An antimicrobial peptide

Description

[0001] DESCRIPTION

[0002] AN ANTIMICROBIAL PEPTIDE

[0003] Technical Field

[0004] The present invention relates to an antimicrobial peptide which is effective against bacteria, fungi and parasites and resistant to proteases.

[0005] Background of the Invention

[0006] Antimicrobial drugs are used in the treatment of bacteria, fungi that cause diseases in humans and animals and infections caused by parasites in the structure of protozoa. Due to the intensive use of antibiotics in the treatment of bacterial infections, antibiotic -resistant bacteria have increased rapidly. Today, many antibiotic-resistant bacteria cause fatal infections. Similarly, resistance to antifungal drugs used in the treatment of fungal infections and antiparasitic drugs used in the treatment of parasites has become significantly widespread. Bacteria have managed to develop resistance to almost all antibiotics that have been developed for nearly a century. The development and introduction into clinical use of a new antibiotic takes years. Bacteria can develop resistance to many newly developed antibiotics in a very short time. Sometimes the resistance development can occur with a single mutation in the gene encoding the protein targeted by the antibiotic. This mutation leads to an amino acid change in the protein, which prevents the drug from binding to the protein target. Another important resistance mechanism is the inactivation of antibiotics by modification with enzymes produced by bacteria. One of the most important examples of these is betalactamase enzymes. These enzymes break down the beta-lactam ring in all penicillin and cephalosporin derivatives, thus rendering these drugs ineffective. Bacteria have developed thousands of types of beta-lactamase enzymes againstdozens of penicillin and cephalosporin derivatives that have been introduced into clinical use so far and have become resistant to almost all of them. Unless novel and effective antibiotics are developed against bacteria without the development of resistance, it is anticipated that millions of people will pass away from infections caused by antibiotic -resistant bacteria in the coming years. As bacteria develop resistance to every new antibiotic developed, the pharmaceutical industry has lost interest in developing new antibiotics.

[0007] In nature, there are antimicrobial effective peptides that are produced in many different species from insects to humans and that protect them against microorganisms. Although these antibiotics in peptide form have been present in nature for hundreds of millions of years, bacteria have not been able to develop resistance to them. One of the most important reasons for this is that the target of peptide antibiotics is cell membranes. While bacteria can rapidly develop resistance to antibiotics whose target is a protein with a mutation that causes a change in this protein and occurs in its gene, they cannot develop resistance to an antibiotic that targets cell membranes with such a mutation. In order to develop resistance to such an antibiotic, the structure of the fats forming the cell membrane must be altered. The synthesis of fats occurs as a result of a complex metabolic pathway. Changing this entire metabolic pathway requires significant genetic changes. As a bacterium that encounters a peptide antibiotic is killed before it has a chance to develop resistance, it is very difficult or impossible to develop resistance to these antibiotics.

[0008] The most important reason why natural peptide antibiotics cannot be made into drugs is that they are sensitive to proteases produced by both bacteria and human cells. Therefore, they have a very short life span and act in close proximity to the cells from which they are secreted. As they are broken down by proteases, they need to be continuously produced at an infection site. Many strategies have been developed to make peptide antibiotics resistant to proteases. Replacing the ends of the peptide with molecules other than amino acids, making the peptide circular,using unnatural derivatives of amino acids, using D-amino acids instead of L-amino acids are among these strategies. However, these strategies have not been very successful and the need for antimicrobial peptides resistant to proteases has continued.

[0009] The European patent document no. EP2994154, an application included in the state of the art, discloses a novel and potent antimicrobial peptide that overcomes the shortcomings of conventional antibiotics and that has improved properties over known antimicrobial peptides. It was found that the polypeptide PIO with the sequence LAREYKKIVEKLKRWLRQVLRTLR is highly effective against (drug-resistant) Gram-positive (e.g. Staphylococcus aureus) and Gram-negative (e.g. Pseudomonas aeruginosa) bacterial species as well as against fungi (e.g. Candida albicans and Aspergillus Niger in vitro). PIO is considerably more effective than any other peptide tested. In addition, PIO can prevent methicillin-resistant S. aureus biofilm formation on plastic as well as biotic (wounded 3-D human skin model) surfaces. Moreover, PIO neutralizes endotoxin lipoteichoic acid (LTA), peptidoglycan (PG) and lipopolysaccharides (LPS), thus reducing the proinflammatory response. PIO is an a-helix structure resulting in an amphipathic structure in which polar amino acids are located at one side of the helix and lipophilic amino acids at the opposite side. Addition of peptides in which proline was added to break the helix eliminated the activity of the molecule. This indicated that the amphipathic nature of the polypeptide is important for its biological activities. However, this invention does not mention that the peptide is shorter than 12 amino acids, is made up of only the R and L amino acids and can be written with a unique formula.

[0010] Summary of the Invention

[0011] An object of the present invention is to obtain an antimicrobial peptide which is effective against bacteria, fungi and parasites and resistant to proteases.Detailed Description of the Invention

[0012] “An Antimicrobial Peptide” realized to fulfil the objective of the present invention is shown in the figure attached, in which:

[0013] Figure 1. HPLC analysis of TN6 and D-TN6 peptides after being kept with proteinase K: A:TN6 peptide before proteinase K treatment: B: TN6 peptide after proteinase K treatment. C: D-TN6 peptide before proteinase K treatment. D: D-TN6 peptide after proteinase K treatment. It can be seen that the TN6 peptide is completely broken down by proteinase K, while the D-TN6 molecule completely preserves its structure.

[0014] The inventive antimicrobial peptide exhibits a wide range of antibacterial, antifungal and antiparasitic activities. The cathelicidin-type peptide, which is produced in different species in nature, was developed inspired by the amino acid sequences of antibiotics that exhibit similarities between species. It is found that the most effective of these inventive peptides on bacteria, fungi and parasites are those composed of arginine (R), a positively charged amino acid, and the hydrophobic amino acid leucine (L). It is found that these peptides starting with R should have 2 to 4 L following the R, and that after repeating this structure three times, it should be completed at the end without an additional amino acid or with R. It is found that for these peptides, which are composed of only two types of amino acids, to be resistant to proteases, either all amino acids must be D (Dextro) amino acids, or they must be composed of one D and one L (Levo) amino acid isomer in a D-L-D-L... sequence. These peptides, which can be easily synthesized in a peptide synthesizer device, have an alpha-helix structure in aqueous medium and are written with the general formula of [R(L2-4)3]Ro-i. Since peptides previously made entirely of L-amino acids are sensitive to proteases, all of them are synthesized from D-amino acids, or amino acids arranged in one D and one L form. Studies have shown that arginine as a positively charged amino acid showsmuch better antimicrobial activity than lysine and leucine as a hydrophobic amino acid shows much better antimicrobial activity than valine and isoleucine.

[0015] The use of D-amino acid instead of L-amino acid does not lead to a loss of activity, on the contrary, it provides an increase in activity. The different peptide antibiotics developed in the study and their activity against some Gram-negative and Gram-positive bacteria and Candida albicans, a fungal species, are shown in Table 1.

[0016] Table 1. Minimal inhibitory concentration (MIC) values of different peptide antibiotics in microgram / ml for various microorganisms. Amino acids shown in bold font are amino acids in D-form and amino acids shown in light font are amino acids in L-form. R: Arginine, L: Leucine, I: Isoleucine, A: Alanine, V: Valine are abbreviated names of amino acids. It shows that the lower the MIC value, the lower the concentration at which the antibiotic is effective.

[0017] Peptide Amino acid sequence S. aureus E. coli P. aeruginosa C. albicans TNI RLLRLLLLRLLR 4 2 32 0.5 D-TN1 RLLRLLLLRLLR (Sequence No 1) 4 8 4 1 TN3-amide RLLRLLRLLL 8 8 4 2 TN3-carboxy RLLRLLRLLL 16 4 32 1 D-TN3 RLLRLLRLLL (Sequence No 2) 8 2 4 0.25 TN3V1 RVLRVLRVLL 8 16 32 4 TN3V9 RVVRVVRVVV 16 64 256 128 TN6 RLLRLLLRLLR 2 2 8 0.5 D-TN6 RLLRLLLRLLR (Sequence No 3) 1 1 2 0.5 TN6I1 RIIRIIIRIIR 16 32 128 128 TN6I2 RILRILIRLIR 2 16 32 128 TN6A1 RALRALARALR 128 128 256 256 TN6A5 RAARAAARAAR >1024 1024 >1024 128 RTN6 RRLLRLLLRLLR 1 2 8 4 D / L-TN6 RLLRLLLRLLR 8 8 16 8 TN6(2) RLLRLLRLLLRLLRLLR 16 16 64 32 TN8 RLLRLLRLLLL 8 4 256 0.5

[0018] As seen in Table 1, it is seen that the antimicrobial activity of TN6, which is made of L-amino acids, increased when it was transformed into a peptide made of D-amino acids called D-TN6. This change also made the peptide resistant to proteases (Figure 1).

[0019] In the invention, one of the antimicrobial peptides is composed of D isomers of the RLLRLLLLRLLR (Sequence No. 1) amino acids. Another of the antimicrobial peptides is composed of D isomers of the RLLRLLRLLL (Sequence No. 2) amino acids. A further antimicrobial peptide is composed of D isomers of the RLLRLLLRLLR (Sequence No. 3) amino acids.

[0020] With the said invention, original peptide antibiotics resistant to proteases were developed. These peptides have been found to have low toxicity against human eukaryotic cells and therefore high safety indices in terms of side effects. These peptides have a wide range of activities. In addition to their activity against various types of Gram-negative and Gram-positive bacteria, they have also been found to be effective against fungi such as Candida species and Aspergillus species, which are in yeast and mold structure, and against parasites such as Leishmania species. Again, studies have shown that the peptide also exhibits high activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum beta-lactamase-producing Gram-negative organisms that cause nosocomial infections and are resistant to many antibiotics. A study performed on mice showed that D-TN6 accelerated the healing of a wound caused by Staphylococcus aureus and did not show any side effects during application.

[0021] Since the inventive peptides consist of only two amino acids and are very short, 12 amino acids or less, they are easy and inexpensive to produce. They can be easily produced in a few hours with an automated peptide synthesizer. The production method can be easily adjusted so as to produce large quantities when needed.Within these basic concepts; it is possible to develop various embodiments of the inventive “An Antimicrobial Peptide”; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.

Claims

CLAIMS1. An antimicrobial effective peptide; characterized in that it is composed of only arginine (R) and leucine (L) amino acids; these amino acids have a dextro (D) isomer and are defined by the general formula [R(L2-4)3]Ro-i.

2. An antimicrobial peptide according to Claim 1 ; characterized in that it is resistant to protease.

3. An antimicrobial peptide according to Claim 1; characterized in that it has an alpha-helix structure in aqueous medium.

4. An antimicrobial peptide according to Claim 1; characterized in that it has 12 amino acids or a smaller number of amino acids.

5. An antimicrobial peptide according to Claim 1; characterized in that it exhibits antibacterial, antifungal and antiparasitic effects.

6. An antimicrobial peptide according to Claim 1 ; characterized in that it is composed of D isomers of the RLLRLLLLRLLR (Sequence No. 1) amino acids.

7. An antimicrobial peptide according to Claim 1 ; characterized in that it is composed of D isomers of the RLLRLLRLLL (Sequence No. 2) amino acids.

8. An antimicrobial peptide according to Claim 1 ; characterized in that it is composed of D isomers of the RLLRLLLRLLR (Sequence No. 3) amino acids.

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