Proline-rich antimicrobial peptide

Abstract

The invention relates to a peptide that can be used as an alternative to existing antibiotics for the inhibition of pathogenic microorganisms and the treatment of infections due to these microorganisms, contributing to the prevention of resistance development arising from antibiotic use by reducing antibiotic use, is safer compared to human-derived natural peptides, has a very low toxicity compared to existing antimicrobial peptides, and also reducing tissue damage and side effects during infection by reducing inflammation due to microbial infections, also having the potential to regulate the immune system and immunomodulation, which will contribute to fight infections by influencing the activation and function of immune cells.

Description

[0001] DESCRIPTION

[0002] PROLINE-RICH ANTIMICROBIAL PEPTIDE

[0003] Technical Field

[0004] The invention relates to a peptide that can be used as an alternative to existing antibiotics in the inhibition of pathogenic microorganisms and the treatment of infections due to these microorganisms, contributes to the prevention of the development of resistance due to the use of antibiotics by reducing the use of antibiotics, is safer compared to natural peptides of human origin, has very low toxicity compared to existing antimicrobial peptides, and also reduces tissue damage and side effects in the infection process by reducing inflammation due to microbial infections, regulates the immune system and has immunomodulation potential to contribute to the fight against infections by affecting the activation and function of immune cells.

[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 to antimicrobial peptides by microorganisms, unlike antibiotics, is much more difficult and takes longer. Antimicrobial peptides used as an alternative to antibiotics have the potential to prevent the development of resistance to existing antibiotics by reducing the use of antibiotics.

[0007] The AMPs detected so far, "LL-37", which is effective against bacteria, and "histatin" and its derivatives, which are effective against fungi, stand out. However, the development of resistance, which may occur due to the long-term use of these peptides of human origin as drugs, will make these peptides, which are part of the human immune system, ineffective against pathogens.

[0008] To date, a large number of antimicrobial peptides have been identified, which are produced naturally by many organisms or produced synthetically by designing a peptide sequence. The most important limiting factors in these peptides are low antimicrobial effect, high cytotoxicity, and hemolytic activity. WO2016024296 A2, EP2905288A1 and US6492328B2 disclose anti-microbial compositions containing synthetic peptides for therapeutic use. These compositions have found application in the treatment of human infections.

[0009] Although very different design methods have been followed in the design of the peptides included in the previous art, a design strategy based on the combination of amino acid sequences of proline-rich peptides has not been reported to date.

[0010] Descriptions of the Figures

[0011] Figure 1. Natural and synthetic peptide sequences used in the amino acid sequence and design of the 385P peptide

[0012] Drosocin-1: GKPRPYSPRPTSHPRPIRV

[0013] Pyrrhocoricin: VDKGSYLPRPTPPRPIYNRN

[0014] Myticalin A6 (16-29): PRYPRWPRHPTIYA

[0015] Figure 2. Cell viability rates according to Methyl Thiazole Tetrazolium (MTT) test results of 385P Peptide

[0016] Figure 3. Scanning Electron Microscope (SEM) images of bacteria with impaired structure and integrity after treatment with the 385P peptide. A: Escherichia coli 0157:147 (ATCC 43894), B: Staphylococcus aureus (ATCC 25223)

[0017] Brief Description of the Invention

[0018] The peptide (385P) amino acid sequence of the invention can be applied as follows:

[0019] SEQ NO 1: GPRPYSPRGSYLPRPYPRWPRHPTSHPRPIRVPIYN

[0020] • In combination with direct or other antimicrobial compounds in the treatment of infections and in commercial packages such as capsules, ampoules, powders,

[0021] • As an antimicrobial coating material to prevent biofilms caused by the colonization of microorganisms in many products such as medical equipment and devices, prostheses, stents, implants, surgical gowns,

[0022] • In various formulations as they have high antimicrobial effect and low cytotoxicity values,

[0023] • In cosmetic formulations containing peptides, pastes, masks, gels, skin lotions, massage creams, toothpastes, cleaning creams, cleaning foams, sprays, creams, soaps, • As an additive and / or antimicrobial food additive in foods such as meat and dairy products, confectionery, beverages, vitamin complexes, baby products, bakery products, directly added to food or on surfaces in contact with food,

[0024] • In order to improve the weight gain, meat quality, milk yield and immunity of livestock in animal feed composition,

[0025] • As a pesticide for the control of bacteria, fungi, and viral pests in agricultural areas.

[0026] Detailed Description of the Invention

[0027] The peptide (385P) amino acid sequence of the invention is as follows:

[0028] SEQ NO 1 : GPRPYSPRGSYLPRPYPRWPRHPTSHPRPIRVPIYN

[0029] The peptide (385P) sequence of the invention is a peptide intended to be used as a therapeutic agent for the prevention and / or treatment of diseases caused by bacterial, viral, fungal, etc. The first amino acid of the sequence, glycine (G), was used to increase stability.

[0030] Data sets were obtained by selecting proline-rich peptides with high activity against pathogenic bacteria from AMP databases (APD, DADP, DBAASP, DRAMP, YADAMP, etc.) for the design of 385P. Among the peptides in the data set, the most frequently repeated sequences of 4-10 amino acids were selected and candidate peptides were obtained with the combination of these sequences. The physicochemical properties of the candidate peptides were scored using many in silico estimation software. For example, molecular weight, net charge and isoelectric point, peptide property calculator (https: / / pepcalc.com / ), hydrophobicity and hydrophobic moment HeliQuest (https: / / heliquest.ipmc.cnrs.fr / cgi- bin / ComputParams.py), conformational states and 3D structure,

[0031] SOPMA, (https: / / npsa-prabi.ibcp. fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html), antimicrobial activity, CAMPR3 (http: / / www.camp.bicnirrh.res.in / ) and hemolytic activity of candidate peptides were estimated using software programs such as HemoPl (https: / / webs.iiitd.edu.in / raghava / hemopi / index.php). Among the candidate peptides, the 385P coded peptide with the highest antimicrobial activity and the lowest hemolytic activity was selected.

[0032] 385P is an antimicrobial peptide formed by the combination of 4-9 amino acid length sequences of proline-rich peptides selected from antimicrobial peptide databases with high antimicrobial activity. For this purpose, it consists of sequences of Drosocin-1, Pyrrhocoricin, Myticalin A6 (16-29) peptides in antimicrobial peptide databases. The amino acid sequence of the 385P peptide and the sequences of different proline-rich antimicrobial peptides forming this sequence are given in Figure 1. Amino acids 2-8 of the 385P peptide were taken from Drosocin-1 (ID: DBAASPR 564), amino acids 9-15 were taken from Pyrrhocoricin (ID: DBAASPR 1205), amino acids 16-23 were taken from Myticalin A6 (16-29) (ID: DBAASPS 18054), and amino acids 33-36 were taken from Pyrrhocoricin (ID: DBAASPR 1205) peptide. Only the ID codes in the DBAASP database of these peptides from which the sequences forming 385P were obtained are given.

[0033] The production of the 385P peptide of the invention was carried out by the chemical synthesis method and also by the recombinant method in Pichia pastoris host cells. Antimicrobial activity tests of the 385P peptide against many pathogenic bacteria and fungi have been performed and it has been determined that it has a strong antimicrobial effect. Antimicrobial activity results of 385P peptide are given in Table 1, hemolysis test results are given in Table 2, cytotoxicity (MTT test) results are given in Figure 2 and SEM results against Escherichia coli O157:H7 (ATCC 43894), Staphylococcus aureus (ATCC 25223) bacteria are given in Figure 3.

[0034] Table 1. Antimicrobial activity results of 385P peptide

[0035] Table 2. Hemolysis values% of 385P peptide

[0036] MIC (Minimum Inhibitory Concentration) analysis is performed to determine the lowest concentration of an antimicrobial agent (antibiotic, peptide, etc.) required to stop the growth of a microorganism. The minimum inhibition concentrations of the 385P peptide were measured using the liquid microdilution method. In MIC analysis, pathogenic microorganism concentrations at the level of 106cfu / mL were used. The lowest peptide concentration at which cell development was inhibited was considered the MIC value. Among the gramnegative bacteria in the test group, the lowest MIC value was found to be 4 pg / mL against A. baumannii (Clinical isolate). This shows that A. baumannii is the bacterium in which the 385P peptide is most effective among the gram-negative bacteria in the test group. MIC values for other bacteria in the gram-negative bacteria class were 8 pg / mL for E. coli (ATCC 25922) and 5. Typhimurium (ATCC 1428), 16 pg / mL for E. coli O157:H7 (ATCC 43894) and K. pneumoniae (Clinical isolate), respectively. Among the gram-negative bacteria in the test group, the bacterium with the lowest effect of the 385P peptide is P. aeruginosa (Clinical isolate), and the MIC value obtained for this bacterium was found to be 32 pg / mL.

[0037] Among the gram-positive bacteria in the test group, the bacterium in which the 385P peptide is most effective is L. monocytogenes (ATCC 7644), and the MIC value detected for this bacterium is 2 pg / mL. MIC values for other bacteria in the gram-positive class were found to be 16 pg / mL for B. cereus (ATCC 33019) and 32 pg / mL for S. aureus (ATCC 25223) and MRS A (ATCC 43300), respectively. The MIC value of the 385P peptide against pathogenic yeast strains such as C. albicans (ATCC 1223) and C. glabrata (EUCC-15M190) was found to be 64 pg / mL. It is seen that the antimicrobial effect of the peptide varies depending on the different types of microorganisms and is effective against yeasts at higher concentrations.

[0038] Hemolytic activity analysis is performed to evaluate whether a substance, especially peptide or drug candidates, damages red blood cells (erythrocytes) and causes hemolysis by breaking down cells. This analysis helps to determine the toxicity level and safety of the tested substance, in particular plays an important role in assessing the risk of a potential candidate for therapeutic use damaging host cells. Hemolytic activity analysis of the 385P peptide was performed according to the principle of spectroscopic measurement of hemoglobin released from erythrocytes. In this analysis, 4% fresh mouse erythrocytes were used. As a result, the hemolysis value of the 385P peptide was found to be 1.04% at the peptide concentration at the level of 4 pg / mL, while it was found to be 3.99% at the peptide concentration at the level of 256 pg / mL. This shows that the effect of the 385P peptide against mouse erythrocytes is less than 5% even at the level of 256 pg / mL. The hemolysis rate of the 385P peptide in 64 pg / mL, which is the highest MIC value against the pathogenic microorganisms tested, was calculated as 2.99%. This reveals that the 385P peptide has a strong effect against pathogenic microorganisms, but also has a low effect against mouse erythrocytes. As a result, it is predicted that if the 385P peptide is used as a drug, its toxicity on the host cell will be low.

[0039] MTT analysis is used to determine the harmful effects (cytotoxic effects) of chemical, peptide, or drug candidates on cells. If the viability of the drug-treated cells decreases, it can be concluded that this substance is toxic. In order to evaluate the toxicity of the 385P peptide, an MTT test was performed against the human lung fibroblast (MRC-5) cell line. According to the results of the MTT analysis presented in Figure 2, cell viability was found to be 100% at concentrations of the 385P peptide up to 80 pg / mL. This finding shows that the 385P peptide does not cause any toxic effect on the cells up to this concentration level and maintains the metabolic activities of the cells. Cell viability was found to be 87% at a peptide concentration of 160 pg / mL and 71% at a peptide concentration of 320 pg / mL. These results showed that 385P does not show toxic effects against human cells at concentrations where it inhibits microorganisms, and the toxic effect may occur at very high concentrations of 385P. SEM analysis is used as an important tool in studies of antimicrobial activity. This method of analysis allows to directly observe the effects of antimicrobial substances on microorganisms by displaying structural changes on the surface of microbial cells at high resolution. SEM images of E. coli O157:H7 (ATCC 43894) and S. aureus (ATCC 25223) bacteria treated with 385P peptide are given in Figure 3. In the images, it was observed that the structure and cellular integrity of both bacteria were significantly impaired. After the application of the 385P peptide, deposits, deformations, and serious deterioration in cell morphology occurred on the surface of the bacteria. These results show that the 385P peptide has a strong antimicrobial effect against the pathogenic bacteria tested and inactivates the bacteria by disrupting the structure of the cell walls.

[0040] The amino acid sequence of the 385P peptide of the invention is unique and is not a natural peptide or a synthetically produced peptide sequence previously detected in any living creature.

[0041] The advantages of 385P over other counterparts are that it has high antimicrobial activity and low cytotoxicity values and a wide spectrum of activity.

[0042] Today, the increase in the number of bacteria resistant to antibiotics used in the treatment of infections has become one of the most important health problems globally. This problem increases the need for new antimicrobial effective compounds. AMPs, which constitute the first line of defense of many living creatures against pathogenic microorganisms, stand out as alternative compounds to antibiotics. AMPs act on various microbial targets through multiple mechanisms, so the development of resistance is less compared to traditional antibiotics. 385P, which is a proline-rich AMP , has high antimicrobial activity since it both disrupts the cell membrane and has an inhibitory effect on intracellular targets, but the risk of resistance development is very low.

[0043] Most of the AMPs identified so far are membrane-active peptides that make the bacterial membrane permeable. However, proline-rich AMPs are an AMP class that inhibits the development of microorganisms by interfering with intracellular processes such as protein synthesis since they have an intracellular mechanism of action. Therefore, 385P, which is a proline-richAMP , has very high antimicrobial activity against microorganisms since it shows its activity on intracellular targets. The most important limiting factors in naturally obtained or synthetically produced peptides are low antimicrobial effect, limited activity spectrum, high cytotoxicity, and hemolytic activity. 385P peptide is a peptide formed by the combination of amino acid sequences of proline-rich peptides. Since amino acid sequences with high antimicrobial activity without hemolytic activity are used in the design of 385P, it has low cytotoxicity and hemolytic activity with higher antimicrobial activity compared to existing AMPs. Therefore, it provides effective inhibition of pathogenic microorganisms without having a toxic effect on the host.

[0044] The advantages of the peptide subject to the invention over other counterparts are that it has high antimicrobial activity, high activity spectrum, and low hemolytic activity values.

[0045] Compared to antibiotics, it takes a more difficult and longer process for microorganisms to develop resistance to antimicrobial peptides. 385P can be used directly against pathogenic microorganisms or in combination with existing antibiotics. This situation may increase the effect of antibiotics used in the treatment of infections, as well as contribute to the use of less antibiotics and the development of less resistance accordingly.

[0046] Peptides, which are effective against bacteria such as LL-37 and against fungi such as histatin, stand out. However, there is a risk of developing resistance due to long-term use of these human-derived peptides. This may lead to the inactivation of these peptides against pathogens after a while. Therefore, since the amino acid sequence of the 385P peptide is unnatural and designed, it is safer compared to human-derived natural peptides.

[0047] 385P has been experimentally shown to be effective against gram-negative and gram-positive bacteria and fungi. Apart from this, it is thought that 385P may have antiviral, antiparasitic, antibiofilm and anticarcinogenic effects.

[0048] Since 385P has low toxicity, it may offer a potentially safe treatment option. It can reduce tissue damage and side effects during infection by reducing inflammation due to microbial infections.

[0049] In addition, apart from inhibiting microorganisms, antimicrobial peptides have an effect on regulating and modulating the immune system. Therefore, it is thought that 385P potentially has the effect of regulating and modulating the immune system and may be effective in combating infections by affecting the activation and function of immune cells.

Claims

CLAIMS1. Proline-rich antimicrobial peptide, characterized in comprising following amino acid sequence:SEQ. NO 1: GPRPYSPRGSYLPRPYPRWPRHPTSHPRPIRVPIYN2. The method for synthesizing the peptide according to claim 1, characterized in using proline-rich sequence of 4-9 amino acids in length3. The method according to claim 2, characterized in usingDrosocin-1: GKPRPYSPRPTSHPRPIRVPyrrhocoricin: VDKGSYLPRPTPPRPIYNRNMyticalin A6 (16-29): PRYPRWPRHPTIYA 4. The peptide according to claim 1, for use in the treatment of infection.

5. The composition, characterized in comprising a peptide according to claim 1.

6. The composition according to claim 5, wherein the composition is an antimicrobial coating.

7. The composition according to claim 5, wherein the composition is a cosmetic product or a therapeutic product.

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