WO2002000836A2

WO2002000836A2 - Spinigerin variants, an antibacterial and antifungal peptide derived from pseudacanthotermes spiniger, preparation method and compositions containing same

Info

Publication number: WO2002000836A2
Application number: PCT/FR2001/002100
Authority: WO
Inventors: Philippe Bulet; Jules Hoffmann; Mireille Lamberty
Original assignee: Centre National De La Recherche Scientifique -Cnrs-; Entomed
Priority date: 2000-06-29
Filing date: 2001-06-29
Publication date: 2002-01-03
Also published as: WO2002000836A3; AU2001270722A1; FR2810986A1; FR2810986B1

Abstract

The invention concerns peptides of formula: β1 ζ2 υ3 β4 β5 ζ6 σ7 υ8 β9 ζ10 ζ11 ζ12 ζ13 β14 υ15 ζ16 β17 ζ18 ζ19 β20 ζ21 ζ22 σ23 β24 ζ25 wherein: β is a basic amino acid, ζ is a hydrophobic amino acid, υ is a negatively charged or polar/large type amino acid, σ 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

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

The present invention relates to new peptides having antibacterial and antifungal properties. The invention also relates to the preparation of these peptides and the compositions containing them which can be used in agriculture and in human or animal therapy. The production of antimicrobial peptides represents, in a wide variety of animal and plant species, an essential immune defense mechanism 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 antimicrobial 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. To date, insect antimicrobial peptides have been mainly characterized from insects having a complete metamorphosis during development, for example Diptera, Lepidoptera and Coleoptera. Among the antibacterial peptides induced in these insects, the following four groups can be distinguished:

- Cationic peptides of 4 Da, forming two amphipathic α helices. In this group, cecropins are particularly classified. - Cationic peptides rich in proline, of size between 2 kDa and 4 kDa which may be glycosylated, such as for example, drosocin, pyrrhocoricin, and lebocins, or non-glycosylated such as, for example, apidaecins and metalnikins . - Several distinct polypeptides, having a molecular weight of 8 to 27 kDa, for the most part cationic and frequently rich in glycine residues such as for example attacines, sarcotoxins II, diptericins and coleoptericin.

- Peptides comprising intramolecular disulfide bridges. In this group, we classify insect defensins 4 kDa, 3 disulfide bridges), drosomycin (4 kDa, 4 disulfide bridges) and thanatin (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.

To Ala alanine

C Cysteine

D Asp aspartic acid

E Glu glutamic acid

F Phe phenylalanine

G Gly glycine

H His histidine

I isoleucine island

K lysine lysine

L Leu leucine

M Met methionine

N Asn asparagine

P Pro proline

Q Gin glutamine

R Arg arginine

S Ser Serine

T Thr threonine

V Val valine

W Trp tryptophan

Y Tyr tyrosine Little work has been undertaken to date on insects with incomplete metamorphosis, in particular polyneoptera. Now it has precisely been isolated, from an insect with incomplete metamorphosis, the termite Pseudacantho terms spiniger, a peptide, which has remarkable characteristics as well as antibacterial and antifungal properties.

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

H V D K K V A D K V L L L K Q L R I M R L L T R L

This molecule, of reduced size, does not contain cysteine residues. The Applicant has studied the spatial conformation of the molecule and the mutations capable of being made on its sequence so as to increase its bactericidal and / or fungicidal properties. The subject of the present invention is therefore a peptide of sequence (I) as follows: βi φ ₂ θ ₃ β ₄ βδ φe σ ₇ θ ₈ βθ φio φn Φ12 Φ13 β 14 Θ15 φie βi7 φis Φ19 β 20 Φ21 Φ22 σ23 β 24

or a fragment thereof, in which:

- β is a basic amino acid,

- φ is a hydrophobic amino acid,

- θ is a negatively charged or polar / broad amino acid, σ is an amino acid chosen from glycine, serine, lanine or threonine,

The peptides of formulas (I) advantageously exhibit an alpha helical conformation. Thus, β 'θ and σ 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 the said sequence. By way of example of such fragments, the invention more particularly contemplates a peptide of formula (I) above, including a group of 1 to 4 amino acids at one and / or the other of the N and / ends or C terminales is deleted.

Such peptides are those in which one or more of the amino acids β1, φ2, Θ3, φ22, σ23, β24, or φ25 are absent. The invention more particularly contemplates a peptide of formula above in which the amino acids β1, φ2, Θ3 and / or φ22, σ23, β24, φ25 are absent, and therefore corresponding to the following formulas: β ₄ βδ φθ σ θs β9 φlO Φl1 φl2 Φl3 β 14 Θ15 φlδ βl7 Φl8 φl9 β 20 21 Φ22 3LZ β 24 φ25 (II) βl φ ₂ Θ ₃ β ₄ β5 6σ7θ8β9φl0φl1 φl2 Φl3 β 14 Θ15 l6 βl7 Φl8 φl9 β 20 φ21 (III) β β ₅ φδ σ ₇ θ ₈ βθ φlO Φl1 Φl2 Φl3 β 14 Θ15 φl6 βl7 Φl8 Φl9 β 20 φ21 (IV)

The following are understood more particularly by: basic amino acid, those chosen from arginine, lysine or histidine, - hydrophobic amino acid, those chosen from valine, leucine, isoleucine, methionine, phenylalanine, tyrosine , or tryptophan,

- negatively charged or polar / broad amino acid, those chosen from aspartic acid, glutamic acid, asparagine, or glutamine.

A preferred example of a peptide according to the invention has 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: KVADKVLLLKQLRIMRLLTRL (SEQ ID NO: 2)

HVDKKVADKVLLLKQLRIMRL (SEQ ID NO: 3) KKVADKVLLLKQLRIMRL (SEQ ID NO: 4) The peptides of the invention can be prepared by chemical synthesis or by genetic engineering.

The invention also relates to functional equivalents of the above peptides, the amino acid sequences of which include deletions, additions and / or conservative modifications at the level of one or more amino acids, since these deletions, additions and / or modification do not modify the antimicrobial activity of the peptide from which they originate, and for example not the alpha helix structure. Mention may in particular be made of the modifications resulting from the post-translational processes such as glycosylation or the degeneration of the genetic code, or else chemical modifications such as amidation, acetylation, acylation, coupling with lipids or sugars, coupling with nucleotides, etc.

The functional equivalents also include peptides of the invention in which one or more of the amino acids of the amino acids are enantiomers, diastereoisomers, natural amino acids of D conformation, rare amino acids including hydroxyproline, methyllysine, dimethyllysine and synthetic amino acids, including ornithine, norleucine, cyclohexylalanine and omega-a inoacids. The invention also covers retropeptides and retro-inversopeptides.

Another subject of the invention is the use of the above peptides or analogues thereof, for preventing or treating a fungal or bacterial infection both in humans and animals and in plants. The subject of the invention is therefore a composition, more particularly antibacterial or antifungal, comprising as active agent at least one peptide as defined above, advantageously combined 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 relates in particular to pharmaceutical applications in humans and animals of these peptides and of compositions containing them, but it is also interested in agronomic applications. In fact, the peptides of the invention are useful for making plants resistant against diseases, in particular fungal and bacterial. A first form of implementation of this agronomic application consists in applying to plants an effective amount of peptide or of a composition containing them. A second form of implementation of this agronomic application consists in transforming plant cells or plants with a nucleic acid sequence capable of expressing the peptide of the invention so as to confer on plants resistance to diseases.

Other advantages and characteristics of the invention will appear from the following examples concerning 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 of the experiments were carried out with adultesess w ithout special interest. Pseudacantho terme s spiniger (Order of isoptera, family of Termitidae, subfamily of Macrotermitinae). These insects come from a breeding carried out at the Joint Research Unit of the CNRS "development, Chemical communication", Dijon.

2) Extraction of the peptides.

The extraction of antimicrobial compounds was carried out from whole insects which were frozen in liquid nitrogen. The termites (200) were 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 μg / ml final concentration) as a protease inhibitor and phenylthiouree (20 μM final concentration) as melanization inhibitor. The extraction was carried out in an ice-water bath with gentle agitation for 30 minutes. After centrifugation (14,000 xg for 30 min at 6 ° C), the clarified supernatant was directly deposited on a Sep-Pak C ₁₈ 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% of acetonitrile in water containing 0 .05% TFA. The fraction eluted with the 40% acetonitrile solution was lyophilized before passing through reverse phase HPLC.

3) Purification of the peptides by HPLC. - Step 1: The Sep-Pak 40% fraction was subjected to reverse phase chromatography on an Aquapore RP-300 C ₁₈ semi-preparative column (250 x 7 mm, Brownlee ™). Balanced 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 in 120 min with a flow rate of 1.3 ml / min. At the end of this step, several fractions shown in FIG. 1 are obtained which exhibit antimicrobial activity on the following germs: E. coli 363, Neurospora crassa, Micrococccus luteus. The active compounds of the FI and F2 fractions were then characterized.

- Step 2: The second purification step was carried out on an Aquapore OD-300 analytical column (220 x 4.6 mm, Bro nlee ™). The 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 narrow bore C ₁₈ column in reverse phase (Delta Pak HPIC ₁₈ , 2x 150 mm, Waters ™). The column was equilibrated in acidified water and developed with the same linear gradient of acetonitrile as that 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 purification steps under controlled temperature, a Garlic PEEK Waters HPLC system (Waters pump model 626) attached to a variable absorbance detector (Waters 486) was used. During the HPLC purification steps, the molecules eluted from 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 (Millipore ™) before analysis of the antimicrobial activity.

4) Analysis by mass spectrometry. It is carried out using the so-called time-of-flight 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 fraction

FI

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. 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 2649.4 and 3000.8 Da respectively. They are in agreement 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 molecule of molecular mass 2649.5 Da corresponds to that of 3000.7 Da devoid of the three residues of the amino-terminal end of the peptide. The sequencing of a neighboring fraction (Fraction F2) containing a molecule of molecular mass 2516.2 Da made it possible to determine a third form of the peptide corresponding to the molecule of molecular mass 3000.7 Da devoid of the four carboxy-terminal amino acids. All of these results are reported in Table 1 below. Table 1

Example 2: synthesis of synthetic spinigerin.

The peptides according to the invention can also be obtained according to a FMOC chemical synthesis process (Atherton and Sheppard RC (1989), Solid Phase Peptide Synthesis (IRL, Oxford, UK). The chemical synthesis was carried out for spinigerin (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 carried out with the synthetic molecule. This molecule has antibacterial properties against Gram negative and Gram positive bacteria, phytopathogenic bacteria and phytopathogenic fungi.

Exempl e detec ti on of antimicrobial ac t ivi ties.

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

1) Detection of antibacterial activity.

For each strain of bacteria used

(Gram positive bacteria and Gram negative bacteria), an isolated colony is suspended in 10 ml of PB medium (Poor Broth, Luria Bertani medium devoid of yeast extract) DIFCO and incubated at 30 ° C. overnight slow agitation. The suspensions of bacteria to be tested are adjusted to an optical density at 600 nm of 0.001 in a fresh culture medium. 10 μl of each fraction are deposited in microtitration 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.

The bacteria are as follows: - Gram positive: B a c i 1 l u s megateriυm,

Mi crococcus l u teus, Staphyl ococcus aureus, Streptococcus pyogenes.

Gram negative: Escheri chi a coli D22, Es cheri ch i a c o l i SBS 363, Kl ebsi el l a pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa.

Table 2 below reports the activity of spinigerin against bacteria. Table 2

* ND: activity not detected up to a concentration of 100 μM.

2) Activity detection test against filamentous fungi and against the Candi da albicans yeast.

~ The antifungal activity was detected by a growth inhibition test in liquid medium. The spores of the fungi to be tested were suspended in a “Potato-Glucose” type culture medium. Preferably, 12 g of Potato Dextrose Broth medium (Difco) are used per 1 1 of demineralized 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 analyzed are deposited in microtitration plates in the presence of 90 μl of culture medium containing the spores (at a final concentration of 10 ⁴ 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 culture medium of the "Sabouraud" type and incubated at 30 ° C for 24 h with slow shaking. The test sample (10 μl) was placed in microtiter plate wells to which 90 μl of a diluted yeast culture have been added, the density of which has been adjusted to OD 600 = 0.001. Growth is evaluated by measuring the absorbance at 600 nm using a microtiter plate reader spectrophotometer.

The filamentous fungi and yeast that have been used are:

Aspergillus fumiga tus (gift from Dr H. Koenig, Hôpital civil, Strasbourg); Nectria haema tococca, Fusari um culmorum, Tri choderma viride (mycotheque of the Catholic University of Leuven, Belgium); Neurospora crassa, Fusarium oxysporum, (mycotheque of the Société Clause, Paris); Candida albicans (donation from Dr H. Koenig, Hôpital civil, Strasbourg).

Table 3 below reports the activity of spinigerin against fungi.

Table 3

Claims

1) Peptide of sequence (I): βl φ ₂ 03 β4 βδ Φδ σ ₇ θ ₈ βg φlO Φl1 Φl2 Φl3 β 14 015 Φl6 βl7 Φl8 Φl9 β 20

or a fragment thereof, in which: - the amino acids βi, β ₄ , βs, βθ, βι, βi7, β20 and β ₂ 4 are independently chosen from basic amino acids, - amino acids φ ₂ , φβ , φio φi 1, φi2 φl3

Φ16, φi8 φi 9, Φ21 / Φ22 and Φ25 are independently chosen from hydrophobic amino acids,

- amino acids Θ3, 08 and Θ ₁₅ are independently chosen from amino acids negatively charged or of polar / broad nature, amino acids σ and σ ₂₃ are independently chosen from glycine, serine, alanine or threonine, optionally at least one of the amino acids βi, φ ₂ , Θ3, φ ₂ 2 (? 23 / β24, or Φ25 is absent.

/

2) 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 of the N and / or C terminal ends is deleted.

4) Peptide according to any one of claims 1 to 3, characterized in that it corresponds to one of the following formulas: β ₄ β ₅ φδ σ ₇ 08 βθ ΦlO Φl1 Φl2 Φl3 βl4 θ- | ₅ φl ₆ βl7 Φl8 Φl9 β20 φ21 Φ22 σ ₂ 3 β24 φ25 (II) βl φ ₂ θ ₃ β ₄ βδ Φθ σ ₇ θ ₈ βg φlO Φ 1 Φl2 Φl3 βl4015 Φl6 βl7 Φl8 Φl9 β20 φ21 (III) β β ₅ φe σ ₇ θs βg Φ10 φ β β β β β β β β β β β β β 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, 1 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 polar / broad in nature are chosen from aspartic acid, glutamic acid, 1 asparagine, or glutamine.

8) Peptide according to any one of the preceding claims, characterized in that it consists of one of the following sequences: H V D K K V A D K V L L L K Q L R I M R L L T R L (SEQ ID NO: 1)

K K V A D K V L L L K Q L R I M R L L T R L (SEQ ID NO:

2)

H V D K K V A D K V 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)

9) 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 combined in said composition with an acceptable vehicle. 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.