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WO2009070777A1 - Modulation de la cyclisation et de la signalisation des récepteurs couplés aux protéines g - Google Patents

Modulation de la cyclisation et de la signalisation des récepteurs couplés aux protéines g Download PDF

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Publication number
WO2009070777A1
WO2009070777A1 PCT/US2008/085053 US2008085053W WO2009070777A1 WO 2009070777 A1 WO2009070777 A1 WO 2009070777A1 US 2008085053 W US2008085053 W US 2008085053W WO 2009070777 A1 WO2009070777 A1 WO 2009070777A1
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WIPO (PCT)
Prior art keywords
receptor
active agent
target
composition
gpcr
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Application number
PCT/US2008/085053
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English (en)
Inventor
Essam Enan
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Tyratech, Inc.
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Publication of WO2009070777A1 publication Critical patent/WO2009070777A1/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
    • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
    • 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
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/16—Ericaceae [Heath or Blueberry family], e.g. rhododendron, arbutus, pieris, cranberry or bilberry
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/18—Euphorbiaceae [Spurge family], e.g. ricinus [castorbean]
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/20—Fabaceae or Leguminosae [Pea or Legume family], e.g. pea, lentil, soybean, clover, acacia, honey locust, derris or millettia
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/22—Lamiaceae or Labiatae [Mint family], e.g. thyme, rosemary, skullcap, selfheal, lavender, perilla, pennyroyal, peppermint or spearmint
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/36—Rutaceae [Rue family], e.g. lime, orange, lemon, corktree or pricklyash
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass

Definitions

  • the invention relates to compositions and methods for altering and / or disrupting G protein-coupled receptor signaling and cycling resulting in the disruption of normal biology or behavior in invertebrates.
  • G protein-coupied receptors form one of the largest families of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a variety of effector systems. Therapeutic agents often target GPCRs because of their capability to bind ligands, hormones, and drugs with high specificity. Agonist activation of GPCRs also initiates processes that desensitize GPCR responsiveness and their internalization.
  • GPCR-targeted compositions target organism tolerance or resistance, as receptor desensitization can mute their effectiveness.
  • the invention can include a method of pest control including the steps of; providing a target pest having at least one target GPCR receptor; contacting the target pest with a composition comprising at least a first active agent and a second active agent, wherein the first active agent is capable of interacting with the target receptor to trigger, disrupt or alter a biological function related to the binding of the target receptor with the first active agent, and wherein the second active agent is capable of interacting with a non-receptor molecule or step associated with cycling of the target receptor, to disrupt the signaling cascade of the target receptor.
  • the active agents in combination can cooperate to amplify the disrupted or altered function resulting from the binding of the target receptor by the first active agent, resulting in control of the pest.
  • the composition can include a third active agent, and the third active agent can be capable of interacting with a GPCR receptor in the target pest, and the interaction can be complementary to the action of the first active agent.
  • the first and third active agents can interact with the same receptor, or different receptors, in some embodiments the complementarity of the first and third active agents can cause an additive effect of the active agents together as compared with an effect of each active agent separately. In some embodiments the complementarity of the first and third active agents can cause a synergistic effect of the active agents together as compared with an effect of each active agent separately, in some embodiments, receptor cycling can include at ieast one of receptor sensitization, receptor desensitization, receptor recycling, iigand release, receptor phosphorylation, and receptor dephosphorylation.
  • Embodiments of the invention can include a pest-contro! composition that has a first active agent capable of disrupting or altering a function of a receptor in a target pest, and a second active agent capable of disrupting cycling of the receptor, and the second active agent can act to amplify an effect of the first active agent.
  • Embodiments of the invention can include a method of making a pest control composition with the steps of: providing a target pest having at least one target receptor; contacting the target pest with a composition including at ieast a first active agent and a second active agent, wherein the first active agent is capable of interacting with the target receptor to disrupt or alter a function related to normal activity of the target receptor, and wherein the second active agent is capable of interacting with a non-receptor molecule or step associated with cycling of the target receptor, to disrupt the cycling of the target receptor.
  • Some embodiments can include measuring the effect of the composition upon the target pest and selecting the at least a first active agent based on the desired properties of the composition.
  • Some embodiments of the invention can include a third active agent, wherein the third active agent can be capable of interacting with a receptor in the target pest, and wherein the interaction can be complementary to the action of the first active agent.
  • the first and third active agents can interact with a same receptor, or with a different receptor.
  • the complementarity of the first and third active agents can cause an additive effect of the active agents together as compared with an effect of each active agent separately.
  • the complementarity of the first and third active agents can cause a synergistic effect of the active agents together as compared with an effect of each active agent separately.
  • Embodiments of the invention can include a pest-co ⁇ troi composition that has an active agent capable of disrupting or altering cycling of a GPCR in a target pest, wherein the active agent acts to amplify an effect of a ligand binding the GPCR.
  • the amplification can result in a prolonged intracellular Ca 2+ cascade as compared with the Ca 2+ cascade that occurs when the receptor is bound without the presence of the active agent.
  • the amplification can result in a prolonged perturbation of intracellular cAMP level as compared with the perturbation in cAMP level that occurs when the receptor is bound without the presence of the active agent.
  • Embodiments of the invention can include a pest-contro! composition that has an active agent capable of disrupting or altering cycling of a GPCR in a target pest, wherein the active agent acts to attenuate an effect of a ligand binding the GPCR.
  • Certain embodiments of the invention can include a method of pest control including the steps of: providing a target pest having at least one target GPCR receptor; contacting the target pest with a composition including at least a first active agent and a second active agent, wherein the first active agent can be capable of interacting with the target receptor to trigger, disrupt or alter a biological function related to the binding of the target receptor with the first active agent, and wherein the second active agent can be capable of interacting with a non-receptor molecule or step associated with the biological pathway triggered, disrupted, or altered as a result of the first active agent's interacting with the target receptor; wherein the active agents in combination can cooperate to amplify the disrupted or altered function resulting from the binding of the target receptor by the first active agent, resulting in control of the pest.
  • the composition can further include a third active agent, wherein the third active agent can be capable of interacting with a GPCR receptor in the target pest, and wherein the interaction can be complementary to the action of the first active agent.
  • the first and third active agents can interact with a same receptor, or with different receptors. Additionally, the complementarity of the first and third active agents can cause an additive effect of the active agents together as compared with an effect of each active agent separately.
  • G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases leads to GPCR desensitization.
  • GRK-mediated receptor phosphorylation promotes the binding of beta-arrestins, which in addition to uncoupling receptors from heterotrimeric G proteins, also target many GPCRs for internalization in clathrin-coated vesicles.
  • Beta-arrestin proteins play a dual role in regulating GPCR responsiveness by contributing to both receptor desensitization and internalization.
  • GPCRs can be resensitized. GPCR sequestration to endosomes is thought to be the mechanism by which GRK-phosphorylated receptors are dephosphoryiated and resensitized.
  • beta-arrestins The identification of beta-arrestins as GPCR trafficking molecules suggested that beta-arrestins can be determinants for GPCR resensitization.
  • other cellular components also play pivotal roles in the de- and re-sensitization (D/R) process, including, for example, GRK, N-ethylmaleimide-sensitive factor (NSF), clathrin adaptor protein (AP-2 protein), protein phosphatases, clathrin, and the like.
  • D/R de- and re-sensitization
  • Intensifying a signal cascade achieved, for example, by inhibition of the desensitization process, or the like, can increase the effects of extracellular stimuli (such as, for example, pharmaceuticals, insecticides, or the like) on the GPCR signaling process.
  • extracellular stimuli such as, for example, pharmaceuticals, insecticides, or the like
  • Embodiments of the invention can include a method to disrupt or alter GPCR D/R by altering or disrupting the various signal cascades triggered through GPCR action.
  • Certain embodiments can disrupt or alter GPCR D/R in various ways, including, for example, the application of compositions containing active agents such as, for example, essential oils, and the like.
  • active agents can include, for example, any of the following compounds from Table 1 , or active components of any of the compositions listed as "blends" in Table 2, or the like:
  • stabilizers, fragrances, and other substances can comprise compositions of the invention.
  • “synergy” is a specific feature of a combination of ingredients, and is shown by a measurable effect of a combination of active ingredients that is above any background level of enhancement that would be due solely to, e.g., additive effects of that combination of ingredients. Effects that can be used to show synergy include but are not limited to: repellent effect of the composition; pesticidal effect of the composition; perturbation of a ceil message or cell signal such as, e.g., calcium, cyclic-AMP, and the like; and diminution of activity or downstream effects of a molecular target.
  • a substantial enhancement of a measurable effect can be expressed as a coefficient of synergy.
  • a coefficient of synergy is an expression of a comparison between measured effects of a composition and measured effects of a comparison composition.
  • the comparison composition can be a component of the composition.
  • the synergy coefficient can be adjusted for differences in concentration of the complete blend and the comparison composition.
  • Synergy coefficients can be calculated as follows.
  • An activity ratio (R) can be calculated by dividing the % effect of the composition (A 8 ) by the % effect of the comparison composition (X n ), as follows:
  • a concentration adjustment factor (F) can be calculated based on the concentration (C n ), i.e., % (wt/wt) or % (vol/vol), of the comparison composition in the composition, as follows:
  • the synergy coefficient (S) can then be calculated by multiplying the activity ratio (R) and the concentration adjustment factor (F), as follows:
  • the synergy coefficient (S) can also by calculated, as follows:
  • a coefficient of synergy of about 1 1 , 1 2, 1 3, 1 4, or 1 5 can be substantial and commerctaliy desirable
  • the coefficient of synergy can be from about 1 6 to about 5, including but not limited to about 1 8, 2 0, 2 5, 3 0, 3 5, 4 0, and 4 5
  • the coefficient of synergy can be from about 5 to 50, including but not limited to about 10, 15, 20, 25, 30, 35, 40, and 45
  • the coefficient of synergy can be from about 50 to about 500, or more, including but not limited to about 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, and 450. Any coefficient of synergy above 500 ss aSso contemplated within embodiments of the compositions
  • synergy can be described as being “greater than” a given number and therefore not necessarily limited to being within the bounds of a range having a lower and an upper numerical limit Likewise, in some embodiments described herein, certain low synergy coefficients, or lower ends of ranges, are expressly excluded Accordingly, in some embodiments, synergy can be expressed as being "greater than” a given number that constitutes a lower limit of synergy for such an embodiment For example, in some embodiments, the synergy coefficient is equal to or greater than 25, in such an embodiment, all synergy coefficients below 25, even though substantial, are expressly excluded
  • synergy or synergistic effect associated with a composition can be determined using calculations similar to those described in Colby, S R , "Calculating synergistic and antagonistic responses of herbicide combinations," Weeds (1967) 15 1 , pp 20-22, which is incorporated herein by this reference In this regard, the following formula can be used to express an expected % effect (E) of a composition including two compounds, Compound X and Compound Y
  • X is the measured actual % effect of Compound X in the composition
  • Y is the measured actual % effect of Compound Y of the composition
  • the expected % effect (E) of the composition is then compared to a measured actual % effect (A) of the composition If the actual % effect (A) that is measured differs from the expected % effect (E) as calculated by the formula, then the difference is due to an interaction of the compounds
  • the composition has synergy (a positive interaction of the compounds) when A > E, Further, there is a negative interaction (antagonism) when A ⁇ E.
  • Formula 5 can be extended to account for any number of compounds in a composition; however it becomes more complex as it is expanded, as is illustrated by the following formula for a composition including three compounds, Compound X, Compound Y, and Compound Z:
  • E' is the expected % of control of the composition
  • X n is the measured actual % effect of an individual compound (Compound X n ) of the composition
  • X n ' is the % of control of an individual compound of the composition
  • A' is the actual measured % of control of the of the composition.
  • the expected % of control (E') for the composition is calculated by dividing the product of the measured actual % of control values (X n ') for each compound of the composition by 100 n"1 .
  • the expected % of control (E') of the composition is then compared to the measured actual % of control (A') of the composition. If the actual % of control (A') that is measured differs from the expected % of control (E') as calculated by the Formula 10, then the difference is due to an interaction of the compounds.
  • the composition has synergy (a positive interaction of the compounds) when A' ⁇ E ⁇ Further, there is a negative interaction (antagonism) when A'> E'.
  • compositions containing two or more compounds in certain ratios or relative amounts can be tested for a synergistic effect by comparing the pesticida! effect of a particular composition of compounds to the pesticidal effect of a component the composition.
  • compositions of the present invention can be used to control pests by either treating a host directly, or treating an area in which the host will be located.
  • host is defined as a plant, human or other animal.
  • the host can be treated, for example, directly by using a cream or spray formulation, which can be applied externally or topically, e.g., to the skin of a human.
  • a composition could be applied to the host, for example, in the case of a human, by using formulations of a variety of persona! products or cosmetics for use on the skin or hair.
  • any of the following could be used: fragrances, colorants, pigments, dyes, colognes, skin creams, skin lotions, deodorants, tales, bath oils, soaps, shampoos, hair conditioners and styling agents.
  • An area can be treated with a composition of the present invention, for example, by using a spray formulation, such as an aeroso! or a pump spray, or a burning formulation, such as a candle or a piece of incense containing the composition.
  • a spray formulation such as an aeroso! or a pump spray
  • a burning formulation such as a candle or a piece of incense containing the composition.
  • various treatment methods can be used without departing from the spirit and scope of the present invention.
  • compositions can be comprised in household products, for example, hard surface cleaners.
  • Embodiments of the invention can include a method for screening a composition for indirect GPCR desensitization inhibitory activity.
  • an indication that the test composition has indirect GPCR desensitization inhibitory activity can be apparent when a test composition has GPCR desensitization inhibitory activity with respect to different GPCRs.
  • an indication that the test composition has indirect GPCR desensitization inhibitory activity can be apparent when GPCR cycling is inhibited without the composition binding the receptor itself.
  • indications of desensitization can include a reduced response to extracellular stimuli, such as, for example, a reduction in GPCR recycling from the plasma membrane to the cell's interior and back to the plasma membrane, or the tike
  • Another indication can be an altered period for the GPCR regulated activation of the Ca 2+ cascade or the cAMP levels in the organism
  • Embodiments of the invention can include a method for screening a composition for indirect GPCR resensitization inhibitory activity
  • an indication that the test composition has indirect GPCR resensitization inhibitory activity can be apparent when a test composition has GPCR resensitization inhibitory activity with respect to different GPCRs
  • an indication that the test composition has indirect GPCR resensitization inhibitory activity can be apparent when GPCR cycling is inhibited without the composition binding the receptor itself
  • indications of resensitization can include a reduced response to extracellular stimuli, such as, for example, a reduction in GPCR recycling from the plasma membrane to the cell's interior and back to the plasma membrane, or the like, or a recovery to normal or static level of Ca 2+ or cAMP
  • Embodiments of the invention can include a method for screening a composition for non-specific GPCR desensitization inhibitory activity
  • the method can include screening a test composition for GPCR desensitization inhibitory activity against two or more different GPCRs
  • an indication that the test composition has non-receptor-specific GPCR desensitization inhibitory activity can be apparent when a test composition has GPCR desensitization inhibitory activity with respect to each of the two or more different GPCRs
  • indications of desensitization inhibitory activity can include a reduced response to extracellular stimuli, such as, for example, a reduction in GPCR recycling from the plasma membrane to the cell's interior and back to the plasma membrane, or the like
  • Another indication can be an altered period for the GPCR regulated activation of the Ca 2+ cascade or the cAMP levels in the organism
  • Embodiments of the invention can include a method for screening a composition for non-specific GPCR resensitization inhibitory activity
  • the method can include screening a test composition for GPCR resensitization inhibitory activity against two or more different GPCRs
  • an indication that the test composition has non-receptor-specific GPCR resensitization inhibitory activity can be apparent when a test composition has GPCR resensitization inhibitory activity with respect to each of the two or more different GPCRs
  • Indications of resensitization inhibition can include a reduced response to extracellular stimuli, such as, for example, a reduction in GPCR recycling from the plasma membrane to the cell's interior and back to the plasma membrane, or the tike.
  • Another indication can be an altered period for the GPCR regulated activation of the Ca 2+ cascade or the cAMP levels in the organism.
  • one cell can be used to screen a test composition for indirect GPCR desensitization inhibitory activity.
  • the cell can express two or more GPCRs that are different from each other such that a detection method can be used for determining whether there is an indication that a test composition has GPCR desensitization inhibitory activity with respect to each of the different GPCRs.
  • a multi-well format can be used to screen a test composition for indirect GPCR desensitization inhibitory activity.
  • each well of the plate can contain at least one cell that includes a GPCR, and the assay can include adding a compound in an amount known to activate that GPCR, and thus affect intracellular Ca 2+ levels, to each well, in some embodiments, at least one test compound can also be added to each well.
  • Ca 2+ level can be tested at various time points after adding the at least one test compound.
  • time points used for testing intracellular Ca 2+ level can extend beyond the time points where an increase in Ca 2+ level can be seen without the presence of the at least one test compound.
  • methods of the invention can identify compounds that prolong agonist effect on GPCRs.
  • cAMP levels can be evaluated to gauge the effect of the at least one test compound on GPCR response.
  • a multi-well format can be used to screen a test composition for indirect GPCR desensitization inhibitory activity.
  • each well of the plate can contain at least one cell that includes a GPCR, and the assay can include adding a compound in an amount less than that required to activate that GPCR, and thus affect intracellular Ca 2+ levels, to each well.
  • at least one test compound can also be added to each well.
  • Ca 2+ level can be tested at various time points after adding the at least one test compound.
  • time points used for testing intracellular Ca level can extend beyond the time points where an increase in Ca level can not be seen without the presence of the at least one test compound
  • methods of the invention can identify compounds that enhance agonist effect on GPCRs.
  • cAMP levels can be evaluated to gauge the effect of the at least one test compound on GPCR response.
  • a cell used in the method can also include at ieast one conjugate comprising a marker moiecuie and a protein associated with the GPCR desensitization pathway of one or more of the GPCRs that are being evaluated.
  • the conjugate can indicate, through the use of the marker molecule, GPCR desensitization inhibitory activity of a test composition with respect to each of the GPCRs that are being used to screen the test composition.
  • the conjugate can comprise, for example, an arrestin protein and a marker molecule, a GPCR and a marker moiecuie, or the like.
  • the cell can comprise a conjugate of an arrestin protein and a marker molecule as well as a conjugate of a GPCR and a marker molecule.
  • a conjugate of an arrestin protein and a marker molecule as well as a conjugate of a GPCR and a marker molecule.
  • such methods can comprise exposing the cell to, for example, a test composition, to an agonist for the first GPCR (when the first GPCR requires agonist for desensitization), and to an agonist for the second GPCR (when the second GPCR requires agonist for desensitization), or the like, then determining whether the composition has GPCR desensitization inhibitory activity with respect to the first GPCR and with respect to the second GPCR.
  • an indication of GPCR desensitization inhibitory activity with respect to the first GPCR and an indication of GPCR desensitization inhibitory activity with respect to the second GPCR can be distinguished by using, for example, a different conjugate for the determination of GPCR desensitization inhibitory activity of the compositions with respect to the different GPCRs, or the like.
  • a cell can include a first conjugate comprising a first GPCR and a first marker molecule and a second conjugate comprising a second GPCR and a second marker molecule.
  • the cell can be possible to expose the cell to the test composition, the agonist for the first GPCR (if needed for desensitization), and the agonist for the second GPCR (if needed for desensitization) simultaneously or non-simultaneously, and determine whether the composition has GPCR desensitization inhibitory activity with respect to the first GPCR and the second GPCR.
  • [004Sj Detection for each of the items/events discussed above can be conducted, for example, at one point in time, over a period of time, at two or more points in time for comparison (e.g., before and after exposure to a test composition), or the [ike
  • An indication of GPCR desensitization inhibitory activity can be determined by, for example, detecting one or more of the items or events discussed above in a cell exposed to the test composition and comparing the results to those obtained by detecting for the same item or event in a control cell, by comparing the results to a predetermined value, or the like
  • Embodiments of the invention can utilize prokaryotic and eukaryotic cells including, for example, bacterial cells, yeast celts, fungal cells, insect cells, nematode cells, plant cells, animal cells, and the like Suitable animal cells can include, for example, HEK cells, HeLa cells, COS cells, U20S ceils, CHO-K1 cells, various primary mammalian cells, and the like An animal model expressing one or more conjugates of an arrestin and a marker molecule, for example, throughout its tissues, within a particular organ or tissue type, or the like, can be used
  • Embodiments of the invention can utilize at least one cell that expresses, for example, a known GPCR, a variety of known GPCRs, an unknown GPCR, a variety of unknown GPCRs, a modified GPCR, a variety of modified GPCRs, and the like
  • the at least one cell can, for example, naturally express the GPCRs, can be genetically engineered to express the GPCRs at varying levels of expression, can be genetically engineered to inducibiy express the GPCRs, or the like
  • the at least one cell can comprise one or more conjugates of a marker molecule and a protein associated with the GPCR desensitization pathway
  • one or more of the cells can comprise a conjugate of an arrestin protein and a marker molecule, or a conjugate of a GPCR and a marker molecule, or the like
  • marker molecules that can be used as a conjugate can include, for example, molecules that are detectable by spectroscopic, photochemical, radioactivity, biochemical, immunochemical, colonmet ⁇ c, electrical, and optical means, including, for example, bioluminescence, phosphorescence, fluorescence, and the like
  • Marker molecules can be, for example, biologically compatible molecules, and the like
  • Suitable marker molecules can include, for example, radioisotopes, epitope tags, affinity labels, enzymes, fluorescent groups, chemiJuminescent groups, and the like
  • the marker molecules are optically detectable, including, for example, optically detectable proteins, such that they can be excited chemically, mechanically, electrically, or radioactively to emit fluorescence, phosphorescence, or bioluminescence
  • Optically detectable marker molecules can include, for example, beta-galactosidase, firefly luciferase, bacterial luciferase, fluorescein,
  • all forms of arrestin can be used. Confocal microscopy can be used to identify such protein-protein interaction and also to study the trafficking of the protein complex.
  • the cell can be transfected with DNA so that the conjugate of arrestin and a marker molecule can be produced within the cell.
  • GPCRs used in embodiments of the invention can also be conjugated with a marker molecule.
  • the carboxyl-terminus of the GPCR can be conjugated with a marker molecule.
  • a carboxyl-terminal tail conjugated or attached to a marker molecule can be used in a carboxyl-terminal tail exchange to provide a modified GPCR.
  • the GPCRs can be antibody-labeled, for example with an antibody conjugated to an immunofluorescence molecule, or the like, or the GPCRs can be conjugated with, for example, a luminescent donor or the like. In some embodiments, the GPCRs can be conjugated with, for example luciferase, Renilla luciferase, or the like.
  • Embodiments of the invention can be used to evaluate the effect of a test compound on GPCR R/D by measuring intracellular second messenger generation.
  • Intracellular effectors can include, for example, cAMP, cyclic GMP, calcium, phosphatidylinositol, a hydrogen ion, an ion transport molecule, and the like.
  • enzymes such as, for example, adenylyl cyclase, phosphodiesterase, phospholipase C, protein kinase, phospholipase A 2 , and the like, can be measured to gauge the effects of test compounds on GPCR R/D.
  • HEK293 cells are transfected with the pcDNA3.1A/5-HisA vector using Lipofectamine (Invitrogen).
  • the vector contains a full-length construct of the C. elegans tyramine receptor.
  • 48 h after transfection cells are selected in a culture medium containing 0.5 mg/ml G418 (Invitrogen). Cells that survive the first round of G418 selection are further subjected to limiting dilution for single clone selection. Clones are selected and then cell stocks are grown for assay purposes.
  • EMEM Eagle's minimum essential medium
  • HEPES N-2-Hydroxyethylpiperazine-N'-2- ethanesulfonic acid
  • Linalool is used as the receptor activator for the assay, and is added to each well on each plate. Sufficient linalool is added to ensure receptor activation and a resulting increase in intracellular Ca 2+ levels.
  • Essential oil test compositions of varying concentrations are added to the welis of each of the four plates (four piates are used per replicate). The assay is conducted at room temperature.
  • [0061 j At time points of 30 seconds, 60 seconds, 90 seconds, 120 seconds, 180 seconds, 240 seconds, 300 seconds, and 600 seconds post-addition of test compound, the assay is terminated and the cells are analyzed to determine intracellular Ca 2+ levels.
  • HEK293 cells are transfected with the pcDNA3.1 ⁇ /5-HisA vector using Lipofectamine (Invitrogen).
  • the vector contains a full-length construct of the C. elegans tyramine receptor.
  • 48 h after transfection cells are selected in a culture medium containing 0.5 mg/ml G418 (Invitrogen). Cells that survive the first round of G418 selection are further subjected to limiting dilution for single clone selection. Clones are selected and then cell stocks are grown for assay purposes.
  • Growth media is replaced with serum free media (i.e., Eagle's minimum essential medium (EMEM) buffered with 10 mM HEPES (N-2-Hydroxyethylpiperazine- fsT-2-ethanesulfonic acid)) 24 hours after plating of the cells.
  • EMEM Eagle's minimum essential medium
  • HEPES N-2-Hydroxyethylpiperazine- fsT-2-ethanesulfonic acid
  • Thymol is used as the receptor activator for the assay, and is added to each well on each plate.
  • the amount of test composition added is less than that required to ensure receptor activation and a resulting increase in intracellular Ca 2+ levels.
  • Test compositions of varying concentrations are added to the wells of each of the four ptates (four plates are used per replicate). The assay is conducted at room temperature.
  • the assay is terminated and the cells are analyzed to determine intracellular Ca 2+ levels.
  • HEK293 cells are transfected with the pcDNA3,1 ⁇ /5-HisA vector using Lipofectamine (Invitrogen).
  • the vector contains a full-length construct of the Drosophila tyramine receptor as well as an arrestin-GFP conjugate.
  • cells are harvested 48 h after transfection.
  • 48 h after transfection cells are selected in a culture medium containing 0.5 mg/ml G418 (Invitrogen). Cells that survive the first round of G418 selection are further subjected to limiting dilution for single clone selection. Clones are selected and then cell stocks are grown for assay purposes.
  • Growth media is replaced with serum free media (i.e., Eagle's minimum essential medium (EMEM) buffered with 10 mM HEPES (N-2-Hydroxyethylpiperazine- N'-2-ethanesulfonic acid)) 24 hours after plating of the cells.
  • serum free media i.e., Eagle's minimum essential medium (EMEM) buffered with 10 mM HEPES (N-2-Hydroxyethylpiperazine- N'-2-ethanesulfonic acid)
  • Each test compound is solvated using 100% dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • Multiple solutions of each compound are prepared at varying concentrations for testing in separate wells of each plate. The solutions are sonicated to increase solubility.
  • Each of the solutions of varying concentrations of the fifteen compounds is added to a well on each of the four plates (four plates are used per replicate). Two plates per replicate are incubated for 30 minutes at room temperature and atmospheric CO 2 . The other two plates per replicate are incubated for 30 minutes at 37C and 5% CO 2 .
  • Agonist is then added to each well.
  • 100 nM isoproterenol (0.4% weight/volume ascorbic acid) is added to one of the 37C plates and one of the RT plates.
  • 100 nM arginine vasopressin is added to one of the 37C plates and one of the RT plates.
  • the assay is terminated using 1 % paraformaldehyde containing 1 ⁇ M DRAQ5 DNA probe to fix the cells.
  • the cells are analyzed using a line scanning, confocal imaging system to quantitate the localization of the arrestin-GFP conjugate for the cells in each well using the Amersham Biosciences granularity analysis GRNO algorithm.
  • This algorithm finds the nucleus of cells and then dilates out a specified distance in which fluorescent spots of arrestin-GFP localization are identified based on size and fluorescent intensity. The average of the fluorescent intensity of the identified grains per cell in an acquired image is determined for each well on the plates.
  • Control wells are used on each plate to determine the basai level of fluorescent spots for the cells on the different plates as well as to determine the maximally stimulated level of fluorescent spots for the cells on the different plates.
  • the cells in the control wells are subjected to the method described above, but no test compound or agonist is added to the wells.
  • the ceils in the "agonist" control welis are subjected to the method described above, including the addition of agonist, but no test compound is added to the wells,
  • Test results achieved via the methods of examples 1 through 3 are used to select test compositions for testing with insect pests. Compositions demonstrating the greatest ability to perturb intracellular calcium cascades are tested with insects to evaluate their pesticida! effectiveness.
  • acetonic solutions about 1% and 10% from a test composition are prepared. Test concentrations in acetone are then added to the inside of glass vials (about 5 ml.) that are marked to about 3 cm above the bottom. The vials are rotated such that the inner surfaces of the vials, except the area between the marks to the neck, are left with a film of test composition. Al! vials are aerated for about 10 sec to ensure complete evaporation of acetone before introducing the flies to the treated vials. After complete evaporation of acetone, about 10 adult sex mixed flies are added to each vial and the vials are stoppered with cotton plugs. Mortality is observed about 24 hr after exposure.
  • compositions of the invention were tested against Aedes aegypti to determine the EC 50 in parts-per-million (PPM).
  • Compositions used included Blend 77 and Blend 182.
  • Blend 182 is similar to Blend 77, however with genistein added. The compositions were applied to the organisms, and the resulting data are presented in Table 3:
  • Blend 77 shows a pesticida! effect, however the addition of genistein, a tyrosine kinase inhibitor, results in a drastic decrease in the EC 50 value, displaying the enhanced effectiveness achieved via modulation of GPCR cycling.
  • Compositions of the invention were tested against Caenorhabditis elegans to determine the EC 50 in ppm.
  • Blend 182 is similar to Blend 77, however with genistein added. The compositions were applied to the organisms, and the resulting data are presented in Table 4:
  • Blend 77 shows a pesticidal effect, however the addition of genistein, a tyrosine kinase inhibitor, results in a drastic decrease in the EC 50 value, displaying the enhanced effectiveness achieved via modulation of GPCR cycling.
  • Test results achieved via the methods of examples 1 through 3 are used to select test compositions for testing with insect pests. Compositions demonstrating the greatest ability to alter an intracellular calcium cascade are tested with insects to evaluate their pesticidal effectiveness.
  • a laboratory culture of Aedes atropalpus adults maintained in continuous culture is used.
  • the culture is derived from collections made from temporary pools near Melbourne, Florida, US.
  • This species is representative of the many /Aedes spp. which bite humans in spring and summer.
  • the genus is capable of transmitting tropical diseases such as dengue and yellow fever.
  • This species of mosquito is a known vector that transmits West Nile virus.
  • the WHO test protocol for mosquito repellents is used (Barnard, D. R. 2000. Repellent and toxicants for personal protection. WHO/CDS ⁇ /HOPES).
  • Two clear PerspexTM mosquito cages (38 cm x 38 cm x 38 cm) containing more than 400 adults of both sexes and various ages are used.
  • the investigators wear gloves to protect hands and introduce their forearms up to the elbow through a cloth sleeve entry port into the cage.
  • an untreated arm is exposed for a 3-rninute period. The number of landings and bites are recorded as bites per 3-minute period.
  • each forearm is treated (1 ml of repellent per forearm) with a different test composition and each forearm is introduced into the cages for a further 3 minutes of evaluation, then removed from the cage. After one hour, the forearms are exposed a second time and landings and bites recorded. This procedure is repeated at hourly intervals, collecting data until the test composition fails to protect against bites. Faiiure occurs when there are two bites within the 3-minute test period.
  • the mosquitoes are attracted to, land on, probe and promptly take blood meals from the unprotected human forearms. After the application of the test compositions, the mosquitoes clearly avoid the forearms in their flight behavior.

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Abstract

La présente invention a trait à des procédés de lutte contre les animaux nuisibles, ainsi qu'à des procédés de fabrication de compositions utilisables dans le cadre de la lutte contre les animaux nuisibles. Des modes de réalisation de l'invention peuvent comprendre des compositions modulant la cascade de signaux initiée par la liaison de ligands aux récepteurs de la surface cellulaire par exemple. L'invention a également trait à des procédés de criblage desdites compositions.
PCT/US2008/085053 2007-11-28 2008-11-26 Modulation de la cyclisation et de la signalisation des récepteurs couplés aux protéines g WO2009070777A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050008714A1 (en) * 2003-04-24 2005-01-13 Essam Enan Compositions and methods for controlling insects
US6849614B1 (en) * 1998-07-28 2005-02-01 Ecosmart Technologies, Inc. Synergistic and residual pesticidal compositions containing plant essential oils
US20060083763A1 (en) * 2004-10-18 2006-04-20 Michael Neale Method and apparatus for insect repellant system
US20060263403A1 (en) * 2003-04-24 2006-11-23 Essam Enan Compositions and methods for controlling insects involving the tyramine receptor
WO2008011054A2 (fr) * 2006-07-17 2008-01-24 Tyratech, Inc. Compositions et procédés pour lutter contre les insectes
WO2008088827A2 (fr) * 2007-01-16 2008-07-24 Tyratech, Inc. Compositions et procédés de lutte antiparasitaire

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6849614B1 (en) * 1998-07-28 2005-02-01 Ecosmart Technologies, Inc. Synergistic and residual pesticidal compositions containing plant essential oils
US20050008714A1 (en) * 2003-04-24 2005-01-13 Essam Enan Compositions and methods for controlling insects
US20060263403A1 (en) * 2003-04-24 2006-11-23 Essam Enan Compositions and methods for controlling insects involving the tyramine receptor
US20060083763A1 (en) * 2004-10-18 2006-04-20 Michael Neale Method and apparatus for insect repellant system
WO2008011054A2 (fr) * 2006-07-17 2008-01-24 Tyratech, Inc. Compositions et procédés pour lutter contre les insectes
WO2008088827A2 (fr) * 2007-01-16 2008-07-24 Tyratech, Inc. Compositions et procédés de lutte antiparasitaire

Non-Patent Citations (1)

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Title
MURRAY B. ISMAN: "Plant essential oils for pest and disease mangement", CROP PROTECTION, vol. 19, 2000, Vancover, BC, Canada, pages 603 - 609, XP009113434 *

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