A pH-activated antimicrobial peptide with protocatechuic acid-resistant enzymatic hydrolysis and its application

Abstract

This invention discloses a pH-activated antimicrobial peptide DK-PCA, which is incorporating protocatechuic acid and exhibits anti-enzymatic properties, and its applications, belonging to the field of biotechnology. The antimicrobial peptide has a backbone consisting of alternating D-type lysine and native lysine, with a myristic acid chain linked to the N-terminus. The amino group of the lysine side chain undergoes dehydration condensation with the carboxyl group of protocatechuic acid, followed by C-terminal amidation, resulting in the sequence C14-DK-K-DK-K-DK(PCA)-K(PCA)-NH2. The antimicrobial peptide exhibits pH-selective activation characteristics: at pH 5.0, it significantly enhances its antibacterial activity against Gram-negative bacteria such as *Escherichia coli* and *Pseudomonas aeruginosa*, with the MIC decreasing to 8 μM; at pH 7.0, it effectively inhibits Gram-positive bacteria such as *Staphylococcus aureus*. It demonstrates excellent resistance to enzymatic degradation and salt ionization, exhibits no significant hemolysis at the effective inhibitory concentration, and has a therapeutic index as high as 22.63. This invention can be used to prepare drugs against Gram-positive / negative bacterial infections, providing a novel antimicrobial candidate molecule with high stability and low toxicity for anti-infection and feed additive applications.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a pH-activated antimicrobial peptide that is infused with protocatechuic acid to resist enzymatic hydrolysis and its application. Background Technology

[0002] Antimicrobial peptides (AMPs), as novel antimicrobial agents, have attracted widespread attention due to their unique mechanism of action and low potential for developing drug resistance. AMPs typically consist of 5 to 100 amino acids and inhibit bacterial growth by disrupting the bacterial cell membrane, showing particular promise in combating drug-resistant bacteria. However, natural antimicrobial peptides face some challenges in clinical applications, especially their high cytotoxicity and instability under physiological conditions, which limits their use as feed additives.

[0003] In recent years, protocatechuic acid (CAS: 99-50-3), as a natural plant compound, has attracted much attention due to its significant anti-inflammatory and antibacterial properties. Studies have shown that protocatechuic acid can improve various disease states by inhibiting bacterial growth and inflammatory responses, demonstrating promising application prospects. Its antibacterial mechanism mainly involves disrupting bacterial cell membranes, inhibiting bacterial metabolism, and interfering with bacterial signal transduction. Therefore, combining protocatechuic acid with antimicrobial peptides is expected to enhance the bioactivity and stability of the antimicrobial peptides.

[0004] Currently, the sensitivity of antimicrobial peptides to proteases is a major obstacle to their application in clinical medicine, drug development, and animal husbandry. Especially in the gut, antimicrobial peptides can be rapidly hydrolyzed by intestinal proteases, leading to the loss of their biological activity. Therefore, enhancing the ability of antimicrobial peptides to resist protease hydrolysis while maintaining their chemical stability and low cytotoxicity is a current research focus. Thus, a novel enzymatically resistant antimicrobial peptide is needed. Summary of the Invention

[0005] In view of the above shortcomings, the present invention provides a protocatechuic acid-integrated pH-activated antimicrobial peptide DK-PCA. The peptide sequence is formed by dehydration condensation of the amino group of lysine with protocatechuic acid to form a stable compound, thereby enhancing its antimicrobial properties against positive and negative bacteria and possessing the ability to resist proteases.

[0006] The technical solution adopted in this invention is as follows: a pH-activated antimicrobial peptide DK-PCA with protocatechuic acid anti-enzymatic properties, the molecular formula of which is shown in formula (Ⅰ).

[0007]

[0008] Equation (Ⅰ).

[0009] The present invention also provides the use of the protocatechuic acid-integrated, pH-activated antimicrobial peptide DK-PCA as described above in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.

[0010] Furthermore, the Gram-negative bacteria mentioned above are Pseudomonas aeruginosa, Salmonella typhimurium, or Escherichia coli.

[0011] Furthermore, the Gram-positive bacteria mentioned above are Staphylococcus aureus, Enterococcus faecalis, or Staphylococcus epidermidis.

[0012] Furthermore, as described above, in an environment of pH=5.0, the antimicrobial peptide DK-PCA enhances its antibacterial activity against Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli.

[0013] The present invention also provides a medicament suitable for treating and / or preventing infections caused by Gram-positive and / or Gram-negative bacteria, said medicament containing the antimicrobial peptide DK-PCA as described above.

[0014] Advantages and beneficial effects of the present invention: The antimicrobial peptide DK-PCA of the present invention exhibits strong biological activity and significant resistance to proteases. By binding to non-natural amino acids to evade protease attack, it can be rapidly activated and enhanced in acidic or inflammatory environments. Antimicrobial activity, hemolytic activity, salt ion and protease stability tests of the antimicrobial peptide of the present invention revealed that DK-PCA has a strong inhibitory effect on common pathogens, with no significant hemolysis at effective inhibitory concentrations. The antimicrobial peptide of the present invention significantly improves its antimicrobial performance against Gram-negative bacteria (especially Escherichia coli) at pH 5.0, reducing the minimum inhibitory concentration (MIC) from 64 µM to 8 µM, and increasing the therapeutic index from 1.00 to 22.63. Regarding the stability of its antimicrobial performance against Gram-positive bacteria (such as Staphylococcus aureus), the MIC remains essentially stable under normal salt ion conditions. Furthermore, after incubation with 8 mg of trypsin at 37°C for 1 hour, the MIC of the antimicrobial peptide of the present invention remains at 8 µM, demonstrating excellent resistance to enzymatic degradation. Attached Figure Description

[0015] Figure 1 This is a flowchart of the solid-phase chemical synthesis method for antimicrobial peptides.

[0016] Figure 2 This is a chromatogram of antimicrobial peptides;

[0017] Figure 3 This is a mass spectrum of antimicrobial peptides. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings.

[0019] Example 1

[0020] The design method is as follows:

[0021] The short peptide consists of one group of myristic acid (C14), two groups of protocatechuic acid (PCA), three groups of D-lysine, and three groups of native lysine. The three groups of D-lysine and the three groups of native lysine are arranged alternately to form the backbone, resulting in the following sequence: D KK- D KK- D KK dehydrated and condensed the amino group of D-lysine at position a of the short peptide with myristic acid, and dehydrated and condensed the side chain amino groups of D-lysine at position d of the short peptide and native lysine at position f of the short peptide with the carboxyl groups of a group of protocatechuic acids to form a stable polypeptide; and then amidated the C-terminus of the polypeptide with -NH2. Its sequence and molecular weight are shown in Table 1.

[0022] Table 1. Amino acid sequences of antimicrobial peptides

[0023]

[0024] Design principles of antimicrobial peptides:

[0025] Protocatechuic acid possesses a structure with two hydroxyl groups and an aromatic ring, which gives it a significant advantage in antibacterial activity. The hydroxyl groups can interact with components in the bacterial cell membrane through hydrogen bonds, enhancing the binding ability of the antimicrobial peptide to bacteria. Furthermore, the hydrophobic nature of the aromatic ring allows it to better embed into the bacterial membrane, disrupting membrane integrity and effectively killing bacteria. Myristic acid provides strong hydrophobicity, contributing to the affinity of the peptide chain for the bacterial membrane; the positive charge of lysine enhances the interaction between the peptide chain and the negative charge of the bacterial membrane, further improving the binding ability. This design combines the characteristics of hydrophobic and hydrophilic amino acids, particularly leveraging the important role of the aromatic ring and hydroxyl groups of protocatechuic acid in antibacterial activity. Under pH-activated conditions, the hydroxyl groups of protocatechuic acid not only enhance the antibacterial activity of the peptide chain but also maintain its stability in the physiological environment.

[0026] Example 2

[0027] Preparation of antimicrobial peptides:

[0028] like Figure 1 As shown, the antimicrobial peptide of this invention was synthesized using a solid-phase chemical synthesis method, and then purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain the target compound, as shown. Figure 2-3 As shown, reversed-phase high-performance liquid chromatography and mass spectrometry were obtained; after further determination of antibacterial activity, hemolytic activity, and stability of salt ions and protease, it was finally named anti-enzymatic pH-activated enhanced antimicrobial peptide DK-PCA.

[0029] Example 3

[0030] Assay for the biological activity of antimicrobial peptides:

[0031] 1. Determination of antibacterial activity: The minimum inhibitory concentration (MIC) of the peptide was determined using the standard microbroth dilution method. Logarithmic-phase bacteria were diluted to 10⁻⁶. 5 CFU / mL. 50 μL of peptides at different concentrations (final peptide concentration 1-64 μM) and an equal volume of bacterial suspension were added to 96-well plates. Wells containing only culture medium served as negative controls, and wells containing both bacteria and culture medium served as positive controls. The 96-well plates were then incubated at 37°C for 18-20 hours. The absorbance was measured at 492 nm using a microplate reader to determine the minimum inhibitory concentration (MIC). Three independent replicates were performed, with each replicate in duplicate. The results are shown in Table 2.

[0032] Table 2. Antibacterial activity (μM) of the antimicrobial peptide DK-PCA

[0033]

[0034] As shown in Table 2, the antimicrobial peptide DK-PCA can enhance its antibacterial activity against common pathogens after activation.

[0035] 2. Determination of hemolytic activity: Fresh human erythrocyte suspension was collected and diluted 10-fold with sterile PBS (pH 7 and 5, respectively). 50 μL of peptides at different concentrations (final peptide concentration 1-64 μM) and an equal volume of erythrocyte suspension were placed in each well of a 96-well plate. The positive control was human erythrocyte suspension treated with 0.1% Triton X-100, and the negative control was untreated human erythrocyte suspension. The 96-well plates were incubated at 37 ℃ for 1 hour. After centrifugation at 4 ℃ for 5 minutes (1000 g), 50 μL of supernatant was collected from the mixture and transferred to a new 96-well plate. The absorbance was measured at 570 nm using a microplate reader. The hemolysis rate was calculated using the following formula:

[0036] Hemolysis rate (%) = [(sample OD)] 570 —Negative control OD 570 ) / (Positive control OD 570 —Negative control OD 570 )]×100%

[0037] The minimum hemolytic concentration is the concentration at which the antimicrobial peptide causes a 10% hemolysis rate. The test results are shown in Table 3.

[0038] Table 3 Hemolytic Activity and Therapeutic Index of Antimicrobial Peptide DK-PCA

[0039]

[0040]

[0041] The antimicrobial peptide DK-PCA has a therapeutic index of 9.51 against Gram-positive bacteria at pH 7.00, but when activated at pH 5.00, the therapeutic index against Gram-negative bacteria increases from 1.00 to 22.63. It can be rapidly activated in acidic or inflammatory environments to enhance its antibacterial properties.

[0042] Example 4

[0043] Determination of the stability of antimicrobial peptide proteases:

[0044] To test the antiprotease activity of the antimicrobial peptide DK-PCA, an equal volume of 16 mg / mL protease was mixed with the antimicrobial peptide DK-PCA (2.56 mM) and incubated at 37°C for 1 hour. The peptide that had not been treated with the protease was used as a control. The minimum inhibitory concentration (MIC) was then determined using the method for determining the MIC (as described in the steps of Example 3).

[0045] Table 4. Minimum inhibitory concentration (µM) of DK-PCA after protease treatment against Escherichia coli 25922 and Staphylococcus aureus 25923.

[0046]

[0047] As shown in Table 4, the antibacterial activity of the three concentrations of protease against Escherichia coli 25922 and Staphylococcus aureus 25923 remained unchanged, indicating that the antimicrobial peptide DK-PCA has strong protease stability.

[0048] Determination of the stability of antimicrobial peptide salt ions:

[0049] For each concentration (300 mM NaCl, 9 mM KCl, 2 mM MgCl2, 16 μM ZnCl2, 12 μM NH4Cl, and 6 μM FeCl3), the subsequent steps were the same as those for the determination of antibacterial activity. The test results are shown in Table 5.

[0050] Table 5. Antimicrobial activity (μM) of antimicrobial peptide DK-PCA against Escherichia coli 25922 and Staphylococcus aureus 25923 under physiological saline conditions.

[0051]

[0052] The experimental results show that the antimicrobial peptide DK-PCA maintains good antibacterial activity even at physiological concentrations of salt ions.

Claims

1. An access to a pH-activatable antibacterial peptide DK-PCA against enzymatic hydrolysis, characterized in that: Its molecular formula is shown in formula (Ⅰ). Equation (Ⅰ).

2. The use of the protocatechuic acid-infused, pH-activated antimicrobial peptide DK-PCA according to claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.

3. The application according to claim 2, characterized in that: The Gram-negative bacteria mentioned are Pseudomonas aeruginosa, Salmonella typhimurium, or Escherichia coli.

4. The application according to claim 2, characterized in that: The Gram-positive bacteria mentioned are Staphylococcus aureus, Enterococcus faecalis, or Staphylococcus epidermidis.

5. The application according to claim 2, characterized in that: At pH 5.0, the antimicrobial peptide DK-PCA enhances its antimicrobial activity against Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli.

6. A drug suitable for treating and / or preventing infections caused by Gram-positive and / or Gram-negative bacteria, characterized in that, The drug contains a protocatechuic acid-integrated, pH-activated antimicrobial peptide DK-PCA as described in claim 1.

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