WO2009070777A1

WO2009070777A1 - Modulation of g protein-coupled receptor cycling and signaling

Info

Publication number: WO2009070777A1
Application number: PCT/US2008/085053
Authority: WO
Inventors: Essam Enan
Original assignee: Tyratech, Inc.
Priority date: 2007-11-28
Filing date: 2008-11-26
Publication date: 2009-06-04

Abstract

Methods of pest control, and methods of making pest control compositions. Embodiments of the invention can include compositions that modulate the signal cascade initiated by the binding of ligands to, for example, cell surface receptors. Methods of screening such compositions are also disclosed.

Description

MODULATION OF G PROTEIN-COUPLED RECEPTOR CYCLING AND SIGNALING

FIELD OF THE INVENTION

[Oθθi] 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.

BACKGROUND OF THE INVENTION

[0002] G protein-coupied receptors (GPCRs) 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.

[0003] Common to most GPCRs is the cyclic process of signaling, desensitization, internalization, resensitization, and recycling to the plasma membrane. This cycle prevents cells from undergoing excessive receptor stimulation or periods of prolonged inactivity. Mechanisms for desensitization of GPCRs include receptor phosphorylation and subsequent endocytosis, which removes the receptor-ligand complex from the cell surface. As a result of this desensitization process, a common limitation of GPCR-targeted compositions is target organism tolerance or resistance, as receptor desensitization can mute their effectiveness.

SUMMARY OF THE INVENTION

[0004] In certain embodiments, 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. In some embodiments 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. In some embodiments of the invention, 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.

[0005] In some embodiments, 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.

[0006] 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.

[0007] 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. In some embodiments, the first and third active agents can interact with a same receptor, or with a different receptor. 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. Likewise, 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. [0008] Embodiments of the invention can include a pest-coπtroi 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. Further, the amplification can result in a prolonged intracellular Ca²⁺ cascade as compared with the Ca²⁺ cascade that occurs when the receptor is bound without the presence of the active agent. In some embodiments, 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.

[0009] 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.

[00101 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.

[0011] In various embodiments, 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. Likewise, 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.

DETAILED DESCRIPTION OF THE INVENTION

[0012] G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs) 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.

[0013] Following desensitization, GPCRs can be resensitized. GPCR sequestration to endosomes is thought to be the mechanism by which GRK-phosphorylated receptors are dephosphoryiated and resensitized. The identification of beta-arrestins as GPCR trafficking molecules suggested that beta-arrestins can be determinants for GPCR resensitization. However, 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. In addition to these molecules, other moieties such as, for example, endosomes, lysosomes, and the like, also influence the D/R process. These various components of the D/R cycle provide opportunities to disrupt or alter GPCR "availability" to extracellular stimuli, and thus attenuate or intensify the effect of those extracellular stimuli upon target organisms. Attenuation, achieved, for example, by inhibition of the resensitization process, or the like, can limit the effects of extracellular stimuli (such as, for example, UV exposure, toxins, or the like) on the GPCR signaling process. 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.

[0014] Components of the GPCR signaling process have been the object of significant study; opportunities and targets for disruption of the signaling process according to the present invention are numerous. Discussions of the components of G-protein signaling are provided in Yu (2006) Heterotrimeric G protein signaling and RGSs in Aspergillus nidulans, J Microbiol 44:145-154; Dong (2007) Regulation of G protein-coupled receptor export trafficking, Biochim Biophys Acta 1768:853-870; Nakahata (2007) Regulation of G Protein-coupled Receptor Function by Its Binding Proteins, Yakugaku Zassht 127:3-14; Yang (2006) Mechanisms of regulation and function of G-protein-coupled receptor kinases, World J Gastroenterol 12:7753-7757; Ma (2007) Beta-arrestin signaling and regulation of transcription; J Cell Sci 120:213-218; New (2007) Molecular mechanisms mediating the G protein-coupled receptor regulation of cell cycle progression, J MoI Signaling 2:2-16; Kiasse (2008) Internalization and desensitization of adenosine receptors, Puήnergic Signalling 4:21 -37; Stewart (2007) Phospholipase C-eta Enzymes as Putative Protein Kinase C and Ca2+ Signalling Components in Neuronal and Neuroendocrine Tissues, Neuroendocrinology 86:243-248; Xu (2007) Regulation of G protein-coupled receptor trafficking, Acta Physiol 190:39-45; Wolfe (2007) Clathrin-Dependent Mechanisms of G Protein-coupled Receptor Endocytosts, Traffic 8:462-470; Schulte (2007) Novel aspects of G-protein-coup!ed receptor signailing - different ways to achieve specificity, Acta Physiol 190:33-38; Torrecilla (2006) Co-ordinated covalent modification of G-protein coupled receptors, Curr Pharm Des 12:1797-1808; Neitzel (2006) Cellular mechanisms that determine selective RGS protein regulation of G protein-coupled receptor signaling, Semin Ceil Dev Biol 17:383-389; Sato (2006) Accessory proteins for G proteins; partners in signaling, Annu Rev Pharmacol Toxicol 46:151 -187; Appert-Coliin (2006) Regulation of g protein-coupled receptor signaling by a-kinase anchoring proteins, Recept Signal Transduct Res 26:631-46; Premont (2007) Physiological roles of G protein-coupled receptor kinases and arrestins, Annu Rev Physiol 69:511-534; Smrcka (2008) G protein beta gamma subunits: Central mediators of G protein-coupied receptor signaling, Cell MoI Life Sci 65:2191-2214; Grandy (2007) Trace amine-associated receptor 1 - Family archetype or iconoclast?, Pharmacol Ther 116:355-390; Gilchrist (2007) G-protein-coupSed receptor pharmacology: examining the edges between theory and proof, Curr Opin Drug Discov Devet 10:446-451 ; Takeishi (2007) Role of diacylglycerol kinase in cellular regulatory processes: a new regulator for cariomyocyte hypertrophy, Pharmacol Ther 115:352-359; Dalrymple (2008) G protein-coupled receptor dimers: functional consequences, disease states and drug targets, Pharmacol Ther 118:359-371 ; Jurado-Pueyo (2008) GRK2-dependent desensitization downstream of G proteins, Recept Signal Transduct Res 28:59-70; Miliigan (1998) New aspects of g-protein-coupled receptor signalling and regulation, Trends Endocrinol Metab 9:13-19. Each of the foregoing is incorporated by reference in its entirety for its disclosure of the mechanisms and components of GPCR signaling, cycling, regulation, and the like.

[Θ0I5] 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. These 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:

[0016] Concentrations, amounts, and other numerical data can be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range, as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "about 1 to about 5" should be interpreted to include not only the explicitly recited values of about 1 to about 5, but aiso include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc., as well as 1 , 2, 3, 4, and 5, individually. In certain embodiments, stabilizers, fragrances, and other substances can comprise compositions of the invention.

[Θ017J In embodiments of the present 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. [0018] Exemplary methods that can be used to determine the synergistic effect of a particular composition are set forth in the following applications, each of which is incorporated in its entirety herein by reference: U.S. Application Serial No. 10/832,022, entitled COMPOSITIONS AND METHODS FOR CONTROLLING INSECTS; U.S. Application Serial No. 11/086,615, entitled COMPOSITIONS AND METHODS FOR CONTROLLING INSECTS RELATED TO THE OCTOPAMINE RECEPTOR; U.S. Application Serial No. 11/365,426, entitled COMPOSITIONS AND METHODS FOR CONTROLLING INSECTS INVOLVING THE TYRAMINE RECEPTOR; U.S. Application Serial No. 11/870,385, entitled COMPOSITIONS AND METHODS FOR CONTROLLING INSECTS; U.S. Application Serial No. 12/009,220, entitled PEST CONTROL COMPOSITIONS AND METHODS; and U.S Application Serial No. 61/082,601 , entitled PEST-CONTROL COMPOSITIONS AND METHODS HAVING HIGH TARGET AND LOW NON-TARGET ACTIVITY.

[0019] In some embodiments, 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. In some embodiments, the synergy coefficient can be adjusted for differences in concentration of the complete blend and the comparison composition.

[0020] Synergy coefficients can be calculated as follows. An activity ratio (R) can be calculated by dividing the % effect of the composition (A₈) by the % effect of the comparison composition (X_n), as follows:

R = A_B/Xn Formula 1

[0021] 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:

F = 100/C_n Formula 2

[0022] The synergy coefficient (S) can then be calculated by multiplying the activity ratio (R) and the concentration adjustment factor (F), as follows:

S = (R)(F) Formula 3

[0023] As such, the synergy coefficient (S) can also by calculated, as follows:

S = [(A_B/X_π)(100)]/C_π Formula 4 [0024] In Formula 4, A_B is expressed as % effect of the blend, X_n is expressed as % effect of the comparison composition (X_n), and C_n is expressed as % (wt/wt) or % (vol/vol) concentration of the comparison composition in the blend

[0025] In some embodiments, a coefficient of synergy of about 1 1 , 1 2, 1 3, 1 4, or 1 5 can be substantial and commerctaliy desirable In other embodiments, 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 In other embodiments, 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 In other embodiments, 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

[0026] Given that a broad range of synergies can be found in various embodiments describe herein, it is expressly noted that a coefficient of 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

[0027] In some embodiments, 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

E = X ₊ Y - (χ*γ/i 00) Formula 5

[0028] In Formula 5, X is the measured actual % effect of Compound X in the composition, and 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 Thus, the composition has synergy (a positive interaction of the compounds) when A > E, Further, there is a negative interaction (antagonism) when A < E.

[0Θ29] 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 = X + Y + Z ~ ((XY + XZ + YZ)/100) + (X^*Y^*Z/10000) Formula 6

[0030J An easy-to-use formula that accommodates compositions with any number of compounds can be provided by modifying Formulas 5 and 6. Such a modification of the formula will now be described. When using Formulas 5 and 6, an untreated control value (untreated with composition or compound) is set at 100%, e.g., if the effect being measured is the amount of target parasites killed, the control value would be set at 100% survival of target parasite. In this regard, if treatment with Compound A results in 80% killing of a target parasite, then the treatment with Compound A can be said to result in a 20% survival, or 20% of the control value. The relationship between values expressed as a percent effect and values expressed as a percent-of-control are set forth in the following formulas, where 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, and A' is the actual measured % of control of the of the composition.

E = 100 ~ E' Formula 7

X_n = IOO=X_n' Formula e

A = IOO - A¹ Formula 9

[0031] By substituting the percent-of-control values for the percent effect values of Formulas 5 and 6, and making modifications to accommodate any number (n) of compounds, the following formula is provided for calculating the expected % of control (E¹) of the composition:

,n-i

Formula 10

[Θ032] According to Formula 10, 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. Thus, the composition has synergy (a positive interaction of the compounds) when A' < E\ Further, there is a negative interaction (antagonism) when A'> E'.

[0033] 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.

[0034] 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. For purposes of this application, 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. For example, 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.

[0035] 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. Of course, various treatment methods can be used without departing from the spirit and scope of the present invention. For example, compositions can be comprised in household products, for example, hard surface cleaners.

[0036] Embodiments of the invention can include a method for screening a composition for indirect GPCR desensitization inhibitory activity. In certain embodiments of the invention, 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. In certain embodiments, 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. In certain embodiments of the invention, 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 Ca²⁺ cascade or the cAMP levels in the organism

[0037] Embodiments of the invention can include a method for screening a composition for indirect GPCR resensitization inhibitory activity In certain embodiments of the invention, 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 In certain embodiments, 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 In certain embodiments of the invention, 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 Ca²⁺ or cAMP

[0038] 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 In certain embodiments of the invention, 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 In certain embodiments of the invention, 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 Ca²⁺ cascade or the cAMP levels in the organism

[0039] 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 In certain embodiments of the invention, 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 In certain embodiments of the invention, 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 Ca²⁺ cascade or the cAMP levels in the organism.

[0040] In an embodiment of the invention, one cell can be used to screen a test composition for indirect GPCR desensitization inhibitory activity. In such an embodiment, 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.

[0041] Sn some embodiments of the invention, a multi-well format can be used to screen a test composition for indirect GPCR desensitization inhibitory activity. In some embodiments, 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 Ca²⁺ levels, to each well, in some embodiments, at least one test compound can also be added to each well. In some embodiments, Ca²⁺ level can be tested at various time points after adding the at least one test compound. In certain embodiments, time points used for testing intracellular Ca²⁺ level can extend beyond the time points where an increase in Ca²⁺ level can be seen without the presence of the at least one test compound, In some embodiments, methods of the invention can identify compounds that prolong agonist effect on GPCRs. In some embodiments of the invention, cAMP levels can be evaluated to gauge the effect of the at least one test compound on GPCR response.

[0042] In some embodiments of the invention, a multi-well format can be used to screen a test composition for indirect GPCR desensitization inhibitory activity. In some embodiments, 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 Ca²⁺ levels, to each well. In some embodiments, at least one test compound can also be added to each well. In some embodiments, Ca²⁺ level can be tested at various time points after adding the at least one test compound. In certain embodiments, 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, in some embodiments, methods of the invention can identify compounds that enhance agonist effect on GPCRs. In some embodiments of the invention, cAMP levels can be evaluated to gauge the effect of the at least one test compound on GPCR response.

[0043] In some embodiments of the invention, 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. In one embodiment, 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. jO044] In some embodiments of the invention, two or more different GPCRs that require agonist for desensitization, or are constitutiveiy desensitized, can be used. In general, 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. In such embodiments, 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. For example, 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. In such an embodiment, it 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

[0046] 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

[0047] 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

[0048] In certain embodiments of the invention, the at least one cell can comprise one or more conjugates of a marker molecule and a protein associated with the GPCR desensitization pathway For example, 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

[0049] For certain embodiments of the invention, marker molecules that can be used as a conjugate can include, for example, molecules that are detectable by spectroscopic, photochemical, radioactivity, biochemical, immunochemical, colonmetπc, 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 In some embodiments of the invention, 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, Texas Red, horseradish peroxidase, alkaline phosphatase, rhodamine-conjugated antibody, and the like. In other embodiments, the optically detectable marker molecules can be inherently fluorescent molecufes, such as fluorescent proteins, including, for example, Green Fluorescent Protein (GFP), and the like.

[0050] in certain embodiments of the invention, all forms of arrestin, both naturally occurring and engineered variants, including, for example, visual arrestin, beta-arrestin 1 , beta-arrestin 2, and the like, can be used. Confocal microscopy can be used to identify such protein-protein interaction and also to study the trafficking of the protein complex.

[0051] In some embodiments, the cell can be transfected with DNA so that the conjugate of arrestin and a marker molecule can be produced within the cell.

[0052] GPCRs used in embodiments of the invention can also be conjugated with a marker molecule. In some embodiments, 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.

[0053] In some embodiments of the invention, 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.

[0054] 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. Additionally, enzymes such as, for example, adenylyl cyclase, phosphodiesterase, phospholipase C, protein kinase, phospholipase A₂, and the like, can be measured to gauge the effects of test compounds on GPCR R/D.

[0055] An embodiment of the present invention is further illustrated by the following specific but non-limiting examples. EXAMPLES

[0056] The following examples provide details of exemplary methods. As disclosed herein, it is within the scope of the present invention to vary the components of these methods within useful ranges. Accordingly, these specific methods are merely representative of certain embodiments of the invention.

[0057] EXAMPLE 1- Effect of test compositions on intracellular Calcium levels

[0058] 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. 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.

[0059] 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 Ca²⁺ levels.

[0060] 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 Ca²⁺ levels.

[0°⁶²l EXAMPLE 2- Effect of test compositions on intracellular Calcium levels

[0063] HEK293 cells are transfected with the pcDNA3.1Λ/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.

[0064] 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.

[0065] 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 Ca²⁺ 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.

[0066] 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 Ca²⁺ levels.

[0067] EXAMPLE 3- Effect of test compositions on intracellular Calcium levels

[0068] HEK293 cells are transfected with the pcDNA3,1Λ/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. For transient transfection, cells are harvested 48 h after transfection. For stable 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.

[0069] 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. Per replicate, two plates are incubated for 10 minutes at room temperature and atmospheric CO₂ and two plates are incubated for 10 minutes at 37C and 5% CO₂.

[0070] Each test compound is solvated using 100% dimethyl sulfoxide (DMSO). 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 CO₂. The other two plates per replicate are incubated for 30 minutes at 37C and 5% CO₂.

[0071] Agonist is then added to each well. For each compound to be tested, 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 μM 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.

[0072] 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,

[0073] EXAMPLE 4- Effect of test compositions on flies

[0074] 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.

[0075] Two 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.

[0076] EXAMPLE 5- Effect of test compositions on Aθdes aecivpti

[0077] Compositions of the invention were tested against Aedes aegypti to determine the EC₅₀ 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:

[0078] Blend 77 shows a pesticida! effect, however the addition of genistein, a tyrosine kinase inhibitor, results in a drastic decrease in the EC₅₀ value, displaying the enhanced effectiveness achieved via modulation of GPCR cycling.

[0Θ79] EXAMPLE 6- Effect of test compositions on Caenorhabditis elegans [0080] Compositions of the invention were tested against Caenorhabditis elegans to determine the EC₅₀ in ppm. Compositions used included Blend 77 and Biend 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 4:

TABLE 4: EC_sn for Caenorhabditis eleQans

Test composition ECsn value in ppm

Biend 77 100

Blend 182 45

10081] Blend 77 shows a pesticidal effect, however the addition of genistein, a tyrosine kinase inhibitor, results in a drastic decrease in the EC₅₀ value, displaying the enhanced effectiveness achieved via modulation of GPCR cycling.

[0082] EXAMPLE 7- Effect of test compositions on pests

[0083] 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.

[0084J 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.

[0085] The WHO test protocol for mosquito repellents is used (Barnard, D. R. 2000. Repellent and toxicants for personal protection. WHO/CDSΛΛ/HOPES). Two clear Perspex™ 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. For the controls, an untreated arm is exposed for a 3-rninute period. The number of landings and bites are recorded as bites per 3-minute period. Subsequently 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.

[0086] 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.

[0087] it wili be evident to those skilled in the art that further modifications can be made to the embodiments described herein without departing from the spirit and scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A method of pest control comprising 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 cycling of the target receptor; wherein the active agents in combination 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.

2. The method of claim 1 , wherein the composition further comprises a third active agent, wherein the third active agent is capable of interacting with a GPCR receptor in the target pest, and wherein the interaction is complementary to the action of the first active agent.

3. The method of claim 2, wherein the first and third active agents interact with a same receptor.

4. The method of claim 2, wherein the first and third active agents interact with different receptors.

5. The method of claim 2, wherein the complementarity of the first and third active agents comprises an additive effect of the active agents together as compared with an effect of each active agent separately.

6. The method of claim 2, wherein the complementarity of the first and third active agents comprises a synergistic effect of the active agents together as compared with an effect of each active agent separately.

7. The method of claim 1, wherein cycling comprises at least one of receptor sensitization, receptor desensitization, receptor recycling, ligand release, receptor phosphorylation, and receptor dephosphorylation.

8. A pest-control composition comprising 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, wherein the second active agent acts to amplify an effect of the first active agent.

9. A method of making a pest control composition comprising the steps of: providing a target pest having at least one target 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 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; 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.

10. The method of claim 9, wherein the composition further comprises a third active agent, wherein the third active agent is capable of interacting with a receptor in the target pest, and wherein the interaction is complementary to the action of the first active agent.

11 The method of claim 10, wherein the first and third active agents interact with a same receptor.

12. The method of claim 10, wherein the first and third active agents interact with different receptors.

13. The method of claim 10, wherein the complementarity of the first and third active agents comprises an additive effect of the active agents together as compared with an effect of each active agent separately.

14. The method of claim 10, wherein the complementarity of the first and third active agents comprises a synergistic effect of the active agents together as compared with an effect of each active agent separately.

15. The method of claim 9, wherein cycling comprises at least one of receptor sensitization, receptor desensitization, receptor recycling, ligand release, receptor phosphorylation, and receptor dephosphoryiation.

16. A pest-control composition comprising 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.

17. The composition of claim 16, wherein the amplification results in a prolonged intracellular Ca²⁺ cascade as compared with the Ca²⁺ cascade that occurs when the receptor is bound without the presence of the active agent.

18. The composition of claim 16, wherein the amplification results in a prolonged perturbation of intracellular cAMP levei as compared with the perturbation in cAMP ieveS that occurs when the receptor is bound without the presence of the active agent.

19. A pest-control composition comprising 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 ϋgand binding the GPCR.

20. A method of pest control comprising 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 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 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.

21. The method of claim 20, wherein the composition further comprises a third active agent, wherein the third active agent is capable of interacting with a GPCR receptor in the target pest, and wherein the interaction is compiementary to the action of the first active agent.

22. The method of claim 21 , wherein the first and third active agents interact with a same receptor.

23. The method of claim 21 , wherein the first and third active agents interact with different receptors.

24. The method of claim 21 , wherein the complementarity of the first and third active agents comprises an additive effect of the active agents together as compared with an effect of each active agent separately.

25. The method of claim 21 , wherein the complementarity of the first and third active agents comprises a synergistic effect of the active agents together as compared with an effect of each active agent separately.

6. The method of claim 20, wherein cycling comprises at least one of receptor sensitization, receptor desensitization, receptor recycling, ϋgand release, receptor phosphorylation, and receptor dephosphoryiation.