Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

• the invention relates to assays based on a novel sweet receptor protein, heterologous expression systems containing nucleic acid constructs forming said sweet receptor protein, and the use of the sweet receptor protein in screening.
• Assays with the novel taste receptor protein may be used to identify agents that can modulate the taste response in humans (sweet taste modulators), and thereby render certain foods more palatable or increase patient compliance in oral pharmaceutics and nutraceutics.
• sweet taste modulators include sweet tastants that elicit a taste response in humans.
• the detection of sweet taste is known to be mediated by a receptor comprised of two subunits, T1 R2 and T1 R3, which are specifically expressed in taste receptor cells, and form a dimeric sweet taste receptor complex (T1 R2/T1 R3 heterodimer). Both subunits belong to the family of so-called "G-protein coupled receptors" or GPCRs, in particular class-C GPCRs.
• the class-C receptors have a heptahelical transmembrane domain (TMD).
• TMD heptahelical transmembrane domain
• the class-C GPCRs also have a large extracellular domain composed of two parts: a "venus flytrap module” (VFTM) that is involved in ligand binding; and a cysteine-rich domain (CRD), that contains nine highly conserved cysteines and that links the VFTM to the TMD.
• VFTM venus flytrap module
• CCD cysteine-rich domain
• a variable length intracellular C-terminal tail completes the class-C receptor.
• T1 R2/T1 R3 heterodimer Activation of the sweet receptor response was thought to require both subunits of the dimeric sweet receptor complex, and to date, all sweeteners tested activate the T1 R2/T1 R3 heterodimer.
• T1R2 homomer or T1 R3 homomer Published tests conducted with the separate subunits of the human sweet receptor (T1R2 homomer or T1 R3 homomer) have shown no activity, while the T1 R2/T1R3 heterodimer responds to a broad spectrum of chemically diverse sweeteners, ranging from natural sugars (sucrose, fructose, glucose, maltose), sweet amino acids (D-tryptophan), and artificial sweeteners (acesulfame-K, aspartame, cyclamate, saccharin, sucralose), to sweet tasting proteins (monellin, thaumatin, brazzein) (compare for example Li et al. (2002), Proc Natl Acad Sci USA 99(7), 4692-6
• T1R2 homomer binding assays are described in US 20050032158. Binding assays show binding only, as opposed to functional receptor activation, and are end-point-based and time consuming compared to faster functional assay that involve kinetic measurements. US 20050032158 further describes functional assays including cell-based assays for T1Rs, which are suitable for the known functional receptors T1 R1/T1 R3 and T1 R2/T1 R3.
• a novel receptor protein corresponding to a heavily truncated sequence of the T1 R2 homomer of the T1 R2/T1 R3 heterodimer receptor complex forms, surprisingly, a functional sweet receptor that binds to a sweet ligand and is able to activate G- proteins.
• T1 R2 "homomer” or “homomeric” polypeptide, protein, or receptor as used herein are meant to encompass the monomer, dimmer or oligomer of the T1 R2 polypeptide or protein, as opposed to the heterodimeric T1R2.T1R3 receptor complex.
• the novel receptor protein T1 R2-TMD was found to have a different agonist spectrum than the full-length T1R2 homomer. The latter was surprisingly found to be able not only to bind to ligands but also to activate downstream signalling.
• cells expressing both T1R2-TMD and a G-protein, but not T1 R3 are contacted with test agents, optionally in combination with known or newly determined sweet tastants, to determine the properties of said agents as sweet taste modulators.
• the assays provided herein may therefore be used to identify a tested agent as sweet tastant or modulator of the sweet response (of the sweet tastant, T1R2, or downstream events).
• the functional effects of the agent on the receptor and G-protein are determined by a suitable functional assay, for example, an assay that measures changes in parameters of the transduction pathways such as intracellular IP3 and Ca 2+ , or by other G-protein specific assays such as labeling with GTP ⁇ S, according to techniques known in the art.
• a suitable functional assay for example, an assay that measures changes in parameters of the transduction pathways such as intracellular IP3 and Ca 2+ , or by other G-protein specific assays such as labeling with GTP ⁇ S, according to techniques known in the art.
• binding assays may be used to determine ligand binding to T1R2-TMD.
• a T1R2-TMD sweet receptor able to bind to and be activated by perillartine comprising one or more of a polypeptide substantially homologous to SEQ ID NO:2, a polypeptide encoded by a nucleotide sequence substantially homologous to a nucleotide sequence set forth in SEQ ID NO:1 as determined by sequence identity, a polypeptide encoded by a nucleotide sequence substantially homologous to a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2 as determined by hybridisation, a polypeptide encoded by a nucleotide sequence substantially homologous to a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2 as determined by nucleotide sequence identity, wherein the substantially homologous polypeptide has a sequence identity of at least 74%; wherein the substantially homologous nucleotide as determined by sequence identity has a sequence identity of at least 65%;
• the T1 R2-TMD receptor does not comprise one or more of
• polypeptide homologous to SEQ ID NO:10 or SEQ ID NO:12 a polypeptide encoded by a nucleotide sequence homologous to a nucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 as determined by sequence identity, a polypeptide encoded by a nucleotide sequence homologous to a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 10 or SEQ ID NO: 12 as determined by hybridisation, a polypeptide encoded by a nucleotide sequence homologous to a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 10 or SEQ ID NO: 12 as determined by nucleotide sequence identity, wherein the polypeptide homologous to SEQ ID NO:10 or SEQ ID NO:12 has a sequence identity of at least 60%; wherein the nucleotide sequence homologous to a nucleotide sequence set forth in SEQ ID NO:
• the invention is directed to a T1R2-TMD sweet receptor selected from the group consisting of a polypeptide substantially homologous to SEQ ID NO:2, a polypeptide encoded by a nucleotide sequence substantially homologous to a nucleotide sequence set forth in SEQ ID NO:1 as determined by sequence identity, a polypeptide encoded by a nucleotide sequence substantially homologous to a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2 as determined by hybridisation, a polypeptide encoded by a nucleotide sequence substantially homologous to a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2 as determined by nucleotide sequence identity, wherein the substantially homologous polypeptide has a sequence identity of at least 74%; wherein the substantially homologous nucleotide as determined by sequence identity has a sequence identity of at least 65%; and wherein the substantially homologous nucleotide
• a nucleic acid encoding a T1 R2-TMD sweet receptor that is able to bind to and be activated by perillartine comprising one or more of a nucleic acid substantially homologous to a nucleotide sequence set forth in SEQ ID NO:1 as determined by sequence identity, a nucleic acid substantially homologous to a nucleotide sequence set forth in SEQ ID NO:1 as determined by hybridisation, a nucleic acid substantially homologous to a nucleotide sequence encoding the T1 R2- TMD sweet receptor, wherein the substantially homologous nucleotide as determined by sequence identity has a sequence identity of at least 65%; wherein the substantially homologous nucleotide as determined by hybridisation hybridises under stringent hybridization conditions at a temperature of 42° C in a solution consisting of 50% formamide, 5*SSC, and 1% SDS, and washing at 65° C in a solution consisting of 0.2 ⁇ SSC and 0.
• nucleic acid does not comprise one or more of
• nucleic acid homologous to a nucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:11 as determined by sequence identity a nucleic acid homologous to a nucleotide sequence set forth in SEQ ID NO:9 or SEQ ID NO:11as determined by hybridisation, a nucleic acid homologous to a nucleotide sequence encoding a polypeptide homologous to the polypeptide of SEQ ID NO: 10 or SEQ ID NO: 12, wherein the homologous nucleotide as determined by sequence identity has a sequence identity of at least 50%, and wherein the homologous nucleotide as determined by hybridisation hybridises under moderately stringent hybridization conditions at a temperature of 42° C in a solution consisting of 50% formamide, 5*SSC, and 1% SDS, and washing at 42° C in a solution consisting of 0.2*SSC and 0.1% SDS.
• nucleic acid selected from the group consisting of a nucleic acid substantially homologous to a nucleotide sequence set forth in SEQ ID NO:1 as determined by sequence identity, a nucleic acid substantially homologous to a nucleotide sequence set forth in SEQ ID NO:1 as determined by hybridisation, a nucleic acid substantially homologous to a nucleotide sequence encoding the T1 R2-TMD sweet receptor, wherein the substantially homologous nucleotide as determined by sequence identity has a sequence identity of at least 65%; wherein the substantially homologous nucleotide as determined by hybridisation hybridises under stringent hybridization conditions at a temperature of 42° C in a solution consisting of
• an expression vector comprising the nucleic acid as defined herein-above.
• a host cell transfected with an expression vector as defined herein-above but that does not contain T1R3.
• the host cell as defined herein-above stably expresses a T1 R2-TMD sweet receptor as defined herein-above and a G-protein.
• the host cell as defined herein-above transiently expresses a T1R2- TMD sweet receptor as defined herein-above and a G-protein.
• a method of producing a T1R2-TMD sweet receptor as defined herein-above comprising culturing host cells having contained therein an expression vector encoding for the T1 R2-TMD sweet receptor, under conditions sufficient for expression, thereby forming the T1R2-TMD sweet receptor and optionally recovering it from the cells.
• a method to identify an agent that modulates sweet taste signaling in taste cells comprises:
• T1 R2-TMD sweet receptor is selected from the group consisting of a polypeptide substantially homologous to SEQ ID NO:2, and a polypeptide encoded by a nucleotide substantially homologous to SEQ ID NO:1 as determined by sequence identity, and a polypeptide encoded by a nucleotide substantially homologous to SEQ ID NO:1 as determined by hybridisation; wherein the substantially homologous polypeptide has a sequence identity of at least 74%; wherein the substantially homologous nucleotide as determined by sequence identity has a sequence identity of at least 65%; wherein the substantially homologous nucleotide as determined by hybridisation hybridises under stringent hybridization conditions at a temperature of 42° C in a solution consisting of
• T1R2 sweet receptor expressing cells do not express a T1R3 receptor.
• the method as defined herein-above utilizes cells that also express a G-protein.
• the G-protein is a chimeric G-protein based on Gaq-Gustducin.
• the G-protein is the chimeric G-protein Galpha16-gustducin 44.
• (ii) is performed by measuring a change in or caused by intracellular messengers.
• the functional response is determined by measuring a change in an intracellular messenger selected from IP3 and calcium 2* .
• the cells are selected from the group consisting of bacterial cells, eucaryotic cells, yeast cells, insect cells, mammalian cells, amphibian cells, and worm cells.
• the cells are mammalian cells.
• the cells are mammalian cells selected from the group consisting of CHO, COS, HeLa and HEK-293 cells.
• (i) further comprises contacting the T1 R2 sweet receptor with a test agent in presence of a sweet tastant.
• the sweet tastant is selected from the group consisting of perillartine and methyl chavicol.
• kit comprising:
• kits defined herein-above.
• the method comprises:
• a method to identify an agent that modulates T1 R2-TMD comprising:
• the ligand is selected from the group consisting of perillartine and methyl chavicol.
• (i) is performed by a method selected from the group consisting of fluorescence spectroscopy, NMR spectroscopy, measuring of one or more of absorbance, refractive index, hydrodynamic methods, chromatography, measuring solubility, biochemical, wherein the methods measure the properties of the T ⁇ R2-TMD polypeptide in a suitable environment selected form the group consisting of solution, bilayer membrane, attached to a solid phase, in a lipid monolayer, bound on a membrane, and in vesicles.
• Useful cells in screens or assays according to the invention are cells that contain no T1 R3.
• Transfected or endogenous T1 R3 can negatively interfere with methods that determine agonist responses of T1 R2-TMD or the change of said responses dependent on another modulator.
• the absence of T1 R3 provides a null background for the determination of T1 R2- TMD activation, so that observed signals can be directly attributed to T1 R2-TMD activity. This allows the identification of agents that specifically modulate T1 R2-TMD, and excludes agents that activate T1R3 which could also include umami tastants, as T1R3 is part of both the sweet and the umami heterodimers.
• Suitable eucaryotic cells include eucaryotic cells that do not contain T1 R3, for example, without limitation, mammalian cells, yeast cells, or insect cells (including Sf9), amphibian cells (including melanophore cells), or worm cells including cells of Caenorhabditis (including Caenorhabditis elegans).
• Suitable mammalian cells that do not contain T1R3, include, for example, without limitation, COS cells (including Cos-1 and Cos-7), CHO cells, HEK293 cells, HEK293T cells, HEK293 T-RexTM cells, or other transfectable eucaryotic cell lines.
• Suitable bacterial cells that do not contain T1 R3 include without limitation E. coli.
• G-protein G alpha 16-gustducin 44 also known as G.sub..alpha.16 gust(ducin)44, G.sub.alpha.16gust(ducin)44, G ⁇ 16gust(ducin)44, Ga16gust(ducin)44, G ⁇ i 6 - g ustduci ⁇ 44 . or as used herein-below, "G16gust44” which provides for enhanced coupling to taste GPCRs, is described in detail in WO 2004/055048.
• G16gust44 chimeric G-proteins based on Gaq-Gustducin described in WO 2004/055048, or other G-Proteins, for example, G16 or G15, may also be used.
• T1 R2-TMD can be expressed in a cell with a G-protein that links the receptor to a signal transduction pathway, for example, the phospholipase C signal transduction pathway, or signal transduction pathways including, for example, the following: adenylate cyclase, guanylate cyclase, phospholipase C, IP3, GTPase/GTP binding, arachinoid acid, cAMP/cGMP, DAG, protein kinase c (PKC), MAP kinase tyrosine kinase, or ERK kinase.
• a signal transduction pathway for example, the phospholipase C signal transduction pathway, or signal transduction pathways including, for example, the following: adenylate cyclase, guanylate cyclase, phospholipase C, IP3, GTPase/GTP binding, arachinoid acid, cAMP/cGMP, DAG, protein
• any suitable reporter gene may be linked to a T1 R2-TMD-activation responsive promoter and used to determine T1 R2-TMD activity, as described in more detail herein- below.
• the vector constructs for expressing the GPCR and/or the G-protein in such cells may be produced in a manner known per se using Polymerase Chain Reactions. After verfication of the sequence, cDNA fragments may be sub-cloned into a suitable vector, for example pcDNA 3.1 mammalian expression vector for mammalian cells, and transiently transfected in a mammalian host cell to enable the correct expression of the gene.
• a suitable vector for example pcDNA 3.1 mammalian expression vector for mammalian cells, and transiently transfected in a mammalian host cell to enable the correct expression of the gene.
• cell lysates may be prepared, analysed by a Western-Blot analysis in order to confirm the correct expression of the protein.
• suitable cells for example mammalian cells including HEK293T cells and HEK T-RexTM, may be transfected to generate cells stably expressing the protein according to techniques well known in the art.
• non-mammalian expression vector/host systems can be used to contain and express sequences encoding the T1R2-TMD GPCR.
• these include, for example, microorganisms including bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (for example baculovirus), or with bacterial expression vectors (for example pBR322 plasmids).
• a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding the GPCR. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding a GPCR can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JoIIa Calif.) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding a GPCR into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
• PBLUESCRIPT Stratagene, La JoIIa Calif.
• PSPORT1 plasmid Life Technologies
• these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
• vectors which direct high level expression of a GPCR may be used.
• vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
• Yeast expression systems may be used for production of a GPCR.
• a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
• such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
• heterologous proteins in insect cell lines is, for example, derivatives of the Lepidopteran baculovirus, Autographa californica multicapsid nucleo-virus (>AcMNPV) can be used.
• AdMNPV Autographa californica multicapsid nucleo-virus
• foreign gene expression is directed by a very strong late viral promoter, either the polyhedrin or p10 promoters, and a wide array of vectors is available that optimises expression and recovery of recombinant proteins.
• These vectors enable expression of both membrane-bound and secreted proteins at high levels, and also many post- translational modifications known to occur in mammalian systems, including N- and O-linked glycosylation, phosphorylation, acylation, proteolysis and secreted vaccine components.
• a number of vectors are commercially available, for example the InsectSelectTM System from Invitrogen.
• cDNAs encoding the desired proteins In order to express cDNAs encoding the desired proteins (GPCR and G-protein), one typically subclones the appropriate cDNA into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and a ribosome-binding site for translational initiation.
• Suitable bacterial promoters are well known in the art, for example, E. coli, Bacillus sp., and Salmonella, and kits for such expression systems are commercially available.
• eukaryotic expression systems for mammalian cells, yeast, and insect cells are commercially available.
• the eukaryotic expression vector may be, for example, an adenoviral vector, an adeno-associated vector, or a retroviral vector.
• the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the protein-encoding nucleic acid in host cells.
• a typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding the protein and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
• the nucleic acid sequence encoding the protein may typically be linked to a membrane-targeting signal such as the N-terminal 45 amino acids of the rat Somatostatin-3 receptor sequence to promote efficient cell-surface expression of the recombinant protein, which is useful for cell-surface receptors. Additional elements may include, for example, enhancers.
• An expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
• the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
• vectors for expression in eucaryotic or procaryotic cells well known in the art may be used.
• vectors include bacterial expression vectors, for example, plasmids including pBR322-based plasmids, pSKF, and pET23D, and fusion expression systems , for example, GST and LacZ.
• Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, for example SV40 vectors, cytomegalovirus vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus.
• exemplary eukaryotic vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, pcDNA3.1 , plRES and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
• Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, dihydrofolate reductase and the like.
• the elements that are typically included in expression vectors may also include a replicon that functions in E. coli, a gene encoding drug resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in non-essential regions of the plasmid to allow insertion of eukaryotic sequences.
• the particular drug resistance gene chosen is not critical, any of the many drug resistance genes known in the art are suitable.
• the prokaryotic sequences are optionally chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
• the GPCR cDNA fragment may be expressed alone or as a fusion protein wherein the GPCR of interest is fused to the E. coli periplasmic maltose-binding protein (MBP) wherein the MBP, including its signal peptide, is linked to the amino terminus of the GPCR.
• MBP E. coli periplasmic maltose-binding protein
• the wild-type GPCR cDNA or the MBP:GPCR fusion cDNA is subcloned into a suitable plasmid, for example pBR322, where in E. coli, GPCR expression is driven by the lac wild-type promoter.
• yeast systems substitute a human GPCR and Ga protein for the corresponding components of the endogenous yeast pheromone receptor pathway. Downstream signaling pathways are also modified so that the normal yeast response to the signal is converted to positive growth on selective media or to reporter gene expression (described by Broach, J. R. and J. Thorner (1996) Nature 384 (supp.):14-16).
• Amphibian cell systems in particular melanophore cells, are described, for example, in WO 92/01810 that describes a GPCR expression system.
• T1 R2-TMD T1 R2-TMD may be overexpressed by placing it under the control of a strong constitutive promoter, for example the CMV early promoter.
• a strong constitutive promoter for example the CMV early promoter.
• certain mutations of conserved GPCR amino acids or amino acid domains can be introduced to render the employed GPCR constitutively active.
• Standard transfection methods can be used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of the protein.
• Any known method for introducing nucleotide sequences into host cells may be used. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing the relevant genes into the host cell capable of expressing the proteins of interest. These methods may involve introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell and include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and the like.
• the T-RexTM expression system (Invitrogen Corp., Carlsbad, CA) may be used.
• the T-RexTM System is a tetracycline-regulated mammalian expression system that uses regulatory elements from the E. coli Tn10-encoded tetracycline (Tet) resistance operon. Tetracycline regulation in the T-RexTM System is based on the binding of tetracycline to the Tet repressor and derepression of the promoter controlling expression of the gene of interest.
• the transfected cells may be cultured using standard culturing conditions well known in the art. It will be apparent to the skilled person that different cells require different culture conditions including approriate temperature and cell culture media.
• the protein may be recovered from the cell culture using standard techniques.
• the cells may be burst open either mechanically or by osmotic shock before being subject to precipitation and chromatography steps, the nature and sequence of which will depend on the particular recombinant material to be recovered.
• the recombinant protein may be recovered from the culture medium in which the recombinant cells had been cultured.
• Modulators ligands, agonists, partial agonists, antagonists, inverse agonists, inhibitors, enhancers
• T1 R2-TMD receptor activity can be identified as described herein below. There now follows a definition of the agents to be identified by said assays.
• a modulator is an agent that effects an increase or decrease of one or more of the following: the cell surface expression of a receptor, the binding of a ligand to a receptor, the intracellular response initiated by an active form of the receptor (either in the presence or absence or an agonist).
• the modulator can itself be an agonist that binds to the receptor, activates it and thereby modulates an increase in the cellular response.
• Modulators include various types of compounds, including small molecules, peptides, proteins, nucleic acids, antibodies or fragments thereof. These can be derived from various sources including synthetic or natural, extracts of natural material, for example from animal, mammalian, insect, plant, bacterial or fungal cell material or cultured cells, or conditioned medium of such cells.
• a ligand is an agent that binds to the receptor; it may be an agonist, partial agonist, enhancer, antagonist, or inverse agonist.
• An agonist is a ligand of the T1R2-TMD receptor that activates the receptor and increases an intracellular response when it binds to a receptor compared to the intracellular response in the absence of the agonist. Additionally or alternatively, an agonist may decrease internalization of a cell surface receptor such that the cell surface expression of a receptor is increased as compared to the number of cell surface receptors present on the surface of a cell in the absence of an agonist.
• a partial agonist is an agonist that only partially activates the receptor in comparison to other agonists that maximally activate the receptor.
• An antagonist is a ligand which binds to the receptor at the same (competitive antagonist) or at a different site (alllosteric antagonist) as an agonist, but does not activate an intracellular response initiated by an active form of a receptor, thereby inhibiting the intracellular response induced by an agonist as compared to the intracellular response in the presence of an agonist and in the absence of an antagonist.
• An inverse agonist binding to a receptor, decreases the constitutive intracellular response mediated by a receptor as compared to the intracellular response in the absence of the inverse agonist.
• An inhibitor decreases the binding of an agonist to the receptor as compared to the binding of the agonist in the absence of inhibitor, and/or decreases the intracellular response induced by an agonist.
• An enhancer increases the binding of an agonist to the receptor as compared to the binding of the agonist in the absence of enhancer, and/or increases the intracellular response induced by an agonist.
• the activity, or changes in activity, of a receptor binding a ligand and transmitting the signal through, for example, a G-protein can be determined by the assays described herein-below.
• Modulators can be identified using a wide variety of in vitro and in vivo assays to determine and compare functional effects/parameters, or alternatively by binding assays.
• the effects of the test agents upon the function of the receptors can be measured by examining a suitable functional parameters. Any physiological change that affects receptor activity can be used to identify modulators.
• Such functional assays are well-known in the art, for example assays using intact cells or tissues isolated from animals based on measuring the concentration or activity or their change of a secondary messenger (including, for example, intracellular calcium (Ca 2+ ), cAMP, cGMP, inositol phospate (IP 3 ), diacylglycerol/DAG, arachinoid acid, MAP kinase or tyrosine kinase), ion flux, phosphorylation levels, transcription levels, neurotransmitter levels, and assays based on GTP-binding, GTPase, adenylate cyclase, phospholipid-breakdown, diacylglycerol, inositol triphosphate, arachidonic acid release, PKC, kinase and transcriptional reporters.
• a secondary messenger including, for example, intracellular calcium (Ca 2+ ), cAMP, cGMP, inositol phospat
• Receptor activation typically initiates subsequent intracellular events, for example, increases in second messengers, for example, IP 3 , which releases intracellular stores of calcium ions.
• IP 3 inositol triphosphate
• IP 3 inositol triphosphate
• IP 3 phospholipase C-mediated hydrolysis of phosphatidylinositol.
• IP 3 in turn stimulates the release of intracellular calcium ion stores.
• All functional assays may be performed by samples containing cells expressing the receptor on their surfaces or on isolated cell membrane fractions. Useful cells are described herein- above. Instead of samples with separate cells or cell membranes, tissues from transgenic animals may be used.
• samples with and without test agent are compared.
• a control (with agonist but without modulator) is assigned a relative receptor activity value of 100.
• a decrease in activity relative to the control identifies an inhibitor, antagonist or inverse agonist, an increase identifies an enhancer.
• an increase or decrease in the measured activity of 10% or more in a sample with test agent compared to a sample without test agent or compared to a sample with test agent but based on cells that do not express T1R2-TMD (mock-transfected cells) can be considered significant.
• an agonist or partial agonist To identify an agonist or partial agonist, a sample with test agent is compared to a positive control with an agonist (for example perillartine or methyl chavicol), or alternatively/additionally, samples with and without test agent are compared in their receptor activity.
• an agonist or partial agonist will have a biological activity corresponding to at least 10% of the maximal biological activity of the positive control sweet tastant when the agonist or partial agonist is present at 100 mM or less, for example it may have a maximal biologial activity comparable to the agonist or higher.
• Maximal biological activity is defined as the maximal achievable receptor response to an agonist, for example perillartine or methyl chavicol, that can be achieved within a given receptor assay format and this response fails to increase further despite application of increasing concentrations of that same agonist.
• an increase in the measured activity of, for example, 10% or more in a sample with test agent is compared to a sample without test agent or is compared to a sample with test agent but based on cells that do not express T1R2-TMD (mock-transfected cells).
• Antagonists show a reduction of agonist-stimulated receptor activity, for example by at least 10%.
• receptor activity in the presence of a known agonist with and without a test agent is compared in samples comprising animals/cells/membranes that overexpress the receptor as described herein-above.
• Inverse agonists show a reduction of constitutive activity of the receptor, for example by at least 10%.
• Intracellular calcium release induced by the activation of GPCRs is detected using cell- permeant dyes that bind to calcium.
• the calcium-bound dyes generate a fluorescence signal that is proportional to the rise in intracellular calcium. The methods allows for rapid and quantitative measurement of receptor activity.
• Cells used are transfected cells that co-express the T1R2-TMD GPCR and a G-protein which allows for coupling to the phospholipase C pathway as described herein-above.
• Negative controls include cells or their membranes not expressing T1R2-TMD (mock transfected), to exclude possible non-specific effects of the candidate compound.
• Day 0 96-well plates are seeded with 8.5K cells per well and maintained at 37°C overnight in nutritive growth media.
• wash buffer consisting of 2.5 mM probenicid dissolved in a Hanks balanced salts solution (HBSS) that has been supplemented with 10 mM Hepes, 200 ⁇ M calcium chloride and 0.1% bovine serum albumin, pH 7.4 at 37°C, is added to each well and the plate is further incubated for 30 minutes at room temperature in the dark.
• HBSS Hanks balanced salts solution
• Buffer solutions are discarded and plate is washed 3 times with 100 ⁇ l wash buffer and cells are reconstituted in 200 ⁇ l of wash buffer and incubated for 15 minutes at 37°C.
• the plate is placed in a fluorescent microplate reader, for example, the Flexstation (Molecular Devices) or the FLIPR (Molecular Devices) and receptor activation is initiated following addition of 20 ⁇ l of a 10X concentrated ligand stock solution. Fluorescence is continuously monitored for 15 seconds prior to ligand addition and for 45 - 110 seconds after ligand addition.
• Useful cells are mammalian cells as described herein-above, for example HEK293T cells and HEK293 T-RexTM cells.
• Cells may be transfected with a GPCR and a G-Protein transiently or stably as is well known in the art.
• GPCR G-Protein transiently or stably as is well known in the art.
• An excellent heterologous expression system is described in detail in WO 2004/055048.
• a calcium flux assay can be performed, for example, as described in example 1 herein-below.
• the identification of a modulator is performed as described above subject to the following modifications.
• the signals are compared to the baseline level of T1R2-TMD activity obtained from recombinant cells expressing T1 R2-TMD in the presence of an agonist but in the absence of a test agent.
• An increase or decrease in T1 R2-TMD activity for example of at least 2 fold, at least 5 fold, at least 10 fold , at least a 100 fold, or more identifies a modulator.
• the identification involves an increase or decrease fluorescence intensity of, for example, 10% or more, when compared to a sample without modulator, or when compared to a sample with modulator but in cells that do not express the T1 R2-TMD polypeptide (mock- transfected cells).
• Adenylate Cyclase activity :
• Assays for adenylate cyclase activity are performed, for example, as described in detail by Kenimer & Nirenberg, 1981 , MoI. Pharmacol. 20: 585-591. Reaction mixtures are incubated usually at 37° C for less than 10 minutes. Following incubation, reaction mixtures are deproteinized by the addition of 0.9 ml of cold 6% trichloroacetic acid. Tubes are centrifuged and each supernatant solution is added to a Dowex AG50W-X4 column.
• the cAMP fraction from the column is eluted with 4 ml of 0.1 mM imidazole-HCI (pH 7.5) into a counting vial in order to measure the levels of cAMP generated following receptor activation by the agonist.
• Control reactions should also be performed using protein homogenate from cells that do not express a T1 R2-TMD polypeptide.
• inositol triphosphate (IP3)/Ca 2+ and thereby receptor activity can be detected using fluorescence.
• Cells expressing a GPCR may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it may be desirable, although not necessary, to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EDTA, to distinguish fluorescence response resulting from calcium release from internal stores.
• a chelating agent such as EDTA
• T1 R2-TMD is expressed in a cell with a G-protein that links the receptor to a phospholipase C signal transduction pathway. Changes in intracellular Ca 2+ concentration are measured, for example using fluorescent Ca 2+ indicator dyes and/or fluorometric imaging.
• a measure of receptor activity is the binding of GTP by cell membranes containing the GPCR. Measured is the G-protein coupling to membranes by detecting the binding of labelled GTP.
• Membranes isolated from cells expressing the receptor are incubated in a buffer containing
• 35S-GTP ⁇ S and unlabelled GDP Active GTPase releases the label as inorganic phosphate, which is detected by separation of free inorganic phosphate in a 5% suspension of activated charcoal in 20 mM H 3 PO 4 , followed by scintillation counting. The mixture is incubated and unbound labelled GTP is removed by filtration onto GF/B filters. Bound and labelled GTP is measured by liquid scintillation counting. Controls include assays using membranes isolated from cells not expressing T1 R2-TMD (mock-transfected), in order to exclude possible nonspecific effects of the test agent. The method is described in detail by Traynor and Nahorski, 1995, MoI. Pharmacol. 47: 848-854.
• an agent is identified as an agonist usually if the activity is at least 50% of that of a known agonist (for example perillartine) when the compound is present at 100 mM or less, for example 10 to 500 ⁇ M, for example about 100 ⁇ M, or if it will induce a level the same as or higher than that induced by a known agonist.
• a known agonist for example perillartine
• Such assays can be performed as described in detail in Hafner, 2000, Biosens. Bioelectron. 15: 149-158.
• the intracellular level of arachinoid acid is employed as an indicator of receptor activity. Such a method is described in detail by Gijon et al., 2000,J. Biol. Chem., 275:20146-20156.
• Intracellular or extracellular cAMP is measured using a cAMP radioimmunoassay (RIA) or cAMP binding protein, for example as described by Horton & Baxendale, 1995, Methods MoI. Biol. 41: 91-105.
• a number of kits for the measurement of cAMP are commercially available, for example the High Efficiency Fluorescence Polarization-based homogeneous assay by LJL Biosystems and NEN Life Science Products.
• the intracellular or extracellular levels of cGMP may measured using an immunoassay. For example, the method described in Felley-Bosco et al., Am. J. Resp. Cell and MoI.
• Biol., 11 :159-164 (1994) may be used to determine the level of cGMP.
• an assay kit for measuring cAMP and/or cGMP as described in US 4,115,538 can be used.
• Negative controls with mock-transfected cells or extracts thereof to exclude possible nonspecific effects of test agents may be used.
• DAG Diacylglycerol
• IP3 inositol triphosphate
• Negative controls with mock-transfected cells or extracts thereof to exclude possible nonspecific effects of test agents may be used.
• Growth factor receptor tyrosine kinases can signal via a pathway involving activation of
• PKC Protein Kinase C
• Gene products induced by PKC show PKC activation and thereby receptor activity.
• gene products include, for example, proto-oncogene transcription factor- encoding genes (including c-fos, c-myc and c-jun), proteases, protease inhibitors (including collagenase type I and plasminogen activator inhibitor), and adhesion molecules (including intracellular adhesion molecule I (ICAM I)).
• proto-oncogene transcription factor- encoding genes including c-fos, c-myc and c-jun
• proteases including collagenase type I and plasminogen activator inhibitor
• adhesion molecules including intracellular adhesion molecule I (ICAM I)
• PKC activity may be directly measured as described by Kikkawa et al., 1982, J. Biol. Chem. 257: 13341 , where the phosphorylation of a PKC substrate peptide, which is subsequently separated by binding to phosphocellulose paper, is measured. It can be used to measure activity of purified kinase, or in crude cellular extracts. Protein kinase C sample can be diluted in 20 mM HEPES/ 2 mM DTT immediately prior to the assay.
• activity can be measured through the use of reporter gene constructs driven by the control sequences of genes activated by PKC activation.
• Negative controls with mock-transfected cells or extracts thereof to exclude possible nonspecific effects of test agents may be used.
• MAP kinase activity can be measured using commercially available kits, for example, the p38 MAP Kinase assay kit by New England Biolabs, or the FlashPlateTM MAP Kinase assays by Perkin-Elmer Life Sciences.
• p42/44 MAP kinases or ERK1/2 can be measured to show T1R2-TMD activity when cells with Gq and Gi coupled GPCRs are used, and an ERK1/2 assay kit is commercially available by TGR Biosciences, which measures the phosphorylation of endogenous ERK1/2 kinases following GPCR activation.
• tyrosine kinase activity through known synthetic or natural tyrosine kinase substrates and labelled phosphate are well known; the activity of other types of kinases (for example, Serine/Threonine kinases) can be measured similarly.
• All kinase assays can be performed with both purified kinases and crude extracts prepared from cells expressing a T1 R2-TMD polypeptide.
• the substrates of kinases that are used can be either full-length protein or synthetic peptides representing the substrate.
• Pinna & Ruzzene (1996, Biochem. Biophys. Acta 1314: 191-225) lists a number of phosphorylation substrate sites useful for detecting kinase activities.
• a number of kinase substrate peptides are commercially available.
• One that is particularly useful is the "Src-related peptide," RRLIEDAEYAARG (commercially available from Sigma), which is a substrate for many receptor and nonreceptor tyrosine kinases.
• Some methods require the binding of peptide substrates to filters, then the peptide substrates should have a net positive charge to facilitate binding.
• peptide substrates should have at least 2 basic residues and a free-amino terminus. Reactions generally use a peptide concentration of 0.7-1.5 mM. Negative controls with mock-transfected cells or extracts thereof to exclude possible nonspecific effects of test agents may be used.
• Transcriptional reporters/ T1 R2-TMD-responsive promoter/reporter gene To identify modulators with reporter gene assays, an at least 2-fold increase or 10% decrease in the signal is significant.
• An agonist stimulates for example at least 2-fold, 5-fold, 10-fold or more when comparing activity in presence and absence of the test agent.
• the intracellular signal initiated by binding of an agonist to T1R2-TMD sets in motion a cascade of intracellular events, the ultimate consequence of which is a rapid and detectable change in the transcription or translation of one or more genes.
• the activity of the receptor can therefore be determined by measuring the expression of a reporter gene driven by a promoter responsive to T1R2-TMD activation.
• a “promoter” as used herein is one or more transcriptional control elements or sequences necessary for receptor-mediated regulation of gene expression, including one or more of basal promoter, enhancers and transcription-factor binding sites necessary for receptor- regulated expression. Promoters responsive to the intracellular signals resulting from agonist binding to T1R2-TMD are selected and operatively linked to a corresponding promoter- controlled reporter gene whose transcription, translation or ultimate activity is readily detectable and measurable.
• Reporter genes may be selected, for example, from luciferase, CAT, GFP, ⁇ -lactamase, ⁇ - galactosidase, and the so-called "immediate early" genes, c-fos proto-oncogene, transcription factor CREB, vasoactive intestinal peptide (VIP) gene, the somatostatin gene, the proenkephalin gene, the phosphoenolpyruvate carboxy-kinase (PEPCK) gene, genes responsive to NF- ⁇ B, and AP-1 -responsive genes (including the genes for Fos and Jun, Fos- related antigens (Fra) 1 and 2, l ⁇ B ⁇ , ornithine decarboxylase, and annexins I and II).
• Promoters will be selected according to the selected reporter gene, as will be apparent to the skilled person. Luciferase, CAT, GFP, ⁇ -lactamase, ⁇ -galactosidase and assays for the detection of their products are well known in the art. Examples of further reporter genes are described herein- below.
• reporter genes are suitable and are rapidly induced (for example within minutes of contact between the receptor and the effector protein or ligand).
• Desirable properties in reporter genes include one or more of the following: rapid responsiveness to ligand binding, low or undetectable expression in quiescent cells; induction that is transient and independent of new protein synthesis; subsequent shut-off of transcription requires new protein synthesis; and mRNAs transcribed from these genes which have a short half-life of several minutes to a few hours.
• the promoter may have one, several or all of these properties.
• the c-fos proto-oncogene is an example of a gene that is responsive to a number of different stimuli and has an rapid induction.
• the c-fos regulatory elements include a TATA box that is required for transcription initiation; two upstream elements for basal transcription, and an enhancer, which includes an element with dyad symmetry and which is required for induction by TPA, serum, EGF, and PMA.
• the 20 bp c-fos transcriptional enhancer element located between -317 and -298 bp upstream from the c-fos mRNA cap site, is essential for serum induction in serum starved NIH 3T3 cells.
• One of the two upstream elements is located at -63 to -57 and it resembles the consensus sequence for cAMP regulation.
• the transcription factor CREB (cyclic AMP responsive element binding protein) is responsive to levels of intracellular cAMP. Therefore, the activation of a receptor that signals via modulation of cAMP levels can be determined by detecting either the binding of the transcription factor, or the expression of a reporter gene linked to a CREB-binding element (termed the CRE, or cAMP response element).
• the DNA sequence of the CRE is TGACGTCA. Reporter constructs responsive to CREB binding activity are described in US 5,919,649.
• VIP vasoactive intestinal peptide
• somatostatin gene and its promoter which is cAMP responsive
• proenkephalin and its promoter which is responsive to cAMP, nicotinic agonists, and phorbol esters
• PPCK phosphoenolpyruvate carboxy- kinase
• reporter genes and their promoters that are responsive to changes in GPCR activity include the AP-1 transcription factor and NF- ⁇ B.
• the AP-1 promoter is characterised by a consensus AP-1 binding site which is the palindrome TGA(C/G)TCA.
• the AP-1 site is also responsible for mediating induction by tumor promoters including the phorbol ester 12-O-tetradecanoylphorbol- ⁇ -acetate (TPA), and are therefore sometimes also referred to as a TRE, for TPA-response element.
• TPA phorbol ester 12-O-tetradecanoylphorbol- ⁇ -acetate
• TRE for TPA-response element.
• AP-1 activates numerous genes that are involved in the early response of cells to growth stimuli.
• AP-1 -responsive genes include the genes for Fos and Jun (which proteins themselves make up AP-1 activity), Fos-related antigens (Fra) 1 and 2, l ⁇ B ⁇ , ornithine decarboxylase, and annexins I and II.
• the NF- ⁇ B promoter/binding element has the consensus sequence GGGGACTTTCC.
• Genes responsive to NF- ⁇ B include for example those encoding IL-1 ⁇ , TNF- ⁇ , CCR5, P-selection, Fas ligand, GM-CSF and l ⁇ B ⁇ .
• Vectors encoding NF- ⁇ B-responsive reporters are known in the art or can be readily formed using ordinary skill in the art, for example, synthetic NF- ⁇ B elements and a minimal promoter, or using the NF- ⁇ B-responsive sequences of a gene known to be subject to NF- ⁇ B regulation. Further, NF- ⁇ B responsive reporter constructs are commercially available from, for example, CLONTECH.
• a given promoter construct can easily be tested by exposing T1R2-TMD-expressing cells, transfected with the construct, to an agonist (for example perillartine).
• an agonist for example perillartine.
• An increase of at least 2-fold in the expression of reporter gene in response to the agonist indicates that the reporter is suitable to measure T1 R2-TMD activity.
• Controls for transcription assays include both cells not expressing T1 R2-TMD, but carrying the reporter construct, and cells with a promoterless reporter construct.
• Agents that modulate T1 R2-TMD activity as shown by reporter gene activation can be verified by using other promoters and/or other receptors to verify T1 R2-TMD specificity of the signal and determine the spectrum of their activity, thereby excluding any non-specific signals, for example non-specific signals via the reporter gene pathway.
• IP Inositol Phosphates
• Phosphatidyl inositol (Pl) hydrolysis may be determined as described in US 5,436,128, which involves labelling of cells with 3H-myoinositol for at least 48 hours or more.
• the labelled cells are contacted with a test agent for one hour, then these cells are lysed and extracted in chloroform-methanol-water. This is followed by separating the inositol phosphates by ion exchange chromatography and quantifying them by scintillation counting.
• fold stimulation is determined by calculating the ratio of counts per minute (cpm) in the presence of tested agent, to cpm in the presence of buffer control.
• fold inhibition is determined by calculating the ratio of cpm in the presence of test agent, to cpm in the presence of buffer control (which may or may not contain an agonist).
• ligand binding may be determined by binding assays that measure the binding of a ligand to the T1R2-TMD receptor.
• Binding assays are well known in the art and can be tested in solution, in a bilayer membrane, optionally attached to a solid phase, in a lipid monolayer, or in vesicles. Binding of a modulator to a T1R2-TMD polypeptide can be determined, for example, by measuring changes in spectroscopic characteristics (for example fluorescence, absorbance, or refractive index), hydrodynamic methods (employing for example shape), chromatography, measuring solubility properties of a T1R2-TMD polypeptide. In one embodiment, binding assays are biochemical and use membrane extracts from cells/tissue expressing recombinant T1R2- TMD polypeptides.
• a binding assay may, for example, be performed as described for T1 Rs by Adler et al. in US20050032158, paragraphs [0169] to [0198], therein called “in vitro binding assays", in distinction from functional assays which in US20050032158 are called “cell based binding assays”.
• T1 R2-TMD receptor polypeptide and nucleic acid and substantially homologous polypeptides and nucleic acids:
• the T1R2-TMD receptor useful in methods according to the invention may be the receptor of SEQ ID NO:2, or alternatively a receptor (or nucleotide sequence to form the T1R2-TMD receptor) which is subtantially homologous and remains functional (i.e. binds to ligands and is activated by ligands).
• Such homologous receptors may be, for example, an allelic variant of SEQ ID NO:2, or the corresponding homologous sequence of a different species including rat (about 77.9% amino acid sequence identity and about 81.2% nucleic acid identity), mouse (about 76.2% amino acid sequence identity and about 80.9% nucleic acid identity), dog (about 74.4% amino acid sequence identity and about 82.6% nucleic acid identity), or any other species having sufficient amino acid sequence identity to the human receptor.
• substantially homologous T1R2-TMD nucleotide or polypeptide sequences may be formed by conservative mutations and/or point mutations and include any conservatively modified variant as detailed below.
• nucleic acid sequences conservatively modified variants means nucleic acids which encode identical or essentially identical amino acid sequences (conservatively substituted amino acids, i.e. lysine switched to arginine and further examples as explained herein-below).
• nucleic acids Because of the degeneracy of the genetic code, a large number of nucleic acids different in sequence but functionally identical encode any given polypeptide/protein. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Each nucleic acid sequence which encodes a polypeptide also describes every possible silent variation of the nucleic acid. Therefore, each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical nucleic acid sequence that will produce an identical polypeptide.
• AUG which is ordinarily the only codon for methionine
• TGG which is ordinarily the only codon for tryptophan
• each silent variation of a nucleic acid which encodes a polypeptide is implicit in each given nucleic acid sequence.
• amino acid substitutions may be introduced using known protocols of recombinant gene technology including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in-vitro transcription which may be used to introduce such changes to the T1 R2-TMD sequence.
• the variants can then be screened for taste-cell-specific GPCR functional activity.
• Conservative substitution tables providing functionally similar amino acids are well known in the art.
• one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): ala/gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gin; ile/leu or val; leu/ile or val; lys/arg or gin or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu.
• An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (1); 5) lsoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
• Another alternative guideline is to allow for all charged amino acids as conservative substitutions for each other whether they are positive or negative.
• Substantially homologous nucleotide or polypeptide sequences have the degree of sequence identity or hybridize under certain stringent hybridization conditions as indicated below.
• a substantially homologous nucleotide sequence has a % sequence identity of at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 98%.
• a substantially homologous polypeptide sequence has a % sequence identity of at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.
• BLAST Basic Local Alignment Search Tool
• blastn is the heuristic search algorithm employed by the programs blastn which is available at http://www.ncbi.nlm.nih.gov.
• Blastn is used, using default parameters of BLAST version 2.2.1.3, including an
• EXPECT statistical significance threshold for reporting matches against database sequences
• DUST filtering DUST filtering
• Blastp is used, using default parameters of BLAST version 2.2.1.3, including an
• Nucleotide sequences are considered substantially homologous provided that they are capable of selectively hybridizing to the nucleotide sequences presented herein, or to their complement, under stringent hybrdisation conditions detailed below.
• Background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened.
• a signal that is less than 10 fold as intense as the specific interaction observed with the target DNA is considered background.
• the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32P. Kit to identify a modulator :
• a kit for example a screening kit or high throughput screening kit, that comprises recombinant cells that express the T1R2-TMD homomer, or a substantially homologous sequence thereto, but that do not express T1 R3; and that comprises an agonist of the T1 R2- TMD homomer, for example perillartine or methyl chavicol.
• the cells further comprise a G-protein for example for calcium signalling.
• G-proteins are known and described herein-above; the skilled person is aware how to introduce them to the cells if necessary.
• a very useful chimeric G-protein is Galpha16- gustducin 44.
• the agonist is provided in suitable concentrations, for example 1 nM to 10 mM, or 0.1 microM to1 milliM, for example 0.1 microM to 100 microM.
• Optional kit components may include a suitable medium for culturing the recombinant cells provided, and a solid support to grow the cells on, for example, a cell culture dish or microtiter plate, these optional components will be readily available to the skilled person.
• the kit may be used as follows:
• Test agents at concentrations from about 1 nM to 100 mM or more are added to the culture medium of defined plates or wells in the presence of the agonist in a suitable concentration
• a change in a functional response of the cells is determined by comparing the response in presence and absence of the test agent, and the test agent is thereby identified as a modulator.
• (iii) may be performed according to any one of the assays described-herein above, in combination with any one of the detection methods that report receptor activity described herein-above. This may require specifically chosen or adapted recombinant cells, which are also described herein-above.
• a suitable assay is, for example, the calcium flux assay to determine activation of T1R2-TMD and its change in response to a test agent.
• a modulator identified by a method described herein-above may easily be confirmed by simple sensory experiments using a panel of flavorists or test persons to taste the identified modulators.
• the compounds are tasted e.g. in water to confirm sweet taste or together with sweet tastants in comparison to a negative control without modulator to confirm a modulator that enhances the sweet taste.
• Transcriptional reporter assays and most cell-based assays described herein-above are well suited for screening libraries for agents that modulate T1R2-TMD activity.
• the assays may be designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to the assays, which are typically run in parallel (for example in microtiter formats on microtiter plates in robotic assays).
• Assays may be run in high throughput screening methods that involve providing a combinatorial chemical or peptide library containing a large number of potential modulators. Such libraries are then screened in one or more assays described herein-above to identify those library agents (particular chemical species or subclasses) that display the activity described herein-above.
• the modulators thus identified can be directly used or may serve as leads to identify further modulators by making and testing derivatives.
• Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N. H.), and Microsource (New Milford, Conn.).
• a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
• a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
• a rare chemical library is available from Aldrich (Milwaukee, Wis.).
• libraries include protein/expression libraries, cDNA libraries from natural sources, including, for example, foods, plants, animals, bacteria, libraries expressing randomly or systematically mutated variants of one or more polypeptides, genomic libraries in viral vectors that are used to express the mRNA content of one cell or tissue.
• each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
• a single standard microtiter plate can assay about 100 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds is possible.
• Types of test agents that may be tested for their T1 R2-TMD modulating effect in the assay methods:
• test agents may be any agent including small chemical compounds, chemical polymers, biological polymers, peptides, proteins, sugars, carbohydrates, nucleic acids and lipids.
• An agent can be a synthetic compound, a mixture of compounds, a natural product or natural sample, for example plant extract, culture supernatant, or tissue sample.
• sweet tastant compounds or compounds that modifiy sweet taste there may be mentioned Theasaponin E1, Acesulfame K, Alitame, Aspartame, CH 401, Dulcin, Erythritol, Guanidine Sweetener, Isomalt, Isomaltosylfructoside, Isoraffinose, NC 174, Neotame, Phenylacetylglycyl-L-Lysine, Saccharin, SC 45647, sodium Cyclamate, Sorbitol, Sucralose, Sucrononic Acid, Suosan, Superaspartame, Methyl alpha-L-arabinoside, Methyl beta-L-arabinoside, Methyl beta-D-Glucoside, Methyl a-D-mannoside, Methyl beta-L- xylopyranoside, Methyl alpha-D-xyloside, Methyl
• Identified sweet tastants may include, for example, artificial sweeteners that are able to elicit a sweet taste sensation. These are of particular interest as they can be used to replace sugar compounds, for example, to reduce calories or to provide consumables that are more healthy for the teeth.
• Consumables include food products, beverages, oral care products, and compositions for admixture to such products, in particular flavour compositions.
• Flavour compositions may be added to processed foods or beverages during their processing, or they may actually be consumables in their own right, e.g. condiments such as sauces and the like.
• Sweet tastants are particularly interesting in confectionary and other sweet consumables including desserts, but also in savoury and sweet-sour consumables.
• consumables examples include confectionary products, cakes, cereal products, baker's products, bread products, gums, chewing gums, sauces (condiments), soups, processed foods, cooked fruits and vegetable products, meat and meat products, egg products, milk and dairy products, cheese products, butter and butter substitute products, milk substitute products, soy products, edible oils and fat products, medicaments, beverages, alcoholic drinks, beers, soft drinks, food extracts, plant extracts, meat extracts, condiments, sweeteners, nutraceuticals, pharmaceutical and non-pharmaceutical gums, tablets, lozenges, drops, emulsions, elixirs, syrups and other preparations for making beverages, instant beverages and effervescent tablets.
• SEQ ID NO:1 corresponds to the nucleotide/nucleic acid sequence encoding the T1 R2-TMD receptor
• SEQ ID NO: 2 corresponds to the polypeptide/amino acid sequence of the T1R2-TMD receptor protein.
• SST TAG SEQ ID NO:3
• SEQ ID NO:1 the nucleotide/nucleic acid sequence encoding the T1 R2-TMD receptor
• SEQ ID NO: 2 corresponds to the polypeptide/amino acid sequence of the T1R2-TMD receptor protein.
• SST TAG SEQ ID NO:3
• SEQ ID NO:1 novel T1R2-TMD protein
• HSV TAG nucleic acid SEQ ID NO:5
• the resulting protein will accordingly comprise the following amino acids in the order indicated: amino acids of SEQ ID NO:4, SEQ ID NO:2, and SEQ ID NO:6.
• SEQ ID NOS: 1 + 2 T1R2-TMD nucleic acid and protein
• SEQ ID NOS: 3 + 4 SST TAG nucleic acid and protein
• SEQ ID NOS: 5 + 6 HSV TAG nucleic acid and protein
• Fluo-4 is a fluorescent indicator for intracellular calcium and allows to determine changes in the calcium concentration, in particular an increase in response to receptor activation occurring after ligand addition (for example pehllartine or methyl chavicol).
• HEK293 cells stably expressing G alpha 16 gustducin 44 and transfected with T1 R2-TMD as described in example 3 were used as host cells.
• Black, clear-bottom 96-well plates were used for all assays. They were seeded the day before with 8500 transfected cells per well and maintained at 37° C overnight in a growth medium appropriate for the cells used.
• Dulbecco's Modified Eagle medium containing high glucose, L-glutamine, pyroxidine hydrochloride, and supplemented with10% fetal bovine serum was used for growth and maintenance of the HEK293 cells.
• C1 buffer solution contains 130 mM NaCI, 5 mM KCI, 10 mM Hepes, 2 mM CaCI2 and 10 mM glucose (pH 7.4).
• the plates were washed 5 times with 100 ⁇ l per well of C1 buffer using an automated plate washer (BioTek) and after washing, the plate was further incubated for 30 minutes at room temperature in the dark to allow for complete de- esterification of the Fluo-4-AM.
• the buffer solutions were discarded, the plate was washed 5 times with 100 ⁇ l C1 wash buffer and finally the cells were reconstituted in 180 ⁇ l of C1 wash buffer.
• the plate was placed in a FLIPR (fluorescence imaging plate reader (FLIPR-Tetra, Molecular Devices)), and receptor activation was initiated following addition of 20 ⁇ l of a 10X concentrated ligand stock solution.
• FLIPR fluorescence imaging plate reader
• RNU relative flourescence units
• Fluorescence Increase Maximum Fluorescence - baseline fluorescence, wherein the baseline fluorescence represents the mean fluorescence calculated for the first 10 to 15 seconds prior to ligand addition.
• the template for the PCR amplification was a full length cDNA for human T1R2 isolated from a cDNA library generated from human fungiform papillae taste tissue. Reaction parameters were: 94° C for 5 min followed by 35 cycles of 94° C for 45 seconds, 54° C for 15 seconds and 68 ° C for 1 minute, followed by a final extension cycle of 68° C for 10 minutes.
• the resulting nucleic acid fragment (compare Seq ID NO:1) was separated by gel electrophoresis, purified and subcloned into the pCR-Topo-ll vector (Invitrogen) and the resulting clones were verified by DNA sequencing to ensure absence of mutations arising from the PCR amplification.
• the T1R2-TMD insert was subcloned into an expression cassette based on pcDNA4-TO (Invitrogen).
• the cloning cassette already contains the first 45 amino acids of the rat somatostatin type 3 receptor at the N-terminus to facilitate cell surface membrane targeting of the transgene (described by Bufe et al. 2002, Nat Genet, 32(3), 397-401 ).
• the C-terminus of this vector encodes the herpes simplex virus (HSV) glycoprotein D epitope, which can be used for immuncytochemistry studies using a specific antibody that binds to this epitope.
• HSV herpes simplex virus
• T1 R2-TMD protein of joined amino acid sequences of Seq ID 2 (T 1 R2- TMD) preceded by Seq ID NO:4 (45 aminoacids of rat somatostatin) and followed by 6 (HSV epiotope) (in amino terminus to C terminus direction).
• T1R2-TMD Transfection of T1R2-TMD into cells, cells stably expressing T1R2-TMD and G16qust44
• Human cell lines that stably express human T1R2-TMD were generated by transfecting a linearised pCDNA 4-T0 vector (Invitrogen) containing the hT1R2-TMD (formed as described in 2) into a G16gust44 expressing cell line, which was formed as described in WO 2004/055048.
• This cell line shows enhanced coupling to taste receptors, is tetracycline inducible, stably expresses the G16gust44 promiscuous G-protein, and is based on the HEK- 293-T-Rex cell line (commercially available from Invirtogen, USA). Transfection is performed as follows:
• the HEK293T/ G16gust44 cells are plated in 6-well black, clear-bottom plates at a density of 900,000 cells per well and grown overnight in selective growth media.
• the media is changed to an antibiotic-free and serum-free growth medium and the cells are transfected using 4 ug of linearized T1R2 TMD vector construct DNA and 0.3 I of Lipofectamine 2000 (Invitrogen.
• the lipofectamine/DNA mixture is incubated on the cells for 3-4 hours and then replaced with an antibiotic-free, serum-containing growth medium.
• the cells are re-plated in selective medium containing DMEM supplemented with 10% FBS, 0.005 mg/ml blasticidin, 0.36 mg/ml G418, and 0.1 mg/ml zeocin (Invivogen) at 37.degrees C.
• zeocin-resistant colonies were selected, expanded, and tested by calcium imaging as described in example 1 for responses to 50 M perillartine. Resistant colonies were expanded, and identified as containing T1R2-TMD by their response to 50 ⁇ M perillartine, which was determined via automated fluorimetric imaging on the FLIPR-
• Tetra instrumentation using the methods described in example 1 following induction of T1 R2-TMD expression with 10 ⁇ g/ml tetracycline.
• the lack of response in the tetracycline-induced T1R2-TMD cells may be due to cellular toxicity arising from tetracycline-induced overexpression of the T1R2-TMD.
• T1 R3 was constitutively overexpressed in the presence of a tetracycline-regulated T1R2 to avoid possible cytotoxic effects of constitutive overexpression of both proteins. Placing one subunit of the heterodimer (T1 R2) in a tetracycline-regulated vector allows to regulate its expression level so that viability and functionality of the stable clonal lines can be optimised accordingly.
• Human cell lines that stably express the human T1 R2/T1 R3 sweet heterodimer were generated by first transfecting a linearized plRES-Puro vector (Clontech ) containing the human T1R3 into a G16gust44 expressing cell line, which was formed as described in WO 2004/055048.
• This cell line shows enhanced coupling to taste receptors, is tetracycline inducible, stably expresses the G16gust44 promiscuous G-protein, and is based on the HEK- 293-T-Rex cell line (commercially available from Invirtogen, USA).
• a linearized pcDNA4-TO vector (Invitrogen) containing human T1R2 cDNA was transfected.
• the cells were re-plated at 10x dilutions up to 1 :150,000 in selective medium containing Gluatamax DMEM (Invitrogen) supplemented with 10% FBS, 0.005 mg/ml blasticidin. 0.36 mg/ml G418, and 0.4 ⁇ g/ml puromycin at 37.degrees C.
• Gluatamax DMEM Invitrogen
• FBS 0.005 mg/ml blasticidin.
• a puromycin-resistant heterogeneous population of T1R3-expressing cells were then transfected with 4 ug of the linearized T1 R2 vector construct DNA and 0.3 I of Lipofectamine 2000 (Invitrogen) .
• the lipofectamine/DNA mixture was incubated on the cells for 3-4 hours and then replaced with an antibiotic-free, serum-containing growth medium.
• the cells were re-plated in selective medium containing Glutamax DMEM supplemented with 10% FBS, 0.005 mg/ml blasticidin. 0.36 mg/ml G418, 0.4 g/ml puromycin, and 0.1 mg/ml zeocin at 37.degrees C.
• Resistant colonies were expanded, and identified as containing the T1 R2/T1 R3 sweet heterodimer by their response to various sweetener compounds including sucrose, sucralose, aspartame and acesulfame K, which was determined via automated fluorimetric imaging on the FLIPR-Tetra instrumentation (Molecular Devices) using the methods described in example 1.
• All potential clones were also evaluated for a functional response to sweet tastants in the presence of 10 ⁇ g/ml tetracycline (to induce overexpression of T1 R2) as well as in the absence of tetracycline induction to identify any clones that are basally expressing low-level but sufficient expression of the T1 R2 receptor to allow for assembly with T1 R3 resulting in a functional sweet heterodimer complex
• the tetracycline-regulated systems such as the T-Rex HEK-293 (Invitrogen) are known to have a low-level basal expression of transgenes due to the inherent leakiness of the system.).
• T1 R2 Signals were lower in cells treated with tetracycline to induce overexpression of the T1 R2.
• the lower response in the tetracycline-induced T1R2/T1R3 cells may be due to cellular toxicity arising from tetracycline-induced overexpression of the T1R2.
• One clonal cell line exhibiting the greatest response to sweet tastants was propagated and used for subsequent comparisons to the T1 R2-TMD stable cell line.
• HEK293T cells stably expressing G16gust44 and stably transfected with T1R2-TMD, which were formed as described in example 3.
• Intracellular calcium responses to 50 ⁇ M perillartine were determined.
• Cells were plated in black, clear-bottom plates (Costar) at a density of 8500 cells/well and maintained in selective growth media (as described in example 3) for 48 hours before determining receptor activity as described in example 1.
• the cells were not induced with tetracycline since the particular clone selected already basally expressed sufficient levels of the T1 R2-TMD to generate a robust increase in intracellular calcium following ligand stimulation.
• the data was calculated as described in example 1 and illustrated the net increase in fluorescence over baseline following stimulation of the cells with 50 ⁇ M perillartine.
• the data represent the mean ⁇ Standard deviation of six replicate experiments.
• the method quantitates T1 R2-TMD activity and allows, for example, to predict the potency of identified modulators including sweet tastants.
• HEK293T cells that stably express G16gust44 and T1R2-TMD are loaded with the calcium dye Fluo-4, and their response to perillartine is measured by using fluorescent calcium signals as described in example 1.
• the data was calculated as described in example 1 (net increase in fluorescence over baseline following stimulation of the cells with increasing doses of perillartine, over a range of 0.1 to 200 micromolar).
• the data includes the mean ⁇ standard deviation (STD) of three replicate experiments.
• the dose-response curve of signals elicited by perillartine in T1R2-TMD expressing cells are compared to the signals obtained in cells that stably express the T1R2/T1 R3 heterodimer.
• the two dose-response curves are found to match closely (see fig-1)
• the EC50 value which represents the agonist concentration that elicits a 50% of maximum response and is indicative of receptor sensitivity (a lower EC50 value indicates greater sensitivity to an agonist), was calculated from this regression anlaysis.
• the calculated EC50 value for T1 R2-TMD is 6.2 micromolar and for the T1 R2/T1 R3 heterodimer is 3.5 micromolar.
• T1 R2-TMD is a biologically relevant receptor.
• Fig. 1 Dose response curves of the T1 R2-TMD homomer (filled-in triangles) and the T1 R2/T1 R3 heterodimer (open triangles).
• Test agents were tested in duplicate at a final concentration of 100 micromolar.
• the signals elicited by the test agents in cells that stably express G16gust44 and T1 R2-TMD containing cells were compared to the signals obtained in cells that stably express G16gust44 and the T1 R2/T1 R3 heterodimer (formed as described in example 4), and as a negative control, cells that stably express G16gust44 were used.
• T1R2-TMD is activated by a compound that does not activate the T1 R2/T1 R3 heterodimer; accordingly assays based on the T1 R2-TMD homomer serve to identify modulators that cannot be identified using assays performed in presence of T1R3 and based on the T1 R2/T1 R3 heterodimer.
• Methyl chavicol (FEMA # 2411 , Estragole; p-Methoxyallylbenzene) is a known flavour described to have a sweet taste and the taste is described as follows: “sweet, herbaceous, anise-fennel odor", “sweet, phenolic, anise, harsh, spice, green, herbal, minty” odor and a “sweet, licorice, phenolic, weedy, spice, celery-like” taste at 10 ppm.

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