A multifunctional active peptide and its application

Abstract

The present invention discloses a group of multifunctional active peptides whose amino acid sequences are GLLHLLHHLLHH and GLLKLLHHLLHH, respectively. The present invention also provides applications for anti-infection, anti-tumor, calcification promotion, and hydroxyapatite adsorption. The present invention has the advantage over the prior art in that the multifunctional active peptides provided by the present invention can simultaneously address the different characteristics of complex diseases. Based on their anti-infection, anti-tumor, calcification promotion, and hydroxyapatite adsorption properties, they can be used to treat a variety of diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a multifunctional active peptide and application thereof. Background Art

[0002] Fluoride, a classic anti-caries agent, can prevent caries through remineralization and inhibition of bacterial metabolism, but its antibacterial effect is relatively poor. Common broad-spectrum oral antimicrobial drugs, such as chlorhexidine, while highly effective, also indiscriminately kill symbiotic bacteria that contribute to caries, disrupting the oral microbial ecosystem. Other anti-caries drug development focuses solely on antimicrobial or remineralization, failing to simultaneously combat cariogenic bacteria and protect tooth enamel.

[0003] In cancer treatment, tumor cells are also human cells, and effective methods are needed to differentiate the effects of drugs on tumor tissue from normal tissue. Furthermore, microbial infection is also associated with tumor progression, and intratumoral microbes are also at the forefront of research. Some studies have clearly shown that intratumoral microbes have a positive effect on tumor suppression. Therefore, while fighting tumors, we must also pay attention to anti-infection.

[0004] In application number 2021111181354, entitled "Antimicrobial Peptide and Its Application", an antimicrobial peptide and its application in a drug for regulating the microecology of bacteria in dental caries are disclosed. It discloses an anti-caries peptide that can target cariogenic bacteria, and the inventors conducted further research based on this. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a group of simple active peptides with multiple functions and their application in simultaneously addressing different characteristics of complex diseases.

[0006] In order to solve the above technical problems, the present invention provides a technical solution: a group of multifunctional active peptides, characterized in that the polypeptide sequence is GLLHLLHHLLHH-NH2 or GLLKLLHHLLHH-NH2.

[0007] In addition, the present invention discloses a pharmaceutically acceptable salt of a multifunctional peptide, wherein the pharmaceutically acceptable salt includes hydrochloride, sulfate, acetate, methanesulfonate, succinate, fumarate, citrate, malate or organic amine salt.

[0008] In addition, the present invention discloses a multifunctional peptide derivative, and the polypeptide derivative is an ester.

[0009] In addition, the present invention discloses a pharmaceutical composition comprising the above-mentioned multifunctional active peptide, polypeptide derivative and pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0010] The preparation is a liquid preparation, a solid preparation or a semisolid preparation, the liquid preparation is a solution or an injection, the solid preparation is a tablet or a capsule, and the semisolid preparation is an ointment or a gel.

[0011] In addition, the present invention discloses a tooth care composition comprising the group of multifunctional active peptides of the present invention.

[0012] In addition, the present invention discloses an antibacterial pharmaceutical composition comprising a group of multifunctional active peptides described in the present invention, wherein the bacteria include at least one of Streptococcus mutans, Lactobacillus casei, Lactobacillus fermentum, Actinomyces viscosus, Actinomyces naeslundii, Streptococcus sanguinis, Streptococcus gattii, Streptococcus salivarius, and Streptococcus salivarius.

[0013] In addition, the present invention discloses the application of a group of multifunctional peptides described in the present invention in anti-infection, anti-tumor, calcification promotion and hydroxyapatite adsorption.

[0014] The advantages of the present invention compared with the prior art are:

[0015] The multifunctional active peptides provided by the present invention can simultaneously address the different characteristics of complex diseases, as shown by:

[0016] Utilize antibacterial properties to exert anti-infective and anti-pathogenic microorganism effects;

[0017] Utilize pH responsiveness to target patient microenvironments such as the acidic microenvironment of caries and tumors to differentiate between normal flora / tissues and pathogenic flora / tissues;

[0018] Utilize calcium ions combined with the adsorption capacity of hydroxyapatite to achieve mineralization and in situ caries prevention effects;

[0019] Utilize the ability to inhibit tumor cells to exert anti-tumor effects;

[0020] There is hope that this group of simple peptides can be used to treat a variety of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the binding of LH12 and LK12 to calcium ions.

[0022] Figure 2 Schematic diagram of the hydroxyapatite adsorption capacity of LH12 and LK12.

[0023] Figure 3 Schematic diagram of the ability of LH12 and LK12 to resist pathogenic bacteria contamination after adsorption on hydroxyapatite.

[0024] Figure 4 This is a schematic diagram of the biosafety of LH12 and LK12.

[0025] Figure 5 Schematic diagram of the tumor suppressor ability of LH12 and LK12. DETAILED DESCRIPTION

[0026] The present invention will be described in further detail below with reference to the accompanying drawings.

[0027] 1. A multifunctional active peptide, denoted as LH12 and LK12 in the present invention, with amino acid sequences of GLLHLLHHLLHH and GLLKLLHHLLHH, and its C-terminus is amidated to enhance antibacterial activity.

[0028] 2. The preparation steps of the antimicrobial peptide are as follows

[0029] 1) Fmoc-His(Trt)-Wang Resin was selected as the resin (support);

[0030] 2) Fully swell the resin with DCM;

[0031] 3) Use appropriate concentration of DBLK (piperidine + DMF) to remove the Fmoc-protecting group;

[0032] 4) Wash with DMF several times to remove DBLK;

[0033] 5) Weigh appropriate condensing agent and activating agent (HBTU, NMM) and the second Fmoc-protected amino acid at the C-terminus (Fomc-Leu-OH) for coupling;

[0034] 6) Ninhydrin test method is used to ensure that the connection is relatively complete;

[0035] 7) Wash with DMF to remove various residual residues and activator condensing agent;

[0036] 8) Coupling is performed according to the GLLHLLHHLLHH amino acid sequence, referring to steps 3 to 7 above;

[0037] 9) After all amino acids are connected, remove the final Fmoc-protecting group using the method of steps 3 to 4;

[0038] 10) using TFA cleavage solution to remove the resin and amino acid protecting groups to obtain a crude product;

[0039] 11) Mass spectrometry was performed to confirm the product was correct (LH12 molecular weight 1438.76 was consistent with the theoretical value);

[0040] 12) The crude product is sent for purification and separation to improve its purity.

[0041] 3. pH responsiveness and antibacterial activity of LH12 and LK12

[0042] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of LH12 and LK12 at different pH levels were measured to determine whether the peptides exhibited pH-responsiveness and antibacterial activity. The microplate dilution method was used to test the MIC and MBC at different pH levels against cariogenic bacteria such as Streptococcus mutans UA159, the Gram-positive pathogen Staphylococcus aureus, and the Gram-negative pathogen Escherichia coli.

[0043] 1) Single colonies of Streptococcus mutans were selected from the collection and cultured in 10 mL of BHI liquid medium in a constant-temperature anaerobic incubator (80% N2, 10% H2, 10% CO2) at 37°C overnight. Single colonies of Staphylococcus aureus and Escherichia coli were cultured in LB liquid medium in a constant-temperature incubator at 37°C overnight.

[0044] 2) Pipette 20 μL of Streptococcus mutans culture medium into 10 mL of BHI liquid medium in a 37°C constant temperature anaerobic culture tank (80% N2, 10% H2, 10% CO2), and culture Staphylococcus aureus and Escherichia coli culture medium in LB liquid medium in a 37°C constant temperature culture tank aerobically. After two generations of subculture, the mid-logarithmic growth stage of the culture medium was diluted to 1×106 CFU / mL with the corresponding citric acid / phosphate buffered medium with pH 5.5 and 7.2 for later use.

[0045] 3) Antimicrobial peptides were diluted 2-fold into a 96-well plate, with 10 μL per well. 90 μL of the corresponding buffered culture medium containing 1 × 106 CFU / mL was added to each well to achieve a final concentration of 2-128 μg / mL of antimicrobial peptides. Sterile water was used as a negative control.

[0046] 4) The 96-well plate was placed in a 37°C constant temperature anaerobic culture tank (80% N2, 10% H2, 10% CO2) or a constant temperature culture tank for aerobic culture for 24 hours.

[0047] 5) MIC is the lowest antimicrobial concentration in the clear wells of the plate.

[0048] 6) Pipette 50 μL of the bacterial solution from the clear well onto a BHI or LB agar plate and culture in an anaerobic incubator (80% N2, 10% H2, 10% CO2) at 37°C or aerobic overnight in a constant temperature incubator for 24 h. The minimum bactericidal concentration (MBC) is the minimum bactericidal concentration at which no colonies grow on the plate.

[0049] As shown in Table 1, both LH12 and LK12 exhibited strong antibacterial activity against a wide range of pathogens, with the modified LK12 exhibiting even greater antibacterial activity. Furthermore, acidic pH enhanced the antibacterial activity of both LH12 and LK12. This demonstrates that LH12 and LK12 possess both antibacterial activity and pH-responsiveness.

[0050]

[0051]

[0052] Table 1: Antibacterial activity of LH12 and LK12 at different pH values

[0053] 4. Pathogen-targeting ability of LH12 and LK12

[0054] The protocol used cariogenic bacteria such as Streptococcus mutans UA159, Lactobacillus casei ATCC393, Lactobacillus fermentum A1753, Actinomycetes viscosus ATCC15987, and Actinomycetes naeslundiiJCM8349, as well as commensal bacteria such as Streptococcus sanguinis JCM5708, Streptococcus gordonii DL1, Streptococcus mitis ATCC6249, and Streptococcus salivarius ATCC27945. The microplate dilution method was used for drug sensitivity testing to detect the antibacterial ability of LH12 against cariogenic bacteria and commensal bacteria.

[0055] 1) Pick a single colony from the collection and place it in 10 mL of BHI liquid culture medium, and culture it overnight in a constant temperature anaerobic culture tank (80% N2, 10% H2, 10% CO2) at 37°C.

[0056] 2) Pipette 20 μL of bacterial solution into 10 mL of BHI liquid medium and subculture for two generations in a constant temperature anaerobic culture tank (80% N2, 10% H2, 10% CO2) at 37°C. Then, take the bacterial solution at the mid-logarithmic growth stage and dilute it with BHI medium to 1×106 CFU / mL for later use.

[0057] 3) Add the antimicrobial peptide to a 96-well plate using a two-fold dilution method, with 10 μL per well. Add 90 μL of BHI medium containing 1 × 106 CFU / mL to each well to achieve a final concentration of 2-128 μg / mL. Sterile water was used as a negative control.

[0058] 4) The 96-well plate was placed in a 37°C constant temperature anaerobic culture tank (80% N2, 10% H2, 10% CO2) and cultured for 24 hours.

[0059] MIC is the lowest antimicrobial peptide concentration in the clear wells of the plate.

[0060] 5) Pipette 50 μL of the bacterial solution from the clear well onto a BHI agar plate and incubate overnight at 37°C in an anaerobic incubator (80% N2, 10% H2, 10% CO2) for 24 hours. The MBC is the lowest peptide concentration at which no colonies grow on the plate.

[0061] 6) As shown in Table 2, LH12 and LK12 had lower MICs and MBCs against cariogenic bacteria, but higher MICs and MBCs against commensal bacteria, indicating that LH12 and LK12 had selective antibacterial activity.

[0062]

[0063] Table 2 MIC and MBC of LH12 and LK12 against pathogenic bacteria and commensal bacteria

[0064] 5. Calcium ion binding and hydroxyapatite adsorption capacity of LH12 and LK12

[0065] 1) I-TASSER software was used to perform molecular simulations of the ion binding abilities of LH12 and LK12.

[0066] 2) Soak the hydroxyapatite sheets with 64 μg / mL of rhodamine-labeled LH12 and LK12 for 30 minutes, then rinse with sterile water to remove unadsorbed fluorescent peptides. Observe the adsorption of the fluorescent peptides using a confocal microscope.

[0067] 3) Pick a single colony from the collection and place it in 10 mL of BHI liquid culture medium, and culture it overnight in a constant temperature anaerobic culture tank (80% N2, 10% H2, 10% CO2) at 37°C.

[0068] 4) Pipette 20 μL of bacterial solution into 10 mL of BHI liquid medium and subculture for two generations in a constant temperature anaerobic culture tank (80% N2, 10% H2, 10% CO2) at 37°C. Then, take the bacterial solution at the mid-logarithmic growth stage and dilute it to 1×106 CFU / mL with BHIS medium containing 1% sucrose for later use.

[0069] 5) Soak the hydroxyapatite sheets in 64 μg / mL of LH12 and LK12 for 30 minutes, then rinse with sterile water to remove unadsorbed peptides. Place the hydroxyapatite sheets and 1 mL of BHIS bacterial solution in a 24-well plate and incubate in a 37°C constant temperature anaerobic culture tank (80% N2, 10% H2, 10% CO2) for 24 hours before observation using a scanning electron microscope.

[0070] like Figure 1 As shown, both LH12 and LK12 have calcium ion binding ability, and the binding site is located in the N segment of the polypeptide; Figure 2 The laser confocal microscopy shows that both LH12 and LK12 can adsorb hydroxyapatite; Figure 3 As shown, the peptide can be adsorbed on the surface of hydroxyapatite, preventing pathogenic bacteria from contaminating the hydroxyapatite surface. The above suggests that LH12 has the ability to bind calcium ions and adsorb to hydroxyapatite, which can prevent pathogenic bacteria from adhering to and forming biofilms, and has calcification potential and in situ antibacterial ability.

[0071] 6. Biosafety and anti-tumor activity of LH12 and LK12

[0072] 1) Defibrinated sheep blood was centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded and washed with PBS. Repeat the centrifugation until the supernatant was clear to obtain red blood cells. The red blood cells were resuspended in ten volumes of PBS to obtain a red blood cell suspension.

[0073] 2) LH12 and LK12 were diluted 2-fold and added to EP tubes, 100 μL per tube. 900 μL of red blood cell suspension was added to each well to achieve a final antimicrobial peptide concentration of 8-128 μg / mL. 100 μL of 1% Triton X-100 was used as a positive control, and 100 μL of PBS was used as a negative control. Incubate at 37°C for 1 hour. After centrifugation at 1000 rpm for 10 minutes, the supernatant was collected and the OD540 was measured. Hemolysis rate (%) = (OD 540处理组 -OD 540阴性对照组 ) / (OD 540 Triton X-100 -OD 540阴性对照组 )×100%.

[0074] 3) Logarithmic-phase tumor cells were trypsinized and diluted in RPMI-1640 medium supplemented with 10% fetal bovine serum to prepare a cell suspension of 1×105 cells / mL. 100 μL of the cell suspension was seeded into each well of a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. After the cells adhered, the culture medium was discarded and 100 μL of culture medium containing LH12 and LK12 was added. A blank control group (culture medium only) and a control group (cells and culture medium only) were also established.

[0075] After 72 hours of continuous culture, 100 μL of culture medium containing CCK-8 was added, incubated for 3 hours, and OD450 was measured. Inhibition rate % = (OD 450处理组 -OD 450空白组 ) / (OD 450对照组 -OD 450空白组 )×100%

[0076] like Figure 4 As shown in the figure, different lowercase letters represent significant differences, P < 0.05. The hemolytic toxicity of LH12 and LK12 is extremely low, and the biosafety is good at 64 μg / mL and below.

[0077] like Figure 5 As shown, different lowercase letters represent significant differences, P < 0.05, showing significant inhibitory effects on tumor cells within this biosafety concentration range, indicating that LH12 and LK12 have anti-tumor capabilities and cause little damage to normal tissues while fighting tumors.

[0078] The contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0079] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A use of a multifunctional peptide, wherein the amino acid sequence of the multifunctional peptide is GLLHLLHHLLHH-NH2, characterized in that: Application in the preparation of drugs that utilize calcium ion binding and hydroxyapatite adsorption capacity to achieve mineralization and in-situ caries prevention effects.

2. A multifunctional peptide, characterized in that: The amino acid sequence of the multifunctional peptide is GLLKLLHHLLHH-NH2, which is a carbon-terminal amidation product of the amino acid sequence.

3. A pharmaceutically acceptable salt of a multifunctional peptide, characterized in that: The multifunctional peptide is the multifunctional peptide according to claim 2; The pharmaceutically acceptable salts include hydrochloride, sulfate, acetate, methanesulfonate, succinate, fumarate, citrate, and malate.

4. A derivative of a multifunctional peptide, characterized in that: The multifunctional peptide is the multifunctional peptide according to claim 2, and the polypeptide derivative is an ester.

5. A tooth care composition, characterized in that Comprising the multifunctional peptide according to claim 2.

6. An antibacterial pharmaceutical composition, characterized in that: Comprising the multifunctional peptide according to claim 2, the pharmaceutically acceptable salt of the multifunctional peptide according to claim 3, or the derivative of the multifunctional peptide according to claim 4; The bacteria include at least one of Streptococcus mutans, Lactobacillus casei, Lactobacillus fermentum, Actinomyces viscosus, Actinomyces naeslundii, Streptococcus sanguinis, Streptococcus grisea, Streptococcus salivarius, and Streptococcus salivarius.

7. An antibacterial pharmaceutical composition according to claim 6, characterized in that: It also contains pharmaceutically acceptable carriers or excipients.

8. A preparation containing the antibacterial pharmaceutical composition according to claim 6, characterized in that: The preparation is a liquid preparation, a solid preparation or a semisolid preparation. The liquid preparation is a solution or an injection, the solid preparation is a tablet or a capsule, and the semisolid preparation is an ointment or a gel.

9. Use of the multifunctional peptide according to claim 2 in the preparation of a drug for antibacterial infection, mineralization and in situ caries prevention by utilizing calcium ion binding and hydroxyapatite adsorption capacity; The bacteria include at least one of Streptococcus mutans, Lactobacillus casei, Lactobacillus fermentum, Actinomyces viscosus, Actinomyces naeslundii, Streptococcus sanguinis, Streptococcus grisea, Streptococcus salivarius, and Streptococcus salivarius.

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