Assays for identifying compounds that modulate bitter taste

Patent metadata

Field	Value
Publication number	US-9347934-B2
Application number	US-201214352620-A
Country	US
Kind code	B2
Filing date	Oct 22, 2012
Priority date	Oct 20, 2011
Publication date	May 24, 2016
Grant date	May 24, 2016

Abstract

The present invention is based on applicants' discovery, disclosed herein, of agonists for the TAS2R receptors TAS2R1, TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R16, TAS2R44, TAS2R46, and TAS2R60. The assignment of agonists to these receptors makes assays for identifying compounds that modulate bitter taste possible. For example, the present invention provides methods of identifying compounds that inhibit the bitter taste due to these agonists. The present invention also provides methods of identifying compounds that selectively inhibit the bitter taste due to these agonists. The present invention further provides methods of identifying compounds that mimic the bitter taste due these agonists. The present invention also provides methods of identifying compounds that enhance the bitter taste due to these agonists.

First claim

What is claimed:

1. A method for identifying a compound that inhibits the bitter taste due to KCl, comprising:
a) providing a first and a second cell,
wherein said first and second cell express one or more bitter taste receptors selected from the group consisting of:
TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R44 and TAS2R60, wherein said first and second cell express the same one or more bitter taste receptors;
b) contacting said first cell with a tastant,
wherein the tastant activates the one or more bitter taste receptor;
c) contacting said second cell with a test compound and the tastant;
d) assaying said first and second cells for bitter taste receptor activation; and
e) comparing the bitter taste receptor activation of said first cell to the bitter taste receptor activation of said second cell,
wherein the test compound is an inhibitor of bitter taste due to KCl, if bitter taste receptor activity of said second cell is less than the bitter taste receptor activity of said first cell, and wherein the tastant is KCl.

2. The method of claim 1 , wherein the bitter taste receptor is complexed to a G protein.

3. The method according to claim 2 , wherein said G protein is a Gq protein, an alpha transducin or an alpha gustducin.

4. The method according to claim 3 , wherein the Gq protein is a Gα15 protein.

5. The method according to any one of claims 1 - 4 , wherein bitter taste receptor activity is determined by measuring intracellular calcium concentration.

6. The method according to claim 5 , wherein the concentration of intracellular calcium is determined using a calcium-sensitive fluorescent dye.

7. The method according to claim 6 , wherein the calcium-sensitive fluorescent dye is selected from the group consisting of Indo-1, Fura-2, Fluo-3, Fluo-4, Rhod-2, Rhod-5N, Calcein, Calcein blue, cytoCalcein Violet, Quin-2, Quest Fluo-8H™, Quest Fluo 8L™, Quest Fluo 8™, Quest Rhod-4™, coelenterazine and Calcium-3.

8. The method according to claim 1 , wherein said first and second cells are present in in vitro cell lines.

9. The method according to claim 1 , wherein said first and second cells are present in panels of in vitro cell lines.

10. A method for identifying a compound that selectively acts on receptors to inhibit the bitter taste due to KCl comprising:
a) providing a first and second panel of cell lines,
wherein each of said first and second panel comprises cell lines that express a bitter taste receptor selected from the group consisting of: TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R44 and TAS2R60;
wherein TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R44 and TAS2R60 are each expressed in at least one cell line, and
wherein the first and second panels comprise the same cell lines;
b) contacting each cell line in the first panel with a tastant,
wherein the tastant activates at least two of the group selected from TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R44 and TAS2R60;
c) contacting each cell line in the second panel with a test compound and the tastant;
d) assaying each cell line for bitter taste receptor activation;
wherein, the test compound is a selective inhibitor of bitter taste due to KCl if the bitter taste receptor activity of at least two of the group selected from TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R44 and TAS2R60 is less in the second panel compared to the first panel, and wherein the tastant is KCl.

11. The method of claim 10 , wherein the bitter taste receptor activity of at least three of the group selected from TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R44 and TAS2R60 is less in the second panel compared to the first panel.

12. The method of claim 10 , wherein the bitter taste receptor activity of at least four of the group selected from TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R44 and TAS2R60 is less in the second panel compared to the first panel.

13. The method of claim 10 , wherein the bitter taste receptor activity of at least five of the group selected from TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R44 and TAS2R60 is less in the second panel compared to the first panel.

14. The method of claim 10 , wherein the bitter taste receptor activity of each member in the group selected from TAS2R4, TAS2R9, TAS2R13, TAS2R14, TAS2R44 and TAS2R60 is less in the second panel compared to the first panel.

15. The method of any one of claims 10 - 14 , wherein the first and second panel comprise TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R8, TAS2R9, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R38, TAS2R39, TAS2R40, TAS2R41, TAS2R43, TAS2R44, TAS2R45, TAS2R46, TAS2R47, TAS2R48, TAS2R49, TAS2R50, TAS2R55, and TAS2R60 bitter taste receptors.

16. The method of claim 10 , wherein the bitter taste receptor is complexed to a G protein.

17. The method according to claim 16 , wherein said G protein is a Gq protein, an alpha transducin or an alpha gustducin.

18. The method according to claim 17 , wherein the Gq protein is a Gα15 protein.

19. The method according to claim 10 , wherein bitter taste receptor activity is determined by measuring intracellular calcium concentration.

20. The method according to claim 19 , wherein the concentration of intracellular calcium is determined using a calcium-sensitive fluorescent dye.

21. The method according to claim 20 , wherein the calcium-sensitive fluorescent dye is selected from the group consisting of Indo-1, Fura-2, Fluo-3, Fluo-4, Rhod-2, Rhod-5N, Calcein, Calcein blue, cytoCalcein Violet, Quin-2, Quest Fluo-8H™, Quest Fluo 8L™, Quest Fluo 8™, Quest Rhod-4™, coelenterazine and Calcium-3.

Assignees

• Chromocell Corp
• Kraft Foods Group Brands Llc

Inventors

• Shekdar Kambiz
• Shah Purvi Manoj
• Gunnet Joseph
• Leland Jane V
• Brown Peter H
• Slade Louise

Classifications

• G01N2333/705

  Assays involving receptors, cell surface antigens or cell surface determinants · CPC title

• G01N33/5044 Primary

  involving specific cell types · CPC title

• G01N33/5041 Primary

  involving analysis of members of signalling pathways · CPC title

• G01N2333/726

  G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH · CPC title

• G01N33/74

  involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors} · CPC title