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WO2012074490A1 - Maîtrise de la croissance bactérienne - Google Patents

Maîtrise de la croissance bactérienne Download PDF

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Publication number
WO2012074490A1
WO2012074490A1 PCT/SG2011/000421 SG2011000421W WO2012074490A1 WO 2012074490 A1 WO2012074490 A1 WO 2012074490A1 SG 2011000421 W SG2011000421 W SG 2011000421W WO 2012074490 A1 WO2012074490 A1 WO 2012074490A1
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WIPO (PCT)
Prior art keywords
bacteria
compound
sulfonylamide
acid synthase
acetohydroxy acid
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PCT/SG2011/000421
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English (en)
Inventor
Jason Kreisberg
Patrick Tan
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Agency for Science Technology and Research Singapore
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Agency for Science Technology and Research Singapore
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Publication of WO2012074490A1 publication Critical patent/WO2012074490A1/fr
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    • 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
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
    • A01N47/28—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N<
    • A01N47/36—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N< containing the group >N—CO—N< directly attached to at least one heterocyclic ring; Thio analogues thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to the discovery that sulfonylurea compounds can prevent the growth of certain bacterial pathogens.
  • a gram-negative rod-shaped bacteria, Bwkholderia pseudomallei is present in muddy soil throughout Southeast Asia and Northern Australia and can readily be isolated from over 50% of the rice paddies in northeastern Thailand. Human or animal infection can occur after exposure to contaminated soil or water via direct contact, inhalation, or even ingestion which was thought to be the route on infection in some cases of near-drowning victims after the 2004 Indian Ocean tsunami.
  • Infection can result in a range of clinical symptoms such as skin abscesses or pneumonia with the most severe outcome being sepsis leading to death in sometimes as little as 2 - 3 days.
  • melioidosis accounts for 20% of all community acquired cases of septicemia; at the Royal Darwin Hospital in Northern Australia, melioidosis is the most common cause of fatal community acquired bacteraemic pneumonia.
  • Case fatality rates from primary disease range from approximately 20% in Northern Australia to nearly 50% in Thailand.
  • Antibiotic resistance in Gram-negative bacteria is an ever-increasing clinical problem yet few new antimicrobial compounds are under development.
  • Targeting microbial amino acid biosynthetic pathways may represent a promising therapeutic approach as mammalian species often lack these enzymes and enzyme-deficient pathogen mutants are often attenuated in vivo.
  • sulfonylurea herbicides Due to their low application rates and low toxicity towards animals, crop-selective sulfonylurea herbicides have been developed for weed control for a wide variety of crops including wheat, corn, barley, and rice. Sulfonylurea herbicides work by inhibiting the activity of acetohydroxy acid synthase (AHAS; also known as acetolactate synthase, ALS), an enzyme required for the biosynthesis of the branched-chain amino acids (BCAA) valine, leucine, and isoleucine.
  • AHAS acetohydroxy acid synthase
  • BCAA branched-chain amino acids
  • SHs can potently inhibit the growth of three clinically important Gram- negative pathogens: Pseudomonas aeruginosa and Acinetobacter baumannii, both major sources of hospital acquired infections; and B. pseudomallei, an emerging infectious disease and a potential bioterror agent.
  • a phylogenetic analysis revealed that many other pathogenic bacteria encode AHAS isozymes which may be sensitive to these inexpensive and non-toxic compounds.
  • Both mutational and structural evidence demonstrate that SHs directly inhibit AHAS in these pathogens.
  • Treatment of infected mice with SHs at doses well below reported levels of toxicity provided significant protection against otherwise acute, lethal challenges of B. pseudomallei or P. aeruginosa.
  • US5998420 discloses a method for treating tuberculosis in a mammal which comprises administering to the mammal a therapeutically effective amount of an inhibitor compound that inhibits an enzyme in the branched chain amino acid biosynthetic pathway in
  • mice infected with Mycobacterium tuberculosis strain ATCC35801 were injected with sulfometuron methyl, a herbicidal compound that inhibits AHAs, which resulted in a significant reduction in growth of Mycobacterium tuberculosis in the lungs.
  • US7666404 discloses a treatment for Burkholderia pseudomallei infection such as melioidosis.
  • the treatment involves inhibiting the expression of one or more transcriptional regulatory proteins and/or synthase enzymes.
  • Inhibitors may include agents or drugs which either bind or sequester synthase substrate(s) or cofactor(s), or inhibit the synthase itself or which inhibit binding of the signal produced by the synthase to the synthase transcriptional regulator, agents which inhibit binding of the transcriptional regulator itself.
  • Inhibitors of synthase and/or synthase enzyme transcriptional regulator may be used in the treatment or amelioration of glanders disease or melioidosis, and diseases associated with Burkholderia infection.
  • Atkins et al.. Infection and immunity, Vol. 70, No.9: 5290-5294 discloses that Burkholderia pseudomallei auxotrophic in the branched chain amino acid biosynthetic pathway are unable to grow without the supply of branched amino acids.
  • the transposon was shown to have interrupted the ilvl gene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneal ly with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-type B. pseudomallei.
  • SHs melioidosis
  • other human pathogens such as melioidosis and other human pathogens.
  • SHs could be used to '"treat" B. pseudomallei containing rice paddies so as to hopefully reduce the levels of Bp in the soil and subsequent number of infections in the local population.
  • the term "about” as used in relation to a numerical value means, for example, +50% of the numerical value, preferably ⁇ 20%, more preferably + 10%, more preferably still ⁇ 5%, and most preferably +1%. Where necessary, the word "about' ' may be omitted from the definition of the invention.
  • composition “comprising" X may consist exclusively of X or may include one or more additional components.
  • treatment is to be construed broadly and includes both therapeutic treatment and prophylactic or preventative measures, and includes all uses which remedy a disease state or one or more symptoms thereof, prevent the establishment of disease, or otherwise prevent, hinder, retard, stabilize, reduce or reverse the onset or progression of disease or other undesirable symptoms in any way whatsoever.
  • FIG. 3 Type III AHAS from B. pseudomallei is sensitive to inhibition by sulfonylurea herbicides.
  • A High-resolution growth curves of parental E. coli or 18G 12 treated with DMSO or SHs were generated with OD600 readings collected every 5 minutes. Valine (75 ⁇ g/ml each) was included in as indicated. Leucine (75 ⁇ g/ml) was always present as the parental E. coli strain is a leucine auxotroph.
  • B A phylogenetic tree of the catalytic subunit of AHAS was constructed using maximal likelihood methods. Bootstrap analysis was performed on 100 trees and nodes appearing more than 75 times are indicated with a circle. The clusters formed by each isozyme are highlighted.
  • C Structural models of chlorimuron ethyl bound to the catalytic subunit of AHAS from B. pseudomallei and P. aeruginosa.
  • Carbon atoms from AHAS are shown in grey whereas chlorimuron ethyl carbon atoms are shown in green.
  • nitrogen atoms are shown in blue, oxygen in red, and sulfur in yellow.
  • the chlorine in the heterocyclic ring of chlorimuron ethyl is also colored green.
  • Hydrogen atoms are not shown. Black dashed lines indicate potential hydrogen bonds.
  • FIG. 4 Sulfonylurea herbicides inhibit growth of pathogenic bacteria.
  • B. thailandensis ATCC 700388 was exposed to increasing doses of DMSO or SHs and high-resolution growth curves obtained with OD600 readings collected even' 10 minutes.
  • B Two reference strains, AYE and 5377, and two local clinical isolates, KAb 1 and KAb 2, of A. baumannii were treated with various SHs and high-resolution growth curves obtained with OD600 readings collected every 12 minutes.
  • CFUs were determined from the microdilution well corresponding to the MIC for B. thailandensis ATCC 700388 and P. aeruginosa PAOl .
  • B Phase contrast images (100x) of the infected cultures. Scale bars, 50 ⁇ .
  • FIG. 8 Various controls along with additional sulfonylurea herbicides that can also dampen the growth of B. thailandensis.
  • B. thailandensis ATCC 700388 was exposed to increasing doses of either chloramphenicol, imazapyr, or SHs and high-resolution growth curves obtained as described earlier with measurements taken every 10 minutes.
  • FIG. 9 Chiorimuron ethyl and metsulfuron methyl slow intracellular replication of B. thailandensis in A549 human respiratory epithelial cells while preserving cellular integrity.
  • A Intracellular CFUs after B. thailandensis-m ' fected A549 cultures were treated with either DMSO or various AHAS inhibitors two hours after infection (multiplicity of infection approximately 3). Note that the chiorimuron ethyl (red) and metsulfuron methyl (blue) data points are nearly identical therefore largely obscuring the chiorimuron ethyl values.
  • B Phase contrast images (100 x ) of infected A549 cells from the indicated time points. Scale bar, 50 ⁇ .
  • sulfonylurea herbicides compounds currently used in agricultural settings to control growth of unwanted species (i.e. weeds) are capable of inhibiting the growth of two human bacterial pathogens, Burkholderia pseudomallei and Pseudomonas aeruginosa.
  • Burkholderia pseudomallei normally lives in the soil and often affects rice farmers, one possible use could be to "treat" infected fields with some of these herbicides so that less people will get sick from the bacteria in the infected fields.
  • the second possible application is the use of these compounds (or derivatives of these compounds) as a therapeutic agent for infection by the above-mentioned pathogens as well as other pathogens.
  • the invention will find utility in controlling the growth of a range of bacteria, including both gram negative and gram positive bacteria.
  • the bacteria do not possess a type I enzyme acetohydroxy acid synthase.
  • a first aspect of the invention provides the use of a sulfonylamide compound or a derivative thereof in the preparation of a medicament for treating and preventing infections caused by bacteria, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase and bacteria having a type III enzyme acetohydroxy acid synthase.
  • a second aspect of the invention relates to a sulfonylamide compound or a derivative thereof for treating and preventing infections caused by bacteria, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase.
  • derivative thereof in connection with a sulfonylamide compound of the invention includes any compound structurally related to said sulfonylamide compound (and optionally formed from said sulfonylamide compound) but still retaining the biological activity of said sulfonylamide compound in all essential respects.
  • derivatives are chemically or biologically modified versions of the compound that are structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound.
  • derivatives mention can be made of functional derivatives containing one or more additional functional groups such as OH, NH2 and the like, or with one or more of the functional groups being removed from or displaced within the compound.
  • Derivatization may involve substitution of one or more moieties within the molecule (e.g., a change in functional group).
  • a hydrogen may be substituted with a halogen, such as fluorine or chlorine, or a hydroxyl group (- OH) may be replaced with a carboxylic acid moiety (-COOH).
  • derivative also includes conjugates, and prodrugs of a parent compound (i.e., chemically modified derivatives which can be converted into the original compound under physiological conditions).
  • the prodrug may be an inactive form of an active agent. Under physiological conditions, the prodrug may be converted into the active form of the compound.
  • Prodrugs may be formed, for example, by replacing one or two hydrogen atoms on nitrogen atoms by an acyl group (acyl prodrugs) or a carbamate group (carbamate prodrugs).
  • derivative is also used to describe all solvates, for example hydrates or adducts (e.g., adducts with alcohols), active metabolites, and salts (e.g. pharmaceutically acceptable salts) of the parent compound.
  • Pharmaceutically acceptable salts are well known in the art and refer to the relatively nontoxic, inorganic and organic acid addition salts of the compounds used in the present invention.
  • S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977) and see also Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991 ).
  • Salts which may be conveniently used in the present invention include physiologically acceptable base salts, for example, derived from an appropriate base, such as an alkali metal (eg sodium), alkaline earth metal (eg magnesium) salts, and ammonium.
  • physiologically acceptable acid salts include hydrochloride, sulphate, mesylate, besylate, phosphate and glutamate. Salts according to the invention may be prepared in conventional manner, for example by reaction of the parent compound with an appropriate base to form the corresponding base salt, or with an appropriate acid to form the corresponding acid salt.
  • the treating or preventing by a sulfonylamide compound or a derivative thereof may be carried out in vivo or in vitro.
  • An example of the latter type of use would be the treatment of water (e.g. stagnant water) and fields (e.g. rice paddies).
  • the sulfonylamide compound (or derivative thereof) may be contacted with the water or field, such as by adding the sulfonylamide compound (or derivative thereof) to the field or water to be treated.
  • a further example of where the invention may find utility in an in vitro context would be the use of a sulfonylamide compound or a derivative thereof in a cleaning or disinfecting composition.
  • Such a cleaning or disinfecting composition may, for example, take the form of a rinse, spray, cream or the like and may, for example, find utility in cleaning or disinfecting objects, surfaces or equipment. It is envisaged that such a cleaning or disinfecting agent may find utility in the domestic environment, in hospitals (e.g. in cleaning hospitals or hospital equipment such as medical devices (e.g. catheters)), in industry and in agricultural settings.
  • a cleaning or disinfecting agent may find utility in the domestic environment, in hospitals (e.g. in cleaning hospitals or hospital equipment such as medical devices (e.g. catheters)), in industry and in agricultural settings.
  • the sulfonylamide compound (or derivative thereof) may be contacted with the object / surface / equipment etc. to be treated.
  • the term "contacting" is to be construed broadly and includes, but is not limited to, soaking, rinsing, flushing, submerging, and washing. It will be appreciated that the contacting should be performed for sufficient time for the sulfonylamide compound (or derivative thereof) to exert a useful cleaning or disinfecting effect. In one specific embodiment, the contacting is for at least 10 seconds, 20 seconds, 40 seconds, 1 minute, 3 minutes, 5 minutes. 8 minutes, 10 minutes, 20 minutes. 30 minutes. 45 minutes, 60 minutes or 120 minutes.
  • the concentration of active components used may vary as desired or necessary to decrease the amount of time the amount of contact time required. Persons skilled in the art easily determine these variations in the concentrations of active components and suitable contact times.
  • a third aspect of the invention relates to a method of treating and preventing infections caused by bacteria comprising administering a sulfonylamide compound or a derivative thereof to a patent in need thereof, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase.
  • the bacteria are selected from bacteria having: (i) a type II enzyme acetohydroxy acid synthase and not a type I enzyme acetohydroxy acid synthase; or (ii) a type III enzyme acetohydroxy acid synthase and not a type I enzyme acetohydroxy acid synthase.
  • the bacteria are gram negative bacteria.
  • examples of gram negative bacteria include Escherichia coli. Salmonella species, other
  • the bacteria are gram positive bacteria.
  • gram positive bacteria include: Bacillus species, Listeria species, Staphylococcus species,
  • Streptococcus species Enterococcus species, and Clostridium species.
  • the bacteria are pathogenic bacteria, for example human pathogenic bacteria.
  • pathogenic refers to bacterial cells capable of infecting and causing disease in a host, as well as producing infection-related symptoms in the infected host, such as fever.
  • disease as used herein is intended to be synonymous with the terms “disorder” and "condition” in that these terms all reflect an abnormal condition of the human or animal body (or a part thereof) that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
  • the bacteria may optionally be selected from the group consisting of Burkholderia pseudomallei, Pseudomonas aeruginosa, Haemophilus influenza, Neisseria ghonorrhea, Bordetella pertussis, Campylobacter jejuni, Bacillus anthracis, Listeria monocytogenes, Staphylococcus epidermidis, Streptococcus pneumoniae.
  • Staphylococcus aureus Neisseria meningitidis, Acinetobacter baumannii, Burkholderia cepacia, Burkholderia cenocepacia and Vibrio cholera.
  • the bacteria are not
  • Mycobacterium and more preferably the bacteria are not M. tuberculosis.
  • bacteria, and the diseases that they cause which may usefully be controlled by the present invention include: Haemophilus influenza (respiratory tract infections), Neisseria ghonorrhea, Bordetella pertussis (whooping cough), Campylobacter jejuni (food poisoning), Bacillus anthracis (anthrax). Listeria monocytogenes (food poisoning), and Staphylococcus epidermidis (hospital acquired infection).
  • the bacterium and disease is not Mycobacterium tuberculosis and a disease caused thereby.
  • control and prevention' as used herein in relation to bacteria / bacterial growth / disease etc. are to be interpreted broadly and include, not onl substantially stopping bacterial growth (e.g. by a bactericidal or bacteriostatic action), but also any reduction or retardation in bacterial growth (e.g. by a bactericidal or bacteriostatic action).
  • the various aspects of the invention may find utility in reducing the rate of increase in the numbers of a population of a particular bacterium, stopping the growth of the population, reducing the number of bacteria in the population, or eliminating the bacteria in the population.
  • a fourth aspect of the invention relates to the use of a sulfonylamide compound or a derivative thereof in the preparation of a medicament for treating and preventing melioidosis or skin abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei.
  • a fifth aspect of the invention relates to a sulfonylamide compound or a derivative thereof for treating and preventing melioidosis or skin abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei.
  • a sixth aspect of the invention relates to a method of treating and preventing melioidosis or skin abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei comprising administering a sulfonylamide compound or a derivative thereof to a patent in need thereof.
  • Pseudomallei is an opportunistic pathogen which can cause disease in susceptible individuals such as those that are immunocompromised or suffer from diabetes mellitus.
  • the patient is immunocompromised or is suffering from diabetes mellitus.
  • the treating and preventing in the first to sixth aspects of the invention may comprise administering a sulfonylamide compound or a derivative thereof to a patient in need thereof by one or more various routes of administration.
  • the route of administration may, for example, be topical, oral, intravenous, cutaneous, subcutaneous, transdermal, transmucosal. intramuscular, intraperitoneal routes.
  • An example of a topical administration would be a disinfecting hand wash or lotion etc. which may, for example, be useful in a domestic environment and hospital environments to prevent the spread of potentially disease causing bacteria.
  • the sulfonylamide compound or a derivative thereof may be administered topically, systematical!)', or locally (e.g. as an implant).
  • medicaments comprising a sulfonylamide compound or a derivative thereof will be within the skill of persons skilled in the art and a wide variety of formulations are contemplated.
  • the compounds can be formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations etc.
  • Such medicaments may comprise, in addition to the sulfonylamide compound (or derivative thereof), a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient(s).
  • Some examples of the kinds of materials that can serve as pharmaceutically acceptable substances include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatine, talc, oils, glycols, esters, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer solutions, coloring agents, releasing agents, coating agents, sweetening and flavoring agents, preservatives and antioxidants.
  • the precise nature of the carrier or other material may depend on the route of administration and will be within the ability and judgement of the skilled person.
  • Medicaments for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • Medicaments for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
  • Suitable auxiliaries can be added, if des!ired.
  • Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone. agar, and alginic acid or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e.. dosage.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate. and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • Formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution. Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyi cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the medicaments employed in the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping, or iyophilizing processes.
  • the medicament is to be administered in a therapeutically effective amount (either as a single dose or as part of a series of doses).
  • a therapeutically effective amount is meant the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient such that beneficial or desired results are achieved.
  • the medicament comprises from at least about 1% to about 50% (and more preferably from at least about 0.5% to about 25%) of the sulfonylamide compound (or derivative thereof) by weight based upon the total weight of the composition of the invention being employed.
  • the sulfonylamide compound (or derivative thereof) may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time.
  • the precise effective amount for a patient will depend upon the severity of the disease state, general health of the patient, age, weight, whether the treatment is prophylactic or to treat an existing infection, the gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • the effective dose for a given situation can be determined by routine experimentation and is within the judgement of the skilled person.
  • cell culture assays and animal studies can be used.
  • the dosage range is between 0.1 to 50mg/kg.
  • patient' refers to patients of human or other mammal and includes any individual it is desired to treat (including prophylatically) using the methods of the invention. However, it will be understood that '"patient” does not imply that symptoms are present.
  • "Mammal” refers to any animal classified as a mammal, including humans, primates, domestic and farm animals (eg. sheep, cows, horses, donkeys, pigs), and zoo, sports, and pet companion animals, laboratory test animals (eg. rabbits, mice, rats, guinea pigs, hamsters) and captive wild animals (eg. foxes, deer, dingoes).
  • Preferred companion animals are dogs and cats.
  • the patient is an immunocompromised patient (e.g. an AIDS patient, a chemotherapy patient, a malnourished individual, an elderly person, or the very young) or agricultural worker (e.g. a farmer, such as a rice paddy farmer).
  • immunocompromised patient e.g. an AIDS patient, a chemotherapy patient, a malnourished individual, an elderly person, or the very young
  • agricultural worker e.g. a farmer, such as a rice paddy farmer.
  • Embodiments of the various aspects of the invention are envisaged whereby more than one (e.g. 2, 3, 4 or more) sulfonylamide compounds (or derivatives thereof) are employed.
  • the more than one sulfonylamide compounds (or derivatives thereof) may be provided in the same formulation / composition etc. or separately (e.g. in the form of a kit of parts). It is envisaged that the more than one sulfonylamide compounds (or derivatives thereof) may be administered or employed simultaneously, separately or sequentially.
  • the one or more sulfonylamide compounds (or derivatives thereof) are used in conjunction with one or more further active agents.
  • the one or more sulfonylamide compounds (or derivatives thereof) and the one or more further active agents may be provided in the same formulation / composition etc. or separately (e.g. in the form of a kit of parts) so that they may be administered or employed simultaneously, separately or sequentially.
  • a “further active agent'” we include compounds which are useful in achieving one or more of the following: (i) treating and preventing infections caused by bacteria, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase and bacteria having a type III enzyme acetohydroxy acid synthase; (ii) treating and preventing melioidosis or skiri abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei: (iii) reducing or substantially preventing the growth of soil bacteria in a crop-planting site; and (iv) reducing or substantially preventing the growth of bacteria in a water supply.
  • the one or more further active agent may act synergistically or additively with the one or more sulfonylamide compounds (or derivatives thereof).
  • suitable “further active agents” may include anti-bacterial agents such as natural and synthetic penicillins and cephalosporins, sulphonamides, erythromycin, kanomycin, tetracycline, chloramphenicol, rifampicin and including gentamicin.
  • sulfametopyrazine sulphadiazine, sulphadimidine, sulphaguanidine, sulphaurea, capreomycin, metronidazole, tinidazole, cinoxacin, ciprofloxacin, nitrofurantoin, hexamine, streptomycin, carbeniciHin, colistimethate, polymyxin B, furazolidone, nalidixic acid, trimethoprim-sulfaniethoxazole, clindamycin, lincomycin, cycloserine, isoniazid, ethambutol, ethionamide, pyrazinamide and the like.
  • the one or more sulfonylamide compounds (or derivatives thereof) is / are the sole active agent(s) in terms of achieving the following: (i) treating and preventing infections caused by bacteria, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase and bacteria having a type III enzyme acetohydroxy acid synthase; (ii) treating and preventing melioidosis or skin abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei; (iii) reducing or substantially preventing the growth of soil bacteria in a crop-planting site; or (iv) reducing or substantially preventing the growth of bacteria in a water supply.
  • sulfonylamide compounds or pharmaceutically acceptable derivatives used in the first to sixth aspects of the invention can be packaged as articles of manufacture comprising:
  • packaging material e.g. blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes and bottles
  • the one or more sulfonylamide compounds or derivative thereof
  • optionally instructions for use e.g.
  • sulfonylamide compounds including derivatives thereof
  • the invention may find utility in treating soil or water. In this way, the transmission of bacterial pathogens, and consequent infections, may be reduced.
  • sulfonylamide compounds including derivatives thereof
  • a seventh aspect of the invention provides a sulfonylamide compound or a derivative thereof for preparing a composition for reducing or substantially preventing the growth of soil bacteria in a crop-planting site, in which the soil bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase, to thereby reduce the bacterial contamination of said crop-planting site.
  • An eighth aspect of the invention relates to sulfonylamide compound or a derivative thereof for reducing or substantially preventing the growth of soil bacteria in a crop-planting site, in which the soil bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase, to thereby reduce the bacterial contamination of said crop-planting site.
  • a ninth aspect of the invention relates to a method of reducing or substantially preventing the growth of soil bacteria in a crop-planting site comprising the step of providing a
  • the soil bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase.
  • the crop-planting site is selected from the group consisting of: a paddy field, a field, an orchard, a glass house, a vineyard, forest, or a tea plantation.
  • the crop may. for example, be an ornamental, vegetable, field, cereal, or fruit crop.
  • the sulfonylamide compound (or derivative thereof) as used in the seventh, eighth and ninth aspects of the invention may be provided in a composition which comprises one or more additional components selected from the group consisting of the following: a fungicide, an insecticide, a pesticide, an acaricides, a nematocide, a herbicide, a plant growth regulator, soil improvement agent and a fertilizer.
  • a fungicide an insecticide
  • a pesticide an acaricides
  • a nematocide a herbicide
  • a plant growth regulator soil improvement agent and a fertilizer.
  • Other moieties which may usefully be incorporated into a composition used in the seventh, eighth or ninth aspect of the invention includes carriers, surfactants, solid diluents and liquid diluents, and the preparation of suitable compositions will be within the abilities of the skilled person.
  • the sulfonylamide compound (or derivative thereof) may be provided in a form suitable for application to the crop-planting site.
  • the sulfonylamide compound (or derivative thereof) may conveniently be provided in the form of a liquid (e.g. as a ready-to-use solution, emulsion or suspension) or solid (e.g. in the form of a powder, wettable powder, soluble powder or granules).
  • the composition comprising the one or more sulfonylamide compounds (or derivatives thereof) may be sprayed on (by hand or by means of a spray race or arch or vehicle- or aircraft-mounted apparatus), applied by granule scattering, administered via watering etc.
  • the amount applied may be varied within a wide range depending on the desired effect, the location of application, the time of application, the interval between treatments, weather and climate and the like.
  • the sulfonylamide compound (or derivative thereof) may be prepared either as a formulation ready for use on the crop planting site, or as a formulation requiring dilution prior to application, but both types of formulation comprise the sulfonylamide compound (or derivative thereof) in admixture with one or more carriers or diluents.
  • the carriers may be liquid, solid or gaseous or comprise mixtures of such substances, and the sulfonylamide compound (or derivative thereof) may be present in a suitable concentration depending upon whether the formulation requires further dilution.
  • a tenth aspect of the invention relates to the use of a sulfonylamide compound or a derivative thereof for preparing a composition for reducing or substantially preventing the growth of bacteria in a water supply, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase, to thereby reduce the bacterial contamination of said water supply.
  • An eleventh aspect of the invention relates to a sulfonylamide compound or a derivative thereof for reducing or substantially preventing the growth of bacteria in a water supply, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase, to thereby reduce the bacterial contamination of said water supply.
  • a twelfth aspect of the invention relates to a method of reducing or substantially preventing the growth of bacteria in a water supply comprising the step of providing a sulfonylamide compound or a derivative thereof to said water supply, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase.
  • the concentration of the sulfonylamide compound is from about 5 to about 100 g / ha, preferably from about 5 to about 20 g /ha, about 10 to about 90 g / ha, about 20 to about 80 g/ha, about 30 to about 70 g / ha, about 40 to about 60 g/ha.. about 15 to about 90 g /ha, about 25 to about 85 g /ha. or about 35 to about 80 g /ha.
  • the sulfonylamide compound is bensulfuron methyl and the concentration is from about 50 to about 85g / ha, preferably from about 60 to about 75 g/ha. In another embodiment, the sulfonylamide compound is metsulfuron methyl and the concentration is from about 5 to about 7g / ha.
  • the sulfonylamide compound is chlorsulfuron and the concentration of is from about 15 to about 25g / ha, preferably from about 10 to about 18 g/ha.
  • the invention envisages various uses of sulfonylamide compounds (including their derivatives).
  • the sulfonylamide compounds including their derivatives.
  • sulfonylamide compound is a sulfonylurea compound.
  • the sulfonylurea compound is a compound of general formula 1 :
  • R ! is H or CH : -NHR a , where R a is an acyl radical,
  • R 2 is Cl-C4-alkoxy, CO-(C,-C 4 -alkoxy), S0 2 -(C C 4 -alkyl), or a halide selected from F.
  • R 3 is H or Ci-C 4 -alkyl
  • R 4 and R " independently of one another are identical or different and are selected from the group consisting of Cj-Ce-alkyl, Q-Cg-alkoxy and C]-C 4 -alkylthio, each radical being optionally substituted by one or more groups selected from halogen, (_VC 4 -alkoxy, Q-C 4 - alkylthio, C 3 -C 6 -cycloalkyl. C2-C 6 -alkenyl, C 2 -C6-alkynyl, C 6 -C 6 -alkenyloxy or C 6 -C 6 - alkynyloxy,
  • R 6 is CH or N .
  • - R' is C]-C 4 -alkyl.
  • n 0 or 1.
  • acyl as used herein includes a carbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, or any other moiety where the atom attached to the carbonyl is carbon.
  • Examples of acyl groups include formyl, alkanoyl (e.g. acetyl), aroyl (e.g. benzoyl), heteroaroyl, propanoyl, 2-methylpropanoyl, butanoyi, propenoyl, and caproyl.
  • alkoxy includes alkoxy moieties having varying numbers of carbon atoms, with examples including methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy. and the like.
  • C1-C4 alkoxy means an alkoxy moiety containing 1, 2, 3 or 4 carbon atoms.
  • C I -C6 alkoxy means an alkoxy moiety containing 1, 2, 3, 4, 5 or 6 carbon atoms.
  • alkylthio refers to those alkyl groups attached to the remainder of the molecule via a sulfur atom.
  • C]-C 4 -alkylthio means an alkylthio moiety containing 1, 2, 3 or 4 carbon atoms.
  • halogen or halo as used herein indicate fluorine, chlorine, bromine, astatine, or iodine.
  • alkyl as used herein includes linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof.
  • Alkyl moieties as used herein, may be saturated, or unsaturated, i.e., the moiety may comprise one, two, three or more independently selected double bonds or triple bonds.
  • alkyl groups include methyl, ethyl, propyl, isopropyl. butyl, iso-. sec- and tert-butyl, pentyl. hexyl, heptyl, 3-ethylbutyl, and the like.
  • the number of carbon atoms in an alkyl group or moiety can vary.
  • CI -C4 alkyl means an alkyl moiety containing 1, 2, 3 or 4 carbon atoms and likewise C1 -C6 alkyl means an alkyl moiety containing 1 , 2, 3, 4, 5 or 6 carbon atoms
  • alkenyl as used herein includes a moiety' that comprises linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof, that comprises one or more double bonds, e.g.. 1, 2. 3. 4. 5, 6 or more, typically 1 or 2.
  • the number of carbon atoms in an alkenyl group or moiety can vary.
  • C2-C6 alkenyl means an alkenyl moiety containing 2, 3, 4. 5 or 6 carbon atoms.
  • cycloalkyl includes cycloalkyl moieties having varying numbers of carbon atoms, with examples including cyclopropyl, cyclobutyl. cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptanyl.
  • C 3 -C 6 -cycloalkyl means a cycloalkyl moiety containing 3. 4, 5 or 6 carbon atoms.
  • C?-C 6 -alkynyl means an alkynyl moiety containing 2, 3, 4, 5 or 6 carbon atoms.
  • CrC 6 -alkenyloxy means an alkenyloxy moiety containing 3, 4, 5 or 6 carbon atoms.
  • C 3 -C 6 -aIkynyloxy means an alkynyloxy moiety containing 3, 4, 5 or 6 carbon atoms.
  • the sulfonylurea compound is selected from the group consisting of bensulfuron methyl, metsulfuron methyl, chlorimuron ethyl, amidosulfuron, chlorsulfuron, cinosulfuron, ethametsulfuron methyl, flazasulfuron, halosulfuron, imazosulfuron, nicosulfuron, primisulfuron, pyrazosulfuron ethyl, sulfometuron methyl, thifensulfuron.
  • BCAA sulfonylurea herbicides
  • pseudomallei clinical isolates was generated. End-sequencing and array-based comparative genomic hybridization (array-CGH) showed that the approximately 12,000-member library demonstrated extensive chromosomal coverage, lacking only regions associated with reference strain-specific genomic islands. Functional expression of B. pseudomallei genes in E. coli was confirmed, both at the level of single genes (metalloprotease production) and multi-gene clusters (O-antigen synthesis). To identify B. pseudomallei genes capable of conferring a growth advantage under nutrient-limiting conditions, the pooled fosmid library was grown in either rich or minimal media and enrichment assessed by array-CGH and endsequencing. Array-CGH repeatedly identified a region enriched after growth in minimal media centered at ilvl. End-sequencing of individually recovered fosmids confirmed this finding and revealed that these fosmids always encoded three genes, ilvIHC.
  • the ilvIHC (BPSL1 196 - 1198) cluster encodes the first two enzymes in the BCAA biosynthetic pathway (Fig. 7A).
  • the catalytic and regulatory subunits of type III AHAS are encoded by //v/ and ilvH, respectively, and acetohydroxvacid isomeroreductase (EC 1.1.1.86) is encoded by ilvC.
  • ilvC acetohydroxvacid isomeroreductase
  • tuberculosis tuberculosis, Streptococcus pneumoniae, and Staphylococcus aureus, only encoded a type III AHAS (Fig. 3B).
  • Fig. 3B To explore the potential interactions of these type III AHASs with SHs, computational structural studies were performed. Using the co-crystal structure of the SH chlorimuron ethyl bound to the catalytic subunit of AHAS from A. thaliana as a starting point, structural models of chlorimuron ethyl binding to the catalytic subunits of AHAS from B. pseudomallei or P. aeruginosa were generated (Fig. 3C).
  • A.baumannii were also tested: two sequenced reference strains, AYE (Fournier PE, et al.
  • Emerobacter 638 5110480 ⁇
  • Emerobacter 6 81 5113032 I
  • Emerobacter (6381 5113663 m
  • Klebsiella pneumoniae (MGH 78578) 5339104 m
  • Mycobacterium bovis (BCG Pastern' 1 1 ⁇ 3P2 ) 4698974 in
  • Neisseria gononhoeae (FA 10901 32S2600 m
  • Neisseria laciamica 020-06 i 1000 ⁇ 32S in
  • Neisseria meningitidis (22491 ) 90765S ni
  • Pseudomonas aeruginosa (PAOl ) 881496 m
  • Salmonella enterica (Typhmiurium LT2 i 1251634 ⁇
  • Salmonella enterica (Typ urium LT2 ) 125 320 I
  • Salmonella enterica (Typlumurium LT2.i 125542 " ⁇
  • Vibrio vulnificus ( CMCP6 ) 11 " 7966 n
  • aeuruginosa isolates including multidrug resistant strains, resulting in MIC50 values for both SHs of 62.5 ⁇ , with no differences seen between the multidrug resistant and sensitive strains (data not shown).
  • the Burkholderia species was also used to test sensitivity to chloramphenicol, a clinically effective antibiotic, under the modified conditions and comparable levels of inhibitory activity were found (Table 1 ). In these as in the other experiments, addition of BCAAs to the minimal media rendered SHs ineffective (data not shown). Table 1. MICs ( ⁇ ) of Selected AHAS Inhibitors and Chloramphenicol in Minimal Media
  • SHs for therapeutic potential two different mouse infection studies were performed. In the first, mice were infected intranasally with a highly virulent strain of B. pseudomallei and orally treated twice daily with either phosphate- buffered saline (PBS) or metsulfuron methyl (50 mg/kg). After eight days, all of the PBS- treated mice had died whereas all of the metsulfuron methyl -treated mice were still alive (Fig.
  • PBS phosphate- buffered saline
  • metsulfuron methyl 50 mg/kg
  • mice were infected with P. aeruginosa intratracheally and then given a single tail-vein injection with either PBS or metsulfuron methyl (200 mg/kg) (Fig. 6D-E). Even with just this single dose regimen, the group treated with metsulfuron methyl showed a much higher rate of survival than the PBS-treated group (p ⁇ 0.01 ; Log-rank test. Yates corrected).
  • SHs constitute a new class of antibiotics with potentially broad applications.
  • a wide variety of low cost, non-toxic SHs are readily available for testing against additional Gram-negative and Gram-positive pathogens.
  • the structural models suggest potential starting points for chemical modifications to increase potency or specificity.
  • BCAA auxotrophs of certain pathogens are attenuated when inoculated into the blood, lungs, or peritoneum (Atkins T, et al. (2002) Infect Immun 70(9):5290-5294: Cuccui J. et al. (2007) Infect Immun 75(3): 1 186- 1 195; Ulrich RL, Amemiya K, Waag DM, Roy CJ, & DeShazer D (2005) Vaccine
  • SHs then might be effective at combating human pathogens lacking a type I AHAS during infections of particular anatomical sites. As the lungs appear to be one of these sites, increasingly common cases of ventilator-associated pneumonia by P. aeruginosa and A. baumannii might be candidates for treatment. When treating immunocompromised patients, bacteriostatic agents might actually be the preferred course of treatment so to avoid overwhelming a poorly functioning immune system with bacterial debris such as endotoxin (Pankey GA & Sabath LD (2004) Clin Infect Dis 38(6):864-870.).
  • Bensulfuron methyl is marketed as Londax to control broadleaf weed growth in rice crops.
  • rates of melioidosis infection are known to correlate with the levels of B. pseudomallei present in the soil, treating
  • B. pseudomallei-contaminated rice paddies with bensulfuron methyl might reduce the burden of melioidosis among rice farmers.
  • BCAAs are not plentiful in the soil.
  • these compounds could be used in the lab to see if they can block the growth of Bp under conditions designed to mimic the nutrient conditions in soil. It could then be determined whether treating a rice paddy known to contain Bp with e.g.
  • bensulfuron methyl (Londax), a sulfonylurea herbicide used to prevent the growth of weeds in rice paddies all across the world, would reduce the levels of Bp in the soil. If treating Bp containing rice paddies with bensulfuron methyl or similar compounds results in decreased levels of Bp, then a larger and longer study could then be carried to see if treatment of rice paddies decreases the actual number of melioidosis cases.
  • High-resolution growth curves were obtained using a Bioscreen C MBR (Oy Growth Curves Ab Ltd) integrated incubator and micro-plate reader. All cultures (250 ⁇ ) were grown aerobically at 37 °C with SHs included as indicated and optical density readings at 600 nm (OD600) collected at regular intervals. Preparation of input inoculums varied depending on the experiment as described in SI Materials and Methods.
  • Protein sequences corresponding to fifty catalytic AHAS subunits from thirty-eight species were aligned (Clustal W2), gaps stripped (BioEdit), and a phylogenetic tree derived using maximal likelihood methods (PHYLIP). Further details are provided in SI Materials and Methods.
  • P. aeruginosa PAOl (PA4696) were modeled using the co-crystal structure of chlorimuron ethyl bound to the catalytic subunit of AHAS from A. thaliana, 1 YBH, as a template. Detailed procedures are provided in SI Materials and Methods.
  • MICs Minimum inhibitory concentrations
  • Spontaneous P. aeruginosa PAOl resistance mutants were selected for on M9 plates containing chlorimuron ethyl. Sequencing ilvIH from the five mutants identified three separate, nonsynonymous changes in ilvl. Expression plasmids containing either wild-type or mutant versions of ilvl were constructed and used to transform parental P. aeruginosa PAOl as described SI Materials and Methods.
  • the fosmid library was created using the CopyControl HTP Fosmid Library Production Kit (Epicentre Biotechnologies). Input DNA consisted of genomic DNA from 39 clinical isolates and the library is composed of approximately 12.000 colonies. To prepare a pooled version of the fosmid library, all colonies were robotically transferred from the 384-well master plates on to Q-trays (Genetix) containing LB agar and chloramphenicol (12.5 ⁇ °/ ⁇ 1). After overnight growth at 37 °C, the colonies were harvested in LB and, after the addition of glycerol (40% final concentration), small, single-use aliquots of the pooled library were stored at -80 °C.
  • fosmid DNA was purified using the FosmidMAX DNA Purification Kit (Epicentre) from a culture of the pooled library grown in LB containing streptomycin ( 100 ⁇ g/ml), trimethoprim (50 ⁇ g ml), chloramphenicol (12.5 ⁇ g/ml) and induction solution (Epicentre) aerobically at 37 °C overnight.
  • streptomycin 100 ⁇ g/ml
  • trimethoprim 50 ⁇ g ml
  • chloramphenicol 12.5 ⁇ g/ml
  • induction solution Epicentre aerobically at 37 °C overnight.
  • the resulting fosmid library DNA or reference genomic DNA from B. pseudomallei K96423 was hybridized to a custom designed tiling array (Nimblegen Systems) in which 50 bp probes were tiled approximately every 35 bp across a single strand of the B.
  • pseudomallei K96423 genome except for highly repetitive regions resulting in an array with 97% genomic coverage.
  • Fosmid DNA was sonicated to fragments approximately 100 to 500 bp in length, fiuorescently labeled (Kreatech BAC Array Labeling Kit), and hybridized to the arrays for 16 h at 60 °C. The arrays were then washed, dried, and scanned using an Axon Genepix 4000B scanner (Molecular Devices) at 5 ⁇ resolutions. The fluorescent images were converted to digital signal intensities using
  • NimbleScan Version 2.4 (Nimblegen).
  • This program uses a random walk method to identify missing regions in a high-density data set and assigns each of these regions a separate statistical significance score while also controlling for the overall statistical significance of the analysis.
  • this program allows the user to specify both an intensity cut-off value below which probes are considered absent and a Bonferroni corrected significance cut-off for entire analysis. For the analysis, an intensity cut-off of mean - 2 x standard deviations and a significance cut-off at 0.001 were used.
  • EPI300 Epicentre
  • EPI300 Epicentre
  • Both the LB and M9 cultures also contained streptomycin (100 ⁇ g ml), trimethoprim (50 ⁇ g ml), chloramphenicol (12.5 ⁇ g/ml) and induction solution (Epicentre).
  • fosmid DNA was purified from both cultures using the FosmidMAX DNA Purification Kit (Epicentre).
  • a custom-made tiling array (Nimblegen) was again used but these differed from the ones used earlier as the probes on these arrays were tiled across both strands of the B. pseudomallei K96423 genome.
  • these experiments were performed as two-color hybridizations in which fosmid DNA was sonicated into fragments approximately 100 to 300 bp in length, fluorescently labeled with either Cy3 or Cy5 (Kreatech Diagnostics aRNA Labeling Kit), and hybridized to the arrays for 17h at 65 °C. Processing and scanning of the arrays was performed as described previously. To determine which regions of the genome were enriched, the ratio of signal from the M9 culture to the LB culture was calculated, log2 transformed, and smoothed using a 1 kb sliding window.
  • glycerol stocks were streaked out to LB agar plates containing streptomycin (100 ⁇ g/ml). trimethoprim (50 ⁇ g/ml), and
  • Fosmid DNA for was purified using the FosmidMAX DNA Purification Kit (Epicentre) and subjected to transposon mutagenesis using the EZ-Tn5 ⁇ KAN-2> Insertion Kit (Epicentre). Approximately 40 kanamycin resistant (50 g/ml colonies were then subjected to
  • fosmid DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen) from overnight cultures grown in LB containing streptomycin (100 ⁇ g/ml), trimethoprim (50 ⁇ g/ml), chloramphenicol ( 12.5 ⁇ g ml), kanamycin (50 ⁇ g ml) and induction solution
  • Catalytic subunits of AHAS were identified by searching through the genomes of select organisms, mostly pathogenic bacteria, using the enzyme commission number 2.2.1.6 (Durfee T, et al. (2008), J Bacteriol 190(7):2597-2606; and Duggleby RG, McCourt JA. & Guddat LW (2008), Plant Physiol Biochem 46(3):309-324). In most genomes, multiple genes have been annotated as AHAS catalytic subunits. To ensure that only actual catalytic subunits were analyzed, only those genes adjacent to a regulatory subunit were included.
  • Nucleotide sequences of fifty AHAS catalytic subunits from thirty-eight species were translated into protein sequences, aligned (ClustalW2), and gaps stripped from the alignment (BioEdit).
  • ClustalW2 aligned
  • BioEdit BioEdit
  • a phylogenetic tree was derived using maximum likelihood methods and subjected to bootstrap analysis 100 times (PHYLIP).
  • Other phylogenetic approaches such as distance-matrix methods or parsimony analysis (PHYLIP) yielded similar trees.
  • P. aeruginosa PAO l PA4696 were modeled as dimers using Modeler 9.7 (Sali A &
  • CHARMm force field (Brooks BR et al. (2009), J Comput Chem 30(10): 1545-1614) was used to generate the conformers of the protein.
  • the docking procedure was started with the random generation of ligand conformations. For each conformation, fitting of the ligand into the active site was carried out by comparing the shape of the ligand to the shape of the active site and, if deemed acceptable, a dock energy was computed between the protein and the ligand. Annealing from 300 K to 700 K was simulated next and the top ten (i.e. the best docking scores) poses of the ligand were rescored. The two poses with the best scores for each molecule were retained and subject to molecular dynamic (MD) simulations.
  • MD molecular dynamic
  • N- and C-termini were capped with acetyl (ACE) and N-methyl (NME) moieties respectively to keep them neutral.
  • MD simulations were performed with the SANDER module of the AMBER9 package (Case DA. et al. (2006) AMBER 9, University of California. San Francisco. AMBER 9, University of California, San Francisco) employing the all-atom Cornell force field (Cornell WD, et al. (1995), J Am Che Soc 1 17(19):5179-5197).
  • Chlorimuron ethyl parameters were generated using antechamber (Wang J, Wolf RM, Caldwell JW, Kollman PA, & Case DA (2004) J Comput Chem 25: 1 157-1174; and Wang J, Wang W. Kollman PA, & Case DA (2006) J Mol Graph Model 25:247-6077).
  • Each system was solvated with a T1P3P water box (Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, & Klein ML (1983) J Chem Phys 79:926-935) whose sides were at least 8 A from any protein atom.
  • PME Particle Mesh Ewald method
  • MICs Minimum inhibitory concentrations were determined in M9 minimal medium based off the two-fold microdilution broth technique described in the Clinical and Laboratory Standards Institute guidelines (M7-A6). In brief, starter cultures in LB broth were grown aerobically at 37 °C for 3 - 4 h. The exponential-phase cultures were then washed twice with M9 salts before resuspending in M9 medium and diluted such that each well of a 96-well microtiter plate was inoculated with approximately 5 x 104 CFUs. The M9 medium used in these experiments contained glucose (0.2%) and thiamine ⁇ 0.5 ⁇ g/ml).
  • MIC assays were performed as described above and the well corresponding to the MIC serially diluted in M9 salts before plating on LB agar to determine the number of CFUs present in the MIC well.
  • the "input" well was prepared along with the rest of the microdilution assay plate but before incubating the plate at 37 °C overnight, the well was immediately harvested, serially diluted and plated to determine "input" CFUs.
  • P. aeruginosa PAOl in LB were washed twice in M9 salts and then plated on to M9 plates containing glucose (0.2%) and chlorimuron ethyl (400 uM). Two days later, five chlorimuron ethyl-resistant colonies were isolated from which genomic DNA was purified and ilvIH sequenced.
  • the E. coli-P. aeruginosa shuttle vector, pUCP28T (West SE, Schweizer HP, Dall C, Sample AK, & Runyen-Janecky LJ (1994) Gene 148(1 ): 81-86), was used to express wild-type or mutated versions of ilvl in P. aeruginosa PAOl .
  • Wild-type ilvl from P. aeruginosa PAOl was PCR amplified to include a C-terminal glycine spacer (5x) and FLAG epitope along with the necessary restriction sites for cloning into the MCS of pUCP28T.
  • Mutant versions of ilvl were generated using the QuikChange II XL Site Directed Mutagenesis Kit (Agilent). Prior to usage, the entire ilvl gene from each of the pUCP28T-based constructs was sequenced.
  • Electrocompetent P. aeruginosa PAOl was generated as previously described (Choi KH, Kumar A, & Schweizer HP (2006) J Microbiol Methods 64(3):391 -397) and the presence of pUCP28T was selected for using trimethoprim (200 ⁇ g/ml).
  • a stationary phase culture of B. thailandensis ATCC 700388 was washed twice with phosphate-buffered saline (PBS) before resuspending in DMEM containing heat-inactivated FBS (10%: Invitrogen) and then used to infect A549 human respiratory epithelial cells at a multiplicity of infection of approximately 10.
  • PBS phosphate-buffered saline
  • FBS heat-inactivated FBS
  • the tissue-culture plates were spun at 200 ⁇ g for 10 min at room temperature prior to incubation at 37 °C.
  • the A549 cells were washed three times with PBS and then cultured in DMEM containing heat-inactivated FBS (10%), gentamicin (250 ⁇ g/ml) to kill the extracellular bacteria, and DMSO (1 %) or AHAS inhibitors (2 mM) as indicated.
  • the A549 cells were washed three times with PBS and lysed by exposure to Triton-X (0.1%) at 37 °C for 20 min. Lysates were harvested by scraping, serially diluted in M9 salts and plated on LB agar to determine the CFUs present in the lysate.
  • mice Female 6-week-old BALB/c mice were intranasally challenged with 1 x l02 CFUs, five-times the established LD50 (Tan GY, et al. (2008) J Med Microbiol 57(Pt 4):508-515). For intranasal inoculation of bacteria, the mice were lightly anaesthetized with 3% isoflurane in oxygen. The desired dose of bacteria in a total volume of 20 ⁇ PBS was then delivered through one nostril with a pipette tip. Solutions of metsulfuron methyl were prepared fresh each day by dissolving the powder in DMSO (500 mg/ml) and then diluting into PBS pH 8.0 (5 mg/ml).
  • mice were fed 200 ⁇ of either PBS pH 8.0 (also containing 1% DMSO) or metsulfuron methyl twice daily using a gavage tube. The treatment was initiated 24 hours before challenge and continued for 10 days after infection. Body weights were measured everyday and routine observations were performed twice daily. All experimental procedures were carried out in a BSL3 laboratory and in accordance with the Animal Care and Use Committee ' s Guidelines, DSO National Laboratories. Singapore, using protocols reviewed and approved by the DSO National Laboratories Biosafety Committee.
  • aeruginosa inoculum (3 x 105 CFU in 50 ⁇ of PBS for survival curves; 2 x 105 CFU in 50 ⁇ of PBS for lung CFUs) was delivered just beneath the cricoid cartilage. The incision was sealed by sterile surgical clips (Braintree Scientific). Metsulfuron methyl (5 mg/ml) was injected through the tail vein immediately after intra-tracheal infection. To determine CFUs present in the lungs, mice were killed by inhalation of carbon dioxide after which the lungs were removed aseptically, suspended PBS (2 ml), and homogenized using a Wheaton overhead stirrer (Wheaton Instruments.). Dilutions of the homogenate were spread on LB agar plates and incubated at 37 °C overnight. Animal protocols were based on guidelines from the National Advisory Committee for Laboratory Animal Research and were approved by the Institutional Animal Care and Use Committee, Singapore.

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Abstract

La présente invention concerne l'utilisation d'un composé sulfonylamide ou d'un dérivé de celui-ci, dans la préparation d'un médicament pour le traitement et la prévention d'infections provoquées par des bactéries, dans lesquelles les bactéries sont choisies parmi le groupe consistant en des bactérie ayant une enzyme acétohydroxy acide synthase de type II et des bactéries ayant une enzyme acétohydroxy acide synthase de type III. L'invention concerne en outre les composés sulfonylamides correspondants ou leurs dérivés.
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CN115108997A (zh) * 2021-03-22 2022-09-27 南开大学 一类具抑菌活性的磺酰脲类化合物的合成与应用
CN116023338A (zh) * 2021-10-25 2023-04-28 南开大学 一类具抑菌剂活性的磺酰脲类化合物的合成及应用

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN115108997A (zh) * 2021-03-22 2022-09-27 南开大学 一类具抑菌活性的磺酰脲类化合物的合成与应用
CN116023338A (zh) * 2021-10-25 2023-04-28 南开大学 一类具抑菌剂活性的磺酰脲类化合物的合成及应用

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