WO2010099983A1

WO2010099983A1 - Methods for isolating agonists and antagonists of taste receptors

Info

Publication number: WO2010099983A1
Application number: PCT/EP2010/001433
Authority: WO
Inventors: Thomas Hofmann; Andreas Dunkel; Alexander TÄUBERT; Wolfgang Meyerhof; Frauke STÄHLER; Jakob Ley
Original assignee: Deutsches Institut Für Ernährungsforschung Potsdam-Rehbrücke; Technische Universität München; Symrise Gmbh & Co. Kg
Priority date: 2009-03-06
Filing date: 2010-03-08
Publication date: 2010-09-10
Also published as: EP2404172A1

Abstract

The invention relates to improved methods for identifying molecules that modulate a taste response, e.g. suppress (antagonist) or enhance (agonist) taste receptor activity. The invention also relates to a pharmaceutical composition, a food stuff, precursor material or additive employed in the production of foodstuffs which comprises an antagonist or agonist identified according to a method of the invention and to a saliva co-factor component useful for the methods according to the invention.

Description

Methods for Isolating Agonists and Antagonists of Taste Receptors

Background of the Invention

Investigators have recently turned their attention to understanding the biological mechanisms of taste. For a review of the literature see, for example, Abe K., Biosci Biotechnol Biochem. (2008);72(7): 1647-56 (Meyerhof, 2005; Behrens and Meyerhof, 2006; Chandrashekar et al., 2006; Sugita, 2006; Behrens, 2007; Roper, 2007; Stahler et al., 2007). The chemosensory receptors for taste have been identified as G protein-coupled receptors

(GPCRs) and ion channels that are expressed on the surface of highly specialized taste sensory cells. Intensive research efforts are currently aimed at further elucidating the coupling of taste receptors and their respective signal transduction pathways, as well as characterizing the ligands that interact with them and are capable of modulating the receptor response such as agonists and antagonists.

Some tastes are aversive, and as such provide humans with a mechanism of protection against poisonous substances. Tastants also affect the palatability of food, beverages, thereby influencing human nutritional habits. They also affect the palatability of other ingestibles such as orally administered pharmaceuticals and nutraceuticals. Understanding the mechanism of taste transduction has implications for the food and pharmaceutical industries. For example, agonists of taste receptors are used as flavour enhancers. If the taste transduction pathway can be manipulated, it may be possible to suppress or eliminate aversive taste to render foods more palatable and e.g. increase patient compliance with oral pharmaceutics.

Taste transduction involves the interaction of molecules, i.e. tastants, with taste receptor- expressing cells which reside in the taste buds located in the papillae of the tongue. Taste buds relay information to the brain on the nutrient content of food and the presence of poisons. Recent advances in biochemical and physiological studies have enabled researchers to conclude that taste transduction is mediated by so-called G-protein coupled receptors (GPCRs) and by ion channels. While sweet and umami taste is mediated mainly by GPCRs, sour and salty compounds are generally tasted via ion channels. However, also sweet and umami tastants have been shown to propagate signals to a downstream non-selective cation channel designated TRPM5 (transient receptor potential-melastatin).

GPCRs are 7 transmembrane domain cell surface proteins that amplify signals generated at a cell surface when the receptor interacts with a ligand (a tastant) whereupon they activate heterotrimeric G-proteins. The G-proteins are protein complexes that are composed of alpha and beta-gamma sub-units. They are usually referred to by their alpha subunits and classified generally into 4 groups: G alpha s, i, q and 12. The G alpha q type couple with GPCRs to activate phospholipase C which leads to an increase in cellular Ca²⁺. There are many Gq-type G- proteins that are promiscuous and can couple to GPCRs, including taste receptors, and these so- called "promiscuous" G-proteins are well known in the art. These G-proteins dissociate into alpha and beta-gamma subunits upon activation, resulting in a complex cascade of cellular events that results in the cell producing second messengers, such as calcium ions, that enable the cells to send a signal to the brain indicating a taste response. Most mammalian taste cells also express the protein gustducin that is a G-protein subunit that is implicated in the taste perception in mammals. See, for example, Palmer RK, MoI Interv. (2007); 7(2):87-98.

In the past, several candidate sour taste receptors were described including acid-sensing ion-channels, proton-sensitive potassium channels, hyperpolarization-activated and cyclic nucleotide-gated ion channels, and, possibly, proton-sensitive G-protein-coupled receptors (see: Ugawa S. et al., Nature, (1998), 395:555-556; Stevens DR, et al., (2001), Nature. 413:631-635; Richter TA, et al., J Neurophysiol. 92:1928-1936 and Breslin PA, Huang L. (2006), Adv Otorhinolaryngol. 63:152-190 (Chandrashekar et al., 2006; Roper, 2007)). Recently, a member of the TRP family, PKD2L1 has been found to be expressed in sour taste receptor cells and serves as a maker for those (Huang et al., 2006; Ishimaru et al., 2006; LopezJimenez et al., 2006).

Regarding salt taste receptors, for example, the salt taste receptor mechanism for NaCl, the Na⁺ and Cl^" ions affect excitability or membrane potentials of taste cells in at least two ways: sodium ions increase the membrane potential of taste cells by passing through amiloride- sensitive channels in the apical membrane (see e.g. Feldman, et al., J. Neurophysiol. 90: 2060- 2064 and Schiffman S. et al., (1983), Proc. Natl. Sci. U.S.A. 80: 6136-6140). The sodium- specific salt taste receptor is the epithelial sodium channel, ENaC, which activity can be reduced by the inhibitory compound amiloride (see also the publication by Canessa CM., et al., (1993), Nature 361 : 467-470 and also by DeSimone, J., (2006), Am. J. Physiol. Gastrointest. Liver Physiol., 291 : G1005-G1010). This sodium ion channel is comprised by α-, β- and γ-ENaC subunits. In addition in humans exists an additional subunit, δ-ENaC which is not present in mice and rats. Delta- β-γ-ENaC forms a functional sodium-permeable ion channel. As amiloride insensitive salt taste receptors a vanilloide-receptor-1 variant (TRPVl) has been described in e.g. Lyall V., (2004), J. Physiol. 558: 147-159 and the TRPML3 ion-channel has been described in WO 2009/008950. In contrast, chloride ions, that also contribute to salt perception, can penetrate the tight junctions between epithelial cells in the taste bud and/or lingual epithelium to modify the membrane potential evoked by sodium ions (Ye et al., (1991), Science 254: 724-726).

The knowledge about compounds that act as taste receptor agonists is prerequisite to the implementation of a method to isolate antagonists which can be structurally related to the agonist or can also have an entirely different structure than the agonist. Once an antagonist is identified it may be the basis to develop further structurally related antagonists, which may be at least as potent in suppressing the taste receptor activity as antagonist originally identified and which may feature additional advantages such as lower toxicity, better solubility, improved stability and so forth. A taste receptor antagonist isolated by such method can also be isolated and modified or combined with other taste receptor antagonists in such a way that it is capable of targeting a broader range of known taste receptors with high affinity to e.g. achieve a more effective suppression of aversive tastes.

When a taste receptor agonist or antagonist is found using an in vitro-scτeen, said agonist or antagonist is typically counter-screened subsequently using human subjects to determine the potency of the agonist or antagonist as perceived in vivo, i.e. by an individual. However, such counter-screens are time- and cost intensive and often also result in false-positive results as the perception is subjective and, thus, varies between subjects and the placebo effect (controls) may result in a false-positive judgment.

Notwithstanding of what has been learned about taste perception, there exists a need for improved in vitro assays to validate known taste modulators and to isolate novel taste receptor agonists and antagonists. Ideally, said assays have an improved predictive power, i.e. wherein the assay can be used as an alternative to sensory studies with, e.g. human subjects. The disclosure of the present patent application allows the implementation of an improved time- and cost effective in vitro screening method that can be used to isolate novel taste receptor modulators and to validate the activity of taste receptor agonists and antagonists identified in in vitro screens, while minimizing the generation of false-positive data.

Summary of the Invention

The present invention provides a novel assay to isolate taste receptor agonists and antagonists which reduces the number of false-positive results. Thus, in a first aspect the invention provides a method for isolating a modulator of a taste receptor comprising the steps: (a) contacting a taste receptor or a host cell expressing said taste receptor with a potential modulator in the presence of saliva or a saliva co-factor component; and

(b) determining the degree by which the potential modulator modulates the activity of said taste receptor. In a further aspect the invention provides a food stuff, precursor material or additive employed in the production of foodstuffs producible according to the method of the invention.

Further provided is a pharmaceutical composition producible according to the method of the invention.

The invention also relates to a saliva co-factor component characterized by having a molecular mass of smaller or equal than 100 kDa and having co-factor activity.

Also provided is the use of a composition comprising L-cysteine for the enhancement of salt-taste.

Description of the Invention Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). The term "identity" or "identical" in the context of polynucleotide, polypeptide or protein sequences refers to the number of residues in the two sequences that are identical when aligned for maximum correspondence. Specifically, the percent sequence identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Alignment tools that can be used to align two sequences are well known to the person skilled in the art and can, for example, be obtained on the World Wide Web, e.g., ClustalW (www.ebi. ac.uk/clustalw) or Align (http://www.ebi.ac.uk/emboss/align/index.htrnl^'). The alignments between two sequences may be carried out using standard settings, for Align EMBOSS ::needle preferably: Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5. Those skilled in the art understand that it may be necessary to introduce gaps in either sequence to produce a satisfactory alignment. The "best sequence alignment" between two polypeptides is defined as the alignment that produces the largest number of aligned identical residues.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps, hi the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The present inventors provide an assay system for determining the potency of taste receptor agonists and antagonists under physiological conditions which is fast and cost effective and which can be carried out without relying on the subjective perception of human subjects. This method enables the effective development of compounds and compositions to modulate tasting components of foods, in particular animal foods, nutrients and dietary supplements and pharmaceutical or homeopathic preparations.

Therefore, in a first aspect the present invention provides a method for isolating a modulator of a taste receptor comprising the steps:

(a) contacting a taste receptor or a host cell expressing said taste receptor, preferably a host cell genetically engineered to express said taste receptor, with a potential modulator in the presence of saliva or a saliva co-factor component; and

(b) determining the degree by which the potential modulator modulates the activity of said taste receptor.

As used herein, "modulator" refers to an agonist or antagonist of taste receptors. A "potential modulator" may be any perceivable chemical substance including polypeptides in a non-purified, partially purified or purified state. The term "potential modulator" is also used to refer to modulators that have shown taste receptor modulating activity in art known in vitro assays not using saliva or a saliva co-factor components and which are to be validated as taste receptor antagonist or taste receptor agonist. Thus, in one preferred embodiment the potential modulator used in the method of the invention has previously been isolated using an in-vitro assay comprised in the art, i.e. one which does not use saliva or saliva co-factor components. Potential agonists and potential antagonists which are not capable of activating and suppressing, respectively, the activity of a taste receptor in the method of the invention may not be perceived by human test individuals in sensory assays. Thus, such potential agonists may not be tasted and such potential antagonists may not be capable of reducing the sensation of the respective tastant in vivo and, thus, represent false- positive results from prior art assays. Accordingly, the methods of the present invention can be used as a screening method for validating bona-fide taste receptor agonists and antagonists. In one preferred embodiment the method of the invention further comprises a step of an in vivo sensory method for measuring taste of potential taste-receptor modulators. Preferably, this further step is carried out prior or after the in vitro steps (a) and (b).

In a preferred embodiment of the method of the invention, the modulator is an antagonist and in step (b) the degree is determined by which the potential antagonist antagonizes said taste receptor activity. If the aim of the method is the identification of an antagonist it is preferred that the method further comprises a step (c) of contacting prior, concomitantly and/or after step (a) said taste receptor or said host cell, preferably a host cell genetically engineered to express said taste receptor with a compound capable of activating said taste receptor, i.e. with a taste receptor agonist. The extent of lowering the taste receptor activity is determined using said agonist, e.g. one of the many compounds known to activate said taste receptor. Several examples of taste receptor agonists are described in the literature (Mombaerts, 2004; Behrens and Meyerhof, 2006; Winnig et al., 2007; Stahler et al., 2008) and additional taste receptor agonists that can be used in the method of the invention are also described herein below. A potential antagonist is considered an antagonist, if it exerts inhibitory activity, if present in the same molar, 2-fold, 5-fold, 10-fold, 50-fold or 100-fold molar concentration as the agonist or if present in lower amounts as the agonist, preferably if present in a 0.5-fold, 0.1-fold, 0.05-fold, 0.001-fold or 0.0001-fold molar concentration of the agonist. In one preferred embodiment of the method of the invention, an antagonist is capable of reducing the taste receptor activity by at least at least: 10%, 15%; 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5% compared to the taste receptor activity observed in the absence of said potential antagonist; or by 100% (i.e. complete inhibition of the activity); preferably if the antagonist is used in the same molar concentration as the agonist.

In another preferred embodiment of the method of the invention, the modulator is a taste receptor agonist and in step (b) of the method the degree is determined by which the potential taste receptor agonist increases said taste receptor activity.

If the modulator is a potential agonist, the activity of the taste receptor activity is in a preferred embodiment determined in the presence and in the absence of said agonist. In one preferred embodiment of the method of the invention, a potential agonist is considered an agonist, if it is capable of increasing the taste receptor activity by at least at least: 10%, 15%; 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%, 100% or more, if compared to the taste receptor activity determined in the absence of said potential agonist. Preferably, an agonist exerts this activity at concentrations of 100 mM or lower, preferably at 50 mM or lower, or more preferably at 10 mM or lower. Said agonist isolatable by the method of the invention can also increase the taste receptor activity by reducing or suppressing the inhibitory effect of the saliva. In other words, the agonist may act by reducing or suppressing an inhibitory effect that saliva has on certain compounds that are only capable of stimulating the taste receptor activity when dissolved in buffer but not when dissolved in saliva or a saliva co-factor component. Thus, to isolate agonists having this activity, in another preferred embodiment of the method of the invention,

(i) prior, concomitantly and/or after step (a) the taste receptor is additionally contacted with a second compound which increases the activity of said taste receptor more in the absence of saliva or a saliva co-factor component than in the presence of saliva or a saliva co-factor component; and (ii) in step (b) the degree is determined by which the potential agonist activates or increases the stimulatory function of said second component on said taste receptor.

The above-outlined embodiment of the method is particularly useful to find suppressors of the inhibitory effect of saliva on agonists (i.e. de-repressors) that increase taste-receptor function in a saliva or saliva co-factor component free assay system. For example, as also described below in more detail, the ENaC salt taste receptor activity is increased when contacted with L-cysteine, if a saliva-free buffer is used in the assay. Thus, L-cysteine can be used as second compound when ENaC is used as the taste receptor. In the above mentioned preferred embodiment of the method of the invention, the potential agonist is isolated if said potential agonist causes said second compound to be active in the presence of saliva, i.e. if said potential agonist activates or increases the function of the second compound to stimulate the taste receptor.

Taste is a particular problem when orally administering pharmaceuticals, which often have an unpleasant taste. In particular in elderly persons, children and chronically ill patients this taste can lead to a lack of compliance with a treatment regimen. In addition in veterinary applications the oral administration of adverse tasting pharmaceuticals can be problematic. Furthermore, taste receptor agonists are useful to modulate the taste of food products. Thus, in another preferred embodiment, the method of the invention comprises a further step (d) of admixing the isolated modulator, i.e. agonist and/or antagonist, with a pharmaceutical, a foodstuff or a precursor material or additive employed in the production of a foodstuff. "Foodstuff' is any substance, usually composed of carbohydrates, fats, proteins and/or water, that can be eaten or drunk by an animal or human for nutrition or pleasure.

Preferably, the additive employed in the production of a foodstuff is a phytochemical or a derivative thereof. Phytochemicals are plant-derived chemical compounds which can improve human health by e.g. reducing the risk of cancer and other diseases, e.g. inflammatory diseases. Preferred phytochemicals are e.g. phenolic compounds, terpenes (i.e. isoprenoids), betalains, organosulfides, indoles and glucosinolates. preferred phytochemicals are selected from the group consisting of apiolejCarnoso^carvacroljdillapiolejrosemarinol, quercetin, gingerol, kaempferol, myricetin, resveratrol, rutin, isorhamnetin, hesperidin naringenin, silybin, eriodictyol, apigenin, tangeritin, luteolin, catechins, (+)-catechin, (+)-gallocatechin, (-)-epicatechin, (-)- epigallocatechin, (-)-epigallocatechin gallate(egcg), (-)-epicatechin 3-gallate, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, theaflavin-3,3'-digallate, thearubigins, pelargonidin, peonidin, cyanidin, delphinidin, malvidin, petunidin, daidzein (formononetin), genistein (biochanin a), glycitein, dihydroflavonols, chalcones, coumestans (phytoestrogens) such as coumestrol, ellagic acid, gallic acid, salicylic acid, tannic acid, vanillin, capsaicin, curcumin, caffeic acid, chlorogenic acid, cinnamic acid, ferulic acid, coumarin, silymarin, matairesinol, secoisolariciresinol, pinoresinol, tyrosol, hydroxytyrosol, oleocanthal, oleuropein, resveratrol, pterostilbene, piceatannol, punicalagins, α-carotene, β-carotene, γ-carotene, δ-carotene, lycopene, neurosporene, phytofluene, phytoene, xanthophylls, canthaxanthin, cryptoxanthin, zeaxanthin, astaxanthin, lutein, rubixanthin, limonene, perillyl alcohol, saponins, phytosterols, campesterol, beta sitosterol, gamma sitosterol, stigmasterol, tocopherols (vitamin e), omega- 3,6,9 fatty acids, gamma-linolenic acid, oleanolic acid, ursolic acid, betulinic acid, moronic acid, betanin - beets, isobetanin, probetanin, neobetanin, indicaxanthin, vulgaxanthin, sulphoraphane, thiosulphonates (allium compounds), allyl methyl trisulfide, diallyl sulfide, indole-3-carbinol, sulforaphane, 3,3'-diindolylmethane or dim, sinigrin - broccoli family, allicin, alliin, allyl isothiocyanate, piperine, syn-propanethial-s-oxide, oxalic acid, phytic acid (inositol hexaphosphate), tartaric acid and anacardic acid. Also the potential modulator of a taste receptor tested in the method of the invention may be a phytochemical or structural derivative thereof.

As used herein, "saliva" refers to the watery and usually frothy substance secreted from salivary glands of humans and animals. Saliva is readily available as it is estimated that a healthy person produces from 0.75 liters per day to 1.5 liters of saliva per day. The saliva of any human or animal may be used. In preferred embodiments, saliva of animals are used that exhibit a greater daily saliva production than humans, such as e.g. cattle. In preferred embodiments of the method of the invention, said saliva and/or saliva co-factor component can be used in diluted form, e.g. diluted with a physiological buffer, such as such as PBS, NaCl-NMDG-ORJ buffer (see examples below) or Cl -buffer (130 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mM CaCl_2, and 5 mM Glucose, pH 7,4). It is preferred that when using a human taste receptor in the method of the invention that also human saliva is used in the method and that if a taste receptor of a particular other species is used that saliva derived from the same other species is used in the method. A "saliva co-factor component" refers to a fraction of saliva that is capable of providing essentially the same "co-factor activity" as non-fractionated saliva. Said saliva co-factor component can act by physically interacting with the respective taste receptor but can also act indirectly, e.g. by binding and/or sequestering tastants, i.e. agonists or antagonists of taste receptors or by binding and/or sequestering other yet unidentified compounds or also by modulating taste modulators. A "saliva co-factor component" provides essentially the same "co- factor activity" as saliva, if it enhances or represses taste of a given taste receptor modulator at least 50%, preferably 60%, 70%, 80%, 90% or 100% of the enhancement or repression that is measured, if non-fractionated saliva is used at the same concentration. This enhancement or repression can be determined in an in vitro assay as described herein. For example, Xenopus laevis oocytes expressing α-, β- and γ-subunits of the ENaC salt taste receptor are contacted in the presence of sodium chloride with L-cysteine dissolved in saliva in a first experiment and dissolved in the saliva co-factor component in a second experiment. As mentioned above, the saliva co-factor component will show a suppression or reduction of the stimulatory effect of L- cysteine on the ENaC taste receptor activity that is at least 50%, preferably 60%, 70%, 80%, 90% or 100% as strong as the suppression or reduction achievable in the first experiment, i.e. when using saliva. A more detailed description of this assay is provided in the examples below.

The saliva co-factor component may comprise more than one chemical entity responsible for the co-factor activity. The saliva co-factor component of the invention is further characterized by being heat stabile. In preferred embodiments said "co-factor activity" is not significantly reduced when said saliva co-factor component is subjected to a heat-treatment at about between 80°C and about 100°C for at least 5 min, most preferably at about 95 °C for about 15 minutes, hi addition, the saliva co-factor component has a molecular mass smaller or equal than 100 kDa, preferably smaller or equal than 10 kDa. To further characterize the saliva co- factor component, standard methods used in the art of fractionation may be employed. A component from a saliva sample can e.g. be isolated using gradient centrifugation, chromatography, e.g. ion-exchange chromatography or RPLC, High pressure liquid chromatography, (HPLC), thin-layer chromatography, column chromatography, filtering, and/or further methods useful to fractionate saliva into different components, preferably using an activity-guided fractionation approach. The various fractions may then be tested for their co- factor activity in an assay as described herein. The various fractions may also be iteratively further separated until a purified "co-factor" or "co-factors" is/are obtained. As used herein "saliva" also refers to a mixture of saliva samples isolated from different donors, such as from different male and/or female donors. Saliva flow rate may be increased by nutritional components. Thus, said saliva and said saliva co-factor component may be collected from stimulated and/or non-stimulated donors. If "stimulated saliva" is collected, salivary flow is induced by chewing e.g. tasteless components like paraffin wax or is induced by an acid such as an organic acid or a pungent compound. If "non-stimulated saliva" is collected saliva is simply spit into collecting tubes without any further stimulation.

In step (a) of the method of the invention, the taste receptor contacted with said potential modulator may be in a host cell or may also be isolated (e.g. useful for in-vitro binding assays)

Host Cells

The host cells used in the method of the present invention may endogenously express the taste receptor of interest or may be genetically engineered to express the taste receptor of interest. Host cells naturally expressing the respective taste receptors may be obtained from primary tissue, e.g. tongue sections, primary cells derived from such tissues or from cell lines established from such primary cells or tissues. Preferred cells which endogenously express said taste receptor are isolated epithelial taste-cells of the tongue. Said isolated primary or established taste-cells can be maintained in culture as in known in the art. In general, as used herein, "genetically engineered" means that the host cell is transgenic for a polynucleotide encoding the taste receptor (for types of taste receptors see below) or a vector containing said polynucleotide. In preferred embodiments "genetically engineered" also refers to the use of a vector which comprises the polynucleotide encoding said taste receptor. Preferably said vector is selected from the group consisting of plasmids (see also below for expression vectors), phagemids, phages, cosmids, artificial mammalian chromosomes, knock-out or knock-in constructs, viruses, in particular adenoviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, lentivirus (Chang, LJ. and Gay, E.E. (2001) Curr. Gene Therap. 1 : 237-251), herpes viruses, in particular Herpes simplex virus (HSV-I, Carlezon, W. A. et al. (2000) Crit. Rev. Neurobiol. 14: 47-67), baculovirus, retrovirus, adeno-associated-virus (AAV, Carter, PJ. and Samulski, RJ. (2000) J. MoI. Med. 6:17-27), rhinovirus, human immune deficiency virus (HIV), filovirus and engineered versions thereof (see, for example, Cobinger G. P. et al. (2001) Nat. Biotechnol. 19:225-30), virosomes, "naked" DNA liposomes, and nucleic acid coated particles, in particular gold spheres. Particularly preferred are viral vectors like adenoviral vectors or retroviral vectors (Lindemann et al. (1997) MoI. Med. 3: 466-76 and Springer et al. (1998) MoI. Cell. 2: 549-58). Liposomes are usually small unilamellar or multilamellar vesicles made of cationic, neutral and/or anionic lipids, for example, by ultrasound treatment of liposomal suspensions. The DNA can, for example, be ionically bound to the surface of the liposomes or internally enclosed in the liposome. Suitable lipid mixtures are known in the art and comprise, for example, DOTMA (l,2-Dioleyloxpropyl-3- trimethylammoniumbromid) and DOPE (Dioleoyl-phosphatidylethanolamin) which both have been used on a variety of cell lines.

Nucleic acid coated particles are another means for the introduction of nucleic acids into host cells using so called "gene guns", which allow the mechanical introduction of particles into cells. Preferably the particles itself are inert, and therefore, are in a preferred embodiment made out of gold spheres.

Polynucleotides useable in preferred embodiments of the method of the present invention to express a taste receptor are operatively linked to expression control sequences allowing expression in prokaryotic and/or eukaryotic host cells. The transcriptional/translational regulatory elements referred to above include but are not limited to inducible and non-inducible, constitutive, cell cycle regulated, metabolically regulated promoters, enhancers, operators, silencers, repressors and other elements that are known to those skilled in the art and that drive or otherwise regulate gene expression. Such regulatory elements include but are not limited to regulatory elements directing constitutive expression like, for example, promoters transcribed by RNA polymerase III like, e.g. promoters for the snRNA U6 or scRNA 7SK gene, the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, viral promoter and activator sequences derived from, e.g. NBV, HCV, HSV, HPV, EBV, HTLV, MMTV or HIV; which allow inducible expression like, for example, CUP-I promoter, the tet- repressor as employed, for example, in the tet-on or tet-off systems, the lac system, the trp, system; regulatory elements directing tissue specific expression, preferably taste bud specific expression, e.g. PLCB2 promoter or gustducin promoter, regulatory elements directing cell cycle specific expression like, for example, cdc2, cdc25C or cyclin A; or the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast α- or a-mating factors. In general, suitable bacterial promoters are well known in the art, e.g., E. coli, Bacillus sp., and Salmonella, and kits for such expression systems are commercially available. Similarly eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available, for example the plasmid vectors pMSG, pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE, pcDNA3.1, pIRES. As used herein, "operatively linked" means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.

In order to express cDNAs encoding the taste receptor(s), one typically subclones receptor 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. In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the receptor-encoding nucleic acid in host cells. A typical expression cassette thus contains a promoter operatively linked to the nucleic acid sequence encoding the receptor and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette 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. The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ, but there are many more known in the art to the skilled person that can be usefully employed.

Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable.

The elements that are typically included in expression vectors 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 nonessential 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.

Standard transfection methods can be used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of the taste receptor, which are then purified using standard techniques. Any of the well-known procedures for introducing foreign polynucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the taste receptor. After the expression vector is introduced into the cells, the transfected cells may be cultured under conditions favouring expression of the taste receptor. In preferred embodiments of the method of the invention the host cell expresses the mature form of the taste receptor. This means that said taste receptor protein when expressed contains all polypeptide elements that allow it to undergo some or all potential post- or cotranslational processes such as proteolytic processing, phosphorylation, lipidation and the like comprised in the state of the art such that said polypeptide or protein can correctly fold and carry out part or all of its wildtype function as a taste receptor once it reaches its "mature form".

Similarly, as also mentioned before, the taste receptor useable in the method of the present invention can form part of a hybrid protein comprising additional polypeptide sequences, for example, polypeptide sequences that function as marker or reporters. Examples of marker and reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo^r, G418^r), dihydro folate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β- galactosidase), and xanthine guanine phosphoribosyl- transferase (XGPRT). As with many of the Standard procedures associated with the practice of the method of the invention, skilled artisans will be aware of additional useful reagents, for example, additional sequences that can serve the function of a marker or reporter. Other hybrids could include an antigenic tag or His tag e.g. to facilitate purification and/or detection. A taste receptor can also be expressed according to the method of the invention which is operatively linked to a heterologous signal sequence. Such signal sequences serve e.g. to insert the taste receptor in the plasma membrane and are well known to someone of skill in the art. If the taste receptor is expressed in a genetically engineered host cell, the host cells that may be used in the method of the present invention include but are not limited to prokaryotic cells such as bacteria (for example, E. coli and B. subtilis), which can be transformed with, for example, recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing polynucleotide molecules encoding a taste receptor according to the invention; simple eukaryotic cells like yeast (for example, Saccharomyces and Pichia), which can be transformed with, for example, recombinant yeast expression vectors containing the polynucleotide molecule of the invention; insect cell systems like, for example, Sf9 or Hi5 cells, which can be infected with, for example, recombinant virus expression vectors (for example, baculovirus) containing the polynucleotide molecules; amphibian cells, e.g. Xenopus oocytes, which can be injected with, for example, plasmids and/or mRNA encoding the taste receptors, preferably salt receptors, most preferably one or more ENaC receptor subunits; plant cell systems, which can be infected with, for example, recombinant virus expression vectors (for example, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (for example, Ti plasmid) encoding a taste receptor; or mammalian cell systems (for example, COS, CHO, BHK, HEK293, VERO, HeLa, MDCK, Wi38, and NIH 3T3 cells), which can be transformed with recombinant taste recptor, preferably salt taste receptor expression constructs containing, for example, promoters derived, for example, from the genome of mammalian cells (for example, the metallothionein promoter) from mammalian viruses (for example, the adenovirus late promoter and the vaccinia virus 7.5K promoter) or from bacterial cells (for example, the tet-repressor binding is employed in the tet-on and tet-off systems). Also useful as host cells are primary or secondary cells obtained directly from a mammal and transfected with a plasmid vector or infected with a viral vector to express the taste receptor.

Taste Receptors In preferred embodiments of the method of the invention the taste receptor is one or more taste receptors selected from the group consisting of a sweet taste receptor, a sour taste receptor, an umami taste receptor, a salt taste receptor, a temperature receptor and a pain receptor. Thus, the method of the invention can be used to determine the effect of a potential modulator upon the activity of one or more of said different taste receptors. Sequences of many taste receptors are widely known and have been previously reported, for example in e.g. WO 2009/008950, and in e.g. (Lindemann et al., 1998; Hoon et al., 1999; Max et al., 2001; Montmayeur et al., 2001 ; Lyall et al., 2004; Huang et al., 2006; Ishimaru et al., 2006; LopezJimenez et al., 2006). The amino acid and polynucleotide sequences of the taste receptors useful for the method according to the invention are also readily available in public databases such as the NCBI PubMed. In addition, the amino acid sequences of various salt taste receptors are also described herein.

Temperature receptors which may be used in the assay may be cold receptors such as the transient receptor potential cation channel, subfamily M, member 8 (TRPM8) receptor which upon activation allows the entry of Na⁺ and Ca²⁺ ions to the cell. Another temperature receptor that may be used is the transient receptor potential cation channel, subfamily V, member 3 and/or 4 (TRPV3 and/or TRPV4) warmth-receptors, which are activated depending on a temperature of between 22 and 40°C. Also the pain receptors designated as transient receptor potential cation channel, subfamily V, member 1 or 2, abbreviated TRPVl and TRPV2 can be used in the method of the invention. TRPVl is activated upon physical and chemical stimuli, including heat greater than about 43⁰C, low pH (acidic conditions), the endocannabinoid anandamide, N- arachidonoyl-dopamine, and capsaicin, the active ingredient of hot chili pepper, while the TRPV2 receptor is activated by temperatures above 52 degrees Celsius and likely also by hot spices. In other preferred embodiments also the transient receptor potential cation channel, subfamily A, member 1, also known as TRPAl receptor may be used in the method as taste receptor. This receptor senses mechanical stress (e.g. pain) and chemical compounds such as allyl isothiocyanate, cinnamaldehyde, farnesyl thiosalicylic acid, formalin, hydrogen peroxide, 4- hydroxynonenal, and acrolein.

As mentioned, taste receptors also include sweet and umami taste receptors. The TlRl, T1R2, and T1R3 receptors are class C GPCRs, characterized by large N-termini, that heterodimerize to form functional taste receptors for sweet or umami sensing. In a preferred embodiment of the method of the invention, the sweet taste receptor is at least one receptor selected from the group consisting of a T1R2/T1R3 heterodimer, a T1R3/T1R3 homodimer receptor and a TRPM5 receptor. For additional information on sweet receptors see also: Reed DR et al., Physiol Behav. (2006); 88(3):215-26 and Galindo-Cuspinera et al., Nature 441, 354- 357.

Also preferred is the method according to the invention, wherein the umami taste receptor is a T1R1-T1R3 heterodimer, mGluR4t, mGluRl and/or a TRPM5 receptor. Umami is one of the five basic tastes sensed by specialized receptor cells present on the human tongue. The umami taste is generally due to the detection of the carboxylate anion of glutamic acid. In another preferred embodiment of the method of the invention, the sour taste receptor is selected from the group consisting of PKD2L1 and PKD1L3. See also Ishimaru Y. et al., PNAS (2006), vol. 103 no. 33, 12569-12574 for a review on sour taste perception and the respective receptors.

In a further preferred embodiment of the method of the invention the salt taste receptor is one or more receptors selected from the group consisting of ENaC, TRPVl and TRPML3. Salt receptors may be sodium specific or sodium nonspecific. Preferably, sodium specific and amiloride sensitive salt taste receptors are used in the method of the invention. The sodium- specific salt taste receptor is an epithelial sodium channel whereas a nonspecific salt taste receptor is a taste variant of the vanilloid receptor- 1 nonselective cation channel, TRPVl. Detailed information on the salt receptor TRPML3 is available from WO 2009/008950. ENaC is a sodium-specific and lithium-specific salt taste receptor that generally comprises the subunits α, β and γ or δ, β and γ.

In a preferred embodiment, the taste receptor used in the method of the invention is a salt taste receptor having salt taste receptor activity and comprising at least one of the following polypeptides or functional derivatives thereof:

(i) the α-subunit of the ENaC receptor having an amino acid sequence according to SEQ ED

NO: 3; (ii) the β-subunit of the ENaC receptor having an amino acid sequence according to SEQ ID

NO: 4; (iii) the γ-subunit of the ENaC receptor having an amino acid sequence according to SEQ ID

NO: 5; (iv) the δ-subunit of the ENaC receptor having an amino acid sequence according to SEQ ID

NO: 6;

(v) the TRPML3 polypeptide according to SEQ ID NO: 7; and (vi) the TRPVl polypeptide according to SEQ ID NO: 8.

It is noted that the above-outlined receptor sequences are preferred and non-limiting embodiments of these receptors. However, also orthologue receptors from species other than

Homo sapiens are known and can be used as well as functional derivatives thereof. In a more preferred embodiment, the functional taste receptor used in the method of the invention comprises one or more of the following polypeptides or functional derivatives thereof:

(a) the polypeptides according to (i), (ii) and (iii) as outlined above;

(b) the polypeptides according to (iv), (ii) and (iii) as outlined above;

(c) the polypeptide according to (v) as outlined above; and/or

(d) the polypeptide according to (vi) as outlined above. As used herein, "salt taste receptor activity" means that said salt receptor is preferably permeable preferably selectively permeable to salt, preferably cations, more preferably to sodium and/or lithium ions.

As used herein, "taste receptor" refers to any functional taste receptor, i.e. an ion channel or a G-protein coupled receptor polypeptide, including multi-subunit receptors, which are capable of binding, or in case of ion-channels, also channeling a tastant and which, upon binding or channeling/sensing of said tastant, causes a change in ion flux, in membrane potential, in current flow, in transcription, in G protein binding, in GPCR phosphorylation or dephosphorylation, in signal transduction, in receptor-ligand interactions or in second messenger concentrations, preferably cAMP, cGMP, IP₃, or intracellular Ca²⁺, in vitro, in vivo, and/or ex vivo and is optionally also capable of eliciting other physiologic effects such as an increase or a decrease of a neurotransmitter or a hormone release. As used herein, a "taste receptor" also includes receptors and ion-channels that are located in the oral cavity and that are sensing the temperature and/or pain, such as heat, cold and hot spices, for example. Preferred temperature and pain sensing taste receptors are described below. In preferred embodiments, a "taste receptor" as used herein also includes functional derivatives of taste receptors. As used throughout this application, the phrase "functional derivative" of a protein or polypeptide generally refers to a modified version of the protein or polypeptide, e.g. one or more amino acids of the protein or polypeptide may be deleted, inserted, modified and/or substituted. The derivative is functional, if, as mentioned also above and below, in an animal that the taste receptor was derived from, the derivative exhibits at least 10%, 20%, 50%, 80% or at least 100% of the physiologic function(s) of the original wild-type taste receptor that the derivative was derived from. For example, a functional taste receptor derivative must still be capable of causing a downstream signaling event upon binding of a respective agonist, e.g. glycyrrhizic acid, neohesperidin dihydrochalcone, monellin, thaumatin or aspartame for sweet taste receptors; hydronium, hydrochloric acid or sodium citrate for sour taste receptors; monosodium glutamate for umami taste receptors; and sodium chloride or lithium chloride for salt taste receptors; wherein preferably said downstream signaling event is a change in membrane potential. In one preferred embodiment, all that is required of a functional derivative of a taste receptor that it has at least 20% (e.g., at least: 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; 100%, 150%, 200%, 500%, 1000%, 10000% or even more) of the ability of the full-length wildtype taste receptor to be stimulated by a respective agonist, e.g. one of the aforementioned agonists.

In the context of a "functional derivative", an amino acid change refers to an insertion, substitution or deletion. A functional derivative preferably comprises one or more amino acid changes in comparison to the naturally occurring polypeptide on which it is based and maintains the functionality of the receptor as outlined above. It is preferred that a functional derivative does not comprise more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more than 100, preferably not more than 10, 20 or 30 and most preferably not more than 20 amino acid changes (i.e. deleted, inserted, modified and/or substituted amino acids) in comparison to the naturally occurring polypeptide on which it is based. It is understood that the number of allowable amino acid changes that can be tolerated without loosing functionality of the receptor may differ. Accordingly, a larger receptor will tolerate more amino acid changes than a smaller receptor. Thus, without referring to the actual number of amino acid changes it is preferred that not more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or more than 20% (most preferably not more than 5%) of all amino acids of the protein or polypeptide are changed (i.e. are deleted, inserted, modified and/or substituted amino acids) in comparison to the naturally occurring polypeptide on which the derivative is based. It is also understood that each receptor will comprise domains that are more critical for receptor function than others. For example, the domains responsible for interaction with G-proteins or the domain recognizing the tastant are more critical to functionality than, e.g. transmembrane domains. Accordingly, it is preferred that amino acids are deleted, inserted, modified and/or substituted outside the regions responsible for G-protein interaction and the domain recognizing the tastant. In a preferred embodiment at least 60%, 70%, 75%, 80%, 85%, 90% or 95% of the deleted, inserted, modified and/or substituted amino acids are outside the regions responsible for G-protein interaction and the domain recognizing the tastant. The receptor families to which the taste receptors usable in the present invention belong are very well known and the skilled person is aware of those areas, which are not likely to influence activity it the amino acid sequence is changed and those regions which should not be mutated to maintain functionality.

Amino acids of the protein or polypeptide may also be modified, e.g. chemically modified. For example, the side chain or a free amino or carboxy-terminus of an amino acid of the protein or polypeptide may be modified by e.g. glycosylation, amidation, phosphorylation, ubiquitination, e.t.c. The chemical modification can also take place in vivo, e.g. in a host-cell, as is well known in the art. For examples, a suitable chemical modification motif, e.g. glycosylation sequence motif present in the amino acid sequence of the protein will cause the protein to be glycosylated. A substitution in a derivative may be a conservative or a non-conservative substitution, preferably a conservative substitution. In some embodiments, a substitution also includes the exchange of a naturally occurring amino acid with a not naturally occurring amino acid. A conservative substitution comprises the substitution of an amino acid with another amino acid having a chemical property similar to the amino acid that is substituted. Preferably, the conservative substitution is a substitution selected from the group consisting of: (i) a substitution of a basic amino acid with another, different basic amino acid; (ii) a substitution of an acidic amino acid with another, different acidic amino acid; (iii) a substitution of an aromatic amino acid with another, different aromatic amino acid; (iv) a substitution of a non-polar, aliphatic amino acid with another, different non-polar, aliphatic amino acid; and

(v) a substitution of a polar, uncharged amino acid with another, different polar, uncharged amino acid.

A basic amino acid is preferably selected from the group consisting of arginine, histidine, and lysine. An acidic amino acid is preferably aspartate or glutamate. An aromatic amino acid is preferably selected from the group consisting of phenylalanine, tyrosine and tryptophane. A non- polar, aliphatic amino acid is preferably selected from the group consisting of glycine, alanine, valine, leucine, methionine and isoleucine. A polar, uncharged amino acid is preferably selected from the group consisting of serine, threonine, cysteine, proline, asparagine and glutamine. In contrast to a conservative amino acid substitution, a non-conservative amino acid substitution is the exchange of one amino acid with any amino acid that does not fall under the above-outlined conservative substitutions (i) through (v). In another meaning of the phrase, a "functional derivative" of a protein having a certain amino acid sequence is a polypeptide having taste receptor activity and having an amino acid sequence which is at least 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical over its entire length to the amino acid sequence of said protein, e.g. to the amino acid sequence specified in SEQ ID NOs: 3-8.

If a functional derivative comprises a deletion, then in the derivative one or several amino acids that are present in the reference polypeptide or protein sequence have been removed. The deletion may, however, not be so extensive that the derivative comprises less than 200 amino acids in total.

In one embodiment a "taste receptor" as used herein can also include fusion proteins that contain either a full-length taste receptor polypeptide or a functional fragment thereof fused to a further amino acid sequence. The further sequence can e.g. add further functional domains or signal peptides. In one example, an N-terminal sequence (SEQ ID NO: 1) from the well- expressed GPCR rhodopsin can be attached to taste receptor sequences to improve taste receptor expression. Such N-terminal tags also allowed easy monitoring of protein expression due to available antibodies against this tag. In a different approach, taste receptors may also be successfully expressed in insect Sf9 cells and used for functional studies using a biochemical GTP [gamma] S binding assay (see Chandrashekar et al., (Id.) 2000). Furthermore, polypeptides and functional fragments of the taste receptor useable in the method of the present invention can be modified, for example, for in vivo use by the addition of blocking agents, at the amino- and/or carboxyl-terminal ends, to facilitate survival of the relevant polypeptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill. The antagonists or agonists of the taste receptors described herein can be used for specific stimulation of a given taste receptor or its functional derivative and identification of substances that antagonize it, respectively. Polynucleotide molecules useful to express a taste receptor in the method of the invention can be synthesized in vitro (for example, by phosphoramidite-based synthesis) or obtained from a cell, such as the cell of a bacteria or a mammal. The polynucleotides can be those of a human but also include orthologous polynucleotides derived from a non-human primate, mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat as long as they encode a functional taste receptor. Combinations or modifications of the polynucleotides within these types of nucleic acids are also encompassed. Means to identify orthologous polynucleotide molecules of the invention are available to a person of skill and comprise the use of BLAST searches and database mining of databases such as the EMBL, NCBI and other databases comprising polynucleotides and amino acid sequences.

In addition, the polynucleotides that can be used in the method of the present invention can encompass segments that are not found as such in the natural state. For example, the invention encompasses recombinant nucleic acid molecules incorporated into a vector (for example, a plasmid or viral vector as described above) or into the genome of said host cell. As mentioned, a taste receptor used in the method of the invention is functional i.e. has taste receptor activity which means that upon binding to one or more tastant molecules (agonists) it triggers an activation pathway in the cell, such as a change in intracellular ion concentration. The cells are preferably mammalian (e.g., human, non-human primate, horse, bovine, sheep, pig, dog, cat, goat, rabbit, mouse, rat, guinea pig, hamster, or gerbil) cells, insect cells, bacterial cells, or fungal (including yeast) cells. Binding assays and bitter substances for taste receptors are described above and below.

The term "contacting" in the context of the present invention means any interaction between the potential antagonist and/or potential agonist with the taste receptor or said host cell, whereby any of the at least two components can be independently of each other in a liquid phase, for example in solution, or in suspension or can be bound to a solid phase, for example, in the form of an essentially planar surface or in the form of particles, beads or the like, in a preferred embodiment a multitude of different compounds are immobilized on a solid surface like, for example, on a compound library chip and the protein of the present invention is subsequently contacted with such a chip, in another preferred embodiment the host cells are genetically engineered with a polynucleotide encoding a taste receptor, or with a vector containing such a polynucleotide, express the taste receptor at the cell surface and are contacted separately in small containers, e.g., micro-titre plates, with various compounds.

As used herein, the term "isolating" an agonist or antagonist refers to the process of selecting, identifying, validating or evolving a taste receptor agonist or antagonist. For example, an antagonist or agonist can be selected out of a group of at least two different potential antagonists or agonists whereby the said selected antagonist or agonist exhibits preferred features compared with the other antagonists or agonists such as, for example, stronger and/or longer or shorter inhibition or activation, respectively, of taste receptor activity.

In one preferred embodiment, the method of the invention is used to enhance the potency of a known antagonist by screening through a library of antagonists that are structurally related to said known antagonist, e.g. that share a common chemical core structure with the known antagonist, and by isolating a structurally related antagonist which shows the largest antagonizing effect in step (b) of the method according to the invention. In other words, in preferred embodiments of the method, the strongest antagonist for a particular taste receptor is isolated from a library comprising potential antagonists that are structurally related to a known antagonist of said receptor.

In step (b) of the methods of the invention "the degree" of inhibition caused by a potential antagonists or "the degree" of activation of a potential agonist is determined. As used herein the phrase "the degree" preferably can have the meaning of determining, if a potential agonist or antagonists modulates taste receptor activity or not. Additionally, the phrase "the degree" also refers to a value that indicates to what extent a potential agonist or antagonist is capable of activating or suppressing taste receptor activity, respectively. In a preferred embodiment of the method of the invention the concentration of the antagonist is determined which suppresses the taste receptor activity by 50% as compared to when the antagonist is absent. In another preferred embodiment of the method of the invention the concentration of the agonist is determined which stimulates the taste receptor activity by 50% compared to when the agonist is absent.

It is preferred that in above stated embodiments of the method of the invention the concentration of the agonist and/or antagonist that is used is larger than 3 mM, 4 mM, 5 mM, 10 mM, 50 mM or larger than 100 mM and most preferably larger than 5 mM.

Agonists and Antagonists

The potential antagonists and agonists, which are employed in the methods of the present invention can be synthesized by methods and standard procedures known to those skilled in the art, i.e. as described in the literature (for example in the standard works, such as Houben-Weyl, Methoden der organischen Chemie [Methods of Organic Chemistry], Georg-Thieme-Verlag, Stuttgart), to be precise under reaction conditions which are known to those skilled in the art and suitable for the said reactions.

The isolated agonist or antagonist is in a preferred embodiment chemically modified in a further step. This chemical modification can be effected by a variety of methods known in the art, which include without limitation the introduction of one or more, preferably two, three or four substituents or the exchange of one or more substituents.

The thus modified agonist or antagonist is then tested again with the method of the present invention and the activation or inhibition of taste receptor activity is determined. If needed the steps of isolating the agonist or antagonist, modifying the compound, contacting the taste receptor or said host cell with said potential agonist or antagonist can be repeated a further or any given number of times as required. The above described method is also termed "directed evolution" of an agonist or antagonist since it involves a multitude of steps including modification and selection, whereby agonizing or antagonizing compounds are selected in an "evolutionary" process optimizing their capabilities with respect to a particular property, e.g. their ability to inhibit or activate the activity of a particular taste receptor, in particular their ability to inhibit the intracellular release of calcium. Preferably, a modified agonist or antagonist is selected that modulates the activity of a given taste receptor at least as good as the identified agonist or antagonist used as basis for the modified agonist or antagonist at the same molar concentration. More preferably, the modified agonist or antagonist shows a stronger modulation of taste receptor activity at the same molar concentration, preferably at least a 10% stronger, 20%, 30%, 40%, 50%, 60, or 70% stronger modulation.

Several taste receptors and their corresponding agonists are known in the art. In one preferred embodiment of the method of the invention, said agonist is selected from the group consisting of:

(i) a saccharide, a sugar alcohol and a sweetener for sweet taste receptors; (ii) a mineral acid, a carbon acid and a salt of carbon acid for sour taste receptors; (iii) glutamic acid, asparaginic acid, nucleic acid and salts thereof for umami taste receptors; and (iv) sodium chloride, lithium chloride, potassium chloride and ammonium chloride for salt taste receptors.

Preferred saccharides are selected from the group consisting of sucrose, trehalose, lactose, maltose, melizitose, melibiose, raffinose, palatinose, lactulose, D-fructose, D-glucose, D-galactose, L-rhamnose, D-sorbose, D-mannose, D-tagatose, D-arabinose, L-arabinose, D- ribose, D-glyceraldehyd, maltodextrin and plant substances and extracts comprising one or more of the aforementioned saccharides (preferably in an amount of at least 5% w/v, more preferably at least 15% w/v), wherein the saccharides, i.e. carbohydrates can be prepared as a natural or artificial, e.g. chemically synthesized composition (in particular honey, invert sugar or high fructose corn sirup). Prefered sugar alcohols are selected from the group consisting of glycerol, erythritol, threitol, arabitol, ribitol, xylitol, sorbitol, mannitol, maltitol, isomaltit, dulcitol und lactitol and preferred sweeteners are selected from the group of artificial sweeteners consisting of magap, sodium cyclamate, acesulfame K, neohesperidin dihydrochalcone, saccharin, saccharin sodium salt, aspartame, superaspartame, neotame, alitame, sucralose, lugduname, carrelame, sucrononate, sucrooctate or are selected from the group of naturally occurring sweeteners consisting of miraculin, curculin, monellin, mabinlin, thaumatin, curculin, brazzein, pentadin, D- phenylalanine, D-tryptophane, or extracts or fractions derived from natural sources containing these amino acids and/or proteins, neohesperidin dihydrochalcone, steviolgylcoside, stevioside, steviolbioside, rebaudiosides (for example rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, dulcoside, rubusoside), suavioside A, suavioside B, suavioside G, suavioside H, suavioside I, suavioside J, baiyunoside 1, baiyunoside 2, phlomisoside 1, phlomisoside 2, phlomisoside 3, phlomisoside 4, abrusoside A, abrusoside B, abrusoside C, abrusoside D, cyclocaryoside A and cyclocaryoside I, oslandin, polypodoside A, strogin 1, strogin 2, strogin 4, selligueanin A, dihydroquercetin-3- acetate, perillartine, telosmoside Al 5, periandrin I-V, pterocaryoside, cyclocaryoside, mukurozioside, trans-anethol, trans-cinnamaldehyde, bryoside, bryonoside, bryonodulcoside, carnosifloside, scandenoside, gypenoside, trilobatin, phloridzin, dihydroflavanol, hematoxylin, cyanin, chlorogenic acid, albiziasaponin, telosmoside, gaudichaudioside, mogroside, hernandulcine, monatin, glycyrrhetinic acid and derivatives thereof (for example their glycosides such as glycyrrhizin) or their potassium, sodium, calcium or ammonium salts and phyllodulcin. In preferred embodiments of the agonists for sour taste receptors, said mineral acid may be phosphoric acid or hydrochloric acid and said carbon acid may be selected from the group consisting of acetic acid, propionic acid, citric acid, tartaric acid, maleic acid, adipinic acid, succinic acid, malic acid, fumaric acid, ascorbic acid (vitamin C) and lactic acid and said salt of a carbonic acid may be a salt of e.g any of the aforementioned caribon acids having a counter ion selected from e.g. sodium, potassium or calcium cations or mixtures thereof.

For umami taste receptors preferred agonists can be selected from mono- or multivalent salts of glutamic acid, asparaginic acid and nucleic acid with sodium, calcium, potassium cations and preferred nucleic acids useful as agonists can be selected from inositolphosphate, guanosine monophosphate and adenosine monophosphate and salts thereof, in particular calcium, sodium and potassium salts thereof. Also agonists described in US provisional applications 60/984,023 and 61/061,273 are preferred umami taste receptor agonists useful for the method of the invention.

Taste Receptor Activity The activity of a taste receptor described herein can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring ligand binding, secondary messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺), ion flux (preferably sodium, lithium, chloride and/or other cations or anions), phosphorylation levels, transcription levels, of reporter constructs neurotransmitter levels, and the like. Such assays can be used in the method of the present invention to determine the activity of the taste receptor. Methods for measuring the activity of sweet taste receptors are comprised in the art (see e.g. Galindo- Cuspinera et al., Nature 441, 354-357). Also methods for determining the activity of sour taste receptors are well known in the art (see e.g. Ishimaru Y. et al., PNAS (2006), vol. 103 no. 33 12569-12574). Salt taste receptor activity can be measured according to methods described in WO 2009/008950 or methods described herein below.

The effects of the test compounds upon the function of the receptors can be measured by examining any of the parameters described above. Any suitable physiological change that results from receptor activity can be used to assess the influence of a test compound on the receptors usable in the methods of this invention. In a preferred embodiment of the method of the invention the taste receptor activity is determined by measuring the intracellular absolute or relative concentration of one or more compounds selected from the group consisting of ions, preferably cations and most preferably sodium ions or lithium ions if the taste receptor is an ion channel; and calcium ions, cAMP, cGMP and/or IP₃, if the taste receptor is a GPCR. When measuring the concentration of these compounds or other downstream functional consequences of taste receptor activity, these compounds/consequences can be measured by any means known to those skilled in the art, e.g., patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers such as transcriptional activation of taste receptor activity- dependent genes; ligand binding assays; voltage, membrane potential and conductance changes; ion, preferably sodium or calcium ion flux assays, for example measuring calcium levels using calcium sensitive dyes such as Fluo-3, Fluo-4 or Fura-2; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP₃); changes in intracellular calcium levels; neurotransmitter release, and the like. These assays may be performed on intact cells expressing a taste receptor polypeptide, on permeabilized cells, or on membrane fractions produced by standard methods. Preferred assays for G-protein coupled receptors include cells that are loaded with ion sensitive dyes to report receptor activity. In assays of the invention for identifying modulator compounds, i.e. agonists and antagonists, changes in the level of ions in the cytoplasm or membrane voltage can be monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Activation of some G-protein coupled taste receptors stimulates the formation of inositol trisphosphate through phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate (Berridge & Irvine (1984) Nature 312: 315-21). IP₃ in turn stimulates the release of intracellular calcium ion stores. Thus, as mentioned, a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as EP₃ can be used to assess G-protein coupled receptor function. Cells expressing such G-protein coupled receptors 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 EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.

Preferably mammalian, amphibian or insect host cells are used in the method of the invention. If the taste receptor is a sweet or umami taste receptor, receptor activity may be measured by expressing the respective taste receptor in a heterologous host cell with a G-protein, such as Gαl5, Ga 16, transducin, gustducin, or a chimeric G-protein that links the receptor to a phospholipase C signal transduction pathway. For example, chimeric Ga 16 proteins harboring 37 and 44 gustducin-specific sequences at their C termini (G16/gust37 and G16/gust44) may be expressed in the host cell. A taste receptor expressed in these host cells responds to known agonist ligands in dose-dependent manner. Thus, preferably, said host cell expresses a chimeric protein comprising sequences of the G-protein subunit G_αi₅ or G_αl6 , preferably G_αi_5> and of gustducin or of transducin. A detailed description of chimeric G-proteins that can be used is described in e.g. Ueda T. et al., Chem. Senses 30 (suppl 1): il6, 2005. One preferred chimeric protein that can be used in a mammalian, amphibian or insect host cell of the method of the invention is the protein Gα_16gust44 according to SEQ ID NO: 2.

Additional ways of measuring G-protein coupled taste receptor activity are known in the art and comprise without limitation transcription assays, which measure, e.g. activation or repression of reporter genes which are coupled to regulatory sequences regulated via the respective G-protein coupled signalling pathway, such reporter proteins comprise, e.g., CAT or LUC; assays measuring internalization of the receptor; or assays in frog melanophore systems, in which pigment movement in melanophores is used as a readout for the activity of adenylate cyclase or phospholipase C (PLC), which in turn are coupled via G-proteins to exogenously expressed receptors (see, for example, McClintock T.S. et al. (1993) Anal. Biochem. 209: 298- 305; McClintock T.S. and Lerner M.R. (1997) Brain Res. Brain, Res. Protoc. 2: 59-68, Potenza MN (1992) Pigment Cell Res. 5: 372-328, and Potenza M.N. (1992) Anal. Biochem. 206: 315- 322). In yet another embodiment, the ligand-binding domains of the receptors can be employed in vitro in soluble or solid-state reactions to assay for ligand binding. Ligand binding to a taste receptor, or a domain of a taste receptor, such as e.g. the extracellular domain, can be tested in solution in the absence and presence of saliva or a saliva co-factor component as outlined in the method of the invention. In preferred embodiments the ligand-binding domains of the taste receptor is in a bilayer membrane attached to a solid phase, in a lipid monolayer or in vesicles. Thereby, the binding of a modulator to the receptor, or domain, can be observed using changes in spectroscopic characteristics, e.g. fluorescence, fluorescence polarization, plasmon resonance, absorbance or refractive index; or hydrodynamic (e.g. shape), chromatographic, or solubility properties, as is generally known in the art.

The methods provided herein may be designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). The skilled person will understand that there are many suppliers of libraries of chemical compounds.

Assays may be run in high throughput screening methods that involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic, or tastant compounds (that are potential ligand compounds). Such libraries are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired modulatory effect upon taste receptor activity. The compounds thus identified can serve as lead compounds to further develop modulators for final products, or can themselves be used as actual modulators. 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. For example, 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. Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art and no more needs to be stated here.

Antagonists identified by method described herein above can be administered directly to a human subject to inhibit or suppress a certain undesired taste. Agonists identified by methods described herein above can be used e.g. as food additives and taste enhancers. Thus, agonists and antagonists isolated by a method of the invention can be formulated with other ingredients or preparations to be taken orally, for example, foods, including animal food, and beverages, pharmaceutical or nutraceutical or homeopathic preparations.

Therefore, another aspect of the invention is a food stuff, precursor material or additive employed in the production of foodstuffs producible according to the invention. In another aspect the invention further provides a pharmaceutical composition producible according to the invention. These pharmaceutical and nutraceutical compositions comprise both products for human and animal consumption. The pharmaceutical composition can e.g. be an oral health care pharmaceutical composition such as but not limited to a mouth wash and/or an antiseptic.

The amount of agonist or antagonist isolatable according to the present invention to be taken orally must be sufficient to effect a beneficial response in the subject, preferably human subject, and will be determined by the efficacy of the particular taste modulators and the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound. Generally, an agonist or antagonist producible according to the present invention is preferably comprised in the neutraceutical or pharmaceutical composition, in food, a food precursor material or food additive in a concentration larger than 0.01 mM.

Salvia Co-Factor Component

In another aspect the invention provides a saliva co-factor component characterized by having a molecular mass of smaller than 100 kDa, preferably smaller or equal than 10 kDa, and by having co-factor activity as defined above. Preferably the co-factor activity is stable against heat of 80°C to 100°C for at least 5 min.

The saliva co-factor component of the present invention is producible by a method comprising the step of: (a) providing saliva and (b) isolating a component from said saliva having saliva co-factor activity and a molecular mass of smaller or equal than 100 kDa, preferably smaller or equal than 10 kDa. A saliva co-factor component of the invention is less frothy than saliva and can, thus, be more easily handled in in-vitro assays.

Preferably, step (a) outlined above is carried out using tangential-flow-filtration and/or stirred-cell-ultrafiltration according to well known methods comprised in the art.

In a further preferred embodiment, the method for producing the saliva composition further comprises a step (b) of (i) heating the saliva and/or the isolated saliva co-factor components for at least 5 min, preferably at least 10, most preferably for at least 15 min, preferably at a temperature of between about 80 and about 100°C. It is further preferred that the method comprises a further step (c) of centrifuging the saliva co-factor components isolated in step (a), the saliva sample used in step (a) and/or the isolated and heated saliva co-factor component of step (b) at a relative centrifugal force of between 2500 and 4500 g, preferably of about 3060 g for at least 30 min preferably at 4°C to remove excessive mucus and dead cells. The method may in a particularly preferred embodiment further comprise a step (d) wherein the pH of the isolated saliva co-factor component is adjusted to 7.4.

It is preferred that said "co-factor activity" is the property of the saliva co-factor component of the invention that the activity of an ENaC receptor contacted with salt, preferably sodium chloride, and L-cysteine dissolved in buffer, preferably sodium chloride-free buffer, is stronger than the activity of said ENaC receptor when contacted with same respective concentration of salt and L-cysteine dissolved in said isolated saliva co-factor component. In other words, said "co-factor activity" is that L-cysteine dissolved in said saliva co-factor component stimulates the activity of an ENaC receptor contacted with salt less strongly than L- cysteine dissolved in a different solvent. In a further aspect the invention provides a method for producing an artificial saliva composition comprising the steps:

(1) sub-fractionating the saliva co-factor component of the present invention into a plurality of sub-fractions having co-factor activity (e.g. using the preferred methods described herein); (2) identifying a compound or compounds comprised in said sub-fractions that have co- factor activity; and (3) synthetically producing the compound or compounds identified in step (2).

In a further aspect the invention also provides a use of a composition comprising L- cysteine for the enhancement of salt-taste. Preferably, said composition further comprises an agonist identified in the method of the invention which is capable of increasing the stimulatory effect of L-cysteine in saliva.

The following figures and examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

Brief Description of the Tables and Figures

Fig. 1 Membrane current changes of oocytes expressing ENaC induced by L-cysteine.

Membrane current traces of at least three oocytes of two to three different frogs were used to record in a perfusion solution containing 15 mM NaCl. Amplitude of current changes of oocytes expressing αβγ-ENaC or δβγ-ENaC after administration of varying concentrations of L-cysteine were normalized to current changes induced by raising the NaCl concentration of the superfusion solution from 15 to 50 mM. Error bars indicate standard deviations.

Fig. 2 Typical membrne current traces of oocytes challanged with L-cysteine. Two-electrode voltage clamp recordings were performed with oocytes expressing αβγ-ENaC or δβγ-

ENaC in a perfusion solution containing 15 mM NaCl. Exposure of oocytes to test compounds is indicated by horizontal bars above the current traces recorded for ENaC expressing oocytes. Scale bars: horizontal 5 min, vertikal 5 μA. Similar results were obtained when using the bona-fide ENaC-agonists L-Arginine and choline chloride

Examples Example 1: Functional expression analyses in Xenopus laevis oocytes

The human α-, β-, γ- and δ-ENaC subunits were cloned in the plasmid pBK-CMV (Stratagene; La Jolla, CA). The full-length α-, β-, and γ-ENaC cRNAs were synthesized using Not I-linearized plasmid-DNAs, while the δ-ENaC cRNA was synthesized from Xba I-linearized plasmid-DNA and using the T3 Message Machine (Ambion, Austin, TX) and Poly(A)Tailing kit (Ambion, Austin, TX). Quality of cRNA was evaluated by denaturating agarose-gel electrophoresis, and yield was estimated by photometric measurements. The RNAs were stored in aliquots at -80°C. In total the mount of 2 ng cRNAs for α-, β-, γ-ENaC (2:1 :1) or δ-, β-, γ- ENaC (1:1 :1) were injected into follicle-free Xenopus laevis oocytes (stages V-VI), which were maintained in sterile NMDG-KulORI solution (in mM: 10 NaCl, 1 KCl, 80 NMDG, 2 CaCl₂, 5 HEPES, 2.5 Na-pyruvate, adjusted to pH 7.5 with HCl) supplemented with 10 U/ml penicillin and 10 μg/ml streptomycin at 16°C for 2-4 d until electrophysiological recordings were performed. Whole cell current recordings were carried out using a conventional two-electrode voltage-clamp circuit (OpusXpress, Molecular Devices, Munich, Germany). Voltage recording and current injection electrodes had dc resistance of 0.5 to 2.5 MΩ. Oocytes were placed in a 200 μl experimental chamber and continuously superfused with NaCl-NMDG-ORI-solution (in mM: 15 NaCl, 75 NMDG, 1 KCl, 2 CaCl₂, 5 HEPES, pH 7.4). Membrane potential was voltage- clamped to -40 mV. Test substances were either dissolved in saliva or in buffer (NaCl-NMDG- ORI). 0.3 ml of solution containing test substances were applied within a time period of 1 min. As reference 50 mM NaCl with a rate of 1 ml / min within a time period of 30 seconds was applied. Amplitudes of current changes of ENaC-expressing oocytes following administration of test substances were normalized to current changes induced by raising the NaCl concentration of the superfusion solution from 15 to 50 mM. Example 2: Isolation of saliva and saliva co-factor components

Whole non-stimulated saliva samples (approximately 7.5 ml) were collected from four to six adult volunteers without any known metabolism disorders. They were asked to spit out saliva directly into sterile polypropylene tubes. Saliva samples were then pooled (also saliva from different individuals i.e. non-pooled saliva can be used) and centrifuged at 3060 g for 30 min at

4°C to remove excessive mucus and dead cells. Test substances were solved in saliva and the pH was adjusted to 7.4. For dilution experiments saliva was diluted in buffer. To measure heat stability of saliva co-factor components, centrifuged saliva was heated at 95 °C in a water bath for 15 min. Fractionation of salvia in different molecular masses was done by tangential-flow- filtration and/or stirred-cell-ultrafiltration according to well known method comprised in the art.

Example 3: Effect of saliva and saliva co-factor components on taste receptor function

In heterologous expression systems the activation and modulation of salt taste "receptors" (for example ENaC), by different compounds are studied. Thereby compounds which act as activators are identified. These compounds are verified for their capacity to modulate salt taste in human subjects. It was observed that some compounds (for example L-cysteine) strongly activate ENaC in a dose-dependent manner in in-vitro assays that were carried out in the absence of saliva, but that these compounds failed to enhances salt taste perception inhuman subjects, i.e. in in-vivo sensory studies involving human subjects. It could now further be shown for the first time that this discrepancy depends on components of saliva which are believed to act as co- factors required by the receptors. For example, as shown in figure 3, in oocytes expressing ENaC receptors, an application of L-cysteine in saliva did not increase ENaC-dependent Na⁺-currents, while L-cysteine dissolved in buffer activated ENaC salt receptors (see Figure 3). Thus, using saliva as solvent abolished the ENaC activation by L-cystein. This is consistent with in vivo sensory studies, where L-cysteine also did not enhance salt taste perception.

In general, salivary components may influence interaction of taste active compounds with their respective "receptors". Using saliva or salivary components in screening systems will help to upgrade the transfer capacity to the in vivo situation. Compounds which activated ENaC only when dissolved in buffer, but not when dissolved in saliva are excluded from further studies. The in-vitro methods of the invention also show a good correlation to in vivo sensory data with respect to the effects of antagonists. For example, the salt-receptor inhibitor amiloride antagonizes the salt receptor ENaC in both the method of the invention, i.e. in an in-vitro assay using saliva or a saliva co-factor component, and in in-vivo sensory studies. The identified active salivary component or saliva itself can be used routinely in screening assays to verify hit candidates, not only for the identification of salt taste strengtheners but also for other taste qualities.

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Claims

1. A method for isolating a modulator of a taste receptor comprising the steps:

(a) contacting a taste receptor or a host cell expressing said taste receptor with a potential modulator in the presence of saliva or a saliva co-factor component; and

2. The method of claim 1, wherein the modulator is an antagonist and wherein in step (b) the degree is determined by which the potential antagonist antagonizes said taste receptor activity and wherein the method further comprises a step (c) of contacting prior, concomitantly and/or after step (a) said taste receptor or said host cell with a compound capable of activating said taste receptor.

3. The method of claim 1, wherein the modulator is an agonist and wherein in step (b) the degree is determined by which the potential agonist increases said taste receptor activity.

4. The method of claim 1 or 3, wherein

(i) prior, concomitantly and/or after step (a) the taste receptor or said host cell is additionally contacted with a second compound which increases the activity of said taste receptor more in the absence of saliva or a saliva co-factor component than in the presence of saliva or a saliva co-factor component; and

(ii) in step (b) the degree is determined by which the potential agonist activates the second compound and thereby increases the activity of said taste receptor.

5. The method of any of claims 1-4, wherein the method comprises a further step of admixing the isolated modulator with a pharmaceutical, a foodstuff or a precursor material or additive employed in the production of a foodstuff.

6. The method of any of claims 1-5, wherein the host cell is a mammalian, amphibian or insect cell.

7. The method of any of claims 1-6, wherein the taste receptor is one or more receptors selected from the group consisting of a sweet taste receptor, a sour taste receptor, an umami taste receptor, a salt taste receptor, a temperature receptor and a pain receptor.

8. The method of claim 7, wherein the sweet taste receptor is at least one receptor selected from the group consisting of a T1R2/T1R3 heterodimer, a T1R3/T1R3 homodimer receptor and a TRPM5 receptor.

9. The method of claim 7, wherein the sour taste receptor is selected from the group consisting of PKD2L1 and PKD 1L3.

10. The method of claim 7, wherein the umami taste receptor is a T1R1-T1R3 heterodimer, mGluR4t, mGluRl and/or a TRPM5 receptor.

11. The method of claim 7, wherein the salt taste receptor is one or more receptors selected from the group consisting of ENaC, TRPVl and TRPML3.

12. The method of any of claims 1-11, wherein the taste receptor is a salt taste receptor having salt taste receptor activity and comprising at least one of the following polypeptides or functional derivatives thereof:

(i) the α-subunit of the ENaC receptor having an amino acid sequence according to SEQ ID NO: 3;

(ii) the β-subunit of the ENaC receptor having an amino acid sequence according to SEQ ID NO: 4;

(iii) the γ-subunit of the ENaC receptor having an amino acid sequence according to SEQ ID NO: 5; (iv) the δ-subunit of the ENaC receptor having an amino acid sequence according to SEQ ID NO: 6; (v) the TRPML3 polypeptide according to SEQ ID NO: 7; and

(vi) the TRPVl polypeptide according to SEQ ID NO: 8.

13. The method of any of claims 7-12, wherein said agonist is one or more compounds selected from the group consisting of: (i) a saccharide, a sugar alcohol and a sweetener for sweet taste receptors;

(ii) a mineral acid, a carbon acid and a salt of carbon acid for sour taste receptors;

(iii) glutamic acid, asparaginic acid, nucleic acid and salts thereof for umami taste receptors; and

(iv) sodium chloride, lithium chloride, potassium chloride and ammonium chloride for salt taste receptors.

14. The method of any of claims 1-13, wherein in step (b) the modulation of the taste receptor activity is determined by determining an intracellular ion concentration.

15. The method of any of claims 1-14, wherein the saliva co-factor component is heat stabile and/or has a molecular mass smaller than 100 kDa.

16. A food stuff, precursor material or additive employed in the production of foodstuffs producible according to any of claims 5-15.

17. A pharmaceutical composition producible according to any of claims 5-15.

18. The pharmaceutical composition according to claim 17, wherein the composition is an oral health care pharmaceutical composition.

19. A saliva co-factor component characterized by having a molecular mass of smaller than 100 kDa and having co-factor activity.

20. A saliva co-factor component characterized by having a molecular mass of smaller than 100 kDa, by having co-factor activity after treatment for 5 minutes at a temperature of between 80°C and 100°C.

21. The saliva co-factor component according to claim 20, wherein the saliva cofactor component is producible by a method comprising the step of:

(a) providing saliva and

(b) isolating a component from said saliva having saliva co-factor activity and a molecular mass of smaller or equal than 100 kDa, preferably smaller or equal than 1O kDa.

22. The saliva co-factor component of claim 21, wherein said method further comprises a step (b) of heating the saliva and/or the isolated saliva co-factor component for at least 5 min.

23. The saliva co-factor component of any of claims 20-22, wherein said "co-factor activity" is that L-cysteine dissolved in said saliva co-factor component stimulates the activity of an ENaC receptor contacted with salt less strongly than L-cysteine dissolved in a different solvent.