WO2013072332A1 - Methods of using antagonists of bitter taste receptors - Google Patents

Abstract

The present invention relates to compositions and methods of using a compound of Formula 1. e.g., (-)-hardwickiic acid, in free or orally acceptable base addition salt form, to mask or decrease a subject's perception of bitter taste and/or enhance perception of sweet taste.

Description

METHODS OF USING ANTAGONISTS OF BITTER TASTE RECEPTORS

This application claims the benefit of US Provisional Application No. 61/559,213 filed 14 November 2011, which is incorporated herein by reference in. its entirety for all purposes.

TECHNICAL FIELD

The field relates to compounds that act as antagonists to the T2R family of bitter taste receptors, in particular to compositions comprising and methods of using [2-(fitran-3- yl)ethyl]-trimethyI-hexahydronaphthalene-carboxylic acid and related compounds in free or orally acceptable base addition salt form to mask or decrease a subject's perception of bitter taste.

BACKGROUND OF THE INVENTION

Human beings have five basic taste modalities: sweet, salty, sour, umarni (savoury), and bitter. While sweet taste may be perceived as attractive, indicative of foods that possess a high caloric content, excessive bitter taste may be perceived as unpleasant, possibly due to associations with plant alkaloids which are generally bitter and frequently toxic. These basic sensor)^ mechanisms of tasting foods have been long known and highly conserved throughout species evolution. However, despite the fact that these sensory mechanisms have long since been known, developing an understanding of the signal transduction pathways underlying these taste modalities has been difficult. There has been relatively little progress made in small molecule development that would allow for specific manipulation, of these same pathways. However, it is known that bitter taste is mediated by the T2R (also known as the TAS2R) family that belongs to the family A of GPCRs including VI r pheremone receptors and opsin, receptors. In humans,, there are about 25 members of the T2R family that may function as bitter taste receptors. While receptor ligands for four of the five of the .25 T2R members (T2R41 , T2R4.2, T2R45, T2R48, T2R60) are at present unknown, T2R41 has been, recently shown, to bind to cyclamate (see for example, WO 201 1/138455). The fact that T2Rs are coexpressed in a. subpopulation of taste receptor cells, at the very least, raises the possibility that T2Rs form heterooligomers like Tl Rs for sweet and. umami taste. The bitter receptor gene family, in vertebrates, retains a relatively conserved family size of about 15-33 functional genes; among mammalian species there are multiple orthologues of those genes. Possibly a contributing factor as to why small molecule development in this area has been slow to develop, is that the region where the most T2R family sequence divergence is observed happens to occur in the extracellular domain where ligand binding may be likely to take place.

Broad spectrum T2R antagonists have been difficult to develop as T2R receptors have shown preference for activation by single specific ligands. For example, mT2R5, hT2R43, and hT2R47 show specificity for single ligands (cyclohexamide, aristolochic acid, and denatoniuni. respectively) (Sainz E.. et al Biochem. J. Immediate Publications, doi, 10.1042/BJ20061744 (2007)). Orthologs hT2R4 and mT2R8 responded to high concentrations of the structurally disparate compounds PROP and denatoniuni, but those two compounds are the only active agonists out of a battery of fifty-five compounds tested (Chaiidreshakar, J. et al. Cell 100, 703-71 1 (2000)). However, two possible exceptions to the high selectivity exhibited by T2Rs appear to be hT2R7 (Sainz E., et al Biochem. J. Immediate Publications, doi, 10.1042/BJ20061744 (2007)) and hT2R14 (Behrens. M., Biochem, Biophys. Res. Comm. 319, 479-484 (2004)). both receptors have been reported to be activated by a variety of unrelated receptor antagonists. However, some experimentation has demonstrated that it is possible that hT2R14 is indeed responsive only to aristolochic acid ((Sainz E.. et al Biochem. J. Immediate Publications, doi, 10.1042/BJ20061744 (2007)). The molecular receptive ranges of human TAS2R bitter taste receptors is reviewed and updated in Meyerhof et al (2010); Chem Senses 35: 157- 170.

Being able to manipulate bitter taste perception would be extremely useful. For example, it has been hypothesized that the lingering aftertaste that accompanies some commercially desirable artificial sweeteners, which show greatly enhanced sweetness compared with sucrose and have very low caloric value, could be due to the sweeteners activation or binding to certain T2R bitter taste receptors. Specifically this can be seen with such sulfonyl amide sweeteners as saccharin and acesulfame K. both of which possess lingering bitter aftertaste (see Kuhn et al (2004); Journal of Neuroscience 24 (45); 10260-10265). Thus, the ability to modulate bitter taste receptors, in the context of artificial sweeteners. could markedly improve the quality of the taste of the food product wherein the artificial sweetener has been incorporated.

Many pharmaceutical products contain active ingredients that taste bitter. The bitterness of the preparation often leads to lack of patient compliance, particularly in the case of drugs administered to children or elderly people, who may receive suspensions or chewable tablets because they have difficulty swallowing tablets or capsules. Masking bitterness is a major technical hurdle in formulating pharmaceuticals, which can be essential to ensure patient compliance.

Certain foods or beverages that are inherently bitter could be improved by the addition of a compound that could modulate bitter taste receptor signal transduction. utraceutical compositions or nutritional supplements may have a bitter taste for example due to the presence of amino acids or minerals. Alleviating bitterness would be useful also for inducing animals to eat food that is healthy, cheap and nourishing, but has a bitter taste.

A major hurdle to finding specific antagonists, however, is the number of the bitterness receptors that exist. To be effective, a compound would need to decrease the perception of bitterness not only at one T2R receptor but via a number of those same T2R family of receptors. Preferably it should not act as an agonist at any of the bitter receptors; even compounds found to be antagonists at one receptor may be agonists at another, so that they enhance rather than reduce the bitter taste.

Consequently, there is an unmet need for a compound that is able to act as a promiscuous or broad spectram bitterness receptor ligand that is able to decrease, or possibly completely abate, bitterness signal transduction from multiple bitterness receptors.

SUMMARY OF THE INVENTION

The inventors have surprisingly discovered that Compounds of the Invention, e.g. of Formula 1 below, or [2-(furan-3-yl)ethyl]-trimethyl-hexahydronaphthalene-carboxylic acid compounds, e.g.. hardwickiic acid and isomers thereof, in free or orally acceptable base addition salt form, are useful to modulate multiple bitter taste receptors (T2R), and the invention thus provides methods and products utilizing Compounds of the Invention to reduce the perception of bitter flavors and/or to enhance the perception of sweet flavors, ; hereinafter described,

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the Invention are compounds of Formula 1

Formula 1

wherein the dotted line denotes a single or double bond,

Ri is H, -CH₃ or (wherein the dotted line is a double bond) is not present;

R₂ is -CH₃, -(CO)OR₉, -CHO, or -C¾OH;

R₃ is -CH₃ or H;

RA is -CH₃ or H;

R₅ is H or -CH3;

R₆ is =CH₂, -CH₃, or HCQ)O ₉ ;

R₇ is -CH₃;

Rg is ^CH₂-(CO)OR₉, =CH-(CO)OR₉; -CH₂-C¾-0(CQ)CH₃

or R₇ and Rg together are furanyl; and

R« is H or Ci.4 alkyl, e.g., methyl;

in free or orally acceptable base addition salt form,.

For example, in particular embodiments, the Compound of the Invention may be selected from, e.g.. Compounds 1 , 2, 3, 4, 5, 6, and 7:

( -Hardwickiic Acid(i) (-)-epl-Hardwickiic Acid (2)

Mo -129,4 (t- OACHCl,) ]_D-?2.S(.e.0.8,CHCi₃)

Compound 3 Compound 4

In a particular embodiment, the Compound of the Invention is a [2-(furan-3-yl)ethyl]- trimethyl-hexahydronaphthalene-carboxylic acid, e.g., selected from hardwickiic acid and isomers thereof, in free or orally acceptable base addition salt form, e.g. selected from (-)- hardwickiic acid (Compound 1), (-)-epi-hardwickiic acid (Compound 2), and the hardwickiic acid structural isomer Compound 7;

(-)-Hardwickiic A«id (l) (-)-epi-Hardwfckik Acid (2)

[a]_D -129.4 (c. OJ. CHCM [aJ_D -72.8 (c. O.S. CHOs)

Compound 7 in free or orally acceptable base addition, salt form..

In a particular embodiment, the compounds of Formula 1 are compounds of Formula l :

wherein R₂, R₃. R$, R?. Rg and Rg are as hereinbefore set forth for Formula 1 , and R§ Is methyl. For example, in particular embodiments the Compound of the Invention may be selected from

a Compound of Formula la wherein R₂ is -COOH,

a Compound of Formula la. wherein R is -CH_3}

a Compound of Formula la wherein ¾ is H,

a Compound of Formula la wherein R₅ is -CH3.

a Compound of Formula la wherein Re is -CH , and

a Compound of Formula la. wherein R? and Rg together are furanyl, e.g., 3-furanyl;

each in free or orally acceptable base addition salt form. By "orally acceptable base addition, salt" is meant a salt formed by reacting a carboxylic acid bitter taste antagonist with a base to form a salt, wherein the cation, provided by the base is safe and palatable for oral ingestion, at concentrations to be administered, e.g., a sodium or potassium salt. In a particular embodiment, the Compound of the Invention, is (-)-hardwickiic acid in. free or orally acceptable base addition salt form. Some Compounds of the Invention occur naturally in some plants. In a particular embodiment for use with the products and methods herein described, the Compounds of the Invention are provided or used in isolated form. By "isolated" as used herein in relation to Compounds of the Invention is meant isolated from or synthesized independently from .an environment where the Compounds of the Invention could naturally occur, e.g., being substantially free of plant alkaloids and/or plant terpenoids which are not Compounds of the Invention but which would normally be present in a plant or a crude plant extract containing a Compound of the Invention. This is important not only because it is desirable where possible to use well characterized materials that can be precisely controlled, but also because these plant alkaloids and terpenoids frequently have a strong bitter taste, which would impair the effectiveness of the Compounds of the Invention.

In one embodiment, the Compound of the Invention is a receptor-binding antagonist (ligand) to one or more T2R bitter receptors. By blocking bitter receptors, such Compounds of the Invention can mask the bitter taste of comestibles, making the comestible less bitter .and more palatable. Moreover, by blocking the bitter receptors, the Compounds of the Invention can. enhance sweet tastes, thereby reducing the need to add additional sweetener, as well as counteracting the bitter aftertaste of some artificial sweeteners. One aspect of the invention therefore provides the use of a compound that can modulate multiple bitter taste receptors (T2R), e.g., a Compound of the Invention, e.g. (-)- hardwickiic acid, in free or orally acceptable base addition salt form, in combination with, a comestible product, e.g., a food, drink, confectionary, oral care, or oral pharmaceutical product, to enhance the taste of the product. Furthermore, the invention contemplates a method of using a Compound, of the Invention in combination with, a comestible product. A Compound of the Invention can. be used as a pretreatment before the ingestion of a bitter substance, or administered contemporaneously with a bitter substance. In one embodiment, the invention provides for Method I, wherein Method I is a method of modulating bitter taste perception in a subject, comprising orally administering an. effective amount of a Compound of the Invention so that bitter taste perception is modulated in said subject, e.g.: 1 , The method of Method I wherein the Compound of the Invention is a Compound of Formula 1 as herein before described, e.g., any of Compounds 1-7, or a Compound of Formula la, or combinations thereof, in free or orally acceptable base addition salt form; 2. The method of Method 1 wherein the Compound of the Invention is a [2-(furan-3- yl)ethyl]-trimethyl-hexahydronaphthalene-carboxylic acid, e.g.. selected from hardwickiic acid and isomers thereof, in free or orally acceptable base addition salt form, e.g. selected from (-)-hardwickiic acid (Compound 1), (-)-epi-hardwickiic acid (Compound 2), and the hardwickiic acid structural isomer Compound 7.

3. Any of the foregoing methods wherein the Compound of the Invention comprises (-)- hardwickiic acid;

in free or orally acceptable base addition salt form;

4. Any of the foregoing methods wherein the Compound of the Invention is a Compound of Formula 1 or la wherein R₂ is -COOH;

5. Any of the foregoing methods wherein the Compound of the Invention is a Compound of Formula 1 or l wherein Rj is -CH3;

6. Any of the foregoing methods wherein the Compound of the Invention, is a Compound of Formula 1 or la wherein R4 is H;

7. Any of the foregoing methods wherein the Compound of the Invention is a Compound of Formula 1 or la wherein R5 is -CHS; 8, Any of the foregoing methods wherein the Compound of the Invention is a Compound of Formula 1 or la wherein R_<¾ is -CH3;

9, Any of the foregoing methods wherein the Compound of the Invention is a Compound of Formula 1 or la wherein R₇ and Rg together are furanyl, e.g., 3-furanyl;

10, Any of the foregoing methods wherein, the Compound of the Invention is obtained or obtainable by extraction from a plant; 11. Any of the foregoing methods wherein the Compound of the Invention is substantially free of plant alkaloids and/or plant terpenoids which are not Compounds of the Invention but which would normally be present in a crude extract from, a plant containing a Compound of the Invention; 12. Any of the preceding methods, wherein the Compound of the Invention modulates bitter taste perception in a subject via binding to one or more bitter taste receptors selected from the group consisting of: h.T2Rl, hT2R10, hT2R14₅ hT2R16. hT2R38, hT2R44, hT2R46. and hT2R50; 1.3. Any of the preceding methods, wherein the Compound of the Invention modulates bitter taste perception in a subject via binding to one or more bitter taste receptors selected from the group consisting of: hT2R10, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46, and. hT2R50; 14, Any of the preceding methods, wherein the Compound of the Invention modulates bitter taste perception in a subject via binding to one or more bitter taste receptors selected from the group consisting of: hT2RJ 0, hT2R14, hT2R16, hT2R38. h.T2R.44, hT2R46, hT2R50; hT2R3, hT2R4. hT2R7, hT2R8. hT2R13, hT2R31 , hT2R39 and h.T2R43; 15. Any of the preceding methods, wherein the Compound of the Invention is an antagonist to at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of the bitter taste receptors in. the group consisting of hT2R10, liT2R14, hT2R16. hT2R38. hT2R44, hT2R46, hT2R50; h!2R3, hT2R4, liI2R7, h.T2R8, hT2Rl 3, hT2R31. hT2R39 and h.T2R43;

16. Any of the preceding methods, wherein the Compound of the Invention is an antagonist to at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of the bitter taste receptors in the group consisting of hT2R10 (SEQ ID No. 15 or SEQ ID No 62), hT2R14 (SEQ ID No 19 or SEQ ID No 64), hT2R16 (SEQ ID No 21 or SEQ ID No 65), hT2R38 (SEQ ID No 29 or SEQ ID No 69), hT2R44 (SEQ ID No 39), hT2R46 (SEQ ID No 43 or SEQ ID No 73). hT2R50 (SEQ ID No 51 or SEQ ID No 74); hT2R3 (SEQ ID No 56), hT2R4 (SEQ ID No 57), hT2R7 (SEQ ID No 59), hT2R8 (SEQ ID No 60), h.T2R13 (SEQ ID No 63), hT2R31 (SEQ ID No 68), hT2R39 (SEQ ID No 70) and hT2R43 (SEQ ID No 72);

17. Any of the preceding methods, wherein the Compound of the Invention is an antagonist to at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of the bitter taste receptors in the group consisting of hT2R10 (SEQ ID No. 15 or SEQ ID No 62), hT2R 14 (SEQ ID No 19 or SEQ ID No 64), hT2R16 (SEQ ID No 21 or SEQ ID No 65), hT2R38 (SEQ ID No 29 or SEQ ID No 69), hT2R44 (SEQ ID No 39), liT2R46 (SEQ ID No 43 or SEQ ID No 73), hT2R50 (SEQ ID No 51 or SEQ ID No 74); hT2R3 (SEQ ID No 56), hT2R4 (SEQ ID No 57), hT2R7 (SEQ ID No 59), hT2R8 (SEQ ID No 60), hT2R13 (SEQ ID No 63), hT2R31 (SEQ ID No 68), hT2R39 (SEQ ID No 70) and hT2R43 (SEQ ID No 72); or variants thereof with at least 80%, 81%, 82%, 82%. 84%. 85%, 86%, 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to hT2R10 (SEQ ID No. 15 or SEQ ID No 62), hT2R14 (SEQ ID No 19 or SEQ ID No 64), hT2R1 (SEQ ID No 21 or SEQ ID No 65), hT2R38 (SEQ ID No 29 or SEQ ID No 69), hT2R44 (SEQ ID No 39), hI2R46 (SEQ ID No 43 or SEQ ID No 73), hT2R50 (SEQ ID No 51 or SEQ ID No 74); hT2R3 (SEQ ID No 56), ttT2R4 (SEQ ID No 57), hT2R7 (SEQ ID No 59), hT2R8 (SEQ ID No 60), hT2R13 (SEQ ID No 63), hT2R31 (SEQ ID No 68), hT2R39 (SEQ ID No 70) and hT2R43 (SEQ ID No 72);

18. Any of the preceding methods, wherein the Compound of the Invention is an antagonist to at least three, e.g., at least six, of the bitter taste receptors in the group consisting of hT2Rl, hT2R!0, taT2R14, hT2R16. hT2R38, hT2R44, hT2R46, and hT2R50; 1 . Any of the preceding methods, wherein the Compound of the Invention is an antagonist to at least three, e.g., at least six. of the bitter taste receptors in the group consisting of hT2R10. hT2R14. hT2R16, h.T2R38, hT2R44, hT2R46. and hT2R50;

5 20. Any of the preceding methods, wherein the Compound of the Invention is an antagonist to at least three, e.g., at least six, of the bitter taste receptors in the group consisting of hT2R10. hT2R14, hT2R16, hT2R38. hT2R44, hT2R46. hT2R50; hT2R3, hT2R4, hT2R7, hT2R8, hT2R13. hT2R31 , hT2R39 and hT2R43;

10 21. Any of the preceding methods, wherein the Compound of the Invention is an antagonist to at least four, e.g., at least eight, of the bitter taste receptors in the group consisting of hT2R10, h.T2R14, hT2R16. HT2R38, h.T2R44, hT2R46, hT2R50; iiT2R3, hT2R4. hT2R7, h^'I^"2R8, hT2R13, hT2R31 , hT2R39 and hT2R43;

15 22. Any of the preceding methods wherein the amount of Compound of the Invention administered is sufficient to provide a concentration in the mouth of at least 1 micromolar, e.g.. approximately 1 -100 micromolar:

23. Any of the preceding methods, wherein the Compound of the Invention, modulates 20 bitter taste signal transduction at a concentration of about 1 -100 micromolar, e.g. at a concentration of about 2-25 micromolar. preferable about 2-30 micromolar, preferably 2, 3, 4, 5. 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19_., 20, 21, 22. 23, 24, 25, 26, 27, 28, 29,

30 micromolar.

25 24. Any of the preceding methods wherein the Compound of the Invention is administered with, a food product.

25. Any of the preceding methods, wherein said food product is coffee, tea. or a soft drink.

30

26. Any of the preceding methods wherein the Compound of the Invention is administered with a pharmaceutical or medicinal product, e.g., a liquid for oral administration, or a chewable or quick-dissolve tablet. 27. Any of the preceding methods wherein, the Compound of the Invention is administered with a nonsteroidal anti-inflammatory agent, a decongestant, an antihistamine, and/or an antibiotic: 28. Any of the preceding methods wherein the Compound of the Invention is administered with a nutritional supplement or nutraceutical:

29. Any of the preceding methods wherein the Compound of the Invention is administered with a sweetener, e.g., an artificial sweetener, e.g., a sulfonyl amide sweetener, e.g.. saccharin, sodium cyclamate or acesulfame potassium;

30. Any of the preceding methods wherein the Compound of the Invention is orally administered prior to oral administration of a substance comprising a T2R bitter receptor agonist;

31. Any of the preceding methods wherein the Compound of the Invention is administered In conjunction with a dental hygiene composition, e.g., a toothpaste or mouth rinse; 32. Any of the preceding methods wherein the Compound of the Invention is administered in conjunction with an animal food product;

33. Any of the preceding methods wherein the Compound of the Invention is added in conjunction with a second bitter masking agent, e.g., an agent selected from the group consisting of: sucralose, zinc gluconate, ethyl maltol. glycine, acesulfame k, aspartame, saccharin, f uctose, xylitol. malitol. isomalt; salt, spray dried licorice root, glycyrrhizin. dextrose, sodium gluconate, sucrose, glucono-delta-lactone, ethyl vanillin, and vanillin, eriodictyol, ferulic acid, homoeriodictyol, sodium ferulate,, trilobatin (disclosed, for example, in WO 2009/140784), hesperitin dihydrochalcone 4-beta-D glucoside (HDG) (disclosed, for example, in WO 2009/140784), sterabin, 4-(2,2,3- trimethylcyclopentyl)butanoic acid (disclosed, for example, in WO2008/119197 and WO 2008/1 191 5), one or more of

4-(2.2.3-trimethylcyclopent-3-enyl)but-2-enoic acid, 4(2,2,3-trimethylcyclopentyl )butanoic acid, 4-(2,2,6-trimethyIcyclohexyl)butanoic acid. 3-(2,2,6~trimethylcyclohexyl)propanoic acid, 2-(3,4-dimethylcycIohexyI)acetic acid, 2-methyl-4-(2_!6,6-trimethylcyclohex-2- enyl)butanoic acid, l-methyl-2-((l .2,2-trimethylbicyclo[3.1.0]hexan-3- yI)methyI)cyclopropanecarboxyIic acid; (E)-3-(I -(2.2,3-trimethylcycIopent-3-enyl)cyclopropyl)acrylic acid, (E)-4-(2,2,3- trimethylcyclohex-3-enyl)but-2-enoic acid, (E)-4-(2,2_f3-trimethylcyclopentyl)but-3-enoic acid, (E)-4-(2,2,3-trimethylcycIopentyl)but-2-enoic acid, l-methyl-2-((l ,2.2- trimethylbicyclo [3.1 , 0]hexan-3 -yl)methyl)cyclopropanecarboxylic acid, 2.2-dimethyl-4- (2,2,3-trimethylcycIopentyl)butanoic acid, 2-((l ,4-dimethylbicyclo[3, 1.0]hexan-2- yl)methyl)-l -methylcyclopropanecarboxylic acid, 2-( l -((i ,2,2- trimethylbicyclo[3.1.0]hexan-3-yl)methyl)cyclopropyI)propanoic acid, 2-(l -((1 - methylbicyclo[3.1.0]hexan-2-yl)methyl)cyclopropyI)acetic acid, 3,3-dimethyI-4-(2,2,3- trimethylcyclopentyl)butanoic acid, and 4-(l-methylbicyclo[3.1.0]hexan-2-yl)butanoic acid, (E}-l -(2-(l-methylbicyclo[3^ .0]hexan-2-yl)%½yl)cyclopropanecarboxylic acid, (E)-l -(2-(7-methyIspiro2.4]heptan-4-yl)vinyl)cycIopropanecarboxylic acid, (E)-4-(l methylbicyclo[3. l .0]hexan-2-y )but-3-enoic acid, (E)-4-(7-methylspiro[2.4]hept-6-en-4- yl)but-2-enoic acid, (E)-4-(7-methylspiro[2.4]heptan-4-yl)but-3-enoic acid, (E)-4-(7- methylspiro[2.4]heptan-4-yl)but-2-enoic acid, (E)-4-(8-methylspiro[2.5]oct-7-en-4-yl)but- 2-enoic acid, 1 -(2-(7-methylspiro[2.4]heptan-4-yl)ethyl)cyclopropanecarboxylic acid, 2- ((6,7-dimethylspiro[2.4]heptan-4-yl)methyl)- 1 -methylcyclopropanecarboxylic acid, 2-(l - (( 1 ,4-dimethylbicyclo[3.1.0]hexan-2-yl)methyl)cyclopropyl)propanoic acid, 2-( 1 -(( 1 - methylbicyclo[3..1 , 0]hexan-2-yl)methyl)cyclopropyl)acetic acid, 2-( 1 -((7- methyIspiro[2.4]heptan-4-yl)methyl)cyclopropyl)acetic acid, and 4-(7- methylspiro[2.4]heptan-4-yl)butanoic acid (WO200S/119196), and one or more of cis-4- propylcyclohexanecarboxylic acid, cis-4-tert-butylcyclohexanecarboxylic acid, cis-4- propylbicyclo[2.2.2]octane-l -carboxylic acid, cis-4-sec-butylcyclohexanecarboxylic acid, cis-4-(4-methylpentyl)cyclohexanecarboxylic acid, cis-4-benzylcyclohexanecarboxylic acid, and cis-4-phenylcyclohexanecarboxylic acid (disclosed, for example, in WO 2009/015504), GIV3727 (disclosed as compound lc in Slack et at (2010) Ciirr Biol.2010 Jim 22; 20(12); 1104-9 -and/or one or more of the bitter taste maskers disclosed for example in WO2010/100158. 34. Any of the preceding methods wherein the Compound of the Invention is added in conjunction with a preservative;

35. Any of the preceding methods wherein the modulation of bitter taste perception by administration of a Compound of the Invention enhances the perception of sweetness or reduces the perception of bitterness of a contemporaneously administered substance;

36. Any of the preceding methods wherein the perception of sweetness of a sweetener, e.g..,. a. natural or artificial sweetener is enhanced;

37. Any of the preceding methods wherein the amount of sweetener necessary to convey a sweet taste to a food or drink is reduced;

38. Any of the preceding methods wherein the amount of sugar, e.g. sucrose, fructose or combinations thereof, necessary to convey a sweet taste to a food or drink is reduced;

39. Any of the preceding methods wherein a bitter taste is masked;

40. Any of the preceding methods wherein the Compound of the Invention reduces the perception of bitterness of another agent;

41. Any of the preceding methods wherein administration of a Compound of the Invention reduces the perception of bitterness of an orally administered pharmaceutical; 42. In another embodiment, the invention provides the use of a Compound of the Invention as a screening assay to identify compounds which are bitter receptor antagonists, for example:

i. a competitive assay to identify novel bitter receptor antagonists, comprising providing a Compound of the Invention, optionally in labeled form, and measuring binding of the Compound of the Invention to a. bitter receptor in the presence and absence of a test compound;

ii. a screening assay for measuring activity of a test compound as a bitter receptor antagonist, wherein a Compound of the Invention, is used as a reference standard; and iii. any of the foregoing methods wherein the bitter receptor is one or more of h.T2Rl, hT2R10, hT2R14, hT2R16, hT2R38_s hT2R44. hI2R46_; and/or hT2R50; or

iv, any of the foregoing methods wherein the bitter receptor is one or more of hT2Rl 0, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46. and/or hT2R50; or

v. any of the foregoing methods wherein the bitter receptor is one or more of hT2R10, hT2R14, hT2R16, hT2R38, h!2R44, hT2R46. h.T2R50, hT2R3, hT2R4, hT2R7. hT2R8, hT2Rl 3, hT2R31 , hT2R39 and/or hT2R43.

The invention also provides a Compound of the Invention in labeled form, e.g., radiolabeled form. In a further embodiment the invention provides Compound of the Invention, optionally in radiolabeled form, in combination or association with suitable diluent or carrier for an assay, e.g., selected from dimethylsulfoxide, ethanol, dichloromethane. methanol, saline buffer solution (optionally comprising sugar and additional ions), and serum free media.

In another embodiment, the invention provides a comestible product, e.g., a food, drink, confectionary, oral care, or oral pharmaceutical product, comprising a taste-enhancing amount of a Compound of the Invention, e.g. (-)-hardwickiic acid, in free or orally acceptable base addition salt form, e.g. providing a combination as set forth in Method I and the following Methods.

In another embodiment, the invention provides a product for addition to a food or drink, comprising a Compound of the Invention, e.g. (-)-hardwickiic acid, in free or orally acceptable base addition salt form, and optionally further comprising flavorings, spices and or sweeteners.

For example, the invention provides a Compound of the Invention, e.g, (-)-hardwickiic acid, in free or orally acceptable base addition salt form, e.g., in combination with a sweetener,, for example a sweetener selected from sugars, e.g., sucrose, lactose and/or fructose; artificial sweetening agents, e.g., selected from steviol glycosides, aspartame, sucralose, neotame, acesulfame potassium, sodium cyclamate, and saccharin; sugar alcohols, e.g. sorbitol and/or xylitol; and combinations of any of the foregoing. In a particular embodiment the invention provides a Compound of the Invention, e.g. (-)- hardwickiic acid, in free or orally acceptable base addition salt form, in combination with an artificial sweetener having a bitter aftertaste, e.g., a sulfonyl amide sweetener, e.g., selected from saccharin, sodium cyclamate and acesulfame potassium. Non-limiting examples of artificial sweeteners include saccharin, sucralose, Ace ^rM and Neotame™. Non-limiting examples of natural sweeteners include tevioside, Luo Han Guo extract, mogrosides. glycyrrhizin, perillartine, naringin dihydrochalcone, neohesperidine dihydrochalcone. mogroside V, rubusoside, rubus extract, and rebaudioside A.

The proportion of compound used will depend on the sweetener, the compound, the use to which the sweetener will be put. and the effect desired. This means that the proportion may vary between very wide limits. The skilled person can easily determine an appropriate proportion in every case, using onl routine experimentation and the ordinary skill of the art. However, as a general, non-limiting guideline, the proportion of compound ma vary between 0.5 ppm and 40 ppm, more particularly in certain embodiments, between about 1 ppm and about 10 ppm of the comestible product.

Yet another aspect of the invention provides for methods of using compounds that modulate multiple bitter taste receptors (T2R or TAS2R) in conjunction with other compounds that also function to mask bitter taste. It is contemplated by the present invention that a Compound of the invention could be used in conjunction with, at least, one other compound that functions to mask bitter taste perception in a free or fixed combination, e.g. in combination with one or more of the bitter-masking agents listed in Method 1.1.33.

Compounds that modulate one or more bitter taste receptors (T2R or TAS2R) are termed "modulator^" and include any substance or compound that alters an activity of a protein of interest, for example, a bitter taste receptor. A substance or compound is a modulator even if its modulating activity changes under different conditions or concentrations or with respect to different forms of a protein of interest, for example, a bitter taste receptor. In specific embodiments, a modulator can alter the structure, conformation, biochemical or biophysical properties or functionality of a bitter taste receptor.

A modulator may for example, alter directly or indirectly the activity of a receptor or the interaction of a receptor with its ligands. The term "modulator" includes genetically modified versions of bitter taste receptors with altered activity as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. The term "modulator" can be an agonist (potentiator or activator) or antagonist (inhibitor or blocker), including partial agonists or antagonists, selective agonists or antagonists, competitive (or unsurmoun table/insurmountable) antagonists and inverse agonists, and can also be an allosteric modulator. In other aspects, a modulator may change the ability of another modulator to affect the function of a protein of interest, for example, a bitter taste receptor. The Compounds of the Invention, are antagonists of one or more of the bitter taste receptors selected from the group of human bitter taste receptors consisting of hT2 10, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46, hT2R50; hT2R3. hT2R4. hT2R7, hT2R8, hT2R13, hT2R31, hT2R39 and hT2R43. The terms "inhibitor", "antagonist" or "blocker" refer to a compound or substance that decreases or blocks one or more activities of a protein of interest, for example, a bitter taste receptor by, for example binding to, partially or totally block stimulation, preventing, delaying activation, inactivating, desensitizing or down-regulating bitter taste receptor and/or taste transduction. An. "inhibitor", "antagonist" or "blocker" attenuates the effect of an. agonist. It may be competitive (or surmountable), i.e. it binds reversibly to a region of the receptor in common with an agonist, but occupies the site without activating the effector mechanism. The effects of a competitive antagonist may be overcome by increasing the concentration of agonist, thereby shifting the equilibrium and increasing the proportion of receptors which the agonist occupies. Alternatively, antagonists may be unsurmountable or insurmountable where no amount of agonist can completely overcome the inhibition once it has been established. Unsurmountable or insurmountable antagonists may bind covalently to the agonist binding site (competitive irreversible antagonists), in which case there is a period before the covalent bond forms during which competing ligands can prevent the inhibition. Other types of unsurmountable antagonists act allosterically at a different site on the receptor or an associated ion channel. In specific embodiments, terms "inhibitor", "antagonist" or "blocker" in the context of a bitter taste receptors, refers to a compound or substance that decreases the downstream, signaling response associated with the bitter taste receptor. In particular embodiments, decreasing taste receptor activity can result in a change in. the amount or distribution of an. intracellular molecule or the activity of an enzyme which is part of the intracellular signaling pathway for the bitter receptor. Examples of the intracellular molecule include, but are not. limited to, free calcium, cyclic adenosine monophosphate (cAMP), inositol mono-, di- or tri-phosphate. Examples of the 5 enzyme include, but are not limited to. adenylate cyclase,. phospholipase-C and G-protein coupled receptor kinase.

Preferably the Compounds of the Invention are isolated antagonists of one or more of the bitter taste receptors selected from the group o human bitter taste receptors consisting of 10 h.T2R10, hT2R14, hT2R16, hT2R38, hT2R44, h.T2R46, hT2R50; hT2R3. hT2R4, hT2R7, hT2R8. hT2R13, hT2R31. hT2R39 and hT2R43.

An "isolated antagonist" of hTAS2R bitter taste receptor activity, respectively, is a substance which is isolated from or synthesized independently from an environment where 15 the antagonist (eg a Compounds of the Invention.) could naturally occur, e.g., being substantially free of plant alkyloids and/or plant terpenoids which, for example, are not Compounds of the Invention but which would normally be present in a plant or a crude plant extract containing a Compound of the Invention.

20 Preferably the isolated antagonist reduces the activity of hTAS2R stimulated by the hTAS2R bitter taste receptor agonist, preferably selected from the group consisting of the agonists (for example, those used in Examples 1 and 2 and as defined in Tables 1, 3, 7 and 7) and structurally related agonists thereof. Preferably this reduction is by at least 10%

(e.g., at least: 10%, 15%; 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%: 99%; 25 99.5%; or 1.00%). preferably, but not necessarily at at the same molar concentration.

The extent of the lowering of the hTAS2R bitter taste receptor activity, caused by the antagonist is determined in the presence of said agonist, e.g. one of the compounds indicated above in any one or more of Tables 1, 3, 6 and 7 or a structurally related 30 compound, which may be added prior, concomitantly or after addition of the antagonist.

Preferably, the identified antagonist exerts this inhibitory activity, if present in the same molar. 2-fold, 5-fold, 10-fold. 50-fold or 100-fold molar concentration as the agonist. Preferably, the antagonist exerts its antagonizing action when it is contacted prior, concomitantly or after, preferably concomitantly, to contacting the hTAS2R polypeptide, the host ceil genetically engineered with a polynucleotide encoding an hTAS2R polypeptide as defined above in. any one or more of Tables 1-3 and 6-7 or a vector containing a polynucleotide as defined above to express hTAS2R polypeptide with said hTAS2R agonist. Preferably, the antagonist is contacted at the same molar concentration as said hTAS2R agonist.

The terms "potentiator", "agonist" or "activator" refer to a compound or substance that increases one or more activities of a protein of interest, for example, a bitter taste receptor, by for example, binding to, stimulating, increasing, opening, activating, facilitating, enhancing activating, sensitising, or up regulating bitter taste receptor signal transduction. In specific embodiments, the terms "potentiator", "agonist" or "activator" in the context of taste receptors, refer to a compound or substance that increases the downstream signaling response associated with the bitter taste receptor. As used herein, the term "partial agonist" means an agonist which, no matter how high a concentration is applied, is unable to produce maximal activation of the receptors. In a preparation with a low receptor reserve, it is therefore unable to produce a maximal response. Because high-efficacy compounds need to occupy relatively few receptors to produce a maximal response, it is possible to inactivate a proportion of the receptors in a tissue (e.g. in the presence of an irreversible antagonist) without depressing the maximum of the concentration-response curve. (The curve is, however, shifted rightward along the x axis). There is said to be a receptor reserve (or, more colloquially, spare receptors) for that particular agonist in that particular tissue. There is no receptor reserve for a drug which acts as a partial agonist in the tissue. The receptor reserve may vary between tissues, depending on the number of receptors in the particular tissue and the efficiency of coupling between them and their effector mechanism, Consequently, a partial agonist in one tissue may appear to act as a full agonist in a tissue with a higher receptor reserve. The molar concentration of an agonist which produces 50% of the maximum possible response for that agonist is termed its EC50 value. Other percentage values (eg EC25. EC ₀ and EC90) are also sometimes used. Where an agonist causes an inhibitory response, the IC50 is the molar concentration which produces 50% of the maximum possible inhibition. In particular embodiments, increasing the bitter taste receptor activity can result in change in the amount or distribution of an intracellular molecule or the activity of an enzyme which is part of the intracellular signaling pathway for the bitter receptor. Examples of the intracellular molecule include, but are not limited to, free calcium, cyclic adenosine monophosphate (cAMP), inositol mono-, di- or tri-phosphate. Examples of the enzyme include, but are not limited to, adenylate cyclase, phospholipase-C, G-protein coupled receptor kinase.

According to yet another aspect of the present invention, a method of identifying a modulator of a bitter receptor function comprises; a) exposing a cell or cell line that stably expresses a bitter taste receptor to a test compound; and b) detecting a change in a function of the bitter taste receptor. In some embodiments, the detecting utilizes an assay that measures intracellular free calcium. In some embodiments, the intracellular free calcium is measured using one or more calcium-sensitive fluorescent dyes, a fluorescence microscope, and optionally a fluorescent plate reader, wherein at least one fluorescent dye binds free calcium. In some other embodiments, the intracellular free calcium is monitored by realtime imaging using one or more calcium-sensitive fluorescent dyes, wherein at least one fluorescent dye binds free calcium. Methods for identifying modulators of human bitter taste receptors can be found in any one or more of the following published patent applications: WO2004/055048, WO2007/121599, WO 2007/147275, WO 2008/086634, WO 2008/1 19195, WO 2009/149577 and WO 2010/015097. all of which are incorporated herein by reference.

The screening method as defined above is useful in identifying potential antagonists of any one or more of the known human bitter taste receptors.

The term "potential antagonist", comprises any perceivable chemical substance or combination thereof having a structure according to the compound formula (I) in a non- purified, partially purified or purified state. The potential antagonist is selected on the basis of its antagonizing behaviour. In a preferred embodiment, the "potential antagonist" is a compound structurally related to the Compound of Formula 1 of the present invention. In a preferred embodiment, the potential bitter taste receptor hTAS2R antagonist is used in the method of the invention has a different structure than any of the preferred bitter taste receptor hTAS2R agonists (bitter substances). In. one preferred embodiment, a modulator identified using a screening method as defined above is a structurally related antagonist to any one of the Compounds of the Invention,

The term "structurally related antagonist" is a substance, which is derived from the compound of Formula Ϊ of the present invention, respectively, by 1. 2, 3, 4, 5 or 6 steps of chemical modification and which lowers the specific hT.AS2R bitter taste receptor activity compared to the activity determined in the presence of an agonist specific for the specific TAS2R receptor, respectively, by at least 10% (e.g. at least 10%, 15%. 20%, 30%, 40%, 50%, 60%. 70%, 80%, 90%, 95%, 98%, 99%, 99,5% or 100%). Preferably, the antagonistic derivative exerts this action, when it is contacted prior, concomitantly or after. preferably concomitantly, to the contacting of the hTAS2Rpolypeptide. the host cell expressing the hIAS2R polypeptide, or the vector comprising the hTAS2R polypeptide, with a hTAS2R agonist.

In one preferred embodiment, the Compound of the Invention is (-)-hardwickiic acid which is used in a method of modulating bitter taste perception in a subject so that bitter taste perception is modulated in the subject.

(-)-Hardwickiic acid is a known diterpenoid compound, which has surprisingly been found to be a natural hTAS2R antagonist. Hardwickiic acid has been found in many plants, e.g. Hardwickia pinnata. Soli da o jimcea, Solidago arguta. Gr ngea maderaspatana, Bacckaris acraei, Clerodendrum neriifolium, Croton oblongifolius, Croton aromaticus, roton sonderianus, Copaifera officinalis, Casearia sylvestris, Ribus nigrum (blackcurrant absolute) and others (see for example, Misra el al (1964), Tetrahedron Letters 49: 3751-9; Henderson el al (1973) 1 (9); 1322-51 ; Perfume & Flavourist (1985) Volume Date 1984 9(6) 39-42 , Chhabra et al (2007) Journal of the Kenya Chemical Society (2007) 4(1 ) 33- 46) and McChesney, Clark and Silveira (1991) J of Natural Products 54(6) 1625-1633). For example, (-)-hardwickiic acid exists, for example, as a 20% w/w of black currant buds absolute extract. It exhibits insecticidal activity against Aphis truccivora, and also antimicrobial activity. While plant alkaloids and terpenoids typically present in extracts comprising hardwickiic acid tend to be very bitter tasting, it is surprisingly found that hardwickiic acid itself, and structurally related compounds, e.g., of Formula 1 supra, are antagonists rather than agonists of the bitter receptors, and moreover exhibit inhibition of a variety of human T2R receptors. This is in contrast to other T2R receptor antagonists that have merely shown affinity to only one or a few receptors. The Compounds of the Invention, particularly (-)-hardwickiic acid, are exceptional in that it is not common for a compound to be a promiscuous or broad spectrum inhibitor of bitter receptors. In another preferred embodiment, there is provided a blackcurrant bud absolute extract as a bitter taste masking agent. Preferably, the w/w content of (-)-hardwickiic acid in the blackcurrant bud absolute extract is about 20% (w/w). Preferably, the blackcurrant bud absolute extract is a fractionated extract. Preferably the blackcurrant bud absolute extract is a Rib s nigrum extract. Preferably the blackcurrant bud absolute extract has a mixture of (- )-hardwickii acid and (-)-epi-hardwickiic acid in the ratio of about 4: 1 (eg from about 120mg to about 29mg respectively). This is the first time that (-)-epi-hardwickiic acid has been isolated from a Blackcurrant source.

Accordingly, there is provided a Compound 2 of Formula 1 ((-)-epi-hardwickiic acid) of the present invention.

Also surprising and unexpected is that inhibition is not dependent on the presence of an agonist. Moreover, pretreatment with an inhibitor is also effective in blocking signal transduction downstream of a. given T2R receptor.

It is contemplated that in certain cases that the efficacy of a Compound of the Invention, e.g., (-)-hardwickiic acid, can be increased when the compound is applied prior to the agonist. The present invention also contemplates a use of a Compound of the Invention, e.g., (-)-hardwickiic acid, wherein the inhibition of T2R receptors is reversible.

The sample isolated from blackcurrant absolute extract (see Example 2) is a mixture of two enantiomers. The minor epi-isomer of (-)-hardwtckiic acid ((-)-epi-hardwickiic acid (Compound 2 of Formula 1)) is also an inhibitor of T2R44, T2R46, T2R50. The epi-isomer differs from the major form of (-)-hardwickiic acid (Compound 1 of Formula 1) in that the epi-isomer is itself a TAS2 14 agonist. Thus the configuration of (-)-hardwickiic acid, e.g., according to Formula la (eg Compound 1 of Formula 1) is generally preferred even though the Examples demonstrate that this configuration acts as a partial agonist for T2R1 and T2R46 (see Figure 1 and Table 7 respectively from Examples 1 and 2).

In a preferred embodiment, there is provided a blackcurrant bud absolute extract comprising a. mixture of (-)-hardwickiic acid and (-)-epi hardwickiic acid in the ratio of about 1 : 1, 2: 1, 3: 1, 4: 1. 5: 1 , 6: 1, 7: 1 , 8: 1, 9: I , 10: L or about I: 2, 1 : 3, 1 : 4, 1: 5, 1 : 6. 1 : 7, 1 : 8, 1 : 9, 1 : 10.

In another preferred embodiment, there is provided a blackcurrant bud absolute extract comprising about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, (w/w) of (-)- hardwickiic acid. The content of hardwickiic acid in. a product can be enriched easily and the ratio can be different depending on the source of raw material and the way of processing. Additionally, the ratio of the isomers may be changed or even reversed if the products produced by biosynthetic methods, such as cell culture or synthetic biology. In another preferred embodiment, there is provided the use of a blackcurrant bud absolute extract as a bitter taste ligand which modulates bitter taste receptor activity by decreasing bitterness signal transduction activity across multiple bitter taste receptors selected from the group consisting of hT2R10, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46, hT2R50; hI2R3, h.T2R4, hT2R7, hT2R8, h.T2R13, h.T2R31 , h.T2R39 and hT2R43.

There is provided the a blackcurrant bud absolute extract according to the previous paragraph wherein the blackcurrant bud absolute extract contains about 20% (w/w) of (-)- hardwickiic acid. There is provided the use of a a blackcurrant bud absolute extract according to either of the two previous paragraphs wherein the blackcurrant bud absolute extract comprises a mixture of (-)-hardwickiic acid and (-)-epi hardwickiic acid in the ratio of about 4:1.. There is provided the use a blackcurrant bud absolute extract according to any one of the three previous paragraphs wherein, the blackcurrant bud absolute extract is a Rihus nigrum extract, "T2R" as used herein refers to the family of taste receptors responsible for the perception of bitter taste in mammals, "T2R" as used herein may refer to one or multiple bitter taste receptors. The term "T2R" is used interchangeably with the term "TAS2R", As discussed above, human bitter taste is mediated by about 25 members of the human. TA.S2 receptor (hTAS2R) gene family. In addition to their role in taste, bitter receptors are also important in a series of physiological contexts. For example, taste receptor agonists elicit a secretory response in entero-endocrine cells in vitro and in animals in vivo, and induce neuronal activation. Therefore, all of the bitter receptor family members are important clinical targets for managing a variety of conditions associated with detection of bitter tastants. Bitter receptors are G protein coupled receptors (GPCRs) expressed at the surface of taste receptor cells and. are coupled to secondary messenger pathways. TAS2R receptors can be coupled to transducing or gustducin for example, through which they can activate both phospodiesterases and a phospholipase C (PLC) p2-dependent pathway to increase intracellular Ca²⁺ concentration. The term "bitter receptor", as used herein, refers to any one of the G protein coupled receptors that is expressed at the surface of a taste receptor cell and that mediates bitter taste perception via secondary messenger pathways. The phrase "functional bitter receptor" refers to a bitter receptor that responds to a known activator or a known inhibitor in substantially the same way as the bitter receptor in a cell that normally expresses the bitter receptor without engineering.

Bitter receptor behavior can be determined, by, for example, physiological activities and pharmacological responses. Physiological activities include, but are not limited to, the sense of bitter taste. In this regard, the perception of the bitter taste can be measured, by, for example, using a 2-alternative forced choice paradigm (2-AFC), coupled with anchored taste intensity ratings in order to quantify the reduction in bitterness (see, for example, the disclosure and teachings in Slack et al (2010) Curr Biol 20(1.2) 1 104-1109). Pharmacological responses include, but are not limited to. a change in. the amount or distribution of an intracellular molecule or the activity of an enzyme which is part of the intracellular signaling pathway for the bitter receptor when a bitter receptor is contacted with a modulator. For example, a pharmacological response may include an increase in intracellular free calcium when the bitter receptor is activated, or a decrease in intracellular free calcium when the bitter receptor is blocked. A representative list of human bitter taste receptors and their accession numbers is provided in Table 2 below.

More specifically, information on the human TAS2R bitter taste receptor polypeptide sequences and polynucleotide sequences for hT2RL hT2R10, hT2R14, hT2R16, hT2R38. hT2R44, hT2R46. hT2R50; hT2R3. hT2R4. hT2R7, hT2R8. hT2R13, hT2R31. hT2R39 and h^'I^'2R43 can be found using the Genbank Accession numbers and Genbank Version numbers listed in Table 2 below (see also US7579453B2 and WO 2005/007891 and Table 1 on pages 17-1 of WO2004/029087). The terms "polypeptide" and "protein" are used interchangeably herein and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification. As used herein, the term protein "variant" is to be understood as a polypeptide which differs in comparison to the polypeptide from which it is derived by one or more changes in the amino acid sequence. The polypeptide from which a variant is derived is also known, as the parent polypeptide. Typically a variant is constructed artificially, preferably by gene- technological means. Typically, the polypeptide from which the variant is derived is a wild-type protein or wild-type protein domain. However, the variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent polypeptide or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent polypeptide. The changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-termmal truncations, or C- terminal truncations, or any combination of these changes, which may occur at one or several sites. In preferred embodiments, a variant usable in the present invention exhibits a total number of up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 15. 20, 25, .30, 35, 40. 45, 50, 55, 60. 65. 70, 75. 80. 85, 90, 95, 100, 110, 120, 130. 140. 150, 160, 170, 1 80. 190. or 200) changes, in the amino acid sequence (i.e. exchanges, insertions, deletions, N-terminal truncations, and/or C -terminal truncations). The amino acid exchanges may be conservative and/or non-conservative. In preferred embodiments, a variant usable in the present invention differs from the protein or domain from which it is derived by up to 1 , 2, 3, 4, 5, 6, 7, 8. 9, 10. 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid exchanges, preferably conservative amino acid changes. Variants may additionally or alternatively comprise deletions of amino acids, which may be N-terminal truncations, C -terminal truncations or internal, deletions or any combination of these. Such variants comprising N-terminal truncations, C-terminal truncations and/or internal deletions are referred to as "deletion variants" or "fragments" in the context of the present application. The terms "deletion variant" and "fragment" are used interchangeably herein. A deletion variant may be naturally occurring (e.g. splice variants) or it may be constructed artificially, preferably by gene-technological means. Typically, the protein or protein domain from, which the deletion variant is derived is a wild-type protein. However, the deletion variants of the present invention may also be derived from homologs. orthologs, or paralogs of the parent polypeptide or from artificially constructed variants, provided that the deletion variants exhibit at least one biological activity of the parent polypeptide. Preferably, a deletion variant (or fragment) has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25. 30. 35, 40. 45, 50, 55, 60, 65, 70. 75, 80, 85, 90, 95, or 100 .amino acids at its N- terminus and/or at its C-terminus and/or internally as compared to the parent polypeptide.

A "variant" as used herein, can alternatively or additionally be characterised by a certain degree of sequence identity to the parent polypeptide from which it is derived. A variant of the present invention may have a sequence identity of at least 50%, 55%, 60%, 65%, 70%, 75% identity to the respective reference polypeptide or to the respective reference polynucleotide. The expression "at least 50%, 55%. 60%, 65%, 70%, 75% sequence identity" is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons.

More precisely, a variant in the context of the present invention exhibits "at least 80% sequence identity" to its parent polypeptide. Preferably, the sequence identity is over a continuous stretch, of 20, 30, 40, 45, 50. 60. 70. 80, 90, 100 or more amino acids. The expression "at least 80% sequence identity" is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to a sequence identity of at least 80%, at least 81 %. at least 82%. at least 83%, at least 84%. at least 85%, at least 86%. at least 87%. at least 88%. at least 89%. at least 90%. at least 1%, at least 92%. at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%), or at least 99% to the respective reference polypeptide or to the respective reference polynucleotide. Preferably, the polypeptide in question and the reference polypeptide exhibit the indicated sequence identity over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids. Preferably, the polynucleotide in question and the reference polynucleotide exhibit the indicated sequence identity over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides. In case where two sequences are compared and the reference sequence is not specified in comparison to which the sequence identity percentage is to be calculated, the sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by SEQ ID. if not specifically indicated otherwise. For example, a peptide sequence consisting of 30 amino acids compared to the amino acids of full length hTAS2R with 299 amino acid residues may exhibit a maximum sequence identity percentage of 10.03% (30/299) while a sequence with a length of 150 amino acids may exhibit a maximum sequence identity percentage of 50.17% (150/299). The similarity of nucleotide and .amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci, USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wusti.edu ) or with the CLUSTAL algorithm (Thompson, J, D., Higgins. D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on http://www.ebi. ac.uk/Tools/clustalw/ or on http://www.ebi.ac.uk Tools/clustalw2/index.html or on http://npsa-pbil.ibcp.fr/cgi-

Preferred parameters used are the default parameters as they are set on http://www.ebi.ac.uk/Tools/clustalw/ or http://www.ebi.ac.uk Tools/clustalw2/index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403- 10. BLAST polynucleotide searches are performed with the BLASTN program, score = 100, word length = 12, to obtain polynucleotide sequences that are homologous to those nucleic acids which encode hTAS2R40, hTAS2R43, h.TAS2R44, hTAS2R46, or hTAS2R47. BLAST protein searches are performed with the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to the hTAS2R40 polypeptide, hTAS2R43 polypeptide, hTAS2R44 polypeptide, hTAS2R46 polypeptide, or hTAS2R47 polypeptide. To obtain gapped alignments for comparative purposes. Gapped BLAST is utilized as described in Altschul el al (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle- LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1 : 154- 162) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

"Hybridization" can also be used as a measure of sequence identity or homology between two nucleic acid sequences. A nucleic acid sequence encoding hT2Rl , hT2R 10, hT2R14. hT2R 16. hT2R38, hT2R44. hT2R46. hT2R50: hT2R3, hT2R4, hT2R7, hT2R8. hT2R13, hT2R3 1 , hT2R39 and hT2R43 ; or a portion of any of these can be used as a hybridization probe according to standard hybridization techniques. The hybridization of a probe for any one of the following hT2R bitter taste receptors: HT2R1 , h.T2R10, hT2R!4, hT2R16, hT2R38, hT2R44. hT2R46, hT2R50; hT2R3, hT2R4. hT2R7, h^'I^'2R8. hT2R13, hT2R31 , liT2R39 and hT2R43, to DNA or RNA from a test source (e.g. a mammalian cell) is an indication of the presence of the DNA or RNA for any one of the following hT2R bitter taste receptors: hT2Rl . hT2R10, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46, hT2R50; hT2R3, h.T2R4, hT2R7, hT2R8. h.T2R13. hT2R31 , hT2R39 and hT2R43 in the test source. Hybridization conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1 - 6.3.6, 1991. "Moderate hybridization conditions" are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 30°C, followed by a wash in IX SSC, 0.1 % SDS at 50°C. "Highly stringent conditions" are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C. Nucleotide sequences encoding any one or more of the following human bitter taste receptors; hT2Rl , h.T2R10, hT2R14, hT2R16, hT2R38, hT2R44, hT2R46. hT2R50; faT2R3, hT2R4, hT2R7, hT2R8, hT2R13, hT2R31 , hT2R39 and hT2R43, can be found in Table 2.

"Conservative substitutions" may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can. be grouped into the following six standard amino acid groups:

(1) hydrophobic: Met, Ala, Val, Leu, He;

(2) neutral hydrophilic: Cys. Ser, Thr; Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys. Arg;

(5) residues that influence chain orientation: Gly, Pro: and

(6) aromatic: Trp, Tyr, Phe, As used herein, "conservative substitutions" are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. Some preferred conservative substitutions within the above six groups are exchanges within the following sub-groups: (i) Ala. Val. Leu and He: (ii) Ser and Thr; (ii) Asn and Gin; (iv) Lys and Arg; and (v) Tyr and Phe. Given the known, genetic code, and recombinant and synthetic DNA techniques, the skilled scientist readily can construct DNAs encoding the conservative amino acid variants. As used herein, "non-conservative substitutions" or "non-conservative amino acid exchanges" are defined as exchanges of an amino acid b another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

As used herein, the term "derivative" of a polypeptide refers to a polypeptide that has been chemically modified so that it comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphoryiated amino acids. The polypeptide from which the derivative derives is also known as the parent polypeptide. This parent polypeptide can be a naturally occurring protein but can also be a protein variant as defined above. Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half- life, or increased water solubility. Chemical modifications applicable to the derivatives 5 usable in the present invention include without limitation: PEGylation. glycosylation of non-glycosylated parent polypeptides, or the modification of the glycosylation pattern present in the parent polypeptide.

A "biological activity" as used herein, refers to any activity a polypeptide may exhibit, 10 including without limitation: enzymatic activity; binding activity to another compound (e.g. binding to another polypeptide, in particular binding to a receptor, or binding to a nucleic acid); inhibitory activity (e.g. enzyme inhibitory activity); activating activity (e.g. enzyme- activating activity); or toxic effects. It is not required that the variant or derivative exhibits such an activity to the same extent as the parent polypeptide. A variant is regarded as a 15 variant within the context of the present application, if it exhibits the relevant activity to a degree of at least 10% of the activity of the parent polypeptide. Likewise, a derivative is regarded as a derivative within the context of the present application, if it exhibits the relevant biological activity to a degree of at least 10% o the activity of the parent polypeptide.

20

The relevant "biological activity" in the context of the present invention is "bitter taste receptor activity", i.e. the ability of the receptors described herein (hT2Rl , hT2R10, hT2R14. hT2R16, hT2R38. hT2R44, hT2R46, hT2R50; hT2R3, hT2R4. hT2R7, hT2R8, h.T2R13, hT2R31. HT2R39 and hT2R43); to be stimulated by bitter substances, such as the 25 bitter receptor agonists as recited herein in Tables 1. 3, 6 and 7 of Examples 1 and 2).

Further agonists of hT2Rl , hT2R10, hT2R14, hT2R 16, hT2R38. hT2R44. hT2R46. hT2R50; hT2R3, hT2R4. hT2R7, hT2R8, hT2R13, h.T2R31, hT2R39 and hT2R43 are listed in WO2004/029087. WO 2006/053771. WO 2005/10231 1. WO2008/1 19526, WO 30 2008/ 128730. WO 2008/1 19527, WO2010/099983, WO201 1/012298 and WO201 1/050955, all of which are incorporated herein by reference. Assays for determining "bitter taste receptor activity" of the polypeptides of any one or more of the following bitter taste receptors, hT2Rl, hT2R10. hT2R14. hT2R16. hT2R38, hT2R44. hT2R46, hT2R50; hT2R3, hT2R4, hT2R7, hT2R8₅ hT2R13, hT2R31 , hT2R39 and hT2R43 are described immediately below and in several other passages of this specification and in the specific Examples,

One way of detecting the bitter taste receptor activity of any one or more of the following bitter taste receptors, hT2Rl , h.T2R10, hT2R14. hT2R16, hT2R38, h.T2R44, hT2R46, hT2R50; hT2R3, h.T2R4, h.T2R7, hT2R8, hT2R13, hT2R31, hT2R39 and hT2R43 is by measuring a change in concentration of an intracellular messenger, e.g. Ca²\ IP₃, or cAMP. All functional assays may be performed by samples containing cells expressing the receptor on their surfaces or on isolated cell membrane fractions. Useful cells are described herein. Instead of samples with separate cells or cell membranes, tissues from transgenic animals may be used. Isolated cells or isolated tissues naturally expressing any one or more of the following bitter taste receptors: hT2Rl , hT2R10. hT2R14, hT2R16, hT2R38, hT2R44, hT2R46. hT2R50; hT2R3. hT2R4. hT2R7, hT2R8, hT2R13, hT2R31 , hT2R39 and hT2R43 can be used in the methods described herein.

Alternatively any one or more of the following bitter taste receptors: hT2Rl , hT2R10, hT2R 14, hT2R16, hT2R38. hT2R44. h^'T2R46, hT2R50; hT2R3, hT2R4, hX2R7, hT2R8, hT2R13, hT2R31 , hT2R39 and hT2R43 can be expressed using stable or transient expression systems. The generation of a stable cell line is well known. Alternatively, cells transiently expressing any one or more of the following bitter taste receptors: hT2Rl, hT2R10, hT2R14, hT2R16. hT2R38, hT2R44, hT2R46. hT2R50: hT2R3, hT2R4, hT2R7, hT2R8, hT2R13, hT2R31 , hT2R39 and h.T2R43, can be used, or for example, HEK293T/Gal6-gustducin 44 cells transiently expressing any one or more of the following bitter taste receptors: hT2Rl, hT2R10, hT2R14, hT2R16, h.T2R38, M2R44, hT2R46, hT2R50; h.T2R3, hT2R4. hT2R7, hT2R8, hT2R13, hT2R31, hT2R39 and hT2R43. Suitable eucaryotic cells include, for example, without limitation, mammalian cells, yeast cells, or insect cells (including Sf9), amphibian cells (including melanophore cells), or worm cells including cells of Caenorhabditis (including Caenorhabditis elegans). Suitable mammalian cells include, for example, without limitation, COS cells (including Cos-1 and Cos-7), CHO cells, HEK293 celts, HEK293T ceils, HEK293 T-RexTM cells, or other transfectable eucaryotic cell lines. Suitable bacterial cells include without limitation E. coli. Cells may be transfected with any one or more of the following bitter taste receptors: h.T2R.l, hT2R10₅ hT2R14, hT2R16, h.T2R38, hT2R44, hT2R46, hT2R50; hT2R3. hT2R4, hT2R7, hT2R8, hT2R13, hT2R31 , hT2R39 and h.T2R43 and a G-protein (which links the bitter taste receptor to a phospholipase C signal ^'transduction pathway) transiently or stably, as is well known in the art. An excellent heterologous expression system that employs the chimeric G-protein G alpha 16-gustducin 44 (also known as G.sub.alpha.16 gust(ducin)44, G.sub.alpha.l6gust(ducin)44, Gctl6gust(ducin)44, GaI6gust(d cin)44, Gal6-gustducin 44, or as used herein-below. "G16gust44") which provides for enhanced coupling to taste GPCRs, is described in detail in WO2004/055048 and in US Patent No, 7919236. Alternatively, other chimeric G-proteins based on Gaq-Gustducin described in WO 2004/055048, or other G-Proteins. for example, G 16 or G 15 may also be used. Any one or more of the following bitter taste receptors: hT2Rl , hT2Rl 0, hT2R14, h^'T2Rl 6, hT2R38, hT2R44, hT2R46, hT2R50; h.T2R3, hT2R4, h.T2R7, hT2R8. hT2R13, h^'T2R3 l, hT2R39 and hT2R43 can be expressed in a cell with a G-protein that links the receptor to a signal transduction pathway, for example, the phospholipase C signal transduction pathway, or signal transduction pathways including, for example, the following: adenylate cyclase, guanylate cyclase, phospholipase C. IP3, GTPase/GTP binding, arachinoid acid, cAMP/cGMP, DAG, protein kinase c (PKC), MAP kinase tyrosine kinase, or ERK kinase. Alternatively, any suitable reporter gene may be linked to a bitter taste receptor-activation responsive promoter and used to determine the specific bitter taste receptor activity, as described in more detail hereinunder.

One preferred way of measuring the "bitter taste receptor activity" of any of these receptors, is the ability to release intracellular calcium in a heterologous cell expression system like, for example, (HEK293T/G16gust44) cells that stably expresses a chimeric G-protein consisting of Gal6 and 44 carboxy-terminal amino acids of a-gustducin, in response to bitter tastants, which is dependent on the expression of polypeptides encoded by the polynucleotides of the present invention. The amount of intracellular calcium, released can be monitored by, for example, the in vitro FLIPR assay described herein but also by the measurement of one of a variety of other parameters including, for example, IP₃ or cAMP level. Additional ways of measuring G-protein coupled receptor activity are known, in the art. and comprise without limitation electrophysiological methods, 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 Lemer 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: 31.5-322).

The Compounds of Formula I of the present invention are useful in offsetting the bitter taste o {bitter tastant compounds which include but are not limited to: Acteoside, Adhumulon.e,Adlupu.lone, Aesculetin, Aesculin, L- Alanine, L-aJanyl-L-aianyl-L- Alanine, L-alanyl-L-isoleucyl-Alanine L-, L-valyl-L-val l-Amarogentin, Amaropanin Amaroswerin. Amygdalin, Angustifoline, Antiacetylhumulone, Antiisohumulone, Arginine, L-Arginyl Leucine, Arginyl Leucy Leucine, Arginyl Proline, Asaronaldehyde, Aspartyl Aspartic acid, Asparasaponin I, Atropine, Benzyl beta-D-arabinoside, Benzyl. beta-L-arabinoside, Benzyl beta-D-fructoside, Benzyl beta-D-galactoside, Benzyl alpha-D-glucoside, Benzyl beta-D- glucoside. Benzyl alpha-D-mannoside, Bitter Peptides. Bitter Peptides from Soy Proteins, Butyl alpha-D-glucoside. Butyl beta-D-giueoside, Caffeine, Carnosifioside II, Camosifloside III.. Carnosifioside IV, Catechin. Epicatechin. EpicatecbJn gallate, Chaconine, alpha-Chaconine, beta2-ChIoramphemcol_s Cholic Acid, Ciehoriin, Cohumulone, Colupuione, Cryptochlorogenic Acid, gamma-lactone, Cucurbitacin B, Cucurbitacin D, Cyclo Alanine-glycine, Cyclo Alanine-phenylanaline, Cyclo Alanine- valine, Cyclo(L-arginylglycyl-L-protyl-L-prolyl-L-phenylalanyl-L-isoIeucyl-L-vaIyl)_} Cyclo Asparagine-phenylalanine, Cyclo Glycine-phenylalanine. Cycloheximide Cyclo Lucine-Tryptophan, Cyclopent(b)azepin-8(l H)-one, 7-Methyl-2,3,6,7-Tetrahydro- Cyclopent(b)azepin-8( 1 H)-one, 2 ,3 ,6,7-tetrahydro-7-hyd.rox.y-7-meth.yl.-C clopent-2-en- 1 - one. 2,5-dih.ydroxy-5-methy!-3-(l -piperidinyl)-

Cyclopent-2-en-l -one, 2,5-dihydroxy-5-methyl-3-(l -pyrrolidinyl.) Cyclopent-2-en-l-one, 2,3-di-l -pyrrolidinyl-Cyclopent-2-en-l -one, 5-hydrox y-5-methyl-2.3-di- 1 -piperidinyl- Cyclopent-2-en-l -one, 5-hydroxy-5-methyl-2_J3-di-l -pyrrolidinyl-Cyclopent-2-en-l-one, 5- methyl-2,3-di- 1 -pyrroIidinyl-Cyclopent-2-en- 1 -one. 5-methylene-2,3-di-l -pyrrolidinyl- CycIopent-2-en-l -one, 3-methyl-2-(l -pyrrolidinyl)-Cyclo Phenyalanine-aspartic acid, Cyclo Proline-alanine, Cycle Proline-asparagine, Cyclo Proline-glycine, Cyclo Proline- isolucine. Cyclo Proline-leucine, Cyclo Proline-methionine, Cyclo Proline-phenylalanine, Cyclo Proline-proline, Cyclo Proline-valine, Cyclo Valine-phenylalanine, Cynaratriol, Cynaropicrin, Cynaropicrin, Daidzein, Daidzin Denatonium benzoate, Denatonium saccharide, Dhurrin, Dihydroxybenzoic Acid, 2,3-Dihydroxybenzoic Acid, 2,4-Ethyl b-L- arabinoside, Ethyl alpha-D-Glucoside. Ethyl beta-D-Glucoside, Eustomoroside, Eustomoside, Gallic Acid, Epigallocatechin, Epigallocatechin gallate. Gaudichaudioside F, Gelidoside, Genistein, Genistin, Gentiopicroside, Gentistic Acid, Gentomoside. Geshoidin, 6 -O-beta-D-GlucosyIgentiopicroside, ucozaluzanin C, Glutamyl Aspartic Acid, Glutamyl Glutamic Acid, Glycyl Leucine, Goitrin, Gramme, Grosshemin, Haematoxylin Tetramethyl Ether Helicin, Heptadeca- 16-ene, 1 -Acetoxy-2,4-Dihydtoxy -Heptadeca- 16-ene, 1 ,2,4- Trihydroxy-Histidine, L-Hulupone, Humulinone, Humulone. Hydroxybenzoic Acid, 4- Hymenoside A. Hymenoside B. Hymenoside C, Hymenoside D, Hymenoside E, Hymenoside F, Isohumulone, cis-Isohumulone, trans-Isoleucine, L-Isolupanine, Isosparteine, beta-Isosparteine, 10,17-Dioxo-beta-Isosparteine, 10-oxo-beta-Lactucin, L- Leucine, L-alanyl-L-alanyI-L-Leucine,N-[(2R)-6-amino-2-[(4S)-2,5-dioxo-4- (phenylmethyl)- 1 -imidazolidinylj-1 -oxohexyl]-L-leucyl-L-methionyl-N-methyl-L- phenylalanyl-, (4-l)-lactam, L-Leucine, glycyl-L-alanyl-Leucine, L-L-Leucine, N-(N2-L- leucyl -L-glutaminyl)-L-Leucine, N-(N-L-leucyl-L-a-glutamyl)-L- Leucine, -[N2-[N2- [N- ( l -L-leucyl-L-proly )-L-phenylalanyl]-L-asparaginyl]-L-glutaminyl]-L-Leucine, N-[N2-[N- rN-(l-L-leucyl-L-prolyl)-L-phenylalanyl]-L-seryl]-L-glutaminyl]-L-Leucine, L-leucyl-L- valyl-Leucy Leucine, Leucyl Phenylalanine, Limonin, Limoninmonolactone, Linamarin, Lotaustralin, Lupine, Lupanine, 13-Hydroxy- Lupanine, 7-hydroxy-Lupinine, Epilupinine Lupoxes B. Lupoxes C, Lupulone, Luputrione, Mellein, 6-Methoxy-Methionine, L- Methyl alpha-L-arabinoside, Methyl beta-L-arabinoside, Methyl beta-D-Glucoside, Methyl alpha- D-Glucoside 2,3-Di-isoleucine, Methyl alpha-D-Glucoside 2,3-Di-leucine. Methyl alpha-D- Glucoside 2,3-Di-L-phenyialanine, Methyl alpha-D-Glucoside 2.3-Di-threonine, Methyl alpha-D-GIucoside 2,3 -Di -tyrosine. Methyl a-D-mannoside, Methyl beta-L- xylopyranoside, Methyl alpha-D-xyloside, Naringin, Neochlorogenic Acid, gamma- Lactone, Neohesperidin. Nuezhenide, Oleonuezhenide. Oleuropein. Olivieroside A, Olivieroside B. Olivieroside C, Perrottetin H, Phenylalanine, L-Phenyl alpha-D-galactoside, Phenyl alpha-D-glucosidc, Phenyl beta-D-glucoside. Phenylthiourea, Phlomisoside II, Piperidine-2-carboxylic acid, 4-[(2-carboxy-2-hydroxyethyl)thio]-Piperidinecarboxylic acid-2. 4-[(2-carboxy-2-hydroxyethyl)thio]-Prehumulone, Prelupulone. Propyl beta-D- fructoside. Propyl alpha-D-glucoside. Propyl beta-D-glucoside, Protocatechuic Acid, Prunasin, Pulcherrimine, Quinidine, Quinine, Quinolizinium-7-olate. Ranitidine, Rebaudioside C, Salicin. Salidroside, Scabraside, Scandenoside R5, Sclareolide. Scopolin, Septemfidoside, Seryl Lysyl Glycyl Leucine, Sinapine, Solanine, alpha-Sparteine, Sparteine, 1 7-oxo-Stevisalioside A, Strychnine, Suavioside CI , Suavioside D2, Suavioside F. Sucrose Octaacetate, S eroside, Swertiamarin, Swertiapunimarin, TaxiphyUin, TFI (Furostan, beta-D-galactopyranoside), Theaflavin. Theaflavin Gallate A, Theaflavin Gal late B. Toraatidine, Tomatine, alpha-Tricyclodehydroisohumulone, Trifloroside, Trihydroxybenzoic Acid, 2,4,6-Tryptophan. L-Uracil, 6-propyl-2-thio-L- Valine, L- arginylglycy!-L-proSyl-L-prolyl-L-phenylalanyl-L-isoleucyl- (BPIa) Valine. and L- Yohimbine.

In particular, the Compounds of Formula 1 of the present invention are useful, e.g., in methods as hereinbefore described, to offset the bitter taste of common food ingredients such as potassium, chloride, ammonium chloride, sodium chloride, magnesium chloride, halide salts, naringin, caffeine, urea, magnesium sulfate, saccharin, acetosulfames, aspirin, potassium benzoate, potassium bicarbonate, potassium carbonate, potassium nitrate, potassium nitrite, potassium sulfate, potassium sulfite, potassium glutamate, food preservatives in their physiologically acceptable salts, antibiotics, unsweetened chocolate, cocoa beans, yogurt, preservatives, flavor enhancers, dietary supplements, supplemental amino acids, gelling agents, pH control agents, nutrients, processing aids, bodying agents, dispersing agents, stabilizers, colorings, coloring diluents, anticaking agents, antimicrobial agents, formulation aids, leavening agents, surface active agents, anticaking agents, nutrient supplements, alkali, acids, sequestrants. denuding agents, general purpose buffers, thickeners, cooked out juice retention agents, color fixatives in meat and meat products, colorfixatives in poultry and poultry products, dough conditioners, maturing agents, yeast foods, mold retardants, emuisifiers, texturizers, binders, water correctives, miscellaneous and general purpose food additives, tableting aids, lye peeling agents, washing water agents, oxidizers, antioxidants, enzymes, extenders, fungicides, cake mixes, coffee, tea, dry mixes, non-dairy creamers, salts, animal glue adjuvant, cheese, nuts, meat and meat products, poultry and poultry product, pork and pork products, fish and fish products, vegetable and vegetable products, fruit and fruit products, smoked products such as meat, cheese fish, poultry, and vegetables, whipping agents, masticatory substances in chewing gums, dough strengthened, animal feed, poultry feed, fish feed, pork feed, defoaming agents, juices, liquors, substances or drinks containing alcohol, beverages including but not limited to alcoholic beverages and non-alcoholic carbonated and/or non-carbonated soft drinks, whipped toppings, bulking agents used in eatables including but not limited to starches, corn solids, polysaccharides and other polymeric carbohydrates, icings, as well as potassium-containing or metal-containing substances with undesirable tastes, which can be masked using a Compound of the Invention.

For example, the invention contemplates one or more comestibles having a bitter ingredient together with a Compound of the Invention in an amount sufficient to reduce or eliminate the bitter taste of the bitter ingredient. For example, in one embodiment the comestibles are breads, biscuits, pancakes, cakes, pretzels, snack foods, baked goods etc, prepared using, for example, potassium bicarbonate or potassium carbonate in place of the sodium salts as leavening agents, comprising a bitter-reducing amount of the Compound of the Invention, e.g. any of the specific compounds disclosed above. For such applications, the Compound of the Invention can be typically present in an amount ranging from about 0.001% to about 1% by weight of the food product, typically l %-50% by weight of a supplement to be added to a food product. In another embodiment, there is provided a method for the production of a food, a food precursor material or additive employed in the production of a foodstuff comprising the step of admixing an antagonist selected from the group consisting of: (i) a compound of Formula 1 which is an isolated compound of Formula 1 in free or orally acceptable base addition salt form; (ii) an antagonist identified in a screening assay as described herein; or (iii) an antagonist structurally related to the antagonists of (i) or (ii); with the comestible product, the food, the food precursor material or the additive employed in the production of the foodstuff. In a further embodiment, there is provided a method for the production of a nutraceutical or a pharmaceutical composition, comprising the step of admixing an antagonist selected from the group consisting of: (i) a Compound of Formula 1 which is an isolated Compound of Formula 1 in free or orally acceptable base addition salt form; (ii) an antagonist identified in a screening assay as described herein; or (Hi) an antagonist structurally related to the antagonists of (i) or (ii); with an active agent and optionally with a pharmaceutically acceptable carrier and/or adjuvant.

In a further embodiment, there is provided the method of the previous paragraph further comprising the step of formulating the pharmaceutical composition into a pharmaceutically acceptable form.

In an even further embodiment, there is provided a wherein the bitter receptor antagonist

(eg the compound of Formula 1 or any one of compounds 1-7) is orally administered prior to oral administratioB of a substance comprising a T2R bitter receptor agonist. The T2R bitter receptor agonists can include but is not limited to any one of the 58 natural bitter compounds disclosed in Meyerhof et at (2010) Chem Senses 35: 157-170. These compounds include limonin, Naringm, Neofaesperidine, Coumarin, Caffeine, Brucine, Thiamine, Riboflavin, Quinine. Simgrin, Campher, Arbutin, Absinthin and others from Table 1 of that publication all of which are incorporated herein by reference. Other natural compounds of interest include but are not limited to sucralose, aceK, saccharin, stevioside, Reb A, Sesquiterpene lactones from lettuce such as laetucopicrin, Amygdalin D found in almonds. Menthol and Cucurbitacins from cucumber and others disclosed in paragraph 1.1.33 above.

In a further embodiment, there is provided a a food, a food precursor material or additive employed in the production of a foodstuff producible according to any of the methods for the production of a food, a food precursor material or additive employed in the production of a foodstuff.

In a further embodiment, there is provided a nutraceutical or a pharmaceutical composition producible by any of the methods for the production of a nutraceutical or a pharmaceutical composition and wherein the nutraceutical or a pharmaceutical composition comprises at least one nutraceutically or pharmaceutically active agent and optionally one or more pharmaceutically acceptable carrier and/or adjuvant.

The Compounds of Formula 1 of the present invention can be used alleviate or reduce the bitter taste of compositions, as part of an ingestible composition. As used herein, an "ingestible composition" includes any substance intended for oral consumption either alone or together with another substance.

The ingestible (or comestible) composition includes both "food or beverage products" and "non-edible products". By "food or beverage products", it is meant any edible product intended for consumption by humans or animals, including solids, semi-solids, or liquids (e.g.. beverages).

The term, "non-edible products includes supplements, nutraceuticals, functional food products (e.g.. any fresh or processed food claimed to have a health-promoting and/or disease-preventing properties beyond the basic nutritional function of supplying nutrients), pharmaceutical and over the counter medications, oral care products such as dentifrices and mouthwashes, cosmetic products such as lip balms and other personal care products. The ingestible (or comestible) composition also includes a pharmaceutical, medicinal or alternatively in a formulation, e.g., a pharmaceutical or medicinal formulation or a food or beverage product or formulation. The Compounds of the present invention can also be provided, individually or in combination, with any ingestible composition known or later discovered.

Typical examples of ingestible or comestible products, include, but not limited to, all food products, food additives, nutraceuticals. pharmaceuticals and any product placed in the mouth including chewing gum, oral, care products, and oral hygiene products including but not limited to. cereal products, rice products, tapioca products, sago products, baker^'s products, biscuit products, pastry products, bread products, confectionery products, dessert products, gums, chewing, gums, flavored or flavor-coated straws, flavor or flavor-coated food/beverage containers, chocolates, ices, honey products, treacle products, yeast products, baking-powder, salt and spice products, savoury products, mustard products, vinegar products, sauces (condiments), tobacco products, cigars, cigarettes, processed foods, cooked fruits and vegetable products, meat and meat products, jellies, jams, fruit sauces, egg products, milk and dairy products, yoghurts, cheese products, butter and butter substitute products, milk substitute products, soy products, edible oils and fat products, medicaments, beverages, carbonated beverages, alcoholic drinks such as beers, wines and spirits, non-alcoholic drinks such as soft drinks, mineral and aerated waters, fruit drinks, fruit juices, coffee, artificial coffee, tea, cocoa, including forms requiring reconstitution including, without limitation, beverage powder, milk based beverage powder, sugar-free beverage powder, beverage syrup, beverage concentrate, instant coffee, instant tea, instant cocoa, and coffee whitener, food extracts, plant extracts, meat extracts, condiments, gelatins, pharmaceutical and non-pharmaceutical gums, tablets, lozenges, drops, emulsions, elixirs, syrups and other preparations for making beverages, and combinations thereof.

Oral care products, by which is meant any product applied to or taken into the oral cavity for the purposes of cleaning, freshening, healing, deodorising the cavity or any part thereof, may include, but are not limited to. toothpastes, tooth gels, tooth powders, tooth whitening products, mouthwashes, lozenges, dental floss, toothpicks, anti-plaque and anti-gingivitis compositions, throat lozenges, throat drops, inflammatory compositions, compositions for treatment of nasal symptoms, cold symptoms and upper gastrointestinal tract distress. compositions for cold relief, for alleviating discomfort of hot flash, and gargle compositions.

In a further embodiment, the invention provides a product and a method substantially as described herein and with reference to the accompanying Tables and Figures. The examples provided in the detailed description are merely examples, which should not be used to limit the scope of the claims in any claim construction or interpretation.

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: (IUPA.C Recommendations)", Leuenberger, H.G.W, Nagel. B. and Kolbl. I I. eds. (1 95), Helvetica Chimica Acta, CH-40H⁾ Basel, Switzerland), 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 impl 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 integer or step. The term "comprising" also means "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y. It must be noted also that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. 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, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety.

EXAMPLES

The invention is described only by way of Example in which reference is made to the following Figures;

Figure 1 shows the chemical structure of Compounds of Formula 1 ; and the chemical structure of Compound 1 ((-)- hardwickiic acid) of Formula 1 and Compound 2 ((-)-epi- hardwickiic acid) of Formula 1 ; Figure 2 shows the chemical structure of Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7 of Formula 1 ; and the Compound of Formula l a; and

Figure 3 shows the inhibition by (-)-hardwickiic acid (15 μΜ) of the response of multiple hTAS2Rs (hTAS2Rl , hTAS2R10. hTAS2R14, hTAS2R16, hTAS2R38, hTAS2R44, hTAS2R46 and hTAS2R50) to the bitter agonists listed in Table 1. The data in. Figure 3 is expressed as a percent of the receptor response to agonist alone. The hTAS2R sequences and the h.TAS2R receptor/agonist combination used to generate the data in Figure 3 are shown in Table 1 below.

Table 1 ; hTAS2R sequence and Agonist (Cas no) for Example 1

Figure hTAS2R TAS2R TAS2R Agonist Agonist CAS number polypeptide nucleotide No

sequence sequence

1 hTASRl SEQ ID No. 1 SEQ ID No. 2 2,38 m.M Cas No 56-75-

AF227129.1 AF227129.1 Chloramphenicol 7

GI.-7262604 GI: 7262604

2 hTASRIO SEQ ID No. 1.5 SEQ ID No. 16 200 Cas No 1421 - AF227136, 1 AF227136.1 pMStrychnine 86-9

GI:7262618 GI-.72626 I 8 HC!

3 hTASRl 4 SEQ ID No. 19 SEQ ID No, 20 0.9 μΜ Cas No 61-68-7

AF227138.1 AF227 38.1 Mefenamic acid

Gl: 7262622 GI:7262622

4 hTASRl 6 SEQ ID No. 21 SEQ ID No. 22 100 μΜ Cas No 1464- AF227139.1 AF227139.1 Phenyl β-D- 44-4

GI: 7262624 GI:7262624 glucopyranoside

5 hTASR38 SEQ ID No. 29 SEQ ID No, 30 1 uM Cas No 51 -52-5

AF49423 1..1 AF49423 1. I 6-n-Propyl-2-

GI:20336 18 GI-.20336518 thiouracil

6 h ASR44 SEQ ID No. 39 SEQ ID No. 40 0.4 μΜ Cas No 13-67- AF494228.1 AF494228.1 Aristolochic 7

GI:20336512 GI:20336512 Acid

7 hTASR46 SEQ ID No. 43 SEQ ID No. 44 1.2 uM Cas No 1421-

AF494227.1 AF494227.1 Strychnine HC1 86-9

Gl:20336510 GI:20336510

8 hTASRSO SEQ ID No. 51 SEQ ID No. 52 10 μΜ Cas No 5508-

AF494235.1 AF494235.1. Andrographolide 58-7

GI:20336526 GI:20336526

9 host cells

(negative

control) Table 2: Human TAS2R Sequence ID Nos. identified by the Genbank Database

Accession No and the Genbank Version No. and NCBI Reference Sequence No.

hTAS2R Genbank Genbank Version NCBI hTAS2R SEQ ID hTAS2R SEQ ID

Accession No Reference No, No. No

(polypeptide (nucleotide sequence) sequence)

TAS2 I AF2271.29 AF227129.1 NM_01 599 SEQ ID No. ! SEQ ID No. 2

01:7262604

TAS2R3 AF227130 AF227130.1 NM_016943 SF.Q ID No. 3 SEQ ID No. 4

GE7262606

TA52K4 AF22 131 AF227131 .1 NM .016944 SEQ lD o, 5 SEQ ID No. 6

GI:7262608

TAS2R5 AF227132 AF22 132.1 1 N JH 8980 SEQ ID No. 7 SEQ ID No. 8

GI:7262610

T.4S2R7 AF227 I 33 AF227133.1 NMJJ2391 SEQ ID No. 9 SEQ ID No. 10

Gt:7262612

TAS2R8 AF227134 AF227134.1 N 0239I SEQ ID No. 1 1 SEQ lD No. 12

01:7262614

TAS2R9 AF227135 AF227135.1 NM_023 17 SEQ ID No. 13 SEQ ID No. 14

01:7262616

TAS2R10 AF2271.36 AF2271.36.1 NMJS2392I SEQ ID No. 15 SEQ lD No. 16

01:7262618

TAS2R13 AF227137 AF227137.1 NN1 023920 SEQ ID No. 17 SEQ lD o. 18

61:7262620

TAS2R14 AF22 138 AF22 138.1 N JK3922 SEQ lD o. 19 SEQ ID No, 20

01:7262622

TAS2R16 AF227139 AF227139.1 NM_016945 SEQ ID No. 21 SEQ ID No. 22

01:7262624

TAS2R20 AF494236 AF494236. I NMJ 76889 SEQ ID No. 23 SEQ ID No. 24

01:20336528

TAS2R30 AF494233 AF494233.1 M_00i09764 SEQ ID No. 25 SEQ lD No. 26

GV.20336522 3

TAS2R3I AF494228 AF494228.1 NMJ76885 SEQ ID No. 27 SEQ ID No. 28

GE203365 I 2

TAS2R38 AF494231 AF494231.1 NMJ 76817 SEQ ID No, 29 SEQ ID No. 30

GE20336518

TAS2R39 AF494230 AF494230.1 NMJ7638I SEQ lD No. 31 SEQ ID No. 32

GI:203365 I 6

TAS2R 0 AF494229 AF494229.1 NM_176882 SEQ ID No. 33 SEQ ID No. 34

GE20336514

TAS2R4! AF494232 AF494232.i NM_1 6883 SEQ ID No. 35 SEQ ID No. 36

GJ:20336520

TAS2R43 AF494237 AF494237.1 NM_176884 SEQ ID No. 37 SEQ ID No. 38

GE20336530

TAS2R 4 AF49422S AF494228.1 NMJ 76885 SEQ ID No. 39 SEQ ID No. 40

Gl:20336S12 TAS2R45 AF494226 AF494266J N _ 176886 SEQ ID No. 41 SEQ' ID No, 42

01:33321829

TAS2R46 AF49 227 AF494227.1 N _ 176887 SEQ !D No, 43 SEQ ID No. 44

GI:20336510

TAS2R47 AF494233 AF494233.1 NM_Q0109764 SEQ ID No, 45 SEQ ID No, 46

GI:20336522 3

TA52R48 AF494234 AF494234.1 NM_176888 SEQ ID No. 47 SEQ ID No. 48

Oi;20336S24

TAS2R49 AF494236 AF494236.1 NM_ 176889 SEQ ID No. 49 SEQ ID No, 50

GI:20336528

TAS2R50 AF494235 AF494235.1 NM_l 76890 SEQ ID No. 51 SEQ ID No. 52

01:20336526

TA82R60 AY 1 14094 AY1 14094, 1 NMJ77437 SEQ ID No. 53 SEQ ID No, 54

01:28603637

For the avoidance of doubt:

SEQ ID No, 1 is the polypeptide sequence and SEQ ID No. 2 is the nucleotide sequence for human TAS2R1 which is disclosed in Genbank Accesssion Number AF227129 and Genbank Version AF227129.1 , GI: 7262604.

SEQ ID No. 3 is the polypeptide sequence and SEQ ID No. 4 is the nucleotide sequence for human TAS2R3 which is disclosed in Genbank Accesssion Number AF227130 and Genbank Version AF227130.1, GI:7262606.

SEQ ID No. 5 is the polypeptide sequence and SEQ ID No. 6 is the nucleotide sequence for human. TAS2R4 which is disclosed in Genbank Accesssion Number AF227131 and Genbank Version AF227131.1 , GE7262608. SEQ ID No. 7 is the polypeptide sequence and SEQ ID No. 8 is the nucleotide sequence for human TAS2R5 which is disclosed in Genbank Accesssion Number AF22 132 and Genbank Version AF227132.1, GI;7262610.

SEQ ID No. 9 is the polypeptide sequence and SEQ ID No. 10 is the nucleotide sequence for human TAS2R7 which is disclosed in Genbank Accesssion Number AF227133 and Genbank Version AF227133.1. GI:7262612.

SEQ ID No. 1 1 is the polypeptide sequence and SEQ ID No. 12 is the nucleotide sequence for human. TAS2R8 which is disclosed in Genbank Accesssion Number AF2271.34 and Genbank Version. AF227134.1, 01:7262614.

SEQ ID No. 13 is the polypeptide sequence and SEQ ID No. 14 is the nucleotide sequence for human TA.S2R9 which is disclosed in Genbank Accesssion Number AF.227135 and Genbank Version AF2271.35.1 , 01:7262616.

SEQ ID No. 1 5 is the polypeptide sequence and SEQ ID No. 16 is the nucleotide sequence for human TAS2R10 which is disclosed in Genbank Accesssion Number AF227136 and Genbank Version AF227136..L GI:7262618. SEQ ID No, 17 is the polypeptide sequence and SEQ ID No, 18 is the nucleotide sequence for human TAS2R 13 which is disclosed in Genbank Accesssion Number AF227137 and

Genbank Version AF227137.1 , GI: 7262620.

SEQ ID No. 19 is the polypeptide sequence and SEQ ID No. 20 is the nucleotide sequence for human TAS2R14 which is disclosed in Genbank Accesssion Number AF227138 and Genbank Version AF227138. 1. GI:7262622. SEQ ID No. 21 is the polypeptide sequence and SEQ ID No. 22 is the nucleotide sequence for human TAS2R16 which is disclosed in Genbank Accesssion Number AF227139 and Genbank Version AF22713 . L GI:7262624.

SEQ ID No. 23 is the polypeptide sequence and SEQ ID No. 24 is the nucleotide sequence for human TAS2R20 which is disclosed in Genbank Accesssion Number AF494236 and Genbank Version AF494236.1, GF20336528.

SEQ ID No. 25 is the polypeptide sequence and SEQ ID No. 26 is the nucleotide sequence for human TAS2R30 which is disclosed in Genbank Accesssion Number AF494233 and AF494233.1. GI:20336522.

SEQ ID No. 27 is the polypeptide sequence and SEQ ID No. 28 is the nucleotide sequence for human TAS2R31 which is disclosed in Genbank Accesssion Number AF4 4228 and Genbank Version AF494228.L GE20336512.

SEQ ID No. 29 is the polypeptide sequence and SEQ ID No. 30 is the nucleotide sequence for human TAS2R38 which is disclosed in Genbank Accesssion Number AF494231 and Genbank Version. AF494231.1 , GF20336518. SEQ ID No. 31 is the polypeptide sequence and SEQ ID No. 32 is the nucleotide sequence for human TAS2R39 which is disclosed in Genbank Accesssion Number AF494230 and Genbank Version AF494230.1 , GI:20336516. SEQ ID No. 33 is the polypeptide sequence and SEQ ID No. 34 is the nucleotide sequence for human TAS2R40 which is disclosed in Genbank. Accesssion Number AF494229 and Genbank Version AF494229.1 , GI:20336514. SEQ ID No. 35 is the polypeptide sequence and SEQ ID No. 36 is the nucleotide sequence for human TAS2R41 which is disclosed in Genbank Accesssion Number AF494232 and Genbank. Version AF494232.1 , GI:20336520.

SEQ ID No. 37 is the polypeptide sequence and SEQ ID No. 38 is the nucleotide sequence for human TAS2R43 which is disclosed in Genbank Accesssion Number AF494237 and Genbank Version AF494237. L GI:20336530.

SEQ ID No. 39 is the polypeptide sequence and SEQ ID No. 40 is the nucleotide sequence for human TAS2R.44 which is disclosed in Genbank Accesssion Number AF494228 and Genbank Version AF494228.1 , GI:20336512.

SEQ ID No. 41 is the polypeptide sequence and SEQ ID No. 42 is the nucleotide sequence for human TAS2R45 which is disclosed in Genbank Accesssion Number AF494266 and Genbank Version AF494266.1 , GE33321829.

SEQ ID No. 4 is the polypeptide sequence and SEQ ID No. 44 is the nucleotide sequence for human TAS2R46 which is disclosed in Genbank Accesssion Number AF494227 and Genbank Version AF494227. L GI:20336510. SEQ ID No. 45 is the polypeptide sequence and SEQ ID No. 46 is the nucleotide sequence for human TAS2R47 which is disclosed in Genbank Accesssion Number AF494233 and. Genbank Version AF494233.1. GE20336522.

SEQ ID No. 47 is the polypeptide sequence and SEQ ID No. 48 is the nucleotide sequence for human TAS2R48 which is disclosed in Genbank. Accesssion Number AF4 4234 and Genbank Version AF494234.1. GI:20336524. SEQ ID No. 49 is the polypeptide sequence and SEQ ID No. 50 is the nucleotide sequence for human TAS2R49 which is disclosed in Genbank. Accesssion Number AF494236 and Genbank Version. AF494236.1 , GI:20336528. SEQ ID No. 51 is the polypeptide sequence and SEQ ID No. 52 is the nucleotide sequence for human TAS2R50 which is disclosed in Genbank Accesssion Number AP494235 and Genbank Version AF494235.L GI:20336526.

SEQ ID No. 53 is the polypeptide sequence and. SEQ ID No. 54 is the nucleotide sequence for human TAS2R60 which, is disclosed in Genbank Accesssion Number AYl 14094 and Genbank Version AYl 14094.1, GI:28603637.

For all of the above described hTAS2Rs which can be employed in a process for isolating binding compounds and for identifying hTAS2R antagonists, with the exception of hTAS2R40, single nucleotide polymorphisms (SNPs) are known. Seventy nine of these are listed in Table 1 on pages 17-1.9 of WO 2004/029087, sixty one of which result in an. amino acid change. Polynucleotide or polypeptides that differ from those disclosed in SEQ ID Nos 1-54 above or SEQ ID Nos 55-74 (provided, below) by, for example, the nucleotide and amino acid changes as indicated in Table 1 on pages 17-19 of WO 2004/029087 can similarly be employed for the screening processes of the present invention.

TAS2R1 (SEQ IP No.l)

MLESHLIIYFLLAVIQFLLGIFTNGIIWVNGIDLIKH MAPL D LLLSC LAVS If LQ LFI FYV 1 1 VI FFI E FI M CS A N C Al LLFI N E LE LW LATWLG VF

YCAKVASVRHPLFIWL RISKLVPWMILGSLLYVSMICVFHS YAGFMVPYFLRKFF SQNATIQKEDTLAIQIFSFVAEFSVPLLIFLFAVLLLIFSLGRHTRQMRNTVAGSRVP

GRGAPISALLS1LSFULYFSHCMIKVFLSSL FHIR .F1 FLFFILVIGIYPSGHSU

LILGNPKLKQNAKKFLLHSKCCQ

TASZBIP fgEQJtPJteJjji)

LRWEGIFIFWVSESVFGVLGNGFIGLVNCIDCA N LSTIG FILTGLAISRIFLIWI IITDGFIQIFSPNIYASGNLIEYISYFWVIGNQSSMWFATSL SIFYFL IANFSNYIFLWLKSRTN VLPF IVFLLISSLLNFAYIAKILNDYKTKNDT VWDLNMY SEYFIKQI LLN LGVIFFFTLSUTCIFLIISLWRHNRQMQSNVTGLRDSN TEAHVKA VLISFIILFILYFIGMAIEISCFTVREN LLLMFGMTTTAIYPWGHSFI LILGNSKLKQASLRVLQQLKCCEKRKN LRVT

GGVIKSIFTFVLIVEFIIGNLGNSFIALVNCIDWVKGRKISSV DR1 LTALAISRIS LVWLIFGS WCVSVFFPALFATEKMFRM LT I TVI NHFSVWLATG LGTFYFL IANFSNSIFLYLKWRVKKVVLVLLLVTSVFLFLNIALINIHINASINGYR R TCSSDSSNFTRFSSLIVLTSTVFIFIPFTLSLAMFLLLIFSMWKHRK MQHTVKI SGDAST AHRGVKSVITFFLLYAIFSLSFFISVWT5ERLEENLIILSQV GMAYPSCH SCVLILG N KLRQAS LSVLLWLRYM f KDGE PSG H KEFRESS

TAS2R1C fSEQ IP No.Zl)

M IPIQ LTV FFHIIYVLESLTIIVQSS LI VAV LG RE W LQ V R R LM P

VD IUSLGISRFCLQWASMLNNFCSYFNLNYVLCNLTITWEFFNILTFWLNSLLTVF YCI VSSFTHHIFLWLRWRILRLFPWILLGSLMITCVTIIPSAIGNYIQIQLLTMEHL PRNSTVTDKLENFHQYQFQAHTVALVIPnLFLASTIFLMASLT QIQHHSTGHCNPS M KA RFTA LRS LAV LFI VFTS YFLTI L1T1 I GTLFD KRC LW VW EAFVY A FI LM H STS L MLSSPTLKRILKGKC

TAS2R38 fSEO IP No.29¾

M LTLTRI RTVS YE V RSTFLFISVLE FAVG FLTN A FVFLVN F W DV V RQALSN5DCVLLCLSISRLFLHGLLFLSAIQLTHFQ LSEPLNHSYQAII LWMIA NQANLWLAACLSLLYCSKLIRFSHTFLICLASWVSRKISQMLLGIILCSCICTVLCVW CFFSRPHFTVTTVLFMNNNTRLNWQN DLNLFYSFLFCYLWSVPPFLLFLVSSG LTV SLGRHMRT KVYTRNSRDPSLEAHI ALKSLVSFFCFFVISSCVAFISVPLULWRDK IGVMVCVGIMAACPSGHAAILISGNA LRRAVMTILLWAQSSL VRADH ADSRTLC TAS2R44 fSEO IP No.39)

TTFIPIIFSSWWLFVIGNFANGFIALVNSIERV RQKISFA

DQILTALAVSRVGLLWVLLLNWYSTVFNPAFYSVEVRTTAYNVWAVTGHFSNWLATSL SIFYLLKIANFS N LIFLH LK VKSVI LVM LLGPLLFLACQLFVINMKEIVRTKEYEG NMT IKLRSAVYLSDATVTTLGNLVPFTLTLLCFLLLICSLC HLK QLHG GSQD PST K V H I KALQTV1 F FLLLC AVYFLS 1 M I S VWS FGS LE N P V FM FC KAI R FSYPS I H P FILIWGNK L QTFLSVLRQVRYWV GE PSSP

TAS2R46 fSEQ IP No.43)

MITFLPIIFSILIWTFV1GNFANGFIALVNSIEWFKRQ ISFA

DQILTALAVSRVGLL VLVLNWYATELNPAFNSIEVRITAYNVWAVI HFSNWLATSL SIFYLL IANFSNLIFLHLKRRVKSVVLVILLGPLLFLVCHLFVINMNQIIWTKEYEG N TW IKLRSAMYLSNTTVTILANLVPFTLTLISFLLLICSLCKHL KMQLHGKGSQD PS KVHI ALQTVTSFLLLCAIYFLSIIMSVWSFESLENKPVFMFCEAIAFSYPSTHP FILIWGN KKLKQTFLSVLWQM RY

TAS2R SO fSEQIP No.SH

MITFLYIFFSIII VLFVLGNFANGFIALVN DWVKRKKISSA

DQILTALAVSRIGLLWALLLNWYLTVLNPAFYSVELRITSYNAWVVTNHFSMWLAANL SIFYLLKIANFSNLLFLHLKRRVRSVILVILLGTLIFLVCHLLVANMDESMWAEEYEG N TGKMKLRNTVHLSYLTVTTLWSFIPFTLSLISFLMLICSLY HLK MQLHGEGSQD LSTKVHIKALQTLISFLLLCAIFFLFLIVSVWSPRRLRNDPWMVSKAVGNIYLAFDS FILIWRT LKHTFLLILCQIRC Example I fIaMss.6 and 7); human TAS2Rs oolvaeo¾jde.seg enees

TAS2R1 fSEO IP No. SSI

LE S H LI IY FLLAVI Q F LLG I FTN G I IVVV N G I D LI K H RK M AP L D L LIS CLAVS R I F LQ LFI FYV N V I V I F F I E F I M CSA N CA I LLF I N E LE LW IATW LGVFYCA K VASV R H P LF I W L M RI S LV PW M I LG S LLYVS I CV F H S KYAG F V PY FLR F FSQ N ATI Q E DTLAI Q I FS FVA E FSV P L LI F LFAV L L LI FS LG RHTRQ R N TVAGS RV PG RG A PI SA LLS I LS F LI LYFS H C I V F LS S L K FH I R R FI F LF F I LVI GIY PSG H S LI LI LG N P K LKQ NA K KF L LH S KCCQ

TAS2R.3 fSEQ ID No, 56)

M M G LTE GVF LI LS GTQ FT LG I LV N C F I E LV N G S S W F KT R M S LS D F I ITT LA L LR I I L LC I I LT D S F LI E FS P N T H D S G I I M Q I I DV SWT FT N H LS I W LATC LGV LY C L K I AS FS H PT F LW L W RVS RV M VW M L LG ALLLS C G STAS LI N E F LY S V FRG I EATR N VTE H F R K R S EYY LI H V LGTLWY L PP LI VS LASYS LLI FS LG R H TR Q M LQ N GTS S R D PTTE AH RAI R I I LS F F FLF LLYFLA F LI AS FG N FLP T M A I GEVM T FY PAG H S FI LI LG N S KL QTFVV M LRC ESG H L PGS KG PIFS

TASZR4(SEQ,IP No,.57)

M I R LFY FS AI I ASV I LN FVG I I M N LFITVV N C KTWV KS H R I S SS D RI L FS LG IT R F L M LG LF LV N TI Y FVSS NT E RS VY LS AFFV LC F F LD S S SVW FVTL L N I LYCV K IT N FQ H SV F L I L K R N I S P K I P R L L LA CV L ISA FTTC LY ITLS QAS P FP E LVTTR N N TS F N I S E G I LS LVVS LV IS S S LQ FI I NVTSAS LLI H S LR R H I Q K M Q K N AT G FW N PQT EA HVGA M K LM VYFLI LYI PYSVATLVQYLP FYAG M D M GTK S ICLIFATLYS PG H S V L I I I T H P L KTTA I LC F K

TAS2R5 $EQ ΪΡ,Νο..5^β)

IS A G L G L L M LVAVV E F L I G LI G N G S LVVWS FR EW I R K F N W S SY N II I LG LA G C R F L LQW L I I L D LS LF P L FQS S RW LRYLS I FWVLVS QAS LW FATF LSV FYC K I TT FD R PAY LW LK Q RAY N LS LWC L LG Y FI I N L L LTVQI G LT FYH P PQ G N S S I RY P F E S W QY LYA F Q LN S G SYLP LVV F LVSS G M LI VS LYT H H M KV H SAG R R DVRA A H 1 TA L KS LG C FL L LH LVYI M A S P F5 ITS KTY P P D LTSV FI W ETL M AAY P S LH S LI LI M G I PRVK QTCQ I LW KTVCAR RCWG P

TAS2R7 (SEQ ID No.59)

A D VQTT L L F LAVG E FSVGI LG N A FI G LV N C D WV R I AS I D LI LTS LA I S R I C L LCVI LL D C F I LV LY P DVYATG E RI I D F FWTLT N H LSI W FATC LS IYY F F KI G N F FH P L F LW M W R I D RV I S WI L LG CVV L SV F I S LPAT E N L N AO F R FCV A R TN LTWS C RV N KTQ HAS T LF L N LAT L LPF CVC L M S F FL LI LS L R R H I R R M Q LS AT GC RD PS T EAHV RA LKAVI S FLLL FIAYY L5 F LIATS S Y FM PET E LAVI FG ESIA LIY PS S H S FI LI LG N N K LR H A S LKVI W V S I L K G R K FQ Q H KQI

TAS2R8 C5EQ,ID No, 60)

M FS PA D N I F I I L I T G E F I LG I LG N GY IA LV N W I D W I K K K I S TV D YI LTN LV IA R I C LI SV M VV N G I VI VLN P D VYT N QQIVI FT FWTFA N YLN M W ITTC L NV FY F L K I A S SS H P L FLW L KW K I D MVV H W I L LG C FAI S L LVS LIAAI V LSC DYRF H A IA H R N IT E M F H V 5 I PY F E P LT L FN L FA I V P FI V S L I S F FL LV RS LW RHT Q I K LYATG S R D P S T E V H V R A I KT M T S FI F F F F LY Y I S S I LM T F S Y L M T KY K LAV E F G E I A A I L Y P L G H S LI L I V L N N K L R Q T F V R M LTC R K I A C M I

.TAS-tRy.fSE9.IP No- «1

M P S A I E A I Y I I L I A G E LT I G I W G N G F I V L V N C I D W L R R D I S LI D I I L I S L A I S R I C L L C V I S L D G F F M L I F P G TY G N S V L V S I V N V V W T F A N N S S I W FTS C I S I FY L L I A N I S H P F F F W L K L K I N K V M L A I L L G S F L I S L I I S V P K N D D M W Y H L F V S H E E N I TW K F K V S K I P G T F K Q LT L N L G A M V P F I LC L I S F F L L L F S LV R H T K Q I R L H AT G F R D P S T E A H R A I KA V I I F I L L L I V Y Y P V F L V M T S S A L I P Q G K LV L M I G D I V T V I F P S S H S F I L I M G N S K L R E A F L K M L R FV K C F L R R R K P F V P

TAS2R10 (SEQ IP No, 62)

L R VV E G I F I F V V V S E S V F G V L G N G F I G LV N C I D C A K N K L S T I G F I LTG LA I S R I F L I W I I I T D G F I Q I F S P N I Y A S G N L 1 E Y I S Y F W V I G N Q S S M W F A TS L S I FY F L K I A N F S N Y I F L W L S R T N V L P F M I V F L L I S S L L N FA Y I A I L N D Y K M K N D T V W D L N Y K S E Y F I Q I L L N L G V I F F FT L S L I T C I F LI I S L W R H N R Q M Q S N VT G L R D S N T E A H V A M V L I S F I I I F I LY F I G M A I E I S C FT V R E N K L L L M F G TTTA I Y P W G H S F I LI L G N S K L K Q A S L RV LQ Q L K C C E K R K N L RVT TAS2R13 fSEQ ID No.63)

M E S A L P S I FT LV I I A E F I I G N LS N G F I V L I N C I D W V S K R E LS S V D K L L I I LA I S R I G L I E I L V S W F L A L H Y L A I FV S G T G L R I I F S W I V 5 N H F N L W LA T I F S I F Y L L K I A S F S S P A F L Y L K W R V N V I I M I L L G T LV F L F L N L I Q I N M H I K D W L D RY E R N TT W N F S M S D F E T F S V S V K FT M T M F S L T P FTVA FI S F L L L I F S I Q K H L Q K M Q I N Y K G H R D P R T K V H T N A L K I V I S F L L F YA S F F L C V L I S W I S E LY Q S TV I Y M L C E T I G V F S P S S H S F L L I LG N A K L R Q A F L LV A A K V WA K R

TAjaRl,4iSeO IP No..64)

G G V I S I FT FV L I V E F I I G N L G N S F I A L V N C I D W V G R I S S V D R I LTA LA I S R I S L V W L I F G S W C V S V F F P A L FAT E K M F R LTN I WTV I H FSVW L AT G LG TFY F L I A N F S N S I F LY L K W R V K V V LV L L L V T S V F L F L N I A L I N I H I N A S I N G Y R R N K T C S S D S S N F T R F S S L I V LTS T V F I F I P FT L S L A M F L L L I F S M W H R K K Q H TV I S G D A S T K A H R G V K S V I T F F L LY A I F S L S F F I S V W TS E R L E E N L I I L S Q V G AY P S C H S C V L 2 L G N K L R Q A S L S V L LW L RY M F D G E P S G H E F R E S S

TAS2R16 fSEO IP No.651

I P I Q LTV F F I I Y V L E S LT I I V Q S S L I V A V L G R E W L Q V R R L PV D M I L I S L G I S R F C L Q W A 5 L N N F C S Y F N L N Y V L C N LT I T W E F F N I L T F W L S L LT V F Y C I K V S S FT H H I F L W L R W R I L R L F P W I L LG S L M I T C VT I I P S A I G N Y I Q I Q L LT M E H L P R N S TVT D K L E N F H Q Y Q F Q A H TV A LV I P F I L F LA S T I F L A S IT K Q I Q H H S TG H C N P S M K A H FT A L R S L AV L F I V FT S Y F LT I L IT I I G T L F D R C W L W V W E A FV Y A F I L H S T S L M L SS P T L K R I L K G K C TAS2R20 (SEP ID No.66)

M M S F L H I V F S I L V VVA F I LG N FAN G F IA L I N F I A W V K R Q K I S S A D Q I I AA LA V S R V G L L W V I L L H W YS TV L N PTS S N L E V I I F I S N A W A VT N H F S I W LAT S LS I FY L L K I V N F S R L I F H H L K R A S V V L V I V L G S L F F LV C H LV M H TY I N V W T E E C E G N VTW I K L R N A M H L S N LTV A M L A N I I P FT LT L I S F L L L I Y S L C K H L K M Q L H G K G S Q D P ST K I H I K A L QT VTS F L I L L A I Y F LC L I I S F W N F M R P K E I V L M LC Q A F G I I Y P S F H S

F 1 L I W G N KT L K QT F IS V L W Q VT C W A K G Q N Q S T P

TAS2R30 (SEQ IP No, 67)

M I T F L P I I F S I L I V V I FV I G N F A N G F I A LV N S I E W V K R Q K I S F V D Q I LTA LA V S R V G L LW V L L L H W Y AT Q L N P A FY S V E V R I T A Y N V W AV T N H F S S W LAT S L S M F Y L L R I A N F S N LI F L R I K R RV S V LV I L L G P L L F LVC H L FV I N D ETV WT^'K EY E G N VT W I K L RS A M Y H S N M T LTM LA N FV P LT LT LI S F L L LI CS LC K H L K K M Q L H G G S Q D PS T K V H I K A L Q TVT S F L L L C A I Y F L S M I I S V C N F G R L E K Q P V F M F C Q A I I F S Y P S T H P F I L I L G N K K L K Q I F L S V L R H V RY W V K D R S L R L

TAS2R?1 (SfQ IP No.68)

M TT F I P I I FS S VV V V LFV I G N FA N G F I A LV N S I E RV K RQ K I S FA D Q I LTA LAV S R V G I L W V L I L N W Y S TV F N P A FY S V E V RTT AY N V W A VT G H F S N W L A T S L S I FY L L K I A N F S N L I F L H L K R R V S V I LV L LG P L L F LA C Q L F V I N M K E I V R T E Y E G N T W I L RS AVY L S D ATVTT LG N LV P FT LT LLC F L L L I C S L C K H L K M Q L H G K G S Q D P S T V H I K A L Q TV I F F L L L C A VY F L S I I S V W S F G S L E N P V F F C A I R F S Y P S I H P F I L I W G N K L K Q T F L S V L R Q V R Y W V G E P S S P

TAS2R38 (SEQ IP No, 69)

M LT LT R I RTV S Y E V RS T F L FI S V LE FAV G F LT N A FV FLV N FW D V V K R Q P LS N S D C V L L C L S I S R L F L H G L L F L S A I Q LT H F Q K L S E P L N H S Y Q A I I M LW M I A N Q A N L W LA AC L S L LY C S L I R F 5 H T F L I C LAS W V S R I S Q L LG I I LC S C I CTV L CV W C F F S R P H FTV TTV L F M N N N T R L N W Q N D L N L FY S F L F CY L W S V P P F L L F L V S S G M LT V S L G R H RT M K V YT R N S R D P S L E A H I A L K S IV S F F C F FV I S S CAA F I S V P L I W R D K I G V M V C V G I M AA C P S G H AA V LI S G N A K L R RA V M TI L LW A Q S S L V RA D H KA D S RT LC

TAS2R39 (SEQ IP No.70)

M L G R C F P P D T E Q Q L R M T K LC D P A E S E L S P F L I T LI LA V L LA E Y L I G I I A N G F I M A I H A A E W VQ N AV STS G R I LV F L S V S R I A LQ S L M M L E ITI S S T S LS FY 5 E D A VYY A F K I S F I F L N F C S L W F A A W LS F F Y FV K I A N F S Y P L F L K L R W R I T G L I P W L L W L S V F I S F S H S M F C I N I CTV Y C N N S F P I H S S N S T K KTY L S E I N V V G LA F F F N LG I V T P I I M F I LTAT L L I L S L K R H T L H M G S N AT G S N D P S M E A H M G A 1 K A I S Y F L 1 L Y I F N AVA L F I Y LS N F D I N S L W N N L C Q I I A AY P A S H S I L L I Q D N P G L R RA W K R L Q L R L H LY P E WTL TAS2R40 fSEQ.lP. No.71)

M ATV N T D AT D K D I S K F K V T FT L V V S G I E C I T G I L G S G F I T A I Y G A E W A R G K T L. PT G D R I M L M L S F S R L L L Q I W M L E N I F S L L F R I V Y N Q N S VY I L F K V I T V F L N H S N L W F A A W L K V FY C L R I A N F N H P L F F L M K R K I I V L M P W L L R L S V L V S L S F S F P L S R D V F N V Y V IM S S I P I P S S N ST E K K Y F S E T N M V N L V F FY N M G I FV P L I M F I L A AT L LI L S L K R H T L H M G S N A T G S R D P S M K A H I G A I K AT S Y F L I L Y I F N A I A L F L S T S N I F D T Y S S W N I L C K I I M A AY PA G H S V Q L I LG N P G L R R A W K R F Q H Q V P L Y L K G QT L

IAS2&43 fSEQ ID No.72)

M I T F L P I I F S S L V V VT F V I G N FA N G F I A L V N S I E S F K R Q K I S FA D Q I LTA LA V S R V G L L W V L L L N W Y S TV L N P A F N S V E V RTTAY N I W A V I N H FS N W LATT L S I FY L L K I A N F S N F I F L H L K R R V K S V I LV M L L G P L L F L A C H L F V I N M N E I V R T K E F E G N M T W K I K L K S A M Y F S N M TV T M V A N LV P FT LT L L S F M L L I C S LC K H L K K M Q L R G K G S Q D P S T K V H I K A L Q T V I S F L L L C A I Y F L S I M I S V W S F G S L E N K PV F M F C K A I R F S Y P S I H P F I L I W G N K K L K Q T F LS V F W Q M RY WV K G E KT S S P

TAS2R46 (SEP ID No.73)

M I T F L P I I F S I L I V VT F V I G N FA N G F I A L V N S I E W F K R Q K I S F A D Q I LTA LA V S R V G L L W V LV L N W Y AT E L N P A F N S I E V RI T AY N V W AV I N H F S N W LAT S LS I F Y L L K I A N F S N L I F L H L K R R V K S V V L V I L L G P L L F LV C H L FV I N M N Q I I W T K E Y E G N M T W K I K L R S A M Y L S N TT V T I LA N L V P FT LT L I S F L L L I C S L C K H L K K M Q L H G K G S Q D P S M K V H I K A L QTVT S F L L L C A I Y F L S I I M S VW S F E S L E N K PV F M F C E A I A F S Y P S T H P F I L I W G N K K L K Q T F L S V L W H V R Y W V K G E K P S S S

TAS2R50 (SEp ID No.74)

M I T F LY I F F S I L I M V L FV L G N F A N G F 1 A LV N F I D W V K R K K I S S A D Q I LTA LAV S R I G L L W A L L L N W Y LTV L N P A FY S V E L R I T S Y N A WV V T N H F S M W LAA N L S I F Y L L K I A N F 5 N L L F L H L K R R V R S V I L V I L L G T L I F LV C H L LV A N M D E S M W A E E Y E G N M T G K M K L R N T V H L S Y L TVTT L W S F I P FT LS LI S F L M L I C S LY K H L K K M Q L H G E G S Q D L S T K V H I K A LQ T L I S F L L L C A I F F L F L I V S V W S P R R L R N D P V V M V S K A V G N I Y L A F D S F I LI W RT K K L K H TF L L I L C Q I R C

EXAMPLE 1: Identification of promiscuous bitter receptor antagonists

Methods: On Day 0, hTAS2R expressing cell lines are pre-plated at a density of 8-15,000 cells per well in DMEM + 10% FBS in black, clear bottom. 96- well plates that had been pre-coated with 0.001 % poly(ethyleneimine) (MW = -60,000, Acros Grgani.es, Morris Plains, NJ). On day 2, high-throughput screening of antagonists is performed via calcium imaging using Fluo-4. Growth medium is discarded and the cells are incubated i the dark for 1 hour at 37° C in 50 μΐ loading buffer consisting of 1.5 μΜ Fluo-4 AM (Invitrogen. San. Diego CA) and 2.5 μΜ probenicid (Sigma-Aldrich, St. Louis, MO, US) in DMEM (no FBS).

After incubation, the plates are washed. 5X with 100 μΐ of assay buffer (described above) and further incubated in. the dark at room temperature for 30 minutes. The cells are then washed 5X with 100 μΐ assay buffer and then, calcium responses are measured in a FLIPR Tetra device (Molecular Devices, Sunnydale, CA). Test compounds are prepared at a. final. concentration, of 15 μΜ in the presence of the appropriate receptor agonist and assessed for their ability to decrease the hTAS2R receptor response to the agonist. Candidate inhibitors that show significant inhibition of the agonist response during primary screening are selected and retested for their ability to inhibit hTAS2R receptor activation. Hits that also cause a decrease in the agonist responses of a non-related GPCR pathway (isoproterenol, pl/p2-adrenergic receptor agonist) are considered as non-specific inhibitors (shown in example as host cells). For these studies, the following hTAS2R receptors and agonists are used: hTAS2Rl - 2.38 mM Chloramphenicol (Cas No 56-75-7)

hTAS2R10 - 200 μΜ Strychnine HC1 (Gas No 1421-86-9)

hTAS2R14 - 0.9 μΜ Mefenamic acid (Cas No 61-68-7)

hTAS2R16 - 100 μΜ Phenyl p-D-glucopyranoside (Cas No 1464-44-4)

hTAS2R38 - 1 μΜ 6-n-Propyl-2- thiouracil. (Cas No 51-52-5)

h.TAS2R44 - 0,4 μΜ Aristoloc ic Acid (Cas No 313-67-7)

h.TAS2R46 - 1.2 μΜ Strychnine HC1 (Cas No 1.421-86-9)

hTAS2R50 - 10 μΜ Andrographolide (Cas No 5508-58-7) Table 3: hTAS2R sequence information and Agonist Cas no for Example 1

Example 1 Results

The data in Figure 3 shows the response of the hTAS2R receptors to agonist + 15 μΜ (-)- hardwickiic acid (GIV237) and are expressed as a percent of the receptor response to agonist alone. It can be seen that (-)-hardwickiic acid effectively inhibits the response to bitter agonists at bitter receptors hTASRl O, hTASR14, hTASR16. hTASR38, hTASR44₅ hTASR46, and hTASRSO. This is an unusual finding,, as most bitter antagonists are specific for particular receptors and do not have activity across multiple receptors. Compounds 3, 4, 5 and. 6 are also tested and show inhibition of bitter receptors at concentrations of 20 micromolar or less. These data was collected using an 88% pure sample of (-)-hardwickiic acid (GIV237) purchased from AnalytiCon (http://www.ac- discovery.com) (Catalogue No. NP-009686). The % purity was determined by HPLC Evaporative Light Scattering Detector (ELSD).

EXAMPLE 2 Identification of promiscuous bitter receptor antagonists If

The data from Example 1 demonstrates that GIV237, which is derived from a botanical source, can inhibit several TAS2R bitter taste receptors (hTAS2 10. hTAS2R14. hTAS2R16. hTAS2R38. hTAS2R44. hTAS2R46. hTAS2R5G) in vitro at a concentration of 15 μΜ. The unique properties of GIV237 makes it an ideal candidate for evaluation in assays employing even more human TAS2Rs to understand in detail its full antagonist (and agonist) properties.

In this second Example, an (-)-hardwickiic acid preparation which was purified from a blackcurrant bud absolute extract was used. The blackcurrant bud absolute extract was purchased from. Biolandes (40420 Le Sen France, Biolandes.com) (Reference No F0424) and it is described in the Biolandes.com product information literature as a natural extract obtained exclusively from Ribes nigrum buds (I CI name; Ribes Nigrum bud extract). It has a GRAS/FEMA number of 2346, an FDA number of 172.510 and a No. COE of 399n.

The blackcurrant bud absolute extract was fractionated using column chromatography as follows: 12 grams of blackcurrant buds absolute were deposited on 5 g of silicagel. This sample was fractionated by column chromatography (330 g of silica gel) using a gradient of hexane/Methyl Tertiary-Butyl Ether (MTBE) (start 100% hexanes to 100% MTBE). The hardwickiic acid was eluted at 80% MTBE to provide 4.1 g of light yellowish semi -so lid. The major component was identified as harwickiic acid (CAS No 1782-65-6, MW 316, C20H28O3) by Gas Chromatography Mass Spectrometry (GCMS).

Proton NMR analyses on the fractionated sample indicated that the sample contained a mixture of two components in a ratio of ca, 4; 1. Pure (-)-hardwickiic acid (Compound 1 of Formula 1) (about 120mg) and a second minor component, (-)-epi-hardwickiic acid (Compound 2 of Formula i) (about 29mg) were generated after repeated HPLC separation. This is the first time that (-)-epi-hardwickiic acid (Compound 2 of Formula 1 ) was isolated from a Blackcurrant source. The C and H NMR data for (-)- hardwickiic acid (Compound 1 of the present invention) and (-)-epi-hardwickiic acid (Compound 2 of the present invention) are provided below in Tables 4 and 5 respectively. In more detail, proton NMR analysis indicated the sample contains a mixture of two components in a ratio of a. 4:1. The optimal conditions for resolving the mixture were determined using an analytical HPLC system under various solvent conditions. Preparative scale separation was carried out on an Agilent 1 100 preparative HPLC system using Phenomenex Luna C-l 8(2) column (21.5 x 250 mm) with an isocratic solvent system of 80% MeOH- water, 25 mL/min, UV 210 nm detection.

Pure hardwickiic acid (120 mg) and the unknown minor component (29 mg) were generated after repeated HPLC separation. Fractions were checked on an analytical HPLC and combined. After removing MeOH, the fractions were lyophilized to afford white powders. The purities were further confirmed by NMR analyses.

Structural identification of Compounds (1) and (2):

The major compound (1), [a]" = -129 (c. 0.8, CHC1₃). was identified to be hardwickiic acid (C20H₂8O3) based on extensive NMR analyses and LC-MS data (315.2, [M-H]^"). The Ή and C NMR spectra showed the presence of two tertiary (C-l 9, 20) and one secondary (C- 17) methyl groups (see Table 4 below).

Additionally, The NMR also displayed signals ascribable to a β-mono-substituted furan ring (CI 1 -C 16). From the analyses of COSY, HSQC and HMBC, 1 was determined to be a furanoid clerodane-type diterpenoid, hardwickiic acid. The stereochemistry was established by NOESY and the optical rotation. The lack of nOe interaction between H-10 and the methyl group at C-5 indicated the trans junction between the two ring systems. A strong nOe observed between the methyl groups at C-5 and C-9 indicated both methyl groups are on the same side of the molecule. The negative specific optical rotation confirmed the absolute stereochemistry of 1 as (-)-hardwickiic acid, a well-known diterpenoid isolated from many sources including Hardwickia pinnata (Misra et al Tetrahedron Letters ( 1 64), (49), 3751 -9). Te Solidago spp (Henderson et al Canadian Journal of Chemistry (1973), 51(9), 1322-51). Ribes nigrum (blackcurrant absolute) (Perfumer & Flavorist (1985). Volume Date 1984, 9(6), 39-42), the genus Croton (Chlabra et al Journal of the Kenya Chemical Society (2007), 4(1), 33-46) and many other sources. The minor compound (2. fa]^D = -72.8 (c. 0.8, CHCU) displayed the same numbers and types of carbon, and protons as in. 1. A molecule formula of CioHigOj was obtained from. LC-MS (315.2. [M-H]^"} and Ή, °C NMR data (see Table 5 below). Both 1 and 2 displayed identical Ή and C signals ascribable to the β -mono-substituted furan ring. However, the remaining NMR signals showed large difference in chemical shifts (see Tables 4 and 5 below). As in 1, compound 2 also displayed 3 methyl groups and α,β-unsaturated carboxylic group, but with different H and C chemical shifts.

The structure of 2 was established by a combination of 2D-NMR including COSY, TOCSY, HSQC, and HMBC. Interpretation of COSY and. TOCSY revealed the proton coupling networks. Based on the proton assignment, an HSQC experiment identified the corresponding carbons.

The stereochemistry was established by phase-sensitive NOESY experiments. When recorded in CDC1₃. strong nOe interaction was observed between H-10 and the methyl group at C-5 indicating a cis fusion between the two ring systems. Strong nOes were also observed between H-10 and 5-CH3 as well as between H-10 and one of the protons at C-l 1 indicated the proximities between these atoms. Unfortunately, the two methyl groups at C-8 and C-9 were degenerate and appeared at δ 0.80 ppm, thus making it impossible to determine the stereochemistry at C-8.

A second NOESY was measured in pyridine-^ in hoping that the overlapped methyl groups would be resolved and provide further information concerning the stereochemistry of the molecule. Fortunately, the two methyl groups were well, separated with 9-CH₃ being at 0.92 ppm and 8-CH3 at 0.76 ppm. Interpretation of the NOESY revealed that nOes were existed between the two methyl, groups at C-8 and C-9 indicating 8-CH3 was β-orientated. The nOes between 7β and 8, 6a and 8 further supported the β orientation of the 8-CH3. The nOes between 8-Ci and 7a (strong), 8-CH3 and 7β (weak) as well as 9-CH₃ and 7β (strong) suggested the B-ring existed in. a chair conformation with 9-CH₃ in an axial and 8- CH₃ in an equatorial positions, respectively. Strong nOes observed between 2β and 9-CH₃, 1 β and 9-CH3 suggested the proximity between these atoms due to the cis junction of the rings. The nOe between 2β and 9-C¾ indicated that the A-ring existed in a half chair conformation with C-l being below the C-2, C-3, C4, C-5 plane. A negative optical rotation led to the conclusion that 2 is (-)-ep?^"-hardwickiic acid. This is the first time that (-)-epi-hardwickiic acid was isolated from a Blackcurrant source, but it was once reported from the axenic cultures of liverwort Scapania mmorea in 1 99 (Geis et al Phytochemistry (1999), 51 (5), 643-649). A closed analogue, named tinotufolin B with the a- methyl group at C-8 was reported from Tinospora tuberculate in 1 93 (Fukuda et al Liebigs Ann. Chem. 1993, 325-327).

Both (-)-hardwickiic acid (1) and (-)-t'/?/-hardwickiic acid (2) adopt a half-chair A-ring and chair B-ring with trans and cis fusions between the ring systems, respectively. Unlike 1, which is more or less flat, 2 has a folded structure. It is well-known that proton and carbon chemical shifts are very sensitive to their environment, and therefore, the change in the conformation has resulted in significant displacement of these.

Table 4 ¹³C and ^lH NMR Data of (-)-Hardwickiic acid (1)

¹³C ^{' !}H ^'

1 1 7.4 t 1.51, 1.68

2 27.5 t 2.18, 2.30

3 140.0 d 6.85 br. f

4 141.6 s ...

5 37.6 s ...

6 35.8 1 1.17 ddd (13.5, 12.4, 4.6)

2.43 dt (13.2, 3.2)

7 27.3 t 1.47 (2H)

8 36.3 d 1.56 m

9 38.8 s —

10 46,7 s 1.38 d (11.8)

! l 38.6 t 1 .61 m (2H)

12 18.1 t 2.18, 2,33

13 125.6 s ...

14 1 10.9 d 6.26 br. dd

15 142,7 d 7.34 t (1.69)

16 138.4 d 7.20 br. s

17 16.0 1 0.83 d (6.43)

18 172.5 s ...

19 20,5 t 1.26 s

20 18.3 t 0.76 s

Recorded in CD£1₃ at 30 °C on Broker Avance-300 and 500 spectrometers. Assignment was based on DEPT, COSY, HSQC, HMBC, and NOESY spectr

4 137.7 s .... 140.7 s —

5 36.4 s ... 37.2 s —

6 36.91 1.17a, 2.74pbr.d (11.8) 37.91 1.27a, 3.26pbr.d (13.1)

7 28.71 1.18.1.32 29.6 t 1.30a, 1.44

8 37.8 d 1.56 m 38.4 d 1.55 m

9 40.3 s 41.0 s

10 45.4 d 1.51 br.d(6.10) 46.1 d 1.56 r.d(6.93)

11 38.5 t 1.54, 1.72 39.2 t 1,56, 1.79

12 18.1 t 2.31 m(2H) 18.81 2.37 (2H) dd (8,8, 8.5)

13 125.7 s 126.8 s

14 111.0 d 6.27 br. d 112,2 d 6.52 s

15 142.7 d 7.35 t (1.65) 143.7 d 7.65 t (1.53)

16 138.4 d 139,5 d

7,22 s 7.58 s

17 16.01 0.80 d (6.80) 16.61 0,76 d (6.70)

18 173.3 s 171.3 s

©

19 33.41 1.26 s 34.31

1.51 s

o

20 18.01 0,80 s 18.5 t 0.92 s

o

NOESY spectra

To investigate the potential antagonist (and agonist) properties of (-)-hardwickiic acid (Compound I of Formula 1), 20 of the 25 hTAS2Rs were expressed individually in HEK293T-G16gust44 cells using the methodology described in Meyerhof et al (2010) Chem Senses 35: 157-170 (doi: 10.1 093/chemse/bjp092) which refers to Ueda et al (2003) J Neurosci 23: 7376-7380 and the methodology disclosed in US Patent No, 7919236. The specific fiTAS2R polypeptide sequences (SEQ ID Nos 55-74) are provided above and in Table 7 below. Changes in TAS2R receptor activity were calculated by measuring changes in intracellular calcium concentration which were detected using a fluorometric imaging plate reader.

The 20 individually expressed hTAS2Rs were challenged with varying concentrations (2 μΜ. 6μΜ, 8.7μΜ, 13μΜ, 21.7μΜ and 26μΜ) of (-)-hardwickitc acid (Compound 1 of Formula 1), in the presence of ^'known agonists for the 21 hTAS2Rs as set out in Tables 6 and 7. The results provided in Table 7 indicate which of the 21 hTAS2Rs are sensitive to (-)-hardwickiic acid and the percentage inhibition of signal activity at the various levels of (-)-hardwickiic acid which were used. These data demonstrate that (-)-hardwickiic acid, is capable of reducing the bitterness of compounds that are agonists for some of the specific human bitter taste receptors studied. It can be seen from Table 4 that (-)-hardwickiic acid, effectively inhibits the response to bitter agonists at bitter receptors hTAS2R3. hTAS2R4, hTAS2R7, hTAS2R8, hTAS2R10, HTAS2R13. hTAS2R14, hTAS2R16, hTAS2R31, hTAS2R38, hTAS2R39, hTAS2R43, hTAS2R46. and hTAS2R50. The data support the inhibitor responses shown for hTAS2R l 0, hTAS2R14, hTAS2R16, hTAS2R38, hTAS2R46 and hTASRSO as demonstrated in Figure 3 from Example 1. This is a very unusual finding, as most bitter antagonists are specific for particular receptors and do not have activity across multiple receptors.

The results also demonstrate that (-)-hardwickiic acid acts as an insurmountable bitter antagonist, can be pre-applied to the receptor in the absence of agonist and still inhibit the receptor and the inhibition is reversible. Hardwickiic acid exists in the trans and cis-isomeric form in the plant source. Both isomeric forms are active but the trans isomer ((-)-hardwickii Acid) is more potent than the cis-isomer. Moreover, unlike the trans isomer, the cis-isomer acts also as an agonist of hTAS2R14 (ECso of circa 30 μΜ).

Table 7: Inhibition of Multiple hTAS2Rs by (-)-hardwickiic acid

Table 7 Legend

Agonist was applied at EC_% concentration (Brockhoff et al (2011) J Neuroscience 31(41 ); 14775-14782. Slack et a! (2010) Curr Biol 20(12) 1 104-1 109;

Agonist of hTAS2Rl , hTAS2R10, hTAS2R40 were applied at the maximal applicable concentration (which did not show unspecific signals)

(a) Insufficient activation by agonist;

(b) - indicates "no inhibition" by (-)-hardwiekiic acid

(c) Student's t test, p <0.05

(d) 26 μΜ (-)-hardwickiic acid elicits minor artefacts

(e) Experiments are performed in triplicate

EXAMPLE 3

Sensory Evaluation

Sensory protocols to assess the effects of the Compounds of Formula 1, especially Compound 1 of Formula 1 ((-)-hardwickiic acid, (hereinafter GIV237) on taste perception are administered in. test subjects with informed writte consent and after prior approval by the Givaudan Institutional Review Board, Human subjects are recruited and trained for the experiments. Initially, the Intrinsic sensory aspects of GIV237 are assessed followed by an examination of its potential to inhibit bitterness. Experiments are done with and without nose clips. For the study to examine the potential of GIV237 to inhibit bitterness, GIV237 is co-administered with bitter tastants to the subject in various concentrations based on the data obtained in Figure 3 and Table 7 and empirically determined in pilot experiments. Bitter compounds are selected based on the results obtained in Figure 3 and Table 7. Two alternative forced choice (2 -AFC) test methods are employed. Experiment 3 a - Effect of the GIV 237 compound on reducing the hitter taste of Acesulfame K and saccharin

Acesulfame K and saccharin, to a lesser extent, are commonly used as sugar substitutes for low calorie food products and their acceptance would be significantly increased by blocking the undesirable bitter tastes associated with them (Schiffman et al (1 95) Brain Res Bull 36; 505-513), Therefore it is useful to determine whether GIV237 can reduce the bitter taste of acesulfame K in vivo using structured human taste trials. Briefly, taste tests are performed with the GIV237 antagonist using a 2 -alternative forced choice method (2- AFC). An Acesulfame K samples with the antagonist was given, to the taste panelists together with the same sample without the antagonist, the panelists are asked to identify the bitterer sample within the pair.

In more detail, for the 2-AFC measurements twenty milliliters each, of acesulfame K (2 mM) and GIV237 (for example at around about 25-30 ppm.) are presented in. random order at room temperature to 22 subjects that had been pre-identified as sensitive to the bitterness of the sulfonamides given that this trait varies in humans. Over two trials (with a. 5 min break between reps) panelists are asked to select the solution perceived as being most bitter (trial 1) or as most sweet (trial 2). After the first trial, the panelists are asked to rate the perceived bitter taste intensity of both solutions using a 0-100 line scale with anchors at the following intervals: 0 = no bitterness, 25 = recognizable, 50 = moderate, 75 = strong, 100 = extreme. In the second trial, panelists are asked to rate the perceived sweet taste intensity of the two solutions following selection of the sample that is most sweet. To detennine if a significant number of panelists identify the sample without the antagonist as being most bitter or sweet, a binomial analysis is used. ANOVA is used to determine whether perceived taste intensity ratings are statistically different between the samples. A p < 0.05 is taken as significant in both statistical tests. In separate sessions, 15 panelists evaluate the effect of the G1V237 compound (25-30 ppm) on sodium saccharin solutions (3 mM), using the same 2-AFC procedure but without the taste intensity ratings. Each panelist performs two replicates of the test.

The results from the sodium saccharin studies are analyzed using beta-binomial statistics and corrected for possible over dispersion in the data. Finally, the effect of a dosage of. for example, about 25-30 ppm GIV237 is examined via triangle test for intrinsic taste or aroma properties that would render it identifiable from a water reference. Briefly,. three samples are presented simultaneously to each panelist. Two samples are identical and one is different (odd). Samples are served to the panelists in a balanced, randomized order. Panelists are asked to evaluate the samples from left to right and select the odd sample. A significance level of p<0.05 is set following binomial statistical analysis.

Experiment 3b - Effect of the GIV 237 compound on reducing the hitter taste of whey proteins

Taste tests were performed with the G1V237 compound using the 2-AFC method as described above but in this case, whey samples with the GIV237 compound are given to the taste panelists together with the same sample without the GIV237 compound and the panelists are asked to identify the bitterer sample within the pair.

Experiment 3c - Effect of the GIV 237 compound on reducing the bitter taste of Green Tea Taste tests were perfonned with the GIV237 compound using the 2-AFC method as described above but in this case, green tea samples with the GIV237 compound are given to the taste panelists together with the same sample without the GIV237 compound and the panelists are asked to identify the bitterer sample within the pair. Experiment 3d - Effect of the GIV237 compound on reducing the bitter taste of Menthol Taste tests were performed with the GIV237 compound using the 2-A.FC method as described above but in this case, menthol samples with the GIV237 compound are given to the taste panelists together with the same sample without the GIV237 compound and the panelists are asked to identify the bitterer sample within the pair.

Experiment 3e -Effect of the GIV237 compound on reducing the bitter taste of Rebaudioside A

Taste tests were performed with the GIV237 compound using the 2-AFC method as described above but in this case. Rebaudioside A samples with the GIV237 compound are given to the taste panelists together with the same sample without the G1V237 compound and the panelists are asked to identify the bitterer sample within the pair.

Experiment 3(f)

The bitter blocking capability of test Compound 1 (GIV237) (25 ppm) in sucralose (3 mM) solution is evaluated. Twenty ml of each solution (sucralose or sucralose with test Compound 1 are presented in random order to 15 bitter-sensitive panelists. In two replications, panelists are asked to select the solution they perceived as more bitter. A beta- binomial analysis is calculated from the forced-choice data to correct for overdispersion. A check is made to see if a significant (p<0.Q5) majority of evaluations select test Compound I (GIV237) (25 ppm) in a 3 mM sucralose solution as being less bitter than a 3 mM sucralose solution. I panelists evaluate bitterness over 2 replications, the data is subjected to a beta-binomial d' analysis to determine if over-dispersion occurs. Not accounting for over-dispersion has the potential to underestimate the significance level at which a test is conducted and therefore can lead to false-positive results. An assessment is made to see i the data showed that test Compound 1 (GIV237) (25 ppm) reduced the perceived bitterness of a 3 mM sucralose solutions. IN SUMMARY

The perception of bitter taste prevents mammals from ingesting food potentially contaminated with hitter-tasting toxins that are numerous and structurally diverse. Bitter taste perception is mediated by a family of heptahelical G protein-coupled receptors, called Taste 2 Receptors (TAS2Rs or T2Rs). In humans, about 25 TAS2Rs exist that have been extensively characterized in terms of the identification of numerous bitter agonists for at least 20-21 of the 25 human TAS2Rs which suggests that TAS2Rs recognize several to many bitter compounds. The present invention demonstrates that a natural compound from edible plants, can act as a broad spectrum (or promiscuous) bitter taste receptor ligand. that is able to modulate bitter taste receptor activity by decreasing or possibly completely abating bitterness signal transduction across multiple bitter taste receptors. This natural compound has utility in reducing the perception of bitter flavours and/or enhancing the perception of sweet flavours.

Claims

Claims:

1, A. method of modulating bitter taste perception in a subject, comprising providing an antagonist which is a compound of Formula 1:

Formula 1

wherein the dotted line denotes a single or double bond,

Ri is H, -CH₃ or (wherein the dotted line is a double bond) is not present;

R₂ is -CH₃, -<CO)OR₉. -CHO, or -C¾OH;

R₃is-CH₃orH;

4 is -CH₃ or H;

R₅isHor -CH3;

e is =C¾, -CH_3S -{CO)OR₉;

R7IS-CH3;

Re is -CH₂-(CO)OR₉, =CH-(CO)OR₉; -CH₂-CH₂-0(CO)CH₃

or 7 and Rg together are furanyl; and

R9IS H or C alkyl;

in. free or orally acceptable base addition salt form.

The method of claim 1 wherein the compound of Formula 1 is selected from ipounds 1,

2, 3, 4, 5.6 and 7:

(-)-Hardwkkik Acid (1) (-)-epi-Hardwkkiic Acid (2) [a]₀ -129,4 (c.0,8, CHCi,) [a]]₃-?2J( 0,8,CHC1₃)

Compound 1

Compound 3 Compound 4

Compound 5

Compound 7

3. The method of claim 1 wherein the compounds of Formula 1 are compounds of

wherein R₂. R , Ri. s, R?₅ Rg and R are as hereinbefore set forth for Formula 1 , and R₆ is methyl, in free or orally acceptable base addition salt form.

4. The method of claim. 3 wherein the Compound of Formula la is selected from

a Compound of Formula la wherein R₂ is -COOH,

a Compound of Formula la wherein R3 is -CH3,

a Compound of Formula l wherein R4 is H,

a Compound of Formula la wherein R5 is -CH3,

a Compound of Formula la wherein R6 is -CH3. and

a Compound of Formula la wherein R and Rg together are furanyl. e.g., 3-furanyi; each in free or orally acceptable base addition salt form.

5, The method of any one of claims 1 -4 wherein the bitter receptor antagonist is (-)- hardwickiic acid in free or orally acceptable base addition salt form.

6. The method of any one of claims 1-5 wherein the bitter receptor antagonist is substantially free of plant alkaloMs and/or plant terpenoids which would normally be present in a crude extract from a plant containing a bitter receptor antagonist which is a Compound of Formula 1.

7. The method of any one of claims 1-6 wherein said the bitter receptor antagonist 5 modulates bitter taste perception in a subject via binding to at least three bitter taste receptors selected from the group consisting of: hX2R10, hT2R14, hT2R16. hT2R38. hT2R44, hT2R46, hT2R50. hI2R4, hT2R7, hT2R8, hT2R13_s hT2R31 , hT2R39 and/or hT2R43.

10 8. The method of any one of claims 1-7 wherein the amount of bitter receptor antagonist administered is sufficient to provide a concentration in the mouth of at least 1 micromolar.

9. The method o any of claims 1 -8 wherein the bitter receptor antagonist is administered with a food or drink product, a pharmaceutical or medicinal product, or a

15 nutritional supplement or nutraceutical. a dental hygiene composition, or an animal food product.

10. The method of any of claims 1 -9 wherein the bitter receptor antagonist is administered with a sweetener and/or one or more additional bitter masking agents.

20

1 1. The method of any of claims 1 -10 wherein the bitter receptor antagonist is orally administered prior to oral administration of a substance comprising a T2R bitter receptor agonist.

25 12. The use o a compound of Formula 1 in a screening assay to identify compounds which are bitter receptor antagonists.

13. A compound of Formula 1 in labeled form.

30 14. A comestible product comprising a taste-enhancing amount of an isolated compound of Formula 1 in free or orally acceptable base addition salt form.

15. The comestible product of claim. 14 wherein the compound of Formula 1 is (-)- hardwickiic acid in free or orally acceptable base addition salt form..

16. A product for addition to a food or drink comprising a bitter receptor antagonist 5 which is an isolated compound, of Formula 1 in free or orally acceptable base addition salt form, and optionally further comprising flavoring, spice, an additional, bitter-masking agent, and/or a sweetener.

17. The product of claim 16 wherein the compound of Formula 1 is (-)-hardwickiic acid 10 in free or orally acceptable base addition salt form.

18. A product according to claim 16 or 17 comprising a sweetener selected from sugar, saccharin, sodium cyclamate and. acesulfame potassium.

15 19. A method for the production of a food, a food precursor material or additive employed in the production of a foodstuff comprising the step of admixing an antagonist selected from, the group consisting of; (i) a compound of Formula 1 which is an isolated compound of Formula 1 in free or orally acceptable base addition salt form; (ii) an antagonist identified in the screening assay according to claim 12; or (iii) an antagonist 20 structurally related to the antagonists of (i) or (ii); with the food, the food precursor material or the additive employed in the production, of the foodstuff.

20. A method for the production of a nutraceutical or a pharmaceutical composition comprising the step of admixing an antagonist selected from the group consisting of: (i) a

25 compound of Formula 1 which is an isolated compound, of Formula 1 in. free or orally acceptable base addition salt form; (ii) an antagonist identified, in the screening assay according to claim 12; or (iii) an antagonist structurally related to the antagonists of (i) or (ii); with an active agent and optionally with a pharmaceutically acceptable carrier and/or adjuvant.

30

21. The method of claim 20 further comprising the step of formulating the pharmaceutical composition into a pharmaceutically acceptable form.

22. A food, a food precursor material or additive employed in the production of a foodstuff producible according to claim 19.

23. A nutraceutical or a pharmaceutical composition producible according to claim 20 or 5 claim 21 , comprising at least one nutraceutically or pharmaceutically active agent and optionally one or more pharmaceutically acceptable carrier and/or adjuvant.

24. The use of a blackcurrant bud absolute extract as a bitter taste ligand which modulates bitter taste receptor activity by decreasing bitterness signal transduction activity across

10 multiple (ie more than one) bitter taste receptors selected from, the group consisting of hT2R10, hT2Rl4, hT2R16_} hT2R38, hT2R44, hT2R46, hT2R50; hT2R3, hT2R4, hT2R7, hT2R8, hT2R13, hT2R31. hT2R39 and hT2R43.

25. The use according to claim 24 wherein the blackcurrant bud absolute extract contains 15 about 20% (w/w) of (-)-hardwickiic acid.

26. The use according to claim 24 or claim 25 wherein the blackcurrant bud absolute extract comprises a mixture of (-)-hardwickiic acid and (-)-epi hardwickiic acid in the ratio of about 4: 1.

20

27. The use according to any one of claims 24-26 wherein the blackcurrant bud absolute extract is a Rihm nigrum extract.

28. A blackcurrant bud absolute extract comprising a mixture of (-)-hardwickiic acid and 25 (-)-epi hardwickiic acid in the ratio of about 4: 1.

29. A blackcurrant bud absolute extract according to claim 28 comprising about 20% (w/w) of (-)-hardwickiic acid.

30 30. A product and a method substantially as described herein and with reference to the accompanying Tables and Figures.