EP0882063A2

EP0882063A2 - Antifungic and antibacterial peptide

Info

Publication number: EP0882063A2
Application number: EP97905217A
Authority: EP
Inventors: Philippe Bulet; Charles Hetru; Jules Hoffmann; Laurence Sabatier
Original assignee: Rhone Poulenc Agrochimie SA
Current assignee: Bayer CropScience SA
Priority date: 1996-02-16
Filing date: 1997-02-17
Publication date: 1998-12-09
Also published as: AU722891B2; FR2745004B1; IL125778A; HUP9900935A2; HUP9900935A3; AU1884397A; US6331522B1; EA000843B1; PL328579A1; KR19990082596A; WO1997030082A3; TR199801582T2; WO1997030082A2; JP2000505440A; CN1216047A; FR2745004A1; IL125778A0; BR9707292A; CA2245518A1; EA199800731A1

Abstract

The invention discloses an antibacterial and antifungic peptide having the formula (I).

Description

Antibacterial and antifungal peptide

The subject of the present invention is a new protein-rich peptide having antibacterial and antifungal properties, compositions which can be used in agriculture and in human or animal therapy containing this peptide as active material. The invention also relates to methods of treating plants using these compositions as well as methods of preparing 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 families of peptides with a broad spectrum of activity. This synthesis is induced by a septic injury or by the injection of a small dose of bacteria. Among the antibacterial peptides induced, the best characterized are the cecropins and defensins of insects. Several other antibacterial peptides have been partially characterized.

Besides the class of insects little is known about other arthropods. Scorpions are animals older than insects on a philogenic level.

It has now been isolated, from an induction in the scorpion Androconus australis, a peptide, which has remarkable characteristics as well as antibacterial and antifungal properties.

More particularly, a first aspect of the invention relates to the peptide of formula I:

Arg Ser Val Cys Arg Gin Ile Lys Ile Cys Arg

Arg Arg Tyr Pro Arg Asn Thr Cys Lys Tyr Tyr Cys Gly Gly

In the following, the molecule of formula I will be called androctonine. This molecule, of reduced size, contains 4 cysteine residues engaged in two intramolecular bridges. Another aspect of the invention relates to a first method for obtaining and isolating the above peptide, which is characterized in that successively: a) the hemolymp of the scorpion Androctonus australis is taken; b) the extraction is carried out by bringing the hemolymph of Androctonus australis obtained previously into contact with an acid medium with stirring, then by centrifugation; c) the supernatant is fractionated with separation by washing of the hydrophilic molecules and elution of the hydrophobic molecules by suitable elements on a separating column d) the extracts are purified. e) characterizing the peptide.

The hemolymph is removed by cuticular incision. It is collected in a tube containing a protease inhibitor. After centrifugation to remove the blood cells, the plasma is stored at -30 ° C.

Preferably, the second step (extraction), the hemolymph of Androctonus australis is brought into contact with an acid medium, consisting of an acid solution of an acid (pH 2). The solution can be a solution of a mineral or organic acid such as, for example, trifluoroacetic acid. The extract obtained is then centrifuged cold at a speed of 30,000 g at 4 ° C, for 25 min.

Preferably the third step (fractionation), the extract is deposited on a reverse phase cartridge to carry out a solid phase extraction. The water-soluble molecules are washed with a dilute acid solution and the hydrophobic molecules are eluted with an appropriate eluent. Good results are obtained with trifluoroacetic acid for washing and an eluent containing increasing amounts of acetonitrile in dilute acid 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 sequencing by degradation of Edman (Acta Chemica Scandinavia 10 (1956) p; 761-768). According to this method, the following structures are obtained:

No signal was detectable in positions 4, 10, 16 and 20. (Edman degradation). The presence of cysteines in these positions has been shown by mass spectrometry, the structure then obtained being as follows: Arg Ser Val Cys Arg Gin Ile Lys Ile Cys Arg

Arg

Àrg Tyr Pro Arg Asn Thr Cys Lys Tyr Tyr Cys Gly Gly

The measured masses of androctonine above are respectively: 3076.65 ± 0.24 Da;

The masses calculated on the basis of the sequence data are respectively: 3080.65 ± 0.24 Da;

The mass defect corresponds to the formation of two intramolecular disulfide bridges.

In order to establish the connectivity of the disulfide bridges, the molecule was cleaved by an enzyme, the endoproteinase Lys-C, which breaks the peptide chain after lysine. The peptides obtained were isolated and mass spectrometry showed that they were two peptides linked by a disulfide bridge. The deduced sequences were Arg Ser Val Cys Arg Gin Ile Lys plus Cys Thr Asn Arg Asn Pro Tyr and Ile Cys Arg Arg Arg Gly Gly Cys Tyr Tyr.

The connectivity of the disulfide bridges is thus established and cysteine 1 is linked to cysteine 4, cysteine 2 to cysteine 3. This result can be schematized as follows:

These good correlations confirm the proposed sequences.

The peptides according to the invention can also be obtained without difficulty according to a second method, by chemical synthesis FMOC (Atherton and Sheppard RC (1989), Solid Phase Peptide Synthesis (IRL, Oxford, UK) followed by renaturation in a 100 mM solution of ammonium acetate at pH 8.5 for 24 h with stirring at room temperature The androctonine obtained has the same chromatographic properties as the native molecule and the connectivity of the disulfide bridges is identical to that of the natural molecule. measured after renaturation (3076, 61 ± 0.67) is very similar to that of the native molecule.The synthetic molecule has the same antibacterial activity as the native molecule on the bacterium Micrococcus liteus. All antibacterial and hemolytic tests are performed with the synthetic molecule.

1. Androctonine has no lytic effect on red blood cells from pork or beef. 2. These molecules have antibacterial properties against bacteria to

Gram negative, and Gram negative (see Table 1), phytopathogenic bacteria and phytopathogenic fungi.

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

Example 1: Isolation and characterization of the peptide the following steps are followed: - extraction and purification: The hemolymph (3.8 ml) is removed by incision from the cuticle. It is transferred to a tube kept cold in the presence of a protease inhibitor (aprotinin) and then centrifuged at 30,000 g for 25 minutes at 4 ° C. The supernatant thus obtained is immediately subjected to the various stages of purification. Fractionation of the extract on Sep-Pak CJ8 cartridges After depositing the extract on Sep-Pak C \ g cartridges, the hydrophilic molecules are eliminated by a simple washing with 5 ml of acidified water with trifluoacetic acid (TFA) at 0.05%.

The elution of the hydrophobic molecules is carried out with solutions containing 10, 40 and 80% acetonitrile in acidified water (TFA 0.05%, 5 ml per cartridge). The fractions collected are called "Elution 10%", "Elution 40%" and

"80% elution" and concentrated in vacuo. The fractions are then reconstituted with water quality HPLC before analysis in HPLC.

Purification by HPLC of molecules with antibacterial activity first stage: The fraction "Elution 40% is analyzed on a reverse phase column

Aquapore OD 300 Ci8 with a linear gradient of 2 to 52% acetonitrile in acidified water (TFA at 0.05%) in 90 minutes (i.e. an increase of 0.44% of acetonitrile per minute) for a flow rate 1 ml / min.

The resulting active fractions are then purified on a "High Pressure Inert" (HPI) column Delta Pak C18 (150 * 3.9 mm).

The elution is carried out in a linear biphasic gradient of acetonitrile from 2 to 11% in acidified water (6 mM HCl) in 10 minutes and from 11 to 21% in 50 min at a flow rate of 1 ml / min. The purity of the active fraction is controlled by capillary electrophoresis before determining the sequence by Edman degradation and analysis by mass spectrometry.

EXAMPLE 2 In Vitro Test: Measurement of the Antibacterial Activity by Microspectrophotometry

For each strain of bacteria used (E.coli; M.luteus), an isolated column is suspended in 10 ml of PB medium (Poor Broth, Luria Bertani medium devoid of yeast extract) DIFCO and incubated at 30 ° C. for one night 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. LOul of each fraction is 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, 50% inhibition is observed at the concentrations, expressed in μM, indicated in the following table:

Table 1

Using the same protocol but with phytopathogenic bacteria, the following results are obtained: Table 2

Using the same protocol but with phytopathogenic fungi, the following results are obtained:

Table 3

* medium supplemented with ImM CaC12 and 20 mM KG

Among the cultures which can be the subject of an antibacterial treatment using a compound according to the invention, mention may be made, for example, of rice, cereals, in particular wheat and barley, as well as arboreal, fruit and vegetable plants. Among the cultures which can be the subject of an antifungal treatment with the aid of a compound according to the invention, mention may be made, by way of examples, of cucurbits, floral crops (petunia), and vegetable crops. (carrots, tomatoes, cabbage).

These results show the excellent antibacterial activity of the peptide according to the invention, which can be applied to the human, animal and plant fields.

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

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

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

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

The surfactant can be an emulsifying, dispersing or wetting agent of ionic or nonionic type or a mixture of such surfactants. Mention may be made, for example, of salts of polyacrylic acids, salts of lignosulfonic acids, salts of phenolsulfonic or naphthalene sulfonic acids, polycondensates of ethylene oxide on fatty alcohols or on fatty acids or on fatty amines , substituted phenols (in particular alkylphenols or arylphenols), ester salts sulfosuccinic acids, taurine derivatives (in particular alkyltaurates), phosphoric esters of polyoxyethylated alcohols or phenols, esters of fatty acids and polyols, the derivatives containing sulfates, sulfonates and phosphates of the above compounds. The presence of at least one surfactant is generally essential when the compound and / or the inert support are not soluble in water and the vector agent for the application is water.

Thus, the compositions for agricultural use according to the invention can contain the active materials 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 fairly diverse forms, solid or liquid.

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

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

As forms of liquid compositions 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, powders wettable (or spray powder), pastes, 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 material.

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

From these concentrates, emulsions with 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 / 1

- alkaline dodecylbenzene sulfonate 24 g / 1

- oxyethylated nonylphenol containing 10 molecules of ethylene oxide 16 g / l

- cyclohexanone 200 g / 1

- aromatic solvent q.s.p.l liter

According to another formula of emulsifiable concentrate,

Example CE 2

- active ingredient 250 g

- epoxidized vegetable oil 25 g

- mixture of alkylaryl sulfonate and poly glycol ether and fatty alcohols 100 g

- diAndhylformamide 50 g

- xylene 575 g

The concentrated suspensions, also applicable in spraying, are prepared so as to obtain a stable fluid product which does not deposit and they usually contain from 10 to 75% of active material, from 0.5 to 15% of surfactants, from 0 , 1 to 10% of thixotropic agents, 0 to 10% of suitable additives, such as defoamers, corrosion inhibitors, stabilizers, penetrating agents and adhesives and, as support, water or an organic liquid in which the active ingredient is sparingly or not very soluble: certain organic solids or mineral salts can be dissolved in the support to help prevent sedimentation or as antifreeze for water.

As an 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 (defoamer) l g

- polysaccharide L5 g

- water 316.5 g

Wettable powders (or spray powder) are usually prepared so that they contain 20 to 95% of active ingredient, and they usually contain, in addition to the solid support, 0 to 30% of a wetting agent, 3 20% of a dispersing agent, and, when necessary, 0.1 to 10% of one or more stabilizers and / or other additives, such as penetrating agents, adhesives, or anti-caking agents, dyes, etc ...

To obtain the sprayable powders or wettable powders, the active ingredients are intimately mixed in the appropriate mixers with the additional substances and ground with mills or other suitable grinders. This gives sprayable powders whose wettability and suspension are advantageous; they can be suspended with water at any desired concentration and these suspensions can be used very advantageously in particular for application to the leaves of plants.

Instead of wettable powders, pasta can be made. The conditions and methods of making and using these pastes are similar to those of wettable powders or spraying powders.

As an example, here are various compositions of wettable powders (or spraying powders):

Example PM 1

- active ingredient 50%

- ethoxy lé fatty alcohol (wetting agent) 2,5%

- ethoxylated phenylethylphenol (dispersing agent) 5%

- chalk (inert support) 42.5%

Example PM 2:

- active ingredient 10%

- branched type oxo synthetic alcohol, Cl 3 ethoxylated with 8 to 10 ethylene oxide (wetting agent) 0.75%

- neutral calcium lignosulfonate (dispersing agent) 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 material 75%

- wetting agent 1.50%

- dispersing agent 8%

- calcium carbonate (inert filler) q.s.p. 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 and nonionic surfactants (wetting agent) 2.5%

- sodium lignosulfonate (dispersing agent) 5% - kaolin 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 can be of the water-in-oil or oil-in-water type and they can have a thick consistency like that of a "mayonnaise".

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

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

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

The rest of the granule is essentially composed of a solid filler and optionally surfactant additives giving the granule properties of dispersibility in water. These granules can be essentially of two distinct types depending on whether the selected filler is soluble or not in water. When the filler is water-soluble, it can be mineral or, preferably, organic. We got excellent results with urea. In the case of an insoluble filler, it is preferably mineral, such as for example kaolin or bentonite. It is then advantageously accompanied by surfactants (at a rate 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 a polynaphthalene alkaline or alkaline earth sulfonate or an alkaline or alkaline earth lignosulfonate, the remainder being nonionic or anionic wetting agents such as an alkali or alkaline earth alkyl naphthalene sulfonate.

Furthermore, although this is not essential, other adjuvants can be added such as anti-foaming agents.

The granule according to the invention can be prepared by mixing the necessary ingredients and then granulation according to several techniques known per se (bezel, fluid bed, atomizer, extrusion, etc.). It generally ends with a crushing followed by sieving to the particle size chosen within the limits mentioned above. Can also be used granules obtained as above and then impregnated with a composition containing the active material.

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% urea pearls are mixed. The mixture is then ground in a pin mill. A powder is obtained which is moistened with approximately 8% by weight of water. The wet powder is extruded in a perforated roller extruder. A granule is obtained which is dried, then crushed and sieved, so as to keep respectively only the granules of a size between 150 and 2000 microns.

Example QD2: Dispersible granules In a mixer, the following constituents are mixed: - active material 75%

- wetting agent (sodium alkylnaphthalene sulfonate) 2%

- dispersing agent (sodium polynaphthalene sulfonate) 8%

- inert filler 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 of size 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 materials, in particular 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 advantageously 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 administration of 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, of Andallic salts or of addition salts with a pharmaceutically acceptable base, in the pure state or in the form of a composition in which it is associated with any other pharmaceutically compatible product, which may be inert or physiologically active. The medicaments according to the invention can be administered by oral, parenteral, rectal or topical route.

As solid compositions for oral administration, tablets, pills, powders (especially in gelatin capsules or cachets) or granules can be used. 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 can also comprise other substances, for example one or more lubricants such as magnesium stearate or talc, a colorant, a coating (dragees) or a varnish.

As liquid compositions for oral administration, use may be made of 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 can also include other substances, for example wetting, sweetening, thickening, flavoring or stabilizing products.

The sterile compositions for parenteral administration may preferably be aqueous or non-aqueous 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 can also contain adjuvants, in particular wetting agents, isotonizers, emulsifiers, dispersants and stabilizers. Sterilization can be done in different ways, for example by aseptic filtration, by incorporating sterilizing agents into the composition, by irradiation or by heating. They can also be prepared in the form of 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 can 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 on the duration of the treatment; they are generally between 50 and 1000 mg per day orally for an adult in one or more drills. In general, the doctor will determine the dosage he considers most appropriate based on age, weight and all other factors specific to the subject to be treated.

The following examples are given without implied limitation illustrating the compositions according to the invention. Example A:

Tablets containing 50 mg of active peptide having the following composition are prepared according to the usual technique:

- androctonine peptide Ml 50 mg

- starch 60 mg - lactose 50 mg

- magnesium stearate 2 mg Example B:

A solution for injection containing 20 mg of active peptide having the following composition is prepared: - androctonine peptide M 2 22.4 mg

- distilled water q.s.p. 2 cπw

Claims

1. Peptide of formula:

2. Antibacterial composition, characterized in that it contains as active material a peptide according to claim 1.

3. Composition according to claim 2, usable for the protection of plants against pathogenic bacteria.

4. Composition according to claim 2, usable for the therapeutic treatment of the human or animal body.

5. Antifungal composition, characterized in that it contains as active material a peptide according to claim 1.

6. Composition according to claim 5, usable for the protection of plants against pathogenic bacteria.

7. Composition according to claim 5, usable for the therapeutic treatment of the human or animal body.

8. Method for the protection of plants against bacterial diseases, characterized in that a peptide according to claim 1 is applied as active material.

9. A method for protecting plants against fungal diseases, characterized in that a peptide according to claim 1 is applied as active material.

10. Process for preparing the peptide according to claim 1, characterized in that, successively: a) the hemolymph of the scorpion Androctonus australis is taken; b) the extraction is carried out by bringing hemolymph or a ground material of Androctonus australis obtained previously into contact with an acidic to neutral medium with stirring, then by centrifugation; c) the supernatant is fractionated with separation by washing of the hydrophilic molecules and elution of the hydrophobic molecules by suitable elements, on a separating column; d) the extracts are purified; e) sequencing is carried out.