Claims
- 1. An assay method for identifying a compound which modulates hT2R4 associated bitter taste comprising:
(i) screening a compound for its effect on quinine or a structurally related compound to induce activation of hT2R4 or a variant, fragment, ortholog, mutant or chimera of said receptor that is activated by quinine; and (ii) determining whether said compound modulates bitter taste based on its effect on the activation of said receptor by quinine or a structurally related compound.
- 2. The assay of claim 1 wherein said hT2R4 has the amino acid sequence contained in SEQ ID NO.: 1.
- 3. The assay of claim 1 wherein said hT2R4 is expressed on a cell membrane.
- 4. The assay of claim 3 wherein said cell membrane is isolated.
- 5. The assay of claim 3 wherein said cell membrane is on an intact cell.
- 6. The assay of claim 3 wherein said cell is an eukaryotic cell.
- 7. The assay of claim 6 wherein said cell is a mammalian cell or insect cell.
- 8. The assay of claim 7 wherein said mammalian cell is selected from an HEK cell, BHK cell, COS cell, and CHO cell.
- 9. The assay of claim 1 wherein the modulatory effect of said compound is further confirmed by a taste test.
- 10. A method for identifying a compound that modulates hT2R4 bitter taste comprising:
(i) obtaining a cell line or cell membrane that expresses a hT2R4 protein, variant, ortholog, chimera or fragment that is and optionally expresses a G protein; (ii) contacting said cell line with quinine or a related compound that activates hT2R4; (ii) contacting said cell line with at least one compound that is to be screened for its possible effect on bitter taste; and (iii) identifying whether said compound modulates bitter taste based on its effect on the activation of said protein, variant, ortholog, chimera or fragment by quinine or a related compound.
- 11. The method of claim 10 wherein said effect on said compound is selected based on its effect on intracellular ion concentration.
- 12. The method of claim 11 wherein said ion is calcium.
- 13. The method of claim 11 wherein said ion is sodium.
- 14. The method of claim 10 wherein said compound is selected based on its effect on cell membrane potential.
- 15. The method of claim 10 wherein said compound is selected based on its effect on transcription of said T2R.
- 16. The method of claim 10 wherein said compound is selected based on its ability to block interaction of said T2R with quinine.
- 17. The method of claim 10 wherein said hT2R4 is operably linked to a sequence that facilitates plasma membrane export.
- 18. The method of claim 10 wherein said cell line is an HEK-293 cell line.
- 19. The method of claim 10 wherein the effect of said compound on said hT2R4 polypeptide is assayed based on change in intracellular cAMP, cGMP or IP3.
- 20. The method of claim 10 wherein said hT2R4 fragment is an extracellular domain or transmembrane region.
- 21. The method of claim 10 wherein the difference in calcium ion concentration is monitored using a reporter molecule.
- 22. The method of claim 10 wherein said reporter molecule is a fluorescent dye selective for calcium.
- 23. The method of claim 22 wherein said dye is Fluo-3, Fluo-4 or Fura-2.
- 24. The method of claim 10 wherein said hT2R4, variant or fragment is attached to a reporter molecule.
- 25. The method of claim 24 wherein said reporter is green fluorescent protein or a variant thereof.
- 26. The method of claim 1 wherein said hT2R4 polypeptide, variant or fragment is contained in solution.
- 27. The method of claim 1 wherein said hT2R4 polypeptide, variant or fragment is in a bilayer membrane, attached to a solid phase, is a lipid monolayer, or is in a vesicle.
- 28. The method of claim 1 wherein the binding of said compound is evaluated based on a change in spectroscopic characteristics, hydrodynamic characteristics, chromatographic characteristics or solubility.
- 29. The method of claim 1 wherein the compound is selected based on its effect on the complexation of said hT2R4, variant or fragment with a G protein.
- 30. The method of claim 10 wherein said compound is selected based on its effect on interaction of said hT2R4 polypeptide, variant or fragment with gustducin or transducin.
- 31. The method of claim 1 wherein said compound is selected using a fluorescence polarization assay.
- 32. The method of claim 1 wherein said hT2R4 polypeptide is attached to a solid phase substrate.
- 33. The method of claim 1 which comprises a high throughput assay.
- 34. An assay method for identifying a compound which particularly modulates hT2R61 associated bitter taste comprising:
(i) screening a compound for its effect on the activation of hT2R61, or a variant, fragment, ortholog, mutant or chimera of said receptor which specifically binds 6-nitrosaccharin, saccharin or a related compound; and (ii) determining whether said compound modulates hT2R61 associated bitter taste based on its effect on the activation of said receptor by 6-nitrosaccharin, saccharin or a structurally related compound.
- 35. The assay of claim 34 wherein said hT2R61 has the amino acid sequence contained in SEQ ID NO.: 5.
- 36. The assay of claim 34 wherein said hT2R61 is expressed on a cell membrane.
- 37. The assay of claim 34 wherein said cell membrane is isolated.
- 38. The assay of claim 34 wherein said cell membrane is on an insect cell.
- 39. The assay of claim 34 wherein said cell is an eukaryotic cell.
- 40. The assay of claim 34 wherein said cell is a mammalian cell or insect cell.
- 41. The assay of claim 34 wherein said mammalian cell is selected from an HEK cell, BHK cell, COS cell and CHO cell.
- 42. The assay of claim 34 wherein the modulatory effect of said compound is further confirmed by a taste test.
- 43. A method for identifying a compound that modulates hT2R61 associated bitter taste comprising:
(i) obtaining a cell line or cell membrane that expresses hT2R61 protein, variant, ortholog, chimera, or fragment that is involved in bitter taste and optionally contains an endogenous or exogenous G protein: (ii) contacting said cell line with 6-nitrosaccharin or a related compound that activates hT2R61; (iii) contacting said cell line with at least one compound that is to be screened for its possible effect on bitter taste; and (iv) identifying whether said compound modulates bitter taste based on its effect on the activation said hT2R61 protein, variant or fragment by 6-nitrosaccharin or a structurally related compound.
- 44. The method of claim 43 wherein said effect on said compound is selected based on changes in intracellular ion concentration.
- 45. The method of claim 44 wherein said ion is calcium.
- 46. The method of claim 44 wherein said ion is sodium.
- 47. The method of claim 43 wherein said compound is selected based on its effect on cell membrane potential.
- 48. The method of claim 43 wherein said compound is selected based on its effect on transcription of said T2R.
- 49. The method of claim 43 wherein said compound is selected based on its ability to block interaction of said T2R with 6-nitrosaccharin.
- 50. The method of claim 43 wherein said hT2R61 is operably linked to a sequence that facilitates plasma membrane export.
- 51. The method of claim 43 wherein said cell line is a HEK-293 cell line.
- 52. The method of claim 43 wherein the effect of said compound on said hT2R61 polypeptide is assayed based on change in intracellular cAMP, cGMP or IP3.
- 53. The method of claim 43 wherein said hT2R61 fragment is an extracellular domain or transmembrane region.
- 54. The method of claim 43 wherein the difference in calcium ion concentration is monitored using a reporter molecule.
- 55. The method of claim 43 wherein said reporter molecule is a fluorescent dye specific for calcium.
- 56. The method of claim 55 wherein said dye is Fluo-3, Fluo-4 or Fura-2.
- 57. The method of claim 43 wherein said hT2R61, variant or fragment is attached to a reporter molecule.
- 58. The method of claim 57 wherein said reporter is green fluorescent protein or a variant thereof.
- 59. The method of claim 43 wherein said hT2R61 polypeptide, variant or fragment is contained in solution.
- 60. The method of claim 43 wherein said hT2R61 polypeptide, variant or fragment is in a bilayer membrane, attached to a solid phase, is a lipid monolayer, or is in a vesicle.
- 61. The method of claim 34 wherein the binding of said compound is evaluated based on a change in spectroscopic characteristics, hydrodynamic characteristics, chromatographic characteristics or solubility.
- 62. The method of claim 34 wherein the compound is selected based on its effect on the complexation of said hT2R61, variant or fragment with a G protein.
- 63. The method of claim 43 wherein said compound is selected based on its effect on interaction of said hT2R61 polypeptide, variant or fragment with gustducin or transducin.
- 64. The method of claim 34 wherein said compound is selected using a fluorescence polarization assay.
- 65. The method of claim 34 wherein said hT2R61 polypeptide is attached to a solid phase substrate.
- 66. The method of claim 34 which comprises a high throughput assay.
- 67. The method of claim 43 wherein said cell line stably expresses said receptor.
- 68. The method of claim 43 wherein said cell line transiently expresses said receptor.
- 69. The method of claim 67 or 68 wherein said cell line is a mammalian or insect cell.
- 70. The method of claim 69 wherein said insect cell is an Sf9 cell.
- 71. The method of claim 43 wherein said assay is a GTPγS binding assay.
- 72. The method of claim 71 wherein said assay uses transducin or gustducin as the G protein.
- 73. The method of claim 72 wherein said receptor is expressed using a baculovirus expression system.
- 74. The method of claim 43 wherein the ability of said compound to inhibit hT2R61 bitter taste is further confirmed by a taste test.
- 75. The method of claim 74 wherein the taste test uses a composition containing saccharin or a related compound.
- 76. An assay method for identifying a compound which particularly modulates hT2R44 associated bitter taste comprising:
(i) screening a compound for its effect on the activation of hT2R44, or a variant, fragment, chimera, ortholog, or mutant of said receptor by denatonium and/or 6-nitrosaccharin; and (ii) determining whether said compound modulates hT2R44 associated bitter taste based on its ability to modulate the activation of said receptor by denatonium and/or 6-nitrosaccharin or a structurally related compound.
- 77. The assay of claim 76 wherein said hT2R44 has the amino acid sequence contained in SEQ ID NO.: 3.
- 78. The assay of claim 76 wherein said hT2R44 is expressed on a cell membrane.
- 79. The assay of claim 78 wherein said cell membrane is isolated.
- 80. The assay of claim 78 wherein said cell membrane is from an insect cell.
- 81. The assay of claim 78 wherein said cell is a eukaryotic cell.
- 82. The assay of claim 81 wherein said cell is a mammalian cell or insect cell.
- 83. The assay of claim 82 wherein said mammalian cell is selected from an HEK cell, BHK cell, COS cell and CHO cell.
- 84. The assay of claim 76 wherein the modulatory effect of said compound is further confirmed by a taste test.
- 85. A method for identifying a compound that modulates hT2R44 associated bitter taste comprising:
(i) obtaining a cell line or cell membrane that expresses a hT2R44 protein, variant, ortholog, chimera or fragment that is involved in bitter taste and optionally contains an endogenous or exogenous G protein; (ii) contacting said cell line with denatonium or a related compound that activates hT2R44; (iii) contacting said cell line with at least one compound that is to be screened for its possible effect on bitter taste; and (iv) identifying whether said compound modulates bitter taste based on its effect on the activation of said T2R protein, variant or fragment by denatonium or a related compound.
- 86. The method of claim 85 wherein said effect on said compound is selected based on changes in intracellular ion concentration.
- 87. The method of claim 86 wherein said ion is calcium.
- 88. The method of claim 86 wherein said ion is sodium.
- 89. The method of claim 85 wherein said compound is selected based on its effect on cell membrane potential.
- 90. The method of claim 85 wherein said compound is selected based on its effect on transcription of said T2R.
- 91. The method of claim 85 wherein said compound is selected based on its ability to block interaction of said T2R with denatonium and/or 6-nitrosaccharin.
- 92. The method of claim 85 wherein said hT2R44 is operably linked to a sequence that facilitates plasma membrane export.
- 93. The method of claim 85 wherein said cell line is an HEK-293 cell line.
- 94. The method of claim 85 wherein the effect of said compound on said hT2R44 polypeptide is assayed based on change in intracellular cAMP, cGMP or IP3.
- 95. The method of claim 85 wherein said hT2R44 fragment is an extracellular domain or transmembrane region.
- 96. The method of claim 85 wherein the difference in calcium ion concentration is monitored using a reporter molecule.
- 97. The method of claim 85 wherein said reporter molecule is a fluorescent dye specific for calcium.
- 98. The method of claim 97 wherein said dye is Fluor-3, Fluo-4 or Fura-2.
- 99. The method of claim 85 wherein said hT2R44, variant or fragment is attached to a reporter molecule.
- 100. The method of claim 99 wherein said reporter is green fluorescent protein or a variant thereof.
- 101. The method of claim 76 wherein said hT2R44 polypeptide, variant or fragment is contained in solution.
- 102. The method of claim 76 wherein said hT2R44 polypeptide, variant or fragment is in a bilayer membrane, attached to a solid phase, is a lipid monolayer, or is in a vesicle.
- 103. The method of claim 76 wherein the binding of said compound is evaluated based on a change in spectroscopic characteristics, hydrodynamic characteristics, chromatographic characteristics or solubility.
- 104. The method of claim 76 wherein the compound is selected based on its effect on the complexation of said hT2R44, variant or fragment with a G protein.
- 105. The method of claim 85 wherein said compound is selected based on its effect on interaction of said hT2R44 polypeptide, variant or fragment with gustducin or transducin.
- 106. The method of claim 76 wherein said compound is selected using a fluorescence polarization assay.
- 107. The method of claim 76 wherein said hT2R44 polypeptide is attached to a solid phase substrate.
- 108. The method of claim 76 which comprises a high throughput assay.
- 109. The method of claim 85 wherein said cell line stably expresses said receptor.
- 110. The method of claim 85 wherein said cell line transiently expresses said receptor.
- 111. The method of claim 109 or 110 wherein said cell line is a mammalian or insect cell.
- 112. The method of claim 111 wherein said insect cell is an Sf9 cell.
- 113. The method of claim 76 wherein said assay is a GTPγS binding assay.
- 114. The method of claim 113 wherein said assay uses transducin or gustducin as the G protein.
- 115. The method of claim 112 wherein said receptor is expressed using a baculovirus expression system.
- 116. The method of claim 85 wherein the ability of said compound to inhibit hT2R44 bitter taste is further confirmed by a taste test.
RELATED APPLICATIONS
[0001] This application claims priority to provisional application U.S. Serial No. 60/303,811 filed on Jul. 10, 2001 and provisional application U.S. Serial No. 60/372,089, filed on Apr. 15, 2002 both of which are incorporated by reference in their entirety herein.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60303811 |
Jul 2001 |
US |
|
60372089 |
Apr 2002 |
US |