WO2011031964A2 - Inhibiteurs de la formation de biofilm - Google Patents

Inhibiteurs de la formation de biofilm Download PDF

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Publication number
WO2011031964A2
WO2011031964A2 PCT/US2010/048422 US2010048422W WO2011031964A2 WO 2011031964 A2 WO2011031964 A2 WO 2011031964A2 US 2010048422 W US2010048422 W US 2010048422W WO 2011031964 A2 WO2011031964 A2 WO 2011031964A2
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WIPO (PCT)
Prior art keywords
inhibitor
squalene
composition
synthesis
phytoene
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PCT/US2010/048422
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English (en)
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WO2011031964A3 (fr
Inventor
Daniel Lopez
Benjamin Hatton
Roberto Kolter
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President And Fellows Of Harvard College
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Publication of WO2011031964A2 publication Critical patent/WO2011031964A2/fr
Publication of WO2011031964A3 publication Critical patent/WO2011031964A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A01N43/80—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,2
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
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    • A—HUMAN NECESSITIES
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    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/46—Deodorants or malodour counteractants, e.g. to inhibit the formation of ammonia or bacteria
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    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
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    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/046—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • A—HUMAN NECESSITIES
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    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
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    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
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    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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Definitions

  • the present invention relates to the field of microbiology and the inhibition of biofilm formation of microbes.
  • bacerial infection remains a major impediment to the utility of medical implants including catheters artificial prosthetics and subcutaneous sensors.
  • Indwelling devices are responsible for over half of all nonsocomial infections, with an estimate of 1 million cases per year in the U.S. alone.
  • Device-associated infections are the results of bacterial adhesion and subsequent biofilm formation at the implantation site.
  • Conventional antibiotic therapies remain ineffective against biofilms. The lack of suitable treatment often leaves extraction of the contaminated device as the only viable option for eliminating the biofilm.
  • implant-associated infections are the result of bacterial adhesion to a biomaterial surface.
  • tissue integration occurs prior to appreciable bacterial adhesion, thereby preventing colonization at the implant.
  • Host defenses are often not capable of preventing further colonization if bacterial adhesion occurs before tissue integration.
  • a 6 hour post-implantation "decisive period" has been identified during which prevention of bacterial adhesion is critical to long-term success of an implant (Poelstra et al., J. Biomed. Mater. Res., 60: 206 (2002)). Over this period an implant is particularly susceptible to surface colonization.
  • Biofilms are remarkably resistant to both the immune response and systemic antibiotic therapies, and thus their development is the primary cause of implant-associated infection.
  • the formation of a pathogenic biofilm ensues from the initial adhesion of bacteria to an implant surface. Inhibiting bacterial adhesion is regarded as the most critical step to preventing implant associated infection.
  • the most common pathogens that cause implant infections are Gram-positive Staphylococcus aureus and Stephylococcus epidermidis.
  • Other bacteria implicated in implant-associated infections are Gram-negative Eschericia coli, Pseudomonas aeruginosa and Proteus group bacteria (e.g., P. mirabilis and P. vulgaris).
  • biofilm producing bacteria cause biofouling of surfaces exposed to aquatic environments.
  • surfaces of ships such as the hull, offshore marine structures such as oil rigs, sea water conduit systems for seaside plants, buoys, heat-exchangers, cooling towers, de-salination equipment, filtration
  • membranes, docks, and the like may all experience some degree of fouling when continually exposed to water. In the case of ships, fouling can inhibit vessel performance and
  • fouling may substantially increase fuel consumption and may necessitate extensive and more frequent maintenance, all of which raise the overall costs of operation.
  • Biofouling can have a direct adverse economic impact when it occurs in industrial process waters, for example in cooling waters, metal working fluids, or other recirculating water systems such as those used in papermaking or textile manufacture. If not controlled, biological fouling of industrial process waters can interfere with process operations, lowering process efficiency, wasting energy, plugging the water-handling system, and even degrade product quality.
  • One aspect of the invention relates to a composition for inhibiting bacterial biofilm formation comprising a carrier and an effective amount of an inhibitor of
  • the inhibitor of squalene/phytoene synthesis inhibits HMG-CoA Reductase. In one embodiment, the inhibitor of
  • the squalene/phytoene synthesis inhibits squalene synthase.
  • the inhibitor of squalene/phytoene synthesis inhibits 1-deoxy-D-xylulose 5-phosphate synthase.
  • the inhibitor of squalene/phytoene synthesis may be selected from the group consisting of a phosphonosulfonate, a statin, zaragozic acid, clomazone, and lapaquistat acetate or a functional derivative thereof.
  • the inhibitor of squalene/phytoene synthesis is a
  • the phosphono sulfonate or a functional derivative thereof.
  • the phosphono sulfonate or a functional derivative thereof.
  • the inhibitor of squalene/phytoene synthesis described in the various embodiments above is a statin or a functional derivative thereof.
  • the statin is selected from the group consisting of mevastatin, lovastatin, atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.
  • the inhibitor of squalene/phytoene synthesis may be zaragozic acid or a functional derivative thereof.
  • the inhibitor of squalene/phytoene synthesis may be clomazone or a functional derivative thereof.
  • the carrier may be a liquid, a solid, semi-solid, slurry or paste.
  • the carrier may be a coating agent.
  • Another aspect of the present invention relates to a solid or semi-solid substrate comprising an inhibitor of squalene/phytoene synthesis.
  • the inhibitor of squalene/phytoene synthesis is deposited or absorbed to a surface of the substrate with a composition described above.
  • the substrate described may be formulated to contain the inhibitor of squalene/phytoene synthesis throughout its entire composition.
  • one or more of the substrates described herein further comprises additional inhibitors of biofilm or antibacterial agents incorporated therein.
  • one or more of the substrates described herein is formed as a device, or part thereof, for implantation into a living subject.
  • Another aspect of the present invention relates to a method for inhibiting bacterial biofilm formation comprising contacting biofilm producing bacteria with an effective amount of an inhibitor of squalene/phytoene synthesis.
  • the inhibitor inhibits HMG-CoA Reductase.
  • the inhibitor is a statin or a functional derivative thereof.
  • the inhibitor inhibits squalene synthase.
  • contacting occurs in vivo.
  • the contacting occurs in a mammal. In one embodiment of the various methods described herein, the contacting occurs in vitro. In one embodiment of the various methods described herein, contacting occurs in a non-living medium.
  • the inhibitor is formulated as an antiseptic. In one embodiment of the various methods described herein, the inhibitor is a phosphono sulfonate. In one embodiment, inhibitor inhibits 1-deoxy-D-xylulose 5-phosphate synthase. In one embodiment, the inhibitor is clomazone or a functional derivative thereof. In one embodiment of the various methods described herein, the inhibitor is zaragozic acid, a statin, or lapaquistat acetate or a functional derivative thereof.
  • the statin is selected from the group consisting of mevastatin, lovastatin, atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.
  • the phosphono sulfonate is selected from the group consisting of BPH-652, BPH-689, BPH-700.
  • contacting occurs in the presence of an additional agent that impacts the growth and/or attachment and/or virulence of a biofilm forming organism.
  • Figures 1A to IB contain data that indicate that yisP affects the pathway to biofilm formation.
  • Figure 1A is a schematic representation of the signaling pathway leading to biofilm formation in B. subtilis. The pathway is triggered by activation of the master regulator SpoOA, via phosphorylation by KinC. Dashed lines represent indirect activation.
  • Figure IB is a schematic representation of putative metabolic pathway to the formation of distinct polysioprenoids in B. subtilis. Enzymes discussed in the text are written in red, next to the reaction they catalyze. Dashed lines represent unknown steps.
  • Figure 2 contains data that show that YisP has squalene synthase activity and is involved in the production of a carotenoid.
  • Figure 2 is a bar graph that represents data from experiments showing the enzymatic activity of purified YisP from B. subtilis under different conditions. FPP at 3.7 ⁇ was used as substrate under the optimal conditions specified in Figure 5. Control reaction was performed with no enzyme added.
  • FIG 3 is a schematic representation of the two distinct biochemical pathways to produce squalene in B. subtilis and S. aureus, and where inhibition of biofilm formation by sterol-lowering drugs occurs within these pathways.
  • Zaragozic acid acts as a competitive inhibitor in both routes, since it acts downstream of the formation of FPP.
  • Statins such as mevastin and lovastatin inhibit the enzyme HMG-CoA reductase and thus, the route to produce squalene in S. aureus.
  • Clomazone inhibits the enzyme 1-deoxy-D- xylulose 5-phosphate synthase and thus, the route to produce squalene in B. subtilis.
  • Figure 4A shows that yisP affects the pathway to biofilm formation upstream of SpoOA.
  • Figure 4A is a schematic representation of the regulatory pathway activated by SpoOA to induce biofilm formation.
  • the active phosphorylated form of SpoOA (SpoOA ⁇ P) inhibits the expression of two regulatory repressors, AbrB and SinR, which in turn inhibit the expression of the structural genes downstream of SpoOA. Dashed lines indicate the regulation is not direct.
  • Figures 5A to 5B contain data that show enzymatic characterization of YisP as a squalene synthase.
  • Figure 5A is a graph of enzymatic activity of purified YisP using farnesyl pyrophosphate (FPP) as a substrate.
  • the V max and K m [FPP] of the equation are presented as ml and m2, respectively.
  • Figure 5B contains numeric data for biochemical characterization of YisP, for example, optimal conditions and different affinities for several substrates (represented as their respective K m values).
  • the typical competitive inhibitor of squalene synthases, zaragozic acid inhibits the activity of the hypothetical squalene synthase YisP (IC 50 represented).
  • YisP acts as a magnesium-dependent squalene synthase, which preferentially uses FPP as a substrate.
  • GPP is geranyl pyrophosphate
  • GGPP is geranylgeranyl
  • Figures 6A to 6B show graphical representation of chemical analyses of extracts from AyisP mutant.
  • Figure 6A is a comparison of the molecular traces of the wild- type strain and the AyisP mutant analyzing the extract from the pellet of cultures using LC- MS. The wild type trace has been offset to overlay the two traces when observed at 280 nm. The major differences are two peaks present in the wild type whose mass were determined to be 738 Da and 600 Da.
  • Figure 6B is a UV-VIS spectrum of the major different peak eluting at 16.9 min on the wild type trace. This spectrum does not match any previously identified molecule and no clear identification could be made.
  • FIG. 7 shows that exogenous polysioprenoids restore biofilm formation to a yisP mutant.
  • Biofilm formation requires the production of an extracellular matrix, which is correlated with the amount of wrinkles observed in the colony (Branda et al. 2001 PNAS 98:11621-11626; Branda et al. 2006 Mol Microbiol 59:1229-1238). Wild type and the AyisP mutant represent positive and negative controls, respectively.
  • Treatment with diverse polyisoprenoids (squalene, ⁇ -carotene, and retinol) partially restored the formation of wrinkles in the AyisP mutant.
  • Figure 8 is a graphical representation of quantitative analysis of biofilm formation in S.
  • Figure 9 is a graphical representation of data obtained from quantitative analysis of the activation of the route of biofilm formation in presence of zaragozic acid.
  • Matrix production was monitored using the reporter P yqx M-yfp, a transcriptional fusion of the operon yqxM-sipW-tasA responsible for the expression of the matrix-associated protein TasA Signal was measured in cultures treated and non-treated with zaragozic acid using flow cytometry.
  • MSgg most of cells show maximun expression of the yqxM operon (profile with peak at the right).
  • zaragozic acid was added, the expression of yqxM operon dramatically dropped in most cells (profile with peak in between). Control is represented by a non-labeled strain (profile with peak to the left). No defect in growth was detected.
  • Table 1 Proteins present in the DRM of B. subtillis and S. Aureus. Proteins associated to the DRM fraction of B. subtilis and S. aureus were sequenced by mass spectrometry are listed. KinC has an asterisk to notice that it was detected in DRM fractions using western blot.
  • Table 4 Bacteria which synthesize squalene from GA3P
  • Table 5 Bacteria which synthesize squalene from HMG-CoA
  • Table 7 Differential Gene expression of the AyisP mutant compared to the wild type.
  • an "effective amount”, as the term is used herein, refers to an amount that inhibits biofilm formation in the intended context. Such context may be in the presence or absence of other agents that inhibit the bacterial growth or biofilm formation/function.
  • Inhibit refers to either partial or complete inhibition of biofilm formation, and is expected to be a reproducibly detectable, statistically significant amount of inhibition, as determined by means known in the art.
  • An "indwelling device” is a device that is invasive, placed in or planted within the body, and is associated with a risk of infection.
  • a “carrier”, as the term is used herein, is an agent or combination of agents formulated to facilitate functional delivery of the inhibitor of squalene/phytoene synthesis to the desired location (e.g., to an external surface, or for in vivo administration).
  • the form the carrier takes will depend upon and/or dictate the intended use of the composition.
  • the carrier may be in liquid form as a solution, dispersion, emulsion, suspension, paste, powder, solid or semi-solid, to result in a composition of similar form.
  • Coating agents are formulations whereby when applied to a surface, a layer or residue of an effective amount of the inhibitor is left deposited on that surface, to thereby inhibit biofilm formation on the surface.
  • coating agents include, without limitation, paints, stains, sealants, waxes, and cleaning products such as disinfectants.
  • the surface is either external or internal, and is exposed to fluid which may contain biofilm forming bacteria.
  • contacting refers to the accomplishment of physical contact of an inhibitor to a bacteria, to an extent which promotes inhibition of biofilm formation by the inhibitor of squalene/phytoene synthesis contained within the agent.
  • multicellular organism is used to refer to an organisms which may be subject to the attack of a biofilm producing organism.
  • the multicellular organisms can be an animal, e.g. mammal. Mammals include rodents (e.g., mice, rats, rabbits, guinea pigs) livestock and pets (e.g., goats, sheep, horses, pigs, cattle, cats, dogs) and primates (humans, chimpanzees, gorillas, etc.).
  • rodents e.g., mice, rats, rabbits, guinea pigs
  • pets e.g., goats, sheep, horses, pigs, cattle, cats, dogs
  • primates humans, chimpanzees, gorillas, etc.
  • aspects of the present invention relate to the finding that biofilm formation in bacteria is dependent upon squalene/phytoene synthesis. Inhibition of this synthesis, e.g. by inhibiting one or more components of the synthetic pathway, inhibits the ability of the bacteria to produce biofilm. Such inhibition has several advantageous applications since the production of biofilm causes a wide variety of detrimental consequences, from increasing resistance of pathogens to host immune defenses, to aggregation of biofouling organisms to non-living surfaces to cause degredation.
  • compositions for inhibiting biofilm formation comprises an effective amount of an inhibitor of
  • squalene/phytoene synthesis and a carrier.
  • the carrier is an agent for delivery of the inhibitor to the location where one desires to inhibit biofilm producing bacteria.
  • squalene synthesis There are two known pathways of squalene synthesis in bacteria, illustrated in Figure 3. Strains such as B. subtillis synthesize squalene from GA3P, whereas strains such as S. aureus synthesize squalene from HMG-CoA. Both pathways converge downstream to produce IPP.
  • the GA3P pathway is characterized in Takahashi et al., (PNAS USA 95: 9879-9884 (1998)).
  • Bacteria that synthesize squalene from GA3P can be inhibited by specific inhibition of this upstream pathway (e.g., inhibition of 1-deoxy-D-xylulose 5-phosphate synthase).
  • Bacteria which are known to synthesize squalene from GA3P are listed in Table 4.
  • the HMG-CoA pathways is characterized in Imogen Wilding et al., (Journal of Bacteriology, 182: 4319-327 (2000)).
  • Bacteria that synthesize squalene from HMG-CoA can be inhibited by specific inhibition of this upstream pathway (e.g., inhibition of HMG-CoA Reductase).
  • the inhibitor of squalene/phytoene synthesis inhibits HMG- CoA reductase.
  • HMG-CoA reductase is inhibited, for example, by statins.
  • Statins are thought to competatively inhibit the bacterial HMG-CoA similarly to their inhibition of human HMG-CoA reductase.
  • a number of statin molecules are known in the art, such as mevastatin, lovastatin (U.S. Patent No. 4,231,938), atorvastatin (U.S. Patent No. 4,681,893; U.S. Patent No. 5,273,995), cerivastatin (U.S. Patent No. 5,006,530; U.S. Patent No.
  • lovastatin and simvastatin are typically administered to a subject in lactone form, and are converted to the active hydroxy acid in the liver. It may be preferable to instead use the hydroxy acid (prodrug) form itself, especially in situations where such processing is not expected to significantly occur.
  • the salts of the specific statin may also be used, e.g. the sodium salt of pravastatin, or the calcium salt of atorvastatin, rosuvastatin, or pitavastatin. It may be useful to include in the compositions and methods described herein, agents which help stabilize and/or solubilize the statin molecule (e.g., a basifying agent, such as magnesium oxide). Such agents are described, for example, in U.S. Patent No.
  • composition of the present invention specifically excludes inclusion of one or more statins (e.g., the specific statins disclosed herein).
  • statins e.g., the specific statins disclosed herein.
  • Other known inhibitors of HMG-CoA reductase can also be used in the compositions and methods described herein.
  • the inhibitor of squalene/phytoene synthesis inhibits 1-deoxy- D-xylulose 5-phosphate synthase.
  • 1-deoxy-D-xylulose 5-phosphate synthase is inhibited, for example, by clomazone (2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone, described in U.S. Patent No. 4,405,357).
  • Clomazone is a potent herbicide.
  • Other known inhibitors of 1-deoxy-D-xylulose 5-phosphate synthase can also be used in the compositions and methods described herein.
  • the inhibitor of squalene/phytoene synthesis inhibits squalene synthase.
  • Squalene synthase is inhibited, for example, by zaragozic acid.
  • Zaragozic acids are a family of natural products produced by fungi. This family of natural products possesses a unique 4,8-dioxabicyclo[3.2.1]octane core. Zaragozic acids are potent inhibitors of S.
  • Squalene synthase is the first committed enzyme in sterol synthesis, catalyzing the reductive condensation of farnesyl pyrophosphate to form squalene (Do et al., Clin. Genet. 75 (1): 19-29 (2009)).
  • zaragozic acid produces lower plasma cholesterol levels in primates (Bergstrom et al., Annu. Rev. Microbiol. 49: 607-39 (1995).
  • zaragozic acid A Treatment of rats with zaragozic acid A caused an increase in hepatic low density lipoprotein (LDL) receptor mRNA levels (Ness et al., Arch. Biochem. Biophys. 311 (2): 277-85 (1994)). Zaragozic acids also inhibit Ras farnesyl-protein transferase (Dufresne et al., J. Nat. Prod. 56 (11): 1923-9 (1993)).
  • LDL low density lipoprotein
  • Squalene synthase is also inhibited by lapaquistat acetate, the active ingredient in TAK 475, is a known squalene synthase inhibitor (Nishimoto et al., British Journal of Pharmacology 139: 911-918 (2003)). Its chemical name is l-[[(3R,55)-1-(3-acetoxy-2,2- dimethylpropyl)-7-chloro-5-(2,3-dimethoxyphenyl)-2-oxo-1,2,3,5-tetrahydro-4,l- benzoxazepin-3-yl]acetyl]piperidine-4-acetic acid (Nishimoto et al., Br J
  • Phosphonosulfonates also referred to in the art as a-phosphonosulfonates, are also inhibitors of squalene synthase.
  • Phosphonosulfonates and their synthesis are known in the art (U.S. Patent No. 5,712,396, U.S. Patent No. 5,618,964, U.S. Patent No. 5,567,841, U.S. Patent No. 5,332,728).
  • the phosphonosulfonates BPH-652, BPH-698, and BPH-700, BMS-187745 and BMS-188494 can be used in the present invention.
  • squalene synthase examples include, without limitation, bisphosphonates (e.g. those disclosed in U.S. Patent No. 5,157,027, U.S. Patent No. 4,871,721), and phosphinylformic acid (U.S. Patent No. 5,025,003).
  • RPR 107393 ⁇ 3- hydroxy-3-[4-(quinolin-6-yl)phenyl]-1-azabicyclo[2-2-2]octane dihydrochloride ⁇ and its R and S enantiomers is another squalene synthase inhibitor (Amin et al., J Pharmacol Exp Ther. 281: 746-752 (1997)).
  • ER-27856 (4-[N-[(2E)-3-(2-Methoxyphenyl)-2-butenyl]-N- methylamino]-l,l-butylidenebisphosphonic acid tris (pivaloyloxymethyl) ester) is another squalene synthase inhibitor (Hiyoshi et al., J Lipid Res. 41: 1136-44 (2003); Hiyoshi et al., J Lipid Res. 44: 128-35(2003)).
  • Quinuclidine derivatives comprising pyrrolidine derivatives are also known squalene synthase inhibitors (U.S.
  • Patent Publication 2004/00730405 N-aryl-substituted cyclic amine derivatives such as those disclosed in U.S. Patent Publication 2004/0072830 and U.S. Patent No. 7,112,593.
  • BMS-187745 chemical name (S)-(-)-4-(3- Phenoxyphenyl)-1-phosphonobutanesulfonic acid
  • BMS-188494 prodrug ester
  • EP2300 compounds such as EP2306 and EP2302, two novel 2-biphenylmorpholine derivatives, also inhibit squalene synthase
  • compositions described herein can be used to prevent biofilm formation, and/or can be applied to existing bacteria in a biofilm to help degrade the biofilm.
  • a functional derivative can be the molecule itself with an additional component, e.g. derivatized to enhance half-life, delivery, solubility, etc.
  • a functional derivative can be a portion of the molecule which retains the desired biological activity (biofilm inhibition).
  • the carrier facilitates functional delivery of the inhibitor of squalene/phytoene synthesis to the desired location where biofilms may form (e.g., to an external surface, or for in vivo administration).
  • the carrier is a coating agent.
  • Coating agents are
  • coating agents include, without limitation, paints, stains, sealants, waxes, and cleaning products such as disinfectants.
  • the coating agent carrier is a polymer coating.
  • the coating agent will be formulated for the specific surface on which the inhibitor is to be delivered (a substrate surface).
  • the coating agent is formulated to adhere to or be absorbed by silicone.
  • the coating agent is formulated to adhere to or be absorbed by a solid polymer (e.g. to a polymeric substrate such as polyvinyl chloride).
  • the coating agent is formulated to adhere to a metal or a metallic surface (e.g., to inhibit fouling by biofilm producing bacteria, such as degredation of components as in rust, or to inhibit clogging).
  • the carrier is an "active coating" such as those developed for deposition and delivery of antibacterial agents to a surface.
  • the carrier itself is a coating agent with antibacterial properties, e.g., a passive coating or as an active coating, each of which themselves, may have other antibacterial properties.
  • a number of synthetic surface and coatings that resist bacterial colonization are known in the art and can be formulated to contain (act as carriers of) the inhibitors of squalene/phytoene synthesis described herein. "Passive coatings" reduce bacterial adhesion by altering the physiochemical properties of the substrate so that conditioning films do not form and/or bacteria-substrate interactions are not favorable.
  • passive coatings are poly(ethylene glcol) (Kingshott et al., Langmuir, 2003, 19, 6912), poly(ethylene oxide) brushes (Kaper et al., J. Biomater. ScL, Polym. Ed., 2003, 14, 313), hydrophilic
  • Active coatings are designed to release high initial fluxes of agents (e.g., antibacterial) during the critical shot term post- implantation period (hours) to inhibit the initial adhesion of bacteria. Coatings which actively release agents (e.g., antibiotics) over a longer time period (weeks to months) have also been developed. Such coatings can be adapted for deposition and release of the inhibitors disclosed herein.
  • agents e.g., antibacterial
  • Coatings which actively release agents e.g., antibiotics
  • Such coatings can be adapted for deposition and release of the inhibitors disclosed herein.
  • One example is controlled delivery from polymer coatings (e.g., polyurethane, silicone, rubber, polyhydroxyalkanoates etc.) (Schierholz et al., Biomaterials, 1997, 18, 839; Rossi et al., Antimicrob. Chemother., 2004, 54, 1013).
  • an additional thin polymer layer can be applied on top of the agent-loaded polymer (e.g, via radio-frequency glow discharge plasma deposition (Kwok et al., J. Controlled Release, 1999, 62, 301).
  • biodegradable polymers e.g., polystyrene
  • lipid-like carriers e.g., poly-D,L-lactic acid, tocopherol acetate, Softisan 649, Dynasan 118
  • a new anti- infective coating of medical implants to prevent biofilm formation on lipid-like carriers e.g., poly-D,L-lactic acid, tocopherol acetate, Softisan 649, Dynasan 118
  • antibiotics to incorporate into the composition of the present invention include, without limitation, vancomycin, tobramycin, cefamandol, cephalothin, carbenicillin, amoxicillin, ciprofloxacin, and gentamicin.
  • the coating agent is formulated to adhere to a biomaterial surface, such as teeth, bone, skin, etc.
  • the coating agent is formulated to adhere to or be absorbed by a fabric, cloth or membrane, such as a bandage or other wound dressing.
  • a membrane is a water treatment membrane.
  • the carrier is formulated for inclusion into a product for application to a body surface, such as personal care product, to thereby inhibit biofilm formation on the body surface.
  • the carrier is formulated to adhere to a device that is to contact a living medium (the medium around or within a multicellular organism), to thereby inhibit biofilm formation on the device.
  • a living medium the medium around or within a multicellular organism
  • biofilm formation on the device For example, to be delivered, contacted into, or otherwise implanted, into a living multicellular organism.
  • Such devices are sometimes referred to in the art as indwelling devices. Examples of such devices include, without limitation, catheters, surgical implants, prosthetic devices, surgery tools, endoscopes, contact lenses, etc.
  • a composition of the present invention may be prepared in solid form.
  • the carrier and inhibitor(s) may be formulated together as a powder or tablet using means known in the art.
  • the tablets may contain a variety of excipient known in the tableting art such as dyes or other coloring agents, and perfumes or fragrances.
  • Other components known in the art such as fillers, binders, glidants, lubricants, or antiadherents may also be included. These latter components may be included to improve tablet properties and/or the tableting process.
  • composition may optionally be prepared as a concentrate for dilution prior to its intended use (e.g., application to a substrate surface).
  • compositions or formulations that contain an effective amount of an inhibitor of squalene/phytoene synthesis, described herein, can be applied to a substrate surface as an antibiofilm coating.
  • a surface may be treated by applying a suitable amount of a coating that comprises one or more squalene/phytoene synthesis inhibitors described herein.
  • the coating composition is applied in an amount which is effective to suppress the settlement and/or growth of biofilm forming bacteria and/or enable their facile release by the application of an external shear stress.
  • the mode of applying the coating may vary.
  • the composition may be applied to a surface using a brush or mechanical sprayer.
  • the surface may be dipped, submerged, or infused with the coating.
  • the present invention relates to the product which is generated by application of the compositions described herein to a substrate surface.
  • the present invention encompasses such substrates described herein which have an effective amount of the inhibitor of squalene/phytoene synthesis deposited on or absorbed to their surface, following application of the composition described herein.
  • the invention does not include live or living substrates, especially human.
  • This includes, without limitation, metal substrates, silicone and other polymeric substrates.
  • This also includes substrates designed for specific products which are particularly susceptible to biofilm formation, e.g, products designed for contact and/or implantation into the body of a multicellular organisms, including without limitation, catheters, medical implants, surgery tools, endoscopes, contact lenses, wound dressings. It further includes substrates designed for products such as components of cooling towers, heat exchanger or warm water systems, pipelines (e.g., oil, gas, water).
  • the inhibitor described herein will be available to at least some of the bacteria such that it may inhibit at least some of the bacteria's squalene/phytoene synthesis. This can occur, for example, through slow release of the inhibitor from the formulation, composition, or substrate described herein into the surrounding environment. Biofouling
  • the invention relates to a composition which comprises a carrier and an effective amount of an inhibitor of squalene/phytoene synthesis as described herein, which is formulated for application to a substrate surface (e.g., non-living) to inhibit biofouling of the surface.
  • a substrate surface e.g., non-living
  • Two parallel lines of coatings research and development aimed at reducing fouling have predominated: biocide containing coatings and low surface energy, "non-stick,” fouling release coatings.
  • Such coatings may optionally contain other additional components to prevent microbial growth and/or biofilm production.
  • Such coatings may be formulated to contain resins (e.g., aldehyde resins), plasticizers, film consumption regulators, solvents.
  • Resins such as aldehyde resins are easily prepared by the alkaline
  • Aldehyde resins useful as vehicle in the present invention are prepared by alkaline condensation of starting compounds of the general formula Ra— CH— (OH)— Rb— CH.dbd.O, where Ra and Rb are non-aromatic organic residues thereby conducting the condensation with elimination of water and other volatile substances in such a way that the final product of condensation preferably contains about 4 to 6 carbon atoms per oxygen atom present in the resin molecule.
  • Phthalate plasticizers such as dioctyl phthalate, dimethyl phthalate or dicyclohexyl phthalate; aliphatic dicarboxylate plasticizers such as diisobutyl adipate or butyl sebacate; glycol ester plasticizers such as diethylene glycol dibenzoate or pentaerythritol alkanoic ester; phosphate plasticizers such as tricresyl phosphate or trichloroethyl phosphate; epoxy plasticizers such as epoxydized soybean oil or epoxydized octyl stearate; and other plasticizers such as trioctyl trimellitate or triacetin.
  • Film consumption regulators are used to retard the rate of dissolution of the surface coating of the present invention. These include, without limitation, chlorinated paraffin, oil, wax, vaseline and liquid paraffin, polyvinyl ether, polypropylene sebacate, partially hydrogenated terphenyl, polyvinyl acetate, polyalkyl (meth)acrylate, alkyd resin, polyester resin, polyvinyl chloride, silicone, epoxy resin, polyurethane resin, urea resin and other hydrophobic polymers having satisfactory compatibility and a low glass transition temperature which retard the rate of dissolution of the paint are useful in the present invention.
  • Other additives which promote film consumption such as monobasic cyclic organic acids such as rosin, monobutyl phthalate or monooctyl succinate; oleic acid and castor oil acid, may also be used.
  • Solvents include, without limitation, hydrocarbons such as xylene, toluene, ethylbenzene, cyclopentane, octane, heptane, cyclohexane or white spirit; ethers such as dioxane, tetrahydrofuran, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol dimethyl ether, ethylene glycol monobutyl ether, ethylene glycol dibutyl ether, diethylene glycol monomethyl ether or diethylene glycol monoethyl ether; esters such as butyl acetate, propyl acetate, benzyl acetate, ethylene glycol monomethyl ether acetate or ethylene glycol monoethyl ether acetate; ketones such as methyl isobutyl ketone or ethyl isobutyl ketone; and alcohols such as n-butanol
  • Another aspect of the present invention relates to a substrate that is formulated to contain the inhibitor of squalene/phytoene synthesis within/throughout its entire composition to thereby inhibit biofilm formation.
  • Such substrates have the inhibitor(s) added during their generation.
  • Such substrates may be created to take any useful form, e.g., solid, semi-solid, or gels.
  • examples are polymers (e.g., polyethylene, silicone, polyvinyl chloride, polypropylene, polystyrene, polytetrafluoroethylene, polyurethane, polyamide, polyacrylamide, resins).
  • the substrate is a formed solid, which has been formed into the desired shape and becomes solid or semi- solid upon curing/hardening during the process of manufacture.
  • the substrate is ductile or plastic, or malleable (e.g., a fabric, a moveable pliable solid, a gel such as a hydrogel)).
  • Substrates which resist or prevent biofilm formation are useful in the production of products which are resistant to biofilm formation, such as surgical implants, artificial heart valves, catheters, membrane filtration devices, materials for wound treatment (e.g., chronic wounds).
  • Such substrates may further comprise additional inhibitors of biofilm formation or antibacterial agents incorporated therein, as described herein and in the art.
  • the substrate of the present invention specifically excludes inclusion of one or more statins (e.g., the specific statins disclosed herein).
  • Another aspect of the present invention relates to a method for inhibiting bacterial biofilm formation comprising contacting a biofilm producing bacteria with an effective amount of an inhibitor of squalene/phytoene synthesis, as described herein.
  • Such methods utilize one or more of the inhibitors of squalene/phytoene synthesis described herein.
  • the inhibitor(s) is in the form of a composition and/or substrate for inhibition of biofilm formation, discussed herein.
  • Such methods may further include the contacting of the biofilm producing bacterial with one or more agents that affects the growth and/or attachment and/or virulence of a biofilm producing bacterial, such as biocidal agents, bacteriostatic agents, antibacterial agents (e.g., antibiotics) and/or other biofilm inhibiting agents (e.g., in addition to the squalene/phytoene synthesis inhibitors disclosed herein) known in the art.
  • agents that affects the growth and/or attachment and/or virulence of a biofilm producing bacterial such as biocidal agents, bacteriostatic agents, antibacterial agents (e.g., antibiotics) and/or other biofilm inhibiting agents (e.g., in addition to the squalene/phytoene synthesis inhibitors disclosed herein) known in the art.
  • the inhibitor may be contained within an agent for delivery of the inhibitor to the desired location for the contacting.
  • the agent can be a composition or formulation, or a substrate described herein.
  • the agent may retain the inhibitor in a form which functions to inhibit biofilm formation upon contact of the bacteria to the agent.
  • the agent may release the inhibitor (e.g., slowly over time) such that it sufficiently contacts the bacteria to thereby inhibit biofilm formation.
  • the contacting occurs in vivo, e.g., as described herein for in vivo uses of formulations, compositions and substrates of the present invention.
  • In vivo contacting includes contacting an external surface of the body of a multicellular organism (e.g., an animal as described herein), as well as contacting internal to the body of a subject.
  • the in vivo contacting occurs in the presence of one or more additional agents that impacts the growth and/or virulence of a pathogenic biofilm forming organism, such as microbiocidal agents, bacteriostatic agents, antibacterial agents (e.g., antibiotics) and/or biofilm inhibiting agents (e.g., in addition to the squalene/phytoene synthesis inhibitors disclosed herein) known in the art.
  • the additional agent may be present in the same formulation, or may be contacted (e.g., administered) separately to the subject.
  • the method excludes the administration (e.g., intravenouse, oral) of statins (e.g., one or more specific statins disclosed herein) with a pharmaceutically acceptable carrier, in vivo, to the subject (e.g., human). In one embodiment, the method excludes the
  • mice administration (e.g., intravenous, oral, topical) of an inhibitor (e.g., of squalene synthase inhibitor such as a phosphono sulfonate disclosed herein) in mice.
  • an inhibitor e.g., of squalene synthase inhibitor such as a phosphono sulfonate disclosed herein
  • In vivo administration may be topical (including ophthalmic, vaginal, rectal, intranasal, epidermal, and transdermal), oral or parenteral.
  • Parenteral administration includes intravenous, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g. , by inhalation or insufflation, or intracranial, e.g., intrathecal or intraventricular, administration.
  • the route of administration may be intravenous (I.V.), intramuscular (I.M.), subcutaneous (S.C.), intradermal (I.D.), intraperitoneal (LP.), intrathecal (I.T.), intrapleural, intrauterine, rectal, vaginal, topical, intratumor and the like.
  • the compounds of the invention can be administered parenterally by injection or by gradual infusion over time and can be delivered by peristaltic means. Administration may be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays, for example, or using suppositories.
  • the compounds of the invention are formulated into conventional oral administration forms such as capsules, tablets and tonics.
  • the pharmaceutical composition is formulated into ointments, salves, gels, or creams, as is generally known in the art.
  • the inhibitor is formulated as an antiseptic.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered can be determined by the skilled practitioner for each individual.
  • the carrier may be a pharmaceutically acceptable carrier.
  • Compositions for in vivo administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • Compositions for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Compositions for oral administration include powders or granules, suspensions or solutions in water or nonaqueous media, capsules, sachets or tablets.
  • Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Common additives such as surfactants, emulsifiers, dispersants, and the like may be used as known in the art to increase the solubility of the inhibitor, as well as other components in a composition or system. Such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.
  • the contacting occurs in vitro.
  • the contacting occurs in a non-living medium, such as on a non-living substrate, as described herein, for out of body use.
  • the inhibitor is formulated as one or more of the compositions described herein (e.g., to inhibit biofouling, etc.).
  • the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").
  • other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the invention ("consisting essentially of). This applies equally to steps within a described method as well as compositions and components therein.
  • the inventions, compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method ("consisting of).
  • the present invention may be as defined in any one of the following numbered paragraphs.
  • a composition for inhibiting bacterial biofilm formation comprising a carrier and an effective amount of an inhibitor of squalene/phytoene synthesis.
  • composition of paragraph 1 wherein the inhibitor of squalene/phytoene synthesis is selected from the group consisting of a phosphonosulfonate, a statin, zaragozic acid, clomazone, and lapaquistat acetate or a functional derivative thereof.
  • composition of paragraph 1 wherein the inhibitor of squalene/phytoene synthesis is a phosphonosulfonate or a functional derivative thereof.
  • composition of paragraph 6, wherein the phosphonosulfonate is selected from the group consisting of BPH-652, BPH-689, BPH-700.
  • composition of paragraph 1, 2, or 5, wherein the inhibitor of squalene/phytoene synthesis is a statin or a functional derivative thereof.
  • composition of paragraph 8, wherein the statin is selected from the group
  • statin consisting of mevastatin, lovastatin, atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.
  • composition of paragraph 1, 3 or 5, wherein the inhibitor of squalene/phytoene synthesis is zaragozic acid or a functional derivative thereof.
  • composition of paragraph 1, 4 or 5 wherein the inhibitor of squalene/phytoene synthesis is clomazone or a functional derivative thereof.
  • composition of paragraphs 1-12, wherein the carrier is a solid, semi-solid, slurry or paste.
  • a solid or semi-solid substrate comprising an inhibitor of squalene/phytoene synthesis.
  • the substrate of paragraph 18 or 19 which is formed as a device, or part thereof, for implantation into a living subject.
  • a method for inhibiting bacterial biofilm formation comprising contacting a biofilm producing bacteria with an effective amount of an inhibitor of squalene/phytoene synthesis.
  • statin is selected from the group consisting of mevastatin, lovastatin, atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.
  • a feature common to all living cells is the presence of a lipid membrane that defines the boundary between the inside and the outside of the cell. Proteins that localize to the membrane serve a number of essential functions. In eukaryotic cells, membrane proteins that mediate signal transduction and protein secretion are often localized in membrane microdomains enriched in certain lipids, such as sterols and sphingolipids. These microdomains are commonly referred to as “lipid rafts” or “membrane rafts” (Pike 2006; Lingwood and Simons 2010).
  • lipid rafts The function of proteins associated with lipid rafts depends on the integrity of these areas. Alterations in the conformation of lipid rafts lead to defects in cell-cell signaling processes and transduction pathways in which these proteins are involved. Thus, disruptions of lipid rafts are associated with a large variety of human diseases including Alzheimer's, Parkinson's, cardiovascular and prion diseases (Michel and Bakovic 2007). Because of their profound importance on cell physiology, these membrane domains are interesting targets for the development of new pharmacological approaches to cure and prevent these diseases.
  • lipid rafts have been identified and characterized in eukaryotic cells.
  • many bacterial membrane proteins involved in cell-cell signaling and signal transduction pathways are distributed heterogeneously across the cytoplasmic membrane (Meile et al. 2006). These observations suggest that specialized membrane microdomains are also a feature of bacterial cells.
  • subtilis flotillin-like protein were inconclusive, finding only that its localization was not dependent on lipids containing phosphatidylglycerol or cardiolipin (Donovan and Bramkamp 2009). Cardiolipin was of particular interest because it had been shown to occur in patches in the B. subtilis membrane (Kawai et al. 2004; Matsumoto et al. 2006; Mileykovskaya and Dowhan 2009). Thus, the function and lipid association of bacterial flotillin-like proteins remains poorly understood.
  • All members of the Flotillin family of proteins are members of a superfamily of proteins that contains "SPFH" or "PHB" domains (named after the proteins Stomatin, Prohibitin, Flotillin and HflK C) (Tavernarakis et al. 1999; Browman et al. 2007).
  • SPFH domain-containing proteins are found associated with lipid rafts and are thought to function in many ways, such as in raft formation, kinase activity enhancement, and ion channel regulation (Morrow and Parton 2005; Kato et al. 2006; Browman et al. 2007).
  • bacteria also encode other SPFH proteins. While these proteins are widely distributed in bacteria, their functions remain poorly understood. The few genetic studies carried out on SPFH proteins have not yielded clear phenotypes; however, they appear to be involved in stress responses such as high salt and antibiotic treatment (Butcher and Helmann 2006).
  • bacteria contain lipid rafts that are functionally similar to those found in eukaryotes in that they harbor and organize proteins involved in signal transduction, small molecule translocation and protein secretion.
  • the lipids associated with the bacterial rafts are probably polyisoprenoids synthesized via pathways that involve squalene synthases; inhibitors of this enzyme interfere with the formation of bacterial lipid rafts.
  • a function for the lipid rafts is demonstrated: a mutant devoid of SPFH proteins is defective in a signal transduction pathway whose sensor kinase is found in the rafts. All of these results are consistent with the idea that the organization of physiological processes into microdomains may be a more widespread feature of membranes than previously appreciated.
  • a lipid synthesis gene involved in signaling biofilm formation in B. subtilis A lipid synthesis gene involved in signaling biofilm formation in B. subtilis.
  • sporulenes While B. subtilis membranes do not contain sterols, structurally similar molecules termed sporulenes have recently been described in this bacterium (Bosak et al. 2008; Kontnik et al. 2008) ( Figure IB). Both ergosterol and sporulenes are synthesized from the common precursor isoprenyl pyrophosphate (IPP). However, while ergosterol is derived from squalene, this is not the case for sporulenes. Instead, sporulenes are synthesized through a pathway involving the product of the sqhC gene, a putative polyisoprenoid cyclase that remains uncharacterized (Bosak et al.
  • IPP isoprenyl pyrophosphate
  • B. subtilis forms floating biofilms (pellicles) when cultures are left standing undisturbed. These pellicles are detectable by visual analysis of the cultures in a biofilm formation assay (described below), also referred to herein as a pellicle formation assay.
  • the cells are held together in the pellicle by an extracellular matrix.
  • This matrix is composed of an exopoly saccharide produced by the products of the eps operon and amyloid-like fibers of the protein TasA, whose formation requires the three gene operon yqxM-sipW-tasA (Branda et al. 2004; Branda et al. 2006; Romero et al. 2010).
  • the AsqhC mutant formed pellicles that were indistinguishable from those formed by the wild type, indicating that sporulenes were not involved in the biofilm formation signaling pathway (data not shown).
  • the product of yisP was shown to be capable of generating a C30 polyisoprenoid by the biofilm formation assay. Deletion of yisP resulted in a complete loss of pellicle forming ability in the assay (data not shown). Pellicle formation was restored in the AyisP mutant by re-introducing a functional copy of the gene into the neutral amyE locus of the chromosome (data not shown).
  • the product of yisP displays squalene synthase activity in vitro.
  • yisP was a gene of unknown function, the activity of its product was characterized to gain some insights as to how it might be involved upstream of SpoOA phosphorylation.
  • the yisP gene was cloned and expressed in Escherichia coli.
  • purified recombinant YisP has the enzymatic features of a phytoene or squalene synthase and preferentially uses farnesyl pyrophosphate as a substrate ( Figure 5) (Lee and Poulter 2008).
  • Motility assays performed indicate that deletion of yisP affects the function of the KinC.
  • the chimera KinC-DegS inhibits motility in response to the signal nystatin, as previously in Lopez et al. (2009 PNAS 106:280-285).
  • the presence of nystatin in the medium reduced colony spreading when plated on swarming agar (data not shown).
  • Deletion of the gene yisP compromised the functionality of the chimeric kinase so the presence of nystatin did not inhibit colony spreading (data not shown).
  • Motility assays were performed according to the protocol published by Kearns et al (2003 Mol Microbiol 49: 581-590).
  • Eukaryotic membranes partition into detergent-resistant (DRM) and detergent- sensitive (DSM) fractions. While it is important to emphasize that the DRM fraction is not to be equated with lipid rafts, there is evidence that this fraction includes many of the proteins thought to be present in lipid rafts (Brown 2002).
  • DRM detergent-resistant
  • DSM detergent- sensitive
  • Membrane fractionation was performed on wild type (WT), AyisP, and WT treated with zaragozic acid (+Z), according to differential sensitivity to detergent solubilization.
  • the membrane fractions sensitive and resistant to detergent solubilization were named DSM and DRM, respectively.
  • Membrane proteins associated with each fraction were visualized in an SDS-PAGE.
  • DRM- associated proteins decreased in AyisP mutant and in the wild-type strain treated with zaragozic acid (+Z).
  • the protein profiles from DRM and DSM were dramatically different, suggesting a heterogeneous distribution of lipids and proteins in B. subtilis membranes.
  • the number and intensity of protein bands in the DRM was greatly decreased in the AyisP mutant or when wild-type cells were treated with zaragozic acid.
  • the DSM fraction of the AyisP mutant still contained significant amounts of protein.
  • the protein profile per se displayed some changes probably as a consequence of the pleiotropic effect caused by the mutation in AyisP itself (data not shown).
  • the AkinC mutant was complemented with the translational fusion KinC-YFP (yellow fluorescent protein) and the protein was detected by immunoblotting using monoclonal antibodies against YFP.
  • KinC was present only in the DRM fraction and was not detected in the AyisP mutant or after treatment with zaragozic acid (data not shown). It is possible that in the absence of lipid rafts, KinC is degraded more quickly because it fails to properly localize in the membrane.
  • the proteins identified in the DRM fraction are not necessarily in lipid rafts. However, some of them could be and would thus be expected to co- localize. [0097] Interestingly, one of the proteins present in the DRM fraction was YuaG, a B. subtilis flotillin-like protein, corroborating the results of Donovan and Bramkamp (2009). In eukaryotic cells Flotillin-1 is localized exclusively in lipid rafts and appears to orchestrate diverse processes related to signal transduction, vesicle trafficking, and cytoskeleton rearrangement (Langhorst et al. 2005; Morrow and Parton 2005).
  • YuaG shares 39% amino acid sequence identity (69% similarity) with Flotillin-1. Because of the sequence and localization similarity of YuaG with Flotillin-1, it was renamed to FloT. The membrane distribution of FloT was visualized by constructing a translational fusion with YFP and expression in B. subtilis. The resulting strain was used to determine cellular localization of FloT-YFP. If the membrane areas where FloT-YFP localized are indeed analogous to eukaryotic lipid rafts, then KinC, present in the DRM fraction, should co-localize with FloT- YFP.
  • B. subtilis expresses two SPFH domain proteins encoded by FloT and YqfA.
  • Various deletion mutant strains were used in a bio film/pellicle formation assay, in the presence and absence of surfactin. The results showed the ability of the AfloT AyqfA double mutant to form pellicles in response to the signaling molecule surfactin, when cultured in LB medium (Lopez et al. 2009).
  • the two SPFH-containing proteins in B. subtilis FloT and YqfA were deleted. This behavior was seen to be dependent of the histidine kinase KinC, since the kinC deficient mutant does not make pellicles when surfactin was added.
  • Pellicles were formed when surfactin was added to the wild-type strain, but not in the fcmC-deficient background. A weak induction of pellicle formation was observed in the double mutant AfloT AyqfA when surfactin was added.
  • the AfloT AyqfA double mutant phenocopies the AkinC mutant, both being unresponsive to surfactin, as observed in analysis of strains that were deleted in floT and yqfA. This indicates that indeed in a cell lacking proteins with SPFH domains, KinC activity is compromised. Interestingly, overexpressing KinC in the AfloT AyqfA double mutant partially restored pellicle formation in the biofilm pellical formation assay (data not shown). A possible explanation for this partial restoration of activity would be that FloT might influence KinC activity by increasing local concentrations or promoting multimerization, similar to what has been observed in eukaryotic cells (Browman et al. 2007).
  • lipid rafts-associated protein FloT The dynamic nature of the lipid rafts was indicated by observing the localization pattern of the lipid rafts-associated protein FloT, by following the distribution of the translational fusion FloT-Yfp along the membrane of whole bacteria during a time frame of 1 minute. The distribution at the membrane was seen to change over the course of 1 minute.
  • Proteins showing sequence similarity to Flotillin-1 are widespread among bacteria. Whether these proteins localize to discrete foci in the membrane in other bacterial species was determined. Fusions of YFP to the Flotilin-1 sequence homologs SA1402 from Staphylococcus aureus and YqiK from E. coli were constructed. Localization of the translational fusion proteins was determined by detecting the fluorophore YFP in the recipient strains. Distribution of the signal was heterogeneous across the membrane of the bacterium. It was observed that both of the fusion proteins displayed a punctate localization in the membrane. In S. aureus the fusion protein localized to a single focus in the bacterial membrane. Consistent with the idea that this microdomain contained a lipid derived from squalene, probably staphyloxantin or a closely related molecule, localization of the protein was lost after treatment with zaragozic acid (data not shown).
  • Proteins associated to the DRM fraction were sequenced and, again, the majority of these proteins function in signal transduction, molecule trafficking, and protein secretion (Table 7).
  • Examples of the proteins present in the DRM fraction are the quorum-sensing regulator involved in virulence, CvfA (Nagata et al. 2008) and the elastin-binding protein EbpS, involved in biofilm formation and tissue colonization (Downer et al. 2002).
  • Flotillin-1 homolog (SA1402) and a KinC homolog WalK were also identified (Dubrac et al. 2007) as well the protease secretion machinery. This last finding is worthy of note because protease secretion in Gram (+) cocci is known to occur from a single point in the membrane called the ExPortal (Rosch and Caparon 2004). It is presumed that the ExPortal is located in the single membrane raft observed in S. aureus. As was observed, treatment with zaragozic acid inhibited protease secretion further supporting the hypothesis that the ExPortal is this single membrane raft (data not shown). A dramatic change in colony color was also observed as a consequence of zaragozic acid treatment (data not shown). This was due to the fact that the yellow carotenoid staphyloxanthin from S. aureus is derived from squalene, thus its synthesis was inhibited by zaragozic acid (Pelz et al. 2005).
  • Squalene synthesis inhibitors inhibit biofilm formation in B. subtilis and S. aureus
  • B. subtilis and S. aureus possess different routes to produce squalene (see Figure 3).
  • S. aureus has the mevalonate route. This route is also present in humans and can be inhibited by molecules, statins, that act on the key enzyme HMG-CoA reductase (Endo 1981; Wilding et al. 2000).
  • B. subtilis has the glyceraldehyde- 3 -phosphate (GA3P) + pyruvate route (Takahashi et al. 1998).
  • Biofilm formation in B. subtilis was observed as a pellicle formed in the surface air-liquid of standing cultures while S. aureus forms biofilms attached to the submerged surfaces (at the bottom of the well plate). Crystal violet staining was used in the S. aureus assay for better visualization.
  • the effects of a range of drug concentrations were investigated, ranging from 0, 3, 6, 9, and 15 ⁇ zaragozic acid in B. subtilis, and from 0, 2, 4, 6, and 10 ⁇ zaragozic acid in S. aureus.
  • Pellicle formation was inhibited by as little as 3 and 2 uM, respectively.
  • the assays for each species are different due to the differences in the biofilms they make; B. subtilis forms floating pellicles, which can be directly visualized while S. aureus forms biofilms attached to submerged solid surfaces that are best visualized when stained.
  • lipid rafts In relation to the function of lipid rafts in bacteria, the compartmentalization of specific proteins in tightly packed membrane areas might facilitate their activity. For example, the formation of protein complexes required for cell-cell communication (like the Opp signal-uptake machinery) or dimerization of membrane kinases (like KinC or WalK), necessary in most cases to activate the cascades of signaling transduction seems more feasible when these proteins are physically located in restricted membrane areas. [00116] On a more practical note, it is possible that lipid rafts can be exploited as a new target to control bacterial infections. The fact that disrupting lipid rafts affects several key physiological processes associated with pathogenesis without killing the cell raises the possibility of "anti-raft" compounds as promising anti-infective agents. Remarkably, small molecules that inhibit raft formation simultaneously targeted diverse processes associated with infections in different bacteria, e.g. biofilm formation and exoprotease production.
  • zaragozic acid as an anti-raft drug does not affect bacterial growth. It is contemplated that without any selective pressure caused by an agent that kills the cells, the use of zaragozic acid to inhibit bacterial infections might not give rise so rapidly to resistance mechanisms that are observed with many commonly used antibiotics.
  • statins to prevent infections.
  • Patients treated to decrease high cholesterol levels have shown a remarkably reduced incidence of post-operative infection if previously treated with statins as cholesterol-lowering drugs.
  • previous treatment with statins strongly reduced the risk of hospitalization for sepsis in patients with chronic kidney disease that were receiving dialysis.
  • individuals receiving treatment with statins showed greatly reduced incidence of bacteremias caused by the pathogen S. aureus in hospitals (Liappis et al. 2001; Gupta et al. 2007; Falagas et al. 2008; Kopterides and Falagas 2009). It is possible that these patients may have inadvertently been protected against bacterial infections.
  • Strains, media and culture conditions were Bacillus subtilis strain NCIB3610 (Branda et al. 2001) and Staphylococcus aureus strains SC-1 and UAMS-1 (Beenken et al. 2003). Additional laboratory strains of E. coli DH5cc, B. subtilis 168 and S. aureus RN4220 were required for cloning purposes. A list of strains used in this study is shown in Table 2.
  • Exoprotease production assay was performed in LB plates supplemented with non-fat powder milk (1%). Plates were incubated at 37° C for two days. MSgg plates were supplemented with polyisopronoids for the AyisP mutant
  • Butcher BG Helmann JD. 2006. Identification of Bacillus subtilis sigma-dependent genes that provide intrinsic resistance to antimicrobial compounds produced by Bacilli. Mol Microbiol 60: 765-782.
  • Inorganic phosphate determination colorimetric assay based on the formation of a rhodamine B-phosphomolybdate complex.
  • PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high-level secretion. Mol Microbiol 8: 727-737. Kontnik R, Bosak T, Butcher RA, Brocks JJ, Losick R, Clardy J, Pearson A. 2008. Sporulenes, heptaprenyl metabolites from Bacillus subtilis spores. Org Lett 10: 3551- 3554.
  • Rafts defined: a report on the Keystone Symposium on Lipid Rafts and Cell Function. J Lipid Res 47: 1597-1598.

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Abstract

L'invention concerne une composition pour inhiber la formation d'un biofilm bactérien comprenant un support et une quantité efficace d'un inhibiteur de synthèse de squalène/phytoène. Les inhibiteurs peuvent inhiber, par exemple, la HMG-CoA réductase, la squalène synthase, la 1-désoxy-D-xylulose 5-phosphate synthase. Des exemples de ces inhibiteurs sont un phosphonosulfonate (par exemple, BPH-652, BPH-689, BPH-700), une statine (par exemple, mévastatine, lovastatine, atorvastatine, cérivastatine, fluvastatine, pitavastatine, pravastatine, rosuvastatine, et simvastatine), l'acide zaragozique, la clomazone, et l'acétate de lapaquistat ou l'un de leurs dérivés fonctionnels. Elle concerne également des substrats comprenant l'inhibiteur de synthèse de squalène/phytoène et des procédés d'inhibition de la formation d'un biofilm bactérien.
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WO2018118768A1 (fr) * 2016-12-19 2018-06-28 Agrobiologics Llc Utilisation d'acides zaragoziques en tant qu'antifongiques en agriculture
US11033604B2 (en) 2018-01-26 2021-06-15 University Of Washington Reagents and methods for treating bacterial infection

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Publication number Priority date Publication date Assignee Title
WO2018118768A1 (fr) * 2016-12-19 2018-06-28 Agrobiologics Llc Utilisation d'acides zaragoziques en tant qu'antifongiques en agriculture
US11033604B2 (en) 2018-01-26 2021-06-15 University Of Washington Reagents and methods for treating bacterial infection
US11826398B2 (en) 2018-01-26 2023-11-28 University Of Washington Reagents and methods for treating bacterial infection

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