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FIELD OF THE INVENTION

Embodiments described here concern a series of active compounds against pathogenic microorganisms, characterized by a low or non-detectable cytotoxicity against the cells of higher organisms, cytostatic or cytotoxic to at least one Gram-negative and/or Gram-positive bacterium and/or a fungus (including yeasts) and/or a virus deriving from reference strains or clinical isolates.

BACKGROUND OF THE INVENTION

Antibiotic-resistance, defined as the emergence (and propagation) of factors of bacterial resistance to antibiotics, is triggered by the selective pressure exerted on microbe populations by an excessive and/or improper use of these drugs.

Starting from the introduction of penicillin in the 1940s, antimicrobial medicines, for example antibiotics, have played an essential role in the treatment of various microbe infections in both man and animals. Apart from the treatment of infectious diseases and nosocomial infections, antimicrobial drugs are essential to reduce the risk of complications connected to complex medical interventions such as hip replacement prostheses, organ transplants, chemotherapy for cancer and treatment for premature babies (COMMUNICATION FROM THE COMMISSION TO THE EUROPEAN PARLIAMENT AND COUNCIL, "Action plan against the rising threats from antimicrobial resistance ^' ", 201 1). Infections from resistant bacteria cause health costs estimated between 10 and 15 billion dollars every year in the US alone, with increased morbidity, mortality and costs associated with sickness (Prevention effectiveness: A guide to decision analysis and economic evaluation. 2nd edition. Editors: Haddix AC, Teutsch SM, Corso PS., New York: Oxford University Press 2003:345-57; Hospital and societal costs of antimicrobialUresistant infections in a Chicago teaching hospital: implications or antibiotic stewardship. Clin Infect Dis 2009; 49: 1175-84). Similarly, the costs caused by drug-resistant infections in Europe have been estimated at 1.5 billion euros per year, due to the increase in health expenses and loss of productivity; patients contaminated by resistant bacteria must be isolated when they receive hospital treatment and this costs 900 million euros and causes 2.5 million additional bed-days a year (Report on a safer health care in Europe: improving patient safety and combating antimicrobial resistance, Committee for the environment, public health and food safety, Brussels 4/5/2015).

Today, this problem has become a real priority in Public Health worldwide, also because of the appearance of pathogens that are simultaneously resistant to several antibiotics (multidrug resistance or heterogeneous resistance), in practice reducing the possibility of effective treatment. It must be stressed that heterogeneous antibiotic-resistance often affects the health structures. These multi-resistant microorganisms deriving from nosocomial infections can be transmitted in communities in various ways, including: ventilation and conditioning systems, water systems, treatment of tissues and lab samples, insufficient hygiene of the staff and the environment, surgical practices and invasive aids (Eggimann, Clin Microbiol Infect 2001 ;7: 91; Pittet, The Lancet 2000;356:1307; Hugonnet, Clin Microbiol Infect 2000;6:350; Pittet, Swiss- NOSO 2001;8:25).

Among the main problems there is methicillin-resistance in S. aureus ("Celbenin " -resistant staphylococci. Br Med J, i: 124-25, 1961), which has reached, in Italy, a constant incidence of around 40% and is one of the highest in Europe. In some European countries and in the US, the frequency of resistance to penicillin of S. pneumoniae reaches more than 20% and in the Far East even exceeds 50%. Recently, for this microorganism a high resistance to macrolides has been found, with values that reach 30% with an increasing trend. For the genus Enterococcus the frequency of vancomycin-resistance has progressively increased all over the world, especially in recent years (around 20% in the US) (in Centers for Disease Control and Prevention, 2002, Morb Mortal Weekly Rep, July 5, 51(26):565-567). In Italy, from a study carried out in 2007, a frequency of vancomycin-resistance emerged of about 2.5% in E. faecalis and about 20% in the less frequent E. faecium (European Antimicrobial Resistance Surveillance System, EARSS, 2007).

For Gram-negative microorganisms, a very heterogeneous distribution of resistances can be observed, with an increasing trend, especially with regard to resistance to fluoroquinolones, aminopenicillins and aminoglycosides in E. coli and K. pneumoniae/ oxytoca, and to carbapenems in P. aeruginosa. {Surveillance of antibiotic-resistance and use of systemic antibiotics in Emilia-Romagna. Dossier 173/2009, Emilia-Romagna Regional Health System). Recently the increase in the incidence of Gram-negative bacteria producing wide-spectrum beta-lactamases for epidemiological/microbiological relapses, relevant for the therapy, has caused considerable interest.

In the last four years, there has been a significant increase in resistance to multiple antibiotics both in E. coli strains and in those of K. pneumoniae in more than 33% of EU countries. There are few treatment options for these patients and are limited to carbapenems even if resistance to carbapenems is dangerously high in some countries, in practice limiting the therapeutic options (Annual epidemiological report, Antimicrobial resistance and healthcare-associated infections 2014, ECDC, www.ecdc.europa.eu).

In a report published by EFSA in 2007 (from "Trends and Sources of Zoonoses and Zoonotic Agents and Food-borne Outbreaks in the European Union in 2008 ", published 26 April 2010) it states that some of the most common zoonotic bacteria coming from animals and foodstuffs in the EU have developed resistance to antimicrobials. In particular, resistance to ampicillin, to sulfonamides and to tetracycline has frequently been found among bacteria tested and, from various countries, resistance to fluoroquinolones, to macrolides or third generation cephalosporins (important antibiotics in the treatment of human infectious pathologies) has been pointed out. These worries are connected to the finding of high levels of resistance to fluoroquinolones in Salmonella strains isolated from poultry and in the strains of Campylobacter isolated from poultry, cattle and pigs intended for consumption and the importance that some of these antibiotics have in the treatment of infectious pathologies in humans.

Living organisms defend themselves from the invasion of extraneous agents using two types of response: a so-called "innate or natural" immunity and an "acquired or specific" immunity. Innate immunity represents a defense mechanism preexisting before the encounter with the antigen. It uses different mechanical and chemical factors (integrity of the epithelia, skin, saliva, gastric secretion), humoral factors (lysozyme, complement, interferon), phagocytic cells (neutrophils, macrophages), dendritic cells, natural killer (NK) cells and commensal bacterial flora.

Antimicrobial peptides represent another innate response to microbe infections (Wiesner J, Vilcinskas A. Antimicrobial peptides: the ancient arm of the human immune system. Virulence. 2010; 1:440-64). They have a double importance since they protect about 80% of animal species and almost all plants, and also play a key role in the immunity of higher animals, supplying a sort of first line of defense that stimulates and cooperates actively with adaptive immune responses (Tossi A, Sandri L. Molecular diversity in gene-encoded, cationic antimicrobial polypeptides. Curr Pharm Des. 2002; 8:743-61). In higher animals, they represent the "effector" molecules in innate immunity (Boman HG. Antibacterial peptides: basic facts and emerging concepts. J Intern Med. 2003; 254: 197-215; Hancock RE. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect. Dis. 2001; 1: 156-164).

Antimicrobial peptides have a wide spectrum of activity: they kill bacterial cells rapidly and are active against numerous antibiotic-resistant strains of clinical importance {Hancock and Chappie, 1999; Scott and Hancock, 2000; Zaslof 2002).

Most antimicrobial peptides act by altering the membrane of the target cells directly ( Thevissen et al, 2000 ) . Other action mechanisms could concern the interaction of the peptides with molecules present in the cell or with membrane receptors, able to inhibit the DNA synthesis or to alter the enzymatic activity and that of the bacterial proteins, events that can cause cell death or can at least inhibit bacterial replication (Park et al, 1998).

Bacterial membranes are rich in anionic phospholipids, such as phosphatidylserine and phosphatidilglycerol: this causes an electrostatic interaction of the positively charged peptide with the membrane itself, which is at the basis of the subsequent effect of perturbing the double layer.

In the case of Gram-negative bacteria, it has been seen that initially the peptide interacts with the polyanionic molecules of lipopoly saccharides of the external membrane and is then able to permeabilize it or to be captured inside it. In the case of Gram-positive bacteria, on the contrary, the peptide is probably attracted by teichoic and teichuronic acids and by other anionic groups that are outside the layer of peptidoglycan. The different composition of the membranes is at the basis of the selectivity that some of these peptides have for bacterial cells.

The membranes of eukaryotic cells are characterized by a high content of zwitterionic phospholipids, such as phosphatidylcholine, sphingomyelin and phosphatidylethanolamine. They are also rich in cholesterol, which is absent from bacteria, which seems to inhibit the action of such peptides, conferring a certain resistance to membranes (Zasloff M.; 2002; Nature; 415:389-395).

Another important factor for selectivity is the value of the membrane potential: a more negative potential inside the cell, typical of bacterial cells (100-150 mV), facilitates the interaction of the peptide with the lipid layer {Bechinger, 1997).

Two general main mechanisms have been proposed to explain the effect consequent to the interaction of peptides with the cytoplasmic membrane;

- a "detergent" effect, in which the amphipathic structure of such molecules is thought to interact with the double lipid layer, destroying its organization and causing the cyto-plasmatic components to emerge;

- the formation of channels, due to the aggregation of the peptide monomers in the double lipid layer (Guerneve et al, 1998) and described in the Shai- Matsuzaki-Huang model (Zasloff M, 2002).

Starting from 1985, numerous antimicrobial peptides have been identified, and in 2014 the considerable figure of 2169 was reached (Wang, G., Li, X. and Wang, Z. (2016) APD3: the antimicrobial peptide database as a tool for research and education. Nucleic Acids Research 44, D1087-D1093).

However, most of these peptides have one or more disadvantages which limit their potential therapeutic use (Stain A. & Raoult D., 2002).

For example, the antimicrobial peptide polymyxin B requires cyclizing for its stabilization and biological activity. Moreover, this peptide causes nephrotoxicity, neurotoxicity and hyperthermia when used in therapeutically effective concentrations (Pike M, Saltiel E. Colistin- and polymyxin-induced nephrotoxicity: focus on literature utilizing the RIFLE classification scheme of acute kidney injury. J Pharm Pract. 2014 Dec;27(6):554-61 ; Moriyama N, Miyoshi M, Imoto T, Maruyama M, Shido O, Watanabe T. Systemic administration of polymyxin B induces hypothermia in rats via an inhibitory effect on metabolic rate. Eur J Pharmacol. 2006 Jul 10;541(l-2):38-43). Another example is given by Temporin L. This is an antimicrobial peptide isolated from Rana temporaria, toxic for eukaryotic cells, including human ones (Rinaldi AC, Mangoni ML, Rufo A, Luzi C, Barra D, Zhao H, Kinnunen PK, Bozzi A, Di Giulio A, Simmaco M. Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles. Biochem J. 2002 Nov 15;368:91-100). Bovine Myeloid Antimicrobial Peptides (BMAPs) have shown toxicity toward endothelial cells in cultures and in general toward almost all cell lines of the hematopoietic line (Risso et al., Cell Immunol., 189: 107-115, 1998). In the same way, Bombinina H2, isolated from the anuran Bombina orientalis, causes strong hemolysis (Csordas and Michl Monatsh Chem 101: 182-189, 1970).

Another example is given by Clavaspirina, an antimicrobial peptide, isolated from pharyngeal tissue of the tunicate Styela clava, rich in histidine, which has proven to be strongly hemolytic toward human and bovine erythrocytes (Lee et al., 2001).

Another example is given by Oxyopinin 4A, an antimicrobial peptide deriving from the toxin of the lynx spider, which despite a potent antimicrobial effect (E. coli MIC 0.5 mM, P. fluorescens MIC 1 mM, S. aureus MIC 10 mM), shows a strong toxicity against erythrocytes already at 7 μΜ (about 21 μg/ml) (Dubovski et al. FEBS J. 2011; 278:4382-93).

The synthetic peptide KFFKFFKFFK, consisting of cationic residues of lysine and hydrophobic residues of phenylalanine, proved to be strongly hemolytic at 40 μg/ml (Vaara M. and Porro M. 1996).

Another synthetic peptide called V681 has shown a strong hemolytic effect (100% hemolysis at 8 mg/ml) despite an exceptional antimicrobial activity (Influence of proline residues on the antibacterial and synergistic activities of alpha-helical peptides, Zhang L, Benz R, Hancock RE. Biochemistry, 1999, 38, 8102-8111).

Similarly, Bombinina H6, isolated from the secretions of the skin of Bombina orientalis, causes strong hemolysis (60%) at a concentration of 4 μΜ.

Document WO-A-2013/038201 describes antimicrobial amphiphilic peptides, preservants or surfactants.

Document WO-A-03/008442 describes peptides with antimicrobial activity, in particular bactericidal and leishmanicidal.

Document WO-A-98/40401 describes cationic peptides used in combination with antibiotic agents to treat infections, in particular bacterial infections.

There is therefore a need to perfect new active compounds against pathogenic microorganisms that can overcome at least one of the disadvantages of the state of the art.

In particular, one purpose of the present invention is to identify those antimicrobial peptides that have high biological activity against microorganisms associated with low or zero toxicity toward eukaryotic cells, a broad spectrum of activity and high structural stability for diverse clinical applications.

Other limitations and disadvantages of conventional solutions and technologies will be clear to a person of skill after reading the remaining part of the present description with reference to the drawings and the description of the embodiments that follow, although it is clear that the description of the state of the art connected to the present description must not be considered an admission that what is described here is already known from the state of the prior art.

SUMMARY OF THE INVENTION

The present invention is set forth and characterized in the independent claims, while the dependent claims describe other characteristics of the invention or variants to the main inventive idea.

In response to the need to identify compounds with high antimicrobial activity and negligible hemo- or cyto-toxicity that represent an effective alternative to the antibiotics currently available on the market for the treatment of antibiotic- resistant infections, the present description describes a series of linear peptides with antimicrobial activity and a wide spectrum of action.

Embodiments refer to a composition comprising one or more peptides, chosen from a group that comprises the peptides having the sequences KIFKKIRKAGIKIAKII (SEQ ID NO.: l), KIFKKILKIL KILKWI (SEQ ID NO.:2), KIFKKIWKILKKILKWI (SEQ ID NO.:3), IIKKIWKIIKKILKWI (SEQ ID NO.: 4), GTWWKKWGWIKTRLGGL (SEQ ID NO.: 5).

According to one embodiment, said composition is for use in medicine.

According to another embodiment, said composition is for the prevention and/or treatment of infectious pathologies of man and animals. According to another embodiment, at least one peptide of said one or more peptides is a linear peptide.

According to another embodiment, at least one peptide of said one or more peptides is composed of one or more D-amino acids.

According to another embodiment, at least one peptide of said one or more peptides is composed of one or more non-natural amino acids.

According to another embodiment, said composition is for the prevention and/or treatment of pathologies caused by microorganisms including bacteria, funguses and/or yeasts, viruses and protozoa.

According to another embodiment, said composition is for the prevention and/or treatment of pathologies caused by bacteria.

The peptides according to possible embodiments described here can be characterized by a length of 17 amino acids, an alpha-helix conformation in a hydrophobic environment or in the presence of membranes.

Moreover, according to possible embodiments described here, the central portion of at least one peptide of said one or more peptides is characterized by the presence of numerous charged amino acids, partly or totally alternated with non- polar amino acids.

The peptides according to embodiments described here can be characterized by the presence of an optimal percentage of hydrophobic, polar and charged amino acids, so as not to disturb the membranes of the eukaryotic cells. Finally, these peptides have high solubility in aqueous solvents.

According to embodiments described here, said peptides can be effectively used as primary agents, adjuvants or synergists in the treatment of pathologies caused by infectious agents attacking different anatomical districts of organisms belonging to the Animal genus, preferably belonging to phylum Chordata.

Moreover, the peptides according to embodiments described here can also be used effectively in the treatment of infectious pathologies of plant organisms. The peptides according to embodiments described here are easy to synthesize, highly effective, proteolytically stable, substantially salt-tolerant, non-hemolytic and non- cytotoxic.

The antibacterial spectrum of the antimicrobial peptides according to embodiments described here against Gram-negative microorganisms such as Escherichia coli (ATCC 25922), Pseudomonas aeruginosa ATCC shows an LD ₉₀ against E. coli comprised between 0.4 and 1.6 μg ml and for P. aeruginosa between 0.2 and 0.8 μg/ml.

Similarly, the results obtained against Gram-positive bacteria are also interesting, in particular against strains of S. aureus and the methicillin-resistant variant (MRSA). The LD ₉₀ vary between 1.6 and 1.3 μg/ml. Equally interesting are the results obtained against strains of MRSA, with LD ₉₀ comprised between 3.2 and 6.3 μg/ml.

The peptides according to embodiments described here have also shown themselves to be effective against fungi and yeast species, in particular against Candida albicans. The values of LD ₉₀ obtained for the different peptides, for both species tested, were comprised between a minimum value of 3.2 and a maximum value of 6.3 μg/ml.

These and other aspects, characteristics and advantages of the present disclosure will be better understood with reference to the following description, drawings and attached claims. The drawings, which are integrated and form part of the present description, show some forms of embodiment of the present invention, and together with the description, are intended to describe the principles of the disclosure.

The various aspects and characteristics described in the present description can be applied individually where possible. These individual aspects, for example aspects and characteristics described in the description or in the attached dependent claims, can be the object of divisional applications.

It is understood that any aspect or characteristic that is discovered, during the patenting process, to be already known, shall not be claimed and shall be the object of a disclaimer.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other characteristics of the present invention will become apparent from the following description of some embodiments, given as a non-restrictive example with reference to the attached drawings wherein:

- fig. 1 shows

(A) A representative image of the three-dimensional structure of the peptides according to embodiments described here, in particular the drawing refers to the peptide KIFKKIRKAGIKIAKII (SEQ ID No. 1);

(B) Representation of the hydrophobic surface in which the dark areas show polar amino acids (Glu, Asp, Lys, Arg, Ser, Thr, Asn, gin, Gly) while the clear areas show non-polar amino acids (Ala, Leu, He, Val, Met, Cys, Phe, Tyr, Trp, Pro);

(C) Representation of the distribution of the electrostatic charge on the surface of the peptide;

- fig. 2 shows Table 1 : List of the sequence of peptides selected;

- fig. 3 shows graphs A, B, C, corresponding to the Circular Dichroism (CD) analysis, respectively CD spectrum in water (H ₂ O), 50% TFE (2,2,2-

Trifluoroethanol) and lOOmM SDS (Sodium Dodecyl Sulfate);

- fig. 4 shows the graphs of the permeabilizing activity of the peptides with sequence SEQ ID No. 3 (distinguished by the acronym ART01) and respectively SEQ ID No. 4 (distinguished by the acronym ART02), while the acronym Ctrl indicates the control; panel A refers to the permeabilizing activity against S. aureus, while panel B refers to the permeabilizing activity against P. aeruginosa.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

We shall now refer in detail to the various embodiments of the present invention, of which one or more examples are shown in the attached drawings. Each example is supplied by way of illustration of the invention and shall not be understood as a limitation thereof. For example, the characteristics shown or described insomuch as they are part of one embodiment can be adopted on, or in association with, other embodiments to produce another embodiment. It is understood that the present invention shall include all such modifications and variants.

Before describing these embodiments, we must also clarify that the present description is not limited in its application to details of the construction and disposition of the components as described in the following description using the attached drawings. The present description can provide other embodiments and can be obtained or executed in various other ways. We must also clarify that the phraseology and terminology used here is for the purposes of description only, and cannot be considered as limitative.

Unless otherwise defined, all the technical and scientific terms used here and hereafter have the same meaning as commonly understood by a person with ordinary experience in the field of the art to which the present invention belongs. Even if methods and materials similar or equivalent to those described here can be used in practice and in the trials of the present invention, the methods and materials are described hereafter as an example. In the event of conflict, the present application shall prevail, including its definitions. The materials, methods and examples have a purely illustrative purpose and shall not be understood restrictively.

All the intervals reported here shall be understood to include the extremes, including those that report an interval "between" two values, unless otherwise indicated.

The present description also includes the intervals that derive from uniting or overlapping two or more intervals described, unless otherwise indicated.

The present description also includes the intervals that can derive from the combination of two or more values taken at different points, unless otherwise indicated.

Terms such as "about", "generally", "substantially" and suchlike shall be understood with their function of modifying a term or value that is not absolute, but is not reported in the state of the art. Such terms shall be defined by the specific circumstances and by the terms that they are intended to modify according to the common acceptance of such terms in the specific field. They shall take into account at least the degree of experimental error expected, the technical error and the instrumental error for a given technique adopted to measure a value.

Embodiments described here concern a series of linear antimicrobial peptides.

The peptides described here can also be obtained by a method according to embodiments described here.

The term "peptide" is defined in the present description as a plurality of amino acid residues joined by peptide bonds. It has the same meaning as polypeptide and protein and can be used interchangeably. The amino acids that make up the peptide can be defined by their chemical name, for example Alanine or Glycine, or by the official abbreviations with three letters, for example Ala, or one letter, for example A. The term "series" is defined in this document as all the possible variants of the peptide according to the present description, in which one or more amino acids of the peptide sequence are replaced by a homologous or similar amino acid, so that the properties of the peptides are maintained, although not necessarily at the same level. For example, a variant can have a bigger or smaller activity and/or a broader spectrum (for example an activity against a wider range of microbes) or can be more specific for a particular microorganism. Preferably, conservative replacements of amino acids are carried out in one or more amino acid residues. The term alpha-helix refers to the three-dimensional structure of the linear peptide as shown for example in fig. 1 : in it we observe consecutive amino acid residues with pairs of binding angles (φ, ψ) comprised between -60° and -45° that are located in the lower left quadrant of a Ramachandran graph. The skeleton of the polypeptide (that is, the sequence of bonds C _a -C-N-C _a of the residues) is tightly rolled around an imaginary central axis, while the side-chains of the amino acid residues protrude outside the central axis.

Collectively or individually, the peptides according to the present description are generally synthetic peptides, synthesized in vitro using chemical methods known in the state of the art. For example, the synthetic peptides are prepared using synthesis procedures on a solid phase, liquid phase, with peptide condensation, or any combination of these techniques. The amino acids that make up the peptides according to the present description can be natural or synthetic. The amino acids used for peptide synthesis can be amino acids in which N- amino-terminal is protected by the acid-unstable group N-tert-t-butyloxycarbonyl (Boc) as in Merrifield's work (J. Amino acid. Chem. Soc, 85: 2149-2154, 1963) or the base-unstable 9-fluorenylmethoxycarbonyl (Fmoc) as described by Carpino and Han (J. Org. Chem., 37:3403-3409, 1972). Both the protected amino acids Boc- or Fmoc- can be obtained from various commercial sources, such as for example Fluka, Sigma-Aldrich Bachem, Advanced Chemtech, Cambridge Biochemical Research.

In general, according to M. Bodansky, Principles of peptide synthesis, (Springer- Verlag, Berlin 1984) or JM Stewart and JD Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., Rockford, Illinois 1984), chemical synthesis methods on a solid phase consist of sequentially adding one or more amino acids to the growing peptide chain. Generally, the amino or carboxyl group of the first amino acid is protected by an optimal protector group. The first protected amino acid is attached to a solid inert support, for example a resin. The protective group is then removed from the residue bonded to the resin and the following amino acids (suitably protected) are added sequentially. After the number of amino acids has been reached, all the remaining protector groups (and any solid support) are removed in sequence or simultaneously, to have the final peptide.

It is possible to add more than one amino acid at a time to the growing chain, for example by coupling (in suitable experimental conditions which prevent the formation of racemes, due to the presence of chiral centers) a protected tripeptide with a suitably protected dipeptide to form, after de -protection, a pentapeptide as described for example by Merrifield in G. Barany and RB Merrifield, Peptides: Analysis, Synthesis, Biology, published by E. and J. Gross Meienhofer, vol. 2, (Academic Press, New York, 1980, pp 3-254).

These peptides can be synthesized by Companies that supply customized peptide synthesis services, by way of non-restricted example, Sigma-Aldrich (St. Louis, MO, USA), SelleckChem (Houston, TX, USA), Invitrogen (Grand Island, NY, USA), Abgent (Oxfordshire, OX144RY, United Kingdom), GeneScript (Piscataway, NJ, USA).

The degree of purity of the peptide compound can be determined with various methods, including identification of the HPLC peaks. Preferably, a peptide that produces a single peak, with height and amplitude equal to at least 75% of the material entering on an HPLC column is preferred. A peptide that produces a single peak which is at least 87%, at least 90%, at least 98% or even more than 98% of the material entering on an HPLC column is even more preferable.

To ensure that the peptide obtained, using one of the synthesis techniques indicated above, is the peptide desired for the uses or formulations described hereafter according to the present description, the composition of the peptide is analyzed with the help of different analytical methods known in the state of the art. The composition can be analyzed for example using a high-resolution mass spectrometer to determine the molecular weight of the peptide. Alternatively, the content of amino acids of a peptide can be confirmed by hydrolyzing the peptide in an acid solution to identify and quantify the components of the mixture using HPLC, or an amino acid analyzer. Equally useful are chromatographic methods on a thin layer which can be used to identify one or more constituents or residues of a desired peptide.

Amino acids can be located in different classes depending on the chemical- physical or structural properties of the side-chain of the amino acid.

The amino acids can be located in different classes: "SMALL" (alanine, glycine) [class 1], "NUCLEOPHILE" (serine, threonine, cysteine) [class 2], "HYDROPHOBIC" (valine, leucine, isoleucine, methionine, proline) [class 3], "AROMATIC" (tyrosine, phenylalanine, tryptophan) [class 4], "ACIDS" (glutamic acid, aspartic acid) [class 5], "STARCHES" (asparagine, glutamine) [class 6], "BASIC" (arginine, lysine, histidine) [class 7].

For example, amino acids can generally be divided into two large groups: hydrophilic or polar amino acids and lipophilic or non-polar amino acids.

Lipophilic amino acids can be selected from the group of lipophilic amino acids consisting of alanine, valine, leucine, isoleucine, proline, tyrosine, phenylalanine, tryptophan, methionine and cysteine; hydrophilic amino acids can be selected from the group of hydrophilic amino acids consisting of asparagine, glutamine, serine, threonine, lysine, arginine, histidine, glutamic acid, aspartic acid, cysteine and tyrosine. In the present description, the terms "lipophilic" or "hydrophilic" refer to amino acids that have a value on the hydrophobicity scale not less than 1.10 or not more than -0.2 respectively, as described for example in the New consensus hydrophobicity scale extended to non-proteinogenic amino acids di A. Tossi, L. Sandri, A. Giangaspero in Peptides, 2002, 416-417.

Moreover, a lipophilic or non-polar amino acid can also refer to an amino acid with a side-chain charged with acid pH {anionic amino acids) (aspartic acid or glutamic acid) or basic pH (cationic amino acids) (lysine, arginine or histidine), or with a non-charged side-chain {aliphatic amino acids) (glycine, valine, leucine, isoleucine or proline), containing an aromatic ring (tyrosine, tryptophan or phenylalanine), ionisable (lysine or arginine) or able to form hydrogen bonds (tryptophan, tyrosine, lysine, arginine, histidine, aspartic acid, glutamic acid).

A hydrophilic amino acid can refer to amino acids containing an atom of sulphur (cysteine or methionine), ionisable (aspartic acid, glutamic acid, histidine, cysteine or tyrosine) or able to form hydrogen bonds (asparagine, glutamine, serine, threonine, cysteine).

For example, amino acids can generally be divided into different classes: small amino acids (alanine, glycine).

In some embodiments, peptides can also consist of 3-50 amino acids, for example 3, 4, 5, 6 up to 50 amino acids. Preferably, the peptides can consist of 7,10,11,13,14,17,21,24,25,28,29,31 amino acids. More preferably, the peptides can consist of 10,11,13,14,17,21,24,25,28 amino acids. Still more preferably, the peptides can consist of 11,13,14,17,21,25 amino acids, for example 17 amino acids.

Embodiments described here concern a composition comprising one or more of the peptides in a group that comprises the individual peptides, or a combination of 1-2-3 up to 5 peptides, with the sequences: KIF KIRKAGIKIA II (SEQ ID NO.: 1), KIFKKILKILKKILKWI (SEQ ID NO.: 2), KIFKKIW ILKKIL WI (SEQ ID NO.: 3), KIIKKIWKIIKKILKWI (SEQ ID NO.: 4), GTWWKKWGWIKTRLGGL (SEQ ID NO.: 5).

Embodiments concern in particular a composition comprising a combination of decaheptapeptides with the following sequences:

Lys-Ile-Phe-Lys-Lys-Ile-Arg-Lys-Ala-Gly-Ile-Lys-Ile-Ala-Lys- Ile-Ile

(KIFKKIRKAGIKIAKII); SEQ ID NO. 1

Lys-Ile-Phe-Lys-Lys-Ile-Leu-Lys-Ile-Leu-Lys-Lys-Ile-Leu-Lys- Trp-Ile (KIFKKILKILKKILKWI); SEQ ID NO. 2

Lys-Ile-Phe-Lys-Lys-Ile-Trp-Lys-Ile-Leu-Lys-Lys-Ile-Leu-Lys- Tφ-Ile

(KIFKKIWKILKKILKWI); SEQ ID NO. 3

Lys-Ile-Ile-Lys-Lys-Ile-Trp-Lys-Ile-Ile-Lys-Lys-Ile-Leu-Lys- Trp-Ile

(KIIKKIWKIIKKILKWI); SEQ ID NO. 4

Ο^-Τ^-Τ -Τ ^8^8-Τφ-Ο^-Τ -Ι1ε^8-Τ1ΐΓ-Α¾-Ε6υ-Ο^-0^-ί6υ (GTWWKKWGWIKTRLGGL); SEQ ID NO. 5

Other embodiments concern a composition comprising the decaheptapeptide with the following sequence:

Lys-Ile-Phe-Lys-Lys-Ile-Arg-Lys-Ala-Gly-Ile-Lys-Ile-Ala-Lys- Ile-Ile

(KIFKKIRKAGIKIAKII); SEQ ID NO. 1

Other embodiments concern a composition comprising the decaheptapeptide with the following sequence:

Lys-Ile-Phe-Lys-Lys-Ile-Leu-Lys-Ile-Leu-Lys-Lys-Ile-Leu-Lys- Trp-Ile (KIFKKILKILKKILKWI); SEQ ID NO. 2

Other embodiments concern a composition comprising the decaheptapeptide with the following sequence:

Lys-Ile-Phe-Lys-Lys-Ile-Trp-Lys-Ile-Leu-Lys-Lys-Ile-Leu-Lys- Trp-Ile (KIFKKIWKILKKILKWI); SEQ ID NO. 3

Other embodiments concern a composition comprising the decaheptapeptide with the following sequence:

Lys-Ile-Ile-Lys-Lys-Ile-Trp-Lys-Ile-Ile-Lys-Lys-Ile-Leu-Lys- Trp-Ile

( IIKKIWKIIKKIL WI); SEQ ID NO. 4

Other embodiments concern a composition comprising the decaheptapeptide with the following sequence:

Gly-Thr-Trp-Trp-Lys-Lys-Trp-Gly-Trp-Ile-Lys-Thr-Arg-Leu-Gly- Gly-Leu (GT WWKK WG WIKTRLGGL) ; SEQ ID NO. 5

Other embodiments concern peptides that can consist of at least 80%, for example 90% or more preferably 91-100%, for example 100%, of L-amino acids.

Other embodiments concern peptides that can consist of at least 80%, for example 90% or more preferably 91-100%, for example 100%, of D-amino acids.

Still other variant embodiments concern peptides that can consist of at least 10%, for example 15% or more preferably 16-100%, for example 66%, of non- natural amino acids.

In possible variant embodiments, it is provided that the net charge of the peptides according to the present description is comprised between +2 and +10, preferably between +3 and +10, more preferably between +4 and +7. By the term net charge we mean, in the present invention, the algebraic sum of the number of cationic and anionic amino acids present in the peptide sequence.

In possible variant embodiments, it is provided that the hydrophobicity of the peptides according to the present description is comprised between 0.1 and 0.9, preferably between 0.2 and 0.7, more preferably between 0.368 and 0.698. By the term hydrophobicity we mean, according to the present invention, the index of mean hydrophobicity of the peptide obtained by the algebraic sum of the individual contributions of the amino acids that make up the peptide, according to the hydrophobicity scale developed by Fauchere, J., and Pliska, V. 1983. Hydrophobic parameters {pi} of amino-acid side chains from the partitioning of N-acetyl-amino-acid amides. Eur. J. Med. Chem. 8: 369-375.

In possible variant embodiments, it is provided that the hydrophobic moment (Hm) of the peptides according to the present description is comprised between 0.1 and 1, preferably between 0.3 and 1, for example between 0.412 and 0.910. The hydrophobic moment is calculated using the formula described by Eisenberg D, Weiss RM, Terwilliger TC, & Wilcox W (1982). Hydrophobic moments and protein structure. In Faraday Symposia of the Chemical Society (Vol. 17, pp. 109-120). Royal Society of Chemistry and using the hydrophobicity scale developed by Fauchere (see previous reference).

In possible variant embodiments, it is provided that the Delta G transferred from the water to the phospholipidic membranes according to the present description is comprised between -471.8 and -1.2, preferably between -20 and -2, even more preferably between -17 and -10, for example between -15.2 and -6.2, calculated as specified in Lomize AL, Pogozheva ID, Lomize MA, Mosberg HI (2006) Positioning of proteins in membranes: A computational approach. Protein Science 15, 1318-1333.

In possible variant embodiments, it is provided that the Boman index of the peptides according to the present description is comprised between -2 and +5, preferably between -1 and +4 and still more preferably between -1 and +1.0, for example between -0.344 and +0.652. By the term "Boman index", the present description defines the sum of the energies of transferring from the water to the cyclohexane of the side-chains of the individual amino acids that make up the peptides, divided by the total number of residues as described by Radzeka and Wolfenden (1988) in "Comparing the polarities of amino acids: side-chain distribution coefficients between vapor phase, cyclohexane, 1-octanol and neutral aqueous solution. ^' " (Biochemistry 27:1664-1670). The values calculated are negative but the sign (+ or -) is inverted.

In other possible variant embodiments, it is provided that the index of intrinsic disorder of the peptides according to the present description is comprised between -100 and 0, preferably between -50 and -0.00000001, even more preferably between -1 and -0.0001, for example between -0.188 and -0.015. This index evaluates the propensity of the peptides to be intrinsically disordered in an aqueous environment. The evaluation of the propensity is based on the algorithm developed by Uversky VN et al. described in Why are "natively unfolded" proteins unstructured under physiologic conditions? Proteins 2000; 41 :415-427.

In other possible variant embodiments, it is provided that the percentage of solubility in water of the peptides according to the present description is comprised between 0% and 99.999%, preferably between 75% and 99.999%, even more preferably between 91.0% and 99.999%. The percentage of solubility estimated is calculated using Wilkinson and Harris' two-parameter solubility model as described in Wilkinson DL and Harrison RG (1991) Bio/Technology 9, 443^148.

Collectively or individually, the peptides according to the present description can have a biological activity toward biota (cell organisms belonging to the Archea, Bacteria or Eukaryote domains).

In some embodiments there is expected to be little or no biological activity for biota belonging to the Eukaryote domain, preferably biota belonging to the subdomain Eumetazoa, still more preferably the biota belonging to the phylum Chordata. By the term "poor activity" we mean biological activity obtained using effective concentrations against biota belonging to the genus Bacteria.

The term "antimicrobial peptide" is used within the present description to define any peptide that has a bactericidal and/or bacteriostatic action and comprises, and is not limited to, any peptide described as antibacterial, antifungal, anti-mycotic, anti-parasitic, anti-protozoal, antiviral, anti-infective and/or germicidal, algaecide, amebicide, microbicide, bactericidal, fungicidal, anti- parasitic and protozoicide.

Collectively or individually, peptides according to the present description can be provided with antimicrobial activity for a wide range of pathogenic agents such as for example bacteria, fungi, yeasts, parasites, protozoa and viruses. A "pathogenic agent" is generally defined as any organism (mono- or multi- cellular, or with a subcellular organization) that causes a disease to an organism or changes the normal homeostasis thereof.

Collectively or individually, antimicrobial peptides according to the present description can be readily synthesized, highly effective, proteolytically stable, substantially salt-insensitive, non-hemolytic and non-cytotoxic.

To identify biologically active peptides, both single peptides and peptide libraries can be constructed, and individual peptides or peptides derived from these libraries can be subjected to screening for toxicity and biological activity, including, but not limited to, anti-mycotic, antibacterial, antiviral, antiprotozoal, and anti-parasitic activity.

The mass of peptides according to the present description can be determined by known procedures, for example, mass spectroscopy. Essentially, this technique provides to ionize a compound and subsequently to separate the ions produced on the basis of its mass/charge ratio. Mass spectrometers have different methods to determine the mass/charge ratio of the ion, for example flight time. Essentially, the ions coming from the source are accelerated by a potential (V) to the same kinetic energy, then they are left to drift along a tube to a detector. If a spectrometer has a length L flight tube, the flight time for a given ion is given by: t = (L ² * m / 2 * z * e * V). Starting from the m/z ratio function of the time it remains in the flight tube, the mass of a given ion can be calculated. The flight tube however has a low resolution (low ability to discriminate two ions with similar m/z) and to overcome this most of these analyzers are equipped with a "Reflectron", that is, a mirror able to reflect the ions and hence to make the ion travel double the road. In this way it is possible to distinguish 2 ions with very similar travel times in the flight tube. The use of the "Reflectron" limits the range of the molecular mass that is analyzable which is comprised between 200 and 5000-10000 Da.

The secondary structure of peptides in solution can be analyzed by a known technique called circular dichroism (see, for example, Fig. 3 and Example 7). It is part of the chiro-optic spectroscopies, that is, those spectroscopic techniques which, using polarized light, emphasize the optical activity of the molecules in question. The results obtained using this technique give information on the percentages of secondary structures present in the polypeptides. Although it is not possible to establish the position in the sequence, this technique can be used for initial screening to choose the solvent system in NMR analysis and as a control of the results obtained from computational calculation.

The antimicrobial activity of a peptide can be determined using a known method, such as broth dilution. Essentially, this method involves the growth of a microorganism in a liquid medium until the logarithmic phase is reached. The peptide to be tested is serially diluted with the growth medium for the bacterium in question in the wells of a multi-well plate. An optimal concentration of the microorganism is added to the wells containing the serially diluted peptide. The plate is incubated in a thermostat at a temperature of 37°C for a sufficient time to grow the microorganism. The growth of the microorganism, evaluated with respect to a negative control (a microorganism grown in the absence of the peptide), is determined by detecting the absorbance of the solution containing the bacterium, for example at 605 nM.

Another known method to determine the antimicrobial activity of a peptide according to the present description is represented by the agar diffusion test. Essentially, the test is carried out on agar plates of 14 cm or 9 cm. In order to inoculate the bacteria on the agar, 4-5 colonies grown on the primary isolation medium are suspended in 4-5 ml of Tryptic Soy Broth (enrichment broth), incubating for 2-6 hours until the broth culture has reached an opacity corresponding to 0.5 of the McFarland nephelometric Scale (1.5 x 10 ⁶ bacteria). The bacterium whose sensitivity is to be assayed is seeded on the surface of the agar. Subsequently, they are deposited on the surface of the perfectly dried agar of the discs, imbued with peptide, using sterile tweezers and, after attaching the discs to the surface, the plate is incubated in a thermostat at a temperature of 37°C for 24 hours. The diameter of the inhibition haloes will allow to define that bacterium which is resistant, intermediate resistant or sensitive for the peptide. In the present description, the cytotoxicity of the peptides was also determined, for example, by determining red blood cell hemolysis and classifying the antimicrobial peptides according to their minimum hemolytic concentration. In this document, MHC _l0 is defined as that concentration of peptide that determines 10% of hemolysis, MHC ₅₀ that peptide concentration that determines 50% of hemolysis and MHC ₉₀ that peptide concentration that determines 90% of hemolysis.

In this description, cytotoxicity of peptides has also been determined against three cell lines of mesenchymal derivation, for example fibroblasts, of endothelial derivation and epithelial derivation, for example cells of the intestinal epithelium or of the bronchial pulmonary epithelium. The cells were cultured, according to known procedures, in the presence and absence of peptides, and membrane damage was evaluated by means of the Trypan Blue vital dye.

According to possible embodiments, it is provided to use a peptide in the manufacture of a medicine useful for the treatment of a subject suffering from an infectious pathology. The term "treatment" refers to the effects of the peptides described here that are capable of conferring a benefit on patients suffering from an infectious pathology, for example improving patient conditions or delaying the progression of the disease. In this document, the term "infectious disease" or its synonym "infection" means the invasion, colonization and/or multiplication of a microorganism inside or on another host organism. By the term "microbial infection" we mean an infectious pathology caused by a pathogen agent, previously defined, for example, a bacterium, a parasite, a protozoa, a virus or a fungus including yeasts. In this document, the term "subject" defines any multicellular organism, including a human being, an animal, plant or insect that can be infected by a microorganism. Preferably, the subject is any animal organism, for example a human or animal that can be infected by a microorganism against which an antimicrobial peptide or one of its variants is active.

A pathogenic bacterium, as previously defined, can be derived from one of the bacterial species selected from the group, but not exclusive to the group, consisting of: Staphylococcus spp, for example, Staphylococcus aureus (for example Staphylococcus aureus ATCC 25923), Enterococcus spp, for example, Enterococcus faecalis; Pseudomonas spp., for example Pseudomonas aeruginosa or Pseudomonas marginalis; Mycobacterium spp, for example Mycobacterium tuberculosis; Listeria spp, for example Listeria monocytogenes; Enterobacter spp; Campylobacter spp;. Salmonella spp; Streptococcus spp, for example Streptococcus group A or B, Streptoccocus pneumoniae, Streptococcus mutans, Helicobacter spp, for example Helicobacter pylori; Neisseria spp, for example Neisseria gonorrea, Neisseria meningitidis. Borrelia burgdorferi, Shigella spp, for example, Shigella flexneri; Escherichia coli (for example Escherichia coli ATCC 25922); Haemophilus spp., for example Haemophilus influenzae; Francisella tularensis, Bacillus spp, for example Bacillus anthracis; Clostridia spp, Clostridium botulinum, Yersinia spp, for example, Yersinia pestis; Treponema spp; Burkholderia spp; for example Burkholderia cepacia, B. mallei and B pseudomallei; Stenotrophomonas spp, for example Stenotrophomonas maltophilia; Erwinia spp, for example Erwinia carotorova.

The biological activity of the peptides according to the present description against microorganisms was determined, for example, by assaying the microdilution broth and counting the colonies in the plate. The present description has determined the ability of peptides to reduce or prevent the growth of those bacteria involved in infectious pathologies of primary clinical importance. For example, Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli, and Gram-positive bacteria, for example, Staphylococcus aureus and its methicillin-resistant variant and Streptococcus mutans.

Pseudomonas aeruginosa is a problematic Gram-negative bacterium because of its invasiveness and heterogeneous resistance to antibacterial chemotherapy drugs. This microorganism is responsible for severe infections and causes significant morbidity in subjects immunocompromised by viral infections such as HIV, antitumor chemotherapy or immunosuppressive therapies. Furthermore, this bacterium is often the etiologic agent of severe infectious pathologies of the lower respiratory tract, urinary tract, skin lesions (wounds, ulcers) in young people, including those with cystic fibrosis, and elderly hospitalized patients. In recent years, the incidence of infections from Pseudomonas in cystic fibrosis has been increasing dramatically.

Escherichia coli is a Gram-negative microorganism belonging to the Enterobacteriaceae family to which bacteria like Shigella, Salmonella, Klebsiella or Proteus belong. Escherichia coli is an important pathogenic agent which often causes infectious pathologies of the urinary tract, bacteriaemia, nosocomial and community pneumonia and various infectious pathologies of the abdominal cavity. The emerging resistance to antibacterial chemotherapy drugs found in Escherichia coli in recent years is becoming a serious health problem. Of particular interest is the resistance correlated to the production of broad-spectrum beta-lactamase which has made this bacterium resistant to cephalosporins and fluoroquinolones, in particular ciprofloxacin. Streptococcus mutans is a Gram-positive bacterium belonging to the Streptococcaceae family, to which bacteria such as Streptococcus pneumoniae and Streptococcus pyogenes belong. It is an optional anaerobic that is commonly found in the human oral cavity. Described for the first time by Clarke in 1924, it is one of the main causes of tooth decay. S. mutans produces an insoluble extracellular polysaccharide (due to bonds 1-3 rather than 1-6) from sucrose. Glucan plays an extremely important role as a mediator of the adhesiveness of S. mutans, as well as a cementing molecule for other microorganisms, and to create a protected site where the microorganism can proliferate undisturbed. The same catalyzing enzyme for the formation of glucan, glucosyltransferase (GTF), is released by S. mutans and also plays the role of adhesiveness mediator because it is able to bind with the surface of the teeth, with that of the bacterial cells and with other glucans.

A fungal pathogen can be derived from one of the selected, but not limited fungi (including yeasts), belonging to the genera Candida spp. (Tor example C. albicans), Epidermophyton spp. Exophiala spp. Microsporum spp. Trichophyton spp. (Tor example T.rubrum and T. inter digitale), Tinea spp. Aspergillus spp.

Blastomyces spp. Blastoschizomyces spp. Coccidioides spp. Cryptococcus spp.

(Tor example Cryptococcus neoformans), Histoplasma spp. Paracoccidiomyces spp. Sporotrix spp. Absidia spp. Cladophialophora spp. Fonsecaea spp.

Phialophora spp. Lacazia spp. Arthrographis spp. Acremonium spp.

Actinomadura spp. Apophysomyces spp., Emmonsia spp. Basidiobolus spp.

Beauveria spp. Chrysosporium spp. Conidiobolus spp. Cunninghamella spp.

Fusarium spp. Geotrichum spp. Graphium spp. Leptosphaeria spp. Malassezia spp. (Tor example Malassezia furfur), Mucor spp. Neotestudina spp. Nocardia spp., Nocardiopsis spp. Paecilomyces spp. Phoma spp. Piedraia spp.

Pneumocystis spp. Pseudallescheria spp. Pyrenochaeta spp. Rhizomucor spp.

Rhizopus spp. Rhodotorula spp. Saccharomyces spp. Scedosporium spp.

Scopulariopsis spp. Sporobolomyces spp. Syncephalastrum spp. Trichoderma spp. Trichosporon spp. Ulocladium spp. Ustilago spp. Verticillium spp.

Wangiella spp.

The biological activity of the peptides according to the present description against a fungus or yeast was determined, for example, by assaying the microdilution broth and counting the colonies in the plate. For example, the ability was determined of an isolated peptide or a peptide belonging to a library of peptides to inhibit the growth and/or to kill fungi (including yeasts) belonging to the genus Candida spp or Malassezia spp. for example Candida albicans or Malassezia furfur.

Malassezia, formerly known as Pityrosporum, is a genus of fungus that lives on the skin of numerous mammals, including man and occasionally causes opportunistic infections. Recently, thanks to the use of molecular biology techniques, it has been observed that this fungus is the pathogenic agent of many human dermatites, including dandruff and seborrheic dermatitis (Sugita T, Tajima M, Takashima M, et al., 2004, "A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects", Microbiol. Immunol. 48 (8): 579-83). It has also been discovered that the skin rashes observable in pityriasis versicolor are due to infection from this fungus (Guillot J, Hadina S, Gueho E, 2008, "The genus Malassezia: old facts and new concepts". Parassitologia 50 (1-2): 77-9).

Collectively or individually, the peptides of the present description can also be useful in the treatment of infections generally associated with the skin, including, but not limited to, ulcers, wounds and skin lesions, cuts or burns. A further aspect of the present description indicates that peptides, collectively or individually, are useful in the treatment of bacterial or pyoderma skin infections.

According to some embodiments, it is provided to use the peptides described here collectively or individually in the treatment of pathologies (clinical or surgical) complicated by bacterial superinfections including, and not limited to, infections associated with the mucous membranes, infections associated with the gastrointestinal tract, urogenital tract, urinary tract infections (for example pyelonephritis, cystitis, urethritis) or respiratory infections, such as chronic bronchitis and cystic fibrosis.

In general, all members of the phylum chordata can be treated with the peptides described here, preferably those belonging to the vertebrate subphylum, more preferably those belonging to the infraphylum Gnathostomata, even more preferably those belonging to the Tetrapoda Superclass, for example those belonging to the class of mammals, birds, reptiles and amphibians. Finally, plants can also be treated with the peptides according to this description.

Various pharmaceutical formulas containing collectively or individually the peptides according to embodiments described here can be prepared using procedures described in the state of art and with known and readily available excipients.

In the present document, by the term "excipient" we mean an optimum compound or mixture thereof for use in a formula prepared for the treatment of a specific infectious pathology or conditions associated with it. For example, an excipient for use in a pharmaceutical formula should not generally cause an adverse reaction in a subject. The excipient, as described above, must not significantly inhibit the important biological activity of the active compound. For example, the excipient does not significantly inhibit the antimicrobial activity of an antimicrobial peptide according to the present description or one of its variants. An excipient can simply provide a buffering activity to maintain the active compound at a suitable pH to exert in this way its biological activity, for example, a phosphate buffered saline. Alternatively or additionally, the excipient can comprise a compound, for example a protease inhibitor, which increases the activity or half-life of the peptide. In another example, the excipient can include or can be itself an additional antimicrobial molecule and/or an anti-inflammatory molecule.

Collectively or individually, the peptides according to the present description can also be formulated as solutions for oral administration or as suitable solutions for parenteral administration, such as for example for intramuscular, subcutaneous, intraperitoneal or intravenous administration.

The pharmaceutical formulas of the peptides according to the present description may also take the form of an aqueous solution, an anhydrous form or a dispersion, or alternatively the form of an emulsion, suspension, pomade, cream or ointment.

Collectively or individually, the peptides according to the present description may be included in oily suspensions, solutions or emulsions in vehicles or in water and may contain agents useful for suspension, stabilization and/or agents promoting dispersion. Collectively or individually, the peptides according to the present description may be formulated in the form of powder obtained by aseptic isolation of a sterile solid or lyophilization of a solution to be reconstituted in solution form with the aid of a suitable vehicle prior to use, for example, water for preparations that can be injected.

Collectively, or individually, the peptides according to the present description may also be administered through the respiratory tract. For administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a mixture in powder of a therapeutic agent and a suitable powder base such as lactose or starch.

Collectively or individually, the peptides according to the present description may be administered in an aqueous solution in the form of an aerosol, or can be inhaled.

Throughout the description and the accompanying claims, the words "include" and "contain" and variations of these words, for example "includes" and "contains", mean "including but not limited to" and does not intend to exclude other fractions, additives, components or whole steps.

Throughout the description and the accompanying claims, the singular includes the plural, unless the context requires otherwise. Moreover, in the parts where the indefinite article is used, the specification is to be understood as plural and singular, unless the context requires otherwise.

By "individually" or "individual" we mean that the embodiments described here include the cited antimicrobial peptide or groups of antimicrobial peptides, and that although the individual peptides or peptide groups might not be listed separately here, the accompanying claims can define said peptides or groups of peptides separately and distinguishably from each other.

By "collectively" or "collective" we mean that the embodiments described here include any number or combination of said antimicrobial peptides or antimicrobial peptide groups, and that, although said numbers or combinations of peptides or peptide groups may not be specifically indicated in this document, the following claims can define said combinations or sub-combinations separately and distinguishably from any other combination of peptides or peptide groups. This disclosure is further described in the following, and non-restrictive, examples.

EXPERIMENTAL PART EXAMPLE 1 : Design of the template characterizing antimicrobial peptides

This example shows the procedure for designing the template used for the in silico selection of peptides with an antimicrobial activity

Results

From the public database DSAAP {The Database of Antimicrobial Activity and Structure of Peptides) 1426 sequences were extracted, having an experimentally proven antimicrobial activity, with the following search criteria: Option = Full Sequence, Length Interval = 10 to 20, N-Terminus = Without N- Terminus Modification, C-Terminus = Without C-Terminus Modification, Unusual Amino Acid = Without Modification, Complexity = Monomer, Target Group = [Gram+, Gram-, Fungus, Virus]. Of these, using visual inspection, 220 sequences were selected which had MIC values for E. Coli (Gram-) and S. aureus (Gram+) of less than 12.5 μg/ml and MIC values for C. albicans (Fungus), where present, of less than 25 μg/ml. The individual amino acids of the antimicrobial sequences were converted into classes 1-7 as defined above. In general it was possible to see that for some positions the amino acid frequencies were highly significant or more often it was possible to identify the type of dominant amino acid residue. For example, the percentage frequencies of the various classes in position 1 were: class 1 ("SMALL") 19%, class 2 ("NUCLEOPHILE") 6%, class 3 ("HYDROPHOBIC") 12%, class 4 ("AROMATIC") 21%, class 5 ("ACIDS") 9%, class 6 ("STARCHES") 2%, class 7 ("BASIC") 30%. In general, irrespective of the position, class 1 has a percentage of 9.5%, class 2 has 6.4%, class 3 has 28.4%, class 4 has 10.2%, class 5 has 3.2%, class 6 has 5.3% and class 7 has 25.2%. The sequences were generated and selected using a proprietary software called STAMP vers. 5.0.1 (Table 1 in fig. 2).

EXAMPLE 2: Synthesis of the peptides

Method

The peptides were synthesized at Sel!eckChem, through its associate in the United States. The synthesis was conducted using known techniques and more specifically synthesis on a solid phase.

Results Thirteen peptides were synthesized using known procedures with a degree of purity comprised between 78% and 95%. The peptides were delivered in ampoules containing 5 mg of lyophilized peptide. The technical reports attached to the peptides did not highlight any problems in the synthesis. Fig. 1 shows the structure of the peptides.

EXAMPLE 3: Determining the spectrum of activity and effectiveness of antimicrobial peptides on Gram-negative bacteria

This example shows the antibacterial spectrum and effectiveness spectrum of the peptides according to embodiments described here against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. The LD ₉₀ was determined for every peptide and for every strain tested.

Methods

a) Serial microdilution

Different peptides were tested for their ability to inhibit bacterial growth in liquid broth. Fifty microliters of peptide at a concentration comprised between about 0.2 raM and 50 raM were added to plates with 96 U-bottomed wells containing the liquid growth medium and one of the following bacteria: Escherichia coli ATCC 25922, P. aeruginosa ATCC 27853. After two hours of incubation at 37°C, the bacteria were transplanted to solid medium. As a growth control, the bacterial cultures were cultured without peptides.

b) Transplantation on agar

The bacterial suspension was put in contact for two hours with different concentrations of the peptide at a temperature of 37°C. After incubation, 20 ml were taken from the wells of each dilution and seeded on solid medium, McConkey agar for the Gram-negative bacteria and Columbia agar supplemented with 5% red corpuscles from cattle for Gram-positive bacteria. The inoculum was uniformly distributed on the agarized medium with the aid of a disposable sterile loop. After incubation for 24 hours at 37°C, the bacterial colonies (CFU) were counted.

Results

The LD ₉₀ expressed in μg/ml were calculated for each of the peptides tested, and the results are shown below: Sequence Escherichia coli Pseudomonas

aeruginosa

KIF IRKAGIKIAKII (SEQ 0.8 0.4

ID No. 1)

KIFK ILKIL KILKWI (SEQ 0.4 0.2

ID No. 2)

KIFKKIWKILKKILKWI (SEQ 0.4 0.4

ID No. 3)

IIKKIWKIIKKILKWI (SEQ 1.6 0.8

ID No. 4)

GTWWKKWGWIKTRLGGL 1.6 0.8

(SEQ ID No. 5)

The peptides according to the embodiments described here are growth and replication inhibitors of Escherichia coli and P. aeruginosa and may therefore be useful in the preparation of formulas for the treatment of infectious pathologies caused by both of the above bacteria.

EXAMPLE 4: Determining the spectrum of activity and effectiveness of antimicrobial peptides on Gram-positive bacteria

This example shows the antibacterial spectrum and effectiveness spectrum of the peptides according to the embodiments described here against S. aureus strains, both methicillin-sensitive and methicillin-resistant. The LD ₉₀ was determined for each peptide and for each strain.

Methods

a) Serial microdilution

Several synthetic peptides were tested for their ability to inhibit bacterial growth in liquid culture. Fifty microliters of peptides at a concentration comprised between about 0.2 iriM and about 50 mM were added to 96-well U- bottom plates containing the liquid growth media and one of the following bacteria: S. aureus ATCC 25923, methicillin-resistant S. aureus. After 2 hours of incubation at 37°C, the bacteria were transplanted on a solid medium. As a control, the bacterial cultures were cultured in the absence of the peptide, b) Transplantation on agar

The bacterial suspension was put in contact for two hours with different concentrations of the peptide at 37°C. After incubation, 20 ml were taken from the wells of each dilution and seeded on solid medium, Columbia agar supplemented with 5% red corpuscles from cattle for Gram-positive bacteria. The inoculum was uniformly distributed on the agarized soil with the aid of a disposable sterile loop. After incubation for 24 hours at 37°C, the bacterial colonies (CFU) were counted.

Results

The LD ₉₀ , expressed in μg/ml, were calculated for each of the peptides tested and the results are as follows:

The peptides according to the embodiments described here are inhibitors of S. aureus growth and replication both in the methicillin-sensitive variant and in the methicillin-resistant variant, and may therefore be useful in the preparation of formulas for the treatment of infectious pathologies caused by S .aureus.

EXAMPLE 5: Method to assay the antimicrobial activity of peptides against fungal strains (yeasts)

Methods

The inoculum was prepared by cultivating Candida albicans ATCC 10231 in Czapek-Dox broth (DIFCO) medium for 48-72 hours. On the day of the assay, the fungal culture was centrifuged at 1700xg for 15 min, the pellet was re- suspended in a phosphate buffer and stirred using an electric vortex to disperse the aggregates of fungal cells. The suspension was diluted until it reached an optical density of 0.5 on the McFarland turbidimetric scale, which contains approximately 1-5 x 10 ⁶ cells/ml. (Branda J. A., Krantz A. Effects of yeast on automated cell counting. Amino acid J Clin Pathol., 2006: 126, 248-254).

Results

The LD ₉₀ , expressed in μg/ml, were calculated for each of the peptides tested and the results are as follows:

The peptides according to the embodiments described here are C. albicans growth and replication inhibitors and may therefore be useful in the preparation of formulas for the treatment of infectious pathologies caused by C. albicans. EXAMPLE 6: Cytotoxicity of antimicrobial peptides

This example evaluates the cytotoxicity of antibacterial peptides by observing and quantifying the hemolysis of red blood cells (RBC) of sheep or the presence of intensely colored cells with Trypan Blue vital dye in the presence and absence of peptide.

Methods

(a) Hemolysis test The red blood cells of sheep, taken immediately prior to the start of the test, were washed in an isotonic buffer until the supernatant became transparent. The washed red blood cells were incubated at different concentrations of peptide for one hour at room temperature. After this period, the red blood cells were pelleted by centrifugation. The supernatant was harvested and analyzed at 450 nm to quantify the hemolysis. The positive control consists of red blood cells lysed with the addition of 0.1% Triton. This sample was used to determine the maximum optical density. The percentage of hemolysis was calculated from the ratio of optical density at 450 nm of the sample and that of the positive control.

The MHC ₅₀ value was calculated as the minimum peptide concentration able to induce cell lysis of 50%.

Results

The HC ₅₀ , expressed in μΜ, were calculated for each of the peptides tested and the results are as follows:

The peptides according to the embodiments described here are non-toxic to erythrocytes and can therefore be incorporated into formulas useful for the treatment of pathologies caused by pathogenic agents.

(b) Cytotoxicity of peptides against epithelial and endothelial cells and fibroblasts

Methods Cell growth was evaluated using a 10% Trypan Blue solution in a Neubauer Brand (Wertheim, Germany) count chamber. The living (non-colored) cells and the dead (colored) cells were counted under the microscope. The cells were seeded in 96-well plates at a concentration of 4.0 x 10 ^J cells per well and incubated for 24 hours at 37°C. The cells were subsequently cultured in the presence and absence of the peptides according to embodiments described here and incubated for 24 hours. After treatment the cells were washed with phosphate buffered saline (PBS), detached with trypsin and EDTA at a concentration of 0.1% and then counted. The experiment was carried out in independent triplicates.

Results

Peptides according to embodiments described here are non-toxic to eukaryotic cells and can therefore be incorporated into formulas useful for the treatment of pathologies caused by pathogenic agents.

EXAMPLE 7: Circular dichroism (CD)

Method

Circular dichroic measurements of the peptides with sequences SEQ ID No. 1 , SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5 were carried out at room temperature in 10 mM sodium phosphate buffer, pH 7.0, 50% TFE and in the presence of lOOmM SDS. The peptides were tested at an average concentration of 1 mg/ml.

Circular dichroism experiments were performed with a Jasco J-700 dicrograph (Tokyo, Japan) and the dichroic signal was processed using a software provided by Jasco and installed on personal computers. The spectra were recorded with a 16-second time constant, making 2 accumulations, at a scan speed of 20 nm/min. Results

Fig. 3 shows the CD spectrum in water (H ₂ 0) (graph A), 50% TFE (2,2,2- Trifluoroethanol) (graph B) and lOOmM SDS (Sodium Dodecyl Sulfate) (graph C). In water the relative curves show a negative minimum peak around 200 nm representing a random coil conformation (in a pure random coil structure a random coil structure is obtained with a weak positive band at 218 nm and an intense negative band at 198 nm), while it assumes an amphipathic alpha-helix conformation following the addition of an organic solvent that lowers polarity by mimicking the hydrophobic environment of the membranes. The TFE50% curves show a positive peak around 195 nm and a negative minimum around 208 nm (a pure alpha-helix structure is characterized by two intense negative bands at 222 nm and 208 nm and a positive band at 192 nm). In the presence of SDS at a concentration of 100 mM, the conformation of the peptides is maintained and/or exalted.

It has also been evaluated that the peptides are structured to form coiled-coils. The ratio between the molar elliptic value [Θ] obtained at 222 nm and that obtained at 208 nm indicates whether there are isolated helixes or aggregates of helixes to form a coiled-coil structure. Generally the threshold value for individual helixes is 0.9, whereas for coiled-coil structures it is 1.10. The results obtained in the different solvents indicate the presence of single helixes (H ₂ O = 0.2-0.4; TFE = 0.5-0.6; SDS = 0.7-0.8).

Finally, the percentage of "alpha-helix" possessed by the peptides in the various solvents was calculated by the following formula:

% helix = ([0] _obs * 100) / ([0] _hellx * (1-2.57/1)) where [0] _O bs is the value obtained experimentally at [Θ] 222nm; [0] _he ii _x is the maximum value obtainable at 222nm which is -39500; 1 is the number of peptide bonds present in the peptide, in this case 16.

The percentage of alpha-helix in water is 3-10% which rises to 7-36% in TFE 50% and settles between 10 and 50%» in SDS 100 mM. The number of amino acids in alpha-helix conformation (binding angles (φ, ψ) of -57° and -47° respectively) varies between 0 and 2 in H ₂ O, between 1 and 6 in TFE50% and between 2 and 8 in SDSlOOmM.

EXAMPLE 8: Determining the Minimum Inhibitory Concentration (MIC)

To evaluate the antimicrobial activity of the peptides with sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, the "Broth Microdilution Susceptibility Test" was performed according to the guidelines suggested by the Clinical and Laboratory Standards Institute (CLSI), using 96-well sterile plates. This type of assay determines the Minimum Inhibitory Concentration (MIC), which corresponds to the minimum concentration at which the peptide can inhibit bacterial growth.

Method

In each row, starting from the second well, 50 μΐ of the appropriate medium (Mueller-Hinton for bacteria, Sabouroud for fungi and yeasts) was dispensed, while a quantity of 100 - x μΐ was put in the first well, where x corresponds to the microliters of the peptide to be assayed, added so as to obtain a double concentration compared to the desired final concentration. Subsequently, serial dilutions were performed by taking 50 μΐ of solution from the first well and taking them into the second, where they were mixed with the medium already present, then from the second to the third and so on for all the wells until the penultimate one. The last well of each row, not containing peptide, thus acts as a positive growth control.

50 μΐ of bacterial or fungal suspension was then added to each well so that the final density in each well was equal respectively to about 2.5 x 105 cells/ml for the bacteria and 5 x 104 cells/ml for the fungi. To verify cell density, an aliquot portion of the added suspension was suitably diluted and plated for the subsequent count of colony forming units (CFUs). The micro-method plates thus prepared were incubated in a thermostat at 37°C to allow bacterial growth, and at 30°C for fungus growth, both visually detectable as turbidity of the medium or as the presence of a bottom in the well. The MIC value, corresponding to the last clear well, indicates the lowest concentration of peptide able to inhibit the growth of microorganisms.

Results

(A) S. aureus

KIFKKIWKILKKILKWI (SEQ ID No. 3): 6.25 μg/ml

KIIKKIWKIIKKILKWI (SEQ ID No. 4): 6.25 g/ml

KIF KIRKAGIKIAKII (SEQ ID No. 1): 200 μg/ml

GTWWKKWGWIKTRLGGL (SEQ ID No. 5): 200 μg/ml

(B) P. aeruginosa

KIFKKIWKILKKILKWI (SEQ ID No. 3): 12.5 μg ml

KIIKKIWKIIKKILKWI (SEQ ID No. 4): 12.5 μ _§ /ηι1

KIFKKIRKAGIKIAKII (SEQ ID No. 1): 12.5 μg/ml

GTWWKKWGWIKTRLGGL (SEQ ID No. 5): 50 μg/ml

( E.coli

KIFKKIWKILKKILKWI (SEQ ID No. 3): 12.5 μg/ml

KIIKKIWKIIKKILKWI (SEQ ID No. 4): 25 μg/ml

KIFKKIRKAGIKIAKII (SEQ ID No. 1): 12.5 μ _§ /ηι1

GTWWKKWGWIKTRLGGL (SEQ ID No. 5): 50 μg/ml (D) Candida albicans

KIFKKIWKILKKILKWI (SEQ ID No. 3): 3.125 μg/ml

KIIKKIWKIIKKILKWI (SEQ ID No. 4): 3.125 μg/ml

KIFKKIRKAGIKIAKII (SEQ ID No. 1): 0.8 μg/ml

GTWWKKWGWIKTRLGGL (SEQ ID No. 5): 12.5 μ^πιΐ

EXAMPLE 9: Permeabilization kinetics

Method

The membrane integrity of the bacterial cells after treatment with peptides having sequences KIFKKIWKILKKILKWI (SEQ ID NO: 3) and KIIKKIWKIIKKILKWI (SEQ ID NO: 4) was evaluated using propidium iodide (PI) (see Fig. 4). PI is a positively charged fluorochrome, belonging to the family of phenanthridine derivatives, which is excluded from the cell in conditions of normal cell vitality. When the membrane integrity is altered, the PI is able to enter the cell and insert itself into the double helix of the DNA, emitting red fluorescence with a maximum of 625 nm. In this way, the percentage of permeabilized cells can be determined based on their positivity to PI (PI +).

To evaluate any possible membrane damage induced by the peptides described here, a bacterial suspension in the exponential growth phase was prepared at a concentration of 1 x 10 ⁶ CFU/ml in MH, and an aqueous solution of PI (Sigma) was added to the final concentration of 10 μg/ml. The peptides were added to the tubes thus prepared in the appropriate concentrations. The samples were acquired by the cytofluorometer after 15, 30 and 60 minutes of treatment with the peptide at 37°C and analyzed for the red fluorescence signal of this probe, as well as for any possible changes in morphological parameters, i.e. front scattering (FS) and side scattering (SS).

All cytofluorometric analyses were performed by a flow-cytometer Cytomics FC 5000 (Beckman-Coulter, Inc., Fullerton. CA) equipped with an argon laser (488 nm, 5 mW). The instrument has a standard configuration: it has two refracted light detectors that measure frontal scattering (FS) and side scattering (SS) and 3 photomultipliers that detect fluorescence at different wavelengths. In particular, the FL3 photomultiplier was used, which detects red fluorescence (610 nm). All the detectors are set to logarithmic amplification; the optical and electronic background noises were eliminated by positioning an electronic threshold at the FS detector, while the cell flow was kept below 300 events per second to prevent the formation of cell aggregates in front of the beam of light. For each sample, 10,000 events were acquired. Data analysis was performed using FCS express V3 software (De Novo Software, CA).

Results

In fig. 4, the data relating to SEQ ID No. 3 are distinguished by the acronym ART01, while the data relating to SEQ ID No. 4 are distinguished by ART02. The acronym Ctrl indicates control.

The permeabilizing activity of peptides with sequences KIFKKIWKILKKILKWI (SEQ ID No. 3) and KIIKKIWKIIKKILKWI (SEQ ID No. 4) at a concentration of 12.5 μ /ηιΐ against S. aureus is clearly visible in fig. 4, panel A. At this concentration the two peptides with sequences SEQ ID No. 3 and SEQ ID No. 4 are able to permeabilize more than 95% of cells after only 15 minutes, 97.41% for per SEQ ID No. 3 and 97.94% per SEQ ID No. 4.

Similarly, the permeabilizing activity of the peptides KIFKKIWKILKKILKWI (SEQ ID NO: 3) and KIIKKIWKIIKKILKWI (SEQ ID NO: 4) against P. aeruginosa was evaluated at a concentration of 12.5 μg/ml. Fig. 4 Panel B shows that at this concentration the peptides having sequences SEQ ID No. 3 and SEQ ID No. 4 can permeabilize more than 90% of the cells after only 15 minutes, 93.55% for SEQ ID No. 3 and 92.77% for SEQ ID No. 4. EXAMPLE 10: Determining the Minimum Inhibitory Concentration (MIC) on scrambled and random control peptides

To evaluate the antimicrobial activity, the "Broth Microdilution Susceptibility Test" was performed according to the guidelines suggested by the Clinical and Laboratory Standards Institute (CLSI), using 96-well sterile plates.

This type of assay determines the Minimum Inhibitory Concentration (MIC), which corresponds to the minimum concentration at which the peptide can inhibit bacterial growth.

The test was carried out on two scrambled peptides, deriving from the peptide with SEQ ID No. 4 (original sequence), and having sequence WIIKIIWKKKKKKILII and WIIIIKKLKIKIWKKKI, and on the two random peptides having sequences KKIWKMKYQHLHVTKSY and KKIWKMKYQHLHVTKSY. The two random peptides keep the same charge as the original sequence of SEQ ID No. 4.

Method

In each row, starting from the second well, 50 μΐ of the appropriate medium (Mueller-Hinton for bacteria, Sabouroud for fungi and yeasts) was dispensed, while a quantity of 100 - x μΐ was put in the first well, where x corresponds to the microliters of the peptide to be assayed, added to obtain a double concentration compared to the desired final concentration. Subsequently, serial dilutions were performed. The last well of each row, not containing peptide, thus acts as a positive growth control. 50 μΐ of bacterial or fungal suspension was then added to each well so that the final density in each well was equal respectively to 2.5 x 105 cells/ml for the bacteria and 5 x 104 cells/ml for the fungi. To verify cell density, an aliquot portion of the added suspension was suitably diluted and plated for the subsequent count of colony forming units (CFUs).

The micro-method plates thus prepared were incubated in a thermostat at 37°C to allow bacterial growth, and at 30°C for fungus growth, both visually detectable as turbidity of the medium or as the presence of a bottom in the well. The MIC value, corresponding to the last clear well, indicates the lowest concentration of peptide able to inhibit the growth of microorganisms.

Results

(A) S. aureus

0. KIIKKIWKIIKKILKWI (SEQ ID No. 4) 6.25 μ^ιηΐ

1. WIIKIIWKKKKKKILII (scrambled peptide) 50 μ^ιηΐ

2. KKIWKMKYQHLHVTKS Y (random peptide, same charge) > 100 μ^πιΐ

3. WIIIIKKLKIKIWKKKI (scrambled peptide) > 100 μ^πιΐ

4. KKIWKMKYQHLHVTKS Y (random peptide, same charge) > 100 μ^ιηΐ

(B) P. aeruginosa

0. KIIKKIWKIIKKILKWI (SEQ ID No. 4) 12.5 μ^πιΐ

1. WIIKIIWKKKKKKILII (scrambled peptide) 100 μ^πιΐ

2. KKIWKMKYQHLHVTKS Y (random peptide, same charge) > 100 μ^ιηΐ

3. WIIIIKKLKIKIWKKKI (scrambled peptide) > 100 μ^πιΐ 25 μ^πιΐ

4. KKIWKMKYQHLHVTKS Y (random peptide, same charge) > 100 μ^πιΐ

It is clear that modifications and/or additions of parts can be made to the new active compounds against pathogenic microorganisms as described heretofore, without departing from the field and scope of the present invention.

It is also clear that, although the present invention has been described with reference to some specific examples, a person of skill in the art shall certainly be able to achieve many other equivalent forms of new active compounds against pathogenic microorganisms, having the characteristics as set forth in the claims and hence all coming within the field of protection defined thereby. SEQUENCE LISTING

<110> ARTA PEPTIDION S.R.L.S.

<120> New Active Compounds Against Pathogenic Microorganisms

<130> G5-0561

<150> ITl 02016000051771

<151> 2016-05-19

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<170> BiSSAP 1.3.6

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<212> PRT

<213> Artificial Sequence

<220>

<223> Antimicrobial peptide <400> 1

Lys He Phe Lys Lys He Arg Lys Ala Gly He Lys He Ala Lys He 1 5 10 15

He

<210> 2

<21 1> 17

<212> PRT <213> Artificial Sequence

<220>

<223> Antimicrobial peptide <400> 2

Lys He Phe Lys Lys He Leu Lys He Leu Lys Lys He Leu Lys Trp 1 5 10 15 He

<210> 3

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antimicrobial peptide <400> 3

Lys He Phe Lys Lys He Trp Lys He Leu Lys Lys He Leu Lys Trp 1 5 10 15 He

<210> 4

<21 1> 17

<212> PRT

<213> Artificial Sequence <220>

<223> Antimicrobial peptide

<400> 4

Lys He He Lys Lys He Trp Lys He He Lys Lys He Leu Lys Trp 1 5 10 15

He

<210> 5

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<213> Artificial Sequence

<220>

<223> Antimicrobial peptide <400> 5

Gly Thr Trp Trp Lys Lys Trp Gly Trp He Lys Thr Arg Leu Gly Gly 1 5 10 15

Leu