EP2408806A1 - Analogues de la temporine-sha et leurs utilisations - Google Patents

Analogues de la temporine-sha et leurs utilisations

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Publication number
EP2408806A1
EP2408806A1 EP10716554A EP10716554A EP2408806A1 EP 2408806 A1 EP2408806 A1 EP 2408806A1 EP 10716554 A EP10716554 A EP 10716554A EP 10716554 A EP10716554 A EP 10716554A EP 2408806 A1 EP2408806 A1 EP 2408806A1
Authority
EP
European Patent Office
Prior art keywords
peptide
leishmania
nucleic acid
peptide according
temporin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10716554A
Other languages
German (de)
English (en)
French (fr)
Inventor
Ali Ladram
Denis Sereno
Feten Abbassi
Bruno Oury
Mohamed Amiche
Pierre Nicolas
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Universite Pierre et Marie Curie
Institut de Recherche pour le Developpement IRD
Original Assignee
Centre National de la Recherche Scientifique CNRS
Universite Pierre et Marie Curie
Institut de Recherche pour le Developpement IRD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Universite Pierre et Marie Curie, Institut de Recherche pour le Developpement IRD filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP2408806A1 publication Critical patent/EP2408806A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance

Definitions

  • the present invention relates to novel antimicrobial peptides, pharmaceutical compositions comprising these peptides and their uses, especially as a drug or disinfectant.
  • the present invention further relates to a transgenic plant expressing these novel peptides.
  • antimicrobial peptides have emerged as very promising for designing new therapeutic agents.
  • Cationic antimicrobial peptides are considered to be one of the key components of the innate immune system that provides the first line of defense for multicellular organisms against pathogens.
  • the interest of these peptides comes on the one hand from their very broad spectrum of activity allowing in particular their use in the treatment of infections by multi-resistant strains.
  • their mode of action is based on rapid permeabilization or fragmentation of the membranes of microorganisms and is therefore unlikely to generate the appearance of resistance mechanisms.
  • Antimicrobial peptides have been identified in plants, insects, amphibians or mammals.
  • the skin of amphibians is an important source of antimicrobial peptides and each frog species has its own peptide repertoire that is usually 10 to 15 PAM.
  • the frogs of the Ranidae family are very numerous and this family currently comprises 16 genera and 338 species. These frogs synthesize and secrete a remarkable diversity of MAP that has been classified into 13 families (Conlon et al, 2008 and 2009).
  • One of these families, the temporins includes small size (generally between 10 and 14 residues) whose sequences are very variable depending on the species. More than 60 members belonging to the temporine family have been identified.
  • the temporins In contrast to the other 12 families of Ranidae peptides, the temporins lack the Rana box motif, a heptapeptide motif cyclized by a C-terminal disulfide bridge (Mangoni, 2006). In addition, most temporins contain a single basic residue which gives them a net charge of +2 at physiological pH.
  • Temporines are generally particularly active against gram-positive bacteria and yeasts, but they also possess antifungal properties (Rollins-Smith et al, 2003) and for some antiviral properties (Chinchar et al, 2004).
  • Leishmaniasis is an extremely widespread disease in the world, mainly in India, South America, Africa and around the Mediterranean. Several million people are infected each year with this parasite. Depending on the species of Leishmania, leishmaniasis can be cutaneous, muco-cutaneous or visceral affections. For example, the visceral form, the most serious and potentially fatal in the absence of treatment, is due to two species of Leishmania: Leishmania infantum and Leishmania donovani.
  • Leishmania present two successive morphological stages: the promastigote stage (free form in the digestive tract of the insect vector, the sandfly) and the amastigote stage (intracellular form infecting mononuclear phagocytes of the mammalian host).
  • the first-line treatment for leishmaniases is the use of antimony derivatives, such as meglumine antimoniate (Glucantime ® ) or sodium stibogluconate (Pentostam ® ).
  • antimony derivatives such as meglumine antimoniate (Glucantime ® ) or sodium stibogluconate (Pentostam ® ).
  • Glucantime ® meglumine antimoniate
  • Pentostam ® sodium stibogluconate
  • antimony derivatives such as meglumine antimoniate (Glucantime ® ) or sodium stibogluconate (Pentostam ® ).
  • Glucantime ® meglumine antimoniate
  • Pentostam ® sodium stibogluconate
  • the object of the present invention is to provide novel antimicrobial peptides, temporin-SHa analogs and having increased antimicrobial activity, especially with respect to bacteria and the parasite Leishmania.
  • these new peptides also have decreased hemolytic activity relative to the temporin-SHa.
  • the present invention firstly relates to a peptide of 13 to 100 amino acids in size, having an antimicrobial activity and comprising the sequence FLX 1 -X 2 -IVX 3 -MLX 4 -KLF, where X 1 is amino acid selected from the group consisting of S, R, H and K, and X 2 , X 3 and X 4 , the same or different, are amino acids selected from the group consisting of G, R, H and K, and wherein, when X is S, at least one of X 2 , X 3 and X 4 residues is selected from the group consisting of R, H and K, and the functional derivatives and pharmaceutically acceptable salts of said peptide.
  • X 1 is an amino acid selected from the group consisting of R, H and K, and X 2 , X 3 and X 4 are G.
  • X 1 is K, and X 2 , X 3 and X 4 are G .
  • the present invention relates to a nucleic acid encoding a peptide according to the invention.
  • the present invention further relates to an expression cassette comprising a nucleic acid according to the invention.
  • the present invention also relates to an expression vector comprising a nucleic acid encoding a peptide according to the invention.
  • the present invention relates to a host cell comprising a nucleic acid, a cassette or an expression vector according to the invention.
  • the present invention also relates to an antibody specifically binding to a peptide according to the invention.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one peptide according to the invention, and a pharmaceutically acceptable carrier and / or excipient.
  • the present invention also relates to a peptide according to the invention as a medicament.
  • the medicament is for treating an infection with a bacterium, a virus, a fungus or a parasite.
  • the parasite belongs to the genus Leishmania.
  • the present invention relates to the use of a peptide according to the invention as a disinfectant, preservative or pesticide.
  • the present invention relates to a medical device or an implant comprising a body having at least one surface covered or including a peptide according to the invention.
  • the present invention relates to a transgenic plant comprising a nucleic acid, a cassette or an expression vector according to the invention, and capable of expressing or expressing a peptide according to the invention.
  • FIG. 1 shows a Schiffer-Edmunson projection of the ⁇ -helix of temporin-SHa.
  • Residues 4, 11, 7, 3 and 10 constitute the polar face of the helix.
  • the residues 8, 1, 12, 5, 9, 2, 13 and 6 constitute the apolar face of the helix.
  • FIG. 2 presents a graph showing the activity of the temporin-SHa (B) and the [K 3 ] temporin-SHa (*) analog on the axenic amastigotes (A) and promastigotes (B) of Leishmania infantum.
  • the percentage of growth of the amastigotes and promastigotes is represented as a function of the peptide concentration.
  • Figure 3 shows a graph showing the cytotoxic activity of temporin-SHa (m) and the [K 3 ] temporin-SHa (*) analog on monocytes. The percentage of monocyte growth is shown as a function of peptide concentration.
  • temporin-SHa formerly called temporin-lSa
  • This temporin is obtained by the post-translational processing of a precursor of 50 residues (entry number in the GenBank database: CAO77282).
  • This precursor has a highly conserved N-terminal domain containing the signal peptide and a region rich in acid residues, as well as a hypervariable C-terminal domain containing the progenitor sequence of the temporin-SHa.
  • the mature form of temporin is obtained after i) proteolytic cleavage at the KR doublet that precedes the progenitor sequence, ii) removal of the C-terminal K residue from the progenitor sequence by a carboxypeptidase and iii) residue amidation C-terminal of temporin thanks to the C-terminal residue G of the progenitor sequence which serves as donor of the amide function (substrate peptidyl-glycine ⁇ -amidating monooxygenase).
  • the mature protein is a peptide of 13 amino acid residues length and having the following sequence FLSGIVGMLGKLF (SEQ ID No.1).
  • the temporins are not structured in aqueous solution but are structured in ⁇ -helix in membrano -mimetic environments.
  • This peptide has antimicrobial activity against Gram-positive and Gram-negative bacteria, yeast and parasite Leishmania infantum (Abbassi et al., 2008).
  • the antiparasitic action of temporin-SHa is exerted on the promastigote forms as well as on the axenic amastigote forms of the parasite with respectively IC 50 of 18.1 ⁇ M and 22.8 ⁇ M.
  • the antimicrobial activity of antimicrobial peptides (MAP), as well as their cytolytic activities towards mammalian cells reflect a subtle equilibrium between several parameters, such as net charge, hydrophobicity and helicity.
  • peptide oligopeptide
  • polypeptide polypeptide or protein
  • C cysteine
  • D aspartic acid
  • E glutamic acid
  • F phenylalanine
  • G glycine
  • H histidine
  • I isoleucine
  • K lysine
  • L leucine
  • M methionine
  • N asparagine
  • P proline
  • Q glutamine
  • R arginine
  • S serine
  • T threonine
  • V valine
  • W tryptophan
  • Y tyrosine
  • microbe or "microbial” as used herein refers to bacteria, fungi, yeasts, viruses and / or parasites.
  • microbial infection refers to an infection caused by bacteria, fungi, yeasts, viruses and / or parasites.
  • IC50 or “inhibitory concentration 50” is the concentration of a compound required to reduce the in vitro growth of a population of microorganisms by 50%.
  • MIC or “minimum inhibitory concentration” corresponds to the minimum concentration of compound which makes it possible to totally inhibit microbial growth, after 18 hours of incubation, generally at 37 ° C., in the presence of said compound.
  • CMB or “minimum bactericidal concentration” is the minimum concentration of compound that can destroy 99.9% of microorganisms after 18 to 24 hours of contact with said compound.
  • lethal concentration 50 refers to the concentration of compound causing the death of 50% of a population cellular.
  • LC50 is a quantitative indicator of the toxicity of a compound. LC50 is used in this document to evaluate the haemolytic activity of PAM and in this case corresponds to the peptide concentration inducing haemolysis of 50% of red blood cells.
  • the present invention relates first of all to a temporin-SHa analog peptide in which one or more amino acids of the polar face of the ⁇ -helix are substituted with basic amino acids.
  • the amino acids constituting the polar face of this helix are the residues 4, 11, 7, 3 and 10 of the temporin-SHa , especially as presented in SEQ ID No.1.
  • the present invention thus relates to a temporin-SHa analog peptide having antimicrobial activity and comprising the sequence FL-Xi-X 2 -IVX 3 -ML-X4-KLF (SEQ ID No.18), where X1 is an amino acid selected from the group consisting of S, R, H and K, and X 2 , X 3 and X 4 , which are the same or different, are amino acids selected from the group consisting of G, R, H and K, and wherein when Xi is S at least one of the residues X 2 , X 3 and X 4 is selected from the group consisting of R, H and K.
  • the peptide of the present invention comprises a sequence selected from the group consisting of
  • FL-Xi-GIVGMLGKLF SEQ ID No. 2
  • FLSX 2 -IVGMLGKLF SEQ ID No.3
  • FLSGIVX 3 -MLGKLF SEQ ID No.4
  • FLSGIVGMLX 4 -KLF SEQ ID No.5
  • FL-Xi-X 2 -IVGMLGKLF SEQ ID NO: 6
  • FL-Xi -GI-VX 3 -MLGKLF (SEQ ID No. 7); FL-Xi -GI-VGMLX 4 -KLF (SEQ ID No.8); FLSX 2 -IVX 3 -MLGKLF (SEQ ID No.9); FLSX 2 -IVGMLX 4 -KLF (SEQ ID No.10); FLSGIVX 3 -MLX 4 -KLF (SEQ ID No.11);
  • FL-Xi-X 2 -IVX 3 -MLGKLF SEQ ID No. 12
  • FL-Xi-X 2 -IVGMLX 4 -KLF SEQ ID No.13
  • FL-Xi-GIVX 3 -MLX 4 -KLF SEQ ID No. 14
  • FLSX 2 -IVX 3 -MLX 4 -KLF SEQ ID No.15
  • FL-Xi-X 2 -IVX 3 -MLX 4 -KLF SEQ ID No.16
  • X 1 , X 2 , X 3 and / or X 4 are basic amino acids selected from the group consisting of R , H and K.
  • X 1 , X 2 , X 3 and / or X 4 are K.
  • Xi is K in SEQ ID Nos. 2 to 16.
  • the peptide has a size of between 13 and 100 amino acids, preferably between 13 and 30, 35, 40, 45 or 50 amino acids. According to another embodiment, the peptide has a size of between 13 and 15, 20 or 25 amino acids. According to a particular embodiment, the peptide has a size of 13 amino acids.
  • the peptide according to the invention may be a precursor of a mature antimicrobial peptide. This precursor then undergoes post-translational modifications making it possible to obtain the mature form of the PAM. It can thus comprise a translocation signal sequence and recognition and / or cleavage sites allowing it to undergo these post-translational modifications.
  • the peptide is a precursor of a mature antimicrobial peptide and comprises the sequence FLGT-1-NLSLCEQERDADEEERRDEPN-ESNVEVEKRFL-Xi-X 2 -I-VX 3 -MLX 4 -KLFGK (SEQ ID No.17 ), where X 1 is an amino acid selected from the group consisting of S, R, H and K, and X 2 , X 3 and X 4 , which are identical or different, are amino acids selected from the group consisting of G, R, H and K, and where when X is S at least one of the residues X 2 , X 3 and X 4 is selected from the group consisting of R, H and K.
  • the amino acids constituting the peptide according to the invention may be of L or D configuration, preferably of L configuration.
  • the peptide according to the invention may have a post-translational modification and / or a chemical modification, in particular a glycosylation, an amidation , acylation, acetylation or methylation.
  • protecting groups may be added at the C- and / or N-terminus.
  • the protecting group at the N-terminus may be acylation or acetylation and the protecting group at the C-terminus may be amidation or esterification.
  • the action of proteases can also be counteracted by the use of amino acids of configuration D, the cyclization of peptide by formation of disulfide bridges, lactam rings or bonds between the N- and C-termini.
  • the peptide according to the invention has an amidation of its C-terminal end.
  • the peptide according to the invention may comprise one or more amino acids which are rare amino acids, in particular hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylsilyl, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid; or synthetic amino acids including ornithine, norleucine, norvaline and cyclohexyl-alanine.
  • amino acids which are rare amino acids in particular hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylsilyl, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid; or synthetic amino acids including ornithine
  • the invention also covers the functional derivatives of a peptide according to the invention as described above.
  • the term "functional derivative” as used herein refers to peptides having substantially the same amino acid sequence, substantially the same helical structure, and substantially the same antimicrobial activity. These functional derivatives can be, for example, retro peptides, retro-inverso peptides, peptides having conservative substitutions and peptides whose side chain of one or more of the amino acids is substituted by groups which do not modify the antimicrobial activity of the peptide of the invention.
  • the term “conservative substitution” as used herein refers to a substitution of one amino acid residue for another that has similar chemical or physical properties (size, charge, or polarity).
  • isoleucine, leucine, alanine, valine, phenylalanine, proline and glycine may be conservatively substituted, as may lysine, histidine and arginine or serine, tyrosine and threonine or cysteine and methionine or asparagine, glutamine and tryptophan or aspartic acid and glutamic acid.
  • the term "functional derivative” also refers to a peptide according to the invention whose sequence is shortened by 1, 2, 3 or 4 amino acids at its C-terminal and / or N-terminal end.
  • the invention also covers the pharmaceutically acceptable salts of a peptide according to the invention.
  • the pharmaceutically acceptable salts may be, for example, salts with pharmaceutically acceptable mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid; salts with pharmaceutically acceptable organic acids such as acetic acid, citric acid, maleic acid, malic acid, succinic acid, ascorbic acid and tartaric acid; salts with pharmaceutically acceptable inorganic bases such as sodium, potassium, calcium, magnesium or ammonium salts; or salts with organic bases which have salifiable nitrogen, commonly used in the pharmaceutical art.
  • the methods for preparing these salts are well known to those skilled in the art.
  • the peptide according to the invention can be obtained by conventional chemical synthesis (in the solid phase or in liquid homogeneous phase) or by enzymatic synthesis (Kullman et al., 1987). It can also be obtained by culturing a host cell, as described hereinafter, comprising a transgene encoding the peptide and expressing said peptide, and extracting said peptide from these host cells or from the culture medium. wherein the peptide has been secreted.
  • the peptide according to the invention is endowed with antimicrobial activity.
  • this activity is greater than that of temporin-SHa with respect to at least one bacterial, viral, fungal or parasitic strain.
  • the peptide according to the invention has an antimicrobial activity superior to temporin-SHa with respect to bacteria and more particularly with respect to Gram-negative bacteria such as Escherichia coli or Pseudomonas aeruginosa.
  • the peptide according to the invention has a MIC against Pseudomonas aeruginosa of less than 10 ⁇ M.
  • the peptide according to the invention has an antimicrobial activity superior to temporin-SHa with respect to parasites, especially with regard to the parasites Leishmania infantum, Leishmania donovani, Leishmania amazonensis, Leishmania major , Leishmania mexicana, Leishmania panamensis, Leishmania tropica, Leishmania braziliensis, Leishmania guyanensis and / or Leishmania peruviana, particularly Leishmania infantum, Leishmania major, Leishmania tropica and / or Leishmania braziliensis, and most preferably vis-à-vis the parasite Leishmania infantum.
  • the peptide according to the invention has an IC50 of less than 15 ⁇ M vis-à-vis the promastigote form of the parasite Leishmania infantum. According to a preferred embodiment, the peptide according to the invention has a lower hemolytic activity than that of temporin-SHa. According to a particular embodiment, the peptide according to the invention has an ⁇ -CL50 for erythrocytes greater than 30 ⁇ M.
  • the present invention also relates to a nucleic acid encoding a peptide according to the invention.
  • nucleic acid means any molecule based on DNA or RNA. It may be synthetic or semi-synthetic molecules, recombinant, possibly amplified or cloned in vectors, chemically modified, comprising non-natural bases or modified nucleotides comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar.
  • the nucleic acid according to the invention may be in the form of DNA and / or RNA, single-stranded or double-stranded.
  • the nucleic acid is an isolated DNA molecule, synthesized by recombinant techniques well known to those skilled in the art.
  • nucleic acid according to the invention can be deduced from the sequence of the peptide according to the invention and the use of the codons can be adapted according to the host cell in which the nucleic acid is to be transcribed. These steps can be performed according to methods well known to those skilled in the art and some of which are described in the reference manual Sambrook et al. (Sambrook et al., 2001).
  • the present invention further relates to an expression cassette comprising a nucleic acid according to the invention operably linked to the sequences necessary for its expression.
  • the nucleic acid may be under the control of a promoter allowing its expression in a host cell.
  • an expression cassette consists of or comprises a promoter for initiating transcription, a nucleic acid according to the invention, and a transcription terminator.
  • expression cassette refers to a nucleic acid construct comprising a coding region and a regulatory region, operably linked.
  • operably linked indicates that the elements are combined in such a way that the expression of the coding sequence (the gene of interest) and / or targeting of the encoded peptide are under control of the transcriptional promoter and / or the transit peptide.
  • the promoter sequence is placed upstream of the gene of interest, at a distance thereof compatible with the control of the expression.
  • the sequence of the transit peptide is generally fused upstream of the sequence of the gene of interest, and in phase with it, and downstream of any promoter. Spacer sequences may be present between the regulatory elements and the gene, as long as they do not prevent expression and / or targeting.
  • said expression cassette comprises at least one enhancer activator sequence operably linked to the promoter.
  • the present invention further relates to an expression vector comprising a nucleic acid or an expression cassette according to the invention.
  • This expression vector can be used to transform a host cell and allow the expression of the nucleic acid according to the invention in said cell.
  • the vector may be a DNA or an RNA, circular or not, single- or double-stranded. It is advantageously chosen from a plasmid, a phage, a phagemid, a virus, a cosmid and an artificial chromosome.
  • the expression vector comprises regulatory elements allowing the expression of the nucleic acid according to the invention.
  • These elements may comprise, for example, transcription promoters, transcription activators, terminator sequences, initiation and termination codons.
  • the methods for selecting these elements based on the host cell in which expression is desired are well known to those skilled in the art.
  • the vector may further comprise elements allowing its selection in the host cell, such as, for example, an antibiotic resistance gene or a selection gene ensuring the complementation of the respective gene deleted in the genome of the host cell.
  • elements allowing its selection in the host cell, such as, for example, an antibiotic resistance gene or a selection gene ensuring the complementation of the respective gene deleted in the genome of the host cell.
  • the expression vector is preferably a plant vector.
  • plant vectors are described in the literature, among which mention may in particular be made of A. tumefaciens T-DNA plasmids pBIN19 (Bevan, 1984), pPZ100 (Hajdukewicz et al., 1994), the pCAMBIA series (R. Jefferson, CAMBIA, Australia).
  • Vectors of the invention may further comprise an origin of replication and / or a selection gene and / or a plant recombination sequence.
  • the vectors can be constructed by conventional molecular biology techniques well known to those skilled in the art.
  • the present invention relates to the use of a nucleic acid, an expression cassette or an expression vector according to the invention to transform or transfect a cell.
  • the host cell may be transiently / stably transfected / transfected and the nucleic acid, cassette or vector may be contained in the cell as an episome or chromosomal form.
  • the present invention relates to a host cell comprising a nucleic acid, a cassette or an expression vector according to the invention.
  • the host cell is a microorganism, preferably a bacterium or a yeast.
  • the host cell is an animal cell, for example a mammalian cell such as COS or CHO cells (US 4,889,803; US 5,047,335).
  • the cell is non-human and non-embryonic.
  • the host cell is a plant cell.
  • plant cell refers to any cell derived from a plant and capable of constituting undifferentiated tissues such as calli, and differentiated tissues such as embryos, plant parts, plant tissues and the like. plants or seeds.
  • the present invention also relates to a method for producing an antimicrobial peptide according to the invention comprising the transformation or transfection of a cell with a nucleic acid, an expression cassette or an expression vector according to the invention; culturing the transfected / transformed cell; and harvesting the peptide produced by said cell.
  • Methods for producing recombinant peptides are well known to those skilled in the art. For example, there may be mentioned the specific modes described in WO 01/70968 for production in a human immortalized cell line, WO 2005/123928 for production in a plant and US 2005-229261 for production in the milk of an animal transgenic.
  • the present invention also relates to a method for producing an antimicrobial peptide according to the invention comprising the insertion of a nucleic acid, a cassette or an expression vector according to the invention into an expression system in vitro also called acellular and the harvest of the peptide produced by said system.
  • an expression system in vitro also called acellular and the harvest of the peptide produced by said system.
  • Many in vitro or acellular expression systems are commercially available and the use of these systems is well known to those skilled in the art.
  • the present invention further relates to a peptide according to the invention as a medicament, in particular as a medicament for the treatment of a microbial infection, namely an infection with a bacterium, a virus, a fungus or a parasite. It also relates to a nucleic acid, a cassette or a vector according to the invention as a medicament.
  • the drug may be for pharmaceutical or veterinary use.
  • the infection is an infection with a parasite, preferably of the genus Leishmania.
  • Infection with a parasite may be cutaneous leishmaniasis, mucocutaneous leishmaniasis or visceral leishmaniasis.
  • the parasite may be selected from the group consisting of Leishmania aethiopica, Leishmania amazonensis, Leishmania arabica, Leishmania aristedes, Leishmania braziliensis, Leishmania infantum, Leishmania colombiensis, Leishmania deanei, Leishmania donovani, Leishmania enriettii, Leishmania equatorensis, Leishmania forattinii, Leishmania garnhami, Leishmania gerbili, Leishmania guyanensis, Leishmania herreri, Leishmania hertigi, Leishmania lainsoni, Leishmania major, Leishmania mexicana, Leishmania naiffi, Leishmania panamensis, Leishmania peruviana, Leishmania pifanoi, Leishmania shawi, Leishmania turanica, Leishmania tropica and Leishmania venezuelensis.
  • the parasite is selected from the group consisting of Leishmania infantum, Leishmania donovani, Leishmania mexicana, Leishmania amazonensis, Leishmania major, Leishmania tropica, Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis and Leishmania peruviana.
  • the parasite is selected from the group consisting of Leishmania infantum, Leishmania major, Leishmania tropica and Leishmania braziliensis.
  • the infection is an infection with the parasite Leishmania infantum.
  • the infection can also be an infection with a parasite of the genus
  • the parasite may be selected from the group consisting of Trypanosoma avium, Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma congolense,
  • Trypanosoma equinum Trypanosoma equiperdum
  • Trypanosoma evansi Trypanosoma lewisi
  • Trypanosoma melophagium Trypanosoma percae
  • the parasite is selected from the group consisting of Trypanosoma brucei, Trypanosoma cruzi and
  • the present invention relates to a peptide according to the invention as an antimicrobial agent.
  • the present invention also relates to a nucleic acid, a cassette or a vector according to the invention as an antimicrobial agent.
  • the present invention relates to a peptide according to the invention as a stimulating agent of the immune system, in particular during a microbial infection.
  • the present invention also relates to a nucleic acid, a cassette or a vector according to the invention as a stimulating agent of the immune system.
  • the peptide according to the invention has chemotactic properties. The peptide induces the recruitment of immune cells to the place of infection and increases the efficacy of the immune response to infections.
  • the present invention also relates to a pharmaceutical composition comprising at least one peptide according to the invention and a pharmaceutically acceptable carrier and / or excipient.
  • the present invention also relates to a pharmaceutical composition comprising at least one nucleic acid, cassette or vector according to the invention and a pharmaceutically acceptable carrier and / or excipient.
  • the pharmaceutically acceptable excipients and carriers that can be used in the composition according to the present invention are well known to those skilled in the art (Remington's Pharmaceutical Sciences, 18 th edition, AR Gennaro, Ed., Mack Publishing Company [1990]; Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of
  • the pharmaceutical composition according to the invention may be suitable for oral, sublingual, cutaneous, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal, rectal, intraocular or intra-auricular administration.
  • the pharmaceutical composition according to the invention is suitable for dermal, oral, intramuscular, intravenous, transdermal or subcutaneous administration.
  • the pharmaceutical composition according to the invention is suitable for topical administration.
  • composition according to the invention may be in the form of tablets, capsules, capsules, granulates, suspensions, emulsions, solutions, gels, pastes, ointments, creams, plasters, potions, suppositories, enemas, injectables, implants, patches, sprays or aerosols.
  • the composition according to the invention comprises from 1 to
  • composition according to the invention comprises from 50 to 100, 150, 200, 250, 500, 750, 1000 or 1500 mg of peptide according to the invention.
  • composition according to the invention may furthermore comprise additional active substances, such as other antimicrobial agents, in particular antimicrobial peptides or antibiotics.
  • additional active substances such as other antimicrobial agents, in particular antimicrobial peptides or antibiotics.
  • the composition may also comprise substances capable of potentiating the activity of the peptide according to the invention.
  • the present invention relates to the use of a peptide according to the invention for the preparation of a medicament for the treatment of a microbial infection.
  • the present invention also relates to the use of a nucleic acid, a cassette or a vector according to the invention for the preparation of a medicament for the treatment of a microbial infection.
  • the present invention relates to a peptide according to the invention for use in the treatment of a microbial infection.
  • the present invention also relates to a nucleic acid, a cassette or a vector according to the invention for use in the treatment of a microbial infection.
  • the treatment can be curative or preventive.
  • the subject to be treated is an animal, preferably a mammal. According to a particular embodiment, the subject to be treated is a human.
  • the present invention also relates to a method of treating a microbial infection comprising administering a therapeutically effective dose of a peptide, nucleic acid, cassette or vector of the invention.
  • terapéuticaally effective dose refers to the amount of peptide, nucleic acid, cassette or vector according to the invention required to observe antimicrobial activity on the bacterium, virus, fungus or the parasite responsible for the infection.
  • the amount of peptide, nucleic acid, cassette or vector according to the invention to be administered as well as the duration of the treatment are evaluated by those skilled in the art according to the physiological state of the subject to be treated, the pathogen and the antimicrobial activity of the peptide vis-à-vis said pathogen.
  • the microbial infection to be treated is a leishmaniasis.
  • An effective dose of peptide according to the present invention may include, but is not limited to, about 1 to 40 mg / kg body weight.
  • the frequency of administration may be for example every 4 to 24 hours, preferably every
  • the duration of the treatment may be, for example, from 1 to 30 days, preferably from 10 to 20 days, and most preferably from 5 to 10 days.
  • the present invention also relates to the use of the peptide according to the invention as a preservative, disinfectant or pesticide.
  • the food products can be treated with a peptide according to the invention in order to eliminate or prevent the risk of infection by micro-organisms and thus improve their preservation.
  • the peptide is used in this case as a preservative.
  • the peptide according to the invention can be used as a pesticide.
  • the peptide is then used to prevent or treat plant infections by phytopathogens.
  • the peptide according to the invention can also be used as a disinfectant.
  • the term "disinfectant" refers to an antimicrobial activity of the peptide on a surface (eg, walls, doors, medical equipment), a liquid (eg, water) or a gas (eg, anesthetic gas) . Biofilms are responsible for nearly 60% of nososomial infections.
  • the peptide according to the invention is used for the elimination of bacterial biofilms. According to a preferred embodiment, the peptide according to the invention is especially used for the disinfection of surgical or prosthetic equipment.
  • the present invention also relates to a medical device or implant comprising a body having at least one surface covered or including a peptide according to the invention.
  • the surface may be covered with a peptide at a density of 0.4 to 300 mg / cm 2 .
  • the peptide may be combined with another active molecule, preferably an antibiotic.
  • the implant can be a vascular implant.
  • the present invention also relates to a method for preparing a medical device or implant comprising applying a peptide layer according to the invention, or putting it in contact with it, on at least one surface of the device. or implant.
  • This type of medical device or implant and its uses and methods of preparation are described for example in the patent application WO 2005/006938.
  • the present invention relates to a food composition comprising at least one peptide according to the invention.
  • the present invention also relates to an agrochemical composition comprising at least one peptide according to the invention.
  • the present invention relates to a transgenic plant comprising a nucleic acid, a cassette or an expression vector according to the invention, capable of expressing or expressing a peptide according to the invention.
  • the introduction of the nucleic acids, cassettes or expression vectors of the invention into a cell or plant tissue, including a seed or plant can be carried out by any technique known to those skilled in the art.
  • Plant transgenesis techniques are well known in the art, and include for example the use of the bacterium Agrobacterium tumefaciens (Hooykaa and Schilperoort, 1992), electroporation, conjugative transfer, biolistic techniques (Russel et al., 1992). ) or microinjection into plant embryos or protoplasts.
  • Other plant transgenesis techniques are well known, or other protocols embodying the above techniques are described in the prior art (Siemens and Schieder, 1996) and can be applied to the present invention.
  • the transgenic plant according to the invention can in particular be obtained according to the method described in the patent application WO 00/055337.
  • the transgenic plant may belong to any plant species. It can be monocotyledonous or dicotyledonous. More particularly, the transgenic plant of the invention is a crop plant intended or not for animal or human food or on which the sandflies, the insect vectors of Leishmania, come to feed such as corn, wheat, rapeseed , soy, alfalfa, flax, rice, sugar cane, beet, tobacco, cotton, sunflower, tomato, cabbage, carrot, potato, or fruit trees such as lemon, apple, apricot, peach or hazel, or plants identified to date as sources of sweet meal for sandflies such as Ricinus communis, Capparis spinosa, Solanum jasminoides, Solanum luteum or Bougainvillea glabra.
  • the expression of the peptide according to the invention allows the transgenic plant to have increased resistance to pathogens, and more particularly to phytopathogens.
  • the use of such a transgenic plant can significantly reduce the spraying or application of pesticides on crops, and thus limit the adverse effects of these products on the environment.
  • the transgenic plant expresses a peptide according to the invention which is administered to an animal, including sandflies or a by ingestion of said plant or its juices.
  • the peptide does not necessarily have an effect on the phytopathogens but has an antimicrobial activity vis-à-vis one or more pathogens of the animal including leishmanias present in the digestive tract of sandflies vectors of leishmaniasis humans and animals or the human to whom it is administered.
  • transgenic plants on which the sandflies take their sweet meal deliver directly into the digestive tract of the insect vector, an antimicrobial peptide of the invention that directly kills the parasite that may be present in the insect vector or by blocking its development by killing bacteria of the intestinal flora of the insect vector, necessary for differentiation and parasitic multiplication.
  • Transgenic plants are an effective means of indirectly controlling the transmission of leishmaniasis.
  • the present invention relates to an antibody specific for the peptide according to the invention.
  • antibody refers in particular to polyclonal or monoclonal antibodies, fragments thereof (e.g., F (ab) '2, F (ab)) fragments, single antibodies, -chain or minibody or any polypeptide comprising a domain of the initial antibody recognizing the peptide according to the invention, in particular the CDRs (regions determining the complementarity).
  • chimeric, humanized or human antibodies may be mentioned.
  • Monoclonal antibodies can be prepared from hybridomas according to methods well known to those skilled in the art. The different techniques for preparing the antibodies are well known to those skilled in the art.
  • the present invention also relates to the use of an antibody according to the invention for detecting a peptide according to the invention. It also relates to the use of an antibody according to the invention for carrying out quantitative measurements of a peptide according to the invention, in particular by immunological assays. These measurements can in particular make it possible to evaluate the expression of the peptide according to the invention in a host cell or a transgenic plant according to the invention. All references cited in this specification are incorporated by reference in this application. Other features and advantages of the invention will appear better on reading the following examples given for illustrative and non-limiting. EXAMPLES
  • Vanhoye et al. Vanhoye et al. (Vanhoye et al, 2004), and using amino acids protected by
  • Escherichia coli ATCC 25922 and ATCC 352148
  • Staphylococcus aureus ATCC 25923
  • Enterococcus faecalis ATCC 29212
  • Bacillus megaterium and Pseudomonas aeruginosa ATCC 27853.
  • a standard inoculum of 10 5 to 10 6 bacteria / mL (exponential growth phase) was prepared.
  • an isolated colony on an LB agar previously seeded with one of the strains was cultured in 10 ml of LB medium.
  • the cultures in a liquid medium were then incubated for 2 to 3 hours with stirring at 37 ° C. so that the bacteria were in an exponential growth phase.
  • Each bacterial suspension was diluted in LB medium to obtain an OD at 630 nm of 0.01 which corresponds to a concentration of 10 5 -10 6 cfu / ml (cfu: colony forming unit).
  • the minimum inhibitory concentration (MIC) of each peptide was determined by a growth inhibition test in a liquid medium.
  • MIC is defined as the lowest concentration of peptide capable of inhibiting the growth of the bacterial strain tested after incubation for 18 h at 37 ° C.
  • the test was performed in a sterile microtiter plate containing 96 wells. A range of increasing concentrations of each peptide (1 to 400 ⁇ M) was previously prepared in sterile milliQ water containing 5% dimethyl sulfoxide (DMSO). DMSO facilitates the solubilization of peptides and shows no antimicrobial activity at the concentration used.
  • 50 ⁇ l of peptide were incubated with 50 ⁇ l of bacterial suspension (10 5 -10 6 cfu / ml). The microplate was then incubated at 37 ° C for 18 h with shaking. Bacterial growth was determined by measuring OD at 630 nm (turbidity) using a plate reader. The tests were performed in triplets for each peptide concentration.
  • the negative growth inhibition control was obtained by replacing the solution containing the peptide with 50 ⁇ l of sterile milliQ water containing 5% DMSO.
  • the positive control allowing to completely inhibit the growth of bacterial strains was obtained by replacing the solution containing the peptide with 50 ⁇ l of formaldehyde at 0.7%.
  • yeast strains Saccharomyces cerevisiae, Candida albicans (ATCC 90028), Candida parapsilosis (ATCC 22019). These strains were previously grown on YPD agar for at least 48 hours. Yeast suspensions were then prepared exactly as for bacteria, and adjusted to 10 5 to 10 6 cfu / ml in liquid YPD medium. The antifungal test corresponds to the growth inhibition test in a liquid medium used for the bacteria (cf above) in which the LB medium has been replaced by a YPD culture medium. The fungal strains were incubated at 30 ° C.
  • the haemolytic activity of antimicrobial peptides was demonstrated using human red blood cells from healthy donors. Haemolysis of globules red results in the release of hemoglobin in the reaction medium whose concentration is determined spectrophotometrically at 450 nm.
  • Red blood cells were separated from plasma and white blood cells by centrifugation of human blood (900 g, 10 min). The pellet containing the red blood cells was washed 3 times with PBS buffer, pH 7.4. After counting with a cell of
  • the test was carried out as follows: 100 .mu.l of the different concentrations of peptide were added to 100 .mu.l of the suspension of red blood cells. After 1 h of incubation at 37 ° C, followed by centrifugation (12000g, 15 sec), the absorbance of the supernatant was measured at 450 nm.
  • the negative control for this test (0% hemolysis) contained 100 ⁇ l of PBS buffer in place of the solution containing the peptide and the positive control (100% hemolysis) contained 100 ⁇ l of 0.1% Triton on the place of the same solution.
  • the LC50 value obtained results from the average of three experiments performed in triplets and corresponds to the peptide concentration inducing 50% hemolysis.
  • the antiparasitic activity of the peptides was evaluated on both forms of the parasite Leishmania infantum, the promastigote form and the amastigote form.
  • the anti-Leishmania activity tests were carried out with an ⁇ NEO- ⁇ LUC line of Leishmania infantum. This line was obtained by transforming the Leishmania infantum strain MHOM / MA / 67 / ITMAP-263 with the pGM ⁇ NEO- ⁇ LUC vector containing the luciferase reporter gene (LUC) and the neomycin resistance gene (NEO) such as as described in Roy et al. (2000). It is maintained in culture in its two forms, promastigotes and amastigotes.
  • LOC luciferase reporter gene
  • NEO neomycin resistance gene
  • Promastigotes of Leishmania infantum were maintained at 26 ° C by one to two weekly passages depending on the number of parasites in the inoculum, in SDM 79 medium, supplemented with 10% to 20% of decomplemented fetal calf serum and 5% mg / mL of pork and in the presence of 100 U / mL of penicillin and 100 ⁇ g / mL streptomycin (Brun & Shonenberger, 1979). From an inoculum of 10 5 cells / ml in log phase growth, the promastigotes reached a cell density of 2 to 3 ⁇ 10 8 parasites / mL in stationary phase, after 7 days of culture in culture flasks. 25 cm 2 . Cell densities were determined by flow cytometry counting in the presence of propidium iodide on a Facscan counter (Excalibur, Becton-Dickinson, Ivry, France).
  • the axenic amastigotes were obtained by differentiation of the promastigotes at 37 ° C ⁇ 0, PC (saturation in H 2 O, 5% CO 2 ), in culture in MAA medium, supplemented with 20% of decomplemented fetal calf serum and 12.5 mg / mL of pork hemoin, in the presence of 100 LVmL of penicillin and 100 ⁇ g / mL of streptomycin (Sereno and Lemesre, 1997). From an inoculum of 5 ⁇ 10 5 cells / ml in log phase growth, the amastigotes reached a cell density of 2 to 3 ⁇ 10 8 parasites / mL in stationary phase, after 7 days of culture in flasks of culture of 25 cm 2 .
  • axenic amastigotes Microscopic observation of axenic amastigotes shows homogeneous forms (round to ovoid) without apparent flagellum and immobile.
  • the axenic amastigotes of various Leishmania species exhibit the same ultrastructural, biological, biochemical, and immunological properties as intracellular amastigotes. Cell densities were also determined by flow cytometry counting according to the same procedure and the same parameters used for promastigotes.
  • the suspension of axenic amastigotes is distributed in a culture microplate in a volume of 80 ⁇ l per well (ie 10 5 parasites / well) into which 20 ⁇ l of each peptide solution (60 to 0.94) are added.
  • ⁇ M final ie a final parasite density of 10 6 parasites / mL.
  • the plate is then incubated at 37 ° C for 72 h.
  • the peptide solution was replaced with 20 ⁇ L of MAA / 20 medium.
  • the positive control was performed with 20 ⁇ L of the solution containing the highest concentration of peptide.
  • the experiments are performed in triplets for each peptide concentration.
  • lysis buffer (Steady GLO, Promega) were added to each well. After incubation for 5 min at room temperature, cell lysis was checked under a microscope. The emitted luminescence was measured by a luminescence reader (Victor,
  • amastigote tests 80 ⁇ l of promastigote suspension (10 5 parasites / well) were mixed with 20 ⁇ l of peptide solution (60 to 0.94 ⁇ M final) in each well of a microtiter plate. Negative and positive controls were performed according to the same protocol as for anti-Leishmania activity tests on amastigotes. The experiments are performed in triplets for each peptide concentration. After 72 hours of incubation at 26 ° C., 50 ⁇ L per well of Steady lysis buffer
  • GIo Promega
  • the cytotoxic activity of the antimicrobial peptides was determined against a THP-I human monocyte line.
  • the cells were maintained in culture in RPMI medium (10% FCS, 100 LVmL penicillin, 100 ⁇ g / mL streptomycin) until an exponential phase of growth. After counting on a Thoma cell, the cell density was adjusted to 6.25 ⁇ 10 5 cells / ml in RPMI 1640 medium. Five-fold concentrated peptide solutions were prepared in RPMI medium (300 to 4.7 ⁇ M).
  • the monocytes are distributed in a volume of 80 ⁇ l of cell suspension per well (ie 5 ⁇ 10 4 monocytes / well or 5 ⁇ 10 5 cells / final ml) and mixed with 20 ⁇ l of peptide solution (60 to 0.94 ⁇ M final). Negative and positive controls were performed according to the same protocol as for anti-Leishmania activity tests. The experiments are performed in triplets for each peptide concentration. The cells are incubated at 37 ° C. under 5% CO 2 for 72 hours.
  • MTT or 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide
  • succinate-tetrazolium reductase which is present in the mitochondrial respiratory chain of metabolically active cells. This formazan blue is detectable by spectrophotometry (570 nm).
  • the plates are then incubated for 4 h at 37 ° C.
  • the enzymatic reaction is stopped by the addition of 100 ⁇ l of a 50% isopropanol / 10% SDS mixture and the microplates are then incubated at room temperature for 30 min, with stirring.
  • OD at 570 nm from each well was then measured (Victor reader, PerkinElmer) to determine IC50.
  • the anti-Leishmania activity of the two temporins was evaluated on the parasite Leishmania infantum, which is mainly responsible for human visceral leishmaniasis in the Mediterranean basin and in Latin America.
  • Culture of the promastigote and axenic amastigote forms of Leishmania infantum (MHOM / MA / 67 / ITMAP-236) expressing the luciferase gene were used.
  • the evaluation of the metabolic activity of parasites is based on the oxidation of luciferin by luciferase in the presence of ATP. This process results in the emission of photons which is proportional to the concentration of the non-ionic parasites.
  • Cirioni O Giacometti A, Ghiselli R, Dell Acqua G, Gov Y, Kamysz W,

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