CN113292636A - Antibacterial hexapeptide and application thereof - Google Patents

Abstract

The invention discloses eight antibacterial hexapeptides and application thereof, wherein the amino acid sequences of the eight antibacterial hexapeptides are respectively as follows: R-W-W-R-W-W-NH₂，R‑W‑W‑R‑W‑F‑NH₂，R‑W‑W‑R‑F‑W‑NH₂，R‑W‑W‑R‑L‑F‑NH₂，R‑W‑W‑R‑F‑F‑NH₂，W‑L‑R‑W‑F‑R‑NH₂，W‑W‑L‑F‑R‑R‑NH₂，R‑W‑W‑R‑I‑W‑NH₂. The eight antibacterial hexapeptides have strong bactericidal performance, ensure smaller cytotoxicity and hemolysis, have low actual cost and can be used for preparing medicines for treating and/or preventing bacterial infection.

Description

Antibacterial hexapeptide and application thereof

Technical Field

The invention relates to the technical field of biology, and in particular relates to eight antibacterial hexapeptides and application thereof.

Background

Abuse of antibiotics has led to the development of bacterial resistance, making bacterial infections a significant safety concern once again threatening human health. Nowadays, diseases caused by drug-resistant bacteria are more and more serious, and the appearance of multiple drug-resistant bacteria and even super bacteria means that the current antibiotic treatment system is about to be broken down. In order to cope with this problem, development of novel antibacterial agents for the treatment of bacterial infections is urgently required.

The drug resistance of bacteria is mainly classified into 4 types:

1. producing an antibiotic that inactivates the enzyme to break down into the interior of the bacteria;

2. altering bacterial cell membrane permeability to prevent the entry of antibiotics;

3. forming efflux pumps to expel the antibiotic;

4. changing the drug target makes the antibiotic ineffective.

An antimicrobial peptide is an antimicrobial biomolecule having a specific amino acid sequence, generally carrying a positive charge and having a secondary structure such as a sigma-helix or a beta-sheet. The electropositivity of the antibacterial peptide enables the antibacterial peptide to be more easily adsorbed and gathered on the surface of a negatively charged bacterial cell membrane, and the secondary structure enables the antibacterial peptide to be inserted into a phospholipid bilayer of the bacterial cell membrane and self-assemble to form a pore structure to kill bacteria. Therefore, the bactericidal mechanism of the antibacterial peptide is to destroy the cell membrane of bacteria, which is not associated with the 4 aspects of the above-mentioned bacteria to acquire drug resistance, so that the antibacterial peptide is not easy to generate drug resistance and is considered as a substitute for the next generation antibiotics.

Currently, most of the antimicrobial peptides contain 10 to 60 amino acids or more (e.g., patent publication No. CN 111892646A, CN 109180794 a). However, the too long sequence of the antimicrobial peptide is likely to generate large biological toxicity on one hand and increase industrial production cost on the other hand, which limits the application of the antimicrobial peptide. However, the reduction of the length of the sequence of the antimicrobial peptide is likely to result in the reduction of the antimicrobial activity of the antimicrobial peptide.

Disclosure of Invention

Aiming at the technical problems and the defects in the field, the invention provides eight antibacterial hexapeptides, which have strong bactericidal performance, ensure smaller cytotoxicity and hemolysis and have low actual cost.

The amino acid sequences of the eight antibacterial hexapeptides are respectively one of the following:

antibacterial peptide 1: R-W-W-R-W-W-NH₂，

Antibacterial peptide 2: R-W-W-R-W-F-NH₂，

Antibacterial peptide 3: R-W-W-R-F-W-NH₂，

Antibacterial peptide 4: R-W-W-R-L-F-NH₂，

Antibacterial peptide 5: R-W-W-R-F-F-NH₂，

Antibacterial peptide 6: W-L-R-W-F-R-NH₂，

Antibacterial peptide 7: W-W-L-F-R-R-NH₂，

And (3) antibacterial peptide 8: R-W-W-R-I-W-NH₂。

The eight antibacterial hexapeptides can show remarkable bactericidal activity due to unique amino acid sequences, show low cytotoxicity and hemolysis at high concentration, and can be applied to medicines for treating diseases such as pneumonia and septicemia caused by bacteria.

The invention also provides application of the eight antibacterial hexapeptides in preparation of medicines for treating and/or preventing bacterial infection.

The invention also provides a medicament suitable for treating and/or preventing bacterial infection, which comprises at least one of the eight antibacterial hexapeptides.

The eight antibacterial hexapeptides provided by the invention have antibacterial/bactericidal universality, and the main action mechanism is to destroy the bacterial cell membrane. The eight antibacterial hexapeptides have positive net charges and can be adsorbed to bacterial cell membranes with negatively charged surfaces. When the eight antibacterial hexapeptides adsorbed on the surface of the cell membrane reach a certain concentration, the amphiphilic polypeptide can be inserted into the phospholipid layer of the cell membrane and self-assembled in the phospholipid layer to generate holes to destroy the cell membrane of bacteria, thereby killing the bacteria. Therefore, the eight antibacterial hexapeptides have broad antibacterial spectrum and have the effect of killing various bacteria.

The bacteria can be gram-positive bacteria and/or gram-negative bacteria, specifically staphylococcus aureus and/or escherichia coli and the like.

The medicament for treating and/or preventing bacterial infection can also comprise a pharmaceutically acceptable carrier and/or an auxiliary material.

Compared with the prior art, the invention has the main advantages that:

1) through a large number of researches, the invention discovers that the eight antibacterial hexapeptides with specific sequences have strong and broad-spectrum antibacterial/bactericidal activity and weak cytotoxicity and hemolytic toxicity, and can be applied to medicines for treating or preventing diseases caused by bacteria.

2) The antibacterial hexapeptide can be artificially synthesized (by adopting the conventional technology such as solid phase synthesis and the like), is convenient to operate, has short peptide chain, is very low in preparation cost, raw material cost and application cost, and has very good application prospect.

Drawings

FIGS. 1 to 8 are graphs showing the results of HPLC (a) and mass spectrometry (b) of antimicrobial peptides 1 to 8, respectively.

Detailed Description

The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.

Example 1

Solid-phase synthesis of antimicrobial peptides 1-8:

swelling of resin

0.6g of 2-Chlorotrityl Chloride Resin having a degree of substitution of 0.4mmol/g was weighed out, the Resin was put into a reaction tube, and methylene Chloride (DCM) (15mL/g) was added thereto, followed by shaking for 30 minutes.

Two, and then the first amino acid

The solvent was filtered off by suction through sand core, 3 times molar excess of Fmoc-L-Leu-OH amino acid was added, 10 times molar excess of Diisopropylethylamine (DIEA) was added, finally a small amount of Dimethylformamide (DMF) was added to dissolve it, and shaking was carried out for 1 hour. Wash alternately 6 times with DMF and DCM.

Third, deprotection

15mL of 20% piperidine DMF solution (15mL/g) was added for 5 minutes, and 15mL of 20% piperidine DMF solution (15mL/g) was removed again for 15 minutes.

Fourth, detection

The piperidine solution is pumped out, dozens of resin particles are taken out and washed by ethanol for three times, ninhydrin, KCN and phenol solution are added, and the mixture is heated for 5 minutes at 105-110 ℃ until the mixture turns dark blue to be a positive reaction.

Fifth, washing

DMF (10mL/g) was taken twice, methanol (10mL/g) was taken twice, and DMF (10mL/g) was taken twice.

Sixthly, condensation

And (3) tripling excessive protected amino acid (Fmoc-L-Gly-OH) and tripling excessive O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU), dissolving with DMF as little as possible, adding into a reaction tube, and immediately adding N-methylmorpholine (NMM) for ten times excessive. The reaction was carried out for 30 minutes.

Seven, washing

DMF (10mL/g) was used once, methanol (10mL/g) was used twice, and DMF (10mL/g) was used twice.

And eight, repeating the two-to-six steps, and sequentially connecting the amino acids in the sequence from right to left.

After nine and the last amino acid had been attached, deprotection was carried out and the resin was washed as follows.

DMF (10mL/g) twice, methanol (10mL/g) twice, DMF (10mL/g) twice, DCM (10mL/g) twice, and suction dried for 10 min.

Ten, cleavage of the polypeptide from the resin

Preparing cutting fluid (10/g) TFA 94.5%; 2.5 percent of water; 2.5 percent of EDT; and (3) TIS 1%.

Resin is filled into a flask or a centrifuge tube, the proportion of the resin to cutting fluid is 10mL/g, and the resin and the cutting fluid are vibrated at constant temperature for a period of time: 120 minutes.

Eleventh, blow-dry washing

Drying the lysate with nitrogen, separating with diethyl ether, washing with diethyl ether for six times, and volatilizing at room temperature. Thus obtaining the crude peptide sequence.

Twelve, purification of the polypeptide by HPLC

The method comprises the following specific operation steps:

1, taking 200mg of crude peptide and putting the crude peptide into a vessel. The mixture was dissolved down with 2-5mL of 50% acetonitrile in water. The sonication may be performed slightly for 2 minutes.

2, the solution was filtered through a 0.45 μm filter.

3, analysis: 3 μ L of crude product was analyzed by analytical grade HPLC. The mobile phase is water and acetonitrile for 30 minutes, gradient elution is carried out, HPLC is balanced for 5 minutes by using initial gradient and then sample injection is carried out, the initial gradient is 95 percent of water, the initial gradient is 5 percent of acetonitrile, the ending proportion is 5 percent of water, and the ending proportion is 95 percent of acetonitrile

4, preparation: and (5) preparing a sample injection for the dissolved sample. Preparative HPLC equilibrated for 10 min, starting gradient water 95%, acetonitrile 5%, ending gradient water 25%, acetonitrile 75% gradient time 40 min. The sample from the detector is collected.

5, identification: the collected samples were sampled for purity and MS identification.

Thirteen, finally, freeze-drying the purified solution to obtain a finished product.

Fourteen, sealing and packaging the white powdery polypeptide, and storing at-20 ℃.

FIGS. 1 to 8 are graphs showing the results of HPLC (a) and mass spectrometry (b) of antimicrobial peptides 1 to 8, respectively.

Example 2

And (5) detecting the antibacterial activity of the antibacterial peptide.

The following standard strains were purchased from Guangdong province microbial strain preservation center.

The antibacterial activity of the antibacterial hexapeptide synthesized in example 1 was examined by a 96-well plate method, and antibacterial peptides gramicidin and polymyxin approved by the FDA were used as gram-positive and gram-negative bacteria controls to evaluate the antibacterial activity of the antibacterial peptides 1 to 8.

The antibacterial activity of the antibacterial peptides was tested as follows.

First, staphylococcus aureus (s. aureus) and escherichia coli (e. coli) were cultured overnight on a sterilized TSA medium plate, and a single colony was picked and inoculated in a sterilized TSB medium at 37 ℃ and 150rpm for 18 hours for overnight culture.

And secondly, respectively preparing 1024 mu G/mL of the antibacterial peptide 1-8 by PBS, diluting the antibacterial peptide 1-8 to 1024, 512, 256, 128, 64, 32, 16, 8 and 4 mu G/mL by PBS according to a 2-fold serial dilution mode, sucking 100 mu L of the antibacterial peptide, adding the antibacterial peptide into 2-9 rows of the 96-well plate, and repeating 6 groups to add B-G rows. The cultured bacteria were diluted to 5X 10^5CFU/mL with TSB, and 100. mu.L of the diluted bacteria solution was added to well B2-D9 to prepare an experimental group. At this time, the concentration of the peptide to be measured (μ g/mL) was as follows:

number of rows	2	3	4	5	6	7	8	9
									Concentration of	512	256	128	64	32	16	8	4

100. mu.L of TSB solution was added to E2-G9 wells as a control, 100. mu.L of PBS and TSB were added to B10-D10 wells as negative controls, and 100. mu.L of PBS and diluted bacterial suspension were added to E10-G10 wells as positive controls. The 96-well plate was sealed with a sealing film, and then placed in a self-sealing bag at 37 ℃ and 150rpm for 18 hours for overnight culture. And (3) respectively measuring the OD600 values of the B2-D9 wells by using a microplate reader, wherein the minimum concentration corresponding to the minimum OD600 value is the MIC value of the bacteria corresponding to the antibacterial peptide.

Table 1 shows the minimum inhibitory concentrations (MIC, μ g/mL, 0-32: +, 32-512: + +) of antibacterial peptides 1-8, brevibacillin, polymyxin against various bacteria

TABLE 1

	Antibacterial peptide 1	Antimicrobial peptide 2	Antimicrobial peptide 3	Antimicrobial peptide 4	Antibacterial peptide 5
						S.aureus	+	+	+	+	+
E.coli	++	++	+	++	++
							Antibacterial peptide 6	Antibacterial peptide 7	Antibacterial peptide 8	Brevibacterium peptide	Polymyxin
S.aureus	++	+	+	+	N/A
						E.coli	++	++	++	N/A	+

The smaller the MIC in Table 1, the stronger the bacteriostatic ability. Table 1 shows that the eight antibacterial peptides of the invention have good bacteriostatic ability on gram-positive bacteria and gram-negative bacteria, and have strong effectiveness and broad spectrum.

Example 3

And (3) detecting the cytotoxicity of the antibacterial peptides 1-8.

This example was conducted to examine the cytotoxicity of the eight antimicrobial polypeptides synthesized in example 1 against human 3T3 cells, and compared with FDA-approved gramicidin.

And (3) detecting cytotoxicity:

first, NIH-3T3 cells were grafted to 96-well plates at a density of 5000/well and cultured for 24 hours to be sufficiently adherent.

And secondly, preparing 200 mu g/mL of antibacterial peptide 1-8PBS solution, and diluting the solution to be 100 mu g/mL by using DMEM medium. After removing the medium from the 96-well plate, culture was continued for 24 hours after adding 200. mu.L of 100. mu.g/mL of drug-containing medium, 5 replicates per group.

And thirdly, washing the drug culture medium by PBS 3 times after sucking off the drug culture medium, adding 20 mu L of thiazole blue (MTT) solution and 100 mu L of DMEM culture medium into each hole, and incubating for 4 hours. The supernatant was aspirated, and 150. mu.L of DMSO was added to dissolve the purple formazan crystals sufficiently. OD 490nm absorbance was read using a microplate reader.

The cytotoxicity is calculated by the formula:

cell viability ═ cell absorbance/PBS cell absorbance × 100%.

Table 2 shows the results of the cytotoxicity tests (drug concentration 100. mu.g/mL) of the antimicrobial peptides 1-8, and the control of Brevibacterium peptide and PBS, wherein the cell survival rate is 80-100%: +, < 80%: ++.

TABLE 2

	Antibacterial peptide 1	Antimicrobial peptide 2	Antimicrobial peptide 3	Antimicrobial peptide 4	Antibacterial peptide 5
						Cell survival rate	+	+	+	+	+
	Antibacterial peptide 6	Antibacterial peptide 7	Antibacterial peptide 8	Brevibacterium peptide	PBS
						Cell survival rate	+	+	+	++	+

In table 2, the higher the cell viability, the lower the cytotoxicity of the antimicrobial peptide. Table 2 shows that the antibacterial peptides 1-8 show obviously smaller cytotoxicity compared with the gramicidin, and provide strong support for patent drugs.

Example 4

And (3) in-vitro hemolytic detection of the antibacterial peptides 1-8.

This example was used to test the hemolytic activity of the eight antimicrobial hexapeptides synthesized in example 1 on erythrocytes and was compared with FDA-approved gramicidin. Blood samples were taken from sterile rabbit blood.

The hemolytic assay comprises the following steps:

first, 1mL of whole blood was collected, centrifuged at 1500rpm for 5 minutes, the supernatant was aspirated and repeated 3 times, and 20mL of PBS was added to prepare a blood cell solution.

Second, 200. mu.g/mL of the antimicrobial peptide 1-8 was prepared, 100. mu.L of the antimicrobial peptide was added to a 96-well plate to serve as an experimental group, 100. mu.L of Triton-X100(1 wt%) and 100. mu.L of PBS was added to the 96-well plate to serve as a positive control and a negative control, and 5 groups were repeated for each sample. To the well plates to which the samples were added, 100. mu.L of each of the blood cell solutions (at this time, the concentration of all the samples was halved, and the concentration of all the experimental groups was 100. mu.g/mL) was added, the well plates were sealed, filled in a self-sealing bag, and incubated at 37 ℃ for 1 hour at 150 rpm.

And thirdly, after the incubation is finished, sucking the mixed solution into a 1.5mL centrifuge tube, centrifuging at 1500rpm for 8 minutes, sucking the supernatant, and testing the OD 570nm value by using an enzyme-labeling instrument.

Hemolysis rate ═ a_m-A_n)/(A_p-A_n)

Wherein A is_mFor OD value of experimental group, A_pPositive control OD value, A_nNegative control OD values.

Table 3 shows the results of hemolytic assay of antimicrobial peptide 1-8 (drug concentration 100. mu.g/mL) with gramicidin and Triton-X100 as control, wherein the hemolytic rate is 0-30%: +, > 30%: ++.

TABLE 3

	Antibacterial peptide 1	Antimicrobial peptide 2	Antimicrobial peptide 3	Antimicrobial peptide 4	Antibacterial peptide 5
						Rate of hemolysis	+	+	+	+	+
	Antibacterial peptide 6	Antibacterial peptide 7	Antibacterial peptide 8	Brevibacterium peptide	Triton-X100
						Rate of hemolysis	+	+	+	++	++

A higher hemolysis rate indicates a greater toxicity of the antimicrobial peptide to blood cells. Table 3 shows that the antibacterial peptides 1-8 show obviously smaller hemolysis compared with the gramicidin, and provide strong support for patent drugs.

Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the above description of the present invention, and equivalents also fall within the scope of the invention as defined by the appended claims.

Sequence listing

<110> Zhejiang university

<120> antibacterial hexapeptide and application thereof

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<170> SIPOSequenceListing 1.0

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Arg Trp Trp Arg Trp Trp

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

Arg Trp Trp Arg Trp Phe

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<400> 3

Arg Trp Trp Arg Phe Trp

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Arg Trp Trp Arg Leu Phe

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<210> 5

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<213> Artificial Sequence (Artificial Sequence)

<400> 5

Arg Trp Trp Arg Phe Phe

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Trp Leu Arg Trp Phe Arg

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Trp Trp Leu Phe Arg Arg

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Arg Trp Trp Arg Ile Trp

1 5

Claims (8)

1. The antibacterial hexapeptide is characterized in that the amino acid sequence of the antibacterial hexapeptide is one of the following:

antibacterial peptide 1: R-W-W-R-W-W-NH₂，

Antibacterial peptide 2: R-W-W-R-W-F-NH₂，

Antibacterial peptide 3: R-W-W-R-F-W-NH₂，

Antibacterial peptide 4: R-W-W-R-L-F-NH₂，

Antibacterial peptide 5: R-W-W-R-F-F-NH₂，

Antibacterial peptide 6: W-L-R-W-F-R-NH₂，

Antibacterial peptide 7: W-W-L-F-R-R-NH₂，

And (3) antibacterial peptide 8: R-W-W-R-I-W-NH₂。

2. Use of an antibacterial hexapeptide according to claim 1 for the preparation of a medicament for the treatment and/or prevention of bacterial infections.

3. Use according to claim 2, wherein the bacteria are gram-positive and/or gram-negative bacteria.

4. The use of claim 2, wherein the medicament further comprises a pharmaceutically acceptable carrier.

5. The use of claim 2, wherein the medicament further comprises a pharmaceutically acceptable excipient.

6. A medicament suitable for the treatment and/or prevention of bacterial infections, characterized in that it comprises at least one of the antibacterial hexapeptides according to claim 1.

7. The medicament according to claim 6, wherein the bacteria are gram-positive and/or gram-negative bacteria.

8. The medicament of claim 6, further comprising a pharmaceutically acceptable carrier and/or adjuvant.

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