LPS-targeted branched peptide and its application

Abstract

The present invention relates to the field of polypeptide technology, and more particularly to an LPS-targeting branched peptide and its applications. The present invention provides a branched peptide with targeted antagonistic activity against LPS. This branched peptide has the ability to kill Escherichia coli and simultaneously neutralize the endotoxin LPS, achieving dual bactericidal and anti-inflammatory effects. Therefore, this branched peptide can be used to treat infections and related diseases caused by Escherichia coli and LPS, and has promising application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, in particular to an LPS-targeted branched peptide and applications thereof. Background Art

[0002] Escherichia coli is a major zoonosis that can cause diarrhea in humans and animals, and in severe cases, can lead to other complications and even death. It is a significant foodborne pathogen, carrying significant risks and lacking effective preventive measures. Antibiotic treatment can exacerbate hemolytic uremic syndrome. Lipopolysaccharide (LPS), a major component of the E. coli cell membrane, is a major pathogenic factor that harms humans and animals, inducing the inflammatory cascade and sepsis. Antibiotics, the primary treatment for infectious diseases, effectively kill bacteria but also release large amounts of LPS into the bloodstream, increasing plasma LPS levels by 20-1000 times and exacerbating the inflammatory response.

[0003] Therefore, developing drugs that can target and antagonize LPS activity and block LPS stimulation of effector cells from the source is expected to open up new avenues for the prevention and treatment of diseases induced by LPS. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides an LPS-targeted branched peptide, the structural formula of which is shown in Formula 1.

[0005] Formula 1

[0006] The molecular formula of the LPS-targeting branched peptide is C 294 H 443 N 87 O 54 S2.

[0007] The molecular weight of the LPS-targeting branched peptide is 6124.45 Da.

[0008] The above LPS-targeting branched peptide is abbreviated as (FKAWRWAWRMKKLAAPS)2-K-(RVQGRWKVRASFFK).

[0009] The present invention screened a large number of LPS-targeting polypeptide molecules through in vitro LPS neutralization ability experiments, including polypeptide molecules such as LL-37, Protegrin-1, CATH-2, PMAP-36 and Indolicidin, and finally found that the above-mentioned LPS-targeting branched peptides have the strongest ability to target and bind to LPS.

[0010] Furthermore, the present invention provides a reagent or kit containing the LPS-targeting branched peptide.

[0011] Furthermore, the present invention provides an antibacterial agent containing the LPS-targeting branched peptide.

[0012] Preferably, the antimicrobial agent is used to inhibit Escherichia coli.

[0013] Furthermore, the present invention provides a medicine containing the LPS-targeting branched peptide.

[0014] In some embodiments, the drug product includes a pharmaceutically acceptable excipient.

[0015] In some embodiments, pharmaceutically acceptable excipients include fillers, excipients, lubricants, wetting agents, diluents, and the like.

[0016] In some embodiments, the drug formulation may be a solid formulation (eg, powder, granules, capsules, tablets, etc.) or a liquid formulation (eg, oral solution, etc.).

[0017] In some embodiments, the pharmaceutical product is used for at least one of the following:

[0018] (1) Inhibit Escherichia coli;

[0019] (2) Targeted antagonism of LPS;

[0020] (3) Reduce the level of LPS-induced cellular inflammatory factors;

[0021] (4) Treatment of sepsis.

[0022] Furthermore, the present invention provides the use of the LPS-targeted branched peptide in preparing products; the products include reagents, kits, antimicrobial agents or medicines.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a branched peptide with targeted LPS antagonism. This peptide not only kills E. coli but also neutralizes the endotoxin LPS, achieving dual bactericidal and anti-inflammatory effects. Therefore, this branched peptide has promising application prospects for treating infections and related diseases caused by E. coli and LPS. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This figure shows the test results of the LPS-targeting branched peptide DL7's ability to neutralize LPS.

[0026] Figure 2 This is a test result diagram of the LPS-targeted branched peptide DL7 regulating LPS-induced cellular inflammatory factors.

[0027] Figure 3 This is a test result of the therapeutic effect of LPS-targeted branched peptide DL7 on septic mice. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, those without specifying specific techniques or conditions are described in accordance with the techniques or conditions described in the literature in this area, or are carried out according to product specifications. Reagents or instruments used are not specified by manufacturer and are conventional products that can be purchased through regular channels.

[0029] Example 1

[0030] In this example, the LPS-targeting branched peptide DL7 was synthesized using solid-phase synthesis using a 12-channel semi-automatic peptide synthesizer. (FKAWRWAWRMKKLAAPS)2-K-(RVQGRWKVRASFFK) was synthesized. The synthesized peptide was purified using a reverse-phase HPLC C18 column (purity >95%). ESI-MS mass spectrometry confirmed the molecular weight of the LPS-targeting branched peptide DL7 to be 6124.45 Da.

[0031] Example 2

[0032] This example measured the antibacterial spectrum of the LPS-targeting branched peptide DL7. The pathogens used in this example were obtained from the China Veterinary Culture Collection (CVCC), the China Industrial Culture Collection (CICC), the American Type Culture Collection (ATCC), and clinical isolates. The specific strains are listed in Table 1.

[0033] The minimum inhibitory concentration (MIC) of the LPS-targeted branched peptide DL7 was determined according to the method developed by the Clinical and Laboratory Standards Institute (CLSI), with slight modifications based on specific circumstances. The details of the procedure are as follows:

[0034] A single colony of the test strain was picked into MH liquid medium, cultured overnight at 37°C with shaking at 250 rpm for activation, and then transferred to MH liquid medium and cultured until the logarithmic growth phase (OD 600 nm = 0.4~ 0.6), and then prepared into 10 5 CFU / mL of bacterial solution was added into a 96-well sterile cell culture plate, 90 μL per well.

[0035] LPS-targeted branched peptide DL7 was diluted with PBS by a two-fold serial dilution method, with 10 μL per well, resulting in final concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / mL, respectively. A negative control group consisted of PBS instead of the test bacterial solution containing LPS-targeted branched peptide DL7, and a blank control group consisted of sterile MH liquid medium. Three replicates were used for each treatment.

[0036] Incubate the culture plate at 37°C for 16-18 hours until the negative control wells become visibly turbid. The lowest concentration that completely inhibits bacterial growth is the MIC value of the LPS-targeted branched peptide DL7 against the test strain. If skipped wells or inconsistent results between replicates occur, retest.

[0037] The results are shown in Table 1. The MIC of LPS-targeted branched peptide DL7 against Escherichia coli is 2-4 μM, and it has no antibacterial effect on Gram-positive bacteria, indicating that LPS-targeted branched peptide DL7 has a targeted inhibitory effect on Escherichia coli.

[0038] Table 1 Antibacterial spectrum of LPS-targeted branched peptide DL7 (μM)

[0039]

[0040] Example 3

[0041] This example determined the in vitro neutralization ability of the LPS-targeting branched peptide DL7. The steps are as follows:

[0042] Escherichia coli 0111:B4 LPS (40 μg / mL) and BODIPY-TR-cadaverine (BC) (10 μM) were mixed in equal proportions in 50 mM Tris buffer (pH = 7.4) and added to a 96-well black plate (180 μL / well). Various peptide concentrations (20 μL) were added to each well, and fluorescence was measured by spectrofluorometry at room temperature (excitation wavelength 580 nm, emission wavelength 620 nm). Changes in relative fluorescence were recorded. Polymyxin and ampicillin were used as controls.

[0043] like Figure 1 As shown, the fluorescence intensity induced by the LPS-targeted branched peptide DL7 was significantly higher than that of the control group, indicating that the LPS-targeted branched peptide DL7 has an excellent ability to neutralize LPS.

[0044] Example 4

[0045] This example measures the ability of the LPS-targeted branched peptide DL7 to regulate LPS-induced cellular inflammatory factors. The steps are as follows:

[0046] The test drugs were diluted to 40 μg / mL and 80 μg / mL (650 μL for each concentration) using DMEM medium. The medium in the 48-well plate was discarded, and after washing twice with PBS, the test drugs were added at 200 μL per well. Each concentration was replicated in triplicate. The blank control group (CK) was treated with only DMEM medium, the negative control (LPS) was treated with DMEM medium, the positive control (Poly) was treated with polymyxin B, and the experimental group was treated with LPS-targeted branched peptide DL7. After 1 hour, 1 μg / mL of LPS was added to all groups except the blank control group. The cells were incubated for 6 hours, and the supernatant was removed (try not to aspirate the cells). The levels of inflammatory factors TNF-α, IL-6, and NO were detected using an ELISA kit.

[0047] The results are as follows Figure 2 As shown, DL7 can significantly reduce the increase in IL-6 levels caused by LPS, especially 80 μg / mL DL7 can neutralize LPS and inhibit LPS from activating cytokines TNF-α, IL-6 and NO, reducing the damage of LPS to the body.

[0048] Example 5

[0049] This example tests the therapeutic effect of LPS-targeted branched peptide DL7 on LPS-induced sepsis in mice. The steps are as follows:

[0050] Twenty SPF-grade Balb / c mice (6 weeks old) were purchased and divided into four groups of five mice each. They were challenged with 2.5 mg / kg LPS to establish a sepsis model. Two hours later, the experimental group was treated with an intraperitoneal injection of 20 mg / kg DL7, while the untreated group ( E. coli ) received no treatment. The positive control group was injected with an equal volume of polymyxin. The negative control group (CK) consisted of normal mice injected with an equal volume of saline. The survival of the mice was observed for seven consecutive days, and a survival curve was plotted.

[0051] The survival rate of mice Figure 3 As shown in Figure 3 . At the same dose, the survival rate of mice treated with the LPS-targeted branched peptide DL7 was significantly higher than that of mice treated with polymyxin. Therefore, the LPS-targeted branched peptide DL7 has a strong ability to target and antagonize LPS in vivo, significantly improving the survival rate of septic mice and demonstrating a favorable therapeutic effect against sepsis.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An LPS-targeted branched peptide for targeted inhibition of Escherichia coli, characterized in that: Its structural formula is shown in Formula 1: ； Formula 1.

2. The LPS-targeted branched peptide for targeted inhibition of Escherichia coli according to claim 1, characterized in that: Its molecular formula is C 294 H 443 N 87 O 54 S2.

3. The LPS-targeted branched peptide for targeted inhibition of Escherichia coli according to claim 1, characterized in that: Its molecular weight is 6124.45 Da.

4. A reagent or kit comprising the LPS-targeting branched peptide for targeted inhibition of Escherichia coli according to any one of claims 1 to 3.

5. An antibacterial agent comprising the LPS-targeted branched peptide for targeted inhibition of Escherichia coli according to any one of claims 1 to 3.

6. The antibacterial agent according to claim 5, characterized in that The antibacterial agent is used for targeted inhibition of Escherichia coli.

7. A medicine containing the LPS-targeting branched peptide for targeted inhibition of Escherichia coli according to any one of claims 1 to 3.

8. The medicine according to claim 7, characterized in that The medicine includes pharmaceutically acceptable excipients.

9. The medicine according to claim 7 or 8, characterized in that The medicine is used for at least one of the following aspects: (1) Targeted inhibition of Escherichia coli; (2) Targeted antagonism of LPS; (3) Reduce the level of LPS-induced cellular inflammatory factors; (4) Treatment of sepsis.

10. Use of the LPS-targeted branched peptide for targeted inhibition of Escherichia coli according to any one of claims 1 to 3 in the preparation of products; the products include reagents, kits, antimicrobial agents or medicines.

Images