WO2023109991A1

WO2023109991A1 - Antimicrobial peptides derived from human ameloblastin protein, effective on microbial biofilms

Info

Publication number: WO2023109991A1
Application number: PCT/CZ2022/050133
Authority: WO
Inventors: Kristyna BOUSOVA; Jiri VONDRASEK; Veronika VETYSKOVA; Petra KASPAROVA; Jan Masak; Olga MATATKOVA; Miroslav Hajek; Lucie Bednarova
Original assignee: Ustav Organicke Chemie A Biochemie Av Cr, V. V. I.; Vysoka Skola Chemicko-Technologicka V Praze
Priority date: 2021-12-16
Filing date: 2022-12-15
Publication date: 2023-06-22
Also published as: CZ309667B6; CZ2021572A3

Abstract

The invention relates to antimicrobial peptides derived from the human protein ameloblastin, intended for therapeutic and biotechnological use, in particular for application onto 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 orthopaedic implants.

Description

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

Field of Art

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

Background Art

Antimicrobial peptides (AMPs) can be categorized as unconventional therapeutic molecules, attractive for their potential as an alternative treatment against the increasing 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. Therefore, AMPs are of vast interest to industry searching to develop commercially available drugs from AMPs. Hence, 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 the microbial infections (Wang et al., Nucleic acid research 44(D1): D1087-D1093, 2016; Cefovsky, V.: EP 3570868, US 10 160 785).

AMPs have been generally characterized as typical 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 to microorganism membrane (Bahar et al., Pharmaceuticals 6(12): 1543-1575, 2013). Although pore formation in the membrane is the generally accepted mechanism of AMPs action, recent studies indicated that some of these peptides have other specific effects on microorganisms that contribute to their selective antimicrobial activities (Zhang, Song et al., Scientific reports 6(1): 1-13, 2016; Correa et al., Biomolecules 8(1): 4, 2019). Furthermore, AMPs can act against cancer or have antiviral properties along with the herein mentioned antimicrobial effects (Felicio, et al., Frontiers in chemistry 5: 5, 2017). In recent years, efforts have been devoted to finding new therapeutic strategies capable to handle the biofilm-associated infections. Bacteria organized in biofilms show a dramatically reduced sensitivity (up to 1000 times) to conventional antibiotics, which results in a high rate of treatment failure and persistence of many types of infections (eg., lung infection in patients with cystic fibrosis, wound infections and infections associated with biomaterials) (Yasir, Willcox et al., Materials 11 (12): 2468, 2018).

Due to a 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 and therefore the development of (chronic) infections associated with them. Additionally, such application offers a solution to overcome the problems related to the systemic transport and toxicity of AMPs (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 their coating onto the implant material. Although the principle of this application is limited, an increasing number of studies describe efficacy against microbial biofilm formation in important fungal and bacterial species (e.g. LL- 37 and magainin) and remarkably high long-term support of coated peptides and stability under various extreme conditions (Martins, et al., Biofouling 37(1): 96-108, 2021).

Naturally occurring AMPs are therefore promising candidates for the treatment of microbial biofilm-related or invasive infections caused by pathogens and resistance to some currently available antifungal or antimicrobial agents. Synthetic peptides and 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 activity of AMPs includes the ability to act at various stages of biofilm formation and with various mechanisms of action. AMPs often exhibit multidrug-resistant activity against bacterial strains (Raheem and Straus 2019, Frontiers in microbiology 10: 2866, 2019). Furthermore, AMPs may involve a mechanism of action effective on bacteria, by which they prevent adhesion of the bacterial surface to the substrate, which decreases the action of intercellular communication, the so-called quorum sensing (QS), or removes the previously formed biofilm; these are so-called antivirulent mechanisms of action (Rasko 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 cell wall synthesis, DNA, RNA and protein synthesis (Di Somma, et al., Biomolecules 10(4): 6520, 2020). The antibiofilm activities of AMPs on biofilms have so far been studied less than the effect of AMPs on suspension populations of microorganisms. The so-called minimum biofilm inhibitor concentration (MBIC), minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) identifiers are used to evaluate the specific ability of AMPs to disrupt the biofilm formation.

Another significant advantage of AMPs acting on biofilms is their specific mode of activity that showed low toxicity for eukaryotic cells providing an opportunity for wide therapeutic applications (Cruz et al., BMC microbiology 18(1): 1-9, 2018). In addition, 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 go beyond their antimicrobial effects, as they can stimulate the immune response to further fight against pathogenic infections. Immune regulation results from the interaction of AMPs 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 prostheses, 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 healing in soft tissues, such as the skin and mucosa (WO9943344A2, Gestrelius). The disclosure 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 cleavage products (peptides). Peptides arising as cleavage products of these proteins are the products 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 analyses of AMBN and its derivatives, peptides with specific antimicrobial activity against bacterial biofilms have been identified within the framework of the present invention.

Disclosure of the Invention

The present invention provides new antimicrobial substances, derived from the human ameloblastin protein (AMBN), which were designed using bioinformatic and molecular modelling methods and prepared in the form of synthetic peptides.

The subject of the invention are antimicrobial peptides of 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), and

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

The present invention also provides the antimicrobial peptides A, B, C and D for use as drugs in medicine, in particular for adjuvant (supplementary, auxilliary) treatment to the treatment of oncological diseases. Oncological diseases herein include the formation of carcinomas especially in the oral cavity, such as benign mesenchymal carcinomas, fibroma, lipoma, haemangioma and the like, malignant carcinomas, fibrosarcoma, myeloma and bone tumours, or neuroectoderm carcinomas.

The invention also provides the 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 replacements (prostheses) used in medicine (made of titanium, ceramic, etc..).

The invention also provides the antimicrobial peptides A, B, C and D for the production of compositions for preventing or removing an infectious agent from the surface of joint, dental and bone replacements and to prevent infection of orthopaedic implants.

The primary role of AMBN is protein matrix formation for the construction of tooth enamel. Studies indicating that proteins and derivatives of the tooth enamel matrix promote healing soft tissue wounds have indicated the possibility of the presence of proteins or peptides with antimicrobial activity.

From the human ameloblastin protein (AMBN, UniProtKB - Q9NP70, AMBN_HUMAN), short proteins, peptides and their mutated analogues were identified and then analysed by bioinformatic tools, 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 analogue of the peptide (A), AP4.9, with one amino acid change, and the peptide of formula (D), AP5.10.1 , is an analogue of the peptide (C), AP5.10., with a change of two amino acids. The peptides A, B, C and D show an antimicrobial effect on microbial biofilms of specific strains of bacteria found in the oral cavity.

Brief description of drawings Figure 1 presents the ECD spectra of the peptides A, SEQ. 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 cm² dmol^-1) per residue.

Examples

Abbreviations:

ACPs Anti Cancer Peptides

AMP Antimicrobial Peptides

APD Antimicrobial Peptides Database

CD circular dichroism

ECD electronic circular dichroism

EDTA ethylene diamine tetra acid

FBS fetal serum albumin

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

HC₅₀ concentration at which half of red blood cells are lysed (hemolysis)

IC50 inhibitory concentration at 50% cytotoxicity

IDP Intrinsically Disordered Protein

MBIC50 minimum concentration for 50% biofilm inhibition

MBIC₈₀ minimum concentration for 80% biofilm inhibition

MBEC50 minimum concentration for 50% biofilm eradication

MIC50 minimum inhibitory concentration to 50% reduction in the increase of suspension cells

MMP metalloproteases

N-Fmoc fluorenylmethyloxycarbonyl (protecting group for organic synthesis)

PBS phosphate buffer

PMSF phenylmethylsulfonyl fluoride

SDS sodium dodecyl sulfate

SDS-PAGE electrophoresis in a polyacrylamide gel

TEV tobacco mosaic virus

TFE trifluoroethanol

RP-HPLC high-performance liquid chromatography on a non-polar sorbent RT room temperature

EDTA ethylenediamine terephthalic acid

MQ ultrafiltered water from Merck

The studies were carried out 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. 1-3). The antimicrobial effect of the identified AMPs is accompanied by very mild cytotoxic activity and almost zero haemolytic activity (Table 4, 5). The peptides A, B, C and D show antimicrobial properties only on biofilms of specific bacterial strains, the antimicrobial activity of AMPs in the solution was not shown to be significant. This fact points to a natural action of AMBN peptides against microbial films present, for example, on tooth enamel. The secondary structure of AMPs is disordered in an aqueous environment, but easily changes to an a-helical structure in the presence of a-helix-inducing compounds such as trifluoroethanol (TFE) (Figure 1 , Table 6). The potential to form an induced helical structure of AMPs indicates the possibility of antimicrobial activity on 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 peptides of the invention showed antimicrobial activity against biofilm, tested by eradication and adhesion.

TABLE 1

The effect of studied antimicrobial peptides A, B, C and D on the suspension growth of Enterococcus faecalis, Staphylococcus aureus and Escherichia coli.

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

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

Example 1

Identification of peptides

Antimicrobial peptides were identified based on the AMBN protein sequence (AMBN, UniProtKB - Q9NP70, AMBN_HUMAN) by combination of different approaches of bioinformatics tools used to predict the antimicrobial activity of short protein sequences. A total of six short segments 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 proposed using rational design with molecular modelling methods. The designed analogues C and D show higher antimicrobial activity on biofilms than peptides A and B.

Example 2 Peptide synthesis

The peptides in this study were chemically synthesized and purified to > 97% purity. The peptides were solubilized in MQ 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 using the solid-phase peptide synthesis technique according to the standard N-Fmoc protocol. The peptides 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). N-Fmoc-protected amino acids (10 equiv) were coupled using 0.4 mol.I^-1 N-methylmorpholine in N,N-dimethylformamide (DMF, 20 equiv) and HBTU (10 equiv) in N-methyl-2-pyrrolidone. Deprotection of the a-amino group was performed using 20% (v/v) piperidine in DMF. Peptides were completely deprotected and cleaved from the resin with TFA/H₂O//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 Inc.) 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 technologies.

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, VSCHT Prague. Control strains of E. faecalis for testing antimicrobial drugs CNCTC 5530 (ATCC 51299) and CNCTC 5483 (ATCC 29212) were samples 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 1260) 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 detected from the same collection. The methicillin-sensitive strain S. aureus DBM 3178 (ATCC 29213) provided by the Institute of Biochemistry and Microbiology, VSCHT Prague and S. aureus M-1 isolated from an infected joint replacement at the Motol Faculty Hospital in Prague were also 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, VSCHT Prague. E. coli strain CCM 4787 (serovar O157: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 peptides of the invention 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-Bertani medium, and cultured at 37 °C and 150 rpm (revolutions per minute) for 24 hours.

Example 4

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

The effect of AMPs 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 = 2.5x107) and subsequently a volume of 30 μl was mixed with 290 μl of the appropriate medium and a solution of the tested peptide in polystyrene microtiter Honeycomb 2 plates (Growth Curves, USA). Cell cultivation takes 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 a drop of at least 50% 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 AMPs on biofilm formation was studied in polystyrene traction plates according to the microtiter procedure described in Vankova et al. (Vankova et al., World J Microbiol Biotechnol, 36(7): 101 , 2020). Briefly, the prepared inoculum (see example 3) was adjusted to OD_600nm = 0.8 (CFU/ml = 2× 10⁸) and then a volume of 210 μl was mixed with 70 μl of the appropriate 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 hours.

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 OD_600nm = 0.6 (CFU/ml = 1.5×10⁸) and pipetted into a 200 μl microtiter plate. 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 at final 200 μl volume. Cultivation was carried out as described above. All experiments contained control samples without the addition of an antimicrobial agent and a blank (samples without cell suspension). All experiments were performed in technical triplicates.

Example 6

Determination of metabolic activity of biofilm cells

The metabolic activity of biofilm cells was determined using resazurin. 25 μl of D-glucose solution (180 g/l in saline), 25 μl of resazurin solution (0.15 g/l in saline) and 100 μl of saline were pipetted onto the washed biofilm. Biofilm was measured fluorometrically 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 on an Infinite M200 Pro Reader plate spectrophotometer (Tecan, Switzerland). The mean background (fluorescence intensity of the blank) was subtracted from the resulting data and the mean and standard deviation were calculated and converted to relative percentages for better comparison of strains and peptides. Experiments were performed in technical triplicates.

Based on the detected metabolic activity, the parameters expressing the antibiofilm effectiveness of the studied substances were determined. The minimum biofilm-inhibiting concentration (MBIC₅₀ or MBIC₈₀) represents the lowest studied concentration of the given substance, which already causes a 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 (MBEC₅₀ or MBEC₈₀) represents the lowest studied concentration of a given substance, which already causes a 50% or 80% reduction in the metabolic activity of mature biofilm cells eradicated by the given substance.

Example 7

Haemolytic essay

The haemolytic activity of the studied antimicrobial peptides was evaluated in vitro using human blood. For haemolysis testing, fresh blood collected in a tube with sodium citrate as an anticoagulant was used. Melittin, 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) in submicromolar amounts. The haemolytic activity of the tested substances was then related to the haemolysis induced by 0.5% Triton X-100, which caused 100% haemolysis at this concentration. The concentration at which half of the red blood cells are lysed (value of haemolytic 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 of 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 peptides (tested antimicrobial peptides in solutions 12.5 to 300 μmol.I^-1, Melitin 0.024 to 50 μmol.I^-1 as a result, binary solution), PBS alone and 0.5% Triton X-100. The microtitre plate with 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

Haemolytic activity of AMP A, B, C and D, measured in the range of 12.5 to 100 (300) μmol.I- ¹ on a human blood sample and expressed as HC₅₀.

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 EGMTM-2 endothelial cell growth medium (Lonza) supplemented with 10% (v/v) inactivated fetal bovine serum (FBS) at 37°C in humid atmosphere with 5% CO₂. Twice a week, when cells reached 80-90% confluence, they were subcultured using 0.25% trypsin/0.53 mmol.I^-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 FBS in moistened atmosphere with 5% CO₂. Twice a week, when the cells reached 80-90% confluence, they were subcultured using 0.25% trypsin/0.53 mmol.I^-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 experiments were performed according to the manufacturer's instructions. 10,000 HUVEC cells (50 μl) were transferred to each well of a white 96-well plate (BD Biosciences™). Cells were then cultured for 24 hours before adding 50 μl of twice- concentrated solutions of test peptides or EGM^TM-2 medium alone (vehicle control) to each well. After an additional 72 h, 100 μl of CellTiter-Glo® reagent (Promega) was added to each well and the 96-well plate was vortexed for 2 min 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 AMPs A, B, C and D, measured in the range of 12.5 to 100 (300) μmol.I^-1 on HCT116 and HUVEC cell lines, expressed as IC₅₀.

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, and bandwidth 1 nm. The recommended sample concentration was 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 ellipticity 0 (deg cm² dmol^-1) per amino acid residue. Secondary analysis content was determined using the online circular dichroism program Dichroweb software (Lee Whitmore and B. A. Wallace, Nucleic Acids Res., 32: W668-W673, 2004). The secondary structure content of all analysed AMPs demonstrated their unstructured character with the potential to form a helical structure with an increasing concentration of TFE. The CD measurement results are shown in Fig. 1 and in Tab. 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.

Industrial Applicability

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

Claims

1. Antimicrobial peptide selected from the group consisting of: 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 peptide selected from the peptides A, B, C and D according to claim 1 , for use as a medicament.

3. Antimicrobial peptide selected from the peptides A, B, C and D according to claim 1 , for use as an antimicrobial medicament.

4. Antimicrobial peptide selected from the peptides A, B, C and D according to claim 1 , for use as an adjuvant treatment in the treatment of oncological diseases related to the formation of cancers in the oral cavity, such as mesenchymal benign carcinoma, fibroma, lipoma and hemangioma, malignant carcinoma, fibrosarcoma, myeloma and bone tumours, or neuroectoderm carcinomas.

5. Antimicrobial peptide selected from the peptides A, B, C and D according to claim 1 , for use in vivo for the prevention of the growth of bacterial contamination growth.

6. Use of the antimicrobial peptide selected from the peptides A, B, C and D according to claim 1 in biotechnological applications ex vivo for prevention of the growth of bacterial contamination.

7. Antimicrobial peptide selected from the peptides A, B, C and D according to claim 1 , for use in vivo for prevention or removal of infectious agents from the surface of joint, dental or bone replacements and/or for prevention or removal of infection of orthopaedic implants.

8. Use of the antimicrobial peptide selected from the peptides A, B, C and D according to claim 1 in biotechnological applications ex vivo for prevention or removal of infectious agents from the surface of joint, dental or bone replacements and/or for prevention or removal of infection of orthopaedic implants.