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WO2005121755A1 - Analyse acellulaire permettant d'identifier le recepteur couple a la proteine g et son ligand - Google Patents

Analyse acellulaire permettant d'identifier le recepteur couple a la proteine g et son ligand Download PDF

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Publication number
WO2005121755A1
WO2005121755A1 PCT/AU2005/000845 AU2005000845W WO2005121755A1 WO 2005121755 A1 WO2005121755 A1 WO 2005121755A1 AU 2005000845 W AU2005000845 W AU 2005000845W WO 2005121755 A1 WO2005121755 A1 WO 2005121755A1
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Prior art keywords
protein
subunit
complex
alexa
gpcr
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PCT/AU2005/000845
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Edward John Mcmurchie
Wayne Richard Leifert
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Commonwealth Scientific And Industrial Research Organisation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4719—G-proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • GPCR Cell free G-protein coupled receptor
  • the present invention relates to compositions and methods for identifying G-protein- coupled receptors (GPCRs), ligands thereof and compounds that modulate G-protein signal transduction. More specifically, the invention relates to an assay system which involves the formation of a functional cell free complex comprising one or more G- protein subunits labeled with an energy donor or an energy acceptor and optionally a GPCR.
  • GPCRs G-protein- coupled receptors
  • the invention relates to an assay system which involves the formation of a functional cell free complex comprising one or more G- protein subunits labeled with an energy donor or an energy acceptor and optionally a GPCR.
  • G-protein-mediated signaling systems have been identified in many divergent organisms, such as mammals and yeast. GPCRs respond to, among other extracellular signals, neurotransmitters, hormones, odorants and light (Watson, S. and Arkinstall, S., The G- protein Linked receptor facts Book, Academic Press, London, 1994).
  • the family of GPCRs has been estimated to include up to a thousand members, fully more than 1.5% of all the proteins encoded in the human genome.
  • the GPCR family members play roles in regulation of biological phenomena involving virtually every cell in the body.
  • ligands and functions of many of these GPCRs are known, a significant portion of these identified receptors are without known ligands.
  • These latter GPCRs also generally have unknown physiological roles.
  • Several GPCRs have been characterised in detail and sequence analysis reveals that they are structurally similar, possessing a number of highly conserved amino acid residues.
  • GPCRs collectively form a large "superfamily" of receptor proteins capable of associating with the plasma membrane such that the N-terminal portion is localized in the extracellular space, the C-terminus is cytoplasmic, with there being seven transmembrane domains (i.e. 7-TM) in the GPCR polypeptide (see Figure
  • 7-TM seven transmembrane domains
  • Individual GPCR types activate particular signal transduction pathways. At least ten different signal transduction pathways are known to be activated via GPCR polypeptides.
  • the secretin receptor sub-family of GPCR polypeptides are activated by a ligand selected from the group consisting of: secretin, glucagon, calcitonin, glucagon-like peptide 1, parathyroid hormone, parathyroid-related peptide, corticotropin-releasing factor (CRF), growth hormone-releasing hormone (GHRH), gastric inhibitory polypeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and insect diuretic hormone (DHR).
  • a ligand selected from the group consisting of: secretin, glucagon, calcitonin, glucagon-like peptide 1, parathyroid hormone, parathyroid-related peptide, corticotropin-releasing factor (CRF), growth hormone-releasing hormone (GHRH), gastric inhibitory polypeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive
  • GPCRs mediate signal transduction across a cell membrane upon the binding of a ligand to an intra-membranous portion or an extracellular portion of a GPCR. Another intracellular portion of a GPCR interacts with G-proteins to modulate signal transduction from outside to inside a cell. A GPCR is therefore said to be "coupled” to G-proteins.
  • G-proteins are composed of three polypeptide subunits: an alpha subunit, which binds and hydrolyzes guanine triphosphate (GTP), and a dimeric beta plus gamma subunit. In the basal, inactive state, the G-proteins exist as a heterotrimer of the alpha, beta and gamma subunits.
  • GDP guanosine diphosphate
  • a ligand binds to a receptor and subsequently causes its activation, there is a resultant decrease in the affinity of the alpha subunit for GDP.
  • the alpha subunit exchanges GDP for GTP and active alpha subunit is reported to disassociate from both the receptor and the beta-gamma dimer.
  • the disassociated, active alpha, and beta and gamma subunits transduce signals to various effectors that are "downstream" in the G-protein signalling pathway within the cell.
  • the G alpha's endogenous GTPase activity returns active alpha subunit to its inactive state, in which it is associated with GDP and reassociation with the beta- gamma dimer occurs.
  • Neurosci., 20: 399-427 In fact, it has been estimated that more than 50% of the drugs in use clinically in humans at the present time are directed at GPCRs, including the adrenergic receptors (ARs).
  • ARs adrenergic receptors
  • ligands to beta ARs are used in the treatment of anaphylaxis, shock, hypertension, hypotension, asthma and other conditions.
  • GPCRs require agonist binding for activation
  • agonist-independent signaling activity has been well documented in the native form of a variety of GPCRs. This spontaneous activation of GPCRs occurs, in both normal and pathological processes, where a GPCR cellular response is generated in the absence of a ligand.
  • native dopamine DIB and prostaglandin EPlb receptors have been found to possess constitutive activity.
  • a number of GPCRs, for example, receptors for thyroid-stimulating hormone have mutants that exhibit agonist-independent activity and cause disease in humans.
  • Increased spontaneous activity and/or basal activity of GPCRs can be decreased by inverse agonism of the GPCRs.
  • Such methods are therapeutically important where diseases cause an increase in spontaneous GPCR activity, or where it is desirable to decrease the basal activity of GPCR.
  • a technology for identifying inverse agonists of native and mutated GPCRs has important pharmaceutical applications.
  • certain constitutively active GPCRs can be tumorigenic, the identification of inverse agonists for these GPCRs can lead to the development of anti- tumor and/or anti-cell proliferation drugs. These compounds have become increasingly important, especially for the treatment of psychological disorders such as depression and bipolar disorder.
  • Conventional assays are not particularly suited to reliably identify inverse agonists as the activity of the GPCRs in response to an inverse agonist cannot be directly measured.
  • GPCRs and G-protein signaling pathways are critical targets for therapeutics, there is a need for fast, effective and reproducible methods for identifying agonists, antagonists and inverse agonists that modulate G-protein signaling, and in particular compounds that regulate this signaling through a GPCR (such as the recently reported family of RGS (regulators of G-protein signalling) and AGS (activators of G-protein signalling) proteins (see Wieland and Mittmann (2003) Pharmacology and Therapeutics 97:95-115)).
  • GPCR such as the recently reported family of RGS (regulators of G-protein signalling) and AGS (activators of G-protein signalling) proteins (see Wieland and Mittmann (2003) Pharmacology and Therapeutics 97:95-115).
  • RGS regulatory of G-protein signalling
  • AGS activators of G-protein signalling
  • these assays require generation of particular downstream effectors (which vary depending on the specific GPCR of interest) in cell based systems.
  • mammalian (or insect) cells are used in these assay systems for convenience.
  • Such cells are relatively fragile, and there are numerous ways in which toxic compounds can interfere with signalling, giving rise to false positives or false negatives in ligand screening.
  • the present inventors have now found that reconstitution of a GPCR/G-protein complex in a cell free environment gives rise to a functional complex in which the G- proteins remain active when the receptor is stimulated by its cognate ligand.
  • the finding that reconstituted GPCR/G-protein complexes can function in this manner has led to the development of a sensitive and adaptable cell-free assay system for detecting GPCR activity.
  • primary event we mean binding of the ligand to the receptor, which in the case of an agonist or inverse agonist, leads to some form of change in the receptor moiety.
  • secondary event we mean the exchange of GDP for GTP which occurs on the G- alpha subunit.
  • tertiary event we mean any rearrangement or change in conformation of one or more G-protein subunits within the complex.
  • tertiary events include dissociation of the G-alpha subunit from the G-beta-gamma heterodimer; changes in the proximity of any one of the G-protein subunits to another G-protein subunit or to the GPCR, and any interaction that occurs between an RGS or AGS protein and a G-protein subunit.
  • the cell free assay system of the present invention enables detection of the tertiary events that occur as a result of stimulation of the GPCR which may occur in the presence or absence of a cognate ligand. Importantly, detection of these tertiary events can be achieved in a homogeneous assay system.
  • This assay system is therefore highly suitable for use in point of care diagnostic applications and for high-throughput drug screening using, for example, microarray, suspension bead, nanoparticle or biochip formats.
  • the present invention provides a method for determining the activity or function of a GPCR, the method comprising providing a cell free complex comprising a GPCR coupled to at least one G- protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor; and detecting a signal associated with a tertiary event involving at least one G- protein subunit in the complex.
  • the GPCR is an adrenergic receptor (for example, an alpha adrenergic or beta adrenergic receptor) or a muscarinic receptor (for example, an Ml, M2, M3 or M4 muscarinic receptor).
  • the GPCR may be an "orphan" GPCR.
  • the GPCR may be derived from any species including animals, plants, and microorganisms.
  • the G-protein coupled receptor/G-protein subunit complex comprises more than one type of G-protein subunit.
  • the complex comprises at least a G-alpha subunit, more preferably at least two G-protein subunits.
  • the complex comprises a G-alpha subunit, a G-beta subunit and a G-gamma subunit or any combination thereof.
  • the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta-gamma dimer.
  • the tertiary event is a change in proximity of the at least one G-protein subunit with respect to another G-protein subunit.
  • the tertiary event is dissociation of the G-alpha subunit from the G-beta-gamma heterodimer.
  • the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa.
  • the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor.
  • the G-beta subunit or G-gamma subunit is labeled with an energy donor and the G-alpha subunit is labeled with an energy acceptor.
  • two or more different energy donor/acceptor pairs can be used so that different changes in G-protein subunits can be detected simultaneously.
  • the tertiary event is an interaction that occurs between the at least one G-protein subunit and a protein that normally interacts with a G-protein subunit.
  • a protein that normally interacts with a G-protein subunit may be, for example, an RGS or AGS.
  • Representative RGS proteins include but are not limited to RGS1, RGS2, RGS4 and RGS 16. Indeed, any of the over 20 RGS proteins expressed in mammals can be employed in a system of the present invention. See Zeng et al., (1998) J. Biol. Chem. 273(52):34687-34690; Xu et al., (1999) J. Biol. Chem. 274(6):3549-3556; and Mukhopadhyay et al, (1999) Proc. Natl. Acad. Sci. USA " 96:9539-9544.
  • the G-protein subunit is labeled with an energy donor and the protein that normally interacts with a G-protein is labeled with an energy acceptor or vice versa.
  • energy donor and acceptor moieties can be used in the context of the present invention, differing in the physical nature of the detectable signal produced by the interaction of the donor and acceptor moieties.
  • the detectable signal may be a fluorescent or electrochemical signal, or may involve nuclear magnetic resonance (NMR) or electron paramagnetic resonance (EPR).
  • the detectable signal is a detectable spectral change.
  • the detectable spectral change may be a change in fluorescent decay time (determined by time domain or frequency domain measurement), fluorescent intensity, fluorescent quenching, fluorescent anisotropy or polarization; fluorescent correlation spectroscopy, a spectral shift of the emission spectrum; a change in time-resolved anisotropy decay (determined by time domain or frequency domain measurement), and the like.
  • Preferred energy donor and acceptors are those which display resonance energy transfer such as fluorescence resonance energy transfer (FRET), luminescence resonance energy transfer (LRET), and bioluminescence resonance energy transfer (BRET).
  • FRET fluorescence resonance energy transfer
  • LRET luminescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • the energy donor and acceptor moieties are fluorescent or luminescent donor and acceptor moieties.
  • Preferred energy donor and acceptor are Terbium/Alexa546, Terbium/fluorescein, Terbium/GFP,
  • TMR Terbiurn/tetramethylrhodarnine
  • Cy3 Terbium/QSY-7
  • Terbium/R phycoerythrin Europium/Cy5, Europium/Allophycocyanin (APC)
  • APC Europium/Alexa 633
  • Alexa 488/AIexa 555 Alexa 568/Alexa 647
  • Alexa 594/Alexa 647 Alexa 647/Alexa 594
  • Cy3/Cy5 BODIPY FL/BODIPY FL
  • Fluorescein/TMR IEDANS/fluorescein, and fluorescein fluorescein.
  • Still another class of useful energy donor and acceptor pairs include fluorophore- quencher pairs in which the second group is a quencher which decreases the fluorescence intensity of the fluorescent group.
  • Suitable quenchers include acrylamide groups, heavy atoms such as iodide and bromate, nitroxide spin labels such as TEMPO, and the like.
  • a suitable quencher may be selected from the group consisting of DABCYL, DABSYL, QSY 7, QSY 9, QSY 21, and QSY 35.
  • any suitable method may be used to label the G-protein subunit or the protein that normally interacts with a G-protein subunit with an energy donor and/or acceptor moiety.
  • the energy donor and/or acceptor moiety is attached by way of a fusion protein or amino acid motif present within the G- protein subunit or the protein that normally interacts with a G-protein.
  • the amino acid motif may be, for example, a lanthanide binding tag motif. This particular embodiment is described in detail in Example 5.
  • the cell free complex is tethered to a support.
  • the complex may be tethered to the support via the GPCR or any one of the G-protein subunits.
  • the complex is tethered to the support via one of the G-protein subunits.
  • the G-protein subunit tethered to the support may be the same as or different to the G-protein subunit which is associated with the detectable signal.
  • the G-alpha subunit is tethered to the support.
  • the G-protein subunit or GPCR may be tethered to the support by any suitable means.
  • the G-protein subunit or GPCR may be labeled with a polyhistidine tag and exposed to a nickel-coated support.
  • other forms of attachment such as the streptavidin-biotin system, the digoxin-antidigoxin system, myc, haemaglutin, GST or FLAG tags may be used.
  • the support may be a support matrix, a solid support such as a dish, tray, plate, multi-well plate, microtiter plate, bead, dendrimer, nanoparticle, nanobead, nano-tube, micro-chip, micro-well or arrays (including addressable arrays and micro-arrays).
  • the support may be a bibulous material including silica gel, hydrogel or other useful gels, cellulosic beads, glass and glass fibres, filter paper or natural and synthetic membranes such as nitrocellulose and nylon membranes.
  • the support may comprise glass, gold, mica, plastic or other polymeric material or gel elements fixed on a solid support.
  • the solid support is part of a biosensor device.
  • a biosensor may be defined as an analytical device which comprises a biological molecular recognition component, which device typically produces an electronic or other addressable signal dependent on the presence and/or concentration of an analyte interacting with the biological recognition component.
  • biosensor devices are well-known and are described, for example, in EP 0 341 927, EP 0 416 730 and EP 0453 224.
  • the cell free complex is stimulated prior to detection of the signal.
  • the cell free complex may be stimulated in any manner.
  • the complex may be stimulated by exposing the complex to a sample comprising a cognate ligand of the GPCR or by exposing the GPCR to an odor or taste.
  • spontaneous stimulation of the GPCR may occur in the absence of a cognate ligand.
  • the present invention also provides a method for determining the amount of a GPCR ligand in a sample, the method comprising providing a cell free complex comprising a GPCR coupled to at least one G- protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor; detecting a signal associated with a tertiary event involving at least one G- protein subunit in the complex; and determining the amount of ligand in the sample based on the level of signal detected.
  • the ligand may be an agonist, antagonist or inverse agonist of the GPCR.
  • the GPCR is a known receptor. In another embodiment, the GPCR is an "orphan" GPCR.
  • the present invention also provides a method of screening for an agent that modulates G-protein interactions, the method comprising exposing a candidate agent to a cell free complex comprising at least one G- protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor; and detecting a signal associated with a tertiary event involving at least one G- protein subunit in the complex.
  • the candidate agent may be an agonist, inverse agonist or antagonist of a ligand of a GPCR.
  • the candidate agent may be a G- protein regulator.
  • the complex comprises at least a G-alpha subunit, more preferably at least two G-protein subunits.
  • the complex comprises a G-alpha subunit, a G-beta subunit and a G-gamma subunit or any combination thereof.
  • the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta-gamma dimer.
  • the tertiary event is a change in proximity of the at least one G-protein subunit with respect to another G-protein subunit.
  • the tertiary event is dissociation of the G-alpha subunit from the G-beta-gamma heterodimer.
  • the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa.
  • the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor.
  • the G-beta subunit or G-gamma subunit is labeled with an energy donor and the G-alpha subunit is labeled with an energy acceptor.
  • two or more different energy donor/acceptor pairs can be used so that different changes in G-protein subunits can be detected simultaneously.
  • the tertiary event is an interaction that occurs between the at least one G-protein subunit and a protein that normally interacts with a G-protein subunit.
  • a protein that normally interacts with a G-protein subunit may be, for example, an RGS or AGS.
  • the G-protein subunit is labeled with an energy donor and the protein that normally interacts with a G-protein is labeled with an energy acceptor or vice versa.
  • the cell-free complex further comprises a GPCR coupled to the at least one G-protein subunit.
  • the GPCR may be a known receptor or an "orphan" GPCR.
  • the candidate agent tested in the method may be a ligand of the GPCR.
  • the candidate agent may be an agonist, inverse agonist or antagonist of a ligand of a GPCR and the method may involve contacting the candidate agent with the complex in the presence of a known ligand of the GPCR.
  • the present invention also provides a method for detecting a GPCR in a sample, the method comprising exposing a sample to a cell free G-protein complex comprising at least one G- protein subunit which is labeled with an energy donor or an energy acceptor, wherein an associated GPCR present in the sample is capable of coupling to the G-protein complex such that stimulation of the GPCR leads to generation of a detectable signal associated with a tertiary event involving at least one G-protein subunit in the complex, and detecting a signal associated with a change in at least one G-protein subunit in the complex.
  • stimulation of the GPCR is achieved by the presence in the sample of a cognate ligand of the GPCR.
  • additional components are included in the sample in order to enhance or improve the assay system.
  • RGS or AGS proteins may be exposed to the complex during the screening process.
  • a detectable signal is preferably associated with a tertiary event following stimulation of the GPCR/G- protein complex.
  • An advantage of this approach is that it allows detection of a generic signal that will be common to a broad range of GPCRs.
  • detection systems that are based on primary events, or on down-stream signalling of G-proteins generally need to be adapted to the specific GPCR of interest.
  • the detectable change involves dissociation of a G-alpha subunit from the G-beta and/or G-gamma subunits.
  • This dissociation may be detected by, for example, attaching energy donor and acceptor labels separately to either the G-alpha subunit or the G-beta-gamma heterodimer.
  • the detectable change involves a change in proximity of one of the G- protein subunits to another G-protein subunit or to the GPCR. This may be detected for example, by attaching energy donor and acceptor labels separately to the GPCR and one of the G-protein subunits or to two G-protein subunits.
  • the detectable change involves an interaction between an RGS or AGS protein and a G-protein subunit.
  • the present invention also provides a cell free complex comprising a GPCR coupled to at least one G-protein subunit, wherein the at least one G-protein is labeled with an energy donor or an energy acceptor; and wherein stimulation of the GPCR leads to a detectable tertiary event involving the at least one G-protein subunit in the complex.
  • the at least one G-protein subunit is a G-alpha subunit.
  • the complex comprises at least two G-protein subunits. More preferably, the complex comprises a heterotrimeric G-protein comprising a G-alpha subunit and a G-beta-gamma dimer. In a further preferred embodiment the G-alpha subunit is labeled with an energy donor and the G-beta subunit or G-gamma subunit is labeled with an energy acceptor or vice versa.
  • the cell free complex is tethered to a support.
  • the cell free complex is tethered to the support via a G-alpha subunit.
  • the support may be selected from the group consisting of a dish, tray, plate, multi-well plate, microtiter plate, bead, dendrimer, nanoparticle, nanobead, nano-tube, micro-chip, micro- well, microarray, silica gel, hydrogel, cellulosic bead, glass, filter paper, nitrocellulose and nylon membrane.
  • the solid support is part of a biosensor device.
  • the present invention also provides a composition comprising a plurality of cell free G- protein coupled receptor protein subunit complexes according to the present invention.
  • the present invention also provides a microarray comprising a plurality of cell free G- protein coupled receptor protein subunit complexes according to the present invention.
  • the present invention also provides a biosensor or bio-diagnostic device comprising at least one cell free G-protein coupled receptor protein subunit complex according to the first aspect and a means for detecting a signal associated with a change in at least one of the G-protein subunits in the complex.
  • the present invention provides a kit for screening for an agent that modulates the activity of a GPCR, the kit comprising a cell free complex comprising at least one G-protein subunit, wherein the at least one G-protein subunit is labeled with an energy donor or an energy acceptor.
  • the cell free complex comprises a heterotrimeric G-protein complex comprising a G-alpha subunit and a G-beta-gamma dimer.
  • the cell free complex further comprises a GPCR coupled to the at least on G-protein subunit.
  • the cell free complex is tethered to a support.
  • At least two components of the cell free complex are labeled, one with an energy donor and one with an energy acceptor.
  • the labels are attached to the at least two G-protein subunits.
  • the labels are fluorescent or luminescent probes.
  • Figure 1 Diagram showing the mode of action of GPCRs (adapted from Marinissen and Gutkind (2001) Trends in Pharmacological Sciences 22:368-375)
  • Figure 2 Schematic representation of a preferred embodiment of the invention showing a cell free GPCR/G-protein complex tethered to a support, wherein binding of a cognate ligand to the GPCR/G-protein complex leads to generation of a detectable signal.
  • Figure 3 Schematic representation showing use of a cell free complex of the present invention to assay for compounds that modulate GPCR signalling.
  • Figure 4 Schematic representation of a preferred method for identifying unknown GPCRs in a sample.
  • Figure 5 Schematic representation of a preferred method for identifying modulators of G-protein signaling, (i) unknown modulators of G-subunit interactions, (ii) RGS moieties that interfere with G subunits, (iii) AGS moieties that interfere with receptor and G protein interactions.
  • Figure 6 Reconstitution of c2A-AR/G-protein complex (transductosome) - dependence on G-protein subunits.
  • Sf9 membranes extracted with 7 M urea (M) expressing the ⁇ 2A-AR were reconstituted with 50 nM Gail (filled squares), 50 nM G il and 50 nM ⁇ l ⁇ 2 (filled circles) or no G-proteins (open and filled triangles) as indicated.
  • ⁇ 2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the partial ⁇ 2A-AR agonist, clonidine (100 ⁇ M).
  • 35 S-GTP (0.2 nM) binding was assayed at the indicated time points at 26°C. Data are the means of duplicate determinations.
  • Figure 7 Concentration dependence of GDP on 35 S-GTP binding in the a2A-AR/G- protein complex.
  • Sf9 membranes extracted with 7 M urea expressing the ⁇ 2A-AR were reconstituted with 52 nM Gail and 3 nM ⁇ l ⁇ 2.
  • ⁇ 2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the partial ⁇ 2A-AR agonist, clonidine (100 ⁇ M), with increasing concentrations of GDP as indicated.
  • S- GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means ( ⁇ SEM) of triplicate determinations. Numbers in parentheses represent fold stimulation.
  • Figure 8 Effect of the a2A-AR agonist on 35 S-GTP binding in the a2A-AR/G-protein complex.
  • Sf9 membranes extracted with 7 M urea expressing the ⁇ 2A-AR were reconstituted with 52 nM Gail and 3.1 nM ⁇ l ⁇ 2.
  • the reconstituted ⁇ 2A-AR membranes (0.4 mg/ml) were incubated with various concentrations of the alpha 2A- AR agonist UK 14303 ("UK") (filled symbols) as indicated + the ⁇ 2A-AR antagonist rauwolscine (500 ⁇ M) (open symbols).
  • 35 S-GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means ( ⁇ SEM) of triplicate determinations. At some points, the SEMs are too small to be seen (as in other figures).
  • Figure 9 Concentration dependence of ⁇ l ⁇ l on 3i 'S-GTP binding in the c ⁇ 2A-ARJG- protein complex.
  • Sf9 membranes extracted with 7 M urea expressing the ⁇ 2A-AR were reconstituted with 52 nM Gail and increasing concentrations of ⁇ l ⁇ 2 as indicated.
  • ⁇ 2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the ⁇ 2A-AR agonist, UK (10 ⁇ M).
  • 35 S-GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means ( ⁇ SEM) of triplicate determinations.
  • Figure 10 Concentration dependence of Goal on 35 S-GTP binding in the 2A-AR/G- protein complex.
  • Sf9 membranes extracted with 7 M urea expressing the ⁇ 2A-AR were reconstituted with 3.1 nM ⁇ l ⁇ 2 and increasing concentrations of Gail as indicated.
  • ⁇ 2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the ⁇ 2A-AR agonist, UK (10 ⁇ M).
  • 35 S-GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means ( ⁇ SEM) of triplicate determinations. Numbers in parentheses represent fold stimulation.
  • FIG. 11 Concentration dependence ofhis-tagged Goal on S-GTP binding in the ct2A ⁇ AR/G-protein complex.
  • Sf9 membranes extracted with 7 M urea expressing the ⁇ 2A-AR were reconstituted with 20 nM ⁇ l ⁇ 2 and increasing concentrations of His6- tagged Gail (Gail (his)) as indicated.
  • ⁇ 2A-AR membranes (0.4 mg/ml) were incubated with (solid symbols) or without (open symbols) the ⁇ 2A-AR agonist, UK (100 ⁇ M).
  • 35 S-GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means ( ⁇ SEM) of triplicate determinations.
  • Figure 12 Dependence of G-protein subunits on UK-stimulated 35S-GTP binding in the cc2A-AR/G-protein complex.
  • Sf9 membranes extracted with 7 M urea expressing the ⁇ 2A-AR were reconstituted with combinations of His6-tagged, non His ⁇ -tagged G- protein subunits, or no addition of G-proteins as indicated.
  • a2A-AR membranes (0.4 mg/ml) were incubated in the absence (open bars)or presence (solid bars) of the ⁇ 2A-AR agonist, UK (100 ⁇ M) as indicated ("UK"). 35 S- GTP (0.2 nM) binding was assayed at 60 minutes. Data are the means (+ SEM) of triplicate determinations.
  • Figure 13 Ni + specificity for capture of His6-tagged G-protein/ a2A-AR transductosome complex.
  • Sf9 membranes extracted with 7 M urea expressing the ⁇ 2A- AR were reconstituted with 10 nM Gail (his) and 20 nM ⁇ l ⁇ 2 (final concentrations in assay).
  • Ni-NTA beads 50 ⁇ l of Ni-NTA beads was added and further incubated for 60 minutes prior to the binding assay.
  • the Ni-NTA beads with captured ⁇ 2A-AR transductosomes were incubated with (solid bars) or without (open bars) the ⁇ 2A-AR agonist, UK (100 ⁇ M) and 35 S-GTP (0.2 nM) binding was assayed at 60 minutes by filtration through Whatman filter paper.
  • FIG. 14 Ni specific capture of His6-tagged G-protein/ a2A-AR transductosome.
  • Sf9 membranes extracted with 7 M urea expressing the ⁇ 2A-AR were reconstituted with 10 nM G il (his) and 20 nM ⁇ l ⁇ 2 (final concentrations in assay).
  • 0 - 50 ⁇ l of Ni- NTA beads were added and further incubated for 60 minutes prior to the binding assay.
  • the Ni-NTA beads with captured ⁇ 2A-AR transductosomes was incubated with (solid symbols) or without (open symbols) the ⁇ 2A-AR agonist, UK (100 ⁇ M) and 35 S-GTP (0.2 nM) binding was assayed at 60 minutes by filtration over Whatman filter paper. Data are the means of duplicate determinations.
  • Figure 15 Saturation binding of [ 3 HJ-Scopolamine. Specific binding of the muscarinic receptor antagonist [ 3 H]-Scopolamine to Sf9 cell membranes overexpressing M 2 -muscarinic receptors. The assay was carried out with triplicate determinations using 5 ⁇ g membrane protein at 28°C for 60min in the absence or presence of lOO ⁇ M atropine. Non-specific binding was less than 5%.
  • Figure 16 Competition of H-Scopolamine binding. Inhibition of specific H- Scopolamine binding in urea-treated Sf9 cell membranes overexpressing M 2 - muscarinic receptors by the non-specific muscarinic receptor antagonist atropine, M ⁇ - muscarinic receptor antagonist pirenzepine or the muscarinic receptor agonist carbachol. The assay was carried out with triplicate determinations using 5 ⁇ g membrane protein at 28°C for 60min with lnM 3 H-scopolamine.
  • FIG. 18 Effect of the M ⁇ -muscarinic receptor agonist carbachol on 35 S-GTP binding in the M R/G-protein complex with 25 ⁇ M GDP.
  • Sf9 membranes extracted with 7 M urea overexpressing M 2 -muscarinic receptors (M 2 -R) were reconstituted with 5 nM Gocii(his) and 5 nM ⁇ 1 ⁇ 2 .
  • the reconstituted M 2 R membranes were incubated with various concentrations of carbachol (filled symbols) as indicated + the non-specific muscarinic receptor antagonist atropine (500 ⁇ M) (open symbol).
  • 35 S-GTP (0.2 nM) binding was assayed at 90 min (28°C) in the presence of 25 ⁇ M GDP, lO ⁇ M AMP and 5ug membrane protein per assay (25 ⁇ l). Data are the means ( ⁇ SEM) of triplicate determinations. The number in parentheses represents the fold stimulation above basal. Appropriate blanks have been subtracted (ie membranes with no G-proteins).
  • Figure 19 Effect of the M 2 -muscarinic receptor agonist carbachol on 3t 'S-GTP binding in the M 2 R/G-protein complex attached to Ni 2+ -NTA coated beads with 25 ⁇ M GDP.
  • Sf9 membranes extracted with 7 M urea overexpressing M 2 -muscarinic receptors (M 2 - R) were reconstituted with 5 nM G ⁇ u(his) and 5 nM ⁇ 1 ⁇ 2 .
  • the reconstituted M 2 R membranes were incubated with lO ⁇ l of Ni-NTA beads and various concentrations of carbachol (filled symbols) as indicated ⁇ the non-specific muscarinic receptor antagonist atropine (500 ⁇ M) (open symbols) 35 S-GTP (0.2 nM) binding was assayed at 90 min (28°C) in the presence of 25 ⁇ M GDP, lO ⁇ M AMP and 20ug membrane protein per assay (lOO ⁇ l). Samples were filtered over Whatman #1 paper filters. The number in parentheses represents the fold stimulation above basal. Appropriate blanks have been subtracted.
  • Figure 20 Effect of the M 2 -muscarinic receptor agonist carbachol on 3 'S-GTP binding in the M 2 R G-protein complex with I ⁇ M GDP.
  • Sf9 membranes extracted with 7 M urea overexpressing M 2 -muscarinic receptors (M 2 R) were reconstituted with 5 nM G ⁇ ( i s ) and 5 nM ⁇ 2 .
  • the reconstituted M 2 R membranes were incubated with various concentrations of carbachol (filled symbols) as indicated ⁇ the non-specific muscarinic receptor antagonist atropine (500 ⁇ M) (open symbol).
  • Figure 21 Effect of the M 2 -muscarinic receptor agonist carbachol on 3 'S-GTP binding in the M 2 R/G-protein complex attached to Ni 2+ -NTA coated beads with I ⁇ M GDP.
  • Sf9 membranes extracted with 7 M urea overexpressing M 2 -muscarinic receptors (M 2 - R) were reconstituted with 5 nM and 5 nM ⁇ r ⁇ 2 .
  • the reconstituted M 2 R membranes were incubated with lO ⁇ l of Ni-NTA beads and various concentrations of carbachol (filled symbols) as indicated + the non-specific muscarinic receptor antagonist atropine (500 ⁇ M) (open symbol) 35 S-GTP (0.2 nM) binding was assayed at 90 min (28°C) in the presence of l ⁇ M GDP, lO ⁇ M AMP and 20ug membrane protein per assay (lOO ⁇ l). Samples were filtered over Whatman #1 paper filters. The number in parentheses represents the fold stimulation above basal. Appropriate blanks have been subtracted.
  • Figure 22 Agonist (UK14304) affinity and Ni 2+ -bead capture of ci 2A -AR-activated 35 S ⁇ GTP:G ⁇ iihis.
  • Various concentrations of UK14304 were incubated with 0.2 mg/ml ⁇ 2A -AR containing reconstituted G ⁇ ⁇ his (50 nM) and ⁇ 2 (50 nM), 5 ⁇ M GDP; 10 ⁇ M AMPNP and 0.2 nM 35 S ⁇ GTP in the absence (D) or presence ( ⁇ ) of 10 ⁇ l Ni(NTA) agarose beads. The mix was incubated for 90 minutes at 27°C with shaking.
  • IC50 values for each of the antagonists was determined as 0.051 ⁇ M (rauwolscine: selective ⁇ 2-AR antagonist); 0.080 ⁇ M (yohimbine: selective ⁇ 2-AR antagonist); 8.3 ⁇ M (prazosin: selective ⁇ l-AR antagonist) and 86.9 ⁇ M (propranolol: ⁇ -AR antagonist).
  • Figure 24 Effect of conjugation of G-protein alpha subunits with TMR fluorescent probes on a2-AR signalling activity in a cell-free, reconstituted assay.
  • Gail (his) and ⁇ l ⁇ 2 G-protein subunits were labelled with the fluorescent probes TMR- NCS or TMR-maleimide.
  • ⁇ 2A-AR membranes (0.05 mg/ml final concentration in assay) were reconstituted with the fluorescent labelled G-protein subunits at 20 nM each (final concentration).
  • lO ⁇ L Ni-NTA beads were added and incubated for 10 minutes prior to the binding assay.
  • the beads with captured transductosomes were incubated in the absence (basal condition, open bars) or presence (solid bars) of the ⁇ 2A-AR agonist, UK (10 ⁇ M) or in the presence (stipled bars) of the ⁇ 2A-AR antagonist yohimbine (lOOuM), 35 S-GTP (0.2 nM) binding was assayed at 90 minutes. Data are the means ( ⁇ SEM) of triplicate determinations.
  • Figure 25 Effect of conjugation of G-protein alpha subunits with Terbium fluorescent probes on a2-AR signalling activity in a cell-free, reconstituted assay.
  • Gail (his) g- protein subunits (derived from a single infection or a triple infection with ⁇ l ⁇ 2) were labelled with the fluorescent probes Tb-mal or Tb-NCS. The subunits were stored in buffer with or without glycerol as indicated.
  • ⁇ 2A-AR membranes (0.05 mg/ml final concentration in assay) were reconstituted with the fluorescent labelled Gail (his) and unlabelled ⁇ l ⁇ 2 G-protein subunits at 20 nM each (final concentration).
  • the reconstituted membranes were incubated in the absence (basal condition, open bars) or presence (solid bars) of the ⁇ 2A-AR agonist, UK (10 ⁇ M). Signalling activity measured by 35 S-GTP (0.2 nM) binding was assayed at 90 minutes. Data are the means of duplicate determinations.
  • Figure 26 Effect of conjugation of G-protein beta/gamma subunits with Terbium fluorescent probes on A ⁇ AR signalling activity in a cell-free, reconstituted assay
  • ⁇ l ⁇ 2 G-protein subunits (derived from a triple infection with G il (his)) were labelled with the fluorescent probes Tb-Mal or Tb-NCS.
  • the subunits were stored in buffer with or without glycerol as indicated.
  • ⁇ 2A-AR membranes (0.05 mg/ml final concentration in assay) were reconstituted with unlabelled Gail (his), Terbium labelled ⁇ l ⁇ 2, unlabelled ⁇ l ⁇ 2 and unlabelled ⁇ 4 ⁇ 2 G-protein subunits at 20 nM each (final concentration).
  • the reconstituted membranes were incubated in the absence (basal condition, open bars) or presence (solid bars) of the ⁇ 2A-AR agonist, UK (10 ⁇ M). Signalling activity measured by 35 S-GTP (0.2 nM) binding was assayed at 90 minutes. Data are the means of duplicate determinations.
  • Figure 27 Effect of conjugation of G-protein alpha and beta/gamma subunits with fluorescent probes on A2AR signalling activity in a cell-free, reconstituted assay.
  • ⁇ 2A- AR membranes (at 0.05mg/ml final assay concentration) were reconstituted with fluorescent labelled Gail (his) and ⁇ l ⁇ 2 G-protein subunits at a 20 nM each (final concentration). The reconstituted membranes were incubated in the absence (basal condition, open bars) or presence (solid bars) of the ⁇ 2A-AR agonist, UK (10 ⁇ M).
  • A Signalling activity following reconstitution with Gail (his) labelled with Tb-mal and ⁇ l ⁇ 2 labelled with TMR (maleimide form).
  • FIG. 28 Time resolved fluorescence resonance energy transfer (TR-FRET) between lOnM G ,n ns labelled with Terbium chelate (Ga, ⁇ profession s Tb) and WnM ⁇ 4j2 labelled with Alexa-546 ( ⁇ Alexa).
  • TR-FRET Time resolved fluorescence resonance energy transfer
  • the excitation and emission filters used were 340nm and 570nm, respectively. Following a 50 ⁇ s delay, emission data were collected for a period of 1400 ⁇ s.
  • G-protein samples were diluted from 1-lO ⁇ M stocks in 50mM TRIS pH 8.0, lOmM MgCl 2 , 50mM NaCl and ImM dithiothreitol to a final volume of lOO ⁇ l in black 96-well plates at 23°C.
  • 5 ⁇ l unlabelled was added (200nM final) as indicated by the vertical line. Background fluorescence at 570nm due to G ⁇ ,ih ⁇ sTb alone is shown (o). There was no significant background fluorescence of ⁇ 4 ⁇ 2 labelled with Alexa-546 under time-resolved conditions (data not shown).
  • FIG. 29 Time resolved fluorescence resonance energy transfer (TR-FRET) between 3nM Go t i fus labelled with Terbium chelate (G , ⁇ , ⁇ s Tb) and 8nM ⁇ 4 f 2 labelled with Alexa-546 ⁇ iAlexa) (•).
  • TR-FRET Time resolved fluorescence resonance energy transfer
  • the excitation and emission filters used were 340nm and 570nm, respectively. Following a 50 ⁇ s delay, emission data were collected for a period of 1400 ⁇ s.
  • G-protein samples were diluted from 1-1 O ⁇ M stocks in 50mM TRIS pH 8.0, lOmM MgCl 2 , 50mM NaCl and ImM dithiothreitol to a final volume of lOO ⁇ l in black 96-well plates at 23°C.
  • 5 ⁇ l unlabelled ⁇ ⁇ 2 was added (200nM final) as indicated by the vertical line. Background fluorescence at 570nm due to G ⁇ h i s Tb alone is shown (o). There was no significant background fluorescence of ⁇ 4 ⁇ 2 labelled with Alexa-546 under time-resolved conditions (data not shown).
  • FIG. 30 Time resolved fluorescence resonance energy transfer (TR-FRET) between 50nM Gauhi labelled with Terbium chelate (G ,i h j S Tb) and 50nM ⁇ 4/2 labelled with Alexa-546 ( ⁇ 4 ⁇ 2Alexa) (•).
  • TR-FRET Time resolved fluorescence resonance energy transfer
  • the excitation and emission filters used were 340nm and 570nm, respectively. Following a 50 ⁇ s delay, emission data were collected for a period of lOO ⁇ s.
  • G-protein samples were diluted from l-10 ⁇ M stocks in 50mM TRIS pH 8.0, lOmM MgCl 2 , 50mM NaCl and ImM dithiothreitol to a final volume of lOO ⁇ l in black 96-well plates at 23°C.
  • A1F 4 " ( ⁇ ) was added (as indicated by the vertical line) to dissociate subunits.
  • Background fluorescence at 570nm due to G ⁇ s Tb (o) or G ⁇ i lh i S Tb + A1F 4 " (D) is shown. There was no significant background fluorescence of ⁇ 4 ⁇ 2 Alexa under time-resolved conditions (data not shown). Data are shown in duplicate.
  • FIG 31 Time resolved fluorescence resonance energy transfer (TR-FRET) between 50nM Gau h i s labelled with Alexa-546 (G u hts Alexa) and lOnM ⁇ 4 ⁇ 2 labelled with Terbium chelate ( ⁇ 4 ⁇ ⁇ Tb) (•).
  • TR-FRET Time resolved fluorescence resonance energy transfer
  • the excitation and emission filters used were 340nm and 570nm, respectively. Following a 50 ⁇ s delay, emission data were collected for a period of lOO ⁇ s.
  • G-protein samples were diluted from l-10 ⁇ M stocks in 50mM TRIS pH 8.0, lOmM MgCl 2 , 50mM NaCl and ImM dithiothreitol to a final volume of lOO ⁇ l in black 96-well plates at 23 °C.
  • A1F " was added to dissociate subunits, as indicated by the vertical line. Background fluorescence at 570nm due to ⁇ 4 ⁇ 2 Tb (o) or ⁇ 4 ⁇ 2 Tb + A1F 4 " (D) is shown. There was no significant background fluorescence of G ⁇ h i s Alexa under time-resolved conditions (data not shown). Data are shown in duplicate.
  • Figure 32 Time Resolved Resonance Energy Transfer between GocuhisTb (the concentrations as indicated next to each plot) + ⁇ 4 ⁇ 2 Alexa.
  • K S (nM) for each concentration are shown in parentheses.
  • the excitation and emission filters used were 340nm and 572nm, respectively. Following a 50 ⁇ s delay, emission data were collected for a period of 900 ⁇ s.
  • TMND 50mM TRIS pH 8.0, lOmM MgCl 2 , 50mM NaCl and ImM dithiothreitol
  • FIG 33 Time resolved fluorescence resonance energy transfer (TR-FRET) between 5 OnM G uhis labelled with Terbium chelate (Gau his Tb) and l50nMRGS4 (his) labelled with Alexa546 (RGS4 his Alexa) (•) or 50nM ⁇ 4 ⁇ labelled with Terbium ( ⁇ Y 2 Tb) and RGS4hisAlexa (A.).
  • TR-FRET Time resolved fluorescence resonance energy transfer
  • G-protein or RGS4 h i S samples were diluted from l-10 ⁇ M stocks in 50mM TRIS pH 8.0, lOmM MgCl 2 , 50mM NaCl and ImM dithiothreitol to a final volume of lOO ⁇ l in black 96-well plates at 23°C.
  • 5 ⁇ l unlabelled Gauhis was added (lOOOnM final) as indicated by the vertical line. Background fluorescence at 570nm due to G ⁇ h isTb (o) or ( ⁇ 4 ⁇ 2 Tb) ( ⁇ ) is shown. There was no significant background fluorescence of RGS4 h i S Alexa under time-resolved conditions (data not shown).
  • FIG. 34 Time Resolved Fluorescent Resonance Energy Transfer (TR-FRET) between RGS4 ⁇ ,i s Alexa (concentrations as indicated next to each plot) + 50nM Gau ht sTb after a 5 minute incubation period.
  • TR-FRET Time Resolved Fluorescent Resonance Energy Transfer
  • the excitation and emission filters used were 340nm and 572nm, respectively.
  • emission data were collected for a period of 900 ⁇ s.
  • G-protein or RGS4hi S samples were diluted from 1- lO ⁇ M stocks in 50mM TRIS pH 8.0, lOmM MgCl 2 , 50mM NaCl and ImM dithiothreitol to a final volume of lOO ⁇ l in black 96-well plates at 23 °C. Experiments were carried out in triplicate.
  • FIG 35 Time resolved fluorescence resonance energy transfer (TR-FRET) between 70nM RGS4] lis Alexa and 40nM Gau if b in the inactive GDP bound state (L), in the activated GTPyS bound state ( ) and in the transitional state mimicked in the presence of aluminium fluoride (a).
  • TR-FRET Time resolved fluorescence resonance energy transfer
  • the excitation and emission filters used were 340nm and 570nm, respectively. Following a 50 ⁇ s delay, emission data were collected for a period of 900 ⁇ s.
  • TMND 50mM TRIS pH 8.0, lOmM MgCl 2 , 50mM NaCl and ImM dithiothreitol
  • Agent as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering (i.e., eliciting or inhibiting) or mimicking a desired physiological function of a GPCR.
  • Antagonist refers to a molecule or substance that binds to or otherwise interacts with a receptor or enzyme to increase activity of that receptor or enzyme.
  • Antagonist refers to a molecule that binds to or otherwise interacts with a receptor or enzyme to inhibit the activation of that receptor or enzyme.
  • Inverse agonist refers to a molecule that binds to or otherwise interacts with a receptor to inhibit the basal activation of that receptor or enzyme.
  • Biosensor refers to an analytical device incorporating a biological material intimately associated with or integrated within a physicochemical transducer or transducing microsystem, which may be optical, electrochemical, thermometric, piezoelectric or magnetic.
  • the usual aim of a biosensor is to produce either discrete or continuous digital electronic signals which are proportional to a single analyte or a related group of analytes.
  • a “cell free” complex” refers to a complex that is not located in the membrane of a functional cell. Preferably the complex is located in a portion of the membrane of the cell from which it is isolated.
  • a “complex" of the present invention may include one or more G-protein subunits and optionally may also include a GPCR.
  • Dye refers to a molecule or part of a compound that absorbs specific frequencies of light, including but not limited to ultraviolet light.
  • the terms “dye” and “chromophore” are synonymous.
  • an "energy donor” is a molecule that, upon absorbing light, can transfer excitation energy to an energy acceptor or a quencher. This energy transfer can occur when the absorption spectrum of an energy acceptor overlaps the emissions spectrum of the energy donor. Other mechanisms also allow energy transfer when the acceptor is a quencher. In both cases, the light emitted by the donor is quenched. However, if the excitation energy is transferred to an acceptor, the acceptor will emit light at its own characteristic emission wavelength spectrum, whereas if the energy is transferred to a quencher, there is no secondary light emission.
  • An "energy acceptor” is a molecule that can accept excitation energy transferred by an energy donor and use the transferred energy to emit light at its own characteristic emission wavelength spectrum.
  • a "quencher” is a molecule that can accept energy from an energy donor, thereby reducing emitted signal of the donor.
  • G-protein coupled receptors and “GPCRs” as used interchangeably herein include but are not limited to all subtypes of the opioid, muscarinic, dopamine, adrenergic, adenosine, rhodopsin, angiotensin, serotonin, thyrotropin, gonadotropin, substance-K, substance-P and substance-R receptors, melanocortin, metabotropic glutamate, or any other GPCR known to couple via G-proteins.
  • This term also includes orphan receptors which are known to couple to G-proteins, but for which no specific ligand is known.
  • the term also encompasses biologically active or immunogenic fragments of GPCRs that are suitable for use in assays for detecting GPCR activity.
  • G-protein subunit as used herein can refer to any of the three subunits, alpha, beta or gamma that form the heterotrimeric G-proteins.
  • the term also refers to a subunit of any class of G-protein, e.g., Gs, Gi /Go, Gq and Gz, and also includes recombinant G- protein constructs such as promiscuous and chimeric G-proteins.
  • Representative G proteins include but are not limited to G q .alpha., G q- beta., G q- gamma., G ⁇ .alpha., G 12/ ⁇ 3 .
  • G alpha. G 12/13- beta., Gum-gamma., Gj-alpha., Gj.beta., Gj-gamma., G s .alpha., G s .beta., Gs-gamma., G-alpha 14 and G-alpha 16 , various G-beta-gamma. dimers, and combinations thereof.
  • a specific subunit e.g., G alpha
  • G alpha is intended to encompass that subunit in each of the different classes, unless the class of G-protein is specifically otherwise specified.
  • Ligand refers to a naturally occurring or synthetic compound that binds to a protein receptor. Upon binding to a receptor, ligands generally lead to the modulation of activity of the receptor.
  • the term is intended to encompass naturally occurring compounds, synthetic compounds and/or recombinanfly produced compounds. As used herein, this term can encompass agonists, antagonists, and inverse agonists.
  • protein that normally interacts with a G-protein subunit means a protein that is normally associated with the G-protein subunit, as the G-protein subunit exists in the cell.
  • proteins can be those that permit the G-protein subunit to mediate its various biological activities, and can also be those having roles that have not been clearly implicated in GPCR activity, yet are found in close spatial proximity to a G-protein subunit at a given point in time.
  • proteins that normally interact with G- protein subunits are RGS and AGS proteins.
  • receptor refers to a protein normally found on the surface of a cell which, when activated, leads to a signaling cascade in a cell.
  • tethered refers to a physical or chemical modification of a biological component that leads to attachment of the component to a support.
  • the signal associated with a change in at least one G-protein is detected by a proximity based assay.
  • a "proximity based" assay refers to an assay that employs an energy donor and an energy acceptor, wherein donors and acceptors are understood to mean members of a class of biological or chemical substances which can interact with one another within spatial vicinity, such as for example photosensitizers and chemiluminescers, photosensitizers and fluorophores, or fluorophores and fluorophores, or fluorophores and quenchers.
  • the transfer of energy may be by means of light, electron radiation or non-radiative transfer and also by reactive chemical molecules.
  • fluorescence quenching as a measurement method, for example, it is known in the art that the fluorescence of a fluorochrome is quenched when a dye that quenches the light emitted by the fluorochrome is about 50 angstroms or closer to the fluorochrome (the spatial vicinity of influence is defined with reference to the Forster radius which differs between respective FRET pairs and is known to be influenced by the sixth power of the difference in distance of FRET pairs).
  • FRET is the phenomenon whereby a fluorescent molecule - the donor - transfers energy by a nonradiative mechanism to a neighbouring chromophore - the acceptor.
  • the absorption spectrum of the acceptor chromophore must overlap with the fluorescence emission spectrum of the donor. When the spectral overlap is more extensive, the efficiency of the FRET process will be higher and consequently, FRET can occur over longer distances.
  • FRET is highly dependent on the proximity between the donor and acceptor, and, in general, only occurs when the molecules are separated by ⁇ 100 A. It is characterized by the F ⁇ rster radius (Ro). Ro values for common donor-acceptor pairs usually vary between 30 and 60 A and can be determined experimentally. They depend on the spectral overlap, relative orientation of the donor and acceptor chromophores, and the quantum yield of the donor fluorophore.
  • the degree of FRET can be determined by any spectral or fluorescence lifetime characteristic of the excited donor moiety. For example, intensity of the fluorescent signal from the donor, the intensity of fluorescent signal from the acceptor, the ratio of the fluorescence amplitudes near the acceptor's emission maxima to the fluorescence amplitudes near the donor's emission maximum, or the excited state lifetime of the donor can be monitored.
  • changes in the degree of FRET are determined as a function of the change in the ratio of the amount of fluorescence from the donor and acceptor moieties, a process referred to as "ratioing".
  • ratioing Changes in the absolute amount of indicator, excitation intensity, and turbidity or other background absorbances in the sample at the excitation wavelength affect the intensities of fluorescence from both the donor and acceptor approximately in parallel. Therefore the ratio of the two emission intensities is a more robust and preferred measure of disassociation than either intensity alone.
  • the donor moiety may be excited by light of appropriate intensity within the excitation spectrum of the donor moiety.
  • the donor moiety preferably emits the absorbed energy as fluorescent light.
  • the acceptor fluorescent protein moiety is positioned to quench the donor moiety in the excited state, the fluorescence energy is transferred to the acceptor moiety which may emit fluorescent light.
  • FRET can be manifested as a reduction in the intensity of the fluorescent signal from the donor moiety, reduction in the lifetime of the excited state of the donor moiety, or emission of fluorescent light at the longer wavelengths (lower energies) characteristic of the acceptor moiety.
  • the fluorescent protein moieties come closer together (or physically separate), and FRET is increased (or decreased) accordingly.
  • Fluorescent molecules and quenchers are well known in the art, as are methods of binding fluorophores and quenchers to other molecules, for example, by coupling through active groups. Fluorophores and quenchers can also be indirectly bound to a target molecule, for example, through binding of a specific binding member that is coupled to a fluorophore.
  • the energy donor and acceptor moieties are fluorescent or luminescent donor and acceptor moieties.
  • Preferred energy donor and acceptor are Terbium/Alexa546, Terbium/FlAsH-(EDT2), Terbium/fluorescein, Terbium/GFP, Terbium tetramethylrhodamine (TMR), Terbium/Cy3, Terbium/QSY-7, Terbium/R phycoerythrin, Europium/Cy5, Europium/Allophycocyanin (APC), Europium/Alexa 633, Alexa 488/Alexa 555, Alexa 568/Alexa 647, Alexa 594/Alexa 647, Alexa 647/Alexa 594, Cy3/Cy5, BODIPY FL/BODIPY FL, Fluorescein TMR, IEDANS/fluorescein, and fluorescein/fluorescein.
  • lanthanides which can be used as energy donors include Dysprosium and Samarium. These energy donors can be used in combination with acceptors such as Alexa dyes. Fluorescent polypeptides can also be used in the invention, including proteins that fluoresce due to either intramolecular re-arrangements or the addition of cofactors that promote fluorescence.
  • green fluorescent proteins of cnidarians which act as their energy-transfer acceptors in bioluminescence, are suitable fluorescent proteins for use in the fluorescent indicators.
  • a green fluorescent protein (“GFP”) is a protein that emits green light
  • BFP blue fluorescent protein
  • YFP yellow fluorescent protein
  • GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, the sea pansy, Renilla reniformis, and Phialidium gregarium. See, Ward et al., (1982) Photochem. Photobiol., 35, 803; and Levine et al, (1982) Comp. Biochem. Physiol., 72B, 77).
  • fluorescent proteins can be used, such as, for example, yellow fluorescent protein from Vibrio flscheri strain Y-l, Peridinin-chlorophyll a binding protein from the dinoflagellate Symbiodinium sp.phycobiliproteins from marine cyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin, or oat phytochromes from oat reconstructed with phycoerythrobilin.
  • These fluorescent proteins have been described in Baldwin et al., (1990) Biochemistry 29, 5509, Morris et al, (1994) Plant Molecular Biology, 24, 673, and Wilbanks et al, (1993) J. Biol. Chem. 268, 1226, and Li et al., (1995) Biochemistry 34, 7923.
  • fluorophore bound to a first component of the GPCR/G-protein complex that can be quenched by a fluorescence quencher bound to a second component of the GPCR/G-protein complex, or the first component of the GPCR/G-protein complex can comprise or bind a quencher and the second component used in the assay is bound to a donor fluorophore.
  • fluorescence quenchers that can be used in the methods of the present invention include DABCYL, DABSYL, QSY 7, QSY 9, QSY 21, and QSY 35.
  • FRET pair for use in the present invention is a Terbium-DPTA-csl24- EDA-fluor (available commercially from Panvera.com), coupled with an Alexa 546 fluor (available commercially from Molecular Probes.com) to report on molecular associations and/or rearrangements occurring at the level of the G-protein subunits consequent upon receptor stimulation.
  • Fluors would have functionalised groups (e.g. isothiocyanate, succinimidyl ester, maleimide and the like) for conjugation to proteins.
  • Terbium can be excited by light at 340nm.
  • the emitted light, Stokes shifted to 550nm, may either be detected at that wavelength or may interact with adjacent Alexa 546 fluor attached to the other G-protein subset if, in the latter case, the molecular arrangement was conductive to such an interaction occurring via FRET and LRET. If such an interaction occurred, light would then be emitted from the Alexa fluor at a wavelength of 570nm.
  • Fluorescence in a sample is measured using a fluorometer or any other instrumentation that incorporates within a means to detect fluorescent radiation over a range of wavelengths.
  • excitation radiation from an excitation source having a first wavelength, passes through excitation optics.
  • the excitation optics cause the excitation radiation to excite the sample.
  • fluorescent moieties on the protein sample emit radiation which have a wavelength that is different from the excitation wavelength.
  • Collection optics then collect the emission from the sample.
  • the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned.
  • a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed.
  • the multi-axis translation stage, temperature controller, auto- focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer.
  • the computer also can transform the data collected during the assay into another format for presentation.
  • the fluorescent probes are separately attached via amine or thiol linkages to either the G-alpha subunit or the G-beta/gamma subunit complex prior to reconstitution of the complete signalling complex.
  • one member of the LRET/FRET pair may be attached to the GPCR under investigation and the other member of the pair attached to one of the G-protein subunits.
  • two or more different FRET pairs are attached to two or more different sets of the GPCR/G-protein complex components, thereby enabling analysis of the interaction between different sets of components simultaneously.
  • probes of a first FRET pair may be attached to the G- alpha subunit and the G-beta/gamma subunit; and probes of a second different FRET pair may be attached to the GPCR and the G-alpha subunit. This allows simultaneous analysis of the interaction between the (i) the G-alpha subunit and the G-beta/gamma subunit; and (ii) the GPCR and the G-alpha subunit or the G-beta/gamma subunit.
  • a FRET pair of the type mentioned above could be attached to one of the G-proteins (particularly the G-alpha subunit) and to an RGS or AGS protein to determine the interaction between that RGS or AGS and the G-alpha subunit by LRET/FRET and to identify those compounds, drugs, products of combinatorial chemistry etc., which could influence or modulate this interaction.
  • one FRET pair is attached to a G-alpha and to a G- beta/gamma dimer
  • another FRET pair is attached to a G-alpha and a RGS or AGS protein to determine simultaneously the interaction between that RGS or AGS and the heterotrimeric G-proteins subunits by LRET/FRET and to identify those compounds, drugs, products of combinatorial chemistry etc., which could influence or modulate this interaction.
  • Luminescent lanthanide chelates have highly unusual spectral characteristics that make them useful non-isotopic alternatives to organic fluorophores, particularly where there are problems of background autofluorescence. Due to their relatively long luminescent emission lifetime resolved signals can be employed. They are also useful donors in fluorescence (luminescence) resonance energy transfer to measure nanometer conformational changes and binding events. Emission primarily arises from electric dipole transitions. Energy transfer is therefore termed lanthanide based or luminescent resonance energy transfer (LRET). LRET also provides the opportunity to undertake homogenous (no separation step) assays as opposed to heterogeneous assays requiring a separation step such as filtering.
  • LRET lanthanide/luminescence
  • LRET-based and FRET-based emission measurements are time-resolved (TR).
  • TR time-resolved
  • Measurement of time-resolved resonance energy transfer can reduce the interference from background fluorescence or luminescence, for example, from the wells that contain the samples.
  • long enough delay times time between pulsed excitation and starting of emission recording
  • all background interferences can be eliminated (for a review see. e.g. Hemmil (1991); Gudgin Dickinson et al, (1995) J PhotochemPhotobiol 27, 3).
  • fluorescence polarization may be detected.
  • Fluorophores that can be directly or indirectly bound to a GPCR or G-protein subunit for fluorescence polarization detection include any fluorophores known in the art or later developed, for example, fluorescein, rhodamine, Alexa dyes, Cy dyes, TMR, JOE, FAM, TAMRA, BODIPY, pyrene, Europium or other lanthanide compounds, and fluorescent proteins such as phycoerythrin, phycocyanin, allophycocyanin, GFP and its derivatives.
  • Fluorescence polarization measurements provide information on molecular orientation and mobility and processes that modulate them, including receptor-ligand interactions, proteolysis, protein-DNA interactions, membrane fluidity and muscle contraction. Because polarization is a general property of fluorescent molecules (with certain exceptions such as lanthanide chelates), polarization-based readouts are somewhat less dye-dependent and less susceptible to environmental interferences such as pH changes than assays based on fluorescence intensity measurements. Experimentally, the degree of polarization is determined from measurements of fluorescence intensities parallel and perpendicular with respect to the plane of linearly polarized excitation light, and is expressed in terms of fluorescence polarization or anisotropy.
  • the present invention provides methods that can be used for a number of different purposes including: (1) determining the function or activity of any given GPCR when stimulated by a cognate ligand (as depicted in Figure 2); (2) screening for new drugs affecting selected GPCRs which may include novel agonists, antagonists or inverse agonists (as depicted in Figure 3); (3) detecting GPCRs in a sample (as depicted in Figure 4); and (4) screening for new drugs affecting G-protein interactions which may include novel agonists, antagonists or inverse agonists (as depicted in Figure 5); .
  • the basic assays described herein and variations thereof can also be used in other applications, as will be apparent to those skilled in the art upon reading the present application.
  • a significant advantage of the assays of the invention is that they can directly detect G- protein activation through GPCRs in a cell free environment either qualitatively or quantitatively, and thus are particularly amenable to high throughput screening of or using large numbers of GPCRs. This can be useful, for example, in determining the receptors activated by a particular drug or receptors that are activated upon exposure to a particular stimulus, such as an odor or taste (e.g., activation of olfactory GPCRs).
  • the assays can be performed in a homogeneous format. Homogeneous assays are single step procedures that require only the mixing of sample with reagents prior to incubation and the reading of results. Separation and washing steps are not required.
  • GPCR polypeptides each of which encompasses biologically active or immunogenic fragments or oligopeptides thereof, can be used for screening compounds that affect GPCR receptor activity by, for example, specifically binding the GPCR and affecting its function, thereby affecting GPCR activity and G-protein activation.
  • Identification of such compounds can be accomplished using any of a variety of drug screening techniques. Of particular interest is the identification of agents that have activity in affecting GPCR function. Such agents are candidates for development of treatments for, conditions associated at least in part with GPCR activity.
  • the screening assays of the invention are generally based upon the ability of the agent to bind to the GPCR polypeptide component of a cell free complex comprising a GPCR and at least one G-protein subunit, and thereby effect a detectable change in one of the G-protein subunits of the complex.
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 40 and less than about 7,800 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably with at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, lipids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts (including extracts from human tissue to identify endogenous factors affecting GPCRs) are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc.
  • the invention also contemplates the use of competitive drug screening assays in which GPCR receptor-specific neutralizing antibodies compete with a test compound for binding of the GPCR polypeptide. In this manner, the antibodies can be used to detect the presence of any polypeptide that shares one or more antigenic determinants with a GPCR polypeptide.
  • reagents may be included in the screening assays described herein.
  • these include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding, protein-DNA binding, and/or reduce non-specific or background interactions.
  • Reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used.
  • Components that modify G-protein function, such as RGS and AGS proteins may also be used.
  • the mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high- throughput screening. Typically between 0.1 and 1 hours will be sufficient.
  • a major asset of the invention is its ability to vastly increase, over current methods, the rate at which compounds can be evaluated for their ability to act as agonists, antagonists, and/or inverse agonists for GPCRs.
  • GPCR genes are identified and characterized, the activity of these receptors in response to various compounds, as well as to methods such as site directed mutagenesis, can be used to gain detailed knowledge about the basic mechanisms at work in these receptors.
  • a fundamental knowledge of the basic mechanisms at work in these receptors will be of great use in understanding how to develop promising new drugs and/or to identify the fundamental mechanisms behind specific tastes, smells and the like.
  • Assays using the cell free complexes of the present invention can identify promising agonists, antagonists and/or inverse agonists for specific GPCRs.
  • the cell free complexes of the invention can be contacted with a candidate therapeutic, and the activity of the G-protein activation through the receptor measured, e.g., by measuring a signal associated with a change in a G-protein subunit of the complex.
  • Agonists can be identified by their ability to increase GPCR activity over basal levels in the system.
  • Antagonists can be identified by contacting the cell free complexes of the assay system with a native ligand or agonist and a candidate antagonist; compounds that inhibit the ability of the ligand or agonist to increase activity of the GPCR are identified as antagonists.
  • G-protein coupled receptors have been shown to exhibit activity in the absence of agonist (so-called spontaneous, constitutive or basal activity).
  • This agonist- independent basal activity can be inhibited by compounds previously considered to be antagonists (e.g., the antipsychotic drugs). Therefore, these compounds are inverse agonists rather than antagonists.
  • cell free complexes are prepared in which two different G-protein subunits are labeled with Fluorescence Resonance Energy Transfer (FRET) or Luminescence Resonance Energy Transfer (LRET) probes. These complexes are then exposed to a candidate GPCR binding compound and the effect of exposure to the compound monitored by detecting a change in the fluorescence or luminescence generated by the probes. A change in the fluorescence or luminescence generated by the probes is caused by a change in the positioning of the G-protein subunits within the complex.
  • FRET Fluorescence Resonance Energy Transfer
  • LRET Luminescence Resonance Energy Transfer
  • functional assays can be used to identify candidate compounds that block activity of a known GPCR agonist (e.g., block the activity of or compete with a known agonist), block activity of a known GPCR antagonist (e.g., block the activity of or compete with a known antagonist) and/or block the activity of a known GPCR inverse agonist.
  • a known GPCR agonist e.g., block the activity of or compete with a known agonist
  • a known GPCR antagonist e.g., block the activity of or compete with a known antagonist
  • block the activity of a known GPCR inverse agonist e.g., block the activity of or compete with a known antagonist
  • cell free complexes which do not contain GPCRs are prepared for the purpose of screening for agonists, antagonists and/or inverse agonists.
  • the cell free complex comprises at least one G-protein subunit that is labeled with an energy donor or energy acceptor.
  • the complex comprises at least two G-protein subunits, more preferably the complex comprises a heterotrimeric G-protein complex comprising a G- alpha subunit and a G-beta-gamma dimer.
  • the G-alpha subunit may be labeled with an energy donor and the G-beta-gamma dimer may be labeled with an energy acceptor.
  • the candidate agent may then be exposed to this complex and any rearrangement or change in conformation in the G-protein complex caused by the candidate agent can be detected by a change in fluorescence or luminescence generated by the energy donor/energy acceptor pair. It will be appreciated that a rearrangement or change in conformation in the G-protein complex caused by the candidate agent indicates that the candidate agent is a potential modulator of GPCR signalling.
  • the cell free complex comprises only a G-protein subunit (for example, a G-alpha subunit) which is labeled with an energy donor.
  • a protein such as an RGS or AGS, labeled with an energy acceptor, is then exposed to the complex under conditions that allow interaction between the G-protein subunit and the RGS or AGS.
  • the candidate agent may then be exposed to this complex and any change in the interaction between the G-protein subunit and the RGS or AGS caused by the candidate agent can be detected by a change in fluorescence or luminescence generated by the energy donor/energy acceptor pair. It will be appreciated that a change in the interaction between the G-protein subunit and the RGS or AGS caused by the candidate agent indicates that the candidate agent is a potential modulator of GPCR signalling.
  • inverse agonists are becoming increasingly important therapeutic agents. Inverse agonism is demonstrated when a drug binds to a receptor that exhibits constitutive activity and reduces this constitutive activity.
  • the assays of the present invention using cell free complexes are particularly well suited to identify promising inverse agonists for specific receptors, as they provide a direct method for detecting a decrease in GPCR activity in response to a compound.
  • cell free complexes can be contacted with a candidate inverse agonist, and the activity of the G-protein activation measured, e.g., by measuring binding of GTP analogs, or rearrangement of G-protein subunits within the complex.
  • Compounds that decrease the basal activity of the receptor in the assay are identified as inverse agonists, and may then be subject to further testing for therapeutic use.
  • the methods of the present invention provide a rational method for designing and selecting compounds including antibodies which interact with and modulate the activity of GPCRs. In the majority of cases these compounds will require further development in order to increase activity. It is intended that in particular embodiments the methods of the present invention includes such further developmental steps. For example, it is intended that embodiments of the present invention further include manufacturing steps such as incorporating the compound into a pharmaceutical composition in the manufacture of a medicament.
  • the method further comprises formulating the identified compound for administration to a human or a non-human animal as described herein.
  • the present invention clearly encompasses the use of any analytical method and/or industrial process for carrying the screening methods described herein into a pilot scale production or industrial scale production of a compound identified in such screens.
  • This invention also provides for the provision of information for any such production.
  • the present invention also provides a process for identifying or determining a modulator of GPCR signalling, said method comprising: (i) performing a method as described herein to thereby identify or determine a compound or modulator; (ii) optionally, determining the structure of the compound or modulator; and (iii) providing the compound or modulator or the name or structure of the compound or modulator such as, for example, in a paper form, machine- readable form, or computer-readable form.
  • step (i) determination of the structure of the compound is implicit in step (i). This is because the skilled artisan will be aware of the name and/or structure of the compound at the time of perfo ⁇ ning the screen.
  • the term "providing the compound or modulator” shall be taken to include any chemical or recombinant synthetic means for producing said compound or modulator or alternatively, the provision or a compound or modulator that has been previously synthesized by any person or means.
  • the compound or modulator or the name or structure of the compound or modulator is provided with an indication as to its use e.g., as determined by a screen described herein.
  • the present invention also provides a process for producing a modulator of GPCR signalling, said method comprising: (i) performing a method as described herein to thereby identify or determine a compound or modulator; (ii) optionally, determining the structure of the compound or modulator; (iii) optionally, providing the name or structure of the compound or modulator such as, for example, in a paper form, machine-readable form, or computer- readable form; and (iv) producing or synthesizing the compound or modulator.
  • the synthesized compound or modulator or the name or structure of the compound or modulator is provided with an indication as to its use e.g., as determined by a screen described herein.
  • An assay system can also be used to classify compounds for their effects on G-protein coupled receptors, such as on orphan receptors, to identify candidate ligands that are the native ligands for these orphan receptors.
  • G-protein coupled receptors such as on orphan receptors
  • Cell free complexes comprising orphan GPCRs can be exposed to a series of candidate ligands, and the ligands with the ability to activate signaling through the receptors can be identified through changes in the G-protein subunits of the complex, eg. by measuring rearrangement of G-protein subunits within the complex.
  • the present invention also provides a rapid and reliable method for determining G- protein activation through receptors known to be involved in drug responses.
  • the present invention thus can also be used to test for drugs, e.g., narcotics, e.g., cocaine, heroin, morphine or designer opiates in foods or bodily fluids, e.g., blood or urine.
  • An exemplary assay for identifying a drug in a substance would comprise the steps of contacting a cell free complex of the invention (e.g., where the GPCR is an opioid receptor) with a sample suspected of containing the drug.
  • the presence of the drug can be verified by detecting, either qualitatively or quantitatively, changes in the G-protein subunits of the complex, eg. by detecting rearrangement of G-protein subunits within the complex.
  • the present invention can also be used for detection of aromas or odorants that stimulate GPCRs.
  • the assays of the present invention can therefore be applied, for example, to detection of aromas in samples of food and wine or to the detection of odors that may be released by patients suffering from diseases.
  • the present invention provides a method for identifying or determining the levels of a GPCR in a sample.
  • combinations of G- protein subunits are used to capture GPCRs in a sample. Detection of the captured GPCR is then achieved by interrogation with a known ligand.
  • G-protein heterotrimeric complexes can be tethered to a support to effectively provide "hooks” or “baits” for capturing different types of GPCRs. Once caught by a degree of specific binding, the captured GPCR can then be interrogated with a range of ligands for identification. For example, a known ligand would identify its cognate, captured receptor via signalling through the G-proteins as per the invention. This aspect of the invention therefore allows detection of changes in GPCR levels and functionality as diagnost c biomarkers for human health and disease.
  • the GPCRs expressed on a variety of cells are obtained from a biological fluid (or even biopsy material) by isolating cells (for example, by differential centrifugation) and performing a detergent extraction in order to solubilise membrane associated GPCRs. A urea step is then performed to remove intrinsic G-proteins leaving the "naked" GPCR available to bind to a multi-addressable, heterotrimeric G- protein array.
  • the GPCRs that are involved in biological responses can be determined using assays of the invention.
  • An assay using an array of cell free complexes of the invention each sample of the array comprising a cell free complex with a particular GPCR, can be exposed to a particular stimulus (e.g., the odor, flavor compound, disease related complex, and the like), and the activity of each sample of the array can be determined. This can identify multiple receptors in a high throughput manner that are involved in the transduction of signals in response to that particular stimulus.
  • the high throughput assays of the invention can be especially useful in determining the spectrum of GPCRs, e.g., olfactory receptors, that are activated or inverse agonized by a specific substance or mixture of substances.
  • GPCRs e.g., olfactory receptors
  • a liquid can be contacted with an array of cell free complexes comprising particular GPCRs, and the GPCRs activated or suppressed can be identified.
  • the complexes of the invention may be immobilised on any of a number of commercially available array platform Hydrogel® based array system on glass slides as a commercially available array platform for this project; however the arraying of the complexes is not limited to the hydrogel covered chip.
  • Proteins are printed onto the HydroGel substrate are captured within the porous structure. Proteins added or captured are kept in their native form and are freely accessible for assay binding reaction. No modifications to the proteins or oligonucleotides are required prior to immobilization, insuring protein integrity and assay dependability.
  • GPCR ligands (which typically involve hormones, neurotransmitters, chemokines and the like) are involved in many physiological and pathophysiological processes associated with but not limited to the cardiovascular system, the central nervous system, the endocrine system, the gastrointestinal system and in inflammation, immunity and cancer, (see Table 1). For example, seventeen or more distinct GPCR ligands profoundly modulate cardiovascular activity by interacting with their respective cognate receptors.
  • the cell free complexes of the present invention enable rapid and efficient measurement of these compounds (ligands) for diagnostic purposes.
  • the nearly 200 functionally known GPCRs directly or indirectly account for nearly 60% of all currently-prescribed prescription drugs generating over $60 billion in sales annually.
  • Members of this therapeutically prolific and valuable superfamily of proteins (receptors) are ubiquitous in human physiology and play vital roles in the most fundamental processes of cells including biochemical communication, growth, differentiation, and survival.
  • the present invention can be used in the discovery of human biomarkers of disease as well as determining therapeutic products targeting known GPCRs and orphan GPCRs.
  • GPCR ligand assays of the present invention have potential applications in areas such as:
  • Foods, beverages and cosmetics e.g., human sensory GPCRs; biosensors
  • Diagnostic applications e.g., detection of GPCR-based ligands
  • Environmental biosensors e.g., detection of GPCR-based toxins
  • Agrichemical products e.g., plant & insect GPCRs
  • Veterinary medicines e.g., animal GPCRs.
  • GPCRs Since many of the natural ligands of GPCRs are hormones, their concentrations in body fluids (e.g., serum samples) are often measured as a diagnostic aid. Concentrations outside of "normal limits” may suggest hypo- or hyper-secretive conditions (e.g., hyperparathyroidism).
  • serum concentrations of many GPCR-based drugs e.g., theophylline
  • Serum concentrations are also measured to confirm cases of suspected toxicity or poisoning.
  • GPCR ligands differ significantly in their chemical composition (eg biogenic amine, peptide, lipid etc.) necessitating a wide range of techniques for their individual analysis and detection.
  • compositions and methods of the invention provide a more rapid and cost-effective approach.
  • the present invention may be adapted for use in a critical care, point of care instrument.
  • Critical care refers to the treatment of patients with acute, life-threatening illness or injury. Care may be provided at the scene of an accident, in an ambulance, in a hospital trauma center or emergency room. Critical care is provided by multidisciplinary teams of healthcare professionals - physicians, nurses, respiratory care technicians and other health personnel. For example, Ambri has developed the SensiDxTM System to ensure that these professionals have a method of critical care testing that will provide fast, accurate and reliable results within minutes.
  • the present invention may be adapted to a similar format.
  • Sf9 insect cells (Invitrogen, Mount Waverley, Vic, Australia) were grown in Sf-900 II SFM serum-free media (Invitrogen) and maintained at 28°C with agitation at 140 oscillations/min in an orbital shaker.
  • Recombinant baculo virus samples encoded either the recombinant porcine ⁇ 2A-adrenergic receptor ( ⁇ 2A-AR) or the G-protein subunits Gail or Gail (his) or ⁇ l or ⁇ 4 or ⁇ 2 or ⁇ 2(his) or the recombinant muscarinic M 2 receptor (M 2 R).
  • Sf9 cells (at 2x10 6 cells/ml) were infected with the appropriate combinations of Gail ( ⁇ His6-tagged) and ⁇ l ⁇ 2 ( ⁇ His6-tagged) or M 2 R or ⁇ 2A-AR baculo virus(es) at a multiplicity of infection ratio of 1-2 and harvested at 72 h. Cell viability following 72 h infection was approximately 40-70% as determined by trypan blue staining.
  • Infected Sf9 cells were collected and centrifuged at lOOOxg for 10 min and suspended 125 mL ice-cold "lysis buffer" (50 mM HEPES pH 8.0, 0.1 mM EDTA, pH 8, 3 mM MgCl 2 , 10 mM ⁇ -mercaptoethanol) with protease inhibitors; 0.02 mg/ml phenylmethyl sulfonyl fluoride (PMSF), 0.03 mg/ml benzamidine, 0.025 mg/ml bacitracin, 0.03 mg/ml Lima bean trypsin inhibitor.
  • PMSF phenylmethyl sulfonyl fluoride
  • membranes were diluted to 4 M urea with "incubation buffer” and protease inhibitors and centrifuged at 100,000xg at 4°C for 30 min.
  • the urea-treated membrane pellet was washed twice in "incubation buffer”.
  • the final urea-treated membrane pellet was resuspended to approximately 1-3 mg/ml protein and rapidly frozen in liquid N2 and stored at -80°C until use.
  • Confirmation and quantification of ⁇ 2A-AR expression was done by [ 3 H] MK912 (Perkin Elmer Life Sciences Inc., Boston, MA, USA) binding.
  • Confirmation and quantification of M 2 R expression was done by [ 3 H]-Scopolamine (Perkin Elmer Life Sciences Inc., Boston, MA, USA) binding.
  • the following fluorescent probes were purchased from Invitrogen (Mount Waverley, Vic, Australia, as agent for Molecular Probes), Alexa Fluor 546 carboxylic acid, succinimidyl ester; Alexa Fluor 546 C 5 maleimide; and TMR (tetramethylrhodamine) maleimide.
  • Tb Terbium
  • CS124-DTPA-Phe-NCS CS124-DTPA-EMCH
  • Tb-mal thiol reactive form
  • G-protein subunits including G ⁇ h i s , ⁇ 4 and ⁇ 2 and ⁇ 2A -AR were from baculovirus cDNA clones expressed in the Sf9 insect cell system.
  • RGS4hi S was expressed from pQE60 (Qiagen, Australia) in M15[ ⁇ REP4] E.coli induced with ImM IPTG for 3 hours.
  • Membranes were stirred on ice for 1 hr to extract. The sample was then centrifuged at 100,000xg for 40 min and the supernatant collected (termed membrane extract) and diluted 5-fold with buffer A (20 mM HEPES, 100 mM NaCl, 1 mM MgCl 2 , 10 mM ⁇ -mercaptoethanol, 0.5% (w/v) polyoxyethylene- 10-lauryl ether and 10 ⁇ M GDP.
  • buffer A (20 mM HEPES, 100 mM NaCl, 1 mM MgCl 2 , 10 mM ⁇ -mercaptoethanol, 0.5% (w/v) polyoxyethylene- 10-lauryl ether and 10 ⁇ M GDP.
  • Ni-NTA nickel-nitrilotriacetic acid
  • the column containing His6-tagged proteins was washed with buffer A (at a volume equating to 5% of the original cell volume) containing 5 mM imidazole and 300 mM NaCl.
  • the column was washed with 50 ml of buffer A (minus the ⁇ -mercaptoethanol for G- proteins labelled with fluorescent labels of the maleimide form) to remove imidazole (and remove ⁇ -mercaptoethanol for the malemide label).
  • the column was warmed to 22°C (room temperature) for 15 min and a five-fold molar excess of the fluorescent label in buffer A (minus ⁇ -mercaptoethanol for the fluorescent labels of the maleimide form) was added.
  • the column containing the labelled G-proteins was incubated at room temperature for 2 hr with occasional mixing.
  • the non His6-tagged G-protein subunits eluted with 1 ml fractions of buffer E (20 mM HEPES pH 8.0, 50 mM NaCl, 10 mM ⁇ - mercaptoethanol, 10 ⁇ M GDP, 1 % (w/v) cholate, 50 mM MgCl 2 , 5 mM imidazole, 10 mM NaF and 30 ⁇ M A1C1 3 .
  • the remaining His6-tagged G-protein subunits were eluted from the nickel column with buffer E containing 150 mM imidazole. Protein expression was confirmed by SDS-polyacrylamide gel electrophoresis and compared with known molecular weight protein standards.
  • Elution fractions containing the appropriate G- protein subunit were pooled (to ⁇ 3ml) and dialysed against 5 x 200 ml buffer F (20 mM HEPES pH 8.0, 3 mM MgCl 2 , 10 mM NaCl, 10 mM ⁇ -mercaptoethanol, 1 ⁇ M GDP and 0.1% (w/v) cholate) using a Slide-a-lyzer (Pierce Chemical Company, Rockford, IL, USA). Following dialysis, the G-protein subunits were stored at -80°C in buffer F or in 50% glycerol in buffer F. Final protein concentration was determined by SDS-polyacrylamide gel electrophoresis of G-protein subunits followed by laser scanning densitometry.
  • Membranes expressing ⁇ 2A-AR were diluted approximately 2-3 fold (to give 0.8 mg protein/ml) with ice-cold reconstitution buffer (50 mM Tris pH 7.6, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT and 0.5 ⁇ M GDP). After addition of the indicated amounts of G-protein subunits in buffer F, the reaction mix was incubated on ice for 60 min.
  • ⁇ 2A-AR membranes For capture of the ⁇ 2A-AR/G-protein complex (termed transductosome), ⁇ 2A-AR membranes at 0.8 mg/ml with reconstituted G-proteins (+His6 tag) were added to the indicated amounts of Ni-NTA beads (45 - 165 ⁇ m diameter in a 50% solution of reconstitution buffer) for a further 60 min on ice. To initiate 35 S-GTP binding, samples were transferred to 26°C and after 5 min various agonists and antagonists (clonidine, UK14303 ("UK”) and rauwolscine (from Sigma, Castle hill, NSW, Australia) were added (final protein was 0.4 mg/ml).
  • GTP ⁇ S35 35 S- GTP, Perkin Elmer Life Sciences Inc., Boston, MA, USA
  • Binding of GTP ⁇ S35 35 S- GTP, Perkin Elmer Life Sciences Inc., Boston, MA, USA was determined following filtration through GF/C filters (transductosome without Ni-NTA beads) or Whatman No.l filters (transductosome with Ni-NTA beads) and two 3 ml washes with ice-cold 20 mM Tris pH 7.4, 25 mM MgCl 2 and 100 mM NaCl. Filters were air-dried and then counted by liquid scintillation counting.
  • TR-LRET luminescent resonance energy transfer
  • other fluorescent measurements were performed using a Victor 3 (Perkin Elmer, Melbourne Australia) multilabel plate counter fitted with a 1500V Xenon Flash light source for time-resolved fluorometric absorbance measurements.
  • excitation was at 340nm with emission being measured at 550 nm and 572 nm with a 500 ⁇ s delay and 1400 ⁇ s counting duration. The time-resolved excitation/emission was gated as indicated in the results.
  • Membranes expressing ⁇ 2A-AR were reconstituted with his-tagged or non his-tagged Gail and ⁇ l ⁇ 2 (or ⁇ 4 ⁇ 2 where indicated) in a buffer containing 50 mM Tris pH 7.6, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 5 ⁇ M GDP and lO ⁇ M AMP-PNP. Either one or both of the G-protein subunits were fluorescent labelled as detailed in the figure legends. The final concentration in the reconstitution mix was 0.1 mg protein/ml for the ⁇ 2A-AR membranes and 40nM for the G-proteins.
  • the reconstituted membranes were transferred to v-shaped 96 well plates and further incubated in the presence or absence of the ⁇ 2A-AR agonist, UK (10 ⁇ M) or the ⁇ 2A-AR antagonist, Yohimbine (lOOuM) for 10 minutes.
  • 35 S-GTP binding was initiated with the addition of [ 35 S]GTP ⁇ S (PerkinElmer Life Sciences, Boston, MA, USA) at a final concentration of 0.2nM.
  • Ni-NTA agarose beads 45-165 ⁇ m diameter in a 50% solution of reconstitution buffer (Qiagen, Clifton Hill, Victoria, Australia) were added to 96 well filtration plates containing 1.2 ⁇ M Durapore® membranes (Millipore Corporation, Bedford, MA, USA). The reconstituted membranes were incubated for 10 minutes with the Ni-NTA beads. Agonists or antagonists were added to the transductosome/bead complex and binding initiated by the addition of [ 3s S]GTP ⁇ S as described above. After 90 minutes at 27°C samples were filtered, washed and counted as described above.
  • TR-LRET Time-resolved luminescence resonance energy transfer
  • 96-well plates in a final volume of lOO ⁇ l at the final concentrations indicated in figures. All dilutions of proteins were done in TMND buffer consisting of 50mM TRIS pH 7.6, lOmM MgCl 2 , lOOmM NaCl, 10 mM dithiothreitol and l ⁇ M GDP.
  • TMND buffer consisting of 50mM TRIS pH 7.6, lOmM MgCl 2 , lOOmM NaCl, 10 mM dithiothreitol and l ⁇ M GDP.
  • A1F 4 " was used (lOmM NaF and 30 ⁇ M A1C1 3 ).
  • Time-resolved luminescent energy transfer was carried out using a Victor 3 V (Perkin Elmer) fluorescence plate reader with a time delay of 500 ⁇ s, counting duration of 1400 ⁇ s and using an excitation wavelength of 340nm and monitoring emission at both 550nm and 570nm.
  • Victor 3 V Perkin Elmer fluorescence plate reader
  • Example 1 Reconstitution and testing of ⁇ 2A-AR/G-protein signalling complex (transductosome).
  • ⁇ 2A-AR membranes were treated with 7 M urea to inactivate endogenous Sf9 cell G- protein ⁇ and ⁇ subunits as described previously (Lim & Neubig, 2001, Biochem. J. 354:337-344). Urea treatment left antagonist [ 3 H]MK912 binding intact. ⁇ 2A-AR membrane expression was approximately 1000 fmol/mg with a Kd of approximately 0.2 nM as determined by [3H]MK912 binding (data not shown).
  • clonidine (a partial ⁇ 2A-AR agonist) was used at 100 ⁇ M.
  • Figure 6 shows that 35 S-GTP binding increases over 60 min in the presence or absence of clonidine.
  • 50 nM Gail to the a2A-AR membranes significantly increases the rate of 35 S-GTP binding in the presence of clonidine.
  • reconstitution of ⁇ 2A-AR membranes with the G-protein subunits 50 nM Gail and 50 nM ⁇ l ⁇ 2 further accelerates the rate of 35 S-GTP binding over 60 min in the presence of clonidine.
  • the concentration of GDP in the 35 S-GTP binding assay is important for obtaining an optimal signal in the receptor activation assay.
  • GDP is required to aid receptor coupling to the heterotrimer in the receptor:G ⁇ ilGDP; ⁇ l ⁇ 2 basal state, whilst GTP is required as the substrate for the receptor activated ⁇ -subunit.
  • the amount of 35 S-GTP bound determines the amount of signal observed.
  • Figure 7 shows that GDP at concentrations between 0.5 - 10 ⁇ M dose- dependently decreased the background binding of 35 S-GTP to Gail as well as the ⁇ 2A- AR activated signal using clonidine.
  • Ni 2+ -coated beads capture the transductosome complex.
  • several volumes of beads were used. As little as 5 ⁇ l Ni-NTA beads captured the transductosome complex as shown in Figure 14, with saturation of bead binding at > 20 ⁇ l beads as determined by 35 S-GTP binding. Additionally, in separate experiments we demonstrated that coating the Ni-NTA beads firstly with Gail (his) followed by addition of ⁇ l ⁇ 2 then ⁇ 2A-AR (with wash steps in between each addition of protein), also resulted in the sequential building up of the transductosome complex (data not shown) .
  • Example 2 Reconstitution and testing of M ⁇ R G-protein signalling complex (transductosome) .
  • M 2 R membranes were treated with 7 M urea to inactivate endogenous Sf9 cell G- protein ⁇ and ⁇ subunits.
  • Figure 15 demonstrates that M 2 R membrane expression was approximately 25.8 pmol/mg with a Kd of approximately 2.1 nM as determined by [3 HJ-Scopolamine binding.
  • Figure 16 shows the EC50 for inhibition of specific binding of the nonspecific muscarinic receptor antagonist [3H]-Scopolamine (at InM) was inhibited by atropine at 5.2 nM (non-specific muscarinic receptor antagonist) compared with the Ml-muscarinic receptor antagonist pirenzepine (1100 nM). Furthermore, the EC50 for carbachol was 39.3 ⁇ M.
  • M 2 R membranes (0.4 mg/ml) were reconstituted with G-proteins (5 nM G ⁇ ( h i s ) and 5 nM ⁇ 2 ) and carbachol (a M 2 R agonist) was used at 25mM.
  • Figure 17 shows that 35 S-GTP binding increased above basal binding following 90 min of incubation.
  • Figure 18 shows that increasing the concentration of carbachol concomitantly increased the binding of 35 S-GTP to the reconstituted transductosomes. Additionally, the muscarinic receptor antagonist, atropine (500 ⁇ M) completely abolished the carbachol-induced 35 S-GTP binding, indicating specificity of the receptor-mediated signal. The ECs 0 for carbachol-induced 35 S-GTP binding was 10.2 ⁇ M resulting in an approximate 8-fold increase of 35 S-GTP binding at maximal stimulation.
  • Example 3 Determination of signalling activity for GPCR-G-protein complex associated with a surface
  • Figure 22 shows the dose-response of the ⁇ 2A-AR - G-protein complex (termed transductosome) to the specific agonist UK 14304 (measured by the sensitivity of the receptor to a specific agonist to be detected by heterotrimeric G-proteins using the 35S-GTP binding assay) when the transductosome was either associated with Ni(NTA) agarose beads or was not.
  • the rank order potency of a number of adrenergic receptor subtype-specific antagonists determined from their respective IC50 values is indicative of the specificity of the ⁇ 2A-AR, further indicating that the receptor signalling complex is unaltered with regard to sensitivity ( Figure 22) or specificity ( Figure 23) when associated with a surface.
  • Example 4 Determination of functionality of fluorescently labeled G-protein subunits in GPCR reconstitution assay
  • Figure 26 demonstrates that labelling of ⁇ protein subunits with Terbium chelate (either NCS or EMCH ie succinimidyl ester or maleimide, respectively) indicated that Terbium labelled proteins are functional. Furthermore, storage of the G-protein subunits in glycerol did not decrease the signalling activity as shown in Figure 24.
  • Terbium chelate either NCS or EMCH ie succinimidyl ester or maleimide, respectively
  • Figure 28 also demonstrates the rapid association of G ⁇ is (labelled with Tb-chelate i.e., G ⁇ u h i s b)- and ⁇ ⁇ 2 labelled with (Alexa 546 i.e., ⁇ 4 ⁇ 2 - Alexa) at lOnM using time resolved-fluorescence resonance energy transfer techniques.
  • the TR-FRET at equilibrium resulted in a 5 -fold increase in signal under the conditions tested.
  • Figure 29 demonstrates that at lower concentrations of G-proteins i.e. at 3nM G ⁇ u h isTb and 8nM ⁇ 4 ⁇ 2 -Alexa, the total TR-FRET signal was lower at equilibrium in comparison to that observed following the addition of lOnM G-proteins (Figure 28)although there was still a 3 -fold increase in the TR-FRET signal.
  • Figure 29 also demonstrates that adding a molar (25x) excess of unlabelled ⁇ 4 ⁇ 2 resulted in a rapid decline of TR-FRET signal indicating dissociation of G-protein subunits.
  • Dissociation of heterotrimeric G-protein subunits can be effected by addition of A1F " as is shown in Figure 30.
  • the addition of A1F 4 " rapidly (less than 1 minute) dissociated the G ⁇ i s Tb and ⁇ 4 ⁇ 2 -Alexa bound subunits at equilibrium, resulting in an approximate 50% decrease in TR-FRET signal. This new steady-state binding remained stable for at least 25 minutes.
  • TR-FRET measurements were done using four concentrations of G ⁇ ilhis-Tb (3nm to 15nm) tested over a range of ⁇ 4 ⁇ 2-Alexa concentrations (Onm -lOOnm).
  • the mean dissociation constant for the interaction was 2.7nm (see Figure 32). This is indicative of a very tight association between these binding partners as has been estimated by other techniques and is also indicative that the TR-FRET technology used here does not perturb the normal high binding affinity of these heterotrimeric G-proteins.
  • the resulting increase in signal was approximately 5 -fold at steady state.
  • Addition of a (20x) molar excess of unlabelled G ⁇ hi s after 6 minutes of G ⁇ hisTb:RGS4 i S Alexa association resulted in a rapid decline of the TR-FRET signal indicating dissociation of G ⁇ h i s Tb and RGS4hj S Alexa as shown in Figure 33.
  • a dose curve of RGS4 h i S Alexa against 50nM G ⁇ h i s Tb demonstrated that increasing concentrations of RGS4 h i S Alexa increased the TR-FRET signal until a stable maximum was reached as shown in Figure 34. From this, a disassociation constant (K d ) of 15nM was calculated indicating a high affinity association between RGS h i S Alexa and G ⁇ ⁇ h i s Tb.
  • TR-FRET measurements were carried out using RGS4hi S Alexa and G ⁇ h i s Tb in various states of activation, inactive in the presence of GDP, activated in the presence of GTP ⁇ S and transitional in the presence of aluminium fluoride (aluminium fluoride has been reported to act as a transition state analogue of GTP; the latter compound being known to influence the properties of the G ⁇ subunit).
  • RGS4 has previously been reported to have greater affinity for the transitional state G ⁇ subunit and this increase in signal could be a result of greater amounts of binding due to increased affinity. Alternatively, this increase could be a result of conformational changes in the G ⁇ h i s Tb subunit that brings labelled residues into closer proximity with labelled residues of RGS4 h i s Alexa resulting in more efficient energy transfer.
  • Optimisation of the fluorescent signal may be accomplished by rational location of the FRET-interacting fluorescent probes in accord with the likely molecular changes occurring in the spatial arrangements and re-arrangements (association and diss- association) of the heterotrimeric G-proteins consequent upon their activation by a liganded GPCR.
  • donor and acceptor TR-FRET pairs may be placed either covalently, or by high affinity binding, to sites on the G-proteins which are known to rearrange during signal transduction. These sites of attachment may be based on the likely changes in the distance between FRET pairs taking into account the Forster radius of the interacting FRET pairs which in part dictates the generation of the FRET signal.
  • Fluorescent probes may be attached by varying means which could include the production of fusion proteins or selected amino acid motifs within the sequence of one or more G-proteins (G-alpha, G-beta, G-gamma and any variants of these which may increase coupling to GPCRs such as chimeric or promiscuous variants, and including other interacting proteins such as RGS, AGS and the like).
  • Fusion proteins or selected amino acid motifs may include, for example, a lanthanide binding tag motif (LBT) as a fusion protein (as described in Nitz et al.
  • spectrally-matched acceptor fluors such as a biarsenical Fluorescein or like molecules may be covalently bound to a tetracysteine motif of the type described in Adams (2002) J Am Chem Soc 124:6063-6067 ', engineered into a known site on a G- protein or other interacting molecule of the type listed above. This would allow significant choice and great precision in localising the FRET pairs to enable optimisation of the signal as well as design of the signal to be one which could selectively increase or decrease upon GPCR activation depending on fluorescent probe localisation.

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Abstract

L'invention porte sur des compositions et des procédés qui permettent d'identifier des récepteurs couplés à la protéine G ('G-protein-coupled receptors' ou GPCR), des ligands de ces derniers et des composés qui modulent la transduction du signal de la protéine G. En particulier, l'invention concerne un système d'analyse qui implique la formation d'un complexe acellulaire fonctionnel comprenant une ou plusieurs sous-unités de la protéine G marquées au moyen d'un donneur d'énergie et d'un accepteur d'énergie et, facultativement, un GPCR.
PCT/AU2005/000845 2004-06-14 2005-06-14 Analyse acellulaire permettant d'identifier le recepteur couple a la proteine g et son ligand WO2005121755A1 (fr)

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DE102009013456A1 (de) * 2009-03-18 2010-09-23 GÖPFERICH, Achim, Prof. Dr. Nanopartikel für die Erkennung von Zellen, durch Bindung an G-Protein gekoppelte Rezeptoren
WO2010112417A1 (fr) 2009-03-30 2010-10-07 Ge Healthcare Uk Limited Procédés pour tester la liaison d'un ligand à des récepteurs couplés à la protéine g
EP2561359A1 (fr) * 2010-04-20 2013-02-27 Addex Pharma SA Polypeptides chimériques utiles dans des procédés de criblage à haut débit proximaux et dynamiques
GB2502306A (en) * 2012-05-22 2013-11-27 Univ Singapore Microparticle sensor
US8703915B2 (en) 2009-06-22 2014-04-22 Heptares Therapeutics Limited Mutant proteins and methods for producing them
US8748182B2 (en) 2007-12-08 2014-06-10 Heptares Therapeutics Limited Mutant proteins and methods for producing them
US8785135B2 (en) 2007-03-22 2014-07-22 Heptares Therapeutics Limited Mutant G-protein coupled receptors and methods for selecting them
US8790933B2 (en) 2007-12-20 2014-07-29 Heptares Therapeutics Limited Screening
WO2014168721A3 (fr) * 2013-04-12 2015-03-19 Tufts Medical Center Procédés et systèmes pour mettre au point et/ou caractériser des agents ligands lipidés solubles
US9081020B2 (en) 2008-02-11 2015-07-14 Heptares Therapeutics Limited Mutant proteins and methods for selecting them
CN107074926A (zh) * 2014-10-14 2017-08-18 蒙特利尔大学 基于Gβγ互作蛋白监测G蛋白激活的生物传感器
EP3697934A4 (fr) * 2017-10-18 2021-06-30 Université de Montréal Systèmes et procédés pour l'évaluation d'une activation des protéines g
US11197906B2 (en) 2016-01-22 2021-12-14 On Target Therapeutics Llc Methods of lowering blink reflex for the treatment of dry eye disease

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US8785135B2 (en) 2007-03-22 2014-07-22 Heptares Therapeutics Limited Mutant G-protein coupled receptors and methods for selecting them
US8748182B2 (en) 2007-12-08 2014-06-10 Heptares Therapeutics Limited Mutant proteins and methods for producing them
US10126313B2 (en) 2007-12-20 2018-11-13 Heptares Therapeutics Limited Methods for screening for binding partners of G-protein coupled receptors
US8790933B2 (en) 2007-12-20 2014-07-29 Heptares Therapeutics Limited Screening
US9260505B2 (en) 2007-12-20 2016-02-16 Heptares Therapeutics Limited Methods for screening for binding partners of G-protein coupled receptors
US9081020B2 (en) 2008-02-11 2015-07-14 Heptares Therapeutics Limited Mutant proteins and methods for selecting them
DE102009013456A1 (de) * 2009-03-18 2010-09-23 GÖPFERICH, Achim, Prof. Dr. Nanopartikel für die Erkennung von Zellen, durch Bindung an G-Protein gekoppelte Rezeptoren
WO2010112417A1 (fr) 2009-03-30 2010-10-07 Ge Healthcare Uk Limited Procédés pour tester la liaison d'un ligand à des récepteurs couplés à la protéine g
US8703915B2 (en) 2009-06-22 2014-04-22 Heptares Therapeutics Limited Mutant proteins and methods for producing them
EP2561359A1 (fr) * 2010-04-20 2013-02-27 Addex Pharma SA Polypeptides chimériques utiles dans des procédés de criblage à haut débit proximaux et dynamiques
GB2502306A (en) * 2012-05-22 2013-11-27 Univ Singapore Microparticle sensor
WO2014168721A3 (fr) * 2013-04-12 2015-03-19 Tufts Medical Center Procédés et systèmes pour mettre au point et/ou caractériser des agents ligands lipidés solubles
US12060397B2 (en) 2013-04-12 2024-08-13 Tufts Medical Center Soluble lipidated ligand agents for treating eye inflammation
US11254720B2 (en) 2013-04-12 2022-02-22 On Target Therapeutics Llc Methods and systems for designing and/or characterizing soluble lipidated ligand agents
US10233219B2 (en) 2013-04-12 2019-03-19 Tufts Medical Center Methods and systems for designing and/or characterizing soluble lipidated ligand agents
JP2017536104A (ja) * 2014-10-14 2017-12-07 ユニベルシテ ドゥ モントリオール Ｇタンパク質活性化を監視するためのＧβγ相互作用タンパク質に基づいたバイオセンサー
CN107074926B (zh) * 2014-10-14 2021-09-21 蒙特利尔大学 基于Gβγ互作蛋白监测G蛋白激活的生物传感器
US10877036B2 (en) 2014-10-14 2020-12-29 Université de Montréal Biosensor based on Gβγ-interacting proteins to monitor G-protein activation
EP3207052A4 (fr) * 2014-10-14 2018-04-25 Université de Montréal Biocapteur faisant appel à des protéines interagissant avec la gbetagamma pour surveiller l'activation de la protéine g
CN107074926A (zh) * 2014-10-14 2017-08-18 蒙特利尔大学 基于Gβγ互作蛋白监测G蛋白激活的生物传感器
US11197906B2 (en) 2016-01-22 2021-12-14 On Target Therapeutics Llc Methods of lowering blink reflex for the treatment of dry eye disease
US12053501B2 (en) 2016-01-22 2024-08-06 Tufts Medical Center Methods for treating symptoms of dry eye disease
EP3697934A4 (fr) * 2017-10-18 2021-06-30 Université de Montréal Systèmes et procédés pour l'évaluation d'une activation des protéines g

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