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WO2007050867A2 - Compositions pesticides et leurs methodes d'utilisation - Google Patents

Compositions pesticides et leurs methodes d'utilisation Download PDF

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Publication number
WO2007050867A2
WO2007050867A2 PCT/US2006/041968 US2006041968W WO2007050867A2 WO 2007050867 A2 WO2007050867 A2 WO 2007050867A2 US 2006041968 W US2006041968 W US 2006041968W WO 2007050867 A2 WO2007050867 A2 WO 2007050867A2
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WIPO (PCT)
Prior art keywords
pest
compound
activity
compounds
animal
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PCT/US2006/041968
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English (en)
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WO2007050867A3 (fr
Inventor
Jeffrey Bloomquist
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Virginia Tech Intellectual Properties, Inc.
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Publication of WO2007050867A2 publication Critical patent/WO2007050867A2/fr
Publication of WO2007050867A3 publication Critical patent/WO2007050867A3/fr

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Classifications

    • 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
    • A01N47/40—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 a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/46—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 a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=C=S groups
    • 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
    • A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
    • A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/10—Aromatic or araliphatic carboxylic acids, or thio analogues thereof; Derivatives thereof
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing within the same carbon skeleton a carboxylic group or a thio analogue, or a derivative thereof, and a carbon atom having only two bonds to hetero atoms with at the most one bond to halogen, e.g. keto-carboxylic acids
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids

Definitions

  • the present invention relates to compounds, compositions, and methods for inhibiting the growth of or for killing pests, including, but not limited to, insects and nematodes. It also relates to methods of identifying compounds having pesticidal activity. More specifically, the invention provides substituted organic acid derivatives and substituted anthracenes having activity against anion transporters, use of those compounds as pesticides, and use of anion transporters to identify compounds having similar structures and pesticidal activities. Description of Related Art
  • Nematodes and insects are economically important pests in the agriculture and animal husbandry industries. Their combined yearly damage in these two fields in the United States alone amounts to well in excess of tens of millions of dollars, m view of this recurring annual economic loss, much research has been performed to identify compounds that have pesticidal activity against these organisms and other related organisms.
  • Various pesticidal compounds ranging from simple chemical compounds produced through chemical syntheses (e.g., carbamates) to toxins produced by bacteria or other living organisms to transgenic crops that are resistant to attacks, have been developed or proposed.
  • chemical syntheses e.g., carbamates
  • toxins produced by bacteria or other living organisms to transgenic crops that are resistant to attacks have been developed or proposed.
  • resistance to pesticides has been encountered. Therefore, there is a continual need for novel pesticides to combat the losses in agriculture and animal husbandry.
  • Nematodes are tiny, worm-like, multicellular organisms found in virtually all habitats throughout the world, including water and soil. While the number of nematode species has been estimated in the hundreds of thousands, only a small portion of those are parasitic and considered to be pests. Plant parasitic nematodes are found associated with most agriculturally important plants. While many are plant species-specific in their targeting, most are promiscuous, capable of parasitizing two or more plant species. The major pathway of infection for soil-dwelling plant- parasitic nematodes is through the root tissue. Their damage to the roots can diminish the plant's ability to take up water and nutrients, and can provide portals of entry for other plant diseases.
  • hydroxystilbenes and salts thereof which are said to have nematicidal activity against pine wood nematodes. See, for example, the Abstract and Summary of the Invention of the '693 patent, hi addition, U.S. Patent No. 5,530,030 to Suga et al. discloses the use of chlorinated hydroxystilbenes or salts thereof as nematicides against the pine wood nematode. See, for example, the Abstract and Summary of the Invention of the '030 patent. U.S. Patent No. 2,920,013 to Shaver also discloses the use of nitrostilbene compounds to treat nematode infestations.
  • U.S. Patent No. 5,662,897 to Miller et al. discloses the use of a combination of a baculovirus and a stilbene compound to infect and kill insects. More specifically, the '897 patent discloses an engineered insect-killing virus. The patent further discloses that the killing effectiveness of the virus can be enhanced by co-treatment of the insect with the virus and a stilbene compound. See, for example, the '897 patent at column 5, lines 65-67, and column 23, lines 41-63.
  • U.S. Patent No. 2,088,651 to Henninger et al. discloses the use of compositions comprising lead arsenate and a stilbene compound to treat fruits for insect infestations. However, in this patent, the stilbene compound is included as a dispersing agent rather than a pesticide.
  • PCT publication WO 2006/060333 of Bloomquist et al. discloses the use of stilbene compounds as nematicides and insecticides, and methods of using anion transporters to identify compounds having such activities. This patent publication links the nematicidal and insecticidal activity of stilbene compounds to inhibition of anion transporter function.
  • chloride channel blockers are not particularly limited in structure, and can include 5-nitro-2-(3- phenylpropylamino)-benzoate (NPPB), N-phenylanthranilic acid, diphenylamine-2- carboxylic acid (DPC), R(+)-methylindaxone, indanyloxyacetic acid 94 (IAA-94), and DIDS.
  • NPPB 5-nitro-2-(3- phenylpropylamino)-benzoate
  • DPC diphenylamine-2- carboxylic acid
  • R(+)-methylindaxone indanyloxyacetic acid 94
  • DIDS DIDS
  • GABA gamma- aminobutyric acid
  • ATs anion transporters
  • Bloomquist J.R.
  • nifiumic acid is an open-channel blocker of CFTR that inhibits Cl(-) permeation by plugging the channel pore. This effect is similar to that of DIDS on the AT.
  • the authors go on to state that nifiumic acid or related agents might be of value in the development of new therapies for autosomal dominant polycystic kidney disease, much as the anion transporter inhibitor furosemide is used as a diuretic (Cabantchik and Greger, 1992).
  • the state of the art is such that stilbene compounds are known to be useful as nematicides and insecticides. Blockers of AT have also been studied as probes of channel function and as drug candidates in mammals.
  • AT blockage has been identified as an ancillary effect of insecticidal compounds known to work primarily on ligand-gated chloride channels.
  • the only publication that shows or suggests that any compound that is active against an AT has any in vivo nematicidal activity is WO 2006/060333, and that publication is primarily focused on stilbene compounds.
  • the present invention provides pesticidal compounds and compositions comprising those compounds. Exemplary embodiments relate to substituted organic acid derivatives and substituted anthracenes.
  • the invention also provides methods of treating plants and other multicellular organisms (e.g., fish, livestock, companion animals, humans) with the pesticidal compounds and compositions of the invention to protect the treated organism from attack by one or more pests, to treat them for infestation by or the parasitic activity of one or more pests, or to eliminate one or more pests from the organism or its environment.
  • the invention provides methods of identifying pesticidal compounds having a structure of a substituted organic acid derivative or an anthracene derivative. The methods use one or more anion transporters (AT) as screening agents to identify pesticidal compounds that bind to and block the activity of the AT.
  • AT anion transporters
  • AT are anion exchangers, anion co-transporters, and voltage-sensitive ion channels.
  • pests, pesticides, and pesticidal compositions relate to numerous organisms that are recognized as causing damage to plants and animals, and in particular to agricultural crops, to animals for human food production, and to humans.
  • pest includes, without limitation, nematodes, protozoa, insects, and Acari (e.g., ticks, mites), and pesticidal compounds are compounds that can kill such organisms.
  • pesticidal compounds and compositions are those that can kill a target organism or slow its growth, development, ability to reproduce, and/or ability to infect or parasitize a host.
  • the invention provides pesticidal compounds having a core structure that is described as a phenylpropylamino benzoic acid, an anthracene, or an indanyloxyacetic acid.
  • a core structure that is described as a phenylpropylamino benzoic acid, an anthracene, or an indanyloxyacetic acid.
  • Non-limiting examples of such compounds are presented in Figures 1, 2, and 3.
  • the compounds provided by the invention have, among other activities described herein or apparent from the present disclosure, pesticidal activity against one or more pests.
  • the compound(s) may have anti-nematode, anti- insect, and/or anti-Acari activity as a result of their effect on one or more AT of these organisms.
  • the present invention provides a composition comprising at least one pesticidal compound of the invention, hi general, the composition comprises one or more compounds having pesticidal activity, and another substance.
  • the other substance is not particularly limited in identity or activity, but is preferably not inhibitory of or detrimental to the pesticidal compound at its useful amount or concentration.
  • the other substance may be water or another liquid solvent, a compatible solid carrier or binder, another biologically active agent, a dispersant, or the like.
  • the present invention provides a method of treating at least one plant or animal susceptible to attack, parasitism, infection, or which is otherwise harmed by one or more pests, hi general, the method of treating comprises contacting at least one pesticidal compound of the invention with at least one target plant or animal, and allowing the compound(s) to remain in contact with the target for a sufficient amount of time for the compound(s) to exert a biological effect, hi embodiments, the method is a method of treating one or more plants, wherein the method comprises contacting at least one pesticidal compound of the invention with the plant, and allowing the compound(s) to remain in contact with the plant for a sufficient amount of time for the compound(s) to kill or inhibit the growth, reproduction, or infectivity of at least one pest, hi other embodiments, the method is a method of treating one or more animals (including humans), wherein the method comprises contacting at least one pesticidal compound of the invention with the animal, and allowing the compound(s) to remain in contact
  • the invention provides a method of treating an environment of a plant or animal that is susceptible to attack, parasitism, infection, or which is otherwise harmed by one or more pests, hi general, the method of treating an environment comprises contacting at least one pesticidal compound of the invention with the environment, and allowing the compound(s) to remain in contact with the environment for a sufficient amount of time for the compound(s) to exert a biological effect.
  • the amount of time maybe an amount sufficient for the compound(s) to kill or inhibit the growth, reproduction, or infectivity of at least one pest in the environment.
  • the present invention provides a method of treating at least one pest, such as a nematode, insect, or Acari, that infects, parasitizes, damages, kills, or otherwise harms one or more plants or animals, including but not limited to humans.
  • the method of treating at least one pest comprises contacting at least one pesticidal compound of the invention with the pest(s), and allowing the compound(s) to remain in contact with the pest(s) for a sufficient amount of time for the compound(s) to exert a biological effect on the pest(s).
  • the biological effect is, in some instances, inhibition of infecting, parasitizing, damaging, or killing the plants or animals. Inhibition can be by reducing the viability or reproduction * of the pest, resulting in ultimate death of the pest, or by direct and relatively quick killing of the pest.
  • the methods of treating typically comprise contacting 1) the target plant, animal, environment, or pest, and 2) at least one pesticidal compound, where the amount of the compound(s) that contacts the plant, animal, environment, and/or pest is sufficient to inhibit the viability, infectivity, and/or activity of the pest, as compared to another of the same type of pest in the absence of the compound(s).
  • the amount is sufficient to kill at least one pest in a biologically relevant time frame, such as the life span or the span of a particular growth stage, of the pest.
  • the invention provides a method of identifying compounds having the structural characteristics of compounds disclosed herein, which also show pesticidal activity against one or more pests.
  • the method comprises contacting at least one anion transporter (AT) with at least one compound, and determining if the compound(s) bind to the AT.
  • the method further comprises determining if the compound alters the function of the AT, for example by inhibiting the transporter activity (as compared to a similar AT under, the same conditions, but in the absence of the compound(s). Alteration of AT activity is indicative of binding of the compound(s) to the AT.
  • the methods of the invention may also identify compounds that activate, enhance, etc. the activity of one or more AT.
  • Such activators may be used for various purposes, including, but not limited to, use as competitors for characterization of certain AT inhibitors, and use as pesticides (disruption of AT activity, whether inhibition or over-activation should disrupt normal cellular activity and result in loss in viability and/or death).
  • the methods of identifying compounds can be practiced in vivo, typically they are performed in vitro, or initially performed in vitro, with confirmatory assays performed in vivo.
  • Figure 1 depicts the chemical structure of 5-Nitro-2-(3- phenylpropylamino)benzoic acid (NPPB).
  • Figure 2 depicts the chemical structure of anthracene-9-carboxylic acid (9- AC).
  • Figure 3 depicts the chemical structure of (+)(2-cyclopentyl-6,7-dichloro- 2-methyl-l-oxo-5-indanyloxy) acetic acid (IAA-94).
  • Figure 4 depicts a bar graph showing the results of various AT blockers against the nematode Heterorhabditis bacteriophora.
  • pest and its various forms are broad terms used to indicate all organisms that are recognized as causing damage to plants and animals. At times in this disclosure, certain pests are referenced specifically, such as by nematodes, insects, and acari. It is to be understood that reference to a particular pest in this disclosure is meant to infer reference to all other pests unless specifically noted as being limited to one or more particular pests.
  • nematode and its various forms are used to indicate all organisms of the phylum Nematoda, including, but not necessarily limited to, all organisms in the various classes that have been traditionally used, are in current use, or are proposed for use in the future.
  • the term thus includes organisms classically referred to as Metazoan organisms. It thus encompasses all organisms in the classes Adenophorea and Secernentea, but is not limited only to those organisms.
  • the use herein of the term nematode is to be understood to encompass all such organisms, without limitation to current or prior taxonomic or phylogenic schemes or labels.
  • the term refers to parasitic nematodes, such as those that infect animals (including humans) and those that infect plants. In other embodiments, the term refers to free-living nematodes, hi yet other embodiments, it refers to both types of nematodes.
  • insects and its various forms is used herein to indicate all organisms within the class Insecta.
  • the term thus includes, but is not necessarily limited to, organisms that are currently classified as members of the class Insecta. It also includes, but is not necessarily limited to, organisms that were previously considered to be members of the class or have been traditionally considered insects.
  • the term "Acari” and all of its forms e.g., acarine
  • the term thus includes, but is not limited to, mites and ticks.
  • the present invention is based, at least in part, on the unexpected realization that certain non-stilbene compounds can act as potent inhibitors of the transporter activity of anion transporters (AT) in pests, and thus can be used as agents to inhibit the growth, reproduction, or activity of such pests, and even kill such pests. It is further recognized that the activity of these compounds can be provided not only when contacted directly with the pest, but when contacted with the environment in which the pest lives, the environment in which the organism that the pest attacks lives, and the organism that the pest attacks.
  • the present invention provides new compounds with pesticidal activity, and provides therapeutic, agricultural, and research fields for treatment of animals (including humans), plants, and the environment. It likewise provides for discovery of new compounds for treatment of animals plants, and the environment.
  • the invention provides pesticidal compounds having a core structure that can be described as a phenylpropylaminobenzoic acid, an anthraceiie-9-carboxylic acid, or an indanyloxyacetic acid.
  • the compounds provided by the invention have, among other activities described herein or apparent from the present disclosure, pesticidal activity against one or more pests through a mechanism of action that includes modulating the activity of one or more AT in the pest.
  • the compound may have anti-nematode, anti-insect, and/or anti- Acari activity as a result of their effect on one or more AT of these organisms.
  • NPPB 5-Nitro-2-(3-phenylpropylamino)benzoic acid
  • 9-AC anthracene-9-carboxylic acid
  • IAA-94 (+)(2-cyclopentyl-6,7-dichloro-2- methyl-l-oxo-5-indanyloxy) acetic acid
  • the structures of the compounds of the present invention can be recognized as non-stilbene compounds, which are structurally different from compounds acting on AT and known in the art to have nematicidal, insecticidal, and acaricidal activity.
  • the pesticidal compounds according to the invention can be substituted at one or more carbons present on the compounds, such as shown in Figures 1-3.
  • one or more carbons on the phenyl or other ring groups can be substituted with one or more substituent, such as, but not limited to, a hydroxyl group, a Nitrogen-containing group ⁇ e.g., NO 2 ), a Sulfur-containing group ⁇ e.g., SO 3 and SO 2 OH) 5 a halide, a carboxyl group, an alkyl group, an aliphatic group, and a substituted or non-substituted ring group.
  • substituent such as, but not limited to, a hydroxyl group, a Nitrogen-containing group ⁇ e.g., NO 2 ), a Sulfur-containing group ⁇ e.g., SO 3 and SO 2 OH) 5 a halide, a carboxyl group, an alkyl group, an aliphatic group, and a substitute
  • each substituent can be the same, two or more (but not all) can be the same, or each can be different from all others. Additionally, if present, a nitrogen atom may be substituted with one or more of the groups listed above.
  • the carbons of the phenyl groups of the compounds can be substituted with any element or group known in the art as suitable for bonding to phenyl carbons.
  • elements or protonated forms of elements such as hydrogen, carbon, nitrogen, oxygen, and sulfur
  • organic groups such as short-chain (1-4 carbon), medium chain (5-12 carbon), and long-chain (13 or greater) carbonyl groups, such as substituted or unsubstituted alkyl, alkenyl, and alkynyl groups
  • substituted and unsubstituted aryl groups such as phenyl groups; nitrogen-containing groups; sulfur-containing groups; metal-containing groups, halide-containing groups, and the like.
  • substituent groups may be substituted in accordance with the listing presented for the phenyl groups of the core structures.
  • a nitrogen atom may be substituted with one or more of the groups listed above.
  • the compounds of the invention include salts of the above-mentioned compounds. [043]
  • the above-described substituents and/or additional substituents can be included on compounds of the invention. Likewise, other modifications and/or substitutions recognized as appropriate by one of ordinary skill in the art are also encompassed by the compounds of the invention.
  • nitrophenylaminobenzoic acids for example, nitrophenylaminobenzoic acids, anthracene-9-carboxylic acids, and dichloro-oxo- indanyloxyacetic acids, as shown below, are encompassed by the compounds of the invention.
  • Variable substitutions X, Y, and Z, as shown below, can be made as deemed appropriate by one of ordinary skill in the art, including the substitutions provided above.
  • the same positions can contain alkyl esters that are cleaved to the corresponding acids, in vivo.
  • a basis of the invention derives from previous studies by the inventor and his colleagues that showed that stilbene compounds can act as inhibitors of the function of AT, and thus affect the growth and activity of nematodes, insects, and Acari.
  • the present invention expands on that initial discovery by providing additional, non-stilbene compounds, having similar activity, and a similar mode of action, against pests.
  • Another basis of the invention derives from the recognition in the art that certain non-stilbene compounds have activity against certain AT, resulting in effects having therapeutic value in humans. Although such compounds are known, each particular compound appears to have been characterized with regard to only one or a few particular AT from humans, and their toxic effects on non-human organisms was not reported and, because of the complexity of biological systems and the evolutionary diversity between species, could not be predicted from the results presented.
  • the invention provides a composition comprising at least one pesticidal compound of the invention and at least one other substance, for example, a carrier.
  • the pesticidal compound can be one or a combination of two or more compounds as disclosed herein.
  • the other substance present in the composition may be any substance or combination of substances, with the caveat that the substance or combination of substances is not inhibitory of or detrimental to the effect of the pesticidal compound at its useful amount or concentration.
  • the other substance(s) maybe inhibitory to the activity of the compound(s) of the invention at the concentration that the two are present in the composition
  • the other substance(s) should not be inhibitory or otherwise detrimental at the concentration of pesticidal compound that is to be contacted with a target (e.g., after dilution of the composition to a working concentration, or at the concentration present after application to a plant, animal, or environment)
  • the compositions are active against one or more nematodes
  • the compositions are active against one or more insects
  • hi embodiments the compositions are active against one or more Acari.
  • the compositions are active against one or more species of two or more of these types of organisms.
  • the amount of compound included in the compositions can be sufficient to kill some, most, or all of one species of nematode (or insect or Acari), yet be less effective against some or all other species of nematodes (or insects or Acari).
  • the present invention provides for use of one or more compounds or compositions of the invention as insecticides, nematicides, and acaricides, and to prepare insecticides, nematicides, and acaricides, which can be broad spectrum agents or specific agents.
  • Exemplary substances that can be included in the compositions in addition to the compounds having pesticidal activity include solids, liquids, and gases. Examples include, but are not limited to, water or other aqueous solutions, and organic liquids, such as alcohols, aldehydes, and ethers, such as DMSO, or combinations thereof.
  • solids include, but are not limited to, whether alone or in combinations, carriers, binders, solubilizing agents ⁇ e.g., salts), dispersants, colorants, gums, inert fillers, minerals, biologically tolerable pharmaceutical additives, ⁇ e.g., excipients), surface active agents, waxes, light protecting agents, preservatives, and other substances that are commonly employed in products for use in agriculture, animal husbandry, and medical and veterinarian settings.
  • solubilizing agents ⁇ e.g., salts
  • dispersants e.g., colorants, gums, inert fillers, minerals, biologically tolerable pharmaceutical additives, ⁇ e.g., excipients
  • surface active agents e.g., waxes, light protecting agents, preservatives, and other substances that are commonly employed in products for use in agriculture, animal husbandry, and medical and veterinarian settings.
  • the compositions may comprise, in addition to the pesticidal compound, one or more other biologically active agent, such as antibiotics, anti-inflammatory agents, anti-viral agents, antifungals, hormones, nutrients, vitamins, and the like, or a pesticide ⁇ e.g., insecticide, nematicide, or acaricide) that is not a compound according to the invention.
  • a pesticide e.g., insecticide, nematicide, or acaricide
  • Further examples include, but are not limited to, synergists that block metabolism, such as monooxygenase inhibitors ⁇ e.g., piperonyl butoxide).
  • the other substance may be a pharmaceutically acceptable substance, such as those that are well known in the medical veterinarian arts.
  • compositions are dispersants or other substances that aid in distribution of compositions in agriculture settings. These can be biologically active or inactive substances that act as fillers or dilution agents. They can also be compounds that improve the solubility of the compounds in aqueous environments, hi preferred embodiments, the compositions comprise one or more substances that improve the solubility of the compound in water or an aqueous liquid.
  • the pesticidal compound(s) of the invention are present in an amount or concentration that is sufficient such that, when applied to a plant, animal, or the environment (including an artificial, in vitro, or laboratory or research environment) shows a measurable effect on the growth, survival, reproduction, or other biological activity of at least one nematode, insect, or Acari.
  • at least one pesticidal compound is present in the composition in a sufficient amount to kill at least some of the nematodes and/or insects and/or Acari within the area treated with the composition (e.g., on the leaves of plants sprayed with the composition).
  • At least one pesticidal compound of the invention is present in the composition in a sufficient amount or concentration to kill a majority, essentially all, or all of the target pests in the area treated.
  • the active compound is present in the composition in an amount that is sufficient such that, when applied to a plant, animal, environment, nematode, insect, or Acari, it is present in a concentration of 1000 parts per million (ppm) or less.
  • the concentration when applied is 500 ppm of less, such as 200 ppm or less or 100 ppm or less, such as 50 ppm, 40 ppm, 25 ppm, 10 ppm, 5 ppm, 2 ppm, 1 ppm, or even less.
  • ppb parts per billion
  • ppb parts per billion
  • 100 ppb, 50 ppb, 10 ppm parts per billion
  • 10 ppb parts per billion
  • a broad range of useful concentrations is envisioned by the invention.
  • a working concentration of from 10 ppb to 10 ppm is suitable.
  • a range of from 10 ppb to 1 ppm is suitable, as is a range of from 100 ppb to 10 ppm, or a range of from 10 ppm to 100 ppm.
  • compositions having much higher concentrations of the compound(s) of the invention are contemplated, with reduction in concentration prior to use, such as by dilution, being recommended.
  • concentration prior to use such as by dilution
  • a concentrated composition of 6 pounds of compound per gallon of solution, 4 pounds of compound per gallon of solution, or 1 pound of compound per gallon of solution can be provided.
  • one of the other substances is another compound having pesticidal activity, including a substance(s) identified using the methods of the present invention.
  • the composition comprises one or more other substances that have biological activity, but not pesticidal activity.
  • the composition may comprise one or more pesticidal compounds and one or more substance that is beneficial for plant or animal growth, such as a fertilizer (e.g., a nitrogen, phosphorous, potassium, calcium, magnesium), or one or more substance that is harmful for plant or animal growth, such as a herbicide (e.g., glyphosphate).
  • a fertilizer e.g., a nitrogen, phosphorous, potassium, calcium, magnesium
  • a herbicide e.g., glyphosphate
  • compositions can be used for treatment of plants, animals, or the environment surrounding selected plants and/or animals. They can be used for treatment to provide a beneficial effect, in vivo or in situ. Alternatively, they can be used for research purposes in vitro to identify suitable concentrations or composition components having a desired activity.
  • the compounds exemplified herein can be used as bases for derivatives having similar activity, but a higher or lower specific activity, solubility, side-effect profile, and the like.
  • the present invention provides a method of treating at least one plant or animal susceptible to attack, parasitism, infection, or which is otherwise harmed or susceptible to harm by one or more pests.
  • the method of treating comprises contacting at least one pesticidal compound of the invention (alone or as part of a composition) with at least one target plant or animal, and allowing the compound(s) to remain in contact with the target for a sufficient amount of time for the compound(s) to exert a biological effect.
  • Contacting may be any action that results in physical contact of at least one molecule of a compound of the invention and a surface of a target (e.g., plant, animal, environment, pest).
  • Contacting thus may comprise exposing the target to the compound(s) through introduction of the compound(s) into the general area of the target, and allowing sufficient time for the compound to contact the target through natural (e.g., diffusion) or facilitated (e.g., mechanical distribution by a person or machine) distribution within the area.
  • natural e.g., diffusion
  • facilitated e.g., mechanical distribution by a person or machine
  • the invention further provides a method of treating an environment of a plant or animal that is susceptible to attack, parasitism, infection, or which is otherwise harmed by one or more pests.
  • the method of treating an environment comprises contacting at least one pesticidal compound of the invention with the environment, and allowing the compound(s) to remain in contact with the environment for a sufficient amount of time for the compound(s) to exert a biological effect, and in particular, an effect on a pest.
  • contacting may comprise any action that results in physical contact of the compound(s) and environment.
  • the biological effect can be any effect that is measurable or detectable, including, but not limited to inhibition of the growth, reproduction, or infectivity of at least one pest in the environment.
  • the effect need not be immediate, but preferably occurs within a time frame that is relevant to the pest of interest, such as within the typical life cycle of the pest, or within the typically time frame of a developmental stage. In preferred embodiments, the effect is evidenced in a short time frame, such as within a 7 days or less.
  • the present invention also provides a method of treating at least one pest that infects, parasitizes, damages, kills, or otherwise harms or has potential to harm one or more plants or animals.
  • the method of treating at least one pest comprises contacting at least one pesticidal compound of the invention (alone or in a composition) with the pest(s), and allowing the compound(s) to remain in contact with the pest(s) for a sufficient amount of time for the compound(s) to exert a biological effect on the pest(s).
  • the method can comprise causing or allowing the pest(s) to ingest or otherwise internalize the compound(s).
  • the biological effect is, in some instances, inhibition of infecting, parasitizing, damaging, or killing the plants or animals. Inhibition can be by reducing the viability or reproduction of the pest, resulting in ultimate death of the pest, or by direct and relatively quick killing of the pest. Time frames for the biological effects are as discussed above.
  • the biological effects can be detected, observed, determined, etc. by any suitable method, such as those commonly known and used in the art.
  • the method kills or inhibits the activity of one or more free-living nematodes, hi embodiments, the method kills or inhibits the activity of one or more plant parasites.
  • the surface is a surface of a leaf, stem, branch, trunk, etc., but can be a surface of a root as well.
  • the surface is skin, hair, or a mucous membrane, although cell surfaces of internal cells are also contemplated.
  • the surface is typically an external surface, although internalization of the compound of the invention is preferred for activity.
  • the surface can be any surface present in the environment.
  • the surface will be soil, rock or another surface generally understood to be a natural part of the earth.
  • contact can include contact with an external portion of a pest, followed by ingestion in some way of the compound by the pest, hi situations where the pest attacks an animal or plant, contact may be through contact of the animal or plant, followed by ingestion by the pest as a result of biting of the animal or plant.
  • the method is a method of treating an environment, the environment can be any environment in which a plant, animal, or pest of interest is found.
  • crops or other plants such as, but not limited to, an agricultural field (e.g., farmland, home garden), a nursery, a tree farm, woods or forest, or aquatic environments, such as freshwater stream, river, pond, or lake, whether it be above or below ground. It also may be a natural environment for animals, such as but not limited to, a farm, ranch, or
  • Non-limiting examples of non-natural environments include zoos, arboretums, research facilities, laboratories, preserves, and structures or equipment contained in these environments.
  • the non-natural environment comprises controlled parameters for research, such as culture dishes and defined growth media.
  • Administering may be by way of any suitable route, including, but not limited to, topical (e.g., by way of a salve, cream, lotion), transdermal (e.g., byway of a patch), injection (e.g., injection, such as subcutaneous, intravenous, intramuscular, and the like), infusion, oral (e.g., by way of lozenge, capsule, pill, powder, liquid suspension), nasal (e.g., by way of aerosol, powder), or mucosal (e.g., by way of suppository, cream, lotion).
  • topical e.g., by way of a salve, cream, lotion
  • transdermal e.g., byway of a patch
  • injection e.g., injection, such as subcutaneous, intravenous, intramuscular, and the like
  • infusion e.g., oral (e.g., by way of lozenge, capsule, pill, powder, liquid suspension), nasal (e.g., by way of aero
  • the method reduces the number of pests to a number less than half of the original number, hi highly preferred embodiments, the method eliminates 75%, 90%, 95%, or 99% or more (preferably completely eliminates) of the pests infecting the animal. Where a single contacting (e.g., administration) does not provide a desired reduction in the number or activity of pests, the contacting may be repeated.
  • the method is a method of treating one or more plants, wherein the method comprises contacting at least one pesticidal compound of the invention with at least one surface of the plant, and allowing the compound(s) to remain in contact with the plant for a sufficient amount of time for the compound(s) to kill or inhibit the growth, reproduction, or infectivity of at least one pest.
  • the method may further comprise ingestion or internalization of the compound(s) in some other way by the pest.
  • the method is a method of treating one or more animals (including humans), wherein the method comprises contacting at least one pesticidal compound of the invention with the animal, and allowing the compound(s) to remain in contact with the animal for a sufficient amount of time for the compound(s) to kill or inhibit the growth, reproduction, or infectivity of at least one pest.
  • the method may further comprise ingestion or internalization of the compound(s) in some other way by the pest.
  • the animal can be any animal, hi some embodiments, it is a mammal. In embodiments, it is specifically a human. It thus can be an animal that is grown or used in an agriculture setting, such as on a farm or ranch.
  • the plant and/or animal treated is one that is parasitized, infected, damaged, or killed by one or more pest, such as an Acari, insect, and/or nematode species, including, but not limited to nematodes.
  • pest such as an Acari, insect, and/or nematode species, including, but not limited to nematodes.
  • the method also may be practiced on at least one plant, animal, and/or environment that, while not currently being parasitized, infected, damaged, or killed by a nematode and/or insect and/or Acari, is known to be susceptible or often parasitized, infected, damaged, or killed by one or more of these organisms.
  • the method can also comprise treating plants and animals that are bitten, damaged, or killed by one or more pests. Inhibition can be by reducing the viability of the organisms, resulting in ultimate death of the organisms, or by direct killing of the organisms. Alternatively, it may simply reduce the activity of the organism, and thus reduce damage caused by that organism. A reduction in activity of one or more pests, while not completely eliminating a source of economic loss, still provides an economic benefit by reducing damage' caused by these organisms. [062] As used herein, contacting is broadly defined, ha some instances, it comprises exposing a target to a compound of the invention. Exposing can be any activity that results in ultimate contact of the compound with the plant, animal, and/or environment.
  • the compound can thus be contacted with the plant, animal, or environment directly or indirectly.
  • Contact can be, for example, by spraying, dusting, dipping, fogging, misting, watering, fumigating, injecting, ingesting, and rubbing. It thus can be by crop dusting. It also can be by broadcasting on an agricultural environment prior to planting of a crop or allowing animals to graze.
  • One non- limiting example of exposing is adding a pesticidal compound to an environment, and permitting natural dispersion of the compound to effect contact.
  • the method can comprise providing an environment containing the compound, and placing the pest in that environment. For example, it can comprise providing a culture dish having the compound of interest attached, adhered, or otherwise associated with the surface of the dish or a medium in the dish, then introducing the pest into the dish.
  • Inhibiting activity is a broad term that generally denotes affecting the normal life and life processes of a pest. It thus can affect the metabolism of a pest. It likewise can affect the growth and/or development of the organism. It can affect the sexual development of the organism.
  • inhibiting can be considered the act of reducing growth of one or more pests from an immature to a mature stage, reducing the ability to reproduce or the rate of reproduction (as compared to untreated pests of the same species in the same environment), reducing the amount of feeding or the ability to metabolize food.
  • inhibiting is the act of causing the death of at least one pest. The act of inhibiting can cause a result in a short period of time or over a prolonged period of time.
  • the action of the compound(s) can be rapid (less than one day), or prolonged (more than two weeks).
  • sufficient effect on activity is seen in one week or less, such as 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. It also may be seen in about 72 hours, 60 hours, 48 hours, 36 hours, 30 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, or less.
  • results may be seen in 60 minutes or less, 30 minutes, 10 minutes, 5 minutes or less, or 2 minutes or less, such as 60 seconds or less, 30 seconds or less, or 15 seconds or less.
  • a direct effect is by direct contact of the compound with the pest
  • an indirect effect is by contact of the compound with a plant, animal, or environment, then contact of a pest with that plant, animal, or environment.
  • direct killing is by contact of the compound with the organism directly from the source of exposure (e.g., direct contact upon spraying from an airplane, a truck-based container, a hand-held sprayer, or a can, such as a hand pump).
  • indirect killing is by application to environment or animal or plant, then contact with a target organism by contact of the target organism with the treated plant or animal or environment. Accordingly, indirect killing can occur at a time that is considerably different than the time at which the compound was exposed to the plant, animal, or environment.
  • Indirect treating is exemplified in one embodiment by treatment of a plant, then eating of plant material by a target organism.
  • the compound may be exposed to the plant, animal, and/or environment as the sole substance provided. It may also be exposed in conjunction with another compound according to the invention.
  • the pesticidal compound(s) may be provided and used as purified (partially, essentially completely, or completely) products, or may be provided as part of one or more compositions. Where one or more compositions are provided, they may be any of those discussed above. Of course, one or more compounds may be provided, each as purified products, all together in a single composition, or some as purified products and others in a composition.
  • the method of treating can comprise performing the exposing step more than one time.
  • the method contemplates a regimen where the plant, animal, or environment is treated multiple times.
  • the time interval between exposures will depend on the plant, animal, and environment, as well as the level of infestation of pests, and the amount or concentration of compound used, hi general, it is preferred that, for agriculture purposes, a single exposure is performed per growing season. Two or more exposures are also contemplated.
  • exposure of a plant, animal, or environment can be performed on a recurring basis, such as once a year, once every four months, once every three months, once every two months, or once a month. Exposure can also be repeated as needed, based on visual observation (e.g., when damage of crops is observed). Practitioners may select the most appropriate exposing regimen for each particular application of the method of the invention.
  • the present invention provides methods of treating multicellular host organisms that are currently parasitized or susceptible to being parasitized by a nematode or insect or Acari.
  • the methods of treatment of these organisms generally comprise contacting the host organism with at least one pesticidal compound of the invention in amount sufficient to disrupt the activity of at least one anion transporter of the pest, the disruption blocking or causing a reduction or cessation of parasitism of the target organism on the multicellular host organism.
  • the compound can be applied directly (either topically or internally) to the host organism or to the environment surrounding the host organism.
  • the method blocks an attack on the host organism by a target organism by interfering with AT function before the target organism can parasitize the host, hi embodiments, the method reduces or eliminates an attack on the host organism after it has begun by interfering with AT function of the target organism that is attacking or parasitizing the host. In embodiments, the method ends an attack or prevents further attack by killing the target organism by disrupting function of at least one AT. hi embodiments where treatment is directed at insect damage, the method does not include treating with a compound that affects a ligand- gated chloride channel of the insect.
  • the present invention also provides methods of protecting a multicellular host organism parasitized by a pest, such as a nematode or insect or Acari, or which is susceptible of being parasitized by a pest. These methods comprise contacting the target organism with at least one pesticidal compound of the invention in amount sufficient to disrupt the activity of at least one anion transporter of the target organism.
  • the method is a method of protecting a host from an insect, the method does not comprise treating with a compound that acts on a ligand-gated chloride channel of the insect.
  • the present invention provides a method of treating where both the host organism (or its environment) and the target organism are treated simultaneously with the same act of contacting. It likewise provides a method of killing target organisms by contacting them with at least one pesticidal compound.
  • compounds are typically applied directly to the target plant or to the soil surrounding the target plant, and the target organism is exposed to the compound as a result of contact with the treated plant or soil.
  • Other methods include treating water in which the target organism lives (during at least a portion of its life cycle), and treating the food of a fish or animal, or treating the fish or animal itself. In certain methods, both the plant, fish, or animal and the target organism are exposed at the same time.
  • the methods of the invention rely, at least in part, on the activity of the compounds of the invention on one or more AT of the target organism, where such activity disrupts normal function of the AT, resulting in impaired anion transport, loss of cellular function, and reduction in viability, or death, of the target organism.
  • the methods of treating may be methods of reducing or blocking initial parasitic activity of pests (i.e., protective or prophylactic methods of treating plants, fish, animals), methods of reducing or ending active attacks, or methods of eliminating pests from plants, fish, or animals.
  • the invention provides for use of compounds and compositions of the invention for production of one or more pesticides, such as insecticides, nematicides, and acaricides.
  • the method can comprise using effective amounts (either as prepared or after reduction in concentration) of the compounds to produce products that can have agricultural, veterinarian, and/or medical use.
  • the invention provides for the use of compounds of the invention to produce veterinarian or medical (therapeutic, prophylactic) products.
  • the present invention likewise provides for the use of the pesticidal compounds and compositions for the treatment of one or more plants, animals, or environments to reduce or eliminate at least one pest.
  • the invention provides for use of pesticidal compounds for treatment of plants, animals, and/or environments to reduce or eliminate at least one nematode and/or at least one insect or Acari.
  • the invention provides methods of screening for compounds, such as compounds having the same general structure as those provided in the figures, that show pesticidal activity, and in particular, activity against nematodes, insects, or Acari.
  • the methods can comprise screening for compounds that affect the activity of at least one AT, have the same general structure as the compounds of the figures, and have pesticidal activity.
  • the methods use one or more AT as screening agents to detect compounds resembling those of the figures and that bind to and affect the activity of the AT.
  • the compounds that can be screened can be generally described as substituted organic acids and substituted anthracenes.
  • the methods generally comprise contacting one or more AT molecules with one or more compounds and determining if the activity of the AT was altered, alteration of activity indicating that one or more of the compounds affected the activity of the AT.
  • the methods of screening are based, at least in part, on the realization that compounds having structures similar to those depicted in the figures can inhibit the activity of AT of pests, resulting in abnormal activity, and even death, of the pests. Accordingly, the invention provides for use of AT to identify compounds having the general structure of the compounds of the claims, and which have pesticidal, and in particular nematicidal, activity.
  • the step of contacting can comprise any activity that results in physical contact of at least one test compound with at least one AT molecule.
  • a test compound is a compound having a structure resembling a structure depicted in the claims, which is being investigated for its potential affect on one or more AT.
  • the act of contacting typically comprises adding the compound to a composition comprising the AT.
  • an AT can be present in a reaction mixture, and the test substance is added to the mixture. Sufficient time is provided for the test substance and the AT to come into contact, then the activity of the AT is assayed.
  • AT assay compositions are known in the art, and any of them maybe used in accordance with the invention.
  • amount of time might vary; however, those of skill in the art are well aware of suitable times for contact to occur within the context of each of the various possible protocols known in the art.
  • Exemplary reaction assays and conditions are provided in the Examples, but other, equally effective conditions and methods are known to those of skill in the art from published journal articles.
  • AT activity can be assayed using electrophysiological methods to measure blockage of ion currents through AT according to Machaca, K., et al, "A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid differentiation", Dev. Biol. 176(1):1-16, 1996.
  • 36 Cl- ion flux assays to determine chloride ion movements through AT by biochemical studies according to Payne, G.T. and Soderlund, D.M, "Activation of gamma-aminobutyric acid insensitive chloride channels in mouse brain synaptic vesicles by avermectin BIa", J.
  • Other assays can include use of fluorescence methods to measure, directly or indirectly, effects on AT function. Effects on AT function can directly alter the fluorescence signal of dyes like MQAE (see, for example, Munkonge F, et al., "Measurement of halide efflux from cultured and primary airway epithelial cells using fluorescence indicators", J. Cyst. Fibros. 2004; Suppl 2:171-6).
  • AT blockage can also upset the acid balance of a cell, leading to a change in intracellular pH, and this effect could be used in a pH-dependent fluorescence-based assay according to, for example, Vieira L.
  • the present invention comprises both in vitro and in vivo screening assays, including those that comprise both in vitro and in vivo aspects.
  • the methods can be practiced in vivo, typically they are performed in vitro, or initially performed in vitro, with confirmatory assays performed in vivo. Where both in vitro and in vivo assays are performed, it is preferred that the in vitro assays precede the in vivo ones.
  • the method of screening according to the invention can comprise contacting one or more AT with one or more test compounds in vitro, followed by contact of one or more AT with one or more test compounds in vivo.
  • the methods of the invention can comprise contacting at least one test compound and at least one living nematode or insect.
  • the effects of the contacting can be determined by observing the activity or viability of the nematode or insect or acarine. It thus can include determining whether a nematode or insect is killed by the contact.
  • the method comprises contacting at least one AT from a known species of nematode, insect, or Acari with at least one compound, and determining the effect of the compound(s) on the AT(s). Where more than one compound is screened at a time, and at least one is determined to have an effect, the compounds are re-screened until each active compound is identified. Likewise, if more than one AT is used in the initial screening, the number of AT used in subsequent screenings is reduced until the loss in activity can be assigned to one or more particular AT. Ultimately, the effect of each active compound (also referred to herein as a lead compound) individually on each AT individually can be tested to find specific relationships between particular compounds and particular AT.
  • a lead compound individually on each AT individually can be tested to find specific relationships between particular compounds and particular AT.
  • a particular active compound is found to have an effect on a particular AT.
  • the method can further comprising correlating the activity on the AT with the inhibitory or killing effect of the compound on a target organism in vivo. Due to the recognition of the molecular basis of the effects of AT-inhibiting compounds on target organism activity and viability, this embodiment of the method provides a confirmatory correlation for the in vivo activity of the active compound. Correlation not only confirms the activity of the lead compound in vivo, but permits one to determine the specificity of the lead compound for various species of target organisms. In vivo inhibition/killing assays are known in the art, and exemplary assays are provided herein.
  • Any suitable in vivo inhibition/killing assay may be used in this embodiment of the method of the invention, the choice of any particular series of steps being well within the competency of those of skill in the art, and being made based on any number of parameters, including, but not limited to, cost, time, availability of reagents and supplies, etc.
  • the method of screening comprises using one or more AT molecules, hi embodiments, it comprises using a single AT molecule (typically, a combination of numerous AT molecules, which are all of the same type/amino acid sequence). In other embodiments, it comprises using two or more AT molecules.
  • a single AT molecule typically, a combination of numerous AT molecules, which are all of the same type/amino acid sequence.
  • it comprises using two or more AT molecules.
  • substances whether it be a test compound, an AT, or any other substance
  • substances are referred to as being present or being present as "a single" substance, it is meant that a substance having a particular identity is present, in one or multiple identical or essentially identical copies.
  • the method comprises using a single AT means that an AT with a particular amino acid sequence is used, and that the AT is present in a single or multiple (up to millions or billions) of copies.
  • test compounds can be contacted with multiple different AT molecules, then re-screened with a subset of those AT molecules to identify which AT molecules are being affected.
  • the method can comprise screening with a single AT molecule. Where more than one AT is used in the method, the method may further comprise exposing the compound to each AT individually to determine which AT is affected.
  • the AT molecule can be provided by purification, at least to some extent, from an organism of interest. Purification techniques are well known in the art, and can be applied to at least partially purify AT activity from lysates of a pest of interest.
  • the method of screening comprises using one or more test compounds. In embodiments, it comprises using a single test compound. In other embodiments, it comprises using two or more test compounds.
  • the method can comprise determining a test compound after a single iteration of the screening process.
  • multiple different test compounds can be contacted with one or multiple different AT molecules, then one or more subsets of the test compounds showing activity on the AT can be re-screened with the same AT, a subset of the AT, one or more different AT, or a mixture of some or all of the same AT and one or more different AT (preferably the same set of AT used in the first screen).
  • One or more sub-subsets can then be contacted with an AT or mixture of AT (preferably from the same set of AT used in the first and/or second screen) to identify which AT molecules are being affected. Re-screening can be repeated until a suitable number of test compounds are determined.
  • the method can comprise screening with a single AT molecule and/or screening a single test compound. Where more than one AT is used in the method, the method may further comprise exposing the compound to each AT individually to determine which AT is affected.
  • the method of screening is a high-throughput method.
  • the method of screening comprises determining if the activity of the AT was altered.
  • An AT has an altered activity if its activity is detectably different in the presence of one or more test compound than in the absence of the compound.
  • the difference can be determined using any of a number of assays, as known in the art and disclosed herein. Comparison can be made between the same AT (e.g., determine activity, then add test compound(s) and determine activity again) or between an AT' in one reaction vessel containing the test compound and an identical AT, in the same composition as the first AT but without the test compound.
  • the methods of the invention may also identify compounds that activate, enhance, etc. the activity of one or more AT.
  • Such activators may be used for various purposes, including, but not limited to, use as competitors for characterization of certain AT inhibitors, and use as pesticides.
  • the disruption of AT activity, whether inhibition or activation, disrupts normal cellular activity and result in loss in the target pest activity, loss in viability, and/or death.
  • the method of screening in its basic form, comprises contacting at least one AT with at least one test compound, and determining if the compound affects the activity of the AT. In embodiments, the method further comprises identifying the test compound(s) that affect the AT.
  • the method can further comprise identifying which AT is affected by which test compound (if more than one test compound is used). Identifying the test compound showing activity can be through any of the various methods used by those of skill in the art. Of course, if the identity of the test compound was known prior to performing the method of screening, it is a simple matter to identify the test compound showing activity. Where the identity was not known prior to practicing the method, it can be determined by mass spectroscopic analysis, chemical degradation, chromatographic techniques, IR spectroscopy, NMR, and the like. Where multiple test compounds were contacted with the AT, the method can comprise identifying one, some, or all of the test compounds showing activity. In such embodiments the method can comprise separating each test compound from each other.
  • the method can further comprise testing one or more test compounds showing in vitro activity for in vivo activity. For example, it can comprise contacting a positive test compound with one or more nematodes in a culture dish. Alternatively, it can comprise contacting a positive test compound with one or more nematodes in a natural environment, such as an agriculture plot. The effect of the positive test compound on the nematode can be determined. Determining can be by visual observation of the activity of the pest, or by assaying any of a number of cellular processes indicative of the health and viability of the pest. If desired, the method can comprise a large test, such as a field test on an agricultural plot.
  • the method of screening can provide the practitioner with a compound of known structure and activity.
  • the invention provides for modification of the positive test compound to engineer a compound having one or more altered activity.
  • the method can comprise engineering a positive test compound to have a higher specificity for a particular AT, to have broader specificity, to have lower toxicity in aqueous environments, etc.
  • Screening of the modified compound can be accomplished using the methods of screening of the invention. Because the method of the invention can identify active compounds, which can then be modified and re-screened, the method of the invention can be a method of identifying lead compounds for use as pesticides, and in particular for treatment of plants, humans, and the environment.
  • the method can include one or more control reactions to determine if one or more of the steps of the assays were performed properly and/or to determine if one or more of the reagents functioned as expected.
  • control reactions to determine if one or more of the steps of the assays were performed properly and/or to determine if one or more of the reagents functioned as expected.
  • free-living or otherwise non-harmful species of nematodes, insects, or Acari are used as a control to determine if the test compound(s) affect the viability of the free-living or non-harmful organism. This information can be beneficial in selecting lead compounds for continued research.
  • Example 1 Toxicity of DDDS, NPPB, 9- AC, and IAA-94 Against M. incognita and C. elegans
  • a stilbene natural product, DST, (Fig. 1) was previously isolated from the symbiotic bacterium Photorhabdus luminescens that lives inside nematodes of the genus Heterorhabditis used as an effective biological control agent in turf.
  • DST has nematicidal activity against a variety of nematode species (24 hr lethality 100% at 100 ppm), including Caenorhabditis elegans and Meloidogyne incognita (Hu et al., 1999).
  • the symbiotic nematodes within which the bacterium lives ⁇ Heterorhabditis spp., in this case H.
  • DIDS In initial petri dish toxicity assays, DIDS proved to be paralytic/lethal to M. incognita (20% mortality at 100 ppm for 24 hr), but not to H. bacteriophora. Thus, DIDS shows a cross resistance pattern similar to that previously observed for DST. These results demonstrate, for the first time, a linkage between nematode lethality and chemistry known to affect AT function.
  • the stilbene nucleus is a simple chemical scaffold, and excellent gains in potency can be envisioned from appropriate chemical modification.
  • other compounds having a similar mode of action have been described, including NPPB, IAA-94, and 9- AC (Figs. 1-3). This Example 1 describes further toxicity studies with these compounds.
  • Toxicity tests were conducted using J 2 juveniles of Meloidogyne incognita adult Caenorhabditis elegans collected from nematode- infested tomato plant roots and culture Petri plates, respectively. Test concentrations of 50, 100, 200, 300, or 400 ppm were prepared in 0.2% dimethylsulfoxide (DMSO) in tap water. Aliquots (300 microliters (ul)) were applied to a 96 well micro plate and a 20 ul aqueous solution containing nematodes was added. The number of nematodes in 20 ul ranged from 17 to 30. Micro plates were sealed with ParafilmTM and incubated at room temperature.
  • DMSO dimethylsulfoxide
  • Table 1-1 Summary of Toxicity of voltage sensitive chloride channel blockers against M. incognita and C. elegans
  • Example 2 AT Blockers Effect on M. incognita Egg Hatching
  • Meloidogyne incognita (plant parasitic nematode) eggs were collected from tomato plant roots and were suspended in water. The solution was either diluted or concentrated to adjust the egg load to about 5000/ml.
  • Example 3 Toxicity of AT Blockers Against The Insect Drosophila melanogaster
  • Toxicity tests were conducted using mixed sex adults of D. melanogaster maintained on artificial diet in the department of entomology, Virginia Tech. Test concentrations of 6.25, 12.5, 25, 50 or 100 ppm were prepared in 0.2% dimethyl sulfoxide (DMSO) for DIDS, 9-AC, NPPB, and IAA-94. The above final concentrations were obtained after mixing them in 10 ml of 10% sugar solution. Similar preparations with 0.2% DMSO alone served as controls. One end of a cotton wick was dipped in 0.5 ml of sugar solution and placed on the glass vial containing 10 adult flies of mixed sex. Each concentration of test chemical had 5 replications.
  • DMSO dimethyl sulfoxide
  • Toxicity tests were conducted using infective juveniles (J3) of H, bacteriophora collected from infested Galleria mellonella larvae.
  • H. bacteriophora is a symbiotic nematode that the inventors and colleagues showed was less sensitive to DDDS in preliminary studies, a cross resistance that suggests modified AT in this nematode. More thorough studies prove that a variety of AT blockers are without activity in H. bacteriophora, with little or no toxicity observed, even after 168 hrs of exposure (Fig. 4). This broad cross resistance supports a role for AT blockers in the action of these compounds. In contrast, the anticholinesterase chlorpyrifos was quite active in killing this resistant nematode.

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  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne des méthodes de protection des plantes et d'autres organismes multicellulaires contre les parasites. Ces méthodes font appel à un acide organique substitué ou à des composés anthracène substitués, qui agissent de façon à modifier l'activité d'un ou de plusieurs transporteurs d'anions dans les parasites. Les nématodes et les insectes comptent parmi les parasites ainsi traités. L'invention concerne également des méthodes permettant de cribler ces composés.
PCT/US2006/041968 2005-10-27 2006-10-27 Compositions pesticides et leurs methodes d'utilisation WO2007050867A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104031859A (zh) * 2014-05-13 2014-09-10 华中农业大学 一种丁香假单胞菌及其筛选方法和在杀灭线虫中的应用
WO2022224026A1 (fr) * 2021-04-20 2022-10-27 Universidade Do Porto Composés à faible toxicité destinés à être utilisés en tant qu'insecticides et procédé de production desdits composés

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5314693A (en) * 1992-02-07 1994-05-24 Kioritz Corporation Pest control chemicals against pine wood nematodes
US6812376B1 (en) * 1999-04-27 2004-11-02 Massachusetts Institute Of Technology Serotonin-gated anion channel

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104031859A (zh) * 2014-05-13 2014-09-10 华中农业大学 一种丁香假单胞菌及其筛选方法和在杀灭线虫中的应用
CN104031859B (zh) * 2014-05-13 2017-04-19 华中农业大学 一种丁香假单胞菌及其筛选方法和在杀灭线虫中的应用
WO2022224026A1 (fr) * 2021-04-20 2022-10-27 Universidade Do Porto Composés à faible toxicité destinés à être utilisés en tant qu'insecticides et procédé de production desdits composés

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