Antimicrobial peptides designed by localization-based motif combination

Abstract

The invention relates to unique peptide sequences with high antimicrobial activity and stability as well as low hemolytic activity, generated by a unique strategy defined by determining the specific motifs of peptides with high antimicrobial activity in the antimicrobial peptide database by a structure-activity relationship based approach and combining the determined motifs with a localization-based approach taking into account the N-terminal, C-terminal or center position in the peptide.

Description

[0001] DESCRIPTION

[0002] ANTIMICROBIAL PEPTIDES DESIGNED BY LOCALIZATION-BASED MOTIF COMBINATION

[0003] Technical Field

[0004] The invention relates to the localization-based motif combination method and peptides developed by this method.

[0005] State of the Art

[0006] Today, antibiotics are used in the treatment of infections. However, the rapid increase in drugresistant pathogenic bacteria and fungi has increased the need for new antimicrobial agents. Antimicrobial peptides (AMP) act on various microbial targets through multiple mechanisms, so the development of resistance is less common compared to traditional antibiotics. Antimicrobial peptides are known to be the most promising alternative agents against rapidly increasing antibiotic resistance. Antimicrobial peptides are natural defense agents produced by many organisms, from bacteria to humans. To date, a large number of antimicrobial peptides produced by many organisms have been identified. These are effective against bacteria, fungi and even viruses. The most important limiting factors in naturally obtained peptides are low antimicrobial effect, limited activity spectrum, high cytotoxicity and high hemolytic activity. In order to overcome the limitations associated with peptides derived from natural sources, researchers have attempted to modify or de novo design peptide sequences. The peptide sequences obtained by modification of natural peptide sequences or de novo designed peptide sequences can be produced by a method such as solid phase peptide synthesis of peptides, recombinant production in a suitable host organism or cell-free protein expression method.

[0007] Descriptions of the Figures

[0008] Figure 1. Bioinformatics Analysis Results of Peptides

[0009] Figure 2. Antimicrobial Activity (Minimum Inhibitory Concentration, MIC) Results of Peptides

[0010] Figure 3. Scanning Electron Microscopy (SEM) image of morphological change in E. coli O157:H7 (ATCC 43894) cell treated with peptides

[0011] Figure 4. Percentage of hemolysis at different concentrations of peptides. Brief Description of the Invention

[0012] There are many different approaches to design of antimicrobial peptides in the literature, such as de novo design, rational design, and linguistic methods. The peptide sequences of the invention were designed by discovering the specific motifs of peptides with high antimicrobial activity in the antimicrobial peptide databases with a structure-activity relationship-based approach and combining the discovered motifs with a localization-based approach, taking into account the their the N-terminal, C-terminal or central position in the original peptide. Therefore, a unique strategy was followed in the design of the peptides of the invention, and as a result, unique peptide sequences were obtained.

[0013] Detailed Description of the Invention

[0014] The present invention relates to peptides containing the following amino acid sequences:

[0015] SEQ. 1 : RVKRFWKKLGQLGKAAVKAVTNA and

[0016] SEQ. 2: GWKKWLRQGIHKAIKVADAIGK

[0017] The 3-letter presentations of the SEQ. 1 AND SEQ. 2 peptides are presented below:

[0018] SEQ. 1 : ArgValLysArgPheTrpLysLysLeuGlyGlnLeuGlyLysAlaAlaValLysAlaValThrAsnAla SEQ . 2 : Gly TrpLy sLy sTrpLeu ArgGlnGly II eHi sLy s Al all eLy s Vai Al a Asp Al all eGlyLy s

[0019] The amino acid sequences of the invention are peptides designed for use as therapeutic agents for the prevention and / or treatment of diseases caused by bacterial, viral, and fungal pathogens. The design of the peptides subject to the invention was performed by motif selection based on structure-activity relationship and with a localisation-based motif combination. Thus, peptides with high antimicrobial activity and stability as well as low cytotoxicity and hemolytic activity are designed within the scope of the invention. The peptides of the invention (SEQ. 1 and SEQ. 2) obtained by this new design strategy are therapeutic agents for the treatment of microbial infections.

[0020] Unlike antibiotics, the development of resistance to antimicrobial peptides by microorganisms is much more difficult and takes longer. The use of antimicrobial peptides as an alternative to antibiotics will reduce the use of antibiotics and prevent the development of resistance to existing antibiotics. Therefore, the peptides of the invention will contribute to the prevention of the development of resistance due to the use of antibiotics by reducing the use of antibiotics. Among antimicrobial peptides, LL-37, which is effective against bacteria, and histatin and its derivatives, which are effective against fungi, stand out. However, the development of resistance that may occur due to long-term use of these human-derived peptides as drugs will render these peptides, which are part of the human immune system, ineffective against pathogens. Therefore, since the amino acid sequence of the peptides of the invention is designed, it is safer than natural peptides of human origin. In addition, since the peptides of the invention have low toxicity, they offer a potentially safe treatment option. The peptides of the invention will be able to reduce tissue damage and side effects during the infection duration by reducing inflammation caused by microbial infections. In addition, peptides have the effect of regulating and modulating the immune system and can contribute to the fight against infections by affecting the activation and function of immune cells. Therefore, the peptides of the invention are potentially an effective option in combating microbial infections.

[0021] Motif-based design methods to develop novel antimicrobial peptides typically involve the identification and optimization of specific sequence motifs known to be important for antimicrobial activity. Approaches to novel antimicrobial peptides design use computational methods such as molecular dynamics simulations, machine learning algorithms, or bioinformatics tools to analyze the structure and activity of peptides. These methods help to identify important structural characteristics or motifs (specific amino acid sequences in peptide which have essential for antimicrobial activity) associated with antimicrobial activity. Peptide databases allow screening of large numbers of peptides with different amino acid sequences and discovery of new motifs not found in natural peptides.

[0022] A peptide design strategy based on the combination of localization-based motifs was followed in the design of the peptides of the invention. Accordingly, peptides effective against pathogenic bacteria, fungi, viruses, etc. were obtained from many AMP databases (APD, DADP, DBAASP, DRAMP, YADAMP, etc.). After each peptide was divided into 3 equal parts, the N-terminal regions, C-terminal regions, and the central regions each of them were grouped separately. The amino acid sequence of each group was loaded into the motif discovery tool of the MEME program (Meme Suit 5.5.1 Motif Discovery Tool) and motifs of a certain length (e.g. 7-10 aa) were determined. The motifs were scored according to their E- values, and the top 10 lowest-scoring motifs in each motif set of N-terminal, central, and C- terminal positions were selected. These selected motifs were then combined according to their specific positions in the peptide sequence to generate potential peptide candidates. The antimicrobial activity of the selected peptides was scored using bioinformatics tools such as CAMPR3 (http: / / www.camp.bicnirrh.res.in / ) and hemolytic activity HemoPl (https: / / webs.iiitd.edu.in / raghava / hemopi / index.php) modules, and 2 of the peptides with the highest antimicrobial activity and the lowest hemolytic activity were selected. The net charge of the SEQ. 1 peptide was 7 and its hydrophobicity was 21%, the net charge of the SEQ. 2 peptide was 5 and its hydrophobicity was 31.5% (Figure 1). The hydrophobicity of antimicrobial peptides influences their antimicrobial activity and specificity. Hydrophobicity is related to the extent to which the peptide acts on the lipid bilayer, and the increase in hydrophobicity increases antimicrobial activity. Antimicrobial peptides first interact with negatively charged cell membranes thanks to their net charge. By changing the net charge of the peptides, their antimicrobial activities can be modified. The high cationic charge causes an increase in the electrostatic interaction between the peptides and the membrane. Searches in AMP databases (e.g. DB AASP database) have shown that the net charges of 80% of peptides with high antimicrobial activity varies between +3 - +8. These results showed that net charge is related to high antimicrobial activity. Since the net charges of the peptides of the invention are in this range (+3 - +8), they are ideal antimicrobial peptides when the above criteria are considered.

[0023] The main advantage of the peptides of the invention is that they have antibacterial / antiviral / antifungal / antiparasitic / antibiofilm and anticarcinogenic effects. They are particularly effective in overcoming drug-resistant microorganisms due to their high selectivity, low cytotoxicity and broad spectrum of antimicrobial activity, compared to several conventional antibiotics that have developed resistance. These peptides can be used alone or in combination with other antimicrobial agents or antibiotics. Thus, these peptides, which have a different mechanism of action from other antimicrobial components, as they exert a synergistic effect with them, can be used together against antibiotic-resistant microbial strains, and the development of new resistance mechanisms can be prevented. Such antimicrobial peptides can be used as antibacterial / antiviral / antifungal / antiparasitic and anticarcinogenic drugs. The peptides of the invention can be used in the treatment of infections directly or in combination with other antimicrobial compounds and in commercial forms such as capsules, ampoules, and powders. The peptides of the invention can also be used as antimicrobial coating materials to prevent biofilms formed as a result of colonization of microorganisms. Therefore, it can be applied to many surfaces such as medical equipment and devices, prostheses, stents, implants, and surgical materials to reduce the risk of infection.

[0024] Since the peptides of the invention have high antimicrobial effect and low cytotoxicity, they can be included in various formulations. Examples of cosmetic formulations in which peptides are included include pastes, masks, gels, skin lotions, massage creams, kinds of toothpaste, cleansing creams, cleansing foams, sprays, creams, and soaps. The peptides of the invention can be used as antimicrobial food additives in foods. In such uses, the peptide can be added directly to the food or applied to food contact surfaces. Examples of their use as food additives include meat and dairy products, confectionery, beverages, vitamin complexes, baby foods, and bakery products. The peptides according to the invention can be incorporated into animal feed as active ingredients. The use of these antimicrobial peptides as feed additives can improve the weight gain, meat quality, milk yield and immunity of livestock by replacing existing antibiotics already in use. The peptides of the invention may also be used as pesticides. In this way, bacteria, fungi, and viral pests in agricultural areas can be controlled.

[0025] It is possible to produce the peptides of the invention using a method such as solid phase peptide synthesis, recombinant production in a suitable host organism, or cell-free expression method. The solid phase peptide synthesis method was used in the production of peptides, which are the subject of the patent application numbered WO2016024296A2, for use in laboratory analysis.

[0026] The minimum inhibitory concentration (MIC) test results of the peptides of the invention are given in Figure 2. Accordingly, it was determined that the SEQ. 1 peptide showed an inhibitory effect at the level of 4-16 pg / mL against the gram-negative bacteria tested, 4-32 pg / mL against the gram -positive bacteria, and 16-128 pg / mL against the pathogenic fungus strains. It was determined that the SEQ. 2 peptide showed an inhibitory effect at the level of 4-32 pg / mL against tested gram-negative bacteria, 4-128 pg / mL against gram-positive bacteria and 16-64 pg / mL against pathogenic fungus strains.

[0027] The SEM (Scanning Electron Microscopy) images of the morphological change in the E. coli O157:H7 (ATCC 43894) cell treated with the SEQ. 1 and SEQ. 2 peptides of the invention are given in Figure 3. Accordingly, it was observed that significant cavities, roughness and depressions occurred on the cell surface after the treatment of E. coli O157:H7 (ATCC 43894) cells at the 1XMIC level of both peptides.

[0028] The hemolytic activity results of the peptides of the invention are given in Figure 4. Accordingly, it was found that the SEQ.l and SEQ. 2 peptides exhibited 3% and 31% hemolytic activity, respectively, against mouse erythrocytes at a concentration of 512 pg / mL. Therefore, it has been determined that the peptides of the invention act on erythrocytes at concentrations much higher than the minimum inhibitory concentrations (MICs) at which they inhibit pathogenic microorganisms.

Claims

CLAIMS1. Antimicrobial peptide, characterized in comprising one of the following sequencesRVKRFWKKLGQLGKAAVKAVTNA (SEQ. 1) or GWKKWLRQGIHKAIKVADAIGK (SEQ. 2).

2. The peptide according to Claim 1, characterized in comprising sequenceRVKRFWKKLGQLGKAAVKAVTNA (SEQ. 1).

3. The peptide according to Claim 1, characterized comprising sequenceGWKKWLRQGIHKAIKVADAIGK (SEQ. 2).

4. Composition comprising an amino acid sequence according to Claim 2.

5. Composition comprising an amino acid sequence according to Claim 3.

6. The composition according to claim 4 or 5 for use in the treatment of infections and for killing pathogenic microorganisms7. Method of synthesizing peptides according to Claim 1, characterized in comprising localization-based motif combination design.