WO2013034666A2 - Novel pheromonal receptor of spodoptera littoralis (lepidoptera, noctuidae) and identification of natural ligand of said receptor and uses thereof - Google Patents

Novel pheromonal receptor of spodoptera littoralis (lepidoptera, noctuidae) and identification of natural ligand of said receptor and uses thereof Download PDF

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WO2013034666A2
WO2013034666A2 PCT/EP2012/067467 EP2012067467W WO2013034666A2 WO 2013034666 A2 WO2013034666 A2 WO 2013034666A2 EP 2012067467 W EP2012067467 W EP 2012067467W WO 2013034666 A2 WO2013034666 A2 WO 2013034666A2
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slitor6
polypeptide
activity
sample
binding
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PCT/EP2012/067467
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French (fr)
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WO2013034666A3 (en
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Sébastien PATINY
Emmanuelle JACQUIN-JOLY
Nicolas MONTAGNE
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Chemcom S.A.
Institut National De La Recherche Agronomique (Inra)
Universite Pierre Et Marie Curie (Paris 6)
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Priority to EP12766609.7A priority Critical patent/EP2753643A2/en
Publication of WO2013034666A2 publication Critical patent/WO2013034666A2/en
Publication of WO2013034666A3 publication Critical patent/WO2013034666A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the insect olfactory receptors present a reversed membrane topology (with the -N end turned into the intracellular space and the -C end external).
  • this point is still questioned and the receptor activation have been mostly detected so far based on calcium exchange between extra- cytosolic and cytosolic spaces or as measurements of the variations of the membrane electrical potential.
  • the present invention provides a new olfactory/pheromonal receptor, namely the SlitOR6 receptor polypeptide, which has an amino acid sequence having more than 60% homology with the amino acids sequence defined in SEQ ID NO.2 shown in Figure 1 b.
  • the invention provides for a SlitOR6 nucleic acid molecule which has the DNA sequence defined in SEQ ID N0.1 shown in Figure 1 a.
  • the invention further provides for a method for determining whether a ligand can modulate the function of the SlitOR6 polypeptide as defined herein, said method comprising the steps of, contacting a cell or cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said SlitOR6 polypeptide with the ligand under conditions permitting activation of said SlitOR6 polypeptide, and measuring a signalling activity of said SlitOR6 polypeptide.
  • the invention further encompasses a method of identifying an agent that modulates the function of a SlitOR6 polypeptide, said method comprising: a) contacting a SlitOR6 polypeptide with a candidate modulator; b) measuring a signalling activity of said SlitOR6 polypeptide in the presence of said candidate modulator; and c) comparing the activity measured in the presence of said candidate modulator to said activity measured in a sample in which said SlitOR6 polypeptide is contacted with Z9,E12-14:Ac at its EC 50 , wherein said candidate modulator is identified as an agent that modulates the function of SlitOR6 when the amount of the activity measured in the presence of the candidate modulator is at least 10% of the amount induced by said Z9,E12-14:Ac present at its EC 50 .
  • the invention further encompasses a method of detecting in a sample the presence of an agent that modulates the function of a SlitOR6 polypeptide, said method comprising a) contacting a SlitOR6 polypeptide with Z9,E12-14:Ac in the presence and in the absence of said sample under conditions permitting the binding of said Z9,E12-14:Ac to said SlitOR6 polypeptide; and b) measuring the binding of said SlitOR6 polypeptide to said Z9,E12-14:Ac, wherein an increase or a decrease in binding in the presence of the sample, relative to the binding in the absence of said sample, indicates the presence of an agent that modulates the function of said SlitOR6 polypeptide in said sample.
  • the invention further encompasses a method of detecting in a sample the presence of an agent that modulates the function of a SlitOR6 polypeptide, said method comprising: a) contacting a SlitOR6 polypeptide with said sample; b) measuring a signalling activity of said SlitOR6 polypeptide in the presence of said sample; and c) comparing said activity measured in the presence of said sample to said activity measured in a reaction in which said SlitOR6 polypeptide is contacted with Z9,E12-14:Ac present at its EC 50 , wherein an agent that modulates the function of said SlitOR6 polypeptide is detected if the amount of the activity measured in the presence of said sample is at least 10% of the amount induced by Z9,E12- 14:Ac present at its EC 5 o- According to the present invention, when using binding methods Z9,E12-14:Ac may be detectably labelled.
  • the step of measuring activation of the receptor comprises electrophysiological measurements (known as electroantennography or single sensillum recording; methods well known of persons skilled in the art) performed on wild-type or genetically engineered individuals, expressing a SlitOR06 polypeptide or one related proteins (displaying homology with SlitOR06 of at least 20%).
  • electrophysiological measurements known as electroantennography or single sensillum recording; methods well known of persons skilled in the art
  • SlitOR6 agonist may be used as attractant
  • SlitOR6 antagonist as an olfactory blocker
  • said agent or composition may be used for the preparation of a pest control solution implying a SlitOR6-related behaviour; wherein said behaviour is preferentially related to the mate recognition system (SMRS).
  • SMRS mate recognition system
  • the present invention also encompasses a composition comprising an isolated SlitOR6 polypeptide and Z9,E12-14:Ac.
  • Another embodiment of the invention relates to the use of SlitOR6 polypeptide fragments or antibodies recognizing a SlitOR6 polypeptide or antibodies recognizing the Z9,E12-14:Ac/SlitOR6 complex, , for the preparation of a pest control solution.
  • Said pest may be Spodoptera littoralis (or a congeneric species).
  • the invention further encompasses a method of pest monitoring or survey using SlitOR6 polypeptide signalling, said method comprising a trapping device using Z9,E12-14:Ac as an attracting lure.
  • the invention further encompasses a method of diagnosing a pest infestation by using traps making use of the attraction mediated by a SlitOR6 polypeptide.
  • the pest for which populations are surveyed may be Spodoptera littoralis and its congeneric species.
  • an SlitOR6 knock-out transgenic animal may be used to study the effect of SlitOR6 in the detection of Z9,E12-14:Ac sources.
  • a SlitOR6 knock-out transgenic animal may be used to study the effect of SlitOR6 on the behaviors induced in Spodoptera littoralis and congeneric species by Z9,E12-14:Ac.
  • SlitOR6 may be also understood as ortholog of SlitOR6 according to the animal model chosen.
  • the SlitOR6 polypeptide may have at least 20% identity or higher identity, such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or even 100% to the polypeptide represented in Figure 1 b but with comparable activity to said SlitOR6 polypeptide.
  • identity or higher identity such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or even 100% to the polypeptide represented in Figure 1 b but with comparable activity to said SlitOR6 polypeptide.
  • a skilled person knows how to evaluate said activity depending on the assay used.
  • SlitOR6 polypeptide refers to a polypeptide having two essential properties: 1 ) a SlitOR6 polypeptide has at least 20% amino acid identity, and preferably 60%, 80%, 90%, 95% or higher, including 100% amino acid identity, to the sequence represented in Figure 1 b; and 2) a SlitOR6 polypeptide has SlitOR6 activity, i.e., the polypeptide binds Z9,E12-14:Ac. Said homology may relate to the whole polypeptide or only part of the polypeptide such as CDR domain (ligand-binding domain of the receptor ⁇ Optimally, a "SlitOR6 polypeptide" also has SlitOR6 signalling activity as defined herein. However, identification of CDR of 7TM receptors is very hazardous and depends of the algorithm applied to define TM.
  • SlitOR6 signalling activity refers to the initiation or propagation of signalling by a SlitOR6 polypeptide.
  • SlitOR6 signalling activity is monitored by measuring a detectable step in a signalling cascade by assaying one or more of the following: stimulation of GDP for GTP exchange on a G protein; alteration of adenylate cyclase activity; protein kinase C modulation; protein kinase A modulation; phosphatidylinositol breakdown (generating second messengers diacylglycerol, and inositol triphosphate); intracellular calcium flux; activation of MAP kinases; modulation of tyrosine kinases; internalization assay; modulation of gene or reporter gene activity; or melanophore assay.
  • a detectable step in a signalling cascade is considered initiated or mediated if the measurable activity is altered by 10% or more above or below a baseline established in the substantial absence of Z9,E12- 14:Ac relative to any of the SlitOR6 activity assays described herein below.
  • the measurable activity can be measured directly, as in, for example, measurement of cAMP or diacylglycerol levels. Alternatively, the measurable activity can be measured indirectly, as in, for example, a reporter gene assay. For most of these assays kits are available in the market.
  • the term Z9,E12-14:Ac refers to a chemical molecule known by a skilled chemist.
  • the terms “candidate compound” and “candidate modulator” refer to a composition being evaluated for the ability to modulate ligand binding to a SlitOR6 polypeptide or the ability to modulate an activity of SlitOR6 polypeptide.
  • Candidate modulators can be natural or synthetic compounds, including, for example, small molecules, compounds contained in extracts of animal, plant, bacterial or fungal cells, as well as conditioned medium from such cells.
  • small molecule refers to a compound having molecular mass of less than 3000 Daltons, preferably less than 2000 or 1500, still more preferably less than 1000, and most preferably less than 600 Daltons.
  • a "small organic molecule” is a small molecule that comprises carbon.
  • the term "change in binding” or “change in activity” and the equivalent terms “difference in binding” or “difference in activity” refer to an at least 10% increase or decrease in binding, or signalling activity in a given assay.
  • condition permitting the binding of Z9,E12-14:Ac to SlitOR6 refers to conditions of, for example, temperature, salt concentration, pH and protein concentration under which Z9,E12-14:Ac binds SlitOR6.
  • Exact binding conditions will vary depending upon the nature of the assay, for example, whether the assay uses viable cells or only membrane fraction of cells. However, because SlitOR6 is a cell surface protein, and because Z9,E12-14:Ac normally interacts with SlitOR6 on the cell surface, favoured conditions will generally include physiological salt (90 mM) and pH (about 7.0 to 8.0). Temperatures for binding can vary from 4°C to 37°C, but will preferably be between room temperature and about 37°C.
  • the concentration of Z9,E12-14:Ac and SlitOR6 polypeptide in a binding reaction will also vary, but will preferably be about 10 ⁇ (e.g., in a reaction with radiolabeled tracer Z9,E12-14:Ac, where the concentration is generally below the K d ) to 1 mM (e.g., Z9,E12- 14:Ac as competitor).
  • tissue that further comprises neurons and glial cells, as well as capillary endothelial cells and blood cells, all contained in a given tissue section or sample.
  • tissue is also intended to encompass non-solid tissues, such as blood.
  • the term "decrease in binding” refers to a decrease of at least 10% in the binding of Z9,E12-14:Ac or other agonist to a SlitOR6 polypeptide as measured in a binding assay as described herein.
  • second messenger refers to a molecule, generated or caused to vary in concentration by the activation of a GPCR-like receptor that participates in the transduction of a signal from that receptor.
  • second messengers include cAMP, diacylglycerol, inositol triphosphates and intracellular calcium.
  • change in the level of a second messenger refers to an increase or decrease of at least 10% in the detected level of a given second messenger relative to the amount detected in an assay performed in the absence of a candidate modulator.
  • the term "aequorin-based assay” refers to an assay for GPCR (GPCR- like receptor) activity that measures intracellular calcium flux induced by activated receptors, wherein intracellular calcium flux is measured by the luminescence of aequorin expressed in the cell.
  • GPCR GPCR-like receptor
  • Such a method is particularly valuable to quantify the Ca++ flux in cells expressing ion channel (like the OR - ORco heterodimers of insect olfaction).
  • binding refers to the physical association of a ligand (e.g., Z9,E12-14:Ac) with a receptor (e.g., SlitOR6).
  • a ligand e.g., Z9,E12-14:Ac
  • a receptor e.g., SlitOR6
  • binding is "specific” if it occurs with an EC 50 or a K d of 100 nM or less, generally in the range of 100 ⁇ to 1000 ⁇ .
  • odorant functional assay Ec50 found are usually around 10, 100 and even 1 mM, said signals may be considered specific (see Figure 3).
  • Odorant binding may be considered specific if the EC 50 or K d is comprised between 1000 ⁇ and 1 nM or less.
  • the term “EC 50 ,” refers to that concentration of an agent at which a given activity, including binding of Z9,E12-14:Ac or other ligand and a functional activity of a SlitOR6 polypeptide, is 50% of the maximum for that SlitOR6 activity measurable using the same assay. Stated differently, the “EC 50 " is the concentration of agent that gives 50% activation, when 100% activation is set at the amount of activity that does not increase with the addition of more agonist. It should be noted that the "EC 50 of an Z9,E12-14:Ac equivalent” will vary with the identity of acid; for example, equivalent Z9,E12-14:Ac molecules can have EC 50 values higher than, lower than or the same as Z9,E12-14:Ac.
  • the EC 50 of a given Z9,E12-14:Ac equivalent is measured by performing an assay for an activity of a fixed amount of SlitOR6 polypeptide in the presence of doses of the Z9,E12-14:Ac equivalent that increase at least until the SlitOR6 response is saturated or maximal, and then plotting the measured SlitOR6 activity versus the concentration of the acid used.
  • Kd is a dissociation constant or the ligand concentration at which half of the receptors are bound by the ligand at equilibrium.
  • IC 50 is the concentration of an antagonist or inverse agonist that reduces the maximal activation of a SlitOR6 receptor by 50%.
  • detectably labelled refers to the property of a molecule, e.g., Z9,E12-14:Ac or an equivalent therefrom, that has a structural modification. Said modification is introduced through the incorporation or addition of a functional group. Said functional group
  • Label can be readily detected. Detectable labels include but are not limited to fluorescent compounds, isotopic compounds, chemiluminescent compounds, quantum dot labels.
  • radioisotopes which can be added to the structure of Z9,E12-14:Ac, or equivalents may be 14 C, or 3 H (as well as eventually also 125 l, 35 S).
  • radioisotopes which can be incorporated to the structure of Z9,E12-14:Ac, or equivalents may be 3 H or 14 C.
  • the various means of detection include but are not limited to spectroscopic, photochemical, radiochemical, biochemical, immunochemical, or chemical means.
  • affinity tag refers to a label, attached to a molecule of interest (e.g., a SlitOR6 polypeptide), that confers upon the labelled molecule the ability to be specifically bound by a reagent that binds the label.
  • Affinity tags include, but are not limited to an epitope for an antibody (known as “epitope tags”), biotin, 6X His, Myc, FLAG and GST. Affinity tags can be used for the detection, as well as for the purification of the labelled species.
  • the term "decrease in binding” refers to a decrease of at least 10% in the amount of binding detected in a given assay with a known or suspected modulator of SlitOR6 relative to binding detected in an assay lacking that known or suspected modulator.
  • the term "delivering,” when used in reference to an agent, means the addition of the agent to an assay mixture, or to a cell in culture.
  • the term also refers to the administration of the agent to an animal. Such administration can be, for example, by injection (in a suitable carrier, e.g., sterile saline or water) or by puffing or other relevant route of drug administration.
  • a suitable carrier e.g., sterile saline or water
  • the term “agent that modulates the function of a SlitOR6 polypeptide” is a molecule or compound that increases or decreases SlitOR6 activity, including compounds that change the binding of Z9,E12-14:Ac or equivalents thereof, and change SlitOR6 downstream signalling activities.
  • transgenic animal refers to any animal, usually an insect
  • the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extra-chromosomally replicating DNA.
  • transgene causes cells to express a recombinant form of one of the subject polypeptide, e.g. either agonistic or antagonistic forms.
  • transgenic animals in which the recombinant gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below.
  • transgenic animal also includes those recombinant animals in which gene disruption of one or more genes is caused by human intervention, including both recombination and antisense techniques.
  • antibody is the conventional immunoglobulin molecule, as well as fragments thereof which are also specifically reactive with one of the subject polypeptides.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described herein below for whole antibodies. For example, F(ab) 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab) 2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
  • the antibody of the present invention is further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for a polypeptide conferred by at least one CDR region of the antibody.
  • the antibodies, the antibody further comprises a label attached thereto and able to be detected, (e.g., the label can be a radioisotope, fluorescent compound, chemiluminescent compound, enzyme, or enzyme co- factor).
  • blocking agent refers to any agent that stops or inhibits olfactory perception by the target. Said perception may be assayed using animal studies, ex vivo or in vitro studies simulating or representative for in vivo olfactory perception.
  • method of monitoring is meant a method by which an infestation or a change in a population dynamic may be assessed, in said situation there exists already measurable indications or signs for the presence and/or development of said infestation or change in the population dynamic.
  • Z9,E12-14:Ac equivalent is meant a molecule which has the same or comparable effect on the SlitOR6 polypeptide receptor compared to Z9,E12-14:Ac.
  • the description provides enough information, in combination with the common general knowledge, so that a skilled person may determine if a compound may be considered as an Z9,E12-14:Ac equivalent or not. It is noteworthy that different radicals containing different chemical function including but not limited to alcohol and acid could be grafted to the general formula described above.
  • Figure 1 a) DNA sequence of the gene coding for the described receptor; b) Amino Acid translation of the above sequence.
  • Figure 2 a) Expression of SlitOR6 in male antennae shown in RT-PCR; b) Expression of SlitOR6 in male antennae pictured by in situ hybridization.
  • Figure 3 EAG dose response curve, after stimulation of Drosophila antennae expressing a SlitOR6 transgene with increasing concentrations of Z9,E12-14:Ac.
  • the diamond series shows response of the neurones expressing conjointly ORco-Gal4 and UAS- SlitOR6.
  • the rectangle series shows response of the neurones expressing UAS-SlitOR6.
  • the triangle series shows response of the neurones expressing OR83b-Gal4;
  • Heptanone is a positive control stimulus and hexane is the solvent for the tested molecules.
  • Figure 4 a) Microscopic observation of cells transfected with the cloned receptor, loaded with a calcium probe (Fluo-4 AM) and stimulated with Z9, E12-14:Ac. b) Fluorescence intensity graphs of the respective transfected cells, each series represents the evolution (through time) of the fluorescence intensity of one cell in the surveyed microscopic field.
  • the invention relates to the identification of a new insect GPCR-like receptor, SlitOR6, and to the fact that Z9,E12-14:Ac is a natural ligand for said SlitOR6 GPCR-like receptor.
  • the interaction is useful for screening assays for agents that modulate the interaction and thus the function of SlitOR6.
  • the known ligand and its interaction with the receptor also provides opportunity for developing attracting lure based on single molecules or blends of at least 2 compounds.
  • Agents that modulate the activity of SlitOR6 can be identified in a number of ways that take advantage of the interaction of said receptor with Z9,E12-14:Ac. For example, the ability to reconstitute SlitOR6/Z9,E12-14:Ac binding either in vitro, on cultured cells or in vivo provides a target for identification of agents that disrupt that binding. Assays based on disruption of binding can identify agents, such as small organic molecules, from libraries or collections of such molecules. Alternatively, such assays can identify agents in samples or extracts from natural sources, including plant, fungal or bacterial extracts or even human tissue samples.
  • Modulators of SlitOR6/Z9,E12-14:Ac binding can then be screened using a binding assay or a functional assay that measures downstream signaling through the said receptor. Both binding assays and functional assays are validated using Z9,E12-14:Ac.
  • SlitOR6/Z9,E12-14:Ac interaction more directly to identify agents that modulate SlitOR6 function measures changes in SlitOR6 downstream signaling induced by candidate agents or candidate modulators.
  • These functional assays can be performed in isolated cell membrane fractions or on cells expressing the receptor on their surfaces.
  • SlitOR6 Assays using the interaction of SlitOR6 and Z9,E12-14:Ac require a source of SlitOR6 polypeptide.
  • the polynucleotide and polypeptide sequence of Spodoptera littoralis SlitOR6 are presented herein in Figure 1 .
  • One skilled in the art can readily amplify a SlitOR6 sequence from a sample containing mRNA encoding the protein through basic PCR and molecular cloning techniques using primers or probes designed from the known sequences.
  • SlitOR6 The expression of recombinant polypeptides is well known in the art. Those skilled in the art can readily select vectors and expression control sequences for the expression of SlitOR6 polypeptides useful according to the invention in eukaryotic or prokaryotic cells. SlitOR6 must be associated with cell membrane or detergents like synthetic liposomes in order to have binding or signalling function. Methods for the preparation of cellular membrane fractions are well known in the art, e.g., the method reported by Hubbard and Cohn, 1975, J. Cell Biol. 64: 461 -479, which is incorporated herein by reference. In order to produce membranes comprising SlitOR6, one need only to apply such techniques to cells endogenously or recombinantly expressing SlitOR6.
  • membrane-free SlitOR6 can be integrated into membrane preparations by dilution of detergent solution of the polypeptide (see, e.g., Salamon et al., 1996, Biophys. J. 71 :283-294, which is incorporated herein by reference).
  • Z9,E12-14:Ac The structure of Z9,E12-14:Ac is well known by a skilled person.
  • the skilled in the art may easily derive equivalent structures and may easily test if said equivalents are able to bind and/or modulate the SlitOR6 receptor.
  • Z9,E12-14:Ac and equivalents thereof may be isolated from natural samples, or chemically synthesized.
  • Methods which can be used to quantify said Z9,E12-14:Ac may be, but are not limited to, a) for extraction and purification: solvent extraction, oil extraction, vapour extraction, C02 supercritical extraction, liquid chromatography, distillation, gas chromatography; b) for quantifying: gas chromatography, liquid chromatography, etc. coupled to mass spectrometry (e.g.).
  • a) for extraction and purification solvent extraction, oil extraction, vapour extraction, C02 supercritical extraction, liquid chromatography, distillation, gas chromatography
  • mass spectrometry e.g.
  • Z9,E12-14:Ac or its equivalents may be used in purified form or used as composition.
  • the amounts of the acetate necessary in a given binding or functional assay according to the invention will vary depending upon the assay, but will generally use 1 nM to 10 ⁇ of labelled and 1 nM to 10mM of unlabelled acetate per assay.
  • the affinities and EC 50 of modified Z9,E12-14:Ac molecules for SlitOR6 may vary relative to those of the original Z9,E12-14:Ac, and the amount necessary for a given assay can therefore be adjusted relative to the normal values. If necessary for a given assay, Z9,E12-14:Ac can be labelled by incorporation or addition of radiolabeled labels as pointed above.
  • Z9,E12-14:Ac is a ligand of the SlitOR6 receptor permits screening assays to identify agonists, antagonists and inverse agonists of receptor activity.
  • the screening assays will have two general approaches.
  • Ligand binding assays in which cells expressing SlitOR6, membrane extracts from such cells, or immobilized lipid membranes comprising SlitOR6 are exposed to a labelled Z9,E12-14:Ac and a candidate compound. Following incubation, the reaction mixture is measured for specific binding of the labelled Z9,E12-14:Ac to the SlitOR6 receptor.
  • Compounds that interfere with or displace labelled Z9,E12-14:Ac can be agonists, antagonists or inverse agonists of SlitOR6 activity. Functional analysis can be performed on positive compounds to determine which of these categories they fit.
  • Binding of a compound may be classified in 3 main categories: competitive binding, non-competitive binding and uncompetitive binding.
  • a competitive binding compound resembles a second (reference) compound and binds to the same binding pocket of a target molecule (here receptor). Upon addition, the competitive binding compound displaces said second compound from said target.
  • a non-competitive binding compound does not bind to the same binding pocket of the target molecule as a second (reference) compound but may interact with the effect of said second compound on said target molecule. The second compound is not displaced upon addition of the non-competitive binding compound.
  • An uncompetitive-binding compound binds to the target molecule when a second compound is already bound.
  • Cooperative binding means that a compound facilitates the binding of another compound which may be a reference compound. The cooperative effect is thus seen in the analysis of the Kd of said other compound.
  • An agonist or partial agonist will have a maximal biological activity corresponding to at least 10% of the maximal activity of Z9,E12-14:Ac when the agonist or partial agonist is present at 100(0) ⁇ or less, and preferably will have 50%, 75%, 100% or more, including 2-fold, 5-fold, 10-fold or more activity than Z9,E12-14:Ac.
  • b) For antagonist or inverse agonist screening cells expressing SlitOR6 or membranes isolated from them are assayed for signalling activity in the presence of Z9,E12- 14:Ac with or without a candidate compound. Antagonists or inverse agonists will reduce the level of Z9,E12-14:Ac-stimulated receptor activity by at least 10%, relative to reactions lacking the antagonist or inverse agonist.
  • ⁇ For inverse agonist screening, cells expressing constitutive SlitOR6 activity or membranes isolated from them are used in a functional assay that measures an activity of the receptor in the presence and in the absence of a candidate compound.
  • Inverse agonists are those compounds that reduce the constitutive activity of the receptor by at least 10%.
  • Overexpression of SlitOR6 may lead to constitutive activation.
  • SlitOR6 can be overexpressed by placing it under the control of a strong constitutive promoter, e.g., the CMV early promoter.
  • binding buffer e.g., 50 mM Hepes pH 7.4; 1 mM CaCI 2 ; 0.5% Bovine Serum Albumin (BSA) Fatty Acid-Free; and 0 , 5 mM MgCI 2
  • BSA Bovine Serum Albumin
  • Z9,E12-14:Ac labelled Z9,E12-14:Ac in the presence or in the absence of increasing concentrations of a candidate modulator.
  • control competition reactions using increasing concentrations of unlabelled Z9,E12-14:Ac can be performed.
  • Z9,E12-14:Ac is measured as appropriate for the given label (e.g., scintillation counting, enzyme assay, fluorescence, etc.).
  • a decrease of at least 10% in the amount of labelled Z9,E12-14:Ac bound in the presence of candidate modulator indicates displacement of binding by the candidate modulator.
  • Candidate modulators are considered to bind specifically in this or other assays described herein if they displace 50% of labelled Z9,E12-14:Ac (sub-saturating Z9,E12-14:Ac dose) at a concentration of 100 ⁇ or less (i.e., ECso is 100 ⁇ or less).
  • binding or displacement of binding can be monitored by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • Surface plasmon resonance assays can be used as a quantitative method to measure binding between two molecules by the change in mass near an immobilized sensor caused by the binding or loss of binding of Z9,E12-14:Ac from the aqueous phase to a SlitOR6 polypeptide immobilized in a membrane on the sensor. This change in mass is measured as resonance units versus time after injection or removal of Z9,E12-14:Ac or candidate modulator and is measured using a Biacore Biosensor (Biacore AB).
  • Biacore Biosensor Biacore Biosensor
  • SlitOR6 can be immobilized on a sensor chip (for example, research grade CM5 chip; Biacore AB) in a thin film lipid membrane according to methods described by Salamon et al. (Salamon et al., 1996, Biophys J. 71 : 283-294; Salamon et al., 2001 , Biophys. J. 80: 1557-1567; Salamon et al., 1999, Trends Biochem. Sci. 24: 213-219, each of which is incorporated herein by reference.). Sarrio et al.
  • SPR can be used to detect ligand binding to the GPCR A(1 ) adenosine receptor immobilized in a lipid layer on the chip (Sarrio et al., 2000, Mol. Cell. Biol. 20: 5164-5174, incorporated herein by reference).
  • Conditions for Z9,E12-14:Ac binding to SlitOR6 in an SPR assay can be fine-tuned by one of skill in the art using the conditions reported by Sarrio et al. as a starting point.
  • SPR can assay for modulators of binding in at least two ways.
  • Z9,E12-14:Ac can be pre-bound to immobilized SlitOR6 polypeptide, followed by injection of candidate modulator at approximately 10 ⁇ /min flow rate and a concentration ranging from 1 nM to 1000 ⁇ , preferably about 100 ⁇ . Displacement of the bound Z9,E12-14:Ac can be quantitated, permitting detection of modulator binding.
  • the membrane-bound SlitOR6 polypeptide can be pre-incubated with candidate modulator and challenged with Z9,E12- 14:Ac. A difference in Z9,E12-14:Ac binding to the SlitOR6 exposed to modulator relative to that on a chip not pre-exposed to modulator will demonstrate binding.
  • Biacore system can be plugged to a system identifying candidate modulator such as mass spectrometry, or gas chromatography.
  • FRET fluorescence resonance energy transfer
  • fluorescence emitted upon excitation of the donor fluorophore will have a different wavelength than that emitted in response to that excitation wavelength when the molecules are not bound, providing for quantitation of bound versus unbound polypeptides by measurement of emission intensity at each wavelength.
  • Donor:Acceptor pairs of fluorophores with which to label the target molecules are well known in the art.
  • a variation on FRET uses fluorescence quenching to monitor molecular interactions.
  • One molecule in the interacting pair can be labelled with a fluorophore, and the other with a molecule that quenches the fluorescence of the fluorophore when brought into close apposition with it.
  • a change in fluorescence upon excitation is indicative of a change in the association of the molecules tagged with the fluorophore: quencher pair.
  • an increase in fluorescence of the labelled SlitOR6 polypeptide is indicative that Z9,E12-14:Ac bearing the quencher has been displaced.
  • a 10% or greater increase in the intensity of fluorescent emission in samples containing a candidate modulator, relative to samples without the candidate modulator indicates that the candidate modulator inhibits SlitOR6: Z9,E12-14:Ac interaction.
  • Bioluminescence Resonance Energy Transfer is a system for monitoring intermolecular interactions in vivo.
  • the assay is based on non-radiative energy transfer between fusion proteins containing Renilla luciferase (Rluc) and e.g. Yellow Fluorescent Protein (YPF) or Green Fluorescent Protein (GFP).
  • the BRET signal is generated by the oxidation of a coelenterazine derivative substrate.
  • Said system may apply a cell-permeable and non-toxic coelenterazine derivative substrate DeepBleuCTM (DBC) and a mutant of the Green Fluorescent Protein (GFP) as acceptor.
  • DBC DeepBleuCTM
  • GFP Green Fluorescent Protein
  • fluorescence polarization measurement is useful to quantitate Z9,E12-14:Ac-receptor binding.
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate. Protein complexes, such as those formed by SlitOR6 associating with a fluorescently labelled Z9,E12-14:Ac, have higher polarization values than uncomplexed, labelled Z9,E12-14:Ac.
  • a candidate inhibitor of the SlitOR6: Z9,E12-14:Ac interaction results in a decrease in fluorescence polarization, relative to a mixture without the candidate inhibitor, if the candidate inhibitor disrupts or inhibits the interaction of SlitOR6 with Z9,E12-14:Ac.
  • Fluorescence polarization is well suited for the identification of small molecules that disrupt the formation of polypeptide or protein complexes. A decrease of 10% or more in fluorescence polarization in samples containing a candidate modulator, relative to fluorescence polarization in a sample lacking the candidate modulator, indicates that the candidate modulator inhibits SlitOR6: Z9,E12-14:Ac interaction.
  • Another alternative for monitoring SlitOR6: Z9,E12-14:Ac interactions uses a biosensor assay.
  • ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute; http//www.ambri. com.au/).
  • AMBRI Australian Membrane Biotechnology Research Institute; http//www.ambri. com.au/.
  • the association of molecules such as SlitOR6 and Z9,E12-14:Ac is coupled to the closing of gramacidin-facilitated ion channels in suspended membrane bilayers and thus to a measurable change in the admittance (similar to impedence) of the biosensor.
  • This approach is linear over six orders of magnitude of admittance change and is ideally suited for large scale, high throughput screening of small molecule combinatorial libraries.
  • a modulator of the interaction need not necessarily to interact directly with the domain(s) of the proteins that physically interact. It is also possible that a modulator will interact at a location removed from the site of ligand-protein interaction and cause, for example, a conformational change in the SlitOR6 polypeptide. Modulators (inhibitors or agonists) that act in this manner are nonetheless of interest as agents to modulate the activity of SlitOR6.
  • any of the binding assays described can be used to determine the presence of an agent in a sample, e.g., a tissue sample, that binds to the SlitOR6 receptor molecule, or that affects the binding of Z9,E12-14:Ac to the receptor.
  • a sample e.g., a tissue sample
  • Z9,E12-14:Ac or ligand binding is measured as appropriate for the binding assay being used.
  • a decrease of 10% or more in the binding of Z9,E12-14:Ac or other ligand indicates that the sample contains an agent that modulates Z9,E12-14:Ac or ligand binding to the receptor polypeptide.
  • Protein chips The methods of the present invention may be applied on protein chips.
  • Said protein chip may be, but is not limited to, a glass slide or a nitrocellulose membrane.
  • Array-based methods for protein chips are well known in the art.
  • the aequorin assay takes advantage of the responsiveness of mitochondrial or cytoplasmic apoaequorin to intracellular calcium release or calcium flux (entrance) induced by the activation of GPCRs (Stables et al., 1997, Anal. Biochem. 252:1 15-126; Detheux et al., 2000, J. Exp. Med., 192 1501 -1508; both of which are incorporated herein by reference). Briefly, SlitOR6-expressing clones are transfected to coexpress mitochondrial or cytoplasmic apoaequorin and Gcc16 or G-olf.
  • Cells are incubated with 5 ⁇ Coelenterazine H or derivates (Molecular Probes) for 4 hours at room temperature, washed in DMEM-F12 culture medium and resuspended at a concentration of 0.5 x 10 6 cells/ml. Cells are then mixed with test agonist peptides and light emission by the aequorin is recorded with a luminometer for 30 sec. Results are expressed as Relative Light Units (RLU). Controls include assays using membranes isolated from cells not expressing SlitOR6 (mock-transfected), in order to exclude possible non-specific effects of the candidate compound.
  • RLU Relative Light Units
  • Aequorin activity or intracellular calcium levels are "changed” if light intensity increases or decreases by 10% or more in a sample of cells, expressing a SlitOR6 polypeptide and treated with a candidate modulator, relative to a sample of cells expressing the SlitOR6 polypeptide but not treated with the candidate modulator or relative to a sample of cells not expressing the SlitOR6 polypeptide (mock-transfected cells) but treated with the candidate modulator.
  • the assay can be used to identify an agonist or inverse agonist of SlitOR6 activity.
  • the assay is performed in the presence of Z9,E12-14:Ac, it can be used to assay for an antagonist.
  • Fluorescence-based assays take advantage of calcium fluxes triggered by receptor activation: either calcium entrance through ORco for instance or calcium release from endoplasmic reticulum.
  • Some fluorophores including but not limited to Fluo3, Fluo4 and Fura2 (Molecular Probes) and Calcium3 kit series (Molecular Device) are known to bind calcium.
  • fluorophore-calcium complexes emit fluorescence at respective specific wavelength.
  • SlitOR6-overexpressing cells are incubated for 30 to 60 minutes with a solution of 1 to 8 ⁇ fluorophore at 37°C.
  • the principle of this assay is to follow depolarization of cell membrane.
  • DiBAC 4 (3) partitions between intra- and extracellular compartments in a membrane potential-dependent manner. With increasing membrane potential (depolarization), the probe further partition into the cell resulting in an increase of fluorescence. Conversely, hyperpolarization leads to a decrease of fluorescence due to a dye extrusion.
  • the DiBAC 4 (3) probe is excited with a wavelength of 488 nm, and emits at a wavelength of 540 nm.
  • Membrane polarization is "changed” if fluorescence intensity increases or decreases by 10% or more in a sample of cells, expressing a SlitOR6 polypeptide and treated with a candidate modulator, relative to a sample of cells expressing the SlitOR6 polypeptide but not treated with the candidate modulator or relative to a sample of cells not expressing the SlitOR6 polypeptide (mock-transfected cells) but treated with the candidate modulator.
  • 100 ⁇ reactions contain 50 mM Tris-Hcl (pH 7.5), 5 mM MgCI 2 , 20 mM creatine phosphate (disodium salt), 10 units (71 ⁇ g of protein) of creatine phosphokinase, 1 mM cc- 32 P-ATP (tetrasodium salt, 2 ⁇ ), 0.5 mM cyclic AMP, G- 3 H-labelled cyclic AMP (approximately 10,000 cpm), 0.5 mM Ro20-1724, 0.25% ethanol, and 50-200 ⁇ g of protein homogenate to be tested (i.e., homogenate from cells expressing or not expressing a SlitOR6 polypeptide, treated or not treated with Z9,E12-14:Ac with or without a candidate modulator).
  • Reaction mixtures are generally incubated at 37°C for 6 minutes. Following incubation, reaction mixtures are deproteinized by the addition of 0.9 ml of cold 6% trichloroacetic acid. Tubes are centrifuged at 1800 x g for 20 minutes and each supernatant solution is added to a Dowex AG50W-X4 column. The cAMP fraction from the column is eluted with 4 ml of 0.1 mM imidazole-HCI (pH 7.5) into a counting vial. Assays should be performed in triplicate. Control reactions should also be performed using protein homogenate from cells that do not express a SlitOR6 polypeptide.
  • Assays should be performed using cells or extracts of cells expressing SlitOR6, treated or not treated with Z9,E12-14:Ac with or without a candidate modulator. Control reactions should be performed using mock-transfected cells, or extracts from them in order to exclude possible non-specific effects of some candidate modulators
  • adenylate cyclase activity is "changed” if it increases or decreases by 10% or more in a sample taken from cells treated with a candidate modulator of SlitOR06 activity, relative to a similar sample of cells not treated with the candidate modulator or relative to a sample of cells not expressing the SlitOR6 polypeptide (mock-transfected cells) but treated with the candidate modulator.
  • a decrease of activity by 10% or more by the candidate modulator of SlitOR6 in a sample treated with a reference compound may be tested.
  • Intracellular cAMP/cGMP is measured using a cAMP/cGMP radioimmunoassay (RIA) or cAMP/cGMP binding protein according to methods widely known in the art.
  • RIA radioimmunoassay
  • cAMP/cGMP binding protein for example, Horton and Baxendale, 1995, Methods Mol. Biol. 41 : 91 -105, which is incorporated herein by reference, describes an RIA for cAMP.
  • kits for the measurement of cAMP/cGMP are commercially available, such as the High Efficiency Fluorescence Polarization-based homogeneous assay marketed by LJL Biosystems and NEN Life Science Products. Control reactions should be performed using extracts of mock-transfected cells to exclude possible non-specific effects of some candidate modulators.
  • Assays should be performed using cells or extracts of cells expressing SlitOR6, treated or not treated with Z9,E12-14:Ac with or without a candidate modulator. Control reactions should be performed using mock-transfected cells, or extracts from them in order to exclude possible non-specific effects of some candidate modulators
  • the level of cAMP is "changed” if the level of cAMP/cGMP detected in cells, expressing a SlitOR6 polypeptide and treated with a candidate modulator of SlitOR6 activity (or in extracts of such cells), using the RIA-based assay of Horton and Baxendale, 1995, supra, increases or decreases by at least 10% relative to the cAMP level in similar cells not treated with the candidate modulator.
  • Phospholipid breakdown, DAG production and Inositol Triphosphate levels are Phospholipid breakdown, DAG production and Inositol Triphosphate levels:
  • Receptors that activate the breakdown of phospholipids can be monitored for changes due to the activity of known or suspected modulators of SlitOR6 by monitoring phospholipid breakdown, and the resulting production of second messengers DAG and/or inositol triphosphate (IP 3 ).
  • DAG and/or inositol triphosphate IP 3
  • Methods of measuring each of these are described in Phospholipid Signalling Protocols, edited by Ian M. Bird. Totowa, NJ, Humana Press, 1998, which is incorporated herein by reference. See also Rudolph et al., 1999, J. Biol. Chem. 274: 1 1824- 1 1831 , incorporated herein by reference, which also describes an assay for phosphatidylinositol breakdown.
  • Assays should be performed using cells or extracts of cells expressing SlitOR6, treated or not treated with Z9,E12-14:Ac with or without a candidate modulator. Control reactions should be performed using mock-transfected cells, or extracts from them in order to exclude possible non-specific effects of some candidate modulators.
  • phosphatidylinositol breakdown, and diacylglycerol and/or inositol triphosphate levels are "changed” if they increase or decrease by at least 10% in a sample from cells expressing a SlitOR6 polypeptide and treated with a candidate modulator in the presence or in the absence of Z9,E12-14:Ac, relative to the level observed in a sample from cells expressing a SlitOR6 polypeptide that is not treated with the candidate modulator.
  • the intracellular signal initiated by binding of an agonist to a receptor sets in motion a cascade of intracellular events, the ultimate consequence of which is a rapid and detectable change in the transcription and/or translation of one or more genes.
  • the activity of the receptor can therefore be monitored by measuring the expression of a reporter gene driven by control sequences responsive to SlitOR6 activation.
  • promoter refers to the transcriptional control elements necessary for receptor-mediated regulation of gene expression, including not only the basal promoter, but also any enhancers or transcription-factor binding sites necessary for receptor-regulated expression.
  • promoters that are responsive to the intracellular signals resulting from agonist binding, and operatively linking the selected promoters to reporter genes whose transcription, translation or ultimate activity is readily detectable and measurable, the transcription based reporter assay provides a rapid indication of whether a given receptor is activated.
  • Reporter genes such as luciferase, Chloramphenicol Acetyl Transferase (CAT), Green
  • Genes particularly well suited for monitoring receptor activity are the "immediate early" genes, which are rapidly induced, generally within minutes of contact between the receptor and the effector protein or ligand.
  • the induction of immediate early gene transcription does not require the synthesis of new regulatory proteins.
  • characteristics of preferred genes useful to make reporter constructs include: low or undetectable expression in quiescent cells; induction that is transient and independent of new protein synthesis; subsequent shut-off of transcription requires new protein synthesis; and mRNAs transcribed from these genes have a short half-life. It is preferred, but not necessary that a transcriptional control element have all of these properties for it to be useful.
  • c-fos proto-oncogene An example of a gene that is responsive to a number of different stimuli is the c-fos proto-oncogene.
  • the c-fos gene is activated in a protein-synthesis-independent manner by growth factors, hormones, differentiation-specific agents, stress, and other known inducers of cell surface proteins.
  • the induction of c-fos expression is extremely rapid, often occurring within minutes of receptor stimulation. This characteristic makes the c-fos regulatory regions particularly attractive for use as a reporter of receptor activation.
  • the c-fos regulatory elements include (see, Verma et al., 1987, Cell 51 : 513-514): a TATA box that is required for transcription initiation; two upstream elements for basal transcription, and an enhancer, which includes an element with dyad symmetry and which is required for induction by phorbol ester 12-0-tetradecanoylphorbol- -acetate (TPA), serum, Epidermal Growth Factor (EGF), and PMA.
  • TNF-cc (Shakhov et al., 1990, J. Exp. Med. 171 : 35-47), CCR5 (Liu et al., 1998, AIDS Res. Hum. Retroviruses 14: 1509-1519), P-selectin (Pan and McEver, 1995, J. Biol. Chem. 270: 23077-23083), Fas ligand (Matsui et al., 1998, J. Immunol. 161 : 3469-3473), GM-CSF (Schreck and Baeuerle, 1990, Mol. Cell. Biol.
  • cells that stably express SlitOR6 polypeptide are stably transfected with the reporter construct.
  • untreated cells are exposed to candidate modulators, or exposed to Z9,E12-14:Ac, and expression of the reporter is measured.
  • the Z9,E12-14:Ac- treated cultures serve as a standard for the level of transcription induced by a known agonist.
  • An increase of at least 10% in reporter expression in the presence of a candidate modulator compare to reporter expression in the absence of any modulator indicates that the candidate is a modulator of SlitOR6 activity.
  • the cells expressing SlitOR6 and carrying the reporter construct are exposed to Z9,E12-14:Ac (or another agonist) in the presence and absence of candidate modulator.
  • Z9,E12-14:Ac or another agonist
  • Controls for transcription assays include cells not expressing SlitOR6 but carrying the reporter construct, as well as cells with a promoterless reporter construct.
  • Compounds that are identified as modulators of SlitOR6-regulated transcription should also be analyzed to determine whether they affect transcription driven by other regulatory sequences and by other receptors, in order to determine the specificity and spectrum of their activity.
  • Z9,E12-14:Ac as a ligand of SlitOR6 provides methods of modulating the activity of a SlitOR6 polypeptide expressed in a cell.
  • SlitOR6 activity is modulated in a cell by delivering to that cell an agent that modulates the function of SlitOR6 polypeptide. This modulation can be performed in cultured cells as part of an assay for the identification of additional modulating agents, or, for example, in an animal, including a human.
  • Agents include Z9,E12-14:Ac and equivalent acids thereof.
  • An agent can be delivered to a cell by adding it to culture medium.
  • the amount to deliver will vary with the identity of the agent and with the purpose for which it is delivered.
  • an amount of Z9,E12-14:Ac that half-maximally activates the receptors (e.g., approximately EC 5 o), preferably without exceeding the dose required for receptor saturation.
  • This dose can be determined by titrating the amount of Z9,E12-14:Ac to determine the point at which further addition of Z9,E12-14:Ac has no additional effect on SlitOR6 activity.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible by methods well known in the art. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
  • candidate modulators may be variants or equivalents of Z9,E12-14:Ac. Therefore, a library of Z9,E12-14:Ac-related compounds may be used
  • Example of such transgenic animals that one can use as tools to study the functional properties of SlitOR6 and to search for modulators is the fly Drosophila melanogaster.
  • Transformed flies can further be screened for electrophysiological or behavioural responses upon stimulation with compound libraries.
  • Kits useful for screening for modulators of SlitOR6 activity can include an isolated SlitOR6 polypeptide (including a membrane- or cell-associated SlitOR6 polypeptide, e.g., on isolated membranes, cells expressing SlitOR6, or, on an SPR chip) and an isolated Z9,E12-14:Ac. When cells included, said cells may be transformed with a polynucleotide encoding said SlitOR6.
  • the kit according to the invention may contain a polynucleotide encoding a SlitOR6 polypeptide and Z9,E12-14:Ac. All kits according to the invention will comprise the stated items or combinations of items and packaging materials therefore. Kits may also include instructions for use.
  • said SlitOR6 polypeptide may comprise one or more additions, insertions, deletions or substitutions relative to the sequence depictured in Figure 1 b.
  • Said SlitOR6 polypeptide may be a truncated SlitOR6 polypeptide; said SlitOR6 polypeptide may comprise additional sequences forming a SlitOR6 fusion protein, wherein said additional sequences may be chosen from the group consisting of glutathione-S-transferase (GST), maltose binding protein (MBP), alkaline phosphatase, thioredoxin, green fluorescent protein (GFP), histidine tags (e.g., 6X or greater His), or affinity tags (e.g., Myc tag, FLAG tag) sequences.
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • GFP alkaline phosphatase
  • thioredoxin alkaline phosphatase
  • GFP green fluorescent protein
  • Example 1 Identification and cloning of a pheromone receptor in Spodoptera littoralis ( Figure 1 a,b,c).
  • the sequences of pheromone receptors have been identified in several species of Moths (see Sakurai et al., 2004, Proc Natl Acad Sci U S A. 2004 Nov 23;101 (47): 16653-16658 for an example). Because of its position of major pest, attention was paid to Spodoptera littoralis. In those Butterflies, the males are attracted to mating areas by the pheromones emitted by females. The male antennae are the main organs involved in the perception of these volatile molecules.
  • cDNA libraries were created based on RNA extracts from freshly excised male antennae. These fragments were sequenced and screened based existing databases of potential orthologues (e.g. Genbank). The fragments displaying the highest homology with known and identified sequences of other species were then used as bases for RACE-PCR, in order to obtain full ORF (i.e. open reading frame). These latter were then PCR amplified and cloned into the relevant expression vectors (for instance pUAST for the in vivo expression).
  • the cloned sequences were phylogenetically compared with those known in other organisms.
  • the resulting tree notably shows the clustering of two groups of receptors (i) the ORco clade, containing the co-receptors identified in the varied compared species; (ii) the pheromone receptors clade including the Spodoptera pheromones receptors as well as those of the closely related species.
  • SlitOR6 is a newly found gene, which phylogenetically groups within the subclade of Moths pheromone receptors. This phylogenetic evidence supports the expected role of the pheromone receptor of that gene.
  • the gene coding for SlitOR6 ( Figure 1 a) and the corresponding protein ( Figure 1 b) were -for the first time- characterized in a functional point of view, using in vivo and in vitro approaches (see subsequent examples).
  • Example 2 Characterization of the receptor expression patterns by in situ hybridization ( Figure 2a, b).
  • the mapping of expression of the characterized receptor (SlitOR6) is of importance, notably to guide the electrophysiological approaches and to illustrate its biological function.
  • Two methods were used to characterise the expression bias of SlitOR6 in males, females and varied tissues: (i) RT-PCR using RNA pool extracts from male and female tissues; (ii) in situ hybridization in section of olfactory organs. Both methods are well known by a person skilled in the art.
  • RT-PCR was realized based on RNA freshly extracted from tissues isolated from varied organs of both males and females ( Figure 4a). In situ hybridization was performed on sections (longitudinal are presented in Figure 4b). Labeled SlitOR6 probes were used to hybridize mRNA in the male antennae.
  • RT-PCR shows that expression of the targeted gene is nearly completely restricted to male antennae. A very faint expression is detectable in the antennae of females also ( Figure 4a).
  • In situ hybridization shows more specifically the expression of the concerned receptor in the olfactory sensillae of the ventral side of antennae.
  • Example 3 Electro-antennography assays on Drosophila antennae expressing SlitOR6 ( Figures 3a, b). SlitOR6 was cloned into the pUAST vector and recombinant plasmid was injected in Drosophila embryos to generate UAS-SlitOR6 lines (BestGene Inc., Chino Hills, CA, USA). UAS-SlitOR6 lines were crossed with a homozygous ORco-GAL4 driver line (Bloomington Drosophila Stock Center, No. 23292).
  • Electro-antennogram (EAG) recordings were performed on UAS-SlitOR6 and ORco-GAL4 parental lines (controls) and on ORco-GAL4/UAS-SlitOR6 lines, according to protocol well known of a person skilled in the art.
  • Example 4 Single-cell calcium-imaging assays performed on Hek 293 cells overexpressing SlitOR6 polypeptide ( Figures 4a, b).
  • cells derived HEK293 cells in the illustrated example: Figure 4a
  • Transfection was made according to the manufacturer's protocol (most assays were realized with Lipofectamin 2000, Invitrogen (c)).
  • Calcium mobilization was recorded with a 10x or 20x magnification objective on a Zeiss Axiovert 200 Mot microscope equipped for fluorescence detection. One image of the same field was taken each second during 60-90 seconds. 50 ⁇ of the two fold concentrated ligand solubilised in the saline buffer was injected 5 seconds after record started.

Abstract

The invention relates to the identification of Z9,E12-14:Ac as a natural ligand of the new olfactory/pheromonal receptor SlitOR6, a G-protein coupled-like receptor (GPCR-like). The invention encompasses the use of the interaction of SlitOR6 polypeptides and Z9,E12-14:Ac as the basis of screening assays for agents that modulate the activity of the SlitOR6 receptor. The invention also encompasses diagnostic and other assays performed based upon the SlitOR6/Z9,E12-14:Ac interaction, as well as kits for performing diagnostic and screening assays.

Description

NOVEL PHEROMONAL RECEPTOR of Spodoptera littoralis (Lepidoptera, Noctuidae) AND IDENTIFICATION OF NATURAL LIGAND OF SAID RECEPTOR AND USES THEREOF
FIELD OF THE INVENTION
The invention relates to a novel olfactory/pheromonal receptor (SlitOR6) of Spodoptera littoralis (Lepidoptera, Noctuidae) and the identification of the natural ligand of SlitOR6 and uses thereof.
BACKGROUND OF THE INVENTION
Transmembrane receptors are proteins responsible for transducing signals within a cell. The insect olfactory receptor can be described as GPCR-like transmembrane receptors. Like their mammalian analogues, they have seven transmembrane domains. Upon binding of a ligand to an extra-cellular portion or fragment of the receptor, a signal is transduced within the cell that results in a change in a biological or physiological property or behaviour of the cell. Insect olfactory receptors (OR), along with coreceptor (ORco) and effectors (intracellular enzymes and channels, likely modulated by G-proteins), are the components of a modular signaling system that connects the state of intra-cellular second messengers to extra-cellular inputs.
Olfactory receptor genes and gene products in insects can modulate various physiological processes and are potential causative agents of many behaviors. The insect olfactory receptors seem to be of critical importance to both the peripheral and central olfactory response within the nervous system, leading to physiological/behavioral response.
Contrary to the other GPCRs of insects, other invertebrates and vertebrates, the insect olfactory receptors present a reversed membrane topology (with the -N end turned into the intracellular space and the -C end external). In addition, even if clues exist, which support a possible coupling with trimeric G proteins, this point is still questioned and the receptor activation have been mostly detected so far based on calcium exchange between extra- cytosolic and cytosolic spaces or as measurements of the variations of the membrane electrical potential.
The insect receptors to pheromones belong to the family of olfactory receptors. The insect olfactory and pheromonal receptors are expressed at the membrane level as heterodimers of a receptor and a coreceptor (ORco, similar to the Drosophila melanogaster OR83b). Within the so constituted couple, each subunit has its own function. The ligand recognition and binding is operated by the OR itself. Following activation of the OR by the ligand reception, the ORco opens and plays the role of an ion (Ca++) channel (Sato et al., 2008, Nature, 452: 1002-1006; Wicher et al ., 2008, Nature, 452: 1007-101 1 ). In addition to its role in signaling through the regulation of the calcium entry in the intracellular space, the ORco also plays the role of chaperone acting in the trafficking of ORs to the membrane and their insertion into the membrane.
There are clues in the literature, that -upon activation by their ligand- the insect OR could interact with G proteins like their mammalian analogues do. G proteins represent a family of heterotrimeric proteins composed of α, β and γ subunits, that bind guanine nucleotides. Following ligand binding, a conformational change is transmitted to the G protein, which causes the a-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the βγ-subunits. The GTP-bound form of the α, β and γ-subunits typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cAMP (e.g. by activation of adenyl cyclase), diacylglycerol or inositol phosphates.
Known and uncharacterized insect olfactory receptors currently constitute major targets for the research of chemicals for the control of insect populations. For some insect olfactory receptors, a possible pheromone-binding role has been assigned. No ligand capable of modulating such receptor has been identified yet in the African cotton leafworm (Spodoptera littoralis Boisduval). There are ongoing efforts to identify new olfactory and pheromonal receptors which can be used to screen for new agonists and antagonists having potential applications in the development of strategies adapted to the control of insect populations.
The sense of smell allows chemical communications between living organisms (invertebrates to mammals) and their environment. Perception and discrimination of thousands of odorants is made through olfaction. Such chemical signalling may modulate social behaviour of most animal species which rely on odorant compounds to identify kin or mate, to locate food or to recognize territory for instance. Smelling abilities are initially determined by neurons of the antennae sensilla, the olfactory sensory neurons (OSN). Therein, odorant molecules bind to olfactory receptor proteins (OR), also known as odorant receptors. These OR constitute a family of G-protein coupled receptors (GPCR)-like family. They are encoded by a large gene family. Whereas in Drosophila melanogaster 62 different OR genes have been identified, 259 such genes have been identified in the red flour beetle genome (Tribolium castaneum (Engsontia et al. 2008, Insect Biochem Mol Biol 38(4): 387- 397). Each olfactory neuron is thought to express only one type of OR, forming therefore the cellular basis of odorant discrimination by olfactory neurons. They are synthesized in the endoplasmatic reticulum, transported and eventually concentrated at the cell surface membrane of the sensilla neurone.
Some insects express also ORs in other body parts such as mouthparts, legs meso- and metasoma or wings. According to the currently available knowledge, the pheromone receptors are more concentrated in the antennae. This receptor subtype can notably be identified based on their gender expression-bias. The phylogenetic approach, comparing the sequence similarity of receptors in closely related species can provide additional clues supporting the identification of the pheromone receptors among the complete olfactory repertoire.
Here we described the cloning of a new receptor, namely SlitOR6, which has been characterized, in the present invention, as a novel Spodoptera littoralis pheromone receptor, which is over-expressed in the antennae of males. The gene sequence of the new receptor SlitOR6 is shown in Figure 1 a. The translated amino acid sequence (Figure 1 b) shows a significant homology with pheromonal receptors characterized in other closely related species.
The (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:Ac) has been described as a component of the Spodoptera littoralis pheromonal bouquet as soon as the early seventies (see pherobase for a reference: www.pherobase.com/). The association between this molecule and an olfactory receptor was however so far unreported.
SUMMARY OF THE INVENTION
The present invention provides a new olfactory/pheromonal receptor, namely the SlitOR6 receptor polypeptide, which has an amino acid sequence having more than 60% homology with the amino acids sequence defined in SEQ ID NO.2 shown in Figure 1 b.
In a preferred embodiment, said SlitOR6 polypeptide has the amino acid sequence defined in SEQ ID NO.2, shown in Figure 1 b. The invention further provides for a nucleic acid molecule, encoding the SlitOR6 polypeptide according to the present invention. The invention further provides for a SlitOR6 nucleic acid molecule having more than 60% homology with the DNA sequence defined in SEQ ID N0.1 shown in Figure 1 a.
In a preferred embodiment, the invention provides for a SlitOR6 nucleic acid molecule which has the DNA sequence defined in SEQ ID N0.1 shown in Figure 1 a.
The invention further provides for a vector, comprising the SlitOR6 nucleic acid molecule according to the present invention. In addition, the invention provides a cell comprising the vector according to the present invention.
The invention further provides a method for determining whether a ligand can specifically bind to the SlitOR6 polypeptide as defined herein, said method comprising the steps of, contacting a cell or cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said SlitOR6 polypeptide with the ligand under conditions permitting binding of said ligand to said SlitOR6 polypeptide, and detecting the presence of any such ligand bound specifically to said SlitOR6 polypeptide, thereby determining whether the ligand binds specifically to said SlitOR6 polypeptide.
The invention further provides for a method for determining whether a ligand can modulate the function of the SlitOR6 polypeptide as defined herein, said method comprising the steps of, contacting a cell or cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said SlitOR6 polypeptide with the ligand under conditions permitting activation of said SlitOR6 polypeptide, and measuring a signalling activity of said SlitOR6 polypeptide.
In a preferred embodiment, said ligand is possibly an agonist or an antagonist of said SlitOR6 polypeptide.
In a further embodiment, the signalling activity is detected by means of a bio-assay, is a measurement of a second messenger selected from the group consisting of measurement of intracellular cAMP concentration, measurement of intracellular Inositol phosphate concentration, measurement of intracellular diacylglycerol concentration, measurement of intracellular calcium mobilisation or measurement of an increase in the SlitOR6 activity. The invention further provides an antibody capable of binding to a SlitOR6 polypeptide which has more than 80% homology with an amino-acid sequence defined in SEQ ID NO.2 shown in Figure 1 b. The present invention relates to the identification of Z9,E12-14:Ac as a natural ligand of SlitOR6 (olfactory/pheromonal receptor). The invention encompasses the use of the interaction of SlitOR6 polypeptides and Z9,E12-14:Ac as the basis of screening assays for agents that modulate the activity of the SlitOR6 receptor polypeptide. The invention also encompasses monitoring, survey and other methods based upon the SlitOR6/Z9,E12-14:Ac interaction, as well as kits for screening methods. In particular, the present invention indicates that Z9,E12-14:Ac has a link towards mate attraction in Spodoptera littoralis.
The invention encompasses methods of identifying an agent that modulates the function of a SlitOR6 polypeptide, among such methods : a) measuring the change of electrical potential on a cell in which SlitOR6 is naturally expressed, contacted with Z9,E12- 14:Ac (for instance by electrophysiological measurement); b) a cell transfected or modified to express a SlitOR06 polypeptide and then contacting such a cell with Z9,E12-14:Ac; c) contacting a SlitOR6 polypeptide with Z9,E12-14:Ac in the presence and in the absence of a candidate modulator under conditions permitting the binding of said Z9,E12-14:Ac to said SlitOR6 polypeptide; and d) measuring the binding of said SlitOR6 polypeptide to said Z9,E12-14:Ac, wherein a decrease in binding in the presence of said candidate modulator, relative to the binding in the absence of the candidate modulator, identifies the candidate modulator as an agent that modulates the function of said SlitOR6 polypeptide. The invention further encompasses a method of identifying an agent that modulates the function of a SlitOR6 polypeptide, said method comprising: a) contacting a SlitOR6 polypeptide with Z9,E12-14:Ac in the presence and in the absence of a candidate modulator, under conditions permitting activation of said SlitOR6 polypeptide by Z9,E12-14:Ac; and b) measuring a signalling activity of said SlitOR6 polypeptide, wherein a change in the activity in the presence of said candidate modulator relative to the activity in the absence of said candidate modulator identifies said candidate modulator as an agent that modulates the function of said SlitOR6 polypeptide.
The invention further encompasses a method of identifying an agent that modulates the function of a SlitOR6 polypeptide, said method comprising: a) contacting a SlitOR6 polypeptide with a candidate modulator; b) measuring a signalling activity of said SlitOR6 polypeptide in the presence of said candidate modulator; and c) comparing the activity measured in the presence of said candidate modulator to said activity measured in a sample in which said SlitOR6 polypeptide is contacted with Z9,E12-14:Ac at its EC50, wherein said candidate modulator is identified as an agent that modulates the function of SlitOR6 when the amount of the activity measured in the presence of the candidate modulator is at least 10% of the amount induced by said Z9,E12-14:Ac present at its EC50.
The invention further encompasses a method of detecting in a sample the presence of an agent that modulates the function of a SlitOR6 polypeptide, said method comprising a) contacting a SlitOR6 polypeptide with Z9,E12-14:Ac in the presence and in the absence of said sample under conditions permitting the binding of said Z9,E12-14:Ac to said SlitOR6 polypeptide; and b) measuring the binding of said SlitOR6 polypeptide to said Z9,E12-14:Ac, wherein an increase or a decrease in binding in the presence of the sample, relative to the binding in the absence of said sample, indicates the presence of an agent that modulates the function of said SlitOR6 polypeptide in said sample.
The invention further encompasses a method for detecting in a sample the presence of an agent that modulates the function of a SlitOR6 polypeptide, said method comprising: a) contacting a SlitOR6 polypeptide with Z9,E12-14:Ac in the presence and in the absence of said sample; b) measuring a signalling activity of said SlitOR6 polypeptide; and c) comparing the amount of said activity measured in a reaction containing a SlitOR6 polypeptide and Z9,E12-14:Ac without said sample to the amount of said activity measured in a reaction containing SlitOR6, Z9,E12-14:Ac and said sample, wherein a change in said activity in the presence of said sample relative to the activity in the absence of said sample indicates the presence of an agent that modulates the function of a SlitOR6 polypeptide in said sample. The invention further encompasses a method of detecting in a sample the presence of an agent that modulates the function of a SlitOR6 polypeptide, said method comprising: a) contacting a SlitOR6 polypeptide with said sample; b) measuring a signalling activity of said SlitOR6 polypeptide in the presence of said sample; and c) comparing said activity measured in the presence of said sample to said activity measured in a reaction in which said SlitOR6 polypeptide is contacted with Z9,E12-14:Ac present at its EC50, wherein an agent that modulates the function of said SlitOR6 polypeptide is detected if the amount of the activity measured in the presence of said sample is at least 10% of the amount induced by Z9,E12- 14:Ac present at its EC5o- According to the present invention, when using binding methods Z9,E12-14:Ac may be detectably labelled. In said methods, Z9,E12-14:Ac may be detectably labelled with a moiety selected from the group consisting of -but not limited to- a radioisotope, a fluorophore, and a quencher of fluorescence. Alternatively, Z9,E12-14:Ac may be detectably labelled with a NMR-detectable moiety.
In one embodiment of any of the preceding methods, the contacting is performed in or on a cell expressing said SlitOR6 polypeptide. According to the present invention, said cell may be, but is not limited to, Human embryonic kidney cells (Hek293) and derived strains, Human cervical cancer cells (HeLa) and derived strains, Chinese hamster cells (CHO), Monkey cells (COS), primary olfactory cells, Xenopus eggs, insect cells (Sf9, S2, HiFive), yeast or bacteria.
In another embodiment of any of the preceding methods, the contacting is performed in or on synthetic liposomes (see Tajib et al., 2000, Nature Biotechnology 18: 649 - 654, which is incorporated herein by reference) or virus-induced budding membranes containing a SlitOR6 polypeptide (see WO0102551 , 2001 , incorporated herein by reference).
In another embodiment of any of the preceding methods, the method is performed using a membrane fraction from cells expressing said SlitOR6 polypeptide.
In a preferred embodiment of either of the preceding methods, the method is performed on a protein chip. In another preferred embodiment of either of the preceding methods, the measuring is performed using a method selected from label displacement, surface plasmon resonance, fluorescence resonance energy transfer, fluorescence quenching, and fluorescence polarization.
In another embodiment of either of the preceding methods, the agent is selected from the group consisting of a peptide, a polypeptide, an antibody or antigen-binding fragment thereof, a lipid, a carbohydrate, a nucleic acid, and a small organic molecule.
According to the present invention, when a functional assay is used, the step of measuring a signalling activity of the SlitOR6 polypeptide may comprise detecting a change in the level of a second messenger. In another embodiment, the step of measuring a signalling activity comprises measurement of guanine nucleotide binding/coupling or exchange, adenylate cyclase activity, cAMP, Protein Kinase C activity, Protein Kinase A activity phosphatidylinosotol breakdown, diacylglycerol, inositol triphosphate, intracellular calcium, calcium flux, arachinoid acid, MAP kinase activity, tyrosine kinase activity, melanophore assay, receptor initialization assay, or reporter gene expression. When the G-protein binding/coupling or exchange is measured, of all Goc subunits possible preferably the behaviour of GTP-binding protein G protein alpha-q subunit (olfactory), also G-q, is studied.
In a preferred embodiment, the measuring of the signalling activity comprises using a fluorescence or luminescence assay. Fluorescence and luminescence assays may comprise the use of Ca2+ sensitive fluorophores including Fluo-3, Fluo-4 or Fura, (Molecular probes); Ca3-kit family (Molecular Device) and aequorin. Furthermore, said assays may apply an automated fluorometric or luminescent reader such as FDSS (Hammamatsu) or FLIPR (Molecular Device).
The invention further encompasses a method of modulating the activity of a SlitOR6 polypeptide in a cell, said method comprising the step of delivering to said cell Z9,E12-14:Ac that modulates the activity of a SlitOR6 polypeptide, such that the activity of SlitOR6 is modulated.
In another embodiment, the step of measuring activation of the receptor comprises electrophysiological measurements (known as electroantennography or single sensillum recording; methods well known of persons skilled in the art) performed on wild-type or genetically engineered individuals, expressing a SlitOR06 polypeptide or one related proteins (displaying homology with SlitOR06 of at least 20%).
In another embodiment of any of the preceding methods, the method is a high throughput screening method.
In another embodiment of any of the preceding methods, the agent is part of a chemical library or animal organ extracts. Said animal organ extracts may be, but are not limited to, extracts prepared from male or female bodies, or body parts (e.g. extracted glands, metasoma section, etc.).
In addition, the present invention encompasses the use of Z9,E12-14:Ac as ligand for a SlitOR6 polypeptide. The invention further encompasses a Z9,E12-14:Ac-related agent identified or detected by any of the preceding methods. Said Z9,E12-14:Ac-related agent may be an agonist, an antagonist, or an inverse agonist for a SlitOR6 polypeptide.
The invention also encompasses a composition comprising a Z9,E12-14:Ac-related agent identified or detected by any of the preceding methods.
Alternatively, these may be used for the preparation of attractants or attractant antagonists. For instance a SlitOR6 agonist may be used as attractant, a SlitOR6 antagonist as an olfactory blocker.
In another preferred embodiment, said agent or composition may be used for the preparation of a pest control solution implying a SlitOR6-related behaviour; wherein said behaviour is preferentially related to the mate recognition system (SMRS).
The present invention also encompasses a composition comprising an isolated SlitOR6 polypeptide and Z9,E12-14:Ac.
The present invention also relates to the use of Z9,E12-14:Ac for the production of a composition comprising an isolated SlitOR6 polypeptide and said Z9,E12-14:Ac.
The present invention further relates to the use of Z9,E12-14:Ac for the production of a) a kit for screening agents that modulate the signaling of a SlitOR6 polypeptide, b) for the production of a kit to screen odorants or odorant antagonists; or c) for the production of a kit for the monitoring of pest infestation in cropping systems. The present invention further encompasses the use of Z9,E12-14:Ac for the preparation of a pest control solution. According to the present invention, said solution may be used for the control of a pest displaying a SlitOR6-related behaviour; wherein said pest is preferentially chosen from the group consisting of Spodoptera littoralis congeneric species. In particular, said pest control solution may be used for the treatment of infestation by those species in which Z9,E12-14:Ac is part of the pheromonal bouquets.
The present invention also relates to an antibody recognizing the Z9,E12-14:Ac/ SlitOR6 complex or fragments thereof.
Another embodiment of the invention relates to the use of SlitOR6 polypeptide fragments or antibodies recognizing a SlitOR6 polypeptide or antibodies recognizing the Z9,E12-14:Ac/SlitOR6 complex,, for the preparation of a pest control solution. Said pest may be Spodoptera littoralis (or a congeneric species).
The invention further encompasses a method of pest monitoring or survey using SlitOR6 polypeptide signalling, said method comprising a trapping device using Z9,E12-14:Ac as an attracting lure. The invention further encompasses a method of diagnosing a pest infestation by using traps making use of the attraction mediated by a SlitOR6 polypeptide.
The invention further encompasses a kit comprising a) an isolated SlitOR6 polypeptide, Z9,E12-14:Ac and packaging materials therefore; b) an isolated polynucleotide encoding a SlitOR6 polypeptide, Z9,E12-14:Ac, and packaging materials therefore; c) a kit comprising a cell expressing a SlitOR6 polypeptide or membranes thereof, Z9,E12-14:Ac and packaging materials therefore. Said cells may be transformed with a polynucleotide encoding said SlitOR6 polypeptide.
According to the present invention, the above-mentioned kits may be used for several purposes. For instance, said kit may be used for screening agents that modulate the signalling of a SlitOR6 polypeptide, for screening attractant, repellent, antagonist or blocking agents. Similar kits can be developed and used for monitoring/surveying the populations of Spodoptera littoralis (and its conspecific species) and the infestations thereof.
The present invention also encompasses the use of a transgenic animal (over)expressing a SlitOR6 polypeptide or an ortholog thereof to study the effect of Z9,E12- 14:Ac on mate recognition and/or attraction behavior. The pest for which those behaviors are studied, may be Spodoptera littoralis and its congeneric species. With the term ortholog is meant a gene with similar function to SlitOR6 in an evolutionary related species. With the term congeneric is meant a species of which phylogenetic affinities with one or some species in the genus Spodoptera Guenee can be demonstrated by a person skilled in the art. The present invention also encompasses the use of a transgenic animal
(over)expressing a SlitOR6 polypeptide or an ortholog thereof to a) disrupt mating; b) to induce sterility of subsequent generations. The pest for which populations are surveyed, may be Spodoptera littoralis and its congeneric species.
Alternatively, an SlitOR6 knock-out transgenic animal may be used to study the effect of SlitOR6 in the detection of Z9,E12-14:Ac sources. In addition, a SlitOR6 knock-out transgenic animal may be used to study the effect of SlitOR6 on the behaviors induced in Spodoptera littoralis and congeneric species by Z9,E12-14:Ac. In the present description, SlitOR6 may be also understood as ortholog of SlitOR6 according to the animal model chosen.
In all the above mentioned methods, agents, uses, compositions or kits of the present invention an equivalent of Z9,E12-14:Ac may be used, referred at, applied, or incorporated which binds specifically to and activates a signaling activity of a SlitOR6 polypeptide represented in Figure 1 b.
In addition, in all the above mentioned methods, uses, compositions or kits of the present invention, the SlitOR6 polypeptide may have at least 20% identity or higher identity, such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or even 100% to the polypeptide represented in Figure 1 b but with comparable activity to said SlitOR6 polypeptide. A skilled person knows how to evaluate said activity depending on the assay used.
In addition, in all the above mentioned methods, uses, compositions or kits of the present invention, the SlitOR6 polypeptide may be a chimera or an active fragment thereof.
As used herein, the term "SlitOR6 polypeptide" refers to a polypeptide having two essential properties: 1 ) a SlitOR6 polypeptide has at least 20% amino acid identity, and preferably 60%, 80%, 90%, 95% or higher, including 100% amino acid identity, to the sequence represented in Figure 1 b; and 2) a SlitOR6 polypeptide has SlitOR6 activity, i.e., the polypeptide binds Z9,E12-14:Ac. Said homology may relate to the whole polypeptide or only part of the polypeptide such as CDR domain (ligand-binding domain of the receptor^ Optimally, a "SlitOR6 polypeptide" also has SlitOR6 signalling activity as defined herein. However, identification of CDR of 7TM receptors is very hazardous and depends of the algorithm applied to define TM.
As used herein, the term "SlitOR6 polynucleotide" refers to a polynucleotide that encodes a SlitOR6 polypeptide as defined herein. As used herein, the term "SlitOR6 activity" refers to specific binding of Z9,E12-14:Ac or an equivalent thereof by a SlitOR6 polypeptide.
As used herein, the term "SlitOR6 signalling activity" refers to the initiation or propagation of signalling by a SlitOR6 polypeptide. SlitOR6 signalling activity is monitored by measuring a detectable step in a signalling cascade by assaying one or more of the following: stimulation of GDP for GTP exchange on a G protein; alteration of adenylate cyclase activity; protein kinase C modulation; protein kinase A modulation; phosphatidylinositol breakdown (generating second messengers diacylglycerol, and inositol triphosphate); intracellular calcium flux; activation of MAP kinases; modulation of tyrosine kinases; internalization assay; modulation of gene or reporter gene activity; or melanophore assay. A detectable step in a signalling cascade is considered initiated or mediated if the measurable activity is altered by 10% or more above or below a baseline established in the substantial absence of Z9,E12- 14:Ac relative to any of the SlitOR6 activity assays described herein below. The measurable activity can be measured directly, as in, for example, measurement of cAMP or diacylglycerol levels. Alternatively, the measurable activity can be measured indirectly, as in, for example, a reporter gene assay. For most of these assays kits are available in the market. As used herein, the term "detectable step" refers to a step that can be measured, either directly, e.g., by measurement of a second messenger (usually an increase in the concentration of the concentration of the intracellular Ca++) or detection of a modified (e.g., phosphorylated) protein, or indirectly, e.g., by monitoring a downstream effect of that step. For example, adenylate cyclase activation results in the generation of cAMP. The increase in the concentration of the concentration of the intracellular Ca++ can be measured directly, e.g., by an assay that uses fluorescent probes such as Fluo4 (e.g.).
As used herein, the term "isolated" refers to a population of molecules (e.g., polypeptides or polynucleotides) the composition of which is less than 50% (by weight), preferably less than 40% and most preferably 2% or less, in contaminating molecules of an unlike nature. When the term "isolated" is applied to a SlitOR6 polypeptide, it is specifically meant to encompass a SlitOR6 polypeptide that is associated with or embedded in a lipid membrane.
As used herein, the term Z9,E12-14:Ac refers to a chemical molecule known by a skilled chemist. As used herein, the terms "candidate compound" and "candidate modulator" refer to a composition being evaluated for the ability to modulate ligand binding to a SlitOR6 polypeptide or the ability to modulate an activity of SlitOR6 polypeptide. Candidate modulators can be natural or synthetic compounds, including, for example, small molecules, compounds contained in extracts of animal, plant, bacterial or fungal cells, as well as conditioned medium from such cells.
As used herein, the term "small molecule" refers to a compound having molecular mass of less than 3000 Daltons, preferably less than 2000 or 1500, still more preferably less than 1000, and most preferably less than 600 Daltons. A "small organic molecule" is a small molecule that comprises carbon.
As used herein, the term "change in binding" or "change in activity" and the equivalent terms "difference in binding" or "difference in activity" refer to an at least 10% increase or decrease in binding, or signalling activity in a given assay.
As used herein, the term "conditions permitting the binding of Z9,E12-14:Ac to SlitOR6" refers to conditions of, for example, temperature, salt concentration, pH and protein concentration under which Z9,E12-14:Ac binds SlitOR6. Exact binding conditions will vary depending upon the nature of the assay, for example, whether the assay uses viable cells or only membrane fraction of cells. However, because SlitOR6 is a cell surface protein, and because Z9,E12-14:Ac normally interacts with SlitOR6 on the cell surface, favoured conditions will generally include physiological salt (90 mM) and pH (about 7.0 to 8.0). Temperatures for binding can vary from 4°C to 37°C, but will preferably be between room temperature and about 37°C. The concentration of Z9,E12-14:Ac and SlitOR6 polypeptide in a binding reaction will also vary, but will preferably be about 10 μΜ (e.g., in a reaction with radiolabeled tracer Z9,E12-14:Ac, where the concentration is generally below the Kd) to 1 mM (e.g., Z9,E12- 14:Ac as competitor).
As used herein, the term "sample" refers to the source of molecules being tested for the presence of an agent that modulates binding to or signalling activity of a SlitOR6 polypeptide. A sample can be an environmental sample, a natural extract of animal, plant yeast or bacterial cells or tissues, a synthetic sample, or a conditioned medium from recombinant cells or a fermentation process. A "tissue" is an aggregate of cells that perform a particular function in an organism. The term "tissue" as used herein refers to cellular material from a particular physiological region. The cells in a particular tissue can comprise several different cell types. A non-limiting example of this would be brain tissue that further comprises neurons and glial cells, as well as capillary endothelial cells and blood cells, all contained in a given tissue section or sample. In addition to solid tissues, the term "tissue" is also intended to encompass non-solid tissues, such as blood.
As used herein, the term "membrane fraction" refers to a preparation of cellular lipid membranes comprising a SlitOR6 polypeptide. As the term is used herein, a "membrane fraction" is distinct from a cellular homogenate, in that at least a portion (i.e., at least 10%, and preferably more) of non-membrane-associated cellular constituents has been removed. The term "membrane associated" refers to those cellular constituents that are either integrated into a lipid membrane or are physically associated with a component that is integrated into a lipid membrane.
As used herein, the term "decrease in binding" refers to a decrease of at least 10% in the binding of Z9,E12-14:Ac or other agonist to a SlitOR6 polypeptide as measured in a binding assay as described herein.
As used herein, the term "second messenger" refers to a molecule, generated or caused to vary in concentration by the activation of a GPCR-like receptor that participates in the transduction of a signal from that receptor. Non-limiting examples of second messengers include cAMP, diacylglycerol, inositol triphosphates and intracellular calcium. The term "change in the level of a second messenger" refers to an increase or decrease of at least 10% in the detected level of a given second messenger relative to the amount detected in an assay performed in the absence of a candidate modulator.
As used herein, the term "aequorin-based assay" refers to an assay for GPCR (GPCR- like receptor) activity that measures intracellular calcium flux induced by activated receptors, wherein intracellular calcium flux is measured by the luminescence of aequorin expressed in the cell. Such a method is particularly valuable to quantify the Ca++ flux in cells expressing ion channel (like the OR - ORco heterodimers of insect olfaction).
As used herein, the term "binding" refers to the physical association of a ligand (e.g., Z9,E12-14:Ac) with a receptor (e.g., SlitOR6). As the term is used herein, binding is "specific" if it occurs with an EC50 or a Kd of 100 nM or less, generally in the range of 100 μΜ to 1000 μΜ. Using odorant functional assay Ec50 found are usually around 10, 100 and even 1 mM, said signals may be considered specific (see Figure 3). Odorant binding may be considered specific if the EC50 or Kd is comprised between 1000μΜ and 1 nM or less.
As used herein, the term "EC50," refers to that concentration of an agent at which a given activity, including binding of Z9,E12-14:Ac or other ligand and a functional activity of a SlitOR6 polypeptide, is 50% of the maximum for that SlitOR6 activity measurable using the same assay. Stated differently, the "EC50" is the concentration of agent that gives 50% activation, when 100% activation is set at the amount of activity that does not increase with the addition of more agonist. It should be noted that the "EC50 of an Z9,E12-14:Ac equivalent" will vary with the identity of acid; for example, equivalent Z9,E12-14:Ac molecules can have EC50 values higher than, lower than or the same as Z9,E12-14:Ac. Therefore, where an equivalent of Z9,E12-14:Ac is used, one of the skill in the art can determine the EC50 for that equivalent according to conventional methods. The EC50 of a given Z9,E12-14:Ac equivalent is measured by performing an assay for an activity of a fixed amount of SlitOR6 polypeptide in the presence of doses of the Z9,E12-14:Ac equivalent that increase at least until the SlitOR6 response is saturated or maximal, and then plotting the measured SlitOR6 activity versus the concentration of the acid used. As used herein, the term "Kd" is a dissociation constant or the ligand concentration at which half of the receptors are bound by the ligand at equilibrium.
As used herein, the term "IC50" is the concentration of an antagonist or inverse agonist that reduces the maximal activation of a SlitOR6 receptor by 50%.
As used herein, the term "detectably labelled" refers to the property of a molecule, e.g., Z9,E12-14:Ac or an equivalent therefrom, that has a structural modification. Said modification is introduced through the incorporation or addition of a functional group. Said functional group
(label) can be readily detected. Detectable labels include but are not limited to fluorescent compounds, isotopic compounds, chemiluminescent compounds, quantum dot labels.
Examples of radioisotopes which can be added to the structure of Z9,E12-14:Ac, or equivalents, may be 14C, or 3H (as well as eventually also 125l, 35S). Examples of radioisotopes which can be incorporated to the structure of Z9,E12-14:Ac, or equivalents, may be 3H or 14C.
The various means of detection include but are not limited to spectroscopic, photochemical, radiochemical, biochemical, immunochemical, or chemical means.
As used herein, the term "affinity tag" refers to a label, attached to a molecule of interest (e.g., a SlitOR6 polypeptide), that confers upon the labelled molecule the ability to be specifically bound by a reagent that binds the label. Affinity tags include, but are not limited to an epitope for an antibody (known as "epitope tags"), biotin, 6X His, Myc, FLAG and GST. Affinity tags can be used for the detection, as well as for the purification of the labelled species. As used herein, the term "decrease in binding" refers to a decrease of at least 10% in the amount of binding detected in a given assay with a known or suspected modulator of SlitOR6 relative to binding detected in an assay lacking that known or suspected modulator.
As used herein, the term "delivering," when used in reference to an agent, means the addition of the agent to an assay mixture, or to a cell in culture. The term also refers to the administration of the agent to an animal. Such administration can be, for example, by injection (in a suitable carrier, e.g., sterile saline or water) or by puffing or other relevant route of drug administration. As used herein, the term "agent that modulates the function of a SlitOR6 polypeptide" is a molecule or compound that increases or decreases SlitOR6 activity, including compounds that change the binding of Z9,E12-14:Ac or equivalents thereof, and change SlitOR6 downstream signalling activities. As used herein, the term "transgenic animal" refers to any animal, usually an insect
(Drosophila e.g.), in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extra-chromosomally replicating DNA. In the typical transgenic animals described herein, the transgene causes cells to express a recombinant form of one of the subject polypeptide, e.g. either agonistic or antagonistic forms. However, transgenic animals in which the recombinant gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below. Moreover, "transgenic animal" also includes those recombinant animals in which gene disruption of one or more genes is caused by human intervention, including both recombination and antisense techniques. As used herein, the term "antibody" is the conventional immunoglobulin molecule, as well as fragments thereof which are also specifically reactive with one of the subject polypeptides. It can alternatively encompass a heavy chain antibody (a.k.a. Camelid antibody). Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described herein below for whole antibodies. For example, F(ab)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. The antibody of the present invention is further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for a polypeptide conferred by at least one CDR region of the antibody. In preferred embodiments, the antibodies, the antibody further comprises a label attached thereto and able to be detected, (e.g., the label can be a radioisotope, fluorescent compound, chemiluminescent compound, enzyme, or enzyme co- factor).
As used herein, the term "blocking agent" refers to any agent that stops or inhibits olfactory perception by the target. Said perception may be assayed using animal studies, ex vivo or in vitro studies simulating or representative for in vivo olfactory perception. With the expression "method of monitoring" is meant a method by which an infestation or a change in a population dynamic may be assessed, in said situation there exists already measurable indications or signs for the presence and/or development of said infestation or change in the population dynamic.
With "Z9,E12-14:Ac equivalent" is meant a molecule which has the same or comparable effect on the SlitOR6 polypeptide receptor compared to Z9,E12-14:Ac. The description provides enough information, in combination with the common general knowledge, so that a skilled person may determine if a compound may be considered as an Z9,E12-14:Ac equivalent or not. It is noteworthy that different radicals containing different chemical function including but not limited to alcohol and acid could be grafted to the general formula described above.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. a) DNA sequence of the gene coding for the described receptor; b) Amino Acid translation of the above sequence.
Figure 2. a) Expression of SlitOR6 in male antennae shown in RT-PCR; b) Expression of SlitOR6 in male antennae pictured by in situ hybridization.
Figure 3. a) EAG dose response curve, after stimulation of Drosophila antennae expressing a SlitOR6 transgene with increasing concentrations of Z9,E12-14:Ac. The diamond series shows response of the neurones expressing conjointly ORco-Gal4 and UAS- SlitOR6. The rectangle series shows response of the neurones expressing UAS-SlitOR6. The triangle series shows response of the neurones expressing OR83b-Gal4; b) chart picturing the screening of 7 molecules close to Z9,E12-14:Ac on Drosophila antennae expressing a SlitOR6 transgene. Heptanone is a positive control stimulus and hexane is the solvent for the tested molecules.
Figure 4 a) Microscopic observation of cells transfected with the cloned receptor, loaded with a calcium probe (Fluo-4 AM) and stimulated with Z9, E12-14:Ac. b) Fluorescence intensity graphs of the respective transfected cells, each series represents the evolution (through time) of the fluorescence intensity of one cell in the surveyed microscopic field.
DETAILED DESCRIPTION OF THE INVENTION The invention relates to the identification of a new insect GPCR-like receptor, SlitOR6, and to the fact that Z9,E12-14:Ac is a natural ligand for said SlitOR6 GPCR-like receptor. The interaction is useful for screening assays for agents that modulate the interaction and thus the function of SlitOR6. The known ligand and its interaction with the receptor also provides opportunity for developing attracting lure based on single molecules or blends of at least 2 compounds.
I. Assays For The Identification Of Agents That Modulate The Activity Of SlitOR6
Agents that modulate the activity of SlitOR6 can be identified in a number of ways that take advantage of the interaction of said receptor with Z9,E12-14:Ac. For example, the ability to reconstitute SlitOR6/Z9,E12-14:Ac binding either in vitro, on cultured cells or in vivo provides a target for identification of agents that disrupt that binding. Assays based on disruption of binding can identify agents, such as small organic molecules, from libraries or collections of such molecules. Alternatively, such assays can identify agents in samples or extracts from natural sources, including plant, fungal or bacterial extracts or even human tissue samples. Modulators of SlitOR6/Z9,E12-14:Ac binding can then be screened using a binding assay or a functional assay that measures downstream signaling through the said receptor. Both binding assays and functional assays are validated using Z9,E12-14:Ac.
Another approach that uses the SlitOR6/Z9,E12-14:Ac interaction more directly to identify agents that modulate SlitOR6 function measures changes in SlitOR6 downstream signaling induced by candidate agents or candidate modulators. These functional assays can be performed in isolated cell membrane fractions or on cells expressing the receptor on their surfaces.
The following description provides methods for both binding and functional assays based upon the interaction of SlitOR6 and Z9,E12-14:Ac.
A. SlitOR6 nucleotides/polypeptides.
Assays using the interaction of SlitOR6 and Z9,E12-14:Ac require a source of SlitOR6 polypeptide. The polynucleotide and polypeptide sequence of Spodoptera littoralis SlitOR6 are presented herein in Figure 1 . One skilled in the art can readily amplify a SlitOR6 sequence from a sample containing mRNA encoding the protein through basic PCR and molecular cloning techniques using primers or probes designed from the known sequences.
The expression of recombinant polypeptides is well known in the art. Those skilled in the art can readily select vectors and expression control sequences for the expression of SlitOR6 polypeptides useful according to the invention in eukaryotic or prokaryotic cells. SlitOR6 must be associated with cell membrane or detergents like synthetic liposomes in order to have binding or signalling function. Methods for the preparation of cellular membrane fractions are well known in the art, e.g., the method reported by Hubbard and Cohn, 1975, J. Cell Biol. 64: 461 -479, which is incorporated herein by reference. In order to produce membranes comprising SlitOR6, one need only to apply such techniques to cells endogenously or recombinantly expressing SlitOR6. Alternatively, membrane-free SlitOR6 can be integrated into membrane preparations by dilution of detergent solution of the polypeptide (see, e.g., Salamon et al., 1996, Biophys. J. 71 :283-294, which is incorporated herein by reference).
B. Z9,E12-14:Ac.
The structure of Z9,E12-14:Ac is well known by a skilled person. In addition, the skilled in the art may easily derive equivalent structures and may easily test if said equivalents are able to bind and/or modulate the SlitOR6 receptor. Z9,E12-14:Ac and equivalents thereof may be isolated from natural samples, or chemically synthesized.
Methods which can be used to quantify said Z9,E12-14:Ac may be, but are not limited to, a) for extraction and purification: solvent extraction, oil extraction, vapour extraction, C02 supercritical extraction, liquid chromatography, distillation, gas chromatography; b) for quantifying: gas chromatography, liquid chromatography, etc. coupled to mass spectrometry (e.g.). A skilled person knows how to perform said methods.
Z9,E12-14:Ac or its equivalents may be used in purified form or used as composition. The amounts of the acetate necessary in a given binding or functional assay according to the invention will vary depending upon the assay, but will generally use 1 nM to 10 μΜ of labelled and 1 nM to 10mM of unlabelled acetate per assay. The affinities and EC50 of modified Z9,E12-14:Ac molecules for SlitOR6 may vary relative to those of the original Z9,E12-14:Ac, and the amount necessary for a given assay can therefore be adjusted relative to the normal values. If necessary for a given assay, Z9,E12-14:Ac can be labelled by incorporation or addition of radiolabeled labels as pointed above.
C. Assays to Identify Modulators of SlitOR6 Activity
The discovery that Z9,E12-14:Ac is a ligand of the SlitOR6 receptor permits screening assays to identify agonists, antagonists and inverse agonists of receptor activity. The screening assays will have two general approaches.
1 ) Ligand binding assays, in which cells expressing SlitOR6, membrane extracts from such cells, or immobilized lipid membranes comprising SlitOR6 are exposed to a labelled Z9,E12-14:Ac and a candidate compound. Following incubation, the reaction mixture is measured for specific binding of the labelled Z9,E12-14:Ac to the SlitOR6 receptor. Compounds that interfere with or displace labelled Z9,E12-14:Ac can be agonists, antagonists or inverse agonists of SlitOR6 activity. Functional analysis can be performed on positive compounds to determine which of these categories they fit.
Binding of a compound may be classified in 3 main categories: competitive binding, non-competitive binding and uncompetitive binding. A competitive binding compound resembles a second (reference) compound and binds to the same binding pocket of a target molecule (here receptor). Upon addition, the competitive binding compound displaces said second compound from said target. A non-competitive binding compound does not bind to the same binding pocket of the target molecule as a second (reference) compound but may interact with the effect of said second compound on said target molecule. The second compound is not displaced upon addition of the non-competitive binding compound. An uncompetitive-binding compound binds to the target molecule when a second compound is already bound. Cooperative binding means that a compound facilitates the binding of another compound which may be a reference compound. The cooperative effect is thus seen in the analysis of the Kd of said other compound.
2) Functional assays, in which a signalling activity of SlitOR6 is measured. a) For agonist screening, cells expressing SlitOR6 or membranes prepared from them are incubated with candidate compound, and a signalling activity of SlitOR6 is measured. The assays are validated using Z9,E12-14:Ac as agonist, and the activity induced by compounds that modulate receptor activity is compared to that induced by Z9,E12-14:Ac. An agonist or partial agonist will have a maximal biological activity corresponding to at least 10% of the maximal activity of Z9,E12-14:Ac when the agonist or partial agonist is present at 100(0) μΜ or less, and preferably will have 50%, 75%, 100% or more, including 2-fold, 5-fold, 10-fold or more activity than Z9,E12-14:Ac. b) For antagonist or inverse agonist screening, cells expressing SlitOR6 or membranes isolated from them are assayed for signalling activity in the presence of Z9,E12- 14:Ac with or without a candidate compound. Antagonists or inverse agonists will reduce the level of Z9,E12-14:Ac-stimulated receptor activity by at least 10%, relative to reactions lacking the antagonist or inverse agonist. c) For inverse agonist screening, cells expressing constitutive SlitOR6 activity or membranes isolated from them are used in a functional assay that measures an activity of the receptor in the presence and in the absence of a candidate compound. Inverse agonists are those compounds that reduce the constitutive activity of the receptor by at least 10%. Overexpression of SlitOR6 may lead to constitutive activation. SlitOR6 can be overexpressed by placing it under the control of a strong constitutive promoter, e.g., the CMV early promoter.
3) Ligand binding and displacement assays. One can use SlitOR6 polypeptides expressed on a cell, or isolated membranes containing receptor polypeptides, along with Z9,E12-14:Ac in order to screen for compounds that inhibit the binding of Z9,E12-14:Ac to SlitOR6. When identified in an assay that measures binding or Z9,E12-14:Ac displacement alone, compounds will have to be subjected to functional testing to determine whether they act as agonists, antagonists or inverse agonists. For displacement experiments, cells expressing a SlitOR6 polypeptide (generally
25,000 cells per assay or 1 to 100 μg of membrane extracts) are incubated in binding buffer (e.g., 50 mM Hepes pH 7.4; 1 mM CaCI2; 0.5% Bovine Serum Albumin (BSA) Fatty Acid-Free; and 0,5 mM MgCI 2) for 1 .5 hrs (at, for example, 27°C) with labelled Z9,E12-14:Ac in the presence or in the absence of increasing concentrations of a candidate modulator. To validate and calibrate the assay, control competition reactions using increasing concentrations of unlabelled Z9,E12-14:Ac can be performed. After incubation, cells are washed extensively, and bound, labelled Z9,E12-14:Ac is measured as appropriate for the given label (e.g., scintillation counting, enzyme assay, fluorescence, etc.). A decrease of at least 10% in the amount of labelled Z9,E12-14:Ac bound in the presence of candidate modulator indicates displacement of binding by the candidate modulator. Candidate modulators are considered to bind specifically in this or other assays described herein if they displace 50% of labelled Z9,E12-14:Ac (sub-saturating Z9,E12-14:Ac dose) at a concentration of 100 μΜ or less (i.e., ECso is 100 μΜ or less). Alternatively, binding or displacement of binding can be monitored by surface plasmon resonance (SPR). Surface plasmon resonance assays can be used as a quantitative method to measure binding between two molecules by the change in mass near an immobilized sensor caused by the binding or loss of binding of Z9,E12-14:Ac from the aqueous phase to a SlitOR6 polypeptide immobilized in a membrane on the sensor. This change in mass is measured as resonance units versus time after injection or removal of Z9,E12-14:Ac or candidate modulator and is measured using a Biacore Biosensor (Biacore AB). SlitOR6 can be immobilized on a sensor chip (for example, research grade CM5 chip; Biacore AB) in a thin film lipid membrane according to methods described by Salamon et al. (Salamon et al., 1996, Biophys J. 71 : 283-294; Salamon et al., 2001 , Biophys. J. 80: 1557-1567; Salamon et al., 1999, Trends Biochem. Sci. 24: 213-219, each of which is incorporated herein by reference.). Sarrio et al. demonstrated that SPR can be used to detect ligand binding to the GPCR A(1 ) adenosine receptor immobilized in a lipid layer on the chip (Sarrio et al., 2000, Mol. Cell. Biol. 20: 5164-5174, incorporated herein by reference). Conditions for Z9,E12-14:Ac binding to SlitOR6 in an SPR assay can be fine-tuned by one of skill in the art using the conditions reported by Sarrio et al. as a starting point.
SPR can assay for modulators of binding in at least two ways. First, Z9,E12-14:Ac can be pre-bound to immobilized SlitOR6 polypeptide, followed by injection of candidate modulator at approximately 10 μΙ/min flow rate and a concentration ranging from 1 nM to 1000 μΜ, preferably about 100 μΜ. Displacement of the bound Z9,E12-14:Ac can be quantitated, permitting detection of modulator binding. Alternatively, the membrane-bound SlitOR6 polypeptide can be pre-incubated with candidate modulator and challenged with Z9,E12- 14:Ac. A difference in Z9,E12-14:Ac binding to the SlitOR6 exposed to modulator relative to that on a chip not pre-exposed to modulator will demonstrate binding. In either assay, a decrease of 10% or more in the amount of Z9,E12-14:Ac bound is in the presence of candidate modulator, relative to the amount of Z9,E12-14:Ac bound in the absence of candidate modulator indicates that the candidate modulator inhibits the interaction of SlitOR6 and Z9,E12-14:Ac. Biacore system can be plugged to a system identifying candidate modulator such as mass spectrometry, or gas chromatography.
Another method of measuring inhibition of binding of Z9,E12-14:Ac to SlitOR6 uses fluorescence resonance energy transfer (FRET). FRET is a quantum mechanical phenomenon that occurs between a fluorescence donor (D) and a fluorescence acceptor (A) in close proximity to each other (usually < 100 A of separation) if the emission spectrum of D overlaps with the excitation spectrum of A. The molecules to be tested, e.g., Z9,E12-14:Ac and a SlitOR6 polypeptide, are labelled with a complementary pair of donor and acceptor fluorophores. While close to each other due to SlitOR6: Z9,E12-14:Ac interaction, fluorescence emitted upon excitation of the donor fluorophore will have a different wavelength than that emitted in response to that excitation wavelength when the molecules are not bound, providing for quantitation of bound versus unbound polypeptides by measurement of emission intensity at each wavelength. Donor:Acceptor pairs of fluorophores with which to label the target molecules are well known in the art.
A variation on FRET uses fluorescence quenching to monitor molecular interactions. One molecule in the interacting pair can be labelled with a fluorophore, and the other with a molecule that quenches the fluorescence of the fluorophore when brought into close apposition with it. A change in fluorescence upon excitation is indicative of a change in the association of the molecules tagged with the fluorophore: quencher pair. Generally, an increase in fluorescence of the labelled SlitOR6 polypeptide is indicative that Z9,E12-14:Ac bearing the quencher has been displaced. For quenching assays, a 10% or greater increase in the intensity of fluorescent emission in samples containing a candidate modulator, relative to samples without the candidate modulator, indicates that the candidate modulator inhibits SlitOR6: Z9,E12-14:Ac interaction.
Bioluminescence Resonance Energy Transfer (BRET) is a system for monitoring intermolecular interactions in vivo. The assay is based on non-radiative energy transfer between fusion proteins containing Renilla luciferase (Rluc) and e.g. Yellow Fluorescent Protein (YPF) or Green Fluorescent Protein (GFP). The BRET signal is generated by the oxidation of a coelenterazine derivative substrate. Said system may apply a cell-permeable and non-toxic coelenterazine derivative substrate DeepBleuC™ (DBC) and a mutant of the Green Fluorescent Protein (GFP) as acceptor. When the donor and acceptor are in close proximity the energy resulting from the catalytic degradation of the DBC is transferred from Rluc to GFP which will then emit fluorescence as its characteristic wavelength. This method allows higher distance between the two tested molecules and is fluorophore-angle independent.
In addition to the surface plasmon resonance and FRET and BRET methods, fluorescence polarization measurement is useful to quantitate Z9,E12-14:Ac-receptor binding. The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate. Protein complexes, such as those formed by SlitOR6 associating with a fluorescently labelled Z9,E12-14:Ac, have higher polarization values than uncomplexed, labelled Z9,E12-14:Ac. The inclusion of a candidate inhibitor of the SlitOR6: Z9,E12-14:Ac interaction results in a decrease in fluorescence polarization, relative to a mixture without the candidate inhibitor, if the candidate inhibitor disrupts or inhibits the interaction of SlitOR6 with Z9,E12-14:Ac. Fluorescence polarization is well suited for the identification of small molecules that disrupt the formation of polypeptide or protein complexes. A decrease of 10% or more in fluorescence polarization in samples containing a candidate modulator, relative to fluorescence polarization in a sample lacking the candidate modulator, indicates that the candidate modulator inhibits SlitOR6: Z9,E12-14:Ac interaction.
Another alternative for monitoring SlitOR6: Z9,E12-14:Ac interactions uses a biosensor assay. ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute; http//www.ambri. com.au/). In this technology, the association of molecules such as SlitOR6 and Z9,E12-14:Ac, is coupled to the closing of gramacidin-facilitated ion channels in suspended membrane bilayers and thus to a measurable change in the admittance (similar to impedence) of the biosensor. This approach is linear over six orders of magnitude of admittance change and is ideally suited for large scale, high throughput screening of small molecule combinatorial libraries. A 10% or greater change (increase or decrease) in admittance in a sample containing a candidate modulator, relative to the admittance of a sample lacking the candidate modulator, indicates that the candidate modulator inhibits the interaction of SlitOR6 and Z9,E12-14:Ac.
It is important to note that in assays of ligand-protein interaction, it is possible that a modulator of the interaction need not necessarily to interact directly with the domain(s) of the proteins that physically interact. It is also possible that a modulator will interact at a location removed from the site of ligand-protein interaction and cause, for example, a conformational change in the SlitOR6 polypeptide. Modulators (inhibitors or agonists) that act in this manner are nonetheless of interest as agents to modulate the activity of SlitOR6. Any of the binding assays described can be used to determine the presence of an agent in a sample, e.g., a tissue sample, that binds to the SlitOR6 receptor molecule, or that affects the binding of Z9,E12-14:Ac to the receptor. To do so, SlitOR6 polypeptide is reacted with Z9,E12-14:Ac or another ligand in the presence or in the absence of the sample, and Z9,E12-14:Ac or ligand binding is measured as appropriate for the binding assay being used. A decrease of 10% or more in the binding of Z9,E12-14:Ac or other ligand indicates that the sample contains an agent that modulates Z9,E12-14:Ac or ligand binding to the receptor polypeptide.
4) Protein chips The methods of the present invention may be applied on protein chips. Said protein chip may be, but is not limited to, a glass slide or a nitrocellulose membrane. Array-based methods for protein chips are well known in the art.
5) Functional assays of receptor activity i. Downstream Pathway Activation Assays: a. Calcium flux - The Aequorin-based Assay.
The aequorin assay takes advantage of the responsiveness of mitochondrial or cytoplasmic apoaequorin to intracellular calcium release or calcium flux (entrance) induced by the activation of GPCRs (Stables et al., 1997, Anal. Biochem. 252:1 15-126; Detheux et al., 2000, J. Exp. Med., 192 1501 -1508; both of which are incorporated herein by reference). Briefly, SlitOR6-expressing clones are transfected to coexpress mitochondrial or cytoplasmic apoaequorin and Gcc16 or G-olf. Cells are incubated with 5 μΜ Coelenterazine H or derivates (Molecular Probes) for 4 hours at room temperature, washed in DMEM-F12 culture medium and resuspended at a concentration of 0.5 x 106 cells/ml. Cells are then mixed with test agonist peptides and light emission by the aequorin is recorded with a luminometer for 30 sec. Results are expressed as Relative Light Units (RLU). Controls include assays using membranes isolated from cells not expressing SlitOR6 (mock-transfected), in order to exclude possible non-specific effects of the candidate compound.
Aequorin activity or intracellular calcium levels are "changed" if light intensity increases or decreases by 10% or more in a sample of cells, expressing a SlitOR6 polypeptide and treated with a candidate modulator, relative to a sample of cells expressing the SlitOR6 polypeptide but not treated with the candidate modulator or relative to a sample of cells not expressing the SlitOR6 polypeptide (mock-transfected cells) but treated with the candidate modulator. When performed in the absence of Z9,E12-14:Ac, the assay can be used to identify an agonist or inverse agonist of SlitOR6 activity. When the assay is performed in the presence of Z9,E12-14:Ac, it can be used to assay for an antagonist.
1 ) a Fluo3, 4, Fura2, and Calcium3 (Molecular Device) based-assay.
Fluorescence-based assays take advantage of calcium fluxes triggered by receptor activation: either calcium entrance through ORco for instance or calcium release from endoplasmic reticulum. Some fluorophores including but not limited to Fluo3, Fluo4 and Fura2 (Molecular Probes) and Calcium3 kit series (Molecular Device) are known to bind calcium. Such fluorophore-calcium complexes emit fluorescence at respective specific wavelength. Thereby, upon activation of a G-protein coupled receptor, calcium released from endoplasmic reticulum or entered through ORco binds to fluorophore leading to specific fluorescence emission. SlitOR6-overexpressing cells are incubated for 30 to 60 minutes with a solution of 1 to 8 μΜ fluorophore at 37°C. After thorough washing with saline buffer, 50 μΙ of the same buffer is poured in each well-containing cells (6 to 1536). Tested agonists are then injected onto such loaded-cells and activation of SlitOR6 is followed by fluorescence measurement. Intracellular calcium levels are modified if fluorescence intensity increases or decreases by 10% or more in a sample of cells, expressing a SlitOR6 polypeptide and treated with a candidate modulator, relative to a sample of cells expressing the C356 polypeptide but not treated with the candidate modulator or relative to a sample of cells not expressing the SlitOR6 polypeptide (mock-transfected cells) but treated with the candidate modulator. 2) Depolarization/hyperpolarization membrane assay (DiBac fluorophore for instance).
The principle of this assay is to follow depolarization of cell membrane. For instance, the anionic probe DiBAC4(3) partitions between intra- and extracellular compartments in a membrane potential-dependent manner. With increasing membrane potential (depolarization), the probe further partition into the cell resulting in an increase of fluorescence. Conversely, hyperpolarization leads to a decrease of fluorescence due to a dye extrusion.
The DiBAC4(3) probe is excited with a wavelength of 488 nm, and emits at a wavelength of 540 nm.
On the day of the experiment, add the glucose to the assay buffer (saline buffer) to a final concentration of 10 mM and the DiBAC4(3) probe to a final concentration of 5 μΜ.
Maintain the assay buffer at 37°C. Remove the cell culture medium and rinse twice each well containing SlitOR6-overexpressing cells with 200 μΙ of pre-heated assay buffer. Place 180 μΙ of Assay buffer containing DiBAC4(3) and incubate cells for 30 min at the appropriate temperature. Cell plates will be ready for assay after these 30 min incubation. Collect baseline for 2 min prior any addition. Add 20 μΙ of candidate modulators to the appropriate well and collect the data for an additional 25 min. Membrane polarization is "changed" if fluorescence intensity increases or decreases by 10% or more in a sample of cells, expressing a SlitOR6 polypeptide and treated with a candidate modulator, relative to a sample of cells expressing the SlitOR6 polypeptide but not treated with the candidate modulator or relative to a sample of cells not expressing the SlitOR6 polypeptide (mock-transfected cells) but treated with the candidate modulator. b. Adenylate Cyclase Assay:
Assays for adenylate cyclase activity are described by Kenimer and Nirenberg, 1981 , Mol. Pharmacol. 20: 585-591 , incorporated herein by reference. That assay is a modification of the assay taught by Solomon et al., 1974, Anal. Biochem. 58: 541 -548, also incorporated herein by reference. Briefly, 100 μΙ reactions contain 50 mM Tris-Hcl (pH 7.5), 5 mM MgCI2, 20 mM creatine phosphate (disodium salt), 10 units (71 μg of protein) of creatine phosphokinase, 1 mM cc-32P-ATP (tetrasodium salt, 2 μθί), 0.5 mM cyclic AMP, G-3H-labelled cyclic AMP (approximately 10,000 cpm), 0.5 mM Ro20-1724, 0.25% ethanol, and 50-200 μg of protein homogenate to be tested (i.e., homogenate from cells expressing or not expressing a SlitOR6 polypeptide, treated or not treated with Z9,E12-14:Ac with or without a candidate modulator). Reaction mixtures are generally incubated at 37°C for 6 minutes. Following incubation, reaction mixtures are deproteinized by the addition of 0.9 ml of cold 6% trichloroacetic acid. Tubes are centrifuged at 1800 x g for 20 minutes and each supernatant solution is added to a Dowex AG50W-X4 column. The cAMP fraction from the column is eluted with 4 ml of 0.1 mM imidazole-HCI (pH 7.5) into a counting vial. Assays should be performed in triplicate. Control reactions should also be performed using protein homogenate from cells that do not express a SlitOR6 polypeptide.
Assays should be performed using cells or extracts of cells expressing SlitOR6, treated or not treated with Z9,E12-14:Ac with or without a candidate modulator. Control reactions should be performed using mock-transfected cells, or extracts from them in order to exclude possible non-specific effects of some candidate modulators
According to the invention, adenylate cyclase activity is "changed" if it increases or decreases by 10% or more in a sample taken from cells treated with a candidate modulator of SlitOR06 activity, relative to a similar sample of cells not treated with the candidate modulator or relative to a sample of cells not expressing the SlitOR6 polypeptide (mock-transfected cells) but treated with the candidate modulator. Alternatively, a decrease of activity by 10% or more by the candidate modulator of SlitOR6 in a sample treated with a reference compound may be tested. c. cAMP/cGMP Assays:
Intracellular cAMP/cGMP is measured using a cAMP/cGMP radioimmunoassay (RIA) or cAMP/cGMP binding protein according to methods widely known in the art. For example, Horton and Baxendale, 1995, Methods Mol. Biol. 41 : 91 -105, which is incorporated herein by reference, describes an RIA for cAMP.
A number of kits for the measurement of cAMP/cGMP are commercially available, such as the High Efficiency Fluorescence Polarization-based homogeneous assay marketed by LJL Biosystems and NEN Life Science Products. Control reactions should be performed using extracts of mock-transfected cells to exclude possible non-specific effects of some candidate modulators.
Assays should be performed using cells or extracts of cells expressing SlitOR6, treated or not treated with Z9,E12-14:Ac with or without a candidate modulator. Control reactions should be performed using mock-transfected cells, or extracts from them in order to exclude possible non-specific effects of some candidate modulators
The level of cAMP is "changed" if the level of cAMP/cGMP detected in cells, expressing a SlitOR6 polypeptide and treated with a candidate modulator of SlitOR6 activity (or in extracts of such cells), using the RIA-based assay of Horton and Baxendale, 1995, supra, increases or decreases by at least 10% relative to the cAMP level in similar cells not treated with the candidate modulator. d. Phospholipid breakdown, DAG production and Inositol Triphosphate levels:
Receptors that activate the breakdown of phospholipids can be monitored for changes due to the activity of known or suspected modulators of SlitOR6 by monitoring phospholipid breakdown, and the resulting production of second messengers DAG and/or inositol triphosphate (IP3). Methods of measuring each of these are described in Phospholipid Signalling Protocols, edited by Ian M. Bird. Totowa, NJ, Humana Press, 1998, which is incorporated herein by reference. See also Rudolph et al., 1999, J. Biol. Chem. 274: 1 1824- 1 1831 , incorporated herein by reference, which also describes an assay for phosphatidylinositol breakdown. Assays should be performed using cells or extracts of cells expressing SlitOR6, treated or not treated with Z9,E12-14:Ac with or without a candidate modulator. Control reactions should be performed using mock-transfected cells, or extracts from them in order to exclude possible non-specific effects of some candidate modulators. According to the invention, phosphatidylinositol breakdown, and diacylglycerol and/or inositol triphosphate levels are "changed" if they increase or decrease by at least 10% in a sample from cells expressing a SlitOR6 polypeptide and treated with a candidate modulator in the presence or in the absence of Z9,E12-14:Ac, relative to the level observed in a sample from cells expressing a SlitOR6 polypeptide that is not treated with the candidate modulator. ii. Transcriptional reporters for downstream pathway activation:
The intracellular signal initiated by binding of an agonist to a receptor, e.g., SlitOR6, sets in motion a cascade of intracellular events, the ultimate consequence of which is a rapid and detectable change in the transcription and/or translation of one or more genes. The activity of the receptor can therefore be monitored by measuring the expression of a reporter gene driven by control sequences responsive to SlitOR6 activation.
As used herein "promoter" refers to the transcriptional control elements necessary for receptor-mediated regulation of gene expression, including not only the basal promoter, but also any enhancers or transcription-factor binding sites necessary for receptor-regulated expression. By selecting promoters that are responsive to the intracellular signals resulting from agonist binding, and operatively linking the selected promoters to reporter genes whose transcription, translation or ultimate activity is readily detectable and measurable, the transcription based reporter assay provides a rapid indication of whether a given receptor is activated. Reporter genes such as luciferase, Chloramphenicol Acetyl Transferase (CAT), Green
Fluorescent Protein (GFP), β-lactamase or β-galactosidase are well known in the art, as are assays for the detection of their products.
Genes particularly well suited for monitoring receptor activity are the "immediate early" genes, which are rapidly induced, generally within minutes of contact between the receptor and the effector protein or ligand. The induction of immediate early gene transcription does not require the synthesis of new regulatory proteins. In addition to rapid responsiveness to ligand binding, characteristics of preferred genes useful to make reporter constructs include: low or undetectable expression in quiescent cells; induction that is transient and independent of new protein synthesis; subsequent shut-off of transcription requires new protein synthesis; and mRNAs transcribed from these genes have a short half-life. It is preferred, but not necessary that a transcriptional control element have all of these properties for it to be useful. An example of a gene that is responsive to a number of different stimuli is the c-fos proto-oncogene. The c-fos gene is activated in a protein-synthesis-independent manner by growth factors, hormones, differentiation-specific agents, stress, and other known inducers of cell surface proteins. The induction of c-fos expression is extremely rapid, often occurring within minutes of receptor stimulation. This characteristic makes the c-fos regulatory regions particularly attractive for use as a reporter of receptor activation.
The c-fos regulatory elements include (see, Verma et al., 1987, Cell 51 : 513-514): a TATA box that is required for transcription initiation; two upstream elements for basal transcription, and an enhancer, which includes an element with dyad symmetry and which is required for induction by phorbol ester 12-0-tetradecanoylphorbol- -acetate (TPA), serum, Epidermal Growth Factor (EGF), and PMA.
The 20 bp c-fos transcriptional enhancer element located between -317 and -298 bp upstream from the c-fos mRNA cap site, is essential for serum induction in serum starved NIH 3T3 cells. One of the two upstream elements is located at -63 to -57 and it resembles the consensus sequence for cAMP regulation.
The transcription factor CREB (cyclic AMP responsive element binding protein) is, as the name implies, responsive to levels of intracellular cAMP. Therefore, the activation of a receptor that signals via modulation of cAMP levels can be monitored by measuring either the binding of the transcription factor, or the expression of a reporter gene linked to a CREB- binding element (termed the CRE, or cAMP response element). The DNA sequence of the CRE is TGACGTCA. Reporter constructs responsive to CREB binding activity are described in U.S. Patent No. 5,919,649.
Other promoters and transcriptional control elements, in addition to the c-fos elements and CREB-responsive constructs, include the vasoactive intestinal peptide (VIP) gene promoter (cAMP responsive; Fink et al., 1988, Proc. Natl. Acad. Sci. 85:6662-6666); the somatostatin gene promoter (cAMP responsive; Montminy et al., 1986, Proc. Natl. Acad. Sci. 8.3:6682-6686); the proenkephalin promoter (responsive to cAMP, nicotinic agonists, and phorbol esters; Comb et al., 1986, Nature 323:353-356); the phosphoenolpyruvate carboxylase (PEPCK) gene promoter (cAMP responsive; Short et al., 1986, J. Biol. Chem. 261 :9721 -9726).
Additional examples of transcriptional control elements that are responsive to changes in GPCR activity include, but are not limited to those responsive to the AP-1 transcription factor and those responsive to NF-κΒ activity. The consensus AP-1 binding site is the palindrome TGA(C/G)TCA (Lee et al., 1987, Nature 325: 368-372; Lee et al., 1987, Cell 49: 741 -752). The AP-1 site is also responsible for mediating induction by tumour promoters such as the phorbol ester 12-0-tetradecanoylphorbol- -acetate (TPA), and are therefore sometimes also referred to as a TRE, for TPA-response element. AP-1 activates numerous genes that are involved in the early response of cells to growth stimuli. Examples of AP-1 - responsive genes include, but are not limited to the genes for Fos and Jun (which proteins themselves make up AP-1 activity), Fos-related antigens (Fra) 1 and 2, ΙκΒα, ornithine decarboxylase, and annexins I and II. The NF-κΒ binding element has the consensus sequence GGGGACTTTCC. A large number of genes have been identified as NF-κΒ responsive, and their control elements can be linked to a reporter gene to monitor GPCR activity. A small sample of the genes responsive to NF-κΒ includes those encoding IL-1 β (Hiscott et al., 1993, Mol. Cell. Biol. 13: 6231 -6240), TNF-cc (Shakhov et al., 1990, J. Exp. Med. 171 : 35-47), CCR5 (Liu et al., 1998, AIDS Res. Hum. Retroviruses 14: 1509-1519), P-selectin (Pan and McEver, 1995, J. Biol. Chem. 270: 23077-23083), Fas ligand (Matsui et al., 1998, J. Immunol. 161 : 3469-3473), GM-CSF (Schreck and Baeuerle, 1990, Mol. Cell. Biol. 10: 1281 -1286) and ΙκΒα (Haskill et al., 1991 , Cell 65: 1281 -1289). Each of these references is incorporated herein by reference. Vectors encoding NF-KB-responsive reporters are also known in the art or can be readily made by one of skill in the art using, for example, synthetic NF-κΒ elements and a minimal promoter, or using the NF-KB-responsive sequences of a gene known to be subject to NF-κΒ regulation. Further, NF-κΒ responsive reporter constructs are commercially available from, for example, CLONTECH.
A given promoter construct should be tested by exposing SlitOR6-expressing cells, transfected with the construct, to Z9,E12-14:Ac. An increase of at least two-fold in the expression of reporter in response to Z9,E12-14:Ac indicates that the reporter is an indicator of SlitOR6 activity.
In order to assay SlitOR6 activity with Z9,E12-14:Ac-responsive transcriptional reporter construct, cells that stably express SlitOR6 polypeptide are stably transfected with the reporter construct. To screen for agonists, untreated cells are exposed to candidate modulators, or exposed to Z9,E12-14:Ac, and expression of the reporter is measured. The Z9,E12-14:Ac- treated cultures serve as a standard for the level of transcription induced by a known agonist. An increase of at least 10% in reporter expression in the presence of a candidate modulator compare to reporter expression in the absence of any modulator indicates that the candidate is a modulator of SlitOR6 activity. An agonist will induce at least as much, and preferably the same amount or more reporter expression than the Z9,E12-14:Ac. Partial agonists may activate the receptor less compared to Z9,E12-14:Ac. This approach can also be used to screen for inverse agonists where cells express a SlitOR6 polypeptide at levels such that there is an elevated basal activity of the reporter in the absence of Z9,E12-14:Ac or other agonists. A decrease in reporter activity of 10% or more in the presence of a candidate modulator, relative to its absence, indicates that the compound is an inverse agonist. To screen for antagonists, the cells expressing SlitOR6 and carrying the reporter construct are exposed to Z9,E12-14:Ac (or another agonist) in the presence and absence of candidate modulator. A decrease of 10% or more in reporter expression in the presence of candidate modulator, relative to the absence of the candidate modulator, indicates that the candidate is an antagonist of SlitOR6 activity. Controls for transcription assays include cells not expressing SlitOR6 but carrying the reporter construct, as well as cells with a promoterless reporter construct. Compounds that are identified as modulators of SlitOR6-regulated transcription should also be analyzed to determine whether they affect transcription driven by other regulatory sequences and by other receptors, in order to determine the specificity and spectrum of their activity. The transcriptional reporter assay, and most cell-based assays, are well suited for screening chemical libraries of chemical compounds for those that modulate SlitOR6 activity. The libraries can be, for example, libraries from natural sources, e.g., plants, animals, bacteria, etc., or they can be libraries comprising randomly or systematically equivalents of Z9,E12-14:Ac. iii. Receptor internalization
Any of the assays of receptor activity, including calcium flux, membrane polarization, melanophore assay, the GTP-binding, GTPase, adenylate cyclase, cAMP, phospholipid- breakdown, diacylglyceorl, inositol triphosphate, PKC, PKA, kinase, receptor internalization and transcriptional reporter assays, can be used to determine the presence of an agent in a sample, e.g., a tissue sample, that affects the activity of the SlitOR6 receptor molecule. To do so, SlitOR6 polypeptide is assayed for activity in the presence and in the absence of the sample or an extract of the sample. An increase in SlitOR6 activity in the presence of the sample or extract relative to the absence of the sample indicates that the sample contains an agonist of the receptor activity. A decrease in receptor activity in the presence of Z9,E12- 14:Ac or another agonist and the sample, relative to receptor activity in the presence of Z9,E12-14:Ac alone, indicates that the sample contains an antagonist of SlitOR6 activity. If desired, samples can then be fractionated and further tested to isolate or purify the agonist or antagonist. The amount of increase or decrease in measured activity necessary for a sample to be said to contain a modulator depends upon the type of assay used. Generally, a 10% or greater change (increase or decrease) relative to an assay performed in the absence of a sample indicates the presence of a modulator in the sample. One exception is the transcriptional reporter assay, in which at least a two-fold increase or 10% decrease in signal is necessary for a sample to be said to contain a modulator. It is preferred that an agonist stimulates at least 50%, and preferably 75% or 100% or more, e.g., 2-fold, 5-fold, 10-fold or greater receptor activation than Z9,E12-14:Ac.
Other functional assays include, for example, microphysiometer or biosensor assays (see Hafner, 2000, Biosens. Bioelectron. 15: 149-158, incorporated herein by reference). II. Population monitoring Based upon the Interaction of SlitOR6 and Z9,E12-14:Ac:
Z9,E12-14:Ac is a compound of the Spodoptera littoralis pheromonal bouquet. SlitOR6 is expressed with a positive bias in the antennae of S. littoralis male (Figures 2a, b). This is suggesting a possible role of this receptor in the pheromonal perception. Homology relationships found between SlitOR6 and moth pheromone receptors in the phylogeny support this hypothesis. The experiments conducted in teams of the inventors show that the molecule (Z9,E12-14:Ac) triggers a response of the receptor SlitOR6.
The interaction of SlitOR6 with Z9,E12-14:Ac can be used as the basis of monitoring infestation of S. littoralis in cropping systems. Monitoring based on SlitOR6-related behaviours (attraction) can have several different forms. Standardized "attract and kill" or mark "and recapture" assays can be designed using
Z9,E12-14:Ac to estimate the population size, its growth and the necessity of using population control systems.
Qualitative assays
Assays that evaluate whether or not the SlitOR6 polypeptide or the mRNA encoding it are wild-type or not can be used diagnostically. In order to diagnose mutation in the olfactory function, RNA isolated from a sample is used as a template for PCR amplification of SlitOR6. The amplified sequences are then either directly sequenced using standard methods, or are first cloned into a vector, followed by sequencing. A difference in the sequence that changes one or more encoded amino acids relative to the sequence of wild-type SlitOR6 can be diagnostic of a mutation characterized by change of SlitOR6 signalling. It can be useful, when a change in coding sequence is identified in a sample, to express the variant receptor or ligand and compare its activity to that of wild type SlitOR6. Among other benefits, this approach can provide novel mutants, including constitutively active and null mutants.
If desired, array or microarray-based methods can be used to analyze the expression or the presence of mutation, in SlitOR6 sequences. Array-based methods for minisequencing and for quantitation of nucleic acid expression are well known in the art.
Modulation of SlitOR6 Activity expressed in a Cell According to the Invention
The discovery of Z9,E12-14:Ac as a ligand of SlitOR6 provides methods of modulating the activity of a SlitOR6 polypeptide expressed in a cell. SlitOR6 activity is modulated in a cell by delivering to that cell an agent that modulates the function of SlitOR6 polypeptide. This modulation can be performed in cultured cells as part of an assay for the identification of additional modulating agents, or, for example, in an animal, including a human. Agents include Z9,E12-14:Ac and equivalent acids thereof.
An agent can be delivered to a cell by adding it to culture medium. The amount to deliver will vary with the identity of the agent and with the purpose for which it is delivered. For example, in a culture assay to identify antagonists of SlitOR6 activity, one will preferably add an amount of Z9,E12-14:Ac that half-maximally activates the receptors (e.g., approximately EC5o), preferably without exceeding the dose required for receptor saturation. This dose can be determined by titrating the amount of Z9,E12-14:Ac to determine the point at which further addition of Z9,E12-14:Ac has no additional effect on SlitOR6 activity. Candidate Modulators Useful According to the Invention
Candidate modulators can be screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of various kinds of compounds. Synthetic compound libraries are commercially available from a number of companies including, for example, Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, Wl). Combinatorial libraries of small organic molecules are available and can be prepared. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible by methods well known in the art. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
As noted previously herein, candidate modulators may be variants or equivalents of Z9,E12-14:Ac. Therefore, a library of Z9,E12-14:Ac-related compounds may be used
Transgenic Animals Useful According to the Invention
Transgenic animals expressing SlitOR6 or variants thereof are useful to study the signalling through SlitOR6, as well as for the study of agents that modulate the activity of SlitOR6. A transgenic animal is a non-human animal containing at least one foreign gene, called a transgene, which is part of its genetic material. Preferably, the transgene is contained in the animal's germ line such that it can be transmitted to the animal's offspring. A number of techniques may be used to introduce the transgene into an animal's genetic material, including, but not limited to, microinjection of the transgene into pronuclei of fertilized eggs and manipulation of embryonic stem cells (U.S. Patent No. 4,873,191 by Wagner and Hoppe; Palmiter and Brinster, 1986, Ann. Rev. Genet, 20:465-499; French Patent Application 2593827 published Aug. 7, 1987, all of which are incorporated herein by reference). Transgenic animals can carry the transgene in all their cells or can be genetically mosaic. According to the method of conventional transgenesis, additional copies of normal or modified genes are injected into the male pronucleus of the zygote and become integrated into the genomic DNA of the recipient animal. The transgene is transmitted in a Mendelian manner in established transgenic strains. Transgenes can be constitutively expressed or can be tissue specific or even responsive to an exogenous drug, e.g., Tetracycline. A transgenic animal expressing one transgene can be crossed to a second transgenic animal expressing a second transgene such that their offspring will carry and express both transgenes.
Example of such transgenic animals that one can use as tools to study the functional properties of SlitOR6 and to search for modulators is the fly Drosophila melanogaster. One can generate UAS-SlitOR6 fly lines that can be further crossed with various Gal4 Drosophila lines to target the expression of SlitOR6 in a specific organ (e.g. the antennae) or a specific cell type (e.g. the "empty neuron" system, see Dobritsa et al., 2003, Neuron, 37: 827-841 for description or the T1 sensilla, see Kurtovic et al. 2007, Nature, 446: 542-546). Transformed flies can further be screened for electrophysiological or behavioural responses upon stimulation with compound libraries.
Knock-Out Animals
Animals bearing a homozygous deletion/insertion in the chromosomal sequences encoding SlitOR6 or variants can be used to study the function of the receptor. Of further particular interest is the identification of the specific role of SlitOR6/ Z9,E12-14:Ac in specific physiological processes.
For example, one of skill in the art can generate a homozygous SlitOR6 knock-out (e.g. a Spodoptera), by creating gene deletions with homologous recombination, or by the use of new technologies such as meganucleases, zing-fingers or TALENs.
Kits Useful According to the Invention
The invention provides kits useful for screening for modulators of SlitOR6 activity. Kits useful according to the invention can include an isolated SlitOR6 polypeptide (including a membrane- or cell-associated SlitOR6 polypeptide, e.g., on isolated membranes, cells expressing SlitOR6, or, on an SPR chip) and an isolated Z9,E12-14:Ac. When cells included, said cells may be transformed with a polynucleotide encoding said SlitOR6. In a further embodiment, the kit according to the invention may contain a polynucleotide encoding a SlitOR6 polypeptide and Z9,E12-14:Ac. All kits according to the invention will comprise the stated items or combinations of items and packaging materials therefore. Kits may also include instructions for use.
According to the present kit, said Z9,E12-14:Ac may be a Z9,E12-14:Ac-related agent having a capacity of binding and/or modulating the SlitOR6 receptor similar to Z9,E12-14:Ac.
According to the present invention, said SlitOR6 polypeptide may be a polypeptide having at least 20% identity or higher identity, such as 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95% or even 100% to the polypeptide represented in Figure 1 b; and which binds specifically Z9,E12-14:Ac or equivalent acids. Alternatively, said SlitOR6 polypeptide may be a fragment of the full length polypeptide as shown in Figure 1 b, wherein the fragment retains at least 50% of the binding activity and level of signaling activation when using Z9,E12-14:Ac. According to the present invention, said SlitOR6 polypeptide may comprise one or more additions, insertions, deletions or substitutions relative to the sequence depictured in Figure 1 b. Said SlitOR6 polypeptide may be a truncated SlitOR6 polypeptide; said SlitOR6 polypeptide may comprise additional sequences forming a SlitOR6 fusion protein, wherein said additional sequences may be chosen from the group consisting of glutathione-S-transferase (GST), maltose binding protein (MBP), alkaline phosphatase, thioredoxin, green fluorescent protein (GFP), histidine tags (e.g., 6X or greater His), or affinity tags (e.g., Myc tag, FLAG tag) sequences.
EXAMPLES
Example 1 : Identification and cloning of a pheromone receptor in Spodoptera littoralis (Figure 1 a,b,c). The sequences of pheromone receptors have been identified in several species of Moths (see Sakurai et al., 2004, Proc Natl Acad Sci U S A. 2004 Nov 23;101 (47): 16653-16658 for an example). Because of its position of major pest, attention was paid to Spodoptera littoralis. In those Butterflies, the males are attracted to mating areas by the pheromones emitted by females. The male antennae are the main organs involved in the perception of these volatile molecules.
In order to characterize pheromones receptors, cDNA libraries were created based on RNA extracts from freshly excised male antennae. These fragments were sequenced and screened based existing databases of potential orthologues (e.g. Genbank). The fragments displaying the highest homology with known and identified sequences of other species were then used as bases for RACE-PCR, in order to obtain full ORF (i.e. open reading frame). These latter were then PCR amplified and cloned into the relevant expression vectors (for instance pUAST for the in vivo expression).
The cloned sequences were phylogenetically compared with those known in other organisms. The resulting tree notably shows the clustering of two groups of receptors (i) the ORco clade, containing the co-receptors identified in the varied compared species; (ii) the pheromone receptors clade including the Spodoptera pheromones receptors as well as those of the closely related species.
SlitOR6 is a newly found gene, which phylogenetically groups within the subclade of Moths pheromone receptors. This phylogenetic evidence supports the expected role of the pheromone receptor of that gene. In addition to its sequencing and phylogenetic characterization, the gene coding for SlitOR6 (Figure 1 a) and the corresponding protein (Figure 1 b) were -for the first time- characterized in a functional point of view, using in vivo and in vitro approaches (see subsequent examples).
Example 2: Characterization of the receptor expression patterns by in situ hybridization (Figure 2a, b). The mapping of expression of the characterized receptor (SlitOR6) is of importance, notably to guide the electrophysiological approaches and to illustrate its biological function. Two methods were used to characterise the expression bias of SlitOR6 in males, females and varied tissues: (i) RT-PCR using RNA pool extracts from male and female tissues; (ii) in situ hybridization in section of olfactory organs. Both methods are well known by a person skilled in the art. RT-PCR was realized based on RNA freshly extracted from tissues isolated from varied organs of both males and females (Figure 4a). In situ hybridization was performed on sections (longitudinal are presented in Figure 4b). Labeled SlitOR6 probes were used to hybridize mRNA in the male antennae.
RT-PCR shows that expression of the targeted gene is nearly completely restricted to male antennae. A very faint expression is detectable in the antennae of females also (Figure 4a). In situ hybridization shows more specifically the expression of the concerned receptor in the olfactory sensillae of the ventral side of antennae.
These tissue expression studies indicate that the SlitOR6 mRNA, and the SliotOR6 polypeptide encoded by this mRNA, is expressed in antennae with a strong bias in favor of the male tissues. Together with other cues (the phylogenetic affinities of the sequence for instance), this shows that the characterized sequence is well part of the pheromone perception machinery.
Example 3: Electro-antennography assays on Drosophila antennae expressing SlitOR6 (Figures 3a, b). SlitOR6 was cloned into the pUAST vector and recombinant plasmid was injected in Drosophila embryos to generate UAS-SlitOR6 lines (BestGene Inc., Chino Hills, CA, USA). UAS-SlitOR6 lines were crossed with a homozygous ORco-GAL4 driver line (Bloomington Drosophila Stock Center, No. 23292). Electro-antennogram (EAG) recordings were performed on UAS-SlitOR6 and ORco-GAL4 parental lines (controls) and on ORco-GAL4/UAS-SlitOR6 lines, according to protocol well known of a person skilled in the art. For the screening experiments (Figure 3b), 1 μg of the different stimuli diluted in hexane was applied during 500 ms: tetradecyl acetate (14:Ac), (Z)-9-tetradecenyl acetate (Z9-14:Ac), (Z)-9-tetradecenol (Z9- 14:0H), (Z)-1 1 -tetradecenyl acetate (Z1 1 -14:Ac), (E)-1 1 -tetradecenyl acetate (E1 1 -14:Ac), (Z,E)-9,1 1 -tetradecadienyl acetate (Z9.E1 1 -14:Ac) and (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:Ac). For the dose-response experiments (Figure 3a), 1 ng to 10 μg of Z9,E12-14:Ac were applied. The positive control stimulus consisted of 10 μΙ of 2-heptanone diluted in paraffin oil (10-3 dilution). Hexane and paraffin oil alone were also tested as negative controls. Experimental group comparisons were made by one-way ANOVA followed by a Tukey post-hoc test.
Assays show that only the stimulation of antennae expressing both SlitOR6 and ORco are producing a response matching a sigmoid curve, which increases as concentration in administrated ligand rise (Figure 3a). Among the 8 ligands tested sole the Z9,E12-14:Ac is eliciting a significantly higher response when applied on cells expressing the couple ORco and SlitOR6 (Heptanone was used as an intern positive control).
Example 4: Single-cell calcium-imaging assays performed on Hek 293 cells overexpressing SlitOR6 polypeptide (Figures 4a, b).
For single-cell calcium imaging assay, cells (derived HEK293 cells in the illustrated example: Figure 4a) were plated into 96 well-plates 30h before the experiment and transfected with plasmids coding for the components of the olfactory heterodimer (20h prior calcium imaging assay): ORco and SlitOR6. Transfection was made according to the manufacturer's protocol (most assays were realized with Lipofectamin 2000, Invitrogen (c)).
After a one hour-incubation at 37°C in a saline buffer containing 4 g/ml Fluo4-AM (Molecular Probe), cells were rinsed twice with Fluo4-AM-free saline buffer (ORS). 50 μΙ of saline buffer was added to each well.
Calcium mobilization was recorded with a 10x or 20x magnification objective on a Zeiss Axiovert 200 Mot microscope equipped for fluorescence detection. One image of the same field was taken each second during 60-90 seconds. 50 μΙ of the two fold concentrated ligand solubilised in the saline buffer was injected 5 seconds after record started.
Recording of fluorescence variation induced upon injection of 10 μΜ of Z9,E12-14:Ac on cells loaded with fluo4-AM, a calcium tracer, and expressing SlitOR6. Cells were observed with a magnification of 10X. Timescale is expressed in seconds. Z9,E12-14:Ac was injected 5 seconds after record started. In the displayed graph on Figure 4b, each series represents the evolution (through time) of the fluorescence intensity of one cell in the surveyed microscopic field. The vertical bar positioned at 5 seconds figures the injection time.
The observations performed according to this approach match the results from EAG and confirm that Z9,E12-14:Ac is the ligand activating SlitOR6.
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Claims

1 . A SlitOR6 polypeptide, which has an amino acid sequence having more than 60% homology with the amino acids sequence defined in SEQ ID NO.2 shown in Figure 1 b.
2. The polypeptide according to the claim 1 , which has the amino acid sequence defined in SEQ ID NO.2, shown in Figure 1 b.
3. A nucleic acid molecule, encoding the SlitOR6 polypeptide according to any of the claims 1 to 2.
4. The nucleic acid molecule according to claim 3, having more than 60% homology with the DNA sequence defined in SEQ ID N0.1 shown in Figure 1 a.
5. The nucleic acid molecule according to claim 4, which has the DNA sequence defined in SEQ ID N0.1 shown in Figure 1 a.
6. A vector, comprising the nucleic acid molecule according to claim 3 or 5.
7. A cell comprising the vector according to the claim 6.
8. A method for determining whether a ligand can specifically bind to the SlitOR6 polypeptide according to any of the preceding claims 1 to 2, said method comprising the steps of, contacting a cell or cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said SlitOR6 polypeptide with the ligand under conditions permitting binding of said ligand to said SlitOR6 polypeptide, and detecting the presence of any such ligand bound specifically to said SlitOR6 polypeptide, thereby determining whether the ligand binds specifically to said SlitOR6 polypeptide.
9. A method for determining whether a ligand can modulate the function of the SlitOR6 polypeptide according to any of the preceding claims 1 to 2, said method comprising the steps of, contacting a cell or cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said SlitOR6 polypeptide with the ligand under conditions permitting activation of said SlitOR6 polypeptide, and measuring a signaling activity of said SlitOR6 polypeptide.
10. The method according to claim 8 or 9, wherein said ligand is possibly an agonist or an antagonist of said SlitOR6 polypeptide.
1 1 . The method according to the claim 9, wherein the signaling activity is detected by means of a bio-assay, is a measurement of a second messenger selected from the group consisting of measurement of intracellular cAMP concentration, measurement of intracellular Inositol phosphate concentration, measurement of intracellular diacylglycerol concentration, measurement of intracellular calcium mobilisation or measurement of an increase in the SlitOR6 activity.
12. An antibody capable of binding to a SlitOR6 polypeptide which has more than 80% homology with an amino-acid sequence defined in SEQ ID NO.2 shown in Figure 1 b.
13. A method of identifying an agent that modulates the function of the SlitOR6 polypeptide according to claim 1 or 2, said method comprising: a) contacting said SlitOR6 polypeptide with Z9,E12-14:Ac in the presence or in the absence of a candidate modulator under conditions permitting the binding of said Z9,E12- 14:Ac to said SlitOR6 polypeptide; and b) measuring the binding of said SlitOR6 polypeptide to said Z9,E12-14:Ac, wherein a decrease or increase in binding in the presence of said candidate modulator, relative to the binding in the absence of said candidate modulator, identifies said candidate modulator as an agent that modulates the function of the SlitOR6 polypeptide.
14. A method of detecting in a sample the presence of an agent that modulates the function of the SlitOR6 polypeptide according to claim 1 or 2 in a sample, said method comprising: a) contacting said SlitOR6 polypeptide with Z9,E12-14:Ac in the presence or in the absence of said sample under conditions permitting the binding of said Z9,E12-14:Ac to said SlitOR6 polypeptide; and b) measuring the binding of said SlitOR6 polypeptide to said Z9,E12-14:Ac, wherein a decrease or increase in binding in the presence of said sample, relative to the binding in the absence of said sample, indicates the presence of an agent that modulates the function of said SlitOR6 polypeptide in said sample.
15. A method of identifying an agent that modulates the function of the SlitOR6 polypeptide according to claim 1 or 2, said method comprising: a) contacting said SlitOR6 polypeptide with Z9,E12-14:Ac in the presence or in the absence of a candidate modulator; under conditions permitting activation of said SlitOR6 polypeptide by Z9,E12-14:Ac, and b) measuring a signalling activity of said SlitOR6 polypeptide, wherein a change in the activity in the presence of said candidate modulator relative to the activity in the absence of said candidate modulator identifies said candidate modulator as an agent that modulates the function of said SlitOR6 polypeptide.
16. A method of identifying an agent that modulates the function of the SlitOR6 polypeptide according to claim 1 or 2, said method comprising: a) contacting said SlitOR6 polypeptide with a candidate modulator; b) measuring a signalling activity of said SlitOR6 polypeptide in the presence of said candidate modulator; and c) comparing said activity measured in the presence of said candidate modulator to said activity measured in a reaction in which said SlitOR6 polypeptide is contacted with Z9,E12-14:Ac at its EC50, wherein said candidate modulator is identified as an agent that modulates the function of said SlitOR6 polypeptide when the amount of said activity measured in the presence of said candidate modulator is at least 10% of the amount induced by said Z9,E12-14:Ac present at its EC50.
17. A method of detecting in a sample the presence of an agent that modulates the function of the SlitOR6 polypeptide according to claim 1 or 2, said method comprising: a) contacting said SlitOR6 polypeptide with Z9,E12-14:Ac in the presence or in the absence of said sample; b) measuring a signalling activity of said SlitOR6 polypeptide; and c) comparing the amount of said activity measured in a reaction containing said SlitOR6 polypeptide and Z9,E12-14:Ac without said sample to the amount of said activity measured in a reaction containing said SlitOR6 polypeptide, Z9,E12-14:Ac and said sample, wherein a change in said activity in the presence of said sample relative to the activity in the absence of said sample indicates the presence of an agent that modulates the function of said SlitOR6 polypeptide in said sample.
18. A method of detecting in a sample the presence of an agent that modulates the function of the SlitOR6 polypeptide according to claim 1 or 2, said method comprising: a) contacting said SlitOR6 polypeptide with said sample; b) measuring a signalling activity of said SlitOR6 polypeptide in the presence of said sample; and c) comparing said activity measured in the presence of said sample to said activity measured in a reaction in which said SlitOR6 polypeptide is contacted with Z9,E12-14:Ac present at its EC50, wherein an agent that modulates the function of said SlitOR6 polypeptide is detected if the amount of said activity measured in the presence of said sample is at least 10% of the amount induced by said Z9,E12-14:Ac present at its EC50.
19. The method of any of claims 13 to 18 wherein Z9,E12-14:Ac is replaced by a chemical equivalent.
20. The method according to claim 13 or 14, wherein said Z9,E12-14:Ac is detectably labelled.
21 . The method according to claim 20, wherein said molecule is detectably labelled with a moiety selected from the group consisting of a radioisotope, a fluorophore, and, a quencher of fluorescence.
22. The method according to any of claims 13 to 21 , wherein said contacting is performed in or on a cell expressing said SlitOR6 polypeptide.
23. The method according to any of claims 13 to 21 , wherein said contacting is performed in or on synthetic liposomes.
24. The method according to any of claims 13 to 21 , wherein said contacting is performed in or on virus-induced budding membranes containing said SlitOR6 polypeptide.
25. The method according to any of claims 13 to 21 , wherein said method is performed using a membrane fraction from cells expressing said SlitOR6 polypeptide.
26. The method according to any of claims 13 to 25, wherein said method is performed on a protein chip.
27. The method according to any of the claims 13 to 25, wherein said method is performed on a receptor incorporated in a Tethered Artificial Lipid Membrane System.
28. The method according to any of claims 13 to 27, wherein said measuring is performed using a method selected from label displacement, surface plasmon resonance, fluorescence resonance energy transfer, fluorescence quenching, and fluorescence polarization.
29. The method according to any of claims 13 to 28, wherein said agent is selected from the group consisting of a peptide, a polypeptide, an antibody or antigen-binding fragment thereof, a lipid, a carbohydrate, a nucleic acid, and a small organic molecule including but not limited to odorant compound and pheromone.
30. The method according to any of claims 15 to 18 and 22 to 29, wherein said step of measuring a signalling activity of said SlitOR6 polypeptide comprises detecting a change in the level of a second messenger.
31 . The method according to any of claims 15 to 18 and 22 to 30, wherein the step of measuring a signalling activity comprises measurement of guanine nucleotide binding/coupling or exchange, Protein Kinase C activity, protein Kinase A activity, phosphatidylinosotol breakdown, diacylglycerol, inositol triphosphate, intracellular calcium, calcium flux, arachinoid acid, MAP kinase activity, tyrosine kinase activity, receptor internalization assay or reporter gene expression.
32. The method according to claim 31 , wherein said signalling activity is measured using a fluorescence or luminescence assay.
33. A method of modulating the activity of a SlitOR6 polypeptide according to claim 1 or 2 in a cell, said method comprising the step of delivering to said cell Z9,E12-14:Ac that modulates the activity of a SlitOR6 polypeptide, such that the activity of SlitOR6 is modulated.
34. The method according to any of claims 13 to 33, wherein said method is a high throughput screening method.
35. The method according to any of claims 13 to 34, wherein said agent is part of a chemical library or living being extracts.
36. An Z9,E12-14:Ac-related agent identified or detected by a method according to any of claims 13 - 35.
37. Use of Z9,E12-14Ac14:Ac alone or in an attracting blend of at least 2 molecules, used for mass trapping of an insect target.
38. Use of Z9,E12-14Ac14:Ac alone or in an attracting blend of at least 2 molecules, used to create mating disruption.
39. Use of Z9,E12-14Ac14:Ac alone or in an attracting blend of at least 2 molecules, used for population survey, monitoring or estimation.
40. A composition comprising an agent identified according to any preceding method claim 8, 9, 13 to 18, used in one of the strategies quoted in claims 37-39.
41 . A kit comprising an isolated SlitOR6 polypeptide according to claim 1 or 2, Z9,E12-14:Ac and packaging materials therefore.
42. A kit comprising an isolated polynucleotide encoding a SlitOR6 polypeptide according to claim 1 or 2, Z9,E12-14:Ac and packaging materials therefore.
43. A kit comprising a cell expressing a SlitOR6 polypeptide according to claim 1 or 2 or membranes thereof, Z9,E12-14:Ac and packaging materials therefore.
44. The kit according to claim 43, wherein said cell is transformed with a polynucleotide encoding said SlitOR6 according to any one of claims 3 to 5.
45. Use of a kit according to any of claims 41 to 44, for screening agents that modulate the signalling activity of a SlitOR6 polypeptide according to claim 1 or 2.
46. Use of a kit according to any of claims 41 to 44, for screening attractants, repellents or anti-attractant agents.
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