JP2013523661A - Low erythrocyte lytic antimicrobial peptide, pharmaceutical composition and use thereof - Google Patents

Abstract

The present invention discloses a low erythrocyte lytic antimicrobial peptide.
[Solution]
The antimicrobial peptide of the present invention has an amino acid sequence represented by (P ₁ ) _M (A ₁ X ₁ X ₂ ) _N (P ₂ ) _X. In the formula, P ₁ is a basic amino acid, A ₁ is an aromatic amino acid or alanine, X ₁ is a basic amino acid or nonpolar amino acid, X ₂ is a basic amino acid or nonpolar amino acid, and P ₂ is a basic amino acid. Furthermore, the numbers of M and X are each independently 0 to 2, and the number of N is 2 to 4. When N> 2, _{A 1} may be a alanine (Ala), the number of the alanine (Ala) is (N-2) does not exceed pieces. The antimicrobial peptide not only has good antibacterial activity, but also has low erythrocyte lytic characteristics.
[Selection] Figure 1

Description

  The present invention relates to antimicrobial peptides, and particularly to low erythrocyte lytic antimicrobial peptides, pharmaceutical compositions and uses thereof.

    With the emergence of bacterial strains that have acquired drug resistance to conventional antibiotics, research on new therapeutic reagents including many animal-derived antimicrobial peptides is underway. It is now known that these antimicrobial peptides play a fairly important role in the defense mechanism inherent in the host. Plants, insects, amphibians and mammals can all have antimicrobial peptides, and the antibiotic properties of these antimicrobial peptides can counteract bacteria, fungi and even some viruses. This type of antimicrobial peptide binds to lipids (> 95 percent) and can quickly separate lipid bilayers and disrupt membrane integrity. On the other hand, it is also possible to increase the small short conduction in the parallel lipid bilayer of bacteria, partially depolarize the cell membrane and destroy the original potential gradient.

  The protective function of this type of antimicrobial peptide in host defense has been demonstrated by Drosophila experiments. If the expression level of the antimicrobial peptide decreases after Drosophila is infected with the microorganism, the survival rate may be greatly reduced. The defective bactericidal killing of pulmonary cystic fibrosis patients and mice in mammals can also demonstrate the protective function of this type of antimicrobial peptide in host defense.

  Antimicrobial peptides expressed in mammals are classified into cysteine-rich defensins (α, β defensins) and various cathelicidins. Cathelicidins contain highly conserved signal sequences and the pro-region cathelin and an antimicrobial sequence with variability located in the C-terminal region. The cathelicidin family is further divided into three groups depending on the amino acid composition and structure. The first group has amphipathic α-helical peptides, such as LL-37, CRAMP, SMAP-29, PMAP-37, BMAP-27 and BMAP-28. The second group is peptides rich in arginine / proline or tryptophan, such as Bac5, Bac7, PR-39 and indolicidin. The third group is a peptide containing cysteine, for example, protegrins. Many cathelicidins have a unique elastase cleavage site between the negatively charged caterin region and the positively charged C-terminal region. This proteolytic reaction at the site of action has been observed in bovine or porcine neutrophils and lymphocytes and is essential for antimicrobial activity. All of these antibacterial peptides have a broad antibacterial potency, but their therapeutic potential is greatly limited due to their different hemolytic activity on erythrocytes. Accordingly, the present invention provides low erythrocyte lytic antimicrobial peptides. This antimicrobial peptide not only has good antibacterial activity, but also has low erythrocyte lytic characteristics.

  Antimicrobial peptides are usually small molecules (<5 kDa) and have a wide range of activities and important relevant properties against microbial infections in the host defense system. Therefore, it can be a starting point for designing a low molecular weight antibacterial complex. In addition, the combination of hydrophobic and charged regions has the potential to fold into an amphiphilic structure and this property is also related to the ability to hydrolyze cell membranes. However, these antibacterial peptides have a wide range of antibacterial potency, but have different hemolytic activity on erythrocytes, thus limiting their therapeutic potential.

  Accordingly, it is an object of the present invention to provide low erythrocyte lytic antimicrobial peptides, compositions and uses thereof.

In the present invention, the technical means employed for solving the problems of the prior art are low erythrocyte lytic antimicrobial peptides, (P ₁ ) _M (A ₁ X ₁ X ₂ ) _N (P ₂ ) _X It has the amino acid sequence shown by. Wherein P ₁ is a basic amino acid, including being selected from the group consisting of arginine (Arg) and lysine (Lys). A ₁ is an aromatic amino acid or alanine, including being selected from the group consisting of tryptophan (Trp), phenylalanine (Phe) and alanine (Ala). X ₁ is a basic amino acid or a non-polar amino acid, and is selected from the group consisting of arginine (Arg), lysine (Lys), valine (Val), leucine (Leu), alanine (Ala) and isoleucine (Ile). including. X ₂ is a basic amino acid or a non-polar amino acid, and is selected from the group consisting of arginine (Arg), lysine (Lys), valine (Val), leucine (Leu), alanine (Ala) and isoleucine (Ile). including. P ₂ is a basic amino acid, including being selected from the group consisting of arginine (Arg) and lysine (Lys). Further, the numbers of M and X are each independently 0 to 2, and when N> 2, A ₁ may be alanine (Ala), and the number of residues of this alanine (Ala) is (N-2). Do not exceed.

  By designing peptides with activity that resists strains of clinical importance for the modification of primary and secondary structure and understanding some important features in this type of peptide structure, the activity of natural antimicrobial peptides Or improve toxicity. This type of antibacterial peptide is a new type of antibacterial peptide with a wide range of uses, and it can be used to make antibiotics with sequences rich in tryptophan. It is an antibacterial peptide that combines antibacterial activity and low hemolytic properties and is designed to resist a wide range of Gram-positive and negative bacteria, protozoa, fungi and enveloped human autoimmune deficiency virus (HIV).

  In a preferred embodiment, the antimicrobial peptide of the invention is selected from the group consisting of SEQ ID NOs: 1-7. The antimicrobial peptide is linear or cyclic in shape and may further comprise acetylation, amidation, formylation, hydroxylation, lipid modification, methylation or phosphorylation modifications.

  By the technical means employed in the present invention, the antimicrobial peptide of the present invention can be used to improve the activity or toxicity of the natural antimicrobial peptide. In the future, it can be applied to provide antibiotics, pharmaceutical compositions or other clinical antimicrobial uses. Furthermore, it has antibacterial activity and low hemolytic properties, and in the future, it can more widely resist microorganisms such as gram positive / negative bacteria, protozoa, fungi and viruses.

  In a preferred embodiment, the antimicrobial peptides and pharmaceutically acceptable carriers of the present invention can be prepared as antimicrobial pharmaceutical compositions. The carrier includes excipients, diluents, thickeners, fillers, binders, disintegrants, lubricants, oleaginous or non-oleaginous bases, surfactants, suspending agents, gelling agents, auxiliary Agents, preservatives, antioxidants, stabilizers, colorants or fragrances. The pharmaceutical composition is in the form of an embedded body, immersion liquid, internal liquid, patch, powder, tablet, injection liquid, suspension, liquid for external use, drops, liniment, coating agent, absorbent, cream, oil It is a medicine, ointment, mud or gel and can be used in mammals by oral, transdermal absorption, injection or inhalation.

  Specific examples employed in the present invention will be further described with reference to the following examples and the accompanying drawings.

FIG. 1 shows the results of measuring erythrocyte lysis of melittin, Pem-2252L and Pem-2254L using human red blood cells.

Peptide design, synthesis, purification and properties The antimicrobial peptides of the present invention and their nomenclature are as shown in Table 1 below. Each amino acid is represented by a three letter alphabet.

C: cyclic, L: linear

  All cyclic and linear peptides are synthesized by solid phase synthesis. Using standard Fmoc, ie (N- (9-fluorenyl) methyloxycarbonyl group), PAL resin (5- (4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy) -valeric acid-methylbenzhydride). Chemically synthesized on a ruamine resin). The Fmoc protecting group of these resins was removed with 20% piperidin / dimethylformamide (piperidine / DMF). The reaction time was approximately 1-1.5 hours, after which it was determined by water and ninhydrin test. Subsequently, 95% trifluoroacetic acid (TFA) was added and mixed for 1 to 1.5 hours to excise the raw peptide from the resin and further purified by reverse phase high pressure liquid chromatography. Here, a semi-preparative C18 reverse phase column was used for reverse phase high pressure liquid chromatography. As the mobile phase for purification, the mixing ratio of acetonitrile and water was changed in gradient with time. Different molecules were separated by different mixing ratios of acetonitrile and water. Detection was performed at wavelengths of 225 and 280 nanometers, and the flow rate was 4 ml / min. The main peptide product obtained was determined for molecular weight by fast atom bombardment mass spectrometry (FAB-MS). The purity of each peptide was determined through a reverse phase high pressure liquid chromatography column.

Measurement of peptide activity by in vitro The measurement of peptide activity by in vitro carried out in this example employed a minimum inhibitory concentration (MIC) test. That is, the minimum peptide concentration required to inhibit or reduce the growth of the organism being measured. The strains used in this example are Escherichia coli (E. coli, ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus aureus (ATCC 29213).

The bacterial culture cultured overnight was diluted with Meuller-Hinton medium to give an inoculum containing 10 ⁵ colonies per milliliter. Furthermore, different concentrations of the polypeptide were added to the diluted bacterial solution. After culturing at 37 ° C. for 18 hours, the turbidity in a phosphate buffer (PBS, pH 7.4) was analyzed. The minimum inhibitory concentration of each peptide was measured three times at different times, and the average value was determined. The results are as shown in Table 2. These results confirmed that Pem-2251L and Pem-2254L have favorable antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. In particular, the minimum inhibitory concentration values of the Pem-2254L peptide against Escherichia coli, Pseudomonas aeruginosa, and S. aureus were 1.565, 1.565, and 3.125 (μg / ml), respectively. In addition, modifications of primary and secondary structures that are generally performed on the premise that the peptide does not affect its biological activity, such as acetylation, amidation, formylation, hydroxylation, lipid modification, methylation or phosphorus Regarding the modification of oxidation, the peptide activity was further measured in vitro. The results can also show its antibacterial properties.

  Subsequently, for the two polypeptides, Pem-2251L and Pem-2254L, which are particularly favorable results in the tests of Table 2, Bacillus subtilis, Staphylococcus in 1 × phosphate buffer. epidermidis, Staphylococcus aureus, Bacillus pumilus, Bacillus cereus, Pseudomonas aeruginosa, and various strains of E. coli Inhibitory concentration values were measured. The results are as shown in Table 3, and the minimum inhibitory concentration values for various different strains were Pem-2251L and Pem-2254L, and it was confirmed that the antibacterial activity was still good.

Analysis of membrane permeability The outer membrane permeability of peptides was determined by measuring the absorption of 1-N-phenylnaphthylamine (NPN). NPN of intact E. coli cells shows weak fluorescence absorption in a liquid environment, but a strong absorption value in a hydrophobic environment. Thus, NPN is hydrophobic and the outer membrane permeability can be measured directly. In general, under normal circumstances, E. coli NPN absorption is small or even not absorbed. In the presence of compounds with several membrane permeabilities, such as ethylenediaminetetraacetic acid, polymyxin B, neomycin or antimicrobial peptides, NPN can partially penetrate the bacterial outer membrane and absorb fluorescence. The value increases. The procedure of this embodiment will be briefly described as follows. One milliliter of the bacterial solution cultured overnight was added to 50 ml of the culture solution, and cultured with shaking at 37 degrees until the OD ₆₀₀ corresponded to 0.4 to 0.6, and the cells were separated at 3500 rpm for 10 minutes. Washing with buffer and suspending the centrifuged cells until OD ₆₀₀ = 0.5. One milliliter was taken in a transparent cuvette and measured for 2-5 seconds. After adding 20 microliters of 0.5 mmol NPN, mixing and equalizing, it was measured again for 2-5 seconds. Furthermore, 10 μl of antimicrobial polypeptide having a different concentration was added, mixed to be equalized, and the fluorescence absorption value was measured (1 to 5 minutes). Of these, the fluorescence absorption value varied with the polypeptide concentration. The polypeptide concentration required to NPN absorption reaches 50% of the maximum value was defined as P _50. Experimental results indicated that Pem-1001L and Pem-2251L peptides have the ability to bind to membranes in the measurement of NPN absorption. The data is as shown in Table 4.

Measurement of erythrocyte lysis The erythrocyte lysis of melittin, Pem-2252L and Pem-2254L was measured using human red blood cells (hRBC). Among them, melittin is a peptide extracted from venom, has high solubility in red blood cells, and was used as a control group in the experimental design. The experimental process consists of washing red blood cells containing ethylenediaminetetraacetic acid (hRBC) three times with phosphate buffer (PBS, pH 7.4) (800 g, 10 min), and finally suspending in PBS, 10% (v / After dilution in v), 50 microliters were dispensed into each tube. Thereafter, the peptide dissolved in PBS buffer was added to 50 μl of 10% hRBC solution, incubated at 37 ° C. for 1 hour (final erythrocyte concentration of 5% v / v), and centrifuged at 800 g for 10 minutes. Pre-treated erythrocytes and different concentrations of peptides were cultured, and their hemolytic ratios (also referred to as lysis ratios) were obtained (of which lysis ratio 0 was PBS instead of antimicrobial peptide, and lysis ratio was 100 Are experimental results using 1% Triton X-100 instead of antimicrobial polypeptide). The results are as shown in Table 5 and FIG. 1, and it was shown that Pem-2252L has a significantly lower hemolytic property to erythrocytes compared to other antibacterial peptides. The dissolution rates at 5 μg / ml, 50 μg / ml and 400 μg / ml concentrations were 0.45%, 1.52% and 16.35%, respectively.

  In summary of the above examples, the antimicrobial peptide of the present invention can improve the activity or toxicity of the natural antimicrobial peptide. In the future, it can be used to provide antibiotics, pharmaceutical compositions or other clinical antibacterial applications. Furthermore, it has antibacterial activity and low hemolytic properties, and in the future, it can more widely resist microorganisms such as gram positive / negative bacteria, protozoa, fungi and viruses.

  The antimicrobial peptide of the present invention further contains a pharmaceutically acceptable carrier, and can be used for preparing an antimicrobial pharmaceutical composition. The carrier is an excipient, diluent, thickener, filler, binder, disintegrant, lubricant, oily or non-greasy base, surfactant, suspending agent, gelling agent, auxiliary Agents, preservatives, antioxidants, stabilizers, colorants or fragrances. The pharmaceutical composition is composed of embedded body, immersion liquid, internal liquid, patch, powder, tablet, injection liquid, suspension, liquid for external use, drops, liniment, coating agent, absorbent, cream. Oil, ointment, plaster or gel. Used in mammals by oral, transdermal absorption, injection or inhalation.

  From the above examples, it can be seen that the antimicrobial peptide provided by the present invention has industrial utility value, so that the present invention meets the important conditions of the patent. However, the above description is only a description of a preferred embodiment of the present invention, and those skilled in the art can make various other improvements based on the above description. However, these modifications still belong to the spirit of the invention and the patent scope defined below.

Claims (14)

(P ₁ ) _M (A ₁ X ₁ X ₂ ) _N (P ₂ ) An antimicrobial peptide having an amino acid sequence represented by _X ,
Wherein P ₁ is selected from the group consisting of basic amino acids;
A ₁ is selected from the group consisting of an aromatic amino acid or alanine;
X ₁ is selected from the group consisting of basic amino acids or nonpolar amino acids;
X ₂ is selected from the group consisting of basic amino acids or non-polar amino acids;
P ₂ is selected from the group consisting of basic amino acids;
Furthermore, the number of M and X is 0-2 respectively independently, and the number of N is 2-4, The antimicrobial peptide. When N> 2, well A ₁ is alanine, the number of said A ₁ is N-2 pieces not exceeding, antimicrobial peptide of claim 1.   The antimicrobial peptide according to claim 1, wherein the antimicrobial peptide is selected from the group consisting of SEQ ID NOs: 1-7.   The antimicrobial peptide of Claim 1 whose shape of the said antimicrobial peptide is linear.   The antimicrobial peptide of Claim 1 whose shape of the said antimicrobial peptide is cyclic | annular.   2. The antimicrobial peptide of claim 1, wherein the antimicrobial peptide further comprises acetylation, amidation, formylation, hydroxylation, lipid modification, methylation or phosphorylation modification.   Use of an antimicrobial peptide that resists microorganisms, wherein the antimicrobial peptide is the antimicrobial peptide of claim 1 and is used for an individual.   The use according to claim 7, wherein the antimicrobial peptide has low erythrocyte lytic properties against the red blood cells of the individual.   8. Use according to claim 7, wherein the microorganism is a gram positive bacterium, a gram negative bacterium, a protozoan, a fungus or a virus.   8. Use according to claim 7, wherein the individual is a mammal.   8. Use according to claim 7, which is administered to the individual by oral, transdermal absorption, injection or inhalation.   An antimicrobial pharmaceutical composition comprising the antimicrobial peptide of claim 1 and a pharmaceutically acceptable carrier.   The carrier is an excipient, diluent, thickener, filler, binder, disintegrant, lubricant, oleaginous or non-oleaginous base, surfactant, suspending agent, gelling agent, auxiliary The pharmaceutical composition according to claim 12, which is an agent, preservative, antioxidant, stabilizer, colorant or fragrance.   The dosage form of the pharmaceutical composition is an embedded body, immersion liquid, oral liquid, patch, powder, tablet, injection liquid, suspension, liquid for external use, drops, liniment, coating agent, absorbent, cream, oil. The pharmaceutical composition according to claim 12, which is a medicine, ointment, mud or gel.