FR2732345A1 - Proline-rich antibacterial peptide(s) from Podisus maculiventris - Google Patents

Abstract

Peptides of formula (I) are new: VDKPDYRPRP-X (I) X = a peptide residue comprising at least one tripeptide motif PRP.

Description

Antibacterial peptide
The present invention relates to novel protein-rich peptides having antibacterial properties, compositions for use in agriculture and in human or animal therapy containing this peptide as an active ingredient. The invention also relates to a process for the preparation of this peptide.

 It has long been known that insects have an effective resistance against bacteria. This defense is largely based on the rapid synthesis of several families of peptides. This defense is due to the rapid synthesis of several peptide families with a broad spectrum of activity. This synthesis is induced by septic injury or by the injection of a low dose of bacteria.

 In addition it is known that insects have developed a significant resistance to fungi.

 The proline-rich peptides, which have remarkable characteristics as well as antibacterial properties, have now been isolated from an induction in the Podisus maculiventrts hemiptera.

More particularly, a first aspect of the invention relates to the peptides of formula I:
VDKPDYRPRP-X
wherein X is a variable peptide residue comprising at least one "PRP" linkage.

Preferably X can be chosen from the group comprising:
X1 = WPRPPSM
X2 = GPRPII
X3 = GPRPIR
X4 = GPRPIIVM
In the following, the molecules of formula I with X = X1, X2, X3 and X4 respectively,
will be called respectively metalnikowine 1 (Met 1), metalnikowine 2 (Met 2),
metalnikowine 3 (Met 3) and metalnikowine 4 (Met 4).

These molecules, of reduced size, contain from 16 to 18 residues rich in proline (P)
(from 5 to 6 prolines) and have 4 to 5 basic residues. They are called in the following
"Métalnikowines".

Another aspect of the invention relates to a method for obtaining and
the isolation of the above peptides, which is characterized in that successively:
a) at least one bacterial inducer is made to act on Podisus maculiventris for the biological synthesis of the molecule;
b) the extraction is carried out by contacting a ground material of Podisus maculi yen tris obtained previously with an acid medium with stirring, then by centrifugation;
c) the supernatant is separated by washing off the hydrophilic molecules and eluting the hydrophobic molecules by appropriate elements on a separating column
d) the extracts are purified.

 e) the peptide is characterized.

 The first step (bacterial induction) is carried out by simple physical aggression such as a wound, for example a sting, or, preferably, and from a hemolymph of adult Podisus maculiventris bugs by anesthetizing them with a anesthetic gas, for example carbon dioxide or any gas having similar effects, and by immunizing them with a needle puncture preferably dipped before each inoculation into a bacterial suspension. Preferably, inoculation is carried out with at least one bacterium selected from the group consisting of bacteria (Gram-positive) and bacteria (Gram-negative). As a representative representative example of bacteria (gram positive), mention may be made of Micrococcus luteus. As a preferred representative example of bacteria (Gram negative), mention may be made of Escherichia coli. More preferably one can simultaneously use at least one bacterium chosen from the group comprising bacteria (Gram-positive) and at least one bacterium chosen from the group comprising bacteria (Gram negative).

Preferably, the second step (extraction) is brought into contact with the ground material.
Podisus maculiventris or the hemolymph of Podisus maculiventris with an acid medium, consisting of an acidic solution of an acid, or neutral (pH of 2 to 7). The solution may be a solution of an inorganic or organic acid such as, for example, trifluoroacetic acid.

The usual addition of a protease inhibitor is not necessary here because of the remarkable natural resistance of the metalnikowine peptide to proteases. The extract obtained is then centrifuged at a speed of 5000 to 20000 rpm for 15 to 60 minutes.

 Preferably the third step (fractionation), the washing of the water-soluble molecules is carried out with a dilute acid solution and the elution of the hydrophobic molecules with a suitable eluent. Good results are obtained with trifluoroacetic acid for washing and an eluent containing increasing amounts of acetonitrile in dilute acidic solution.

 Preferably the fourth step (purification) is carried out with a suitable eluent which may be different or identical to that of the previous phase.

Preferably, in the last step (characterization), the nature of the peptide is analyzed according to the method of degradation sequencing of Edman (Acta Chemica
Scandinavia 10 (1956) p; 761-768). According to this method the following structures are obtained: VDKPDYRPRPWPRPPS M
VDKPDYRPRPGPRPII
VDKPDYRPRPGPRPIR VDKPDYRPRPGPRPIIVM
The measured masses of the above metalnikowines are respectively 2094 ::::: 0.3Da; 1876.11 + 0.3 Da; 1919 f 0.5 Da; 2234 + 0.3 Da;
The masses calculated on the basis of the sequence data are respectively:
2094.3 Da; 1876.17 Da; 1919.2 Da; 2234.5 Da.

 These good correlations confirm the proposed sequences.

 These molecules have antibacterial properties against Gram-negative germs.

 According to another aspect, the subject of the invention is an antibacterial composition that can be used for protecting plants against bacterial diseases.

 The following examples illustrate the obtaining and the antibacterial properties of the peptides and compositions according to the invention.

Example 1 Isolation and Characterization of the Peptide
we proceed according to the following steps:
- natural induction of biological synthesis:
Podisus maculiyentris are immunized by thoracic puncture near the wing insertion with a fine dive needle prior to each inoculation into a bacterial suspension of Escherichia coli 1106 (gram negative) killed in heat for 2 minutes. at 100 ° C. The insects are kept at 20 ° C. for 24 hours.

- extraction and purification:
The hemolymph (1.2 ml) is removed by incision of the cuticle. It is transferred to a cold-maintained tube in the presence of a protease inhibitor (aprotinin) and then centrifuged at 2000 G (10,000 rpm) for 25 minutes at 4 ° C. The supernatant thus obtained is immediately subjected to the various purification steps. .

Splitting the extract on Sep-Pak C18 cartridges
After depositing the extract on Sep-Pak C18 cartridges, the hydrophilic molecules are removed by simply washing with 5 ml of water acidified with 0.05% trifluoroacetic acid (TFA).

 The elution of the hydrophobic molecules is carried out with 20, 50 and 80% acetonitrile solutions in acidified water (0.05% TFA, 5 ml per cartridge).

The collected fractions are named "Elution 20%", "Elution 50%" and "Elution 80%" and concentrated under vacuum. The fractions are then reconstituted with water quality
HPLC before HPLC analysis.

HPLC Puntification of Antibacterial Activity Molecules
first stage:
The fractions "Elution 20%" and "Elution 50%" are analyzed separately on two columns of molecular sieving series (SEC 2000 + SEC 3000, Beckman) under 30% acetonitrile in the presence of TFA 0.05% to one. devit of 0.5 ml / min.

 Fractions with antibacterial activity are then purified by HPLC reverse phase chromatography.

 Final purification: Two steps are necessary for the purification of antibacterial molecules.

 a) The fractions exhibiting this activity were firstly purified on an Aquapore OD 300 C 18 reverse phase column with a linear gradient of acetonitrile of 2 to 52% in the acidified water (0.05% TFA) ) in 90 minutes (an increase of 0.44% acetonitrile per minute) for a flow rate of 1 ml / min.

b) the resulting active fractions of a) are then purified on a column
Aquapore OD 300 C18.

 The elution is carried out in a linear biphasic gradient of acetonitrile of 2 to 17% in acidified water (0.05% TFA) in 10 minutes and 17 to 27% in 45 min at a flow rate of 1 ml / min. . Metalnikowin 1 is obtained for a percentage of 19.4% acetonitrile, metalnikowin 2 at a percentage of 20.7% acetonitrile, metalnikowin 3 at a percentage of 20.7% acetonitrile and metalnikowine 4 at a percentage of 19.7% acetonitrile.

 The purity of the active fraction is monitored by capillary electrophoresis prior to sequence determination by Edman degradation and mass spectrometry analysis.

Example 2 In Vitro Test: Measurement of the antibacterial activity by microspectrophotometry
From each bacterial strain used (E.coli; Mluteus) an isolated column was suspended in 10 ml of PBF medium (Poor Broth, Luria Bertani medium without yeast extract) DIFCO and incubated at 30 ° C overnight. with slow stirring.

 The bacteria to be tested are brought to an optical density at 600 nm of 0.001 in a fresh culture medium. 10 μl of each fraction are deposited in microtiter plates in the presence of 100 μl of the bacterial suspension. After 24 hours of incubation at 25 ° C, the growth is evaluated by measuring the absorbance at 600 nm using a microtiter plate reader.

Under these conditions, a 50% inhibition is observed at the concentrations, expressed in μM, indicated in the following table:

<tb><SEP> Metalnikowine
<tb><SEP> Bacteria <SEP> Met <SEP> 1 <SEP> Met <SEP> 2 <SEP> Met <SEP> 3 <SEP> Met <SEP> 4
<tb> E.Coli <SEP> 1106 <SEP> 10 <SEP> 12 <SEP> 14 <SEP> 21
<tb> E.Coli <SEP> D31 <SEP> 18 <SEP> 11 <SEP> 15 <SEP> 9
<tb> E.Coli <SEP> D22 <SEP> 21 <SEP> 13 <SEP> 12 <SEP> 31
<Tb>
These results show the excellent antibacterial activity of the peptide according to the invention, which can be applied to the human, animal and plant domains.

 Among the cultures that can be treated with an antibacterial treatment using a compound according to the invention, examples that may be mentioned include rice, cereals, in particular wheat and barley, as well as arboreal, fruit and vegetable plants.

 The subject of the present invention is also compositions which can be used as antibacterial agents, containing as active ingredient (s) one or more compounds according to the invention as described above, in admixture with solid or liquid carriers, which are acceptable. in agnculture and surfactants also acceptable in agriculture. In particular, the inert and conventional supports and the usual surfactants can be used. These compositions cover not only the compositions ready to be applied to the crop to be treated by means of a suitable device, such as a spraying device, but also the commercial concentrated compositions which must be diluted before application to the crop.

 These compositions may also contain any kind of other ingredients such as, for example, protective colloids, adhesives, thickeners, thixotropic agents, penetration agents, stabilizers, sequestering agents, etc. More generally the compounds used in the invention can be combined with all solid or liquid additives corresponding to the usual techniques of formulation.

 In general, the compositions according to the invention usually contain from about 0.05 to about 95% (by weight) of a compound according to the invention (hereinafter referred to as active ingredient), one or more solid or liquid carriers. and, optionally, one or more surfactants.

 By the term "carrier" in the present specification is meant an organic or inorganic material, natural or synthetic, with which the compound is combined to facilitate its application on the plant, on seeds or on the ground. This support is therefore generally inert and must be acceptable in agriculture, especially on the treated plant. The support may be solid (clays, natural or synthetic silicates, silica, resins, waxes, solid fertilizers, etc.) or liquid (water, alcohols, especially butanol, etc.).

 The surfactant may be an emulsifier, dispersant or wetting agent of ionic or nonionic type or a mixture of such surfactants. There may be mentioned, for example, salts of polyacrylic acids, salts of lignosulfonic acids, salts of phenolsulphonic or naphthalenesulphonic acids, polycondensates of ethylene oxide with fatty alcohols or with fatty acids or with fatty amines. substituted phenols (especially alkylphenols or arylphenols), salts of sulfosuccinic acid esters, taurine derivatives (especially alkyltaurates), phosphoric esters of polyoxyethylated alcohols or phenols, acid esters and polyols, sulfates, sulfonates and phosphates-based derivatives of the foregoing compounds.

The presence of at least one surfactant is generally essential when the compound and / or the inert carrier are not soluble in water and the application carrier is water.

 Thus, the compositions for agricultural use according to the invention can contain the active ingredients according to the invention within very wide limits, ranging from 0.05% to 95% (by weight). Their content of surfactant is advantageously between 5% and 40% by weight.

 These compositions according to the invention are themselves in quite diverse forms, solid or liquid.

 As forms of solid compositions, mention may be made of dusting powders (with a compound content of up to 100%) and granules, in particular those obtained by extrusion, by compacting, by impregnation of a granulated support, by granulation with from a powder (the content of compound in these granules being between 0.5 and 80% for the latter cases), tablets or effervescent tablets.

 The peptide according to the invention can also be used in the form of dusting powders; a composition comprising 50 g of active material and 950 g of talc can also be used; a composition comprising 20 g of active material, 10 g of finely divided silica and 970 g of talc can also be used; these constituents are mixed and milled and the mixture is applied by dusting.

 As forms of compositions which are liquid or intended to constitute liquid compositions during application, mention may be made of solutions, in particular water-soluble concentrates, emulsifiable concentrates, emulsions, concentrated suspensions, aerosols and powders. wettable (or powder spray), pasta, gels.

 The emulsifiable or soluble concentrates most often comprise 10 to 80% of active material, the emulsions or solutions ready for application containing, for their part, 0.001 to 20% of active ingredient.

 In addition to the solvent, the emulsifiable concentrates may contain, when necessary, 2 to 20% of appropriate additives such as stabilizers, surfactants, penetrating agents, corrosion inhibitors, dyes or adhesives previously cited.

 From these concentrates, emulsions of any desired concentration can be obtained by dilution with water, which are particularly suitable for application to crops.

As an example, here is the composition of some emulsifiable concentrates:
Example CE 1:
active ingredient 400 g / l
alkaline dodecylbenzene sulfonate 24 g / l
- Nonylphenol oxyethylated to 10 molecules
of ethylene oxide 16 gel
- cyclohexanone 200 gel
- aromatic solvent qsp1 liter
According to another emulsifiable concentrate formula, use is made of:
Example CE 2
- active ingredient 250 g
- epoxidized vegetable oil 25 g
- Alkylaryl sulfonate mixture and
of polyglycol ether and fatty alcohols 100 g
dimethylformamide 50 g
- xylene 575 g
The concentrated suspensions, also applicable in spraying, are prepared in such a way as to obtain a stable fluid product which does not settle and usually contain from 10 to 75% of active substance, from 0.5 to 15% of surfactants, from 0 to 1 to 10% of thixotropic agents, 0 to 10% of suitable additives, such as antifoams, corrosion inhibitors, stabilizers, penetrants and adhesives and, as carrier, water or an organic liquid in which the active ingredient is little or not soluble: some organic solids or mineral salts may be dissolved in the support to help prevent sedimentation or as antifreezes for water.

For example, here is a concentrated suspension composition:
Example SC 1:
active ingredient 500 g
- polyethoxylated tristyrylphenol phosphate 50 g
polyethoxylated alkylphenol 50 g
- sodium polycarboxylate 20 g
- ethylene glycol 50 g
- organopolysiloxane oil (antifoam) 1 g
- polysaccharide 1.5 g
- water 316.5 g
Wettable powders (or spray powder) are usually prepared to contain from 20 to 95% active ingredient, and usually contain, in addition to the solid support, from 0 to 30% of a wetting agent, at 20% of a dispersing agent, and, when necessary, from 0.1 to 10% of one or more stabilizers and / or other additives, such as penetrating agents, adhesives, or anti-caking agents, dyes, etc.

 In order to obtain the spraying powders or wettable powders, the active ingredients in the appropriate mixers are intimately mixed with the additional substances and milled with suitable mills or other mills. Spray powders are thus obtained, the wettability and suspension of which are advantageous; they can be suspended with water at any desired concentration and these suspensions can be used very advantageously especially for application to the leaves of plants.

 In place of the wettable powders, pasta can be made. The conditions and methods of production and use of these pastes are similar to those of wettable powders or powders to be sprayed.

By way of example, here are various compositions of wettable powders (or powders for spraying):
Example PM 1
- active ingredient 50%
- ethoxylated fatty alcohol (wetting agent) 2.5%
- ethoxylated phenylethylphenol (dispersing agent) 5%
- chalk (inert support) 42.5%
Example PM 2:
- active ingredient 10%
- oxo synthetic alcohol of branched type, in
C13 ethoxylated with 8 to 10 ethylene oxide
(wetting agent) 0.75%
- neutral calcium lignosulfonate (agent
dispersant) 12%
- calcium carbonate (inert filler) qs 100%
Example PM 3:
This wettable powder contains the same ingredients as in the previous example, in the following proportions:
- active ingredient 75%
- wetting agent 1.50%
- dispersing agent 8%
- calcium carbonate (inert filler) qs 100%
Example PM 4 ::
- active ingredient 90%
- ethoxylated fatty alcohol (wetting agent) 4%
- ethoxylated phenylethylphenol (dispersing agent) 6%
Example PM 5:
- active ingredient 50%
- mixture of anionic surfactants and
nonionic (wetting agent) 2.5%
- sodium lignosulfonate (dispersing agent) 5%
- kaolinic clay (inert support) 42.5%
The aqueous dispersions and emulsions, for example the compositions obtained by diluting with water a wettable powder or an emulsifiable concentrate according to the invention, are included in the general scope of the present invention. The emulsions may be of the water-in-oil or oil-in-water type and may have a thick consistency such as that of a "mayonnaise".

 The compounds according to the invention may be formulated in the form of water-dispersible granules also included in the scope of the invention.

 These dispersible granules, having an apparent density generally of between approximately 0.3 and 0.6, have a particle size generally of between approximately 150 and 2000 and preferably between 300 and 1500 microns.

 The active ingredient content of these granules is generally between about 1% and 90%, and preferably between 25% and 90%.

 The rest of the granule is essentially composed of a solid filler and optionally surfactant adjuvants conferring on the granule properties of dispersibility in water. These granules can be essentially of two distinct types depending on whether the charge retained is soluble or not in water. When the filler is water soluble, it may be inorganic or, preferably, organic. Excellent results have been obtained with urea. In the case of an insoluble filler, it is preferably mineral, such as kaolin or bentonite. It is then advantageously accompanied by surfactants (in a proportion of 2 to 20% by weight of the granule) of which more than half is, for example, constituted by at least one dispersing agent, essentially anionic, such as polynaphthalene. an alkali or alkaline earth metal sulphonate or an alkaline or alkaline earth lignosulfonate, the remainder being nonionic or anionic wetting agents such as an alkaline or alkaline earth alkyl naphthalene sulphonate.

 On the other hand, although not essential, other adjuvants such as antifoam agents may be added.

 The granulate according to the invention can be prepared by mixing the necessary ingredients and then granulating according to several techniques known per se (dredger, fluid bed, atomizer, extrusion, etc ...). It is generally finished by crushing followed by sieving at the particle size chosen within the limits mentioned above. Granules obtained as above and then impregnated with a composition containing the active ingredient can also be used.

 Preferably, it is obtained by extrusion, operating as indicated in the examples below.

Example GDI: Dispersible granules
In a mixer, 90% by weight of active material and 10% of urea beads are mixed. The mixture is then milled in a pin mill. A powder is obtained which is moistened with about 8% by weight of water. The wet powder is extruded in a perforated roll extruder. A granule is obtained which is dried, then crushed and sieved so as to keep only the granules with a size of between 150 and 2000 microns respectively.

Example GD2: Dispersible granules
In a mixer, the following constituents are mixed:
- active ingredient 75%
- wetting agent (sodium alkylnaphthalene sulphonate) 2%
- dispersing agent (sodium polynaphthalene sulphonate) 8%
- inert load insoluble in water (kaolin) 15%
This mixture is granulated in a fluid bed, in the presence of water, then dried, crushed and sieved so as to obtain granules with a size of between 0.15 and 0.80 mm.

 These granules can be used alone, in solution or dispersion in water so as to obtain the desired dose. They can also be used to prepare combinations with other active substances, especially antibacterials, the latter being in the form of wettable powders, or aqueous granules or suspensions.

 As regards the compositions suitable for storage and transport, they more preferably contain from 0.5 to 95% (by weight) of active substance.

 The invention also relates to a method for the therapeutic antibacterial treatment for humans or animals by administering an effective dose of the peptide according to the invention, in free form or, where appropriate, in the form of addition salts with an acid, metal salts or addition salts with a pharmaceutically acceptable base, in pure form or in the form of a composition in which it is combined with any other pharmaceutically compatible product, which may be inert or physiologically active. The medicaments according to the invention may be administered orally, parenterally, rectally or topically.

 As solid compositions for oral administration may be used tablets, pills, powders (especially in gelatin capsules or cachets) or granules. In these compositions, the active product according to the invention is mixed with one or more inert diluents, such as starch, cellulose, sucrose, lactose or silica. These compositions may also comprise other substances, for example one or more lubricants such as magnesium stearate or talc, a dye, a coating (dragees) or a varnish.

 As liquid compositions for oral administration, it is possible to use pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or the like. paraffin oil. These compositions may also comprise other substances, for example wetting agents, sweeteners, thickeners, flavoring agents or stabilizers.

 Sterile compositions for parenteral administration may be preferably aqueous or nonaqueous solutions, suspensions or emulsions.

As solvent or vehicle, water, propylene glycol, polyethylene glycol, vegetable oils, in particular olive oil, suitable organic esters can be used. These compositions may also contain adjuvants, in particular wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. Sterilization can be carried out in various ways, for example by aseptic filtration, by incorporating sterilizing agents into the composition, by irradiation or by heating. They can also be prepared as sterile solid compositions, which can be dissolved at the time of use in a sterile injectable medium.

 The compositions for rectal administration are suppositories or rectal capsules, which contain, in addition to the active peptide, excipients such as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

 The sterile compositions for topical administration may be, for example, creams, ointments, lotions, eye drops, mouthwashes, nasal drops or aerosols.

 In human therapy, the peptide according to the invention is particularly useful in antibacterial treatments. The doses depend on the desired effect and the duration of the treatment; they are generally between 50 and 1000 mg per day orally for an adult in one or more perises.

 In general, the doctor will determine the dosage he considers the most appropriate based on age, weight and all other factors specific to the subject to be treated.

 The following examples are given as non-limiting examples of the compositions according to the invention.

Example A
50 mg of active peptide having the following composition are prepared according to the usual technique:
- metalnikowine peptide M1 50 mg
starch 60 mg
- lactose 50 mg
- magnesium stearate 2 mg
Example B:
An injectable solution containing 20 mg of active peptide having the following composition is prepared:
- metalnikowin M 2 peptide 22.4 mg
- distilled water qs 2 cm3

Claims (8)

 VDKPDYRPRP-X wherein X is a variable peptide residue comprising at least one "PRP" linkage.  1. Peptide of formula: Peptide according to claim 1, characterized in that X is selected from the group  comprising:  X1 = WPRPPSM  X2 = GPRPII  X3 = GPRPIR  X4 = GPRPIIVM 3. Antibacterial composition characterized in that it contains as active ingredient  according to one of claims 1 and 2. 4. Composition according to claim 1 usable for the protection of plants against  pathogenic bacteria. 5. Process for the protection of plants against bacterial diseases, characterized in that  a peptide according to one of claims 1 and 2 is applied as an active ingredient. 6. Process for the preparation of the peptide according to one of claims 1 and 2, characterized in that  that, successively:  a) at least one bacterial inducer of Podisus maculiventris is  biological synthesis of the molecule;  b) the extraction is carried out by contacting hemolymph or a pulverized Podisus  maculiventris obtained previously with an acidic medium to neutral with stirring,  then by centrifugation;  c) the supernatant is fractionated by washing off the hydrophilic molecules and  elution of the hydrophobic molecules by appropriate elements, on a separating column;  d) the extracts are purified;  e) the sequencing is carried out. 7. Method according to claim 6, characterized in that the inductor is used  bacterial bacterium (Gram positive). 8. Method according to claim 6, characterized in that the inductor is used  bacterial a bacterium (Gram negative).