategy for this type of parent peptide. This strategy is highly efficient in enhancing the antibacterial activity of the parent peptide and keeps the safety of the derived peptide at the same level as the parent peptide. At the same time, this structural modification strategy also enables the derived peptide to have a certain degree of resistance to trypsin hydrolysis while possessing highly efficient antibacterial activity.

[0011] In this invention, the parent peptide or derived peptide is encoded using nucleotides; or the parent peptide or derived peptide is prepared using a solid-phase peptide synthesizer. Based on the amino acid sequence disclosed in this invention, the specific preparation method is a conventional technique.

[0012] This invention discloses the application of the above-mentioned trypsin inhibitory peptide with antibacterial activity in the preparation of trypsin inhibitor drugs and / or antibacterial drugs.

[0013] This invention discloses the application of the above-mentioned trypsin inhibitory peptide with antibacterial activity in the preparation of a bifunctional drug that inhibits trypsin and has antibacterial properties.

[0014] A trypsin inhibitor and / or antibacterial agent, wherein the active ingredient of the trypsin inhibitor and / or antibacterial agent comprises the above-disclosed ingredients of the present invention.

[0015] A method for inhibiting trypsin and / or for antibacterial purposes is disclosed in this invention, which utilizes the above-described method for inhibiting trypsin and / or for antibacterial purposes.

[0016] In this invention, the trypsin inhibitor includes a drug with trypsin inhibitory activity; the antibacterial drug includes a drug with bactericidal ability against drug-resistant bacteria.

[0017] In this invention, the bacteria include one or more of the following: Gram-negative strains, Gram-positive strains, fungi, and yeasts.

[0018] In this invention, the drug includes an oral drug.

[0019] In this invention, the amino acid sequence of the parent peptide is SEQ ID NO: 1, and the amino acid sequence of the derived peptide is SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; wherein: SEQ ID NO: 1 is as follows: AALKGCWTKSIPPRPCF SEQ ID NO: 2 is as follows: ALKRALKRCWTKSIPPRPCF SEQ ID NO: 3 is as follows: VKWKVKWKCWTKSKPPKPCK SEQ ID NO: 4 is as follows: VKWKVKWKCWTKSKPPKPK The terminus of the aforementioned parent peptide or derived peptide is an amino group (NH2).

[0020] Antimicrobial peptides (AMPs) generally contain positively charged amino acids, thus exhibiting positive charge. They interact with the anionic cell membranes of microorganisms through carpet-like, barrel-shaped, or ring-shaped action, leading to bacterial death. This mechanism differs significantly from traditional antibiotics because AMPs exert their antibacterial effect through electrostatic attraction to the bacterial cell membrane surface, resulting in rapid bacterial death. This also reduces the likelihood of AMPs inducing antibiotic resistance while exerting their bactericidal effect. However, due to the low selectivity of AMPs for cell membranes, some AMPs exhibit non-selective cytotoxicity and hemolytic activity. Furthermore, because AMPs are rich in lysine and arginine, they are easily degraded by trypsin or chymotrypsin. This invention discloses novel trypsin inhibitor peptides, also known as BBI-like peptides, which possess potent trypsin inhibitory activity and exhibit both trypsin inhibitory and antimicrobial activity, demonstrating multifunctional properties. This type of peptide also exhibits low cytotoxicity and hemolytic activity. Attached Figure Description

[0021] Figure 1 The results of circular dichroism (CD) spectroscopy for OSTA-1872 and its derivative peptides in two different environments: (A) 10 mM NH4Ac buffer as an aqueous environment, and (B) 50% TFE / NH4Ac solution as a simulated microbial film environment.

[0022] Figure 2 This is the mass spectrum of OST1-1872.

[0023] Figure 3 This is the mass spectrum of OST1-2369.

[0024] Figure 4 This is the mass spectrum of OSTA-2483.

[0025] Figure 5 This is the mass spectrum of OSTA-2380.

[0026] Figure 6 The figures show the Morrison inhibition curves for OTI-1872, OTI-2369, OTI-2483, and OTI-2380. The Ki values ​​were calculated using the Morrison formula in Prism9, where trypsin Km = 41.07 μM, [S] = 42.86 μM, and Et = 0.0020 μM.

[0027] Figure 7 For OST1-2369 E. coli 13846 and E. coli The kinetic time-kill curves for 2340 are shown; blue, red, and green represent peptide solutions with concentrations of MIC, 2×MIC, and 4×MIC, respectively. The growth control is orange (no treatment); the vector control is purple (representing bacteria treated with 1% DMSO). The Y-axis represents the logarithmic CFU / mL for all groups, and the X-axis represents time from 0 to 180 minutes. Error bars represent the standard error (SEM) of six replicates in two independent experiments.

[0028] Figure 8 For OST1-2483 E. coli 13846 and E. coli The kinetic time-kill curves for 2340 are shown; blue, red, and green represent peptide solutions with concentrations of MIC, 2×MIC, and 4×MIC, respectively. The growth control is orange (no treatment); the vector control is purple (representing bacteria treated with 1% DMSO). The Y-axis represents the logarithmic CFU / mL for all groups, and the X-axis represents time from 0 to 180 minutes. Error bars represent the standard error (SEM) of six replicates in two independent experiments.

[0029] Figure 9 For OST1-2380 E. coli 13846 and E. coli The kinetic time-kill curves for 2340 are shown; blue, red, and green represent peptide solutions with concentrations of MIC, 2×MIC, and 4×MIC, respectively. The growth control is orange (no treatment); the vector control is purple (representing bacteria treated with 1% DMSO). The Y-axis represents the logarithmic CFU / mL for all groups, and the X-axis represents time from 0 to 180 minutes. Error bars represent the standard error (SEM) of six replicates in two independent experiments.

[0030] Figure 10 For peptide pairs E. coli Membrane permeability of NCTC 13846; all test peptides were set at concentrations of 1×MIC, 2×MIC, and 4×MIC. Positive and negative controls were bacteria treated with 8 μM Melittin and 5% TSB, respectively. Error bars represent the standard error (SEM) of six replicates from two independent experiments; significant differences were calculated by comparing the negative control and the sample control.

[0031] Figure 11 For peptide pairs E. coli Membrane permeability of BAA 2340; all test peptides were set at concentrations of 1×MIC, 2×MIC, and 4×MIC. Positive and negative controls were bacteria treated with 8 μM Melittin and 5% TSB, respectively. Error bars represent the standard error (SEM) of six replicates from two independent experiments; significant differences were calculated by comparing the negative control and the sample control.

[0032] Figure 12 The hemolytic activity of OSTA-1872 and its analogues against equine erythrocytes was measured at concentrations ranging from 1 to 512 μM. The percentage of hemolysis was calculated by comparing the effects with the positive control (1% Triton X-100) and the negative control (PBS).

[0033] Figure 13 The antiproliferative activity of OSTA-1872 and its analogues against HaCaT cells was measured. 1% Triton X-100 and 1% DMSO (in PBS) were used as positive and solvent controls, respectively. Error bars represent the standard error (SEM) of the mean. Data were obtained from nine replicates of three independent experiments. Detailed Implementation

[0034] This invention discloses an antibacterial trypsin inhibitory peptide, with the parent peptide being OTI-1872. This peptide exhibits strong inhibitory activity against trypsin and possesses certain antibacterial activity. Further sequence modification of the parent peptide yields derivative peptides that, while optimizing antibacterial activity, still maintain strong trypsin inhibitory activity similar to that of the parent peptide. These derivative peptides are OTI-2369, OTI-2483, and OTI-2380, all of which demonstrate enhanced antibacterial activity and strong trypsin inhibitory activity.

[0035] In the present invention, the amino acid sequence of the parent peptide is SEQ ID NO: 1, and the amino acid sequences of the derivative peptides are SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. The methods for preparing these peptides are conventional techniques. The parent peptide or derivative peptide can be encoded by nucleotides; or the parent peptide or derivative peptide can be prepared using a solid-phase peptide synthesizer.

[0036] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention exemplarily, and are not used to limit the present invention. The raw materials used in the present invention are all existing products, and the specific preparation operations and performance tests are all conventional techniques.

[0037] The statistical analysis of the bioactivity assay experiments was performed using Prism 9. One-way / two-way analysis of variance (ANOVA) was used to analyze the statistical significance of the differences. The data points are the means of independent experiments, and the error bars represent the standard error of the mean (SEM). Ns represents no significant difference, * represents p < 0.05, ** represents 0.001 < p < 0.01, *** represents 0.0001 < p < 0.001, and **** represents p < 0.0001.

[0038] Example 1 Table 1 shows the amino acid sequences of the parent peptide OSTI-1872, derivative peptides OSTI-2369, OSTI-2483, and OSTI-2380, with the amine group NH2 at the sequence terminus.

[0039] Table 1 BBI peptide sequences and their physicochemical properties

[0040] Example 2 Solid-phase peptide synthesis (SPPS) Following standard techniques, the parent peptide and its derivatives (Table 1) were synthesized using an automated solid-phase peptide synthesizer (Protein Technologies, Tucson, AZ, USA). A brief description follows: 0.375 mmol of each amino acid was required. After weighing, each amino acid was placed in a flask. The amount of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurammonium hexafluorophosphate (HBTU, coupling reagent) was 2.5 times that of the amino acid. Each coupling reaction was catalyzed using 0.25 mmol of Rink Amide MBHA resin (Novabiochem, Germany), the weight of which was equal to the molar mass of the peptide (mmol) divided by the loading (mmol / g). After the reaction, the peptide was passed through a solution containing 94% trifluoroacetic acid (TFA), 2% deionized water (ddH2O), 2% thioanisole (TIS), and 2%... The product was cleaved from the resin using a mixed solution of 1,2-ethylenedithiol (EDT); the cleaved peptide was dissolved in a mixed solution of buffer A (trifluoroacetic acid / distilled water, 0.05 / 99.95, v / v) and buffer B (trifluoroacetic acid / acetonitrile / distilled water, 0.05 / 80.00 / 19.95, v / v / v), lyophilized for 48 hours, and stored at -20°C.

[0041] Figure 1 The results of circular dichroism (CD) spectroscopy of OSTA-1872 and its derived peptides in two different environments: (A) 10 mM NH4Ac buffer as an aqueous environment, and (B) 50% TFE / NH4Ac solution as a simulated microbial membrane environment; the peptides exhibited α-helical, β-chain and coil conformations in both aqueous and simulated membrane environments.

[0042] Synthetic peptides were identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Voyager DE, Perseptive BioSytems, Framingham, MA, USA). A CHCA solution (10 mg / ml) containing 70% acetonitrile, 30% water, and 0.1% TFA was used as a reference solution for peptide molecular weight analysis. Two μl of the HPLC fraction and one μl of the CHCA solution were loaded onto a sample plate, and the molecular weight of the target peptide was determined based on the mass-to-charge ratio (M / Z) in the mass spectrum. Figure 2 , Figure 3 , Figure 4 as well as Figure 5 The mass spectra of the parent peptide OTI-1872, the derived peptides OTI-2369, OTI-2483, and OTI-2380 are shown below:

[0043] Example 3: Assay of Trypsin Inhibitory Activity In this experiment, the substrate for trypsin was Phe-Pro-Arg-AMC (Bachem, Merseyside, UK). The peptide was dissolved in phosphate-buffered saline (PBS) to prepare a series of solutions (1-1000 μM), which were then added to black 96-well plates. Each well contained 180 μL of the substrate (diluted to 50 μM with PBS) and 10 μL of trypsin (diluted to 0.1 μM with 1 mM HCl). The operation was performed in the dark. The final volume per well was 210 μL. Fluorescence intensity was measured immediately after trypsin addition, and fluorescence intensity data were recorded every 30 seconds using a FLUOstar OPTIMA plate reader (BMG Labtech, Ortenberg, Germany) for a total of 30 minutes. An emission wavelength of 460 nm and an excitation wavelength of 395 nm were used, and the temperature was set to 37°C. The Morrison equation in Prism 9 software was used to plot the trypsin inhibitory activity curves (working concentration of substrate, [S] = 42.86 μM; working concentration of trypsin, Et = 0.0020 μM; trypsin kinetic constant, Km = 41.07 μM). The inhibitory activity of the peptides against trypsin was detected by functional experiments, and the results are shown in Table 2; the Morrison inhibition plots of the tested peptides are shown in Figure 6.

[0044] Table 2. Inhibitory activity of OSTI-1872 and its derivative peptides against trypsin.

[0045] Example 4: Antibacterial Activity Assay The minimum inhibitory concentration (MIC) represents the lowest peptide concentration that can inhibit the growth of visible bacteria. The minimum bactericidal concentration (MBC) represents the lowest peptide concentration that can kill all bacteria.

[0046] Ten microorganisms were selected for antimicrobial testing, including Escherichia coli (E. coli). E. coli ATCC CRM 8739), ( E. coli , BAA 2340), ( E. coli NCTC 13846), Pseudomonas aeruginosa ( P. aeruginosa ATCC CRM9027), Staphylococcus aureus ( S. aureus ATCC CRM 6538), Candida albicans ( C. albicansATCC10231), Enterococcus ( E. faecium (NCTC 12697), methicillin-resistant Staphylococcus aureus (MRSA, NCTC12493), Klebsiella pneumoniae (NCTC 12697), and methicillin-resistant Staphylococcus aureus (NCTC 12493). K. pneumoniae ATCC CRM 43861), Acinetobacter baumannii ( A. baumannii (BAA 747). Bacterial culture media include tryptone soybean liquid (TSB), tryptone soybean agar (TSA), nutrient solution (NB), and nutrient agar (NA); yeast culture media include yeast extract peptone glucose liquid medium (YPD-B) and yeast extract peptone glucose agar (YPD-A).

[0047] Microbial strain samples taken from a -20°C freezer were inoculated into bottles containing appropriate culture media, including: nutrient solution (NB) and nutrient agar (NA) for culturing: *Escherichia coli* (… E. coli ATCC CRM 8739), ( E. coli ,BAA 2340), ( E. coli NCTC 13846), Pseudomonas aeruginosa ( P. aeruginosa ATCC CRM 9027), methicillin-resistant Staphylococcus aureus (MRSA, NCTC 12493), Klebsiella pneumoniae ( K. pneumoniae ATCC CRM43861); Tryptone Soy Broth (TSB) and Tryptone Soy Agar (TSA) are used to culture: Staphylococcus aureus ( S. aureus ATCC CRM 6538), Enterococcus ( E. faecium NCTC 12697), Acinetobacter baumannii ( A. baumannii (BAA 747); then these bottles were placed in a shaking incubator and cultured at 37°C with shaking at 120 rpm / min for 18 hours (yeast was cultured at 26°C). Then, 0.5 mL of the culture was transferred to a McFarland flask containing 20 mL of culture medium and cultured a second time under the same conditions. When the optical density (OD) at 550 nm reached an appropriate value, the secondary culture was diluted 200-fold with fresh culture medium to achieve a working cell density of 5 × 10⁶ cells / mL. 5 CFU / ml.

[0048] Dimethyl thionamide (DMSO) was selected to dissolve peptides at different concentration ranges (100 μM – 51200 μM). Different media were then loaded into 96-well plates. The experimental group consisted of 99 μl of diluted culture medium and 1 μl of peptide solution. The solvent control group contained 99 μl of diluted culture medium and 1 μl of DMSO. The positive control group contained 1 μl of norfloxacin (for bacteria, 20 μg / ml) or 1 μl of amphotericin B (for yeast, 10 μg / ml) and 99 μl of diluted culture medium. 100 μl of sterile culture medium served as the negative control group. 100 μl of diluted culture medium was used as the growth control group. The 96-well plates containing samples were incubated for 20–24 hours, and the optical density (OD) values ​​were acquired at 550 nm using a Synergy HT plate reader. Data processing used the following formula: Survival rate (%) = (sn) / (gn), where s represents the absorbance value of the experimental group, n represents the absorbance value of the negative control group, and g represents the absorbance value of the growth control group. The lowest concentration in the experimental group where no visible bacterial growth was observed was determined as the minimum inhibitory concentration (MIC). Under the same conditions as the MIC determination, cultures from the experimental groups were inoculated into petri dishes containing the corresponding solid culture medium for incubation. The minimum bactericidal concentration (MBC) was the lowest concentration of peptide at which no biological growth was observed.

[0049] The in vitro antibacterial activity of the synthetic peptides was evaluated by measuring the MIC / MBC of a representative group of microorganisms. The results are shown in Table 3. These peptides showed antimicrobial activity against Gram-negative bacteria, including two drug-resistant strains. E. coli It showed significantly enhanced antibacterial activity; the antibacterial effect against Gram-positive strains of MRSA was also enhanced.

[0050] Table 3. MIC and MBC values ​​(μM) of peptides against selected microorganisms.

[0051] Existing technologies disclose that OST1 (amino acid sequence AALKGCWTKSIPPKPCF-NH2) has inhibitory activity against trypsin, but it lacks antibacterial properties. Referring to the experiments described above, OST1... E. coli The MIC and MBC values ​​of ATCC CRM 8739 and MRSA (NCTC 12493) are both >128μM.

[0052] Example 5: Experiment to determine the bactericidal efficiency of peptides The bactericidal efficiency experiment aimed to determine the time-kinetic bactericidal ability of the peptide against bacteria. Based on conventional bactericidal requirements (below 10 μM), the selected strain was drug-resistant *Escherichia coli*. E. coli , NCTC 13846) and ( E. coli, BAA2340). The culture method for these strains was the same as that used in the antibacterial activity assay. Then, 198 μL of bacterial suspension (5 × 10⁻⁶) was... 5 CFU / mL peptide solutions were treated with MIC, 2×MIC, and 4×MIC solutions (2 μL) in three sterile tubes. Bacterial-peptide mixed media were diluted 10-fold, 100-fold, and 1000-fold, and the four different concentrations were inoculated into solid medium NA petri dishes at time points of 0, 5, 10, 15, 30, 60, 90, 120, and 180 minutes. All colonies were counted after incubation at 37°C for 24 hours. The control group consisted of 200 μL of untreated bacterial medium as a growth control, and the solvent control group consisted of 198 μL of bacterial medium treated with 2 μL of DMSO. The effects of time-killing assays on peptide efficacy were investigated. E. coli The bactericidal kinetics of strains NCTC 13846 and BAA 2340 were studied. The peptide concentrations in this experiment were 1×MIC, 2×MIC, and 4×MIC. Results are shown in [Table missing]. Figure 7 , Figure 8 as well as Figure 9 Among them, OTI-2369 and OTI-2483 showed immediate bactericidal activity against both test bacterial strains at concentrations of 2×MIC and 4×MIC.

[0053] Example 6: SYTOX Green Membrane Permeation Experiment SYTOX Green nucleic acid dye (ThermoFisher Scientific, USA) cannot penetrate intact membranes, but it can easily penetrate the damaged membranes unique to dead cells, making it an effective indicator for detecting dead bacteria. Bacteria were cultured overnight on a shaker at 37°C using TSB. E. coli NCTC 13846 / E. coli(BAA 2340). After culturing for 2 hours, the bacteria were centrifuged at 4°C and 1000 × g for 10 minutes. The culture medium was then discarded, and the bacteria were washed twice with 5% TSB (in 0.85% NaCl). The bacterial density reached the logarithmic growth phase when the OD value measured in 5% TSB solution reached 0.70. Next, 40 μL of peptide solutions (concentrations of 2.5×MIC, 5×MIC, and 10×MIC) and 50 μL of bacterial suspension were added to black 96-well plates and incubated at 37°C for 2 hours. Afterward, 10 μL of SYTOX Green nucleic acid dye (5 μM) was added to the plate, and the mixture was incubated at 37°C in the dark for 5 minutes. The final peptide concentrations in the 96-well plates were MIC, 2×MIC, and 4×MIC. Fluorescence intensity was measured using a Synergy HT reader (BioTek, USA). The excitation and emission wavelengths were set to 485 nm and 528 nm, respectively. Bacterial culture medium containing 5% TSB was used as a negative control; melittin peptide solution (8 μM) was used as a positive control; and 5% TSB was used as a blank control.

[0054] Through the SYTOX Green permeability test (see...) Figure 10 and Figure 11 The cell membrane permeability of peptides and their analogues was studied. The peptides tested at MIC, 2×MIC, and 4×MIC concentrations... E. coli (NCTC 13846) and E. coli (BAA2340) was treated. Overall, the experimental results showed that, compared with the positive control peptide, all tested peptides were significantly reduced compared with the three strains. E. coli After 2 hours of co-incubation, all strains produced extremely low membrane perforation rates, even at a peptide dosage concentration of 4×MIC, demonstrating that the killing effect of this type of modified peptide on the experimental bacteria is not a traditional membrane perforation mechanism.

[0055] Example 7 Hemolysis Experiment The purpose of the hemolysis assay was to evaluate the in vitro lysing activity of the peptide on equine erythrocytes. Erythrocytes were obtained from defibrinated equine blood and washed with PBS to a 4% suspension. A stock solution of the peptide was prepared by dissolving it in DMSO and then diluted with PBS to working concentrations ranging from 2 μM to 1024 μM (total DMSO less than 1%). Next, 100 μL of the peptide solution and 100 μL of the 4% erythrocyte suspension were added to 36 tubes and incubated at 37°C for 2 hours. These tubes were then centrifuged at 930 × g for 10 minutes, and the supernatant from each tube was carefully transferred to a 96-well plate. Erythrocyte lysis in the plate was detected at 470 nm using a Synergy HT plate reader (BioTek, USA). A 4% erythrocyte suspension treated with 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) served as a positive control, while a negative control consisted of a 4% erythrocyte suspension and 1% DMSO solution (diluted with PBS). The results were calculated using the following formula: Hemolysis rate (%) = (sn) / (pn), where s represents the optical density value of the experimental group, n represents the optical density value of the negative control, and p represents the optical density value of the positive control.

[0056] Assessing the cytotoxicity of peptides is a crucial step in a comprehensive evaluation. The hemolytic activity of four peptides was evaluated in vitro using horse erythrocytes (Figure 12). Both the parent peptide and its derivatives caused hemolysis of no more than 10% at the highest concentrations.

[0057] Example 8: MTT Cell Proliferation Inhibition Assay The MTT assay was used to assess the safety of the peptide's antiproliferative activity against normal cells. HaCaT cells, a commercially available adult skin keratinocyte cell line, were used. Resuscitated cells were cultured at 37°C for 3–5 days until adherence to the flask walls. The culture medium was then removed, and the flask walls were gently washed twice with 10 ml PBS, followed by removal of the PBS. Next, cells were detached from the flask walls using 3–4 ml of trypsin solution, followed by the addition of 10 ml of FBS to terminate the digestion reaction. The cell suspension was transferred to 15 ml tubes and centrifuged at 130 × g for 6 minutes at 18°C. The supernatant was removed from the tubes, and 4 ml of complete growth medium was added to prepare a cell stock suspension. An equal volume of trypan blue and cell culture medium was mixed and transferred to a counting chamber for cell density determination under a microscope. Based on the cell density, the cell suspension was diluted with fetal bovine serum (FBS) to a standard cell density of 1 × 10⁻⁶ cells / mL. 5Cells / ml. Then, 100 μl of cell suspension was transferred to each well of a 96-well plate. After 24 hours of incubation, 100 μl of serum-free medium was used as fresh medium to replace the previous FBS, and the cells were starved in an incubator for 4 hours. Peptides were extracted from 10... -9 M to 10 -4 The working concentration of MTT was prepared in serum-free medium. The sample group contained 100 μl of the working concentration peptide solution. The control group contained 1 μl of DMSO and 99 μl of serum-free medium. Both the blank and growth groups contained 100 μl of serum-free medium. After treatment, the 96-well plates were incubated for 24 hours. Then, 10 μl of MTT was added to each well, and incubation was continued for 2 hours. Finally, the medium in each well was removed, 100 μl of DMSO was added, and after shaking for 10 minutes, cell viability in each well was detected at 570 nm using a BioTek microplate reader. Results of the MTT cell proliferation inhibition assay (…) Figure 13 The results showed that peptides did not exhibit a significant IC50 effect within the concentration range of 0.1–100 μM. 50 The value indicates that they have no significant cytotoxic effect on the proliferation inhibition of normal human cells.

[0058] Example 9: Salt Ion and Serum Sensitivity Test In the antibacterial activity assay, the sensitivity of the peptide to salt ions and serum was investigated. The bacteria tested were *Escherichia coli* (E. coli). E. coli (ATCC CRM 8739). Different concentrations of salt (150 mM NaCl, 4.5 mM KCl, 6 µM NH4Cl, 1 mM MgCl2, 2.5 mM CaCl2, and 4 mM FeCl3) were used to determine the effect of cationic substances on the antibacterial activity of peptides in bacterial culture. The stability of the antibacterial effect of peptides against bacteria was studied in the presence of different salt ions and horse serum. The results (Table 4) showed that all tested peptides maintained a certain degree of stable antibacterial effect under horse serum and salt ion treatment.

[0059] Table 4. Results of peptides after co-incubation with salt ions and horse serum E. coli MIC / MBC value (μM) of the strain

[0060] Example 10: Trypsin Sensitivity Test The peptide was dissolved in PBS to prepare a stock concentration of 12800 μM. Trypsin solutions were prepared at concentrations of 0.25 mg / ml and 0.5 mg / ml (the molecular weight of trypsin is approximately 24K, corresponding to molar concentrations of approximately 10417 μM and 20833 μM, respectively, close to and twice the stock concentration of the peptide). Equal volumes of trypsin and peptide solutions were then added to tubes and incubated at 37°C for 1 hour, followed by incubation at 60°C for 0.5 hours to inactivate collagenase. A control group of the peptide solution was incubated together with the mixture. Subsequent procedures were then performed as described in the previous antimicrobial activity assay.

[0061] After incubation with trypsin, the antibacterial activity of the peptides was tested. The results showed that all analogs had some resistance to trypsin degradation, and the MIC / MBC values ​​of OTI-2483 and OTI-2380 indicated that this ability was positively correlated with their trypsin inhibitory activity (Table 5).

[0062] Table 5. Effects of peptides and trypsin treatment on... E. coli MIC / MBC value (μM) of the strain

[0063] The rapid development of resistance to traditional antibiotics foreshadows a global crisis, prompting extensive and in-depth research into alternatives. Antimicrobial peptides (AMPs), also known as host defense peptides, have evolved over a long period and are key components of the innate immune systems of many organisms. However, the presence of proteases in the host limits the therapeutic application of peptides, as they are easily inactivated or degraded by these enzymes. In vivo toxicity is another concern; many clinically tested antimicrobial peptides are only suitable for topical application due to their susceptibility to protease degradation, rapid renal clearance, and systemic toxicity. If ingested orally, these peptides may be digested. Enzymes in the digestive tract, such as trypsin and pepsin, can cause proteolytic hydrolysis of antimicrobial peptides. Furthermore, systemic administration of these peptides can lead to proteolytic hydrolysis, cytotoxicity to blood cells, and a short half-life.

[0064] This invention discloses a novel BBI peptide, named OTI-1872, which exhibits potent inhibitory activity against trypsin and antibacterial activity. In particular, this peptide is effective against drug-resistant bacterial strains. E. coli (BAA 2340) and E. coli (NCTC13846) showed better performance than non-drug-resistant bacterial strains. E. coli(ATCC CRM 8739) Enhanced antibacterial activity. Therefore, optimizing the antibacterial activity of the parent BBI peptide to construct a bifunctional peptide with potent inhibitory and antibacterial activity offers a possibility for novel antibacterial drugs against drug-resistant bacteria and provides a solution to the problem of antimicrobial peptide degradation by trypsin or trypsin-like proteases.

[0065] This invention further designed several derived peptides: OTI-2369, OTI-2483, and OTI-2380. All derived peptides exhibited potent antitrypsin activity, especially OTI-2369. The results of the peptides' antibacterial effects indicate that the derived peptides help enhance the antibacterial activity of BBI peptides, particularly against Gram-negative bacteria. E. coli Regarding the activity against Gram-positive bacteria MRSA, the bactericidal efficiency of the modified peptides was evaluated using a time-based bactericidal assay. OSTI-2369 and OSTI-2483 showed immediate bactericidal effects against the test strains at concentrations of 2×MIC and 4×MIC, while OSTI-2483 showed limited bactericidal activity against the tested strains at the MIC concentration. E. coli (BAA 2340) also showed an immediate sterilization effect, which is quite different from classic AMPs and unexpected.

[0066] The permeability assay aimed to investigate the antibacterial mechanism of the parent peptide and its analogues. The results showed that the derived peptide exhibited improved permeability compared to the parent peptide OSTI-1872.

[0067] The safety of the peptides was assessed using hemolysis and MTT assays. Antimicrobial peptides (AMPs) are known to interact with the membranes of eukaryotic cells and disrupt their structure, particularly erythrocytes, potentially leading to cytotoxicity, especially hemolysis. The membrane-targeting nature of antimicrobial peptides results in low selectivity, allowing them to potentially attack host cells and exert cytotoxic and hemolytic activity. Surprisingly, similar to the parent peptides, the derived peptides also exhibited low hemolytic activity against mammalian erythrocytes and low proliferative inhibitory activity against normal HaCaT cells. These BBI peptides avoid the high cytotoxicity problem of many existing antimicrobial peptides, which has hindered their clinical application as novel antimicrobial drugs.

[0068] In salt ion and horse serum assays, the peptides of this invention maintained stable antibacterial activity. Trypsin was selected as the protease to evaluate the stability of the peptides' antibacterial activity after treatment. Although the peptide-to-trypsin ratio was close to 2:1, the results showed that OTI-2369 and OTI-2483 retained their antibacterial activity after incubation with trypsin. These results indicate that these bifunctional peptides are resistant to trypsin degradation to some extent, providing a possibility for the development of oral antimicrobial peptides.

[0069] In summary, this invention discloses a novel peptide, named OTI-1872, belonging to the BBI family. This peptide exhibits strong trypsin inhibitory and antibacterial activity, showing higher sensitivity against drug-resistant bacterial strains. Derivative peptides OTI-2369, OTI-2483, and OTI-2380 demonstrate even better antibacterial activity and strong trypsin inhibitory activity, with high bactericidal rates, showing potential clinical applications. Simultaneously, the peptides of this invention showed excellent safety in erythrolysis activity and normal cell proliferation activity (using HaCaT cells). Regarding environmental sensitivity, the tested peptides exhibited high stability in salt ions, serum, and trypsin. These data strongly suggest that these peptides have promising clinical applications as potential antibacterial agents.

Claims

1. A trypsin inhibitory peptide with antibacterial activity, characterized in that, The trypsin inhibitory peptide with antibacterial activity includes one or more of the parent peptide with the amino acid sequence SEQ ID NO: 1 and derivative peptides of the parent peptide.

2. The trypsin inhibitory peptide with antibacterial activity according to claim 1, characterized in that, The amino acid sequence of the derivative peptide of the parent peptide is SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

3. The trypsin inhibitory peptide with antibacterial activity according to claim 2, characterized in that, The terminus of the parent peptide or its derivative peptide is an amino group.

4. A nucleotide encoding the parent peptide or derived peptide of claim 1.

5. The method for preparing the trypsin inhibitory peptide with antibacterial activity according to claim 1, characterized in that, The parent peptide or derived peptide may be encoded using the nucleotides described in claim 3; or the parent peptide or derived peptide may be prepared using a solid-phase peptide synthesizer.

6. The use of the antibacterial trypsin inhibitor peptide according to claim 1 or 2 in the preparation of trypsin inhibitor drugs and / or antibacterial drugs.

7. The use of the trypsin inhibitory peptide with antibacterial activity as described in claim 1 or 2 in the preparation of a bifunctional drug for both trypsin inhibition and antibacterial activity.

8. The application according to claim 6 or 7, characterized in that, Trypsin inhibitors include drugs with trypsin-inhibiting activity; antibacterial drugs include drugs that have bactericidal ability against drug-resistant bacteria.

9. A trypsin inhibitor and / or antibacterial agent, characterized in that, The active ingredient of the trypsin inhibitor and / or antibacterial drug includes the trypsin inhibitor peptide with antibacterial activity as described in claim 1 or 2.

10. A method for inhibiting trypsin and / or an antibacterial method, characterized in that, The trypsin inhibitory peptide with antibacterial activity described in claim 1 or 2 is used for trypsin inhibition and / or antibacterial purposes.

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