WO2012085177A1

WO2012085177A1 - Nemaucin, an antibiotic produced by entomopathogenic xenorhabdus cabanillasii

Info

Publication number: WO2012085177A1
Application number: PCT/EP2011/073738
Authority: WO
Inventors: Maxime Gualtieri; Philippe Villain-Guillot; Alain Givaudan; Sylvie Pages
Original assignee: Nosopharm; Institut National De La Recherche Agronomique
Priority date: 2010-12-22
Filing date: 2011-12-22
Publication date: 2012-06-28

Abstract

Antibiotic compounds purified from Xenorhabdus cabanillasii strain CNCM I-4418 and methods for producing said compounds and pharmaceutical compositions comprising said compounds and a pharmaceutically acceptable carrier for use in the treatment of fungal disease. Xenorhabdus cabanillasii strain CNCM I-4418.

Description

NEMAUCIN, AN ANTIBIOTIC PRODUCED BY

ENTOMOPATHOGENIC XENORHABDUS CABANILLASII

The present invention relates to new antibiotic compounds, to a strain of Xenorhabdus cabanillasii producing said antibiotic compounds and to the use of these compounds in the treatment of microbial disease in humans and animals.

Antimicrobial resistance is a major public health problem with a significant impact on morbidity, mortality and healthcare-associated costs. The problem has been worsened by the restriction of antibiotic drug discovery and development programs. Nowadays, the most relevant multiresistant bacterial pathogens are methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended spectrum β-lactamase formers (ESBL), multiresistant Pseudomonas and Acinetobacter species. For these bacteria, only a few of the existing antibiotics are efficient. There is an urgent need for new antibacterial compounds to ensure that bacterial infections can be effectively treated in the future. Living organisms have proven to be the most reliable source of bioactive chemicals with antimicrobial activity. Environmental microbes continue to be outstanding resources for the identification of new molecules. In this context, the discovery of new groups of antimicrobial peptides makes natural antibiotics the basic element of a novel generation of drugs for the treatment of bacterial and fungal infections. Antimicrobial peptides comprise a diverse class of molecules used in host defense by bacteria, plants, insects, and animals. Two examples of polypeptide antibiotics are polymyxin B and gramicidin S produced respectively by Bacillus polymyxa and Bacillus brevis.

The present application is based on research on an underexploited bacterial genus: Xenorhabdus. Gram-negative bacterial strains of the genus Xenorhabdus are known to be symbiotically associated with soil dwelling nematodes of the Steinernematidae family. After entering the insect larvae via natural openings, nematodes release bacteria from their intestine to the host's hemocoel. Bacteria are involved in killing the insect host by producing insecticidal proteins and inhibitors of the insect immune system. The bacteria proliferate in the corpse of the host and favour the reproduction of the nematode by degrading the insect biomass and by producing antibiotics that inhibit the development of the other microorganisms present in the corpse of the insect (bacteria, fungi). Boemare et al. (Boemare N.E. et al, Appl. Environ. Microbiol. 58, 3032-3037 (1992)) classified the antibiotic activities of Xenorhabdus into two categories: antimicrobial molecules with broad spectrum of activity, and bacteriocins with very narrow spectrum of activity and active only against bacteria closely related to X. nematophila. Only a few families of compounds have been described from Xenorhabdus in the literature: xenocoumacins (Gregson R.P. et al, WO 86/01509; Mclnerney B.V. et al, J. Nat. Prod. 54, 774-84 (1991)), xenorhabdins (Rhodes S.H. et al,WO 84/01775; Mclnerney B.V. et al, J. Nat. Prod. 54, 785-95 (1991)), indole derivatives (Webster J.M. et al, US patent 5,569,668; Li J. Et al, J. Nat. Prod. 58, 1081-6 (1995); Li J. et al, Can. J. Microbiol. 43, 770-3 (1997)), puromycin (Webster J.M. et al, Entomopathogenic hematology. 99-114 (2002)), benzylidenacetone (Ji D. et al, FEMS Microbiol. Lett. 239, 241-8 (2004)), proteinaceous bacteriocins (Boemare N.E. et al, Appl. Environ. Microbiol. 58, 3032-7 (1992); Thaler J.O. et al, Appl. Environ. Microbiol. 61, 2049-52 (1995)), xenematide (Lang G. et al, J. Nat. Prod. 71 (6), 1074-1077 (2008) and PAX (Gualtieri M. et al, J.Antibiot. 62(6) 295-302 (2009)).

In the present application, we describe new antimicrobial compounds, Nemaucins, produced by fermentation of a Xenorhabdus cabanillasii strain, and purified by cation exchange chromatography and reversed phase chromatography. The chemical structure of Nemaucins was obtained from the analysis of homo and heteronuclear 2D NMR and MS- MS experiments.

These molecules showed strong activity against Gram-positive and Gram-negative human bacterial pathogens, including some multi-resistant strains.

Summary

A first object of the present invention is a compound of formula (I):

wherein

Xaai is aspartic acid or glutamic acid,

Xaa₂ is arginine, histidine or lysine,

Xaa₃ is serine, threonine, asparagine or glutamine,

Xaa₄ is selected in the group consisting of

R is H, OH or CH2-OH,

n is 1, 2, 3, 4 or 5.

Preferably, Xaai is aspartic acid.

Preferably, Xaa₂ is histidine.

Preferably, Xaa₃ is asparagine.

Preferably, Xaa₄ is

Preferably, R is OH.

Preferably, n=4.

In preferred embodiments the compound of the present invention is the compound of formula (II):

(Π)

wherein n is 1, 2, 3, 4 or 5.

Preferably, n=4.

Preferably, the compounds of the present invention are for use as a medicament. Preferably, the compounds of the present invention are for use as an antibiotic agent.

Preferably, the compounds of the present invention are for use in treatment of microbial disease. Preferably, the compounds of the present invention are for use for treatment of a microbial disease caused by a microorganism selected from Staphylococcus, Enterococcus, Streptococcus, Bacillus, Clostridium, Chlamydia, Mycobacterium, Pasteurella, Escherichia, Enterobacter, Klebsiella, Citrobacter, Proteus, Serratia, Pseudomonas, Acinetobacter, Shigella, Salmonella, Stenotrophomonas, Yersinia, Burkholderia, Morganella, Moraxella, Propionibacterium, Listeria, Corynebacterium, Bifidobacterium, Lactobacillus, and Brucella.

More preferably, the compound of the present invention is for treatment of a microbial disease caused by a microorganism selected from Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Enterobacter clocae, Klebsiella pneumoniae, Klebsellia oxytica, Klebsiella singaponasis, Citrobacter koseri, Citrobacter freundii, Proteus penneri, Serratia marescens and Pseudomonas aeruginosa.

Another object of the present invention is a composition comprising a compound of the present invention in association with another antibiotic compound.

The present invention is also directed to a pharmaceutical composition comprising:

An effective amount of a compound of formula (I)

wherein

Xaai is aspartic acid or glutamic acid,

Xaa₂ is arginine, histidine or lysine,

Xaa₃ is serine, threonine, asparagine or glutamine,

Xaa₄ is selected in the group consisting of

R is H, OH or -CH2-OH,

n is 1, 2, 3, 4 or 5,

A pharmaceutically acceptable

Preferably, Xaai is aspartic acid.

Preferably, Xaa₂ is histidine.

Preferably, Xaa₃ is asparagine.

Preferably, Xaa₄ is

Preferably, R is OH

Preferably, n=4.

(Π)

wherein n is 1, 2, 3, 4 or 5. Preferably, n=4.

A further object of the present invention is a method for producing a compound according to formula (II) comprising the following steps:

Growing Xenorhabdus cabanillasii strain CNCM 1-4418 in a liquid culture medium, - Purifying a compound according to formula (II)

wherein n=4. In preferred embodiments, the compound of formula (II) wherein n=4, is purified from the culture supernatant after removal of the Xenorhabdus cabanillasii cells.

The present invention is also directed to the Xenorhabdus cabanillasii strain deposited at CNCM on 15-12-2010 having the accession number CNCM 1-4418.

Another object of the present invention is a culture supernatant from the

Xenorhabdus cabanillasii strain of the present invention having antibiotic activity.

A further object of the present invention is an extract from the Xenorhabdus cabanillasii strain of the present invention having antibiotic activity. Description of the invention

In a first aspect, the present invention is related to Xenorhabdus cabanillasii strain JM26 deposited at CNCM (Collection Nationale de Cultures de Microorganismes) in the name of INRA (Institut National de la Recherche Agronomique) on 15-12-2010 having the accession number CNCM 1-4418.

It has been found that Xenorhabdus cabanillasii strain CNCM 1-4418 produces a compound exhibiting antibiotic or antimicrobial activity.

When Xenorhabdus cabanillasii strain CNCM 1-4418 is grown in a liquid culture medium, the antibiotic compound of the present invention is secreted into the culture supernatant. In another aspect, the present invention is related to a culture supernatant from Xenorhabdus cabanillasii strain CNCM 1-4418 exhibiting antibiotic activity. For the preparation of a culture supernatant having antibiotic activity Xenorhabdus cabanillasii strain CNCM 1-4418 is grown in a liquid culture medium under standard conditions, the bacterial cells are removed and the supernatant is recovered. The bacterial cells may for example be removed by centrifugation or filtration.

In another aspect, the invention is related to extracts from Xenorhabdus cabanillasii strain CNCM 1-4418 showing antibiotic activity. Cell extracts from Xenorhabdus cabanillasii may be prepared according to any appropriate method known to the skilled person.

The present invention also encompasses culture supernatant and extracts from

Xenorhabdus cabanillasii strain CNCM 1-4418 for use as medicament.

The present invention also encompasses culture supernatant and extracts from Xenorhabdus cabanillasii strain CNCM 1-4418 for use as an antibiotic agent.

The terms "antibiotic", "antibiotic activity" and "antimicrobial activity" as used herein refer generally to an effect in which a reduction, inhibition or a halt in the growth of a microorganism is achieved. Antibiotic activity may be tested according to any known method such as a microdilution method. The substances and compounds of the present invention inhibit or kill microorganism and more particularly kill or inhibit bacteria. Interestingly, the substances and compounds of the present invention inhibit or kill multidrug resistant bacteria which exhibit resistance to known antibiotics.

Xenorhabdus cabanillasii strain CNCM 1-4418, culture supernatant from this strain and cell extracts derived from this strain exhibit antibiotic activity against different microorganism including human bacterial pathogens.

More particularly, Xenorhabdus cabanillasii strain CNCM 1-4418, culture supernatant from this strain and cell extracts derived from this strain exhibit antibiotic activity against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Enterobacter clocae, Klebsiella pneumoniae, Klebsellia oxytica, Klebsiella singaponasis, Citrobacter koseri, Citrobacter freundii, Proteus penneri, Serratia marescens and Pseudomonas aeruginosa.

Compounds having antibiotic activity as described above have been purified from the culture supernatant of Xenorhabdus cabanillasii strain CNCM 1-4418.

In another aspect, the present invention is related to a compound of formula (I):

(I) wherein

Xaai is aspartic acid or glutamic acid,

Xaa₂ is arginine, histidine or lysine,

Xaa₃ is serine, threonine, asparagine or glutamine,

Xaa₄ is selected in the group consisting of

R is H, OH or CH2- n is 1, 2, 3, 4 or 5, a salt, a hydrate or a solvate thereof. These compounds have been named "Nemaucins". The term "Nemaucins" refers to the compounds of formula I, a salt, a hydrate or a solvate thereof.

Preferably, Xaai is aspartic acid.

Preferably, Xaa₂ is histidine.

Preferably, Xaa₃ is asparagine.

Preferably, Xaa₄ is

Preferably, R is OH

Preferably, n=4.

(Π)

wherein n is 1, 2, 3, 4 or 5,

a salt, a hydrate or a solvate thereof.

Preferably, n=4. This compound has been named "Nemaucin".

The term "Nemaucin" refers to the compound of formula II wherein n=4, a salt, a hydrate or a solvate thereof.

In preferred embodiments, the antibiotic compounds according to the present invention are for use as a medicament, for use as an antibiotic agent, for use as an antimicrobial agent, or for use in the treatment of microbial disease in particular of bacterial infection caused by pathogenic bacteria.

The compounds of the present invention are preferably for use in the treatment of bacterial infection and in particular in the treatment of hospital-acquired infections or nosocomial bacterial infections.

The invention is also related to the use of compounds of formula (I) for the manufacture of a medicament for treatment of microbial infection or microbial disease. The invention is also related to the use of compounds of formula (I) for the manufacture of an antibiotic.

The present invention also provides methods for treating microbial/bacterial infection and/or microbial/bacterial disease including administering an effective amount of a compound of formula (I) to a human or to a patient in need thereof. In a preferred embodiment, the invention relates to methods for treatment of bacterial disease and or bacterial infection.

In one embodiment, the patient is a mammal, i.e., a living mammal. In one embodiment, the patient is a human, i.e., a living human, including a living human child and a living human adult.

The term "treatment," as used herein in the context of treating a condition, refers generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with subjects who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term "treatment."

The term "therapeutically-effective amount," as used herein, refers to the amounts of the active agents (compounds of formula (I) or a salt, hydrate, or solvate thereof) that is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

The term "treatment" includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, the active agents of the present invention may also be used in further combination therapies, e.g., in conjunction with other agents, for example, other antimicrobial or antibiotic agents, etc.

Another aspect of the present invention is a pharmaceutical composition comprising:

- an effective amount of a compound of formula (I)

(I) wherein

Xaai is aspartic acid or glutamic acid,

Xaa₂ is arginine, histidine or lysine,

Xaa₃ is serine, threonine, asparagine or glutamine,

Xaa₄ is selected in the group consisting of

R is H, OH or CH2-OH,

n is 1, 2, 3, 4 or 5,

and

a pharmaceutically acceptable carrier.

Preferably, Xaai is aspartic acid.

Preferably, Xaa₂ is histidine.

Preferably, Xaa₃ is asparagine.

Preferably, Xaa₄ is

Preferably, R is OH

Preferably, n=4. In preferred embodiments the compound is the compound of formula (II):

wherein n is 1, 2, 3, 4 or 5,

a salt, a hydrate or a solvate thereof. Preferably, n=4.

As used herein, "pharmaceutically acceptable carriers" includes any and all solvents, dispersion media, coatings, and the like that are physiologically compatible.

The compositions of the invention may be in a variety of forms. These include for example liquid, semi-solid, and solid dosage forms, but the preferred form depends on the intended mode of administration and therapeutic application.

These pharmaceutical compositions are preferably for oral, topical, percutaneous or parenteral administration.

The compositions as described herein may be orally administered.

As solid compositions for oral administration, tablets, pills, powders (gelatine capsules, sachets) or granules may be used. In these compositions, the active ingredient according to the invention is mixed with one or more inert diluents, such as starch, cellulose, sucrose, lactose or silica, under an argon stream. These compositions may also comprise substances other than diluents, for example one or more lubricants such as magnesium stearate or talc, a coloring, a coating (sugar-coated tablet) or a glaze. Preferred oral compositions include coated and uncoated tablets, hard and soft gelatin capsules, sugar-coated pills, lozenges, pellets, and powders.

As liquid compositions for oral administration, there may be used pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil. These compositions may comprise substances other than diluents, for example wetting, sweetening, thickening, flavouring or stabilizing products.

In preferred embodiments, the pharmaceutical compositions for oral use contain Nemaucin together with the usual excipients as diluting agents like mannitol, lactose and sorbitol; binding agents like starchs, gelatins, sugars, cellulose derivatives, natural gums and polyvinylpyrrolidone; lubricating agents like talc, stearates, hydrogenated vegetable oils, polyethylenglycol and colloidal silicon dioxide; disintegrating agents like starcks, celluloses, alginates, gums and reticulated polymers; coloring, flavoring and sweetening agents.

In other embodiments, the compositions comprise Nemaucin according to the present invention with carriers or excipients suitable for topical administration. Preferred compositions for topical administration of Nemaucin according to the invention include ointments, pomades, creams, gels, and lotions.

The pharmaceutical compositions for topical use contain Nemaucin together with the usual excipients like white petrolatum, white wax, lanolin and derivatives thereof, stearylic alcohol, propylenglycol, sodium lauryl sulfate, ethers of the fatty polyoxy ethylene alcohols, sorbitan monostearate, glyceryl monostearate, propylene glycol monostearate, polyetilene glycols, methylcellulose, hydroxymethylpropylcellulose, sodium carboxymethylcellulose, colloidal aluminium and magnesium silicate, sodium alginate. Any topical preparation may be used in the present invention, for instance ointments, pomades, creams, gels and lotions.

The doses of Nemaucin depend on the desired effect, the duration of the treatment and the route of administration used.

In preferred embodiments, the pharmaceutical compositions according to the present invention are for use as an antimicrobial agent, for use as antibiotic or for use in the treatment of microbial disease in particular of microbial disease caused by bacteria.

The pharmaceutical compositions of the present invention are preferably for use in the treatment of bacterial infection and in particular for use in the treatment of hospital- acquired infections or nosocomial bacterial infections.

Preferably, the compound of the present invention is for use for treatment of a microbial disease caused by a microorganism selected from Staphylococcus, Enter ococcus, Streptococcus, Bacillus, Clostridium, Chlamydia, Mycobacterium, Pasteurella, Escherichia, Enter obacter, Klebsiella, Citrobacter, Proteus, Serratia, Pseudomonas, Acinetobacter, Shigella, Salmonella, Stenotrophomonas, Yersinia, Burkholderia, Morganella, Moraxella, Propionibacterium, Listeria, Corynebacterium, Bifidobacterium, Lactobacillus, and Brucella.

More preferably, the compound of the present invention is for treatment of a microbial disease caused by a microorganism selected from Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Enterobacter clocae, Klebsiella pneumoniae, Klebsellia oxytica, Klebsiella singaponasis, Citrobacter koseri, Citrobacter freundii, Proteus penneri, Serratia marescens and Pseudomonas aeruginosa. The compounds of the present invention may be combined with other active compounds exhibiting an antimicrobial/antibiotic activity. The pharmaceutical compositions encompassed by the present invention may also contain a further therapeutic agent for the treatment of bacterial disease or bacterial infection.

Another aspect of the present invention is a method for producing a compound according to formula (II) comprising the following steps:

Growing Xenorhabdus cabanillasii strain CNCM 1-4418 in a liquid culture medium, - Purifying a compound according to formula (II):

wherein n=4.

The active compounds according to the present invention may be purified from the Xenorhabdus cabanillasii cells of the present invention. Advantageously, the active compounds of the present invention may be purified from the culture supernatant after removal of the Xenorhabdus cabanillasii cells. For the preparation of a culture supernatant having antifungal activity Xenorhabdus cabanillasii strain CNCM 1-4418 is grown in a liquid culture medium under standard conditions, the bacterial cells are removed and the supernatant is recovered. The bacterial cells may for example be removed by centrifugation or filtration.

Further purification of the active compounds according to the present invention may be carried out by any known method including cation-exchange chromatography, reversed-phase chromatography and/or reverse phase HPLC.

In preferred embodiments, the active compound is purified from the culture supernatant of Xenorhabdus cabanillasii strain CNCM 1-4418 by successive cation- exchange chromatography, reversed-phase chromatography and reverse phase HPLC.

Figures

Figure 1 : HPLC chromatogram of the Xenorhabdus cabanillasii growth medium

Figure 2: Bactericidal effects of Nemaucin on growing S. aureus CIP 76.24

Figure 3 : Viability of hTERT HME-1 at 8 concentrations of Nemaucin

Figure 4: Viability of PC-3 at 8 concentrations of Nemaucin Figure 5 : ESI-MS experiments

Figure 6: UV absorption spectra of Nemaucin

Figure 7: Peptidic part of Nemaucin

Figure 8: MSMS fragmentation of Nemaucin

Figure 9: Chemical structure of Nemaucin

EXAMPLES

A. Materials and methods

Producing organism

Xenorhabdus cabanillasi CMCM 1-4418 was grown on Luria-Bertani medium (LB, composed of bactotryptone 10 g/L, yeast extract 5 g/L and NaCl 10 g/L) for liquid culture and on LB-agar for solid cultures. The phase status (I or II) of this strain was determined by culturing on NBTA (Nutrient agar (Difco) 31 g/L, bromothymol blue 25 mg/L and 2, 3, 5-triphenyl tetrazolium chloride 1% 40 mg/L) and measuring antibacterial activity against Micrococcus luteus. Xenorhabdus exhibit two colony forms or variants when cultured in vitro. Modifications of the outer membrane induce differential adsorption of dyes by variants. Phase I variants absorb dyes and are blue on NBTA plates, while phase II colonies are red. Phases I and II of strains are indicated as suffixes {l\ and 12, respectively) attached to strain designations. This strain was maintained at 15°C on NBTA medium.

Antibacterial susceptibility testing methods

The following reference strains were used for evaluation of antimicrobial activity: Pseudomonas aeruginosa CIP 76.110, Escherichia coli CIP 76.24, Staphylococcus aureus CIP 76.25, and clinical isolates obtained from patients with infection at the University Hospital of ines. Vancomycin and polymyxin (Sigma-Aldrich) were provided as standard powders by the manufacturers. MIC and MBC determination procedures were determined as recommended by the CLSI (National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically— Sixth Edition: Approved Standard M07-A6. NCCLS, Villanova, PA, USA. (2003)).

Antibiotics were tested at final concentrations (prepared from serial twofold dilutions) ranging from 0.01 to 100 μg/mL. The minimal inhibitory concentration (MIC) was defined as the lowest antibiotic concentration which yielded no visible growth. The test medium was Mueller-Hinton broth (MHB), the inoculum was 5xl0⁵ CFU/mL. The inoculated microplates were incubated at 37°C for 18 h before reading. Minimal bactericidal concentrations (MBCs) were established by extending the MIC procedure to the evaluation of bactericidal activity. After 24 hours, 10 μΙ_, were drawn from the wells, serially diluted and then spotted onto suitable agar plates. The plates were incubated at 37°C overnight. The MBC was read 18 h later as the lowest concentration of antibiotic which resulted in 0.1 % survival in the subculture. All the experiments were done in triplicates.

Protein binding.

The impact of serum protein binding was assessed by examining the impact of human serum albumin (HSA) on the activity of Nemaucin in vitro. The MICs for S. aureus CIP 76.25 and E. coli CIP 76.24 were determined in MHB + 4% HSA. Concentrations of HSA used for in vitro studies were selected on the basis of normal physiological concentration. A reduced potency (higher MIC) in MHB + HSA was presumed due to drug binding to serum protein.

Cytotoxicity test

100 μΙ_, of cell suspension of PC-3 (Human prostatic carcinoma) or hTERT HME-1 (Human normal mammary epithelium) prepared in RPMI + 10% SVF + 1% glutamine were inoculated into 96-well plates. The inoculating cell number was 3500 cells per well for PC-3 and 7500 cells per well for hTERT HME-1. The microplates were incubated at 37°C for 24h with 5% of C0₂. After 24h of culture, the medium was removed by aspiration and 100 μΐ of RPMI + 10% SVF + 1% glutamine with 8 concentrations of Nemaucin (from 0.78 μg/ml to 100 μg/mL) were added to each well. Microplates were incubated at 37°C for 48h with 5% of C0₂. After 48h of culture, the medium was removed by aspiration and 100 μΙ_, of RPMI + MTT (0.5 mg/mL) were added to each wells. Microplates were inoculated at 37°C during 150 min. Next, all the medium was removed by inverting and tapping the plate and 100 μΙ_, of DMSO were added in each plate. The spectrophotometric absorbance was then measured at 550 nm.

The Lethal Dose 50 corresponds to DL50 = ((50-(Y2-((Yl-Y2)/(Xl- X2))*X2))/((Y1-Y2)/(X1-X2))). XI represents the concentration for which viability was superior to 50%, X2 represents the concentration for which viability was inferior to 50%, Yl represents the percentage of viability at the concentration XI and Y2 the percentage of viability at the concentration X2.

Experiments were done in triplicate.

Bactericidal effects of Nemaucin on growing S. aureus

Bacterial killing curves were carried out by inoculating S. aureus CIP 76.24 with Nemaucin concentrations equal to the MIC, two-fold the MIC and four-fold the MIC. Vancomycin was used at four-fold its MIC. The S. aureus inoculums was prepared from colonies grown overnight in MHB. Antibiotics concentrations in the flask were adjusted in MHB according to the desired concentration. Culture tubes containing 10 mL were inoculated with S. aureus at an approximate inoculum of 10⁵cfu/mL. Samples were drawn and bacteria were counted at 0, 1, 2, 3, 4, 6 and 24h of incubation at 37°C. Thus, after vortexing the culture tubes, two 50 μΐ. samples were removed and serially diluted with MHB. After each dilution step, 20 μΐ. was plated onto LB agar plates, which were incubated for 24 h at 37°C. Afterwards the colonies were counted and back-extrapolated to the original volume to determine the initial concentration (cfu/mL).

NMR and MS analysis

The purified compound was analyzed by Mass Spectroscopy and NMR to determine its chemical structure.

The NMR study was carried out on Bruker Avance 500 and 700 MHz spectrometers equipped with cryoprobes. The sample (5-7 mM) was solubilized in water

(95/5 H20/D20 v/v) at pH 4.3. Protons chemical shifts are expressed with respect to sodium 4,4 dimethyl-silapentane-1 -sulfonate, according to IUPAC recommendations.

LC-MS was first performed in order to obtain the m/z value of the protonated molecules of

Nemaucin. MS-MS fragmentation was then carried out on the Nemaucin. ESI-LC-MS data were obtained in the positive mode on a Waters alliance LC-MS system (Waters ZQ mass detector, Waters photodiode array detector 2696, Waters alliance HPLC systems 2790).

The HPLC column used was a C18 column (Waters Symmetry C18 5μιη 4.6X150 mm) maintained at 35 °C. Solvents were (A) water + 0.1% TFA (B) acetonitrile + 0.1% TFA and the flow rate was 1 mL/min. The mobile phase composition was 100% A at 0 min, ramped to 30% B at 30 min. Samples were dissolved in solvent A (100 L). Sample injection volume was 10 iL. UV- Visible detection was by absorbance at 200-400 nm.

Solvent flow to the MS was diverted to waste for the first 5 min to minimise salt build-up.

MS-MS fragmentation data were obtained on a Waters Micromass Q-Tof micro mass spectrometer.

B. Results

Fermentation

Xenorhabdus cabanillasii CNCM 1-4418 was cultivated for 48 h, at 28°C with shaking in a 2 L Erlenmeyer flask containing 500 mL of medium broth composed of bactopeptone 10 g/L, K₂HP0₄ 5 g/L, MgS0₄, 7H₂0 1 g/L, (NH₄)₂S04 2g/L, CaC0₃ 2 g/L, and NaCl 10 g/L. The culture was inoculated with 0.1% (v/v) of a 24 h preculture in the same medium. The antibiotic production was controlled by analytical HPLC. (Figure 1) Isolation

Bacterial cells were removed by low-speed centrifugation (6000 ^χ g, 10 min at 4°C) and supernatant was sterilized onto 0.22 μιη pore size filter. Supernatant was added (1 : 1; v/v) to a 0.1 M NaCl-0.02 M Tris buffer (pH 7), and subjected to cation-exchange chromatography on a Sep Pack CarboxyMethyl cartridge (Acell™ Plus CM, Waters). Unbound material was removed by washes with a 0.1 M NaCl-0.02 M Tris buffer (pH 7) and the antibiotic activity eluted with 0.5 M NaCl-0.02 M Tris buffer (pH 7). This eluate was acidified with 0.1% (v/v) trifluoroacetic acid (TFA) and was then subjected to reverse- phase chromatography on a Sep Pack C18 cartridge (Sep-Pak Plus CI 8, Waters). Unbound material was removed by washing with H₂0-TFA 0.1% and the antibiotic pool was eluted with acetonitrile. The eluate was freeze-dried then resuspended in water (1 :5; v/v). Pure compounds were isolated from the crude extract by reverse phase HPLC using a C18 column (Waters ; Symmetry Symmetry CI 8; 5μπι; 4.6X150 mm), a linear gradient of H₂0, 0.1% TFA-acetonitrile starting from 0% to 30% in 30 min, a flow rate of 1 mL/min and an UV detection from 200 to 400 nm, yielding pure Nemaucin with the following FIPLC- retention time: 27.16 min.

Biological properties

Nemaucin compounds demonstrate strong antibacterial activities. The Nemaucins were tested for antimicrobial activity against a wide range of bacteria involved in nosocomial and animal infection. Regarding reference strains (Table 1), Nemaucin has strong activity against S. aureus CIP 76.24 and E. coli CIP 76.25 (inferior to ^g/mL) but has moderate activity against P. aeruginosa CIP 76.110. Nemaucin was also tested against multi-resistant bacteria including Gram-positive and Gram-negative bacteria. They also have a very strong activity against Gram-positive multi-resistant bacteria such as MRSA, VRE and Gram-negative multi-resistant bacteria such as ESBL Enterobacter cloacae, E. coli, Klebsiella pneumonia, Klebsiella oxytoca, Klebsiella singaponasis or carbapenem resistant K. pneumoniae.

Nemaucin has a wide spectrum antibacterial activity including multi-resistant bacteria. Table 1: MIC ^g/mL) of Nemaucin, Polymyxin, and Vancomycin against bacterial strains, including multiresistant clinical isolates (Nimes University Hospital)

Bacteria Strain Resistance Nemaucin Polymyxin Vancomycin

Staphylococcus

CIP 76.25 - 0.08 - 1.56 aureus

Staphylococcus

21840 MRSA 0.05 - 1.56 aureus

Staphylococcus

20681 MRSA 0.05 - 1.56 aureus

Staphylococcus

20364 MRSA 0.05 - 1.56 aureus

Staphylococcus

23305 MRSA 0.05 - 1.56 aureus

Staphylococcus

16666 MRSA 0.05 - 1.56 aureus

Enterococcus

N5 VRE 0.05 - >50 faecalis

Escherichia

CIP 76.24 - 0.78 0.31 - coli

Escherichia

PEC21 ESBL/SHV 0.78 0.31 - coli

Escherichia

NEC20 ESBL/TEM 0.39 0.02 - coli

Escherichia

MEC12 ESBL/TEM 0.78 0.07 - coli

Escherichia ESBL/CTX

MEC63 0.78 0.31 - coli M15

Escherichia ESBL/CTX

EC Abwa 0.78 0.15 - coli M15

Escherichia ESBL/CTX

AEC7 0.78 0.31 - coli M14

Enterobacter

11370 0.78 0.62 - clocae

Enterobacter

BEB 1 1.56 1.25 - clocae

Enterobacter

BEB 4 0.78 0.08 - clocae

Enterobacter

41206 1.56 0.08 - clocae

Enterobacter

36265 0.78 0.08 - clocae

Klebsiella

PEB1 0.39 0.62 - pneumoniae

Klebsiella

MEB 11 0.78 0.62 - pneumoniae

Klebsiella

NEB 2 0.78 0.39 - pneumoniae Klebsiella

NEB 4 0.78 5.00 - pneumoniae

Klebsiella

KPC2 475 KPC 1.56 1.25 - pneumoniae

Klebsiella

PEB 4 0.78 0.63 - oxytica

Klebsiella

NEB 9 0.78 0.63 - singaponasis

Citrobacter

BEB 6 0.39 0.07 - koseri

Citrobacter

37553 1.56 0.15 - freundii

Proteus

12042 0.78 >12.5 - penneri

Serratia

31674 >12.5 0.03 - marescens

Serratia

31626 >12.5 0.01 - marescens

Pseudomonas

CIP 76.110 - >12.5 0.62 - aeruginosa

Pseudomonas

5618 MDR >12.5 0.62 - aeruginosa

Pseudomonas

401681 MDR >12.5 0.31 - aeruginosa

Pseudomonas

35170 MDR >12.5 0.62 - aeruginosa

Pseudomonas

42162 MDR >12.5 0.62 - aeruginosa

Pseudomonas

512232 MDR >12.5 0.62 - aeruginosa

As evidenced by its value of MIC and MBC, Nemaucin is bactericidal (Table 2). The Bactericidal effects of Nemaucin on growing S. aureus CIP 76.24 was determined. At twofold and four-fold the MIC, Nemaucin kills 100% of bacteria within 6 h, making this molecule a fast and powerful bactericidal agent. (Figure 2)

Their activities are reduced by a factor of 2 in presence of HSA, suggesting weak interaction between HSA and Nemaucin. (Table 2)

Table 2: MIC (in μg/mL), MBC (in μg/mL) and MIC in presence of 4% of HSA (in μg/mL) against reference bacteria (*MIC/MBC)

S.aureus CIP 76.25 E.coli CIP 76.24

Nemaucin 0.04 μ /ι 1/0.08 1πά 0.78/0.78*

Nemaucin

0.08 1.56

+4% (w/v) HSA The LD50 of Nemaucin A was 36.8 ± 3.54 μ§/ιηΙ. against Human prostatic carcinoma cells (PC-3) and 28.42 ± 3.61 μg/mL against Human normal mammary epithelium cells (hTERT HME-1) (Table 3). 100 % of cell viability was observed up to 12^g/mL. (Figures 3 and 4)

Table 3: LD 50 of Nemaucin A against PC-3 and hTERT HME-1

Physico-chemical properties of nemaucins

One compound referred to as Nemaucin was isolated, purified to homogeneity as a white powder and characterized by mass spectrometry. ESI-MS experiments revealed the molecular weight of Nemaucin. (Figures 5 and 6)

Nemaucin A : White powder; UV MeOHmax nm 214; ESI-MS (m/z) 1303 [M+H]⁺;

Chemical structure elucidation

The 1H spectrum revealed the features of a peptidic moiety with amide signals in the 9.1-6.9 ppm area, alpha proton signals in the 4.8-3.5 ppm, beta proton signals in the 3.7-2.0 ppm and methyl signals at 1.2-1.1 ppm. To identify the spin systems of the different residues an homonuclear data set (COSY, TOCSY, NOESY and ROESY) was recorded, as well as 1H-¹³C data set (HSQC, HSQC-TOCSY, HMBC) and a 1H-¹⁵N HSQC in natural abundance. The combined analysis of all these data allowed us to identify spin systems of the peptidic sequence which contains at least 6 residues, including 2 unusual, derived from Threonine (Thr) and Proline (Pro) alterations.

The Thr derived residue located in position 5 of the sequence results from the change of the γ oxygen atom for a nitrogen atom, giving rise to an unusual β amino group. Moreover, this amino group is involved in a peptidic bond. This is clearly supported first, by the TOCSY and COSY data and confirmed by the 1H-¹⁵N HSQC which shows the two H-N cross peaks. Such an unusual residue was referred to later as Thn.

The Pro derived residue in position 6 was shown to contain an additional -CH(OH)- CH2- moiety between the alpha and carbonyl carbons. The molecular weight of such a residue is 141.1 Da (159.1 Da in its carboxylic and amino form). It was referred to later as P141. The 1H-¹³C HSQC and HSQC-TOCSY data were particularly helpful to identify its spin system. The configuration of the asymmetric carbons of these two non-natural residues was not determined. By using sequential NOEs, the peptidic sequence was found to contain 6 residues as following:

Asp^His^Asp^Asn⁴ Thn⁵-P141⁶-CH2-CH2-R'

Proton and 1H-¹³C HSQC spectra were recorded at different pH ranging from 4.34 to 2.15. Of the two Asp residues only the Asp¹ was sensitive to the ionisation meaning that its γ carboxyl group is free. The signals of the Asp³ spin system were insensitive to pH suggesting that its γ carboxyl group was involved in an amide bond. The NOE between the β amide of Thn⁵ and the β protons of Asp³ indicates that the side chains of these two residues were bound by a peptidic bond to form a cyclic peptide.

We observed that the P141⁶ residue is linked to a seventh one, the amide signal of which was a triplet. Its spin system contains a -HN-CH₂-CH₂- moiety (COSY, TOCSY, HSQC- TOCSY).

Chemical structure of the peptidic part of Nemaucin was confirmed by MSMS fragmentation. (Table 5 and Figure 8)

Table 4: Chemical shifts of the identified peptidic part (water, 280 K)

Table 5: Key fragmentations of the 1303 [M+H]⁺ ions of Nemaucin A.

The 1H NMR spectra also showed 18 methylenes including 9 CH₂ bonded to nitrogen. After a detailed examination of the 2D-NMR spectra (COSY, HSQC, HMBC, TOCSY), the following structure is suggested for the R'-part:

The assignment of 1H in D₂0 is:

1H NMR (500 MHz, D₂0) δ 3.38-3.21 (18H, m, H-2, 7, 10, 15, 18, 23, 26, 31, and 34), 1.75-1.58 (16H, m, H-8, 9, 16, 17, 24, 25, 32, and 33)

The complete chemical structure of Nemaucin in shown in Figure 9. This report describes the production, the purification and the characterization of a novel peptide antibiotic produced by Xenor abdus cabamllasii. Its significant antibacterial activity, including against multi-resistant bacteria, increases the potential interest of this molecule for human and animal health applications.

REFERENCES

Appl. Environ. Microbiol. 58, 3032-3037 (1992)

J. Nat. Prod. 54, 774-84 (1991)

J. Nat. Prod. 54, 785-95 (1991)

J. Nat. Prod. 58, 1081-6 (1995)

Can. J. Microbiol. 43, 770-3 (1997)

J.Antibiot. 62(6) 295-302 (2009)

Entomopathogenic Nematology. 99-114 (2002)

FEMS Microbiol. Lett. 239, 241-8 (2004)

Appl. Environ. Microbiol. 58, 3032-7 (1992)

Appl. Environ. Microbiol. 61, 2049-52 (1995)

J. Nat. Prod. 71 (6), 1074-1077 (2008) PATENT REFERENCES

WO 86/01509

WO 84/01775

US 5,569,668

Print Out (Original in Electronic Form)

0-1 Form PCT/RO/134 (SAFE)

Indications Relating to Deposited

Microorganism(s) or Other Biological

Material (PCT Rule 13bis)

0-1 -1 Prepared Using PCT Online Filing

Version 3 . 5 . 000 . 225 MT/FOP

20020701/0 . 20 . 5 . 20

0-2 International Application No.

PCT/EP201 1/073738

0-3 Applicant's or agent's file reference 359890D28953

1 The indications made below relate to

the deposited microorganism(s) or

other biological material referred to in

the description on:

1-1 page 6

1-2 line 4

1-3 Identification of deposit

1 -3-1 Name of depositary institution CNCM Collection nationale de cultures de micro-organismes

1 -3-2 Address of depositary institution Institut Pasteur, 28 , rue du Dr Roux,

75724 Paris Cedex 15 , France

1 -3-3 Date of deposit 15 December 2010 ( 15 . 12 . 2010 )

1 -3-4 Accession Number CNCM 1-4418

1-4 Additional Indications Xenorhabdus cabanillasii JM 26

1-5 Designated States for Which

Indications are Made All designations

FOR RECEIVING OFFICE USE ONLY

0-4 This form was received with the

international application: Yes

(yes or no)

0-4-1 Authorized officer

Krista Delimon

FOR INTERNATIONAL BUREAU USE ONLY

0-5 This form was received by the

international Bureau on:

0-5-1 Authorized officer

Claims

1. Compound of formula (I):

(I) wherein

Xaai is aspartic acid or glutamic acid,

Xaa₂ is arginine, histidine or lysine,

Xaa₃ is serine, threonine, asparagine or glutamine,

Xaa₄ is selected in the group consisting of

R is H, OH or -CH2- n is 1, 2, 3, 4 or 5.

2. Compound according to claim 1 wherein Xaai is aspartic acid.

3. Compound according to anyone of claims 1-2 wherein Xaa₂ is histidine.

4. Compound according to anyone of claims 1-3 wherein Xaa₃ is asparagine.

5. Compound according to anyone of claims 1-4 wherein Xaa₄ is

6. Compound according to anyone of claims 1-5 wherein R is OH.

7. Compound according to anyone of claims 1-6 wherein n=4.

8. Compound according to anyone of claims 1-7 of formula (II):

(Π)

wherein n is 1, 2, 3, 4 or 5.

9. Compound according to claim 8 wherein n=4.

10. Compound according to anyone of claims 1-9 for use as a medicament.

11. Compound according to anyone of claims 1-10 for use as an antibiotic agent.

12. Composition comprising a compound according to anyone of claims 1-11 and an antibiotic compound.

13. Method for producing a compound according to formula (II)

comprising the following steps:

a. Growing Xenorhabdus cabanillasii strain CNCM 1-4418 in a liquid culture medium,

b. Purifying a compound according to formula (II)

wherein n=4.

14. Xenorhabdus cabanillasii strain deposited at CNCM having the accession number CNCM 1-4418.

15. Culture supernatant from the Xenorhabdus cabanillasii strain of claim 14 having antibiotic activity.