FR2810986A1 - ANTIBACTERIAL AND ANTIFUNGAL PEPTIDES, PROCESS FOR THEIR PREPARATION AND COMPOSITIONS CONTAINING SAME - Google Patents

Abstract

The invention concerns peptides of formula: beta 1 phi 2 DOLLAR g(b)4 beta 5 phi 6 sigma 7 theta 8 beta 9 phi 10 phi 11 phi 12 phi 13 beta 14, theta 15 phi 16 beta 17 phi 18 phi 19 beta 20 phi 21 phi 22 sigma 23 beta 24 phi 25 wherein: beta is a basic amino acid, phi is a hydrophobic amino acid, theta is a negatively charged or polar/large type amino acid, sigma is an amino acid selected among glycine, serine, alanine or threonine. The invention also concerns antibacterial and antifungal compositions for use in man, animal or plants comprising at least said peptides.

Description

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 ANTIBACTERIAL AND ANTIFUNGAL PEPTIDES, PROCESS FOR THEIR PREPARATION AND COMPOSITIONS CONTAINING THEM.

 The present invention relates to novel peptides having antibacterial and antifungal properties. The invention also relates to the preparation of these peptides and the compositions containing them that can be used in agriculture and in human or animal therapy.

 The production of antimicrobial peptides represents, in a large variety of animal and plant species, an essential mechanism of immune defense against infections. In particular, insects have a very effective resistance against bacteria and fungi. This response is largely based on the rapid synthesis of several families of broad-spectrum antimicrobial peptides. This synthesis is induced by septic injury or by the injection of a low dose of bacteria. To date, antimicrobial insect peptides have been mostly characterized from insects having complete metamorphosis during development, for example, dipterans, lepidopterans and coleopterans. Among the antibacterial peptides induced in these insects, the following four groups can be distinguished: Cationic peptides of 4 kDa, forming two amphipathic α-helices. In this group, cecropins are especially classified.

 Cationic peptides rich in proline, of size between 2 kDa and 4 kDa which can be

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 glycosylated, such as, for example, drosocine, pyrrhocoricin, and lebocins, or non-glycosylated such as, for example, apidaecins and metalnikowines.

 Several distinct polypeptides having a molecular weight of 8 to 27 kDa, most of them cationic and frequently rich in glycine residues, such as, for example, attacines, sarcotoxins II, diptericins and coleopterin.

 Peptides comprising intramolecular disulfide bridges; In this group are classified 4 kDa insect defensins, 3 disulfide bridges), drosomycin (4 kDa, 4 disulfide bridges) and thanatine (2 kDa, 1 disulfide bridge).

 In the peptide sequences reported below, the amino acids are represented by their one-letter code, but they can also be represented by their three-letter code according to the nomenclature below.

Ala alanine
Cysteine Cysteine
D Asp aspartic acid
E Glu glutamic acid
F Phenylalanine
Gly glycine
H his histidine
I Isoleucine Island
K Lily lysine
Leucine leucine
M Met methionine
N Asn asparagine
P Pro proline

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Gln glutamine
R Arg arginine
Serine serine
TH threonine
Valine Valley
W Trptophan Trp
Tyr Tyrosine Y
Little work has been done to date on incompletely metamorphosed insects, especially Polynesia. However, it has now been isolated from an insect with incomplete metamorphosis the termite Pseudacanthotermes spiniger, a peptide, which has remarkable characteristics as well as antibacterial and antifungal properties.

 This peptide, which will also be designated hereinafter spinigerin, corresponds to the formula below also given in the annex under the number ID NO: 1.

1 y2 e3 4 5 <))6 a7 e8 9 10 11 12 <t)l3 P 14 015 016 P17 018 19 20 021 022 023 P 24 #25 ou un fragment de celle-ci, dans laquelle : - ss est un acide aminé basique, HVDKKVADKVLLLKQLRIMRL LTRL
This molecule, of reduced size, does not contain cysteine residues. The Applicant has studied the spatial conformation of the molecule and the mutations that may be carried out on its sequence so as to increase its bactericidal and / or fungicidal properties. The subject of the present invention is therefore a peptide of the following sequence (I):

Wherein a ss is an acid or a fragment thereof, wherein: ss is an acid or a moiety thereof; basic amine,

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- is a hydrophobic amino acid, - 0 is a negatively charged amino acid or of polar / broad nature, - a is an amino acid selected from glycine, serine, alanine or threonine,
The peptides of formulas (I) advantageously have an alpha helix conformation. Thus, ss, #, # and # are chosen so that said peptides of formula (I) have such a conformation.

 Fragments of the above sequence are understood to mean a peptide consisting of at least 10 and preferably at least 20 successive amino acids of said sequence.

 By way of example of such fragments, the invention more particularly envisages a peptide of formula (I) above of which a group of 1 to 4 amino acids at one and / or the other of the N and / or or C terminals is deleted.

P4 P5 06O>08P9 01O011 <J>12 <>13 P 14 015 016 Pi7 018 019 P 20 021 022 <*23 P 24 025 (II) Pl 2 03 p4 5 6 cr? 6a 9 10 11 12 13 14 61516 Pl7<{)l8<))19 P 20 021 (III) 45667e891011121314e15161718192021 ( IV)
On entend plus particulièrement par : - acide aminé basique, ceux choisis parmi l'arginine, la lysine ou l'histidine, Such peptides are those in which one or more of the amino acids ss1, 2, 63, <]) 22, # 23, ss24, or # 25 are absent. The invention more particularly envisages a peptide of the above formula whose amino acids ss1, 2, # 3 and / or 22, # 23, ss24, # 25 are absent and therefore corresponding to the following formulas:

P4 P5 06O> 08P9 01O011 <J> 12 <> 13 P 14 015 016 Pi7 018 019 P 20 021 022 <* 23 P 24 025 (II) Pl 2 O 3 p 4 5 6 cr? 6a 9 10 11 12 13 14 61516 P17 <{) 18 <)) 19 P 20 021 (III) 45667e891011121314e15161718192021 (IV)
In particular, the term "basic amino acid" means those chosen from arginine, lysine or histidine,

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 hydrophobic amino acid, those chosen from valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, or tryptophan, negatively charged amino acid or of polar / broad nature, those chosen from aspartic acid , glutamic acid, asparagine, or glutamine.

A preferred example of a peptide according to the invention is of the following sequence ID NO: 1: HVDKKVADKVLLLKQLRIMRL LTRL or a fragment thereof as defined above, and more particularly the sequences SEQ ID NO: 2,3, 4 below: KKVADKVLLLKQLRIMRLLTRL (SEQ ID NO: 2) HVDKKVADKVLLLKQLRIMRL (SEQ ID NO: 3) KKVADKVLLLKQLRIMRL (SEQ ID NO: 4)
The peptides of the invention may be prepared by chemical synthesis or genetic engineering.

 The invention also relates to functional equivalents of the above peptides, the amino acid sequences of which comprise deletions, additions and / or conservative modifications at the level of one or more amino acids, provided that these deletions, additions and / or modification do not modify the antimicrobial activity of the peptide from which they are derived, and for example not the alpha helical structure.

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 In particular, modifications resulting from post-translational processes such as glycosylation or degeneracy of the genetic code, or chemical modifications such as amidation, acetylation, acylation, coupling with lipids or sugars, coupling with nucleotides, etc.

 The functional equivalents also comprise peptides of the invention in which one or more of the amino acids of the amino acids are enantiomers, diastereoisomers, natural amino acids of conformation D, rare amino acids, in particular hydroxyproline, methyllysine, dimethyllysine and synthetic amino acids, including ornithine, norleucine, cyclohexylalanine and omega-amino acids.

The invention also covers retropeptides and retro-inversopeptides.

 The invention also relates to the use of the above peptides or analogs thereof, for preventing or treating a fungal or bacterial infection in humans and animals as well as in plants.

The invention therefore relates to a composition, more particularly antibacterial or antifungal, comprising as active agent at least one peptide as defined above, advantageously associated in said composition with an acceptable vehicle.

 The vehicle is chosen according to the type of application of the composition for pharmaceutical or agronomic purposes.

 The invention particularly relates to pharmaceutical applications in humans and animals of

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 these peptides and compositions containing them, but it is also interested in agronomic applications. Indeed, the peptides of the invention are useful for making plants resistant against diseases including fungal and bacterial. A first form of implementation of this agronomic application is to apply to the plants an effective amount of peptide or a composition containing them. A second form of implementation of this agronomic application consists of transforming plant cells or plants with a nucleic acid sequence capable of expressing the peptide of the invention so as to confer on plants a resistance to diseases.

 Other advantages and characteristics of the invention will become apparent from the following examples relating to the purification and identification of spinigerin, the synthesis of synthetic spinigerin and its antibacterial and antifungal activity.

 Example 1 Materials and Methods Used for the Purification and Identification of Spinigerin

 1) Insects.

 All the experiments were carried out with adults of the species Pseudacanthotermes spiniger (Order of the Isoptera, family Termitidae, subfamily Macrotermitinae). These insects come from a breeding carried out at the Mixed Unit Research CNRS development, Chemical Communication, Dijon.

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 2) Extraction of the peptides.

 Extraction of antimicrobial compounds was performed from whole insects that were frozen in liquid nitrogen. The termites (200) have been reduced to a fine powder in a mortar containing liquid nitrogen.

The peptides were extracted at pH3 with 100 ml of trifluoroacetic acid (TFA, 0.1%) containing aprotinin (10 g / ml final concentration) as protease inhibitor and phenylthiourea (20 M final concentration) as Melanization inhibitor. The extraction was carried out in a water-bath with gentle stirring for 30 minutes. After centrifugation (14,000 × g for 30 min at 6 ° C.), the clarified supernatant was directly deposited on a Sep-Pak C18 Vac cartridge (5 g of phase, Waters). The cartridge was conditioned with methanol and equilibrated with acidified water (0.05% TFA), the elutions were carried out with solutions of 5%, 40% and 80% acetonitrile in water containing 0%. , 05% TFA. The fraction eluted with the 40% acetonitrile solution was lyophilized before passage on reverse phase HPLC.

 3) Purification of peptides by HPLC.

 Step 1: The 40% Sep-Pak fraction was subjected to Aquapore RP-300 C18 semi-preparative column reversed phase chromatography (250 x 7 mm, BrownleeTM). Equilibrated with a solution of 2% acetonitrile in acidified water. The fractions were separated using a linear gradient of 2% to 60% acetonitrile in acidified water over 120 min with a flow rate of 1.3 ml / min. At the end of this step, we obtain several

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 fractions shown in Figure 1 exhibiting antimicrobial activity on the following germs: E. coli 363, Neurospora crassa, Micrococcus luteus. The active compounds of the F1 and F2 fractions were then characterized.

 Step 2: The second purification step was carried out on an Aquapore OD-300 analytical column (220 × 4.6 mm, BrownleeTM). Elution was carried out using a biphasic gradient of acetonitrile in acidified water, from 2 to 18% for 10 min and from 22 to 42% for 100 min with a flow rate of 0.8 ml. min.

 Step 3: The fraction containing the antimicrobial molecule was purified to homogeneity using a reverse phase C18 narrow boron column (Delta Pak HPIC18, 2x 150 mm, Waters'). The column was equilibrated in acidified water and developed with the same linear gradient of acetonitrile as described above in step 2.

 All the purification steps at room temperature were carried out on a Beckman Gold HPLC system equipped with a Beckman 168 photoarray detector. For the controlled temperature purification steps, an HPLC all PEEK Waters system (Waters 626 pump model) attached to a variable absorbance detector (Waters 486) was used. During the HPLC purification steps the eluted molecules of the column were detected by their absorbance at 225 nm. The fractions were collected manually, dried under vacuum (Speed-Vac, Savant) and reconstituted in MilliQ water (MilliporeTM) before analysis of the antimicrobial activity.

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 4) Analysis by mass spectrometry.

 It is carried out according to the so-called flight time measurement technique after matrix-assisted laser desorption (MALDI TOF technique) using a Bruker (Bremen) BIFLEX mass spectrometer.

 5) Sequence analysis.

 The peptide sequences are analyzed according to the Edman degradation sequencing method using an Applied Biosystems 473A sequencer.

 6) Identification of the peptides of the F1 fraction.

 Despite the presence of two peptide compounds, the purified fraction active against the microbial strains Eschericia coli, Micrococcus luteus and Neurospora crassa, was subjected to sequencing. The analysis of the PTH-amino acid signals revealed two sequences, one corresponding to a truncated form of the other in its N-terminal part. The molecular weights calculated from the sequences obtained are respectively 2649.4 and 3000.8 Da. They are consistent with the molecular weights measured by MALDI-TOF mass spectrometry of 2649.5 Da and 3000.7 Da confirming that the sequences are complete. The molecular mass molecule 2649.5 Da corresponds to that of 3000.7 Da devoid of the three residues of the amino-terminal end of the peptide. Sequencing of a nearby fraction (Fraction F2) containing a molecule of molecular weight 2516.2 Da

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 allowed to determine a third form of the peptide corresponding to the molecular weight molecule 3000.7 Da lacking the four carboxy-terminal amino acids.

All of these results are reported in Table 1 below.

<Tb>
<Tb>

Table <SEP> 1
<tb> Number <SEP> of <SEP> Mass <SEP> molecular <SEP> Sequence <SEP> in <SEP> amino acids <SEP>
<tb> the <SEP> fraction <SEP> (Da)
<tb> Calculated / Measured
<tb> F1 <SEP> 3000.8 <SEP> / <SEP> 3000.7 <SEP> HVDKKVADKVLLLKQLRIMRLLTRL
<tb> 2649.4 <SEP> / <SEP> 2649.5 <SEP> --- KKVADKVLLLKQLRIMRLLTRL
<tb> F2 <SEP> 2517.2 / <SEP> 2516.7 <SEP> HVDKKVADKVLLLKQLRIMRL ----
<Tb>

Example 2 Synthesis of synthetic spinigerine

 The peptides according to the invention can also be obtained according to a FMOC chemical synthesis method (Atherton and Sheppard RC (1989) Solid Phase Peptide Synthesis (IRL, Oxford, UK) Chemical synthesis was carried out for spinigerine (HVDKKVADKVLLLKQLRIMRLLTRL). The peptide obtained has the same chromatographic properties as the native molecule.The mass measured is identical to that of the native molecule.The synthetic molecule has the same antibacterial activity as the native molecule on the bacterium Micrococcus luteus.

 All antibacterial tests are performed with the synthetic molecule. This molecule has antibacterial properties against Gram-negative and Gram-positive bacteria, phytopathogenic bacteria and phytopathogenic fungi.

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 Example 3 Detection of antimicrobial activities.

 The method was used for the detection of antimicrobial molecules during the various purification steps, for the determination of the spectrum of activity of the peptide and for the determination of the minimum inhibitory concentration (MIC) at which the peptide was active. . The MIC was expressed as a concentration range [a] - [b] where [a] was the minimum concentration at which growth was observed and [b] the concentration at which no growth was observed . Examples of the specific activity of spinigerin with respect to filamentous fungi and yeasts are given in Tables 2 and 3 below.

 1) Detection of antibacterial activity.

 For each bacterial strain used (Gram-positive bacteria and Gram-negative bacteria), an isolated colony is suspended in 10 ml PBDE medium (Poor Broth, Luria Bertani medium without yeast extract) DIFCO and incubated at 30 ° C. C overnight with slow stirring. The bacterial suspensions to be tested are adjusted to an optical density at 600 nm of 0.001 in a fresh culture medium. 10 l of each fraction are deposited in microtiter plates in the presence of 100 l of the bacterial suspension. After 24 hours of incubation at 25 C, the growth is evaluated by measurement

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 Absorbance at 600 nm using a microtiter plate reader.

 The bacteria are: - Gram positive: Bacillus megaterium, Micrococcus luteus, Staphylococcus aureus, Streptococcus pyogenes.

 Gram negative: Escherichia coli D22, Escherichia coli SBS 363, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa.

Table 2 below reports the activity of spinigerine against bacteria.

<Tb>
<Tb>

Table <SEP> 2
<tb> Bacteria <SEP> Spinigerine <SEP> MIC <SEP> (M)
<tb> Bacteria <SEP> to <SEP> Gram <SEP> positive
<tb> Bacillus <SEP> megaterium <SEP> 0.8-1.5
<tb> Micrococcus <SEP> luteus <SEP> 0.8-1.5
<tb> Staphylococcus <SEP> aureus <SEP> ND *
<tb> Bacteria <SEP> to <SEP> Gram <SEP> Negative
<tb> Escherichia <SEP> coli <SEP> D22 <SEP> 3-6
<tb> Escherichia <SEP> coli <SEP> SBS <SEP> 363 <SEP> 3-6
<tb> Klebsiella <SEP> pneumoniae <SEP> 50-100
<tb> Salmonella <SEP> typhimurium <SEP> 3-6
<tb> Pseudomonas <SEP> aeruginosa <SEP> 50-100
<Tb>
* ND: activity not detected up to a concentration of 100 M.

 2) Activity detection test against filamentous fungi and yeast Candida albicans.

 The antifungal activity was detected by a growth inhibition test in a liquid medium. The spores of the fungi to be tested were suspended in a culture medium of the potato glucose type. Preferably, 12 g of Potato medium are used.

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 Dextrose Broth (Difco) for 1 liter of demineralised water. Two antibiotics were added to the culture medium: tetracycline (final concentration of 10 μg / ml) and cefotaxime (100 μg / ml). 10 μl of each fraction to be assayed are plated in microtiter plates in the presence of 90 μl of culture medium containing the spores (at a final concentration of 104 spores / ml). The incubation was carried out in a humid chamber at 30 ° C. for 48 hours. The fungal growth was observed under a light microscope after 24 h and quantified after 48 hours by measuring the absorbance at 600 nm using a microtiter plate reader spectrophotometer.

 The different yeast strains were incubated in a Sabouraud culture medium and incubated at 30 ° C. for 24 h with slow stirring. The sample to be tested (10 l) was placed in microtiter plate wells into which was added 90 l of a diluted yeast culture whose density was adjusted to OD 600 = 0.001. The growth is evaluated by measuring the absorbance at 600 nm using a microtiter plate reader spectrophotometer.

The filamentous fungi and yeast that were used are as follows:
Aspergillus fumigatus (gift from Dr. H. Koenig, Civil Hospital, Strasbourg); Nectria haematococca, Fusarium culmorum, Trichoderma viride (mycoteca of the Catholic University of Leuven, Belgium); Neurospora crassa, Fusarium oxysporum, (Mycoteque of the Clause Society, Paris); Candida albicans (gift from Dr. H. Koenig, Civil Hospital, Strasbourg).

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Table 3 below reports the activity of spinigerine against fungi.

<Tb>
<Tb>

Table <SEP> 3
<tb> fungi <SEP> Spinigerine <SEP> MIC <SEP> (M)
<tb> Neurospora <SEP> crassa <SEP> 3-6
<tb> Fusarium <SEP> culmorum <SEP> 1.5-3
<tb> Nectria <SEP> haematococca <SEP> 0.4-0.8
<tb> Trichoderma <SEP> viride <SEP> 1,5-3
<tb> Candida <SEP> albicans <SEP> 3-6
<Tb>

Claims (3)

1) peptide sequence (I): 1 02 63 4 5 z6 <? 7 08 9 <{> 10 <t> 11 <t> 12 013 P 14 615 <P16 17 18 <19 P 20 # 21 # 22 # 23 ss24 # 25 or a fragment thereof, wherein: the amino acids ss1, ss4, ss5, ss9, ss14, ss17, and ss24 are independently selected from the basic amino acids, - amino acids # 2, # 6, # 10, # 11, # 12, # 13, # 16, # 18, # 19, # 21, # 22 and 25 are independently selected from hydrophobic amino acids, - amino acids # 3, # 8 and 615 are independently selected from negatively charged amino acids or of polar / broad nature, - amino acids # 7 and # 2 are independently selected from glycine, serine, alanine or threonine, less amino acids ss1, # 2, 63, # 22, # 23, 24, or # 25 are absent. 2) A peptide according to claim 1, characterized in that it has an alpha helix configuration. 3) Peptide according to one of claims 1 or 2, characterized in that a group of 1 to 4 amino acids at one and / or the other end N and / or C terminal is deleted. 4) Peptide according to any one of claims 1 to 3, characterized in that it corresponds to one of the following formulas: ## STR1 ## P22 623 24 <P25 (II) <Desc / Clms Page number 17>  1 02 e3 4 5 z6 67 08 9 010 11 012 13 P14 015 16 17 018 019 20 021 (III) 4 5 06 67 08 59 10 011 012 013 514 e15 016 P17 18 019 20 021 (IV).  5) Peptide according to any one of claims 1 to 4, characterized in that the basic amino acids are chosen from arginine, lysine or histidine.  6) Peptide according to any one of claims 1 to 5, characterized in that the hydrophobic amino acids are chosen from valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, or tryptophan.  7) Peptide according to any one of claims 1 to 6, characterized in that the negatively charged amino acids or of polar / broad nature are selected from aspartic acid, glutamic acid, asparagine, or glutamine. 9) An antibacterial or antifungal composition, characterized in that it comprises as active agent at least one peptide according to any one of claims 1 to 8, advantageously associated in said composition with an acceptable vehicle.  8. Peptide according to claim 1, characterized in that it consists of one of the following sequences: ## STR1 ## 2) H V D K K V A D K V L L L L K Q L R I M R L (SEQ ID NO: 3) K K V A D K V L L L K Q L R I M R L (SEQ ID NO: 4) <Desc / Clms Page number 18>  10) Use of a peptide according to any one of claims 1 to 8 for the preparation of a composition for the treatment or prevention of a fungal or bacterial infection in humans or animals.  11) Use of a peptide according to any one of claims 1 to 8 for the preparation of a composition for the treatment or prevention of a fungal or bacterial infection in plants.