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
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to new antimicrobial agents, and more particularly low molecular weight peptides derived from the defensin of the Mediterranean mussel or Mytilus galloprovincialis. It also relates to a process for their preparation and their applications, in particular their use for the preparation of a medicament.
• N-terminal natural defensin exhibited the most interesting profile of antimicrobial activity.
• WO 98/40091 describes antimicrobial peptides whose sequence is derived from the amino acid sequences of natural defensins and bactenecins.
• WO 01/09174 describes antimicrobial peptides derived from plant defensins by substitution of one or more amino acids.
• WO 00/68625 describes cyclic peptides called theta defensins, which have antimicrobial activity. These peptides are derived from natural or modified linear peptides and cyclized by the formation of a peptide bond between the N-terminal amine and the C - terminal carbon. Peptides cyclized in this way do not offer a grip on the action of exopeptidases and are therefore likely to have a resistance to proteol is superior to that of natural peptides.
• the present invention aims to provide new molecules with an interesting spectrum of antimicrobial activity and having unexpected properties compared to those of molecules of the prior art.
• the molecules according to the invention can be easily prepared at a cost which makes it possible to envisage industrial and commercial production.
• the subject of the invention is new cyclic peptides, or comprising a cyclic fragment, as well as certain of their chemical derivatives, which exhibit antimicrobial activity, in particular antibacterial and antifungal, these peptides comprising an unnatural bridge, preferably a disulfide bridge. , these peptides being derived from the defensin of Mytilus galloprovincialis noted MGD l '
• MGD 1 Mytilus galloprovincialis
• the first peptide according to the invention corresponds to the sequence SEQ ID NO: l
• a subject of the invention is also homologs of the peptide SEQ ID NO: 1 and certain chemical derivatives of this peptide.
• the term “homologs” means peptides whose amino acid sequence has at least 60% similarity to the sequence
• SEQ ID NO: 1 even more preferably 70%, even more preferably still 80%, preferably at least 90%, and even more favorably at least 95%, or better, 98% similarity with the sequence SEQ ID NO: l, it being understood that the sequences concerned comprise a cysteine residue at each of their ends, these terminal cysteine residues being linked by a disulfide bridge.
• X% between a peptide P and the sequence SEQ ID NO: l it is meant that: when the sequence of P is aligned opposite SEQ ID NO: l, in the same direction, X% of the amino acids of P are identical to the corresponding amino acid of SEQ ID NO: l or are replaced by an amino acid of the same class, it being understood that if the sequences are not the same length, we will place a space between the amino acids of the sequence concerned.
• the degree of homology can be assessed by methods well known to those skilled in the art (e.g., WILBUR WJ et al Proceedings of the National Academy of Sciences USA 80, 726-730 (1983); MYERS et al, Comput. Appl Biosci.
• the homology with SEQ ID NO: l extends to peptides comprising from 6 to 15 amino acids and the sequence of which, once aligned with SEQ ID NO: l, with the spaces placed between the acids suitable amines, has a similarity included in the values indicated above.
• the homology extends to peptides comprising from 7 to 12 amino acids, even more preferably from 8 to 11 amino acids.
• amino acids of the same class is meant an amino acid having substantially identical chemical properties.
• this term is understood to mean amino acids having substantially the same charge, and / or the same size, and / or the same hydrophilicity or hydrophobia, and / or the same aromaticity.
• Such amino acid groupings generally include:
• proline phenylalanine.
• Other amino acid substitutions can be envisaged within the meaning of the present invention in which an amino acid is replaced by another amino acid of the same class, that is to say having comparable properties, the amino acid of substitution being an unnatural amino acid.
• a particular example of homolog is constituted by the functional fragments of the peptides according to the invention: these are peptides corresponding to one of the sequences according to the invention in which one or more amino acids are removed from the sequence but which retain a bactericidal activity comparable to that described for MGD1, in particular a minimum inhibitory concentration of less than 75 ⁇ M with respect to at least one bacterial microorganism.
• the preferred functional fragments of the peptides according to the invention are those for which at most two amino acids are removed. Even more preferably, those for which an amino acid is removed.
• substitutions of amino acids of the same class allow the peptide to retain its antimicrobial activity.
• Appropriate substitutions can be determined by testing the antimicrobial properties of the peptides obtained using activity tests such as those described below.
• the invention relates more specifically to molecules having a minimum inhibitory concentration of less than 75 ⁇ M with respect to at least one bacterial microorganism.
• the substitution of histidine and leucine of SEQ ID NO: 1 by two lysines made it possible to obtain a peptide of sequence SEQ ID NO: 2 comprising a disulfide bridge between the two cysteines, this cyclic peptide having an antibacterial power greater than that of the cyclic peptide of sequence SEQ ID NO: 1.
• SEQ ID NO: 2 C (S) GGWKRKRC (S)
• the invention therefore also relates to a peptide of sequence
• SEQ ID NO: 2 in which the two cysteine residues are engaged in a disulfide bridge, as well as its pharmaceutically acceptable salts, the functional fragments of this peptide, the homologous peptides and the chemical analogs of this peptide and certain chemical derivatives of this peptide.
• the invention extends to peptides whose amino acid sequence has at least 60% similarity to a peptide corresponding to the sequence SEQ ID NO: 2, even more preferably 70%, even more preferably still 80%, preferably at least 90%, and even more favorably at least 95%, or better, 98% similarity with the sequence SEQ ID NO: 2, said sequence comprising a cysteine residue at each of its ends, these terminal cysteine residues being linked by a disulfide bridge.
• -Xi and X 2 identical or different, represent an amino acid chosen from glycine, valine, alanine, lysine,
• - X 3 represents an amino acid chosen from tryptophan and tyrosine
• - X, X 5 and X 7 identical or different, represent an amino acid chosen from histidine, arginine, lysine,
• - X 6 represents an amino acid chosen from leucine, isoleucine, lysine.
• a subject of the invention is also the derivatives of the peptides of formula (I) chosen from its pharmaceutically acceptable salts, the functional fragments of these peptides, the homologous peptides and the chemical analogs of these peptides and certain chemical derivatives of these peptides.
• at least one of the independent conditions below is met:
• - Xi represents glycine or lysine
• - X 2 represents glycine or lysine
• - X 6 represents leucine or lysine
• - X 7 represents arginine
• a subject of the invention is also peptides comprising a sequence according to formula (I), in particular the sequence SEQ ID NO: 1 or the sequence SEQ ID NO: 2, in which the two cysteine residues are linked by a disulfide bridge, or peptides comprising a functional fragment of these peptides, a homolog, a chemical analog of these peptides or a chemical derivative of these peptides.
• terminal amino acids preferably the terminal cysteines of the peptide according to formula (I) comprise, for one, a free terminal amine function, and for the other, a free terminal carboxyl function, these two functions each being capable of being involved in a bond peptide with C - terminal acid, respectively the N - terminal amine of another peptide fragment.
• the subject of the invention is peptides derived from MGD 1, comprising the sequence according to formula (I) in which the two cysteine residues are linked by a disulfide bridge.
• the invention relates more particularly to peptides corresponding to the sequence SEQ ID NO: 3, in which the two cysteine residues at 25 and 33 are linked by a disulfide bridge, the pharmaceutically acceptable salts of these peptides according to the invention, the functional fragments of these peptides, the homologous sequences, the chemical analogs of these peptides and certain chemical derivatives of these peptides.
• SEQ ID NO: 3 GFGCPNNYQCHRHCKSIPGRC (S) GGYCGGWHR LRC (S) TCYRCG
• the invention therefore also extends to peptides having a homology of at least 60%, preferably 70%, even more preferably 80%, preferably in at least 90% and even more favorably at least 95%, or better, at least 98% with the sequence SEQ ID NO: 3 and in which the two cysteines at 25 and 33 form a disulfide bridge.
• the peptides concerned preferably comprise from 15 to 70 amino acids, even more preferably from 15 to 55 amino acids and advantageously from 15 to 50 amino acids.
• the subject of the invention is the peptides corresponding to formula (II) below: wherein Yi and Y 2 represent a peptide fragment comprising from 1 to 60 amino acids, preferably from 1 to 40 amino acids and even more preferably from 1 to 15 amino acids; Xi, X 2 , X 3 , X4, X5, X 6 , X 7 have the same definition as above, the pharmaceutically acceptable salts of these peptides, the functional fragments of these peptides, the homologous sequences, the chemical analogs of these peptides and certain chemical derivatives of these peptides.
• Yi and Y 2 respectively have a homology of at least 60%, preferably 70%, even more preferably 80%, preferably 90% and even more favorably 95% or better 98% with the N-terminal, respectively C-terminal fragment of MGD 1.
• the term salts refers both to the amine salts of a carboxyl function of the peptide chain as well as to the acid addition salts with an amino group of this same polypeptide chain.
• the salts of a carboxyl function can be formed with an inorganic or organic base.
• Inorganic salts include, for example, alkali metal salts such as sodium, potassium and lithium salts; alkaline earth salts such as for example calcium, barium and magnesium salts; ammonium salts, ferrous, ferric salts, zinc, manganese, aluminum, magnesium salts.
• Salts with organic amines include those formed for example with trimethylamine, triethylamine, tri (n-propyl) amine, dicyclohexylamine, triethanolamine, arginine, lysine, histidine, ethylenediamine, glucosamine , methylglucamine, purines, piperazines, piperidines, caffeine, procaine.
• Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid; salts with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid , malic acid, ascorbic acid, benzoic acid.
• mineral acids such as, for example, hydrochloric acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid
• salts with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid , malic acid, ascorbic acid, benzoic acid.
• Chemical derivatives of the peptides according to the invention include the alpha-amino peptide compounds, the N-alpha acyl substituted derivatives of the RCO- form, in which R represents a linear, branched alkyl, alkenyl, alkynyl, aryl or aralkyl group , or cyclic comprising from 1 to 50, preferably from 1 to 8 carbon atoms.
• the preferred N-alpha acyl group is the acetyl group.
• Such amine-terminal substituents can increase the activity of the peptide by slowing down or preventing the enzymatic degradation of the peptides in vivo.
• peptides according to the invention include derivatives substituted on the C-terminal acid function by a group chosen from -NH 2 , alkyloxy, alkylthio or alkylamino of the form -OR, -SR or -NHR, in which R represents a group chosen to facilitate the in vivo penetration of the peptides.
• R can represent an alkyl, alkenyl, alkynyl, aryl chain or an aralkyl group, linear, branched or cyclic comprising from 1 to 50, preferably from 8 to 50 carbon atoms.
• the peptides according to the invention are amidated on their C-terminal function with -NH 2 .
• Another kind of chemical derivative of the peptides according to the invention includes derivatives carrying a pliarmacophore substituent, such as a fluorescent group, a photoactivable group, a radiolabelled group or any other group allowing detection by spectroscopy and quantitative evaluation. of the peptide according to the invention in a biological sample without degradation of the biological sample.
• a pliarmacophore substituent such as a fluorescent group, a photoactivable group, a radiolabelled group or any other group allowing detection by spectroscopy and quantitative evaluation.
• the amino acids of which the peptides are made up are L enantiomers.
• one or more amino acids of the peptide sequence can be replaced by its D enantiomer.
• one or more peptide amide bonds can be replaced by an isostere bond such as: -CH 2 NH-, CH 2 S-, -CH 2 CH 2 -, -CH ⁇ CH- (cis and trans), -COCH 2 -, - CH (OH) CH 2 - and -CH SO-.
• the peptides derived from the peptides according to the invention in which the two cysteine residues forming the disulfide bridge have been replaced by two tryptophan residues forming a di-tryptophan bridge or by a lanthionine residue forming a mono-sulfide bridge, or by two carrying amino acids, for one of a free acid function (for example a glutamic acid) and for the other a free ine function (for example a lysine) both engaged in a lactam bridge. It is also possible, according to a variant of the invention, to provide that the N-terminal and C-terminal ends of the peptide are engaged in an amide bond in order to cyclize this peptide.
• the peptides which are the subject of the present invention can be easily obtained by any means known to those skilled in the art, in particular by synthesis.
• the means used to obtain the peptides which are the subject of the invention will be illustrated in the examples.
• a subject of the invention is also the linear peptides which are precursors of the peptides with cyclic fragments according to the invention.
• the subject of the invention is linear peptides whose sequence corresponds to formula (I) or to formula (II), and more particularly the peptides of sequence SEQ ID NO: 1 and SEQ ID NO: 2 these peptides linear being precursors of the cyclized peptides comprising the disulfide bridge between the extreme cysteines of SEQ ID NO: 1 or SEQ ID NO: 2.
• the subject of the invention is also the linear precursor sequences of the functional fragments of the peptides according to the invention, homologous sequences, chemical analogs of the peptides according to the invention and of certain chemical derivatives of these peptides as defined above.
• a subject of the invention is also the linear peptides whose sequences numbered 4, 5, 6, 7, 19, 20, 21 and 22 are given in the examples.
• the invention extends to linear peptides comprising up to 30 amino acids, homologs of SEQ ID NO: 1 or of SEQ ID NO: 2, preferably to peptides comprising of 6 to 20 amino acids, even more preferably of 6 to 15, more preferably still from 7 to 12 amino acids.
• the invention extends to linear peptides whose amino acid sequence has at least 60% similarity to a peptide corresponding to the sequence SEQ ID NO: 1 or to the sequence SEQ ID NO: 2, even more preferably 70%, even more preferably still 80%, preferably at least 90%, and even more favorably at least 95%, or better, 98% similarity with the sequence SEQ ID NO: 1 or the sequence SEQ ID NO: 2 .
• a linear peptide which is a precursor of the peptides with a cyclic fragment according to the invention can be obtained by synthesis or by expression in a cell expressing MGDl isolation then modification: cutting of certain peptide bonds and isolation of the fragments comprising SEQ ID NO: 1.
• the linear peptides which are precursors of the peptides with a cyclic fragment according to the invention can be obtained by expression from a nucleic acid molecule encoding the corresponding peptide.
• a subject of the invention is therefore also the nucleic acid molecules coding for a peptide of formula (I) or of formula (II), in particular the nucleic acid molecules coding for a peptide of sequence SEQ ID NO: 1 and its homologs , and those encoding the peptide of sequence SEQ ID NO: 2 and its homologs.
• a nucleic acid molecule coding for a linear peptide according to the invention can be obtained either by chemical synthesis or by cloning from a cell containing a MGD 1 gene or from a cell coding a cDNA corresponding to l 'MGD mRNA l.
• a nucleic acid molecule according to the invention encoding a precursor of the peptides with a cyclic fragment according to the invention, can be prepared by chemical synthesis on the basis of the amino acid sequence of these peptides and on the knowledge possessed by l skilled in the art of codons corresponding to each amino acid molecule.
• a nucleic acid molecule encoding a precursor of the peptides with a cyclic fragment according to the invention can be cloned into an appropriate vector, in particular into an expression vector and the corresponding linear peptide can be expressed in a host cell in vitro.
• an appropriate vector in particular into an expression vector and the corresponding linear peptide can be expressed in a host cell in vitro.
• a subject of the invention is therefore also vectors containing a nucleic acid molecule encoding a precursor of the peptides with a cyclic fragment according to the invention and host cells comprising these vectors.
• vectors and host cells capable of being used in the present invention are well known to those skilled in the art and are commercially available.
• the peptides with a cyclic fragment according to the present invention represent a broad spectrum antimicrobial activity. They are likely to reduce or inhibit the survival or proliferation capacity of many microorganisms: bacteria, fungi, viruses, protozoa. In particular, among the bacteria there may be mentioned Escherichia, Salmonella, Staphylococcus and Listeria. Among the fungi: Cryptococcus, Candida and Fusarium oxysporum.
• Antimicrobial activity can manifest itself in the form of two distinct actions: microbicide inhibition (bactericide, fungicide, virucide or parasiticide) which consists in destroying or irreversibly damaging the target microorganism; microbiostatic inhibition (bacteriostatic, virostatic, fungistatic or parasitostatic) which consists in reducing or inhibiting the proliferation capacities of the micro-organism without necessarily destroying it.
• microbicide inhibition bactericide, fungicide, virucide or parasiticide
• microbiostatic inhibition bacteriostatic, virostatic, fungistatic or parasitostatic
• the peptides with a cyclic fragment according to the invention being endowed with antimicrobial activity make it possible to reduce or inhibit microbial proliferation or to destroy microorganisms in a given environment.
• microorganism is meant in particular: viruses, bacteria, fungi, protozoa, helminths and other parasites.
• the term “environment capable of being treated with the peptides according to the invention” denotes in particular: the tissues and bodily fluids of humans and animals; liquids such as water or aqueous solutions, such as for example disinfectant solutions, solutions for contact lenses, solutions for washing the skin and mucous membranes, solutions for washing the eyes; hygiene; food substances; objects such as for example food utensils, surgical instruments; gases such as for example anesthetic gases used in surgery; a space, such as the walls or floors of public or private spaces such as a hospital, a school, a retirement home.
• liquids such as water or aqueous solutions, such as for example disinfectant solutions, solutions for contact lenses, solutions for washing the skin and mucous membranes, solutions for washing the eyes; hygiene; food substances; objects such as for example food utensils, surgical instruments; gases such as for example anesthetic gases used in surgery; a space, such as the walls or floors of public or private spaces such as a hospital, a school, a retirement home.
• a treatment method according to the invention consists in treating an environment, excluding the human or animal body, with an effective amount of a peptide with a cyclic fragment according to the invention, so as to reduce or inhibit the capacity of the or microorganisms to proliferate or survive.
• the peptides according to the invention can be used as preservatives in food, cosmetics, hygiene products or any other industry using preservatives, as disinfecting agent.
• the subject of the invention is also a disinfectant composition
• a disinfectant composition comprising a peptide with a cyclic fragment according to the invention in a support suitable for its destination.
• the peptides with a cyclic fragment according to the invention can also be used as a medicament. They can be used for the preparation of a therapeutic composition intended for the treatment of infections by microorganisms, in particular against bacteria and fungi.
• the subject of the invention is also a pharmaceutical composition comprising a peptide with a cyclic fragment according to the invention in a vehicle suitable for its administration to humans or animals.
• Food products may, to improve their conservation or to eliminate or prevent the risk of infection by microorganisms, be treated with cyclic fragment peptides according to the invention.
• Certain food products such as seafood and poultry often harbor endemic pathogenic microorganisms.
• Fruits, vegetables and seeds can be treated to prevent rotting after harvest.
• the quantity of peptide with a cyclic fragment to be administered to humans, or to animals or to any environment capable of being treated depends on the activity specific to this peptide, activity which can be measured by means which will be explained in the examples. It also depends on the degree of the infection to be treated.
• the peptide with a cyclic fragment according to the invention can be administered alone or in combination with other antimicrobial agents.
• AMS 422. Abimed AMS 422.
• the peptides were deprotected and released from the resin by treatment with trifluoroacetic acid (TFA). They were then lyophilized and their purity estimated by HPLC on a C ⁇ 8 column (Waters) with elution in water-acetonitrile in the presence of 0.1% TFA and detection at 260 and 220 nm. Their molecular mass was determined by mass spectrometry in Maldi-Tof (Matrix assisted laser desorption ionization-time offlighi) on a Voyager Elite DE-RP device, DHB Matrix.
• TFA trifluoroacetic acid
• the activity of synthetic peptides was tested against Gram + bacteria Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis and Bacillus megaterium. The activities were determined according to the protocol described by HANCOCK et al. (http://www.interchg.ubc.ca/bobh/methods.htm) Minimum Inhibitory Concentration (MIC)
• the MIC was determined for each peptide by inhibition of growth in a liquid medium.
• the peptide was diluted 2 by 2 in the wells of a Microtest plate in a volume of 10 ⁇ l.
• the bacteria were grown in MUELLER HINTON BROTH liquid medium (MHB Difco). During the growth phase exponential, a solution of optical density 0.001 at 600 nm was made and 100 ⁇ l of this suspension were added to each well. The incubation lasted 24 h at 30 ° C., then the optical densities of each well were measured at 600 nm. The MIC value corresponds to the first well for which the optical density had not changed.
• Minimum Bactericidal Concentration (CMB) Minimum Bactericidal Concentration
• the CMB was determined on Petri dishes containing MUELLER HINTON AGAR (MHA Difco) solid medium by spreading the content of the wells corresponding to the first 3 dilutions for which no bacterial growth had been observed after 24 h of incubation. The dishes were incubated for 24 h at 30 ° C. The CMB value corresponds to the lowest concentration of the peptide for which no colony has developed.
• the antifungal activity was determined by calculation of the MIC in a growth inhibition test of the parasitic fungus of shrimp, Fusarium oxysporum, in the liquid phase, according to the protocol described by FELHBAM et al. (J. Biol. Chem., 1994, 269: 33159-63). Each peptide was diluted 2 by 2 in the wells of a Microtest plate in a volume of 10 ⁇ l. 90 ⁇ l of spores resuspended in POTATO DEXTROSE BROTH medium (DIFCO) at a final concentration of 10 4 spores / ml were added to each well.
• DIFCO POTATO DEXTROSE BROTH medium
• Defensin MGDl It is a small cationic protein of 4 kDa comprising 39 amino acids, isolated from the plasma and hemocytes of the mold Mytilus galloprovincialis (Hubert et al. 1996, Eur. J. Biochem. 240_; 302- 6; Charlet et al, 1996, J. Biol. Chem. 271: 21808-13; Mitta et al, 1999, J. Cell. Sci, 112; 4233-42).
• the MGDl sequence is illustrated in Figure 1.
• the three-dimensional structure of the molecule established by proton magnetic resonance (Yang et al. 2000 Biochemistry, 39: 14436-47) illustrated in Figure 2 consists of an ⁇ helix and 2 antiparallel ⁇ sheets giving it a structure commonly called stabilized Cs ( ⁇ ) also observed in scorpion toxins.
• the molecule has a distribution of positive charges localized over 3 domains and a hydrophobic side chain.
• This peptide has antibacterial activity against Gram + bacteria and antifungal activity.
• Seventeen peptides corresponding to fragments of the MGD 1 sequence were synthesized according to methods well known to those skilled in the art set out above. These peptides have been tested for their antimicrobial activity and their antifungal activity. The results of these tests are set out in Tables I and II.
• Sequences 14 and 15 show no activity. - The elements corresponding to the ⁇ sheets (devoid of the loop) do not contain intrinsic activity: sequence 16.
• SEQ ID NO: 4 SGGYC (S) GG HRLRC (S) SEQ ID NO: 5 C (S) GGYSGGWHRLRSTSYRC (S) G SEQ ID NO: 6 SGGYO s) GGWHRLRC (s) TSYRSG SEQ ID NO: 7.
• K The substitution of histidine and leucine by 2 lysines (K) increases the activity (SEQ ID NO: 2).
• the measured biological activity of the active molecule relates to the Gram + bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillus megaterium and Bacillus epidermidis) and to the filamentous fungus (Fusarium oxysporum) (Table 2).
• This activity is localized on the loop formed by amino acids 25-33 of primary structure (CGGWHRLRC) provided that an unnatural disulfide bridge is made between amino acids 25 and 33.
• the antibacterial power of this structure is increased by strengthening its cationic nature by substitution of histidine 29 and leucine 31 with two lysines.
• Table II are collected the results of MIC and MBC activity (in ⁇ M) of the peptide fragments derived from MGDl defensin on a few bacterial strains (Gram +) and on a filamentous fungus.
• Tests have been undertaken using the sequences SEQ ID NO: 1 and SEQ ID NO: 2 in order to determine the impact on the biological activity of these peptides of an amino acid substitution by another amino acid of the same class or not and a decrease in the size of these peptides. These sequences have been numbered 19 to 24. Their sequence and their biological activity are described in Table III.
• sequences 19 to 23 are endowed with an interesting activity, although weaker for sequence 23.
• sequence No. 24 does not exhibit significant biological activity.
• Sequences 19 to 23 constitute another object of the invention.

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