WO2022238627A1

WO2022238627A1 - Quorum sensing inhibitors and composition containing same

Info

Publication number: WO2022238627A1
Application number: PCT/FR2021/050830
Authority: WO
Inventors: Soizic Prado; Séverine AMAND; Kok Gan CHAN
Original assignee: Museum National D'histoire Naturelle; University Of Malaya (Um)
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2022-11-17

Abstract

The present invention relates to novel quorum sensing inhibitors and compositions containing said inhibitors. These inhibitors disturb the establishment of bacterial biofilms, "swarming" and the production of secondary metabolites of bacteria, in particular pathogenic bacteria, by a mechanism for inhibiting bacterial communication, referred to as quorum sensing.

Description

Quorum sensing inhibitors and composition containing them

The present invention relates to novel quorum sensing inhibitors and compositions containing said inhibitors. These inhibitors disrupt the establishment of bacterial biofilms, the "swarming" and the production of secondary metabolites of bacteria, in particular pathogens, by a mechanism of inhibition of bacterial communication known as quorum sensing.

Bacteria are probably the first form of life to appear on Earth.

They colonize the air, the earth, the sea, the organisms. Although a significant number of them are useful, some are pathogenic and can cause disease or are harmful to the environment and agriculture. Fortunately, there are molecules such as antibiotics that can control their proliferation and virulence, or even eradicate them. We can cite the most famous of them, penicillin, but also compounds of the macrolide type or, among the most powerful of them, vancomycin.

However, antibiotic resistance has become a public health issue, because the massive use of antibiotics since their discovery has led bacteria to put into play defense mechanisms that make them more resistant to conventionally used antibiotics. Another peculiarity of bacteria is their ability to create superstructures called biofilm. In fact, in nature, bacteria are subject to rapid and extreme changes in their environment. They have thus developed many strategies enabling them to adapt to these changes.

Some are, for example, capable of coordinating their genetic expression in order to organize themselves in the form of biofilms, aggregates of bacterial cells attached to a surface and coated with a polymeric matrix, highly resistant to external attacks and in particular resistant to antibiotics and detergents.

When a biofilm forms in an organism, the bacteria it contains can become inaccessible to antibiotics and lead to chronic infections, which most often occur in the lungs, urinary tract or surgical lesions. Biofilm protects bacteria and allows them to survive in harsh environmental conditions. Biofilm bacteria can resist the host's immune response and are much more resistant to antibiotics and disinfectants than planktonic bacterial cells. The ability to form a biofilm is now recognized as a characteristic specific to several microorganisms.

Similarly, the formation of a biofilm on an abiotic surface (industrial equipment or medical equipment, for example) makes it very difficult to clean and disinfect it.

This is particularly problematic in the case of medical equipment, such as catheters and joint prostheses, because the biofilms which can form on their surface are very tolerant to biocides and can lead to the release of planktonic bacteria which cause systemic infections and be a source of nosocomial illnesses in the hospital.

The fine mechanisms concerning the communication between bacteria called quorum sensing allowing the establishment of a biofilm are not yet fully elucidated, but the main stages of the formation of said biofilm are now known as described by and Y. Tremblay and al in Can J Vet Res. 2014 Apr; 78(2): 110-116. Quorum sensing

(QS) is a bacterial communication system that allows them to synchronize their genetic expression in order to regulate various mechanisms such as the secretion of toxins, adhesion, the formation of biofilms or even virulence. This is genetically controlled.

Much research to date aims to disrupt or avoid the establishment of these bacterial biofilms. In application WO2017/197303, new methods and compositions based on apicidin are described for inhibiting the detection of QS, also called quorum quenching, in bacterial infections and new methods and compositions based on apicidin for treatment of staphylococcal infection. QS controlling agents also include a sorbent material, a sorbent mineral or a non-porous mineral such as, for example, phyllosilicate clays, silica, calcite, zeolites, diatomaceous earth, smectite, activated carbon, a nanoparticle or a combination of any of the foregoing. Said compositions avoiding the establishment of bacterial biofilms and the deterioration of food products but also the prevention of vibriosis in fish or molluscs.

Mention will also be made of application WO2015/004305, the subject of which is a peptide possessing QS inhibition activities. Said peptide exhibits a broad spectrum of N-acyl-homoserine lactone (AHL) degradation activity having 4-14 carbon atoms in their side chains, possibly substituted, is active at a pH between 3 and 9, is resistant to proteinase K and chymotripsin, and does not interact with β-lactam antibiotics.

Although these compounds are interesting, due to the broad spectrum of bacteria endowed with QS activity, it remains necessary to have a large panel also of new molecules or compositions likely to have quorum sensing inhibition activities, able to act alone or in synergy or potentiate existing molecules also exhibiting activities making it possible to avoid the establishment of bacterial biofilms capable of causing diseases, ravaging crops or destroying the flora and fauna contaminated by said biofilms .

Thus there remains the need to find molecules or compositions making it possible to limit the formation and proliferation of biofilms and to have a molecular arsenal complementary to the existing one.

The inventors of the present application have demonstrated that compounds of the furan-3(2H)-one type exhibited characteristics responding to the problems cited above, and which could be useful as inhibitors of quorum sensing, of

"swarming", of the production of secondary bacterial metabolites and the formation of bacterial biofilms, but also as a potentiator of compounds already known as quorum sensing inhibitors or even antibacterials used against bacterial biofilms. A significant advantage of these compounds is that their mechanism of action does not lead to selection pressure and therefore potentially limits resistance problems.

The present invention thus relates to a compound of general formula (I):

(I) in which

- R ₁ independently represents a group -C(=O)-OH, -C(=O)-NH ₂ , -C(=O)-SH, -

C(=O)-OR, -C(=O)-NHR, -C(=O)-N(R) ₂ , -C(=O)-SR; where R independently represents a C ₁ -C ₁₀ alkyl group, saturated or unsaturated, linear or branched, which may be substituted by at least one group -OH, -CN, -SO ₃ H, -SH, -NH ₂ , -

NHAIkyl, -N(Alkyl) ₂ in which the alkyl group is a C ₁ -C ₄ alkyl, halogen, alkyloxy in which the alkyl group is a C ₁ -C ₄ alkyl;

- R ₂ independently represents a C ₁ -C ₁₀ alkyl group, saturated or unsaturated, linear or branched, which may be substituted by at least one -OH, -CN, -SO ₃ H, -SH, -NH ₂ group, -

NHAIkyl, -N(Alkyl) ₂ in which the alkyl group is a C ₁ -C ₄ alkyl, halogen, -O-H ₂ PO ₃ , alkyloxy in which the alkyl group is a C ₁ -C ₄ alkyl;

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

- R ₄ independently represents a C ₁ -C ₁₀ alkyl group having at least one unsaturation; and in which the R ₁ and R ₂ groups can also form a cycle of the lactone, lactam or thiolactone type.

In a preferred embodiment, the compound of formula (I) is such that:

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

- R ₄ independently represents a C ₁ -C ₁₀ alkyl group having at least one unsaturation; and the R ₁ and R ₂ groups can also form a cycle of the lactone, lactam or thiolactone type; with the exclusion of compounds where R ₄ comprises two contiguous unsaturations in α of the carbon substituted by the groups O, R ₃ and R ₄ and where R ₁ is the group -C(=O)-OH. A subject of the invention is also a compound of formula (Ia):

(the) in which

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

In a preferred embodiment of the invention, the compound of formula (la) is such that:

- R ₂ independently represents a C ₁ -C ₁₀ alkyl group, saturated or unsaturated, linear or branched, which may be substituted by at least one -OH, -CN, -SO ₃ H, -SH, -NH ₂ group, - NHAIkyl, -N(Alkyl) ₂ in which the alkyl group is a C ₁ -C ₄ alkyl, halogen, -O-H ₂ PO ₃ , alkyloxy in which the alkyl group is a C ₁ -C ₄ alkyl;

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

- R ₄ independently represents a C ₁ -C ₁₀ alkyl group having at least one unsaturation; and the R ₁ and R ₂ groups can also form a cycle of the lactone, lactam or thiolactone type; with the exclusion of compounds where R ₄ comprises two contiguous unsaturations in α of the carbon substituted by the groups O, R ₃ and R ₄ and where R ₁ is the group -C(=O)-OH.

A subject of the invention is also a compound of formula (Ib):

(Ib) in which

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

- R ₄ independently represents a C ₁ -C ₁₀ alkyl group having at least one unsaturation; and in which the R ₁ and R ₂ groups can also form a cycle of the lactone, lactam or thiolactone type. In a preferred embodiment of the invention, the compound of formula (Ib) is such that:

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

In another embodiment of the invention for the compounds of formulas (I), (Ia) and

(lb),

- R ₁ independently represents a -C(=O)-OH, -C(=O)-OR group; where R independently represents a C ₁ -C ₁₀ alkyl group, saturated or unsaturated, linear or branched, which may be substituted by at least one group -OH, -CN, -SO ₃ H, -SH, -NH ₂ , -

- R ₂ independently represents a C ₁ -C ₄ alkyl group, saturated or unsaturated comprising unsaturation, linear or branched, which may be substituted by at least one -OH group,

-CN, -SO ₃ H, -SH, -NH ₂ , -NHAIkyl, -N(Alkyl) ₂ in which the alkyl group is a C ₁ -C ₄ alkyl, halogen, -OH ₂ PO ₃ , alkyloxy in which the alkyl group is C ₁ -C ₂ alkyl;

- R ₃ independently represents a methyl group; - R ₄ independently represents a C ₁ -C ₆ alkyl group having at least two unsaturations. and the R ₁ and R ₂ groups can also form a cycle of the lactone type.

In another preferred embodiment of the invention for the compounds of formulas

(I), (la) and (Ib),

- R ₁ independently represents a -C(=O)-OH, -C(=O)-OR group;

R independently represents a C ₁ -C ₄ alkyl group, saturated or unsaturated, linear or branched, which may be substituted by at least one group -OH, -CN, -SO ₃ H, -SH, -NH ₂ , -

NHAIkyl, -N(Alkyl) ₂ in which the alkyl group is a C ₁ -C ₄ alkyl, halogen, alkyloxy in which the alkyl group is a C ₁ -C ₂ alkyl;

-SO ₃ H, -SH, -NH ₂ , -NHAIkyl, -N(Alkyl) ₂ in which the alkyl group is a C ₁ -C ₄ alkyl,

CN, halogen, alkyloxy in which the alkyl group is methyl;

- R ₃ independently represents a methyl group;

- R ₄ independently represents a C ₁ -C ₆ alkyl group having two unsaturations and the R ₁ and R ₂ groups can also form a cycle of the lactone type.

In one embodiment of the invention, the preferred compounds of the invention are independently selected from the following structural formulas:

5

In the context of the present invention, if the structural formula of one of the compounds according to the invention comprises several chiral centers whose stereochemistry is not mentioned, it is then a racemic mixture.

In an equally preferred embodiment of the invention, the compounds are independently selected from the following structural formulas:

The compounds of the invention can be used in the form of a composition, thus another object of the invention corresponds to a composition comprising individually or as a mixture the compounds of formula (I):

(I)

in which

NHAIkyl, -N(Alkyl) ₂ in which the alkyl group is a C ₁ -C ₄ alkyl, halogen, alkyloxy in which the alkyl group is a C ₁ -C ₄ alkyl; - R ₂ independently represents a C ₁ -C ₁₀ alkyl group, saturated or unsaturated, linear or branched, which may be substituted by at least one -OH, -CN, -SO ₃ H, -SH, -NH ₂ group, -

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

Preferably, the composition comprises individually or as a mixture the compounds of formula (I) in which:

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

- R ₄ independently represents a C ₁ -C ₁₀ alkyl group having at least one unsaturation; and in which the R ₁ and R ₂ groups can also form a cycle of the lactone, lactam or thiolactone type; with the exclusion of compounds where R ₄ comprises two contiguous unsaturations in α of the carbon substituted by the groups O, R ₃ and R ₄ and where R ₁ is the group -C(=O)-OH.

More preferentially, the composition comprises individually or as a mixture the compounds of formulas (Ia) and (Ib): (la) (lb)

in which

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

More preferably, the composition comprises individually or as a mixture the compounds of formulas (Ia) and (Ib):

(la) (lb)

in which

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

- R ₄ independently represents a C ₁ -C ₁₀ alkyl group having at least one unsaturation; and in which the R ₁ and R ₂ groups can also form a cycle of the lactone, lactam or thiolactone type; and said composition is such that the ratio between the compounds (Ia)/(Ib) varies from 100/0 to 0/100 excluding the 50/50 ratio.

Even more preferably, the composition comprises individually or as a mixture the compounds of formulas (Ia) and (Ib):

(la) (Ib)

in which

- R ₁ independently represents a -C(=O)-OH, -C(=O)-OR group; where R independently represents a C ₁ -C ₄ alkyl group, saturated or unsaturated, linear or branched, which may be substituted by at least one group -OH, -CN, -SO ₃ H, -SH, -NH ₂ , -

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

- R ₄ independently represents a C ₁ -C ₆ alkyl group having at least one unsaturation; and in which the R ₁ and R ₂ groups can also form a cycle of the lactone type.

(la) (Ib)

in which

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

- R ₄ independently represents a C ₁ -C ₆ alkyl group having at least one unsaturation; and in which the R ₁ and R ₂ groups can also form a cycle of the lactone type; and said composition is such that the ratio between the compounds (Ia)/(Ib) varies from 100/0 to 0/100 excluding the 50/50 ratio. The also preferred compounds of formula (I) of the invention may be present in a composition individually or as a mixture have the following developed structures:

5

The also preferred compounds of formulas (Ia) and (Ib) of the invention which may be present in a composition individually or as a mixture have the following developed structures:

In particular, the compounds of formula (I) can be independently or as a mixture:

5-((1E,3E)-Hexa-1,3-dienyl)-5-methyl-4-oxo-2-((E)-propenyl)-4,5-dihydro-furan-3- carboxylic acid named also MV53 represented by the following formula:

but also the form (la) and (Ib)

- 5-((1E,3E)-Hexa-1,3-dienyl)-2-(2-methoxy-propyl)-5-methyl-4-oxo-4,5-dihydro-furan-3- carboxylic acid also mentioned MV45 represented by the following formula:

but also the form (la) and (Ib)

2-((1E,3E)-Hexa-1,3-dienyl)-2,6-dimethyl-6,7-dihydro-furo[3,2-c]pyran-3,4-dione also named MV46 represented by the following formula:

but also the form (la) and (Ib)

The compounds of general formula (I), (la) and (Ib) can be extracted from fungi but also prepared by chemical synthesis, particularly from the following compounds of formulas (I), (la) and (Ib):

The modification of a secondary alcohol by nucleophilic substitution or exchange reaction into a -NHAIkyl, -N(Alkyl) ₂ function in which the alkyl group is a C ₁ -C ₄ alkyl, alkyloxy, lactone or for the other compounds according to the invention which can derive from a secondary alcohol function, is well known to those skilled in the art in the field of chemistry.

Compounds MV53, MV45 and/or MV46 can also be extracted, for example, from the endophytic fungus Paraconiothyrium variabile as described in Example 1.

Thus the present invention also relates to an extract of Paraconiothyrium variabile titrated in MV53 and/or MV45 and/or MV46 comprising at least 35% by weight of

MV53 and/or MV45 and/or MV46.

In the compositions according to the invention, the compounds of type (I), (Ia) or (Ib) according to the invention are present within the composition in the amount of 0.1% to 5%, preferably between 0. 1% and 1%, by total weight of said composition. The compositions according to the invention may also comprise an anionic surfactant such as alkali metal, in particular sodium, alkyl sulphates, a nonionic surfactant such as an ethoxylated alkylphenol, but also a cationic surfactant or else a mixture of one or more of these agents, in particular a mixture of one or more cationic surfactant(s) and one or more nonionic surfactant(s) or one or more surfactant(s) anionic surfactant(s) and one or more nonionic surfactant(s). Said surfactant being present between 2 to 50% by weight of surfactant relative to the total weight of the composition.

The compositions according to the invention may also comprise at least one bactericidal acid surfactant, such as in particular an alkyl polyether carboxylic acid, an alkenyl polyether carboxylic acid, an alkylaryl polyether carboxylic acid or a phosphoric ester of an alkoxylated nonionic surfactant, under acidic or partially neutralized form. Said bactericidal acid surfactant being present between 2 to 50% by weight of surfactant relative to the total weight of the composition.

The compositions according to the invention may also comprise an inorganic salt such as copper oxide or hydroxide is chosen from copper hydroxide, copper oxychloride, copper carbonate, cuprous oxide or mixtures thereof. . Said mineral salt being present between 20% to 60% by weight of mineral salt relative to the total point.

Depending on the formulation of said composition, stabilizers or desiccants may also be present.

The composition according to the invention also comprises a vehicle suitable for the compounds according to the invention and optionally for the additional agent(s) that it contains and for the use for which it is intended.

By virtue of their action on the inhibition of the biofilm, the compounds according to the invention make it possible to potentiate antibacterial agents; thus, according to another embodiment, the composition according to the invention further comprises at least one additional agent chosen from:

- at least one antimicrobial agent; antimicrobials belong to a large family of substances, including antibiotics, which kill or slow the growth of microbes such as bacteria;

The antimicrobial agents can be chosen from synthetic or natural substances; preferably, natural substances such as terpenes derived from plants will be used.

- at least one antibiotic, including:

Antibiotics inhibiting the synthesis of the bacterial wall:

• bacitracin;

• penicillins: amoxicillin;

• cephalosporins;

• glycopeptides such as vancomycin.

Compounds attacking the membrane of bacterial cells:

• polymyxin;

• gramicidin;

Antibiotics that block the action of DNA gyrase: aminocoumarins, quinolones and fluoroquinolones.

Other molecules block DNA replication by introducing covalent bridges between neighboring bases, such as mitomycin or actinomycin.

Specific bacterial RNA polymerase inhibitors that block gene transcription and messenger RNA synthesis. Among these antibiotics, we find in particular rifampicin; aminoglycosides or aminoglycosides (examples: streptomycin, gentamicin, amikacin); phenicols (examples: chloramphenicol, thiamphenicol); cyclins (examples: tetracycline, doxycycline, aureomycin); macrolides and ketolides

(examples: erythromycin, azithromycin); puromycin; fusidic acid.

Antibiotic compounds target different steps of this folate pathway: Sulfonamides and sulfanilamide; trimethoprim.

- at least one antiseptic:

By antiseptic is meant a disinfectant for human or animal body use. This concerns in particular:

- biguanides such as chlorhexidine,

- iodine derivatives such as povidone iodine,

- chlorine derivatives such as sodium hypochlorite,

- alcohols such as ethanol,

- dyes such as eosin, Milian's solution, fluorescein or methylene blue,

- oxidants such as hydrogen peroxide, potassium permanganate and

- quaternary ammoniums such as benzalkonium chloride.

- at least one biocide: a biocide is a substance or any mixture consisting of one or more active substances, containing or generating them, which is intended to destroy, repel or render harmless harmful organisms, to prevent their action or to combat them in any other way by an action other than a simple physical or mechanical action; in the context of the present invention, the pest is a bacterium;

They are classified into four major groups:

Disinfectants

protective products

Pest control products

Other products

- at least one detergent:

A detergent (or surfactant, detergent, surfactant) is a chemical compound, generally derived from petroleum, with surfactant properties, which makes it capable of removing dirt. Cleansing is a fundamental element of hygiene, since it eliminates a large part of the bacteria present in particular on the skin, and on the utensils used for the preparation and consumption of meals.

A distinction is made between detergents:

• anionic (alkylbenzene sulphonates, alkyl sulphates, alkylarylsulphates);

• cationic (amine hydrochlorides, quaternary ammoniums), mainly used in industrial and hospital environments, due to their disinfectant properties; • ampholytes, ionizing negatively (anions) or positively (cations), depending on the environmental conditions;

• non-ionic (hydroxyls)

Lecithins, which are phosphoaminolipids found in all plant and animal tissues, are natural surfactants.

Examples of synthetic detergents:

• sodium dodecylbenzene sulphonate: C ₁₈ H ₂₉ SO ₃ Na;

• SDS (sodium dodecyl sulphate): NaSO4(CH ₂ ) ₁₁ CH ₃ ;

• CTAB (hexadecyltrimethylammonium bromide or cetrimonium bromide):

(CH ₃ ) ₃ N(CH ₂ ) ₁₅ CH ₃ Br;

• CTAC (hexadecyltrimethylammonium chloride or cetrimonium chloride):

(CH ₃ ) ₃ N(CH ₂ ) ₁₅ CH ₃ Cl;

• Triton X-100;

• Brij 35.

- at least one bacteriophage;

Bacteriophages, or phages, more rarely bacterial viruses, are viruses that only infect bacteria.

- at least one mineral or organic fungicide: copper oxychloride, copper hydroxide, copper carbonate, cuprous oxide, folpel, maneb, mancozeb, propineb, zineb, cymoxanil, metiram-zinc, mineral and paraffinic oils

(for example, the commercial product SuffOil X ^® ), bicarbonates, for example of potassium.

The compounds of general formula (I), (Ia) and (Ib) and the composition according to the invention are particularly suitable for inhibiting the formation of bacterial biofilm and the mechanisms associated therewith such as quorum sensing, cell mobility

("swarming"), bioluminescence and the production of secondary metabolites of bacteria, in particular of pathogenic bacteria.

In particular, the bacteria responsible for biofilm formation can be:

Actinobacillus pleuropneumoniae

Aeromonas hydrophila Trueperella pyogenes Bartonella henselae Bordetella bronchiseptica Bordetella parapertussis Brucella melitensis Burkholderia pseudomallei Campylobacter coli Campylobacter jejuni Clostridium perfringens Corynebacterium pseudotuberculosis Corynebacterium renale Enterococcus faecalis Enterococcus faecium Erysipelothrix rhusiopathiae Escherichia coli

Francisella novicida Francisella tularensis group Bacillus cereus Haemophilus parasuis Histophilus somni Leptospira Listeria monocytogenes Mannheimia haemolytica Mycobacterium Mycoplasma

Pasteurella multocida Pseudomonas aeruginosa Riemerella anatipestifer Salmonella Staphylococcus Streptococcus Yersinia

In the context of the present invention, a biofilm can be mono- or multi-species.

According to one embodiment, the composition according to the invention is a phytosanitary composition acting against the formation of bacterial biofilm and used for: • Protect plants or plant products against all harmful organisms or prevent their action;

• Ensure the conservation of plant products.

According to another embodiment, the present invention relates to the compounds of general formula (I), (Ia) and/or (Ib) or to the composition according to the invention for their use for the prevention and/or treatment of bacterial infections due to biofilm-forming bacteria in human or animal patients; in particular, these are infections due to enterohaemorrhagic strains of E coli, Listeria monocytogenes,

Campylobacter thermophiles such as C. jejuni and C. coli, and salmonella.

The compounds of general formula (I), (Ia) and/or (Ib) or to the composition according to the invention are of particular interest in the veterinary field for the treatment of bacterial infections targeting pets and animals. livestock where the use of antibiotics is avoided as much as possible.

The bacterial biofilm can still cause inconveniences such as the corrosion of industrial equipment, ships, the obstruction of pipes, the contamination of water, the contamination of products in particular in the food industry, or even infections linked to presence of biofilms on implantable medical devices.

Thus, the present invention also relates to the use of the compounds of general formula (I), (Ia) and/or (Ib) or of the composition according to the invention for cleaning, that is to say preventing the formation and promote the elimination of bacterial biofilms, particularly in industrial equipment, pipes, in the food industry, or even in medical devices.

tricks

Figure 1: Profile of the extract of Paraconiothyrium variabile obtained by reverse phase liquid chromatography (measurement of the ionic current) for a culture on a solid biomalt medium (top curve), on a solid malt extract agar medium (second curve from the top ), obtained from the mycelium of a liquid culture on a biomalt medium (third curve starting from the top), obtained after extraction of the aqueous phase of the liquid culture on biomalt medium (bottom curve).

Figure 2: retention time of the three compounds according to the invention in HPLC.

Figure 3: assay of violacein production by C. violaceum in the presence of compounds MV45 or MV53.

Figure 4: assay of violacein production by C. violaceum in the presence of compounds MV45 or MV53.

Figure 5: assay of violacein production by C. violaceum in the presence of compounds MV45 or MV53.

Figure 6: assay of pyocyanin production by P. aeruginosa in the presence of compounds MV45 or MV53.

Figure 7: diagram of QS systems (QUORUM SENSING SIGNAL-RESPONSE SYSTEMS IN

GRAM-NEGATIVE BACTERIA. KAI PAPENFORT & BONNIE L. BASSLER NATURE REVIEWS

MICROBIOLOGY 14, 576-588 (2016) D01:10.1038/NRMICR0.2016.89).

Figure 8: bioluminescence test on E. coli [pSB401] in the presence of compounds MV45 or

MV53.

Figure 9: bioluminescence test on E. coli [pSB1075] in the presence of compounds MV45 or MV53.

Figure 10: swarming test in the presence of compounds MV45 or MV53.

Examples

Example 1 - demonstration of the presence of compounds MV45, MV53 and MV46 in a culture of Paraconiothyrium variabile and purification

The compounds according to the invention MV53 and MV45, as well as the compound MV46 are identified and purified from a crude extract from the culture of Paraconiothyrium variabile (LCP

5644).

Different culture conditions are tested.

1.1- Cultivation of Paraconiothyrium variabile (PV)

-Preculture: it is carried out after inoculation of spores on a Petri dish and left

12 days ; from this culture the mycelium is recovered (by scraping) and the spores are suspended in sterile water (approximately 10 ⁶ spores per ml).

-Culture media: Solid biomalt medium: agar = 30 g, biomalt syrup = 50 g, Qsp 1L of water at the rate of 40mL of medium per kneaded dish (round dish). 18 boxes are inoculated with the spore suspension (3 ml of the spore solution per box).

Liquid biomalt medium: biomalt syrup = 50 g, qsp 1 L of water, 1 Erlenmeyer flask of 500 ml prepared with 250 ml of culture medium, 3 ml of spore suspension are added to the medium.

Solid agar malt extract medium: agar = 20 g, malt extract = 20 g, glucose = 20 g, peptone = 1 g, Qsp 1L of water at the rate of 40mL of medium per petrie dish (round dish).

18 boxes are inoculated with the spore suspension (3 ml of the spore solution per box).

-Culture conditions:

For the 3 cultures, the incubation temperature is 25° C. for 15 days, static for the solid media, with stirring for the liquid medium (1500 rpm) with a 12 hour day/night alternation.

1.2- Extraction

- from culture on solid biomalt medium:

The cultures of the 18 dishes are cut into small pieces and distributed in 2 Erlenmeyer flasks of 500 mL, addition of 200 mL of distilled ethyl acetate in each and sonication for

IhOO, then filtration. This step is repeated 3 times then the organic phases are combined and concentrated under reduced pressure at 40°C. 454 mg of crude extract are obtained.

- from culture on solid malt extract agar medium:

IhOO, then filtration. This step is repeated 3 times then the organic phases are combined and concentrated under reduced pressure at 40°C. 215 mg of crude extract are obtained.

- from culture on liquid biomalt medium:

The liquid biomalt culture is centrifuged at 13400 rpm for 20 mins. The supernatant is separated from the culture pellet. The culture pellet which contains the mycelium is placed in a 100 mL Erlenmeyer flask, addition of 60 mL of distilled ethyl acetate and sonication for

1 hour, then filtration. This step is repeated 3 times. The organic phases are combined and concentrated under reduced pressure at 40°C. 44 mg of crude extract are obtained. The culture supernatant (aqueous phase) is extracted with 250 mL of ethyl acetate in a separatory funnel (liquid-liquid extraction). The organic phase is recovered and the extraction is repeated 3 times. The organic phases are combined and dried with anhydrous sodium sulphate. After filtration, the extract is concentrated under reduced pressure at 40°C.

65 mg of crude extract are obtained.

1.3-Comparison of the content of compounds in the extracts according to their mode of cultivation

The solutions are prepared at a concentration of lmg/ml in methanol for each extract then centrifuged and passed through HPLC/MS:

HPLC method:

UHPLC ultimate 3000 chain from thermoscientific, Acclaim polar advantage II column from thermoscientific, 2.2 μm, 2.1 x 100 mm, flow rate 0.3 ml/mins, Eluent A: ultra pure water +

0.1% A. formic / Eluent B: Acetonitrile + 0.08% A. formic, injection volume 2μL,

Oven temperature = 40°C, sample changer temperature 5°C.

MS method:

Bruker compact ESI-QTOF device, in positive mode, mass range <50-1300>, source

ESI, capillary voltage: 3500 volts, gas flow: 8L/min, drying temp: 200°C, nebulizer gas pressure: 2.4 bars.

gradient:

Results

The curves obtained are presented in Figure 1.

For the same concentration of extract, the solid biomalt medium is richer in each of the compounds MV45, MV53 and MV46.

1.5-Purification of compounds of interest:

Beforehand, the retention time of each of the compounds was determined by HPLC coupled to a UV detector under the following conditions: -From the Paraconiothyrium variabile extract grown on solid biomalt medium, a 10mg/ml solution in methanol is prepared.

HPLC method:

HPLC 321 chain from Gilson, Eclipse XDB-phenyl column from Agilent, 5 μm, 4.6 x 150 mm, flow rate 0.8 ml/min, Eluent A: ultra pure water/ Acetonitrile 95/05 and Eluent B:

Acetonitrile, injection volume 10 μL, oven temperature = 20°C, manual injection.

Wavelength 210, 220 and 254 nm.

gradient:

The retention times measured are (see Figure 2):

MV46, Tr = 10.6 mins

MV 45, Tr = 14.5 mins

MV 53, Tr = 16.9 mins

This method has been adapted to semi-preparative HPLC to purify the 3 compounds:

HPLC method:

HPLC 1260 infinity chain from Agilent, Eclipse XDB-phenyl column from Agilent, 5 μm,

21.2 x 150 mm, flow rate 10 ml/min, Eluent A: ultra pure water/Acetonitrile 95/05 and Eluent B:

Acetonitrile, injection volume 100 μL, room temperature, manual injection.

Wavelength 210, 254 and 300 nm.

gradient:

The solution injected is at 0.97 g/ml, 2 injections are carried out (volume of injection=100 μL) ie 194 mg of extract injected in total.

The masses of products recovered relative to the quantity of extract injected are as follows:

MV46 = 55 mg/g of extract;

MV 45 = 140 mg/g of extract; and MV 53 = 159 mg/g extract.

Example 2 - Biological Activity: Demonstration of Anti-Qorum Sensing Activities

2.1-Bacterial strains and culture conditions

All the bacteria used for this study were cultured on Luria-Bertani (LB) medium (Scharlab, Barcelona, Spain) at 37°C, with shaking at 220 rpm, except the strain

C. violaceum CV026 which was grown at 28°C. The biosensors used in this study are described below.

Table of bacterial strains and plasmids used in this study.

Bacterial growth was estimated using a method described by (Hayouni et al.,

2008). A bacterial pre-culture was carried out then diluted to an optical density of 0.1

(OD600 nm) before being added to a 96-well microtiter plate (230 μL). 20 μL of the compounds to be tested at a concentration of 10 μg mL-1 were then added. Bacteria were then incubated at their optimum temperature and optical density

OD600 nm was determined every 30 min for 24 h after reading on a Tecan Infinite M200 microplate reader (Switzerland).

2.2-Activity tests

- Violacein production test

The CV026 biosensor was constructed by mutagenesis of the C. violaceum strain with the transposon mini-Tn5 in order to obtain a double insertion of Tn5, violacein negative, rendering the strain incapable of producing AHLs. The synthesis of violacein can then be restored only by exogenous addition to the biosensor of synthetic C6-HSL (McClean et al.,

1997).

The quantitative analysis of violacein production was carried out according to the method previously described by (Chong et al., 2011) and after modification. Briefly, a preculture of C. violaceum CV026 was adjusted to an OD600nm of 1.2. 0.125 µg mL ^-1 of synthetic C6-HSL (Sigma-Aldrich, USA) was then added before being transferred

(100 μL) in a 96-well microtiter plate containing 10 μL of samples dissolved in dimethyl sulfoxide (DMSO) at 10 pg mL ^-1 . The microplate was then incubated for 16 h at 28° C. with shaking at 220 rpm. After drying at 60° C. until the medium evaporated, 100 μL of DMSO were added to each well in order to dissolve the crystallized violacein. The plate was then incubated for an additional 2 hours at 28°C with shaking. The absorbance of each of the wells was measured at OD590nm with the Tecan Infinite M200 microplate reader (Switzerland). All experiments were performed in triplicate. DMSO (Merck KGaA, Germany) and synthetic furanone C-30 (Sigma-Aldrich, USA) were used as negative and positive controls, respectively.

-Pyocyanin test

The pyocyanin was extracted from the supernatant of the culture of P. aeruginosa PAO1 previously cultured for 24 h as described previously (Chong et al., 2011). Briefly, 500 μL of samples solubilized at 10 μg mL ⁻¹ in DMSO were added to 4.5 mL of pre-culture. The whole was then diluted to reach an OD600nm of 0.1 and incubated at 37°C for 24 hours. The cell culture was extracted with 3 mL of chloroform (Merck

KGaA, Germany) and shaken for 5 min, followed by centrifugation at 9000 rpm for

10 minutes. The chloroform phase was then transferred and 1 ml of 0.2 M hydrochloric acid

(Merck KGaA, Germany) has been carefully added. After centrifugation at 9000 rpm for 10 min, the upper phase of the mixture was removed and the absorbance was read at

520 nm using the Tecan Infinite M200 microplate reader (Switzerland). All samples for this test were made in triplicate. DMSO (Merck KGaA, Germany) and synthetic furanone C-30 (Sigma-Aldrich, USA) served as negative and positive controls, respectively.

-Bioluminescence test

For the bioluminescence test, the method used is that based on the method previously described by (Winzer et al., 2000) with a few modifications. Cultures of E. coli [pSB401] and E. coli [pSB1075] were grown overnight with shaking in LB medium supplemented with 20 µg mL-1 tetracycline at 37°C. In a 96-well microtiter plate, 20 μL of samples to be tested were added to 230 μL of the bacteria culture previously diluted to an OD600nm of 0.1. Synthetic 3-oxo-C6-HSL (0.001 pg mL-1) and 3-oxo-C10-HSL (0.0125 pg mL-1) (Sigma-Aldrich, USA Unis) were added to cultures of E. coli [pSB401] and E. coli [pSB1075], respectively. The luminescence and turbidity of the biosensors were then read every 30 min for 24 h with the Tecan Infinite M200 microplate reader (Switzerland) at

DQ600nm. All experiments were performed in triplicate.

-Motility test (swarming test)

The strain motility tests were carried out according to the method described above

(Chen et al., 2007) with slight modifications. The medium used for the test consists of 0.6% (w/v) BactoTM agar (BD, USA), 0.6% (w/v) BactoTM Peptone (BD, USA), 0 .2% (w/v) yeast extract (BD, USA) and 0.5% (w/v) glucose (Merck, USA) in 1 L of distilled water. 150 μL of samples to be tested were then mixed with 5 mL of agar before being poured into 6-well plates. The plates were then left in the open air for 15 minutes before being inoculated with 1 μL of a preculture of P. aeruginosa PAO1 at an OD600nm of 0.1 deposited in the center of the surface of the agar. The plates were incubated without shaking at 37°C for 16 h. All experiments were performed in triplicate. DMSO (Merck KGaA, Germany) and synthetic furanone C-30 (Sigma-Aldrich, USA) served as negative and positive controls.

2.3-Results

-Assay of violacein production by C. violaceum

The production of violacein is under the control of the QS and its assay makes it possible to estimate the potential inhibitor or activator of the QS of a compound.

Figures 3, 4 and 5 show that the compound MV-45 very strongly inhibits the production of violacein from a concentration of 10 pg/ml relative to furanone at the same concentration, QS-inhibiting compound which serves as a positive control. MV-45 has a somewhat stronger inhibitory effect at higher doses. With regard to the compound MV-53, it has a dose-dependent inhibitory effect, but it fails to inhibit as strongly as furanone at the maximum concentration used (15 μg/ml).

These two compounds are therefore QS inhibitors and compound MV45 appears to be more potent than compound MV53.

-Assay of pyocyanin production by the pathogenic bacterium P. aeruginosa The production of pyocyanin by the pathogenic bacterium P. aeruginosa is under the control of the QS and its assay makes it possible to estimate the potential inhibitor or activator of the QS of a compound. From FIG. 6, it can be seen that MV-45 also has a strong inhibitory power on the QS of P. aeruginosa at a concentration of 10 pg/ml. MV-53 has an inhibitory effect comparable to the positive control, furanone.

The compounds were then subjected to a bioluminescence assay with two lux biosensors, Escherichia coli [pSB401] (Figure 8) and E. coli [pSB1075] (Figure 9). 3-oxo-C6-

Synthetic HSL was added to E. coli [pSB401], while E. coli [pSB1075] requires synthetic 3-oxo-C10-HSL to induce bioluminescence. The addition of compounds MV 45 and MV53 had a significant impact on the bioluminescence activity of both biosensors

(figures 8 and 9). These results therefore suggest that MV45 and MV53 interfere with QS systems involving short- and long-chain N-acylhomoserine lactones (AHLs).

Moreover, since these biosensors present a defective lux1 synthase gene, the QS mechanisms of the compounds do not seem to involve the inhibition of AHL synthesis.

-Swarming test with compounds MV45 and MV53

The swarming test determines the ability of bacteria to communicate in order to colonize a space. This aspect is also under the control of the QS. A dilution of bacterial culture is transplanted into a Petri dish containing a soft agar allowing the bacteria to diffuse/swarm on the surface of the medium. When these are unable to coordinate, they do not diffuse. In FIG. 10, it is observed that compound MV-45 inhibits the swarming of P. aeruginosa at a concentration of 10 pg/ml, as effectively as furanone, a positive control compound at a concentration of 20 pg/ml. Compound MV-53 at a concentration of 15 μg/ml also has a swarming inhibitory effect comparable to the effect of furanone.

2.4-Conclusion

In conclusion, two compounds extracted from Paraconiothyrium variabile have been shown to exhibit anti-QS activity. The MIC of these compounds was determined at 70 pM, which is clearly above the active concentrations on the QS. There is therefore no antimicrobial effect at active concentrations. This is interesting because it would cause no selection pressure and therefore potentially no resistance. References

Hayouni, E. A., Bouix, M., Abedrabba, M., Leveau, J.-Y., and Hamdi, M. (2008). Mechanism of action of Melalueca armillaris (sol. ex gaertu) sm. essential oil on six lab strains as assessed by multiparametric flow cytometry and automated microtiter-based assay. Food Chem. Ill, 707-718. doi: 10.1016/j.foodchem.2008.04.044

McClean, K.H., Winson, M.K., Fish, L, Taylor, A., Chhabra, S.R., Camara, M., et al. (1997). Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143, 3703-3711. must:

10.1099/00221287-143-12-3703

Chong, Y.M., Yin, W.F., Ho, C.Y., Mustafa, M.R., Hadi, A.H., Awang, K., et al. (2011). Malabaricone

C from Myristica cinnamomea exhibits anti-quorum sensing activity. J.Nat. production 74, 2261-2264. doi: 10.1021/npl00872k

Chen, B.G., Turner, L, and Berg, H.C. (2007). The wetting agent required for swarming in

Salmonella enterica serovar typhimurium is not a surfactant. J. Bacteriol. 189, 8750-8753. must:

10.1128/JB.01109-07

Claims

1. Compound of formula (I):

(I)

in which

NHAIkyl, -N(Alkyl) ₂ in which the alkyl group is C ₁ -C ₄ alkyl, halogen, -O-

H ₂ PO ₃ , alkyloxy in which the alkyl group is a C ₁ -C ₄ alkyl;

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

2. Compound of formula (la):

(the)

in which

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

3. Compound of formula (lb):

(lb) in which

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

4. Compound according to any one of the preceding claims, in which,

- R ₃ independently represents a methyl group;

- R ₄ independently represents a C ₁ -C ₆ alkyl group having at least two unsaturations; and in which the R ₁ and R ₂ groups can also form a cycle of the lactone type.

5. Compound according to any one of the preceding claims, in which,

- R independently represents a C ₁ -C ₄ alkyl group, saturated or unsaturated, linear or branched, which may be substituted by at least one -OH, -CN, -SO ₃ H, -SH, -NH ₂ group, - NHAIkyl, -N(Alkyl) ₂ in which the alkyl group is a C ₁ -C ₄ alkyl, halogen, alkyloxy in which the alkyl group is a C ₁ -C ₂ alkyl;

CN, halogen, alkyloxy in which the alkyl group is methyl;

- R ₃ independently represents a methyl group;

- R ₄ independently represents a C ₁ -C ₆ alkyl group having two unsaturations; and in which the R ₁ and R ₂ groups can also form a cycle of the lactone type.

6. Compound according to any one of the preceding claims represented by the following formulas:

7. Compound according to any one of the preceding claims represented by the following formulas:

8. Composition comprising individually or as a mixture the compounds of formula

(la) and (Ib):

(la) (Ib)

in which

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

9. Composition according to Claim 8, characterized in that the compounds of formula (Ia) and (Ib) are such that:

- R ₃ independently represents a C ₁ -C ₄ alkyl group;

- R ₄ independently represents a C ₁ -C ₆ alkyl group having at least one unsaturation and in which the R ₁ and R ₂ groups can also form a cycle of the lactone type; and wherein the ratio between the compounds (Ia)/(Ib) varies from 100/0 to 0/100 excluding the 50/50 ratio.

10. Composition according to claim 8 or claim 9 comprising individually or as a mixture the compounds of formula (Ia) and (Ib) according to:

11. Composition according to any one of claims 8 to 10 also comprising an antibacterial agent.

12. Composition according to any one of claims 8 to 11 also comprising an anionic surfactant such as alkali metal, in particular sodium, alkyl sulphates, a nonionic surfactant such as an ethoxylated alkylphenol, a cationic surfactant or else a mixture of one or more of these agents, in particular a mixture of one or more cationic surfactant(s) and one or more nonionic surfactant(s) or of one or more several anionic surfactant(s) and one or more nonionic surfactant(s).

13. Composition according to any one of claims 8 to 12 also comprising at least one bactericidal acid surfactant, such as in particular an alkyl acid polyether carboxylic acid, an alkenyl polyether carboxylic acid, an alkylaryl polyether carboxylic acid or an alkoxylated nonionic surfactant phosphoric ester, in acidic or partially neutralized form.

14. Composition according to any one of claims 8 to 13 also comprising an inorganic salt such as copper oxide or hydroxide is chosen from copper hydroxide, copper oxychloride, copper carbonate, copper cuprous oxide or mixtures thereof.

15. Paraconiothyrium variabile extract comprising at least 35% by weight of MV53 and/or MV45 and/or MV46.

16. Compound according to any one of claims 1 to 7 or composition according to any one of claims 8 to 14 or extract according to claim 15 for their use for the prevention and / or treatment of bacterial infections due to bacteria forming a biofilm.

17. Use of a compound according to any one of claims 1 to 7 or composition according to any one of claims 8 to 14 or extract according to claim 15 for inhibiting the formation of bacterial biofilm.

18. Use according to claim 17 for preventing the formation and promoting the elimination of bacterial biofilms in industrial equipment, pipes and medical devices.