CZ2021572A3 - Antimicrobial peptides derived from the human ameloblastin protein, effective on microbial biofilms - Google Patents

Abstract

The solution concerns antimicrobial peptides derived from the human protein ameloblastin, intended for therapeutic and biotechnological use, especially for application to layers, so-called biofilms, to prevent the growth of specific strains of bacterial microflora, to prevent or remove infectious agents from the surface of joint, dental and bone replacements and to prevent infection of orthopedic implants.

Description

Antimicrobial peptides derived from the human ameloblastin protein, effective on microbial biofilms

Field of technology

This application relates to new antimicrobial peptides (AMP) derived from the human ameloblastin protein (AMBN), intended for therapeutic and biotechnological use, especially for application to layers, so-called biofilms, to prevent the growth of specific strains of bacterial microflora.

Current state of the art

Antimicrobial peptides (AMPs) can be categorized as unconventional therapeutic molecules, attractive for their potential as an alternative treatment against the growing number of infections caused by antibiotic-resistant bacteria. AMPs stand out for their effectiveness, broad spectrum of antimicrobial activity, non-developing resistance in microorganisms and low accumulation in tissues. For these reasons, AMPs are of great interest to pharmaceutical companies trying to develop commercially available drugs from AMPs. Therefore, extensive research has been conducted in the last twenty years to evaluate the potential of antimicrobial peptides isolated from various natural sources, such as vertebrates, plants, insects and bacteria, to treat microbial infections (Wang et al., Nucleic acids research 44(D1): D1087-D1093, 2016; Čeřovský, V.: EP 3570868, US 10,160,785).

AMPs have generally been characterized as typically short (<100 amino acids), positively charged, amphiphilic peptides with a broad spectrum of antimicrobial activity against bacteria and fungi or yeasts, with the ability to interact with and/or penetrate the membranes of these microorganisms (Bahar et al., Pharmaceuticals 6(12): 1543-1575, 2013). Although pore formation is the generally accepted mode of action of AMPs, recent research indicates that some of these peptides have other specific effects on microorganisms that contribute to their selective antimicrobial activity (Zhang, Song et al., Scientific reports 6(1): 1- 13, 2016; Correa, et al. Biomolecules 8(1): 4, 2019). At the same time, AMPs can act against cancer or have antiviral properties along with antimicrobial effects (Felício, et al., Frontiers in chemistry 5: 5, 2017).

In recent years, increasing efforts have been devoted to identifying new therapeutic strategies capable of coping with biofilm-associated infections. Biofilm-organized bacteria show dramatically reduced sensitivity (up to 1000-fold) to conventional antibiotics, causing high rates of treatment failure and persistence of many types of infections (eg, lung infections in cystic fibrosis patients, wound infections, biomaterial-associated infections) (Yasir, Willcox et al., Materials 11(12): 2468, 2018).

Due to the frequent formation of biofilms on medical implants, one of the currently interesting applications of AMPs is their immobilization on such devices in order to prevent the formation of bacterial biofilms as well as the development of (chronic) infections associated with biofilms. Additionally, this application offers a solution to overcome the problems related to the systemic transport and toxicity of AMP (Hemmati, et al., Molecular Biotechnology: 1-18, 2021). An important benefit of using AMPs as covalent coatings for implantable therapeutic materials is their long-term stability and activity after coating. Although the principle of this use is limited, more and more studies describe the efficacy against microbial biofilm formation in important fungal and bacterial species (e.g. LL-37 and magainin) and the remarkably high long-term activity of coated peptides and stability under various extreme conditions (Martins, et al .,Biofouling 37(1): 96-108, 2021).

Thus, naturally occurring AMPs are promising candidates for the treatment of infections associated with microbial biofilm or invasive infections caused by pathogens resistant to currently used antifungal or antimicrobial agents. Synthetic peptides a

- 1 CZ 2021 - 572 A3 peptidomimetics based on natural AMPs or their derivatives have the potential to become new (systemic) therapeutics because they overcome the main limitations of standard antibiotics used today. Some AMPs can kill bacteria through membrane disruption and/or pore formation or inhibition of bacterial cell division. Another potential of AMPs includes the ability to act at different stages of biofilm formation and with different mechanisms of action, AMPs often show multidrug resistance activity against bacterial strains (Raheem and Straus 2019, Frontiers in microbiology 10: 2866, 2019). Furthermore, AMPs can be effective against bacteria by the mechanism by which they prevent bacterial cells from adhering to the surface of the substrate, thereby reducing intercellular communication, the so-called quorum sensing (QS), or removing the previously formed biofilm; these are so-called antiviral mechanisms of action (Raško et al., Sperandio, Nature Reviews Drug Discovery 9(2): 117-128, 2010). The antibiofilm activity of AMPs may also be mediated by downregulation of genes involved in motility and inhibition of a number of cell biological processes, such as synthesis of cell walls, DNA, RNA and proteins (Di Somma, et al., Biomolecules 10(4): 6520, 2020). The antibiofilm activities of AMPs on biofilms have been studied less than the effect of AMPs on suspended populations of microorganisms. The so-called minimum biofilm inhibitor concentration (MBIC), minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) are used to evaluate the specific ability of AMP to disrupt biofilm formation.

Another great advantage of AMPs acting on biofilms is their specific mode of activity showing low toxicity to eukaryotic cells, which provides an opportunity for wide therapeutic use (Cruz et al., BMC microbiology 18(1): 1-9, 2018). Moreover, AMPs often show synergy with classical antibiotics, neutralize endotoxins and are very active in animal models. Resistance to AMPs is relatively rare due to their affinity for the negatively charged part of the bilayer lipid structure of bacterial membranes. The kinetics of bacterial growth inhibition by AMP is then faster compared to most conventional antibiotics (de Breij, et al., Science translational medicine 10(423), 2018). The benefits of AMPs may even extend beyond their antimicrobial effects, as they can enhance the immune response to further fight pathogenic infection. Immune regulation results from the interaction of AMP with host cell receptors (Di Somma, et al., Biomolecules 10(4): 6520, 2020).

Since many inorganic matrices (titanium, ceramics) are used in the field of dental restorations, it is possible to cover these materials with a layer of functional peptides with specific activity against bacterial strains in the oral cavity, causing typical infections. A logical solution is offered by the analysis of proteins naturally occurring in the oral cavity, which show possible antimicrobial activity, especially against biofilm. Enamel matrix proteins are well known as precursors to enamel formation (Bartlett, International Scholarly Research Notices, 2013) and have previously been reported in the literature to induce enamel formation. Enamel matrix proteins and their derivatives are also known to promote wound healing in soft tissues such as skin and mucous membranes (WO 9943344 A2, Gestrelius). The description of the support of tissue healing with the help of these proteins indicated a possible wider antimicrobial activity of the proteins of the tooth enamel matrix or its grafts (peptides). Peptides arising as grafts of these proteins are the product of the enzymatic action of proteases, the most important of which are kallikrein-4 (KLK4) and enalysin (MMP20) (Bartlett, International Scholarly Research Notices, 2013). One of the significant proteins of the tooth enamel matrix is ameloblastin (AMBN). It is a phosphoprotein, classified by computational and biophysical methods as intrinsically disordered (IDP) (Stakkestad, Lyngstadaas et al., Frontiers in physiology 8: 531, 2017) and thus well accessible for cleavage by proteases. Based on theoretical and experimental analyzes of AMBN and its derivatives, peptides with specific antimicrobial activity against bacterial biofilms, which are presented in this invention, were identified.

The essence of the invention

The present invention provides new antimicrobial substances, derived from the human ameloblastin protein (AMBN), which have been targeted using bioinformatic analyzes and

- 2 CZ 2021 - 572 A3 molecular modeling and then prepared in the form of synthetic analogues.

The subject of the invention are antimicrobial peptides with the formulas:

AP 4.9. QGSTIFQIARLISHGPMG (A, SEQ ID NO:1),

AP 4.9.1. QGHTIFQIARLISHGPM (B, SEQ ID NO:2),

AP 5.10. STIFQIARLISHGPMPQNKQSPG (c, SEQ ID NO:3), a

AP 5.10.1 STIFQIARLISHGAMAQNKQSP (D, SEQ ID NO:4).

The present invention also includes said antimicrobial peptides A, B, C and D for use as drugs in medicine, in particular as an adjunct to the treatment of oncological diseases.

Oncological diseases include the formation of cancers especially in the area of the oral cavity, such as benign mesenchymal cancers fibroma, lipoma, hemangioma and the like, malignant cancers fibrosarcoma, myeloma and bone tumors, or neuroectoderm cancers.

The present invention also includes the aforementioned antimicrobial peptides A, B, C and D for use in biotechnological applications to prevent the growth of bacterial contamination, especially to prevent or remove infectious agents from the surface of joint, dental or bone prostheses used in medicine (titanium, ceramic, etc. .).

The present invention further includes the indicated antimicrobial peptides A, B, C and D for the production of a means to prevent or remove an infectious agent from the surface of joint, dental and bone replacements and to prevent infection of orthopedic implants.

The primary role of AMBN is as a protein matrix for the construction of tooth enamel. Studies indicating that proteins and derivatives of the tooth enamel matrix support wound healing in soft tissues have indicated the possible presence of proteins or peptides with antimicrobial activity.

From the human ameloblastin protein (AMBN, UniProtKB - Q9NP70, AMBN_HUMAN), the sequences of short segments, peptides and their mutated analogs were identified and then analyzed using bioinformatic analyses, which were designated as (A): AP4.9, (B): AP 4.9. 1., (C): AP 5.10. and (D): AP 5.10.1. The peptide of formula (B), AP4.9.1, is an analog of peptides (A), Ap4.9, with a single amino acid change, and the peptide of formula (D), AP5.10.1, is an analog of peptides (C), AP5.10., with by changing two amino acids. Peptides A, B, C and D show an antimicrobial effect on microbial biofilms of specific strains of bacteria found in the oral cavity.

Clarification of drawings

Figure 1 presents the ECD spectra of peptides A, SEQ ID NO: id. NO. 1; B, SEQ. id. No.: 2; C, SEQ. id. No.: 3; D, SEQ. id. No.: 4 in the presence of 0%, 10%, 30% and 50% (v/v) TFE in MQ water. CD spectra were expressed as molar ellipticity Q (deg cm ² dmol ^-1 ) per residue.

Examples of implementation of the invention

List of given abbreviations:

ACPs

AMP

APD

CD

ECD spectrum

HBTU peptides with an effect against cancer (from English Anti Cancer Peptides) antimicrobial peptides (from English Antimicrobial Peptides) database of antimicrobial peptides (from English Antimicrobial Peptides Database) circular dichroism electronic circular dichroism

O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

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HC ₅₀ IC _5O IDP MBIC50 MBEC50 MIC ₅₀ MMP N-Fmoc PBS PMSF SDS SDS-PAGE TEV RP-HPLC RT EDTA

MQ water concentration at which half of the red blood cells are lysed (hernolysis) inhibitory concentration at 50% cytotoxicity intrinsically disordered protein (intrinsically disordered protein) minimum concentration for 50% biofilm inhibition minimum concentration for 50% biofilm eradication minimum inhibitory concentration to 50 % reduction in the growth of suspension cells metalloprotease fluorenylmethyloxycarbonyl (protecting group for organic syntheses) phosphate buffer phenylmethylsulfonyl fluoride sodium dodecyl sulfate polyacrylamide gel electrophoresis tobacco mosaic virus high-performance liquid chromatography on non-polar sorbent at room temperature ethylenediamine terephthalic acid ultrafiltered water from Merck

Studies were conducted on bacterial strains: Enterococcus faecalis DBM 3075, CNCTC 5530, CNCTC 5483, M-1; Staphylococcus aureus CNCTC 5670 (ATCC 12600), CNCTC 6271 (ATCC 43300), 3178 (ATCC 29213); and Escherichia coli DBM 3125 (ATCC 10536), DBM 3138, B (Table No. 1 to 3). The antimicrobial effect of the identified AMPs is accompanied by very mild cytotoxic activity and almost zero hemolytic activity (table no. 4 and 5). Peptides A, B, C and D show antimicrobial properties only on biofilms of specific bacterial strains, the antimicrobial activity of AMP in solution was not shown to be significant. This fact suggests a possible natural action of AMBN peptide grafts against microbial films present, for example, on tooth enamel. The secondary structure of AMPs is disordered in an aqueous environment, but easily transitions to an α-helical structure in the presence of α-helix-inducing compounds such as trifluoroethanol (Figure 1, Table 6). The potential to form an induced helical structure of AMPs indicates the possibility of antimicrobial activity through the mechanism of helical pore formation in the cell membrane and its disruption. However, a stronger factor is apparently the specific effect of peptides on the formation of specific bacterial strains forming biofilms. The presented peptides showed significant antimicrobial activity against biofilm, tested by eradication and adhesion.

Table 1: Effect of studied antimicrobial peptides A, B, C and D on the suspension growth of Enterococcus faecalis, Staphylococcus aureus and Escherichia coli species. (MIC50 - minimum concentration for 50% inhibition)

MIC 50 (pmol.l- ¹ ) AND B C D E. faecalis DBM 3075 ★ * 4 CNCTC 5530 ♦ Λ FC 4 CNCTC 5483 * FC 4 M-1 * * 4 * S. aureus CNCTC 5670 * 4 200 200 DBM 3138 * and 4 * CNCTC 6271 ★ -λ 4 4 M-1 * * 4 4 E. coli DBM 3125 ★ -λ 300 150 DBM 3138 ★ * 4 CCM 4787 * λ * 300 B * ★ * 4

*not determined in the studied concentration range (50 to 300 pmol.l ¹ )

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Table 2: Effect of peptides A and B on biofilm formation and eradication of mature biofilm of Enterococcus faecalis, Staphylococcus aureus and Escherichia coli.

MBICsd A (pmol.l' ¹ ) MBECso B (pmol.l ¹ ) MBICbo MBECso MBICso MBICeo MBECso MBECso E. faecalis DBM 3075 200 300 50 150 200 150 ft CNCTC 5530 300 * ft * 300 ft ft ft CNCTC 5483 300 * ft ft 300 ft ft ft M-1 300 * ft ft 300 ft ft * S. aureus CNCTC 5670 150 * 300 ft 200 ft 50 ft DBM 3138 300 * ft ft 150 ft ft ft CNCTC 6271 200 * ft ft 200 ft 300 ft M-1 * ft ft ft ft ft ft E. coli DBM 3125 * 100 ft 150 ★ 100 ft DBM 3138 150 150 150 ft 150 ft 160 ft CCM 4787 300 * ft ft ft ft ft ft B 50 100 200 ft 150 150 150 ft

*not determined in the studied concentration range (50 to 300 pmol.l ¹ )

Table 3: Effect of peptides C and D on biofilm formation and eradication of mature biofilm of Enterococcus ίο faecalis, Staphylococcus aureus and Escherichia coli.

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D (pmol I' ¹ )

MBICso MBÍCsa MBECm MBECa- MBICw MBICao MBECsc MBECeo E. DBM 200 300 AND * 200 300 300 AND faecalis 3075 CMCTC AND AND AND * AND AND AND AND 5530 CNCTC AND * AND AND * AND AND t 5483 M-1 * * 200 * 300 * 300 AND S. CMCTC 200 AND AND AND 300 AND • AND aureus 5670 DBM AND AND AND ★ * AND AND AND 3138 CNCTC 300 300 300 AND 300 300 300 AND 6271 M-1 300 * AND AND 300 AND AND AND E. coli DBM AND AND AND * 300 AND AND AND 3125 DBM 200 AND AND AND 150 AND AND AND 3138 CCM AND AND 300 AND 300 AND 200 AND 4787 B 150 150 4 AND 50 200 * *

*not determined in the studied concentration range (50 to 300 pmol.l ¹ )

Example 1

Identification of peptides

Antimicrobial peptides were identified from the AMBN sequence (AMBN, UniProtKB - Q9NP70, AMBN HUMAN) by combining different approaches of bioinformatics tools, used to predict the antimicrobial activity of short protein sequences. A total of six short sections were predicted by the analysis, from which two peptides A and C subsequently showed significant antimicrobial activity on biofilms. From these peptides, two of their analogues designated as B and D were then designed using rational design using molecular modeling methods. The designed analogues C and D show higher antimicrobial activity on biofilms than peptides A and B.

Example 2

Peptide synthesis

All peptides in this study were chemically synthesized and purified to >97% purity. Peptides were solubilized in distilled water and their concentrations were determined by amino acid analysis.

AMBN-derived peptides (AMBN, UniProtKB - Q9NP70, AMBN HUMAN), designated as A, B, C, and D, were synthesized by solid-phase peptide synthesis technique according to the standard V-Fmoc protocol. They were prepared on TentaGel S RAM resin (431 mg with 0.24 mmol/g substitution) on a PS3 automated peptide synthesizer (Protein Technologies, Tucson, AZ). V-Fmoc-protected amino acids (10 equiv) were coupled using 0.4 mol.1 ¹ V-methylmorpholine in V,V-dimethylformamide (DMF, 20 equiv) and HBTU (10 equiv) in V-methyl-2 -pyrrolidone. Deprotection of the α-amino group was performed using 20% (v/v) piperidine in DMF. Peptides were completely deprotected and cleaved from the resin with TFA/H 2 O//triisopropylsilane (92.5:5:2.5) for 2 h and then precipitated with tert-butyl methyl ether. All peptides were purified by RP-HPLC (Jasco lne.) on a (5 µm, 250 x 20 mm) YMC-Pack ODS-AM column. The identity and purity of the synthetic peptides were confirmed using an Agilent 1260 HPLC (Agilent Technologies) coupled to an Agilent 6530 ESI-TOF (Agilent Technologies) with Agilent Jet Stream technology. The purity of all

-6CZ 2021 - 572 A3 peptides was >97%.

Example 3

Microorganisms used and cultivation conditions

Three types of microorganisms that are found in the oral cavity or cause tooth decay have been studied:

Enterococcus faecalis DBM 3075 is a clinical isolate from the Bulovka University Hospital in Prague and was provided by the Institute of Biochemistry and Microbiology, VŠCHT Prague. Control strains of E. faecalis for testing antimicrobial drugs CNCTC 5530 (ATCC 51299) and CNCTC 5483 (ATCC 29212) were obtained from the Czech National Collection of Type Cultures. E. faecalis M-1 is a clinical isolate from the Motol University Hospital in Prague.

Staphylococcus aureus CNCTC 5670 (ATCC 12600) represents the type strain obtained from the Czech National Collection of Type Cultures in Prague. A methicillin-resistant strain of S. aureus CNCTC 6271 (ATCC 43300) was also obtained from the same collection. Furthermore, the methicillin-sensitive strain S. aureus DBM 3178 (ATCC 29213) provided by the Institute of Biochemistry and Microbiology, VŠCHT Prague and S. aureus M-1 isolated from an infected joint replacement at the Motol Faculty Hospital in Prague were studied.

Control strains for antibiotic testing Escherichia coli DBM 3125 (ATCC 10536) and DBM 3138 (ATCC 8739) were provided by the Institute of Biochemistry and Microbiology, VŠCHT Prague. E. coli strain CCM 4787 (serovar 0157:H7) and E. coli CCM 7372 (strain B) obtained from the Czech collection of microorganisms of the Masaryk University in Brno were also studied.

Before carrying out the experiments studying the antimicrobial and antibiofilm effect of the studied peptides on microbial cells, each microorganism was inoculated into a liquid medium, in the case of E. faecalis and S. aureus into tryptone-soy broth and in the case of E. coli into Luria-Bertanni medium, and cultured at 37 °C and 150 rpm (revolutions per minute) for 24 h.

Example 4

Study of the effect of AMP on the suspension growth of microorganisms

The effect of AMP on the suspension growth of microorganisms was assessed using cultivation in a Bioscreen C microculture device (LabSystems, Finland), as described by Matatkova et al. (Matatkova et al., Int J Anal Chem, 8195329, 2017). Briefly, the prepared inoculum (see example 3) was adjusted to an optical density of OD600nm = 0.1 (CFU/ml = 2.5x10 ⁷ ) and was subsequently mixed in a volume of 30 µl with 290 µl of the appropriate medium and the solution of the tested peptide in a polystyrene microtiter Honeycomb 2 plate (Growth Curves, USA). Cell cultivation took place in a microcultivation device for 24 h at 37 °C and 150 rpm. The optical density of the contents of the wells was measured every 30 min, and growth curves reflecting the suspension growth of cells in each well of the microtiter plate were compiled from the resulting data. Each experiment contained control samples without the addition of antimicrobial agents, and all experiments were performed in technical triplicates. From the measured data, the minimum inhibitory concentration (MIC50) was subsequently determined, i.e. the lowest concentration of the studied substance that already caused at least a 50% decrease in the growth of suspension cells after 24 h of cultivation.

Example 5

Study of the effect of AMP on biofilm formation and eradication of already mature biofilm of microorganisms

The effect of AMP on biofilm formation was studied in polystyrene microtitre plates according to the procedure described in Vaňková et al. (Vaňkova et al., World J Microbiol Biotechnol, 36(7):101,

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2020). Briefly, the prepared inoculum (see example 3) was adjusted to OD600nm = 0.8 (CFU/ml = 2x10 ⁸ ) and was subsequently mixed in a volume of 210 µl with 70 µl of the respective growth medium and the solution of the studied peptide in a TPP96 polystyrene microtiter plate ( TPP, Switzerland). The biofilm was formed statically at 37 °C for 24 h.

The effect of AMP on the eradication of an already mature biofilm was studied analogously to the mentioned procedure. First, the biofilm itself was cultured without added antimicrobial agent. In this case, the inoculum was adjusted to OD600nm = 0.6 (CFU/ml = 1.5x10 ⁸ ) and pipetted into the wells of the microtitre plate in a volume of 200 µl. Cultivation took place for 24 h at 37 °C statically. The formed biofilm was washed with physiological solution and the appropriate solutions of the studied peptides and growth medium were pipetted to it to a final volume of 200 µl. Cultivation was carried out as described above. All experiments contained control samples without the addition of antimicrobial agent and blank (samples without cell suspension). All experiments were performed in technical triplicate.

Example 6

Determination of metabolic activity of biofilm cells

Metabolic activity of biofilm cells was determined using resazurin. 25 µl of D-glucose solution (180 g/l in physiological solution), 25 µl of resazurin solution (0.15 g/l in physiological solution) and 100 µl of physiological solution were pipetted to the washed biofilm. The biofilm was measured fluorimetrically immediately, or in the case of E. coli incubated for 30 min at 37 °C and then measured. Fluorescence intensity was determined at 545/575 nm in an Infinite M200 Pro Reader plate spectrophotometer (Tecan, Switzerland). The average background (fluorescence intensity of the blank) was subtracted from the resulting data and the mean and standard deviation were calculated, which were converted to relative percentages for a better comparison of strains and peptides. Experiments were performed in technical triplicates.

Based on the determined metabolic activity, the parameters expressing the antibiofilm effectiveness of the studied substances were determined. The minimum biofilm-inhibiting concentration (MBIC50, or MBICxq) represents the lowest studied concentration of the given substance, which already causes 50%, or 80% decrease in the metabolic activity of the cells of the biofilm formed in the presence of the given substance. Analogously, the minimum biofilm-eradicating concentration (MBIC50, or MBECxq) represents the lowest studied concentration of the given substance, which already causes 50% or 80% decrease in the metabolic activity of mature biofilm cells eradicated by the given substance.

Example 7

Hemolytic essay

The hemolytic activity of the studied antimicrobial peptides was evaluated in vitro using human blood. For hemolysis testing, fresh blood collected in a tube with sodium citrate as an anticoagulant was used. Melitin, an amphipathic peptide from honey bee venom, was used as a positive control because it exhibits lytic activity against a variety of cells (including red blood cells) at submicromolar concentrations. The hemolytic activity of the tested substances was then related to the hemolysis induced by 0.5% Triton X-100, which caused 100% hemolysis at this concentration. The concentration at which half of the red blood cells are lysed (value of hemolytic activity HC50) is an indicator of the toxicity of the tested peptides.

Whole blood (1 ml) diluted in PBS (9 ml) was centrifuged (800 x g, 15 min, RT), and then the red cell pellet was washed five times with 10 ml PBS (800 x g, 15 min, RT). After removing the supernatant, the washed red blood cell pellet was diluted with PBS to a concentration of 0.5% (v/v) and 50 µl of the resulting suspension was transferred to a 96-well microtiter plate. Then 50 µl of twice concentrated peptide solutions (tested antimicrobial peptides in concentrations of 12.5 to 300 pmol.l ^-1 , Melitin 0.024 to 50 pmol.l ^-1 as a result, two-fold dilution), PBS alone and 0.5% were added

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Triton X-100. The microtitre plate with the samples was incubated for 60 min at 37°C and centrifuged (1000 x g, 15 min, RT). 40 µl of supernatant from each well was then transferred to a transparent 384 well plate. The absorbance of the supernatant was measured at 415 nm using a microtiter plate reader (Cytation 3, BioTek, USA).

Table 4: Hemolytic activity of AMP A, B, C and D, measured in the range of 12.5 to 100 (300) pmol.l ¹ on a human blood sample and expressed as HC50.

Peptide

AND

B

C

D Melitin

Hemolytic activity HC50 (pmol.l ^-1 ) »> 300 » 300 >» 300 - 300 0.79

Example 8

Cytotoxicity assays using HUVEC and HCT 116 human endothelial cells

Cell cultures:

Primary human HUVEC (human umbilical vein endothelial cells, Lonza) cell culture was maintained in EGM™-2 endothelial cell growth medium (Lonza) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) at 37° C in a humidified atmosphere with 5% CO2. Twice a week, when the cells reached 80 to 90% confluence, they were subcultured using 0.25% thypsin/0.53 mmol.l ^-1 EDTA solution for the next passage.

The human colon cancer cell line HCT 116 (ATCC) was maintained in McCoy's 5A growth medium (Sigma Aldrich) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere with 5% CO2. Twice a week, when the cells reached 80 to 90% confluence, they were subcultured with 0.25% thypsin/0.53 mmol.l ^-1 EDTA solution for the next passage.

Cytotoxicity tests:

The CellTiter-Glo® Luminiscent Cell Viability Assay kit (Promega) was used to determine the cytotoxicity of the tested antimicrobial peptides. Cells were cultured as described above and the experiment was performed according to the manufacturer's instructions. 10,000 HUVEC cells (50 μΐ) were transferred to each well of a white 96-well plate (BD Biosciences™). Cells were then cultured for 24 hours before adding 50 μΐ of twice-concentrated solutions of test peptides or EGM™-2 medium alone (vehicle control) to each well. After an additional 72 hours, 100 μΐ of CellTiter-Glo® reagent (Promega) was added to each well and the 96-well plate was agitated for 2 minutes at 400 rpm on an orbital shaker in the dark. Subsequently, the luminescence signal was allowed to stabilize at room temperature for 10 min. Luminescence was recorded using a microtiter luminometer (Cytation 3, BioTek, USA). In this test, the intensity of luminescence directly correlates with the number of living cells. Data obtained were normalized and IC50 values were calculated by non-linear regression analysis assuming a sigmoidal concentration-response curve with a variable Hill slope (GraphPadPRISM® 7 software).

Table 5: Cytotoxic activity of AMP A, B, C and D, measured in the range of 12.5 to 100 (300) pmol.l ^-1 on HCT116 and HUVEC cell lines, expressed as IC50.

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Cytotoxic activity IC 50 (pmol.l· ¹ )

Peptide/cell line HCT116 HUVEC AND »> 100 »> 300 B >» 100 >300 C >» 100 »> 300 D >» 100 - 300

Example 9

Circular Dichroism (CD) Measurement

Electronic circular dichroism (ECD) spectra of all AMPs were measured with a Jasco J-815 CD spectrometer (Jasco Corporation, Tokyo, Japan) in the spectral range of 200–300 nm using a 0.1 cm long quartz cell at RT. The experimental setup was as follows: step resolution 0.5 nm, speed 10 nm/min, response time 16 s, bandwidth 1 nm. The sample concentration was kept constant at 0.1 mg/mL in MQ water (pH = 7.5). The samples were also measured with a mixture of trifluoroethanol (TFE) (0%, 25% and 50% v/v TFE). After baseline correction, the final spectra were expressed as molar elliptic 0 (degcm ² dmol ^-1 ) per amino acid residue. Secondary structure content was determined using the online circular dichroism analysis program Dichroweb software (Lee Whitmore and BA Wallace, Nucleic Acids Res., 32: W668-W673, 2004). The secondary structure content of all analyzed AMPs demonstrated their unstructured character with the potential to form a helical structure with increasing TFE concentration. The results of CD measurements are shown in Fig. 1 and in Tab. No. 6.

Table 6: Calculated incidence (%) of secondary structural content. Peptides A, B, C and D were studied by CD spectroscopy in the presence of 0%, 10%, 30% and 50% (v/v) TFE in MQ water.

Secondary structure, % AMP TFE, % alpha helix beta sheet loop unstructured AND 0 6% 32% 24% 38% AND 25 9% 31% 24% 36% AND 50 25% 23% 23% 30% B 0 6% 35% 23% 0% B 25 7% 35% 23% 35% B 50 34% 14% 23% 28% C 0 6% 37% 23% 35% C 25 8% 36% 23% 34% C 50 34% 13% 24% 30% D 0 5% 38% 22% 35% D 25 6% 40% 22% 32% D 50 25% 26% 19% 29%

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Industrial applicability

New antimicrobial peptides are considered for selective use on microbial biofilms, 5 to prevent the growth of bacterial and fungal contamination, for example joint replacements or other implants. The application of antimicrobial peptides is applicable in the field of medicine and biotechnology.

Claims (5)

1. Antimicrobial peptides having the formulas: QGSTIFQIARLISHGPMG A, SEQ. id. NO. 1, QGHTIFQIARLISHGPM B, SEQ. id. No.: 2, STIFQIARLISHGPMPQNKQSPG C, SEQ. id. NO: 3, and STIFQIARLISHGAMAQNKQSP D, SEQ. id. No.: 4. 2. Antimicrobial peptides A, B, C and D according to claim 1 for use as drugs, in particular as antimicrobial drugs. 3. The use of antimicrobial peptides A, B, C and D according to claim 1 as an adjunct in the treatment of oncological diseases related to the formation of cancers in the oral cavity, such as mesenchymal benign cancers fibroma, lipoma and hemangioma, malignant cancers fibrosarcoma, myeloma and bone tumors, or neuroectoderm carcinomas. 4. The use of antimicrobial peptides A, B, C and D according to claim 1 in biotechnological applications in the prevention of the growth of bacterial contamination, especially in the prevention or removal of infectious agents from the surface of joint, dental or bone substitutes used in medicine. 5. Use of antimicrobial peptides A, B, C and D according to claim 1 for the production of an agent effective in preventing or removing an infectious agent from the surface of joint, dental and bone replacements and in preventing infection of orthopedic implants.