T1R hetero-oligomeric taste receptors, cell lines that express said receptors, and taste compounds

Patent metadata

Field	Value
Publication number	US-10352929-B2
Application number	US-201615244135-A
Country	US
Kind code	B2
Filing date	Aug 23, 2016
Priority date	Aug 6, 2003
Publication date	Jul 16, 2019
Grant date	Jul 16, 2019

Abstract

The invention relates to compounds that specifically bind a T1R1/T1R3 or T1R2/T1R3 receptor or fragments or sub-units thereof. The present invention also relates to the use of hetero-oligomeric and chimeric taste receptors comprising T1R1/T1R3 and T1R2/T1R3 in assays to identify compounds that respectively respond to umami taste stimuli and sweet taste stimuli. Further, the invention relates to the constitutive of cell lines that stably or transiently co-express a combination of T1R1 and T1R3; or T1R2 and T1R3; under constitutive or inducible conditions. The use of these cells lines in cell-based assays to identify umami and sweet taste modulatory compounds is also provided, particularly high throughput screening assays that detect receptor activity by use of fluorometric imaging.

First claim

What is claimed:

1. A method of identifying one or more non-naturally occurring compounds that putatively enhance sweet taste perception comprising
(i) screening one or more non-naturally occurring compounds in a first binding assay that identifies whether said one or more non-naturally occurring compounds specifically bind to a T1R2/T1R3 receptor composed of hT1R2/hT1R3,
(ii) conducting a second binding assay using compounds identified in the first binding assay to determine whether said identified non-naturally occurring compounds bind to rT1R2/rT1R3,
(iii) selecting non-naturally occurring compounds that putatively enhance sweet taste perception based on their specific binding to a T1R2/T1R3 receptor composed of hT1R2/hT1R3 in the binding assay of step (i) and their absence of specific binding to rT1R2/rT1R3 in the binding assay of step (ii), and
(iv) screening compounds which are selected in step (iii) for their ability to enhance sweet taste in a human or non-human animal taste test and further wherein said method includes one or more of the following steps:
a. further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to hT1R2/hT1R3 and rT1R2/r3-h3 but not to rT1R2/rT1R3 or to hT1R2/h3-r3;
b. further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to hT1R2/hT1R3 and r2-h2/rT1R3 but not to rT1R2/rT1R3 or to h2-r2/hT1R3;
c. further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the Venus Flytrap Domain (VFD) of T1R2 of the hT1R2/hT1R3 and hT1R2/rT1R3 receptor;
d. further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to amino acid residues 144 and 302 of the human N-terminal Venus flytrap domain of the T1R2 subunit of the T1R2/T1R3 receptor;
e. further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the Transmembrane Domain (TM) of T1R2 of the hT1R2/hT1R3 receptor;
f. further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the human N-terminal extracellular domain of the T1R3 subunit of the T1R2/T1R3;
g. further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the Venus Flytrap Domain (VFD) of T1R3 of the hT1R2/hT1R3 receptor;
h. further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the to the Transmembrane Domain (TM) of T1R3 of the hTIR2/hTIR3 receptor; or
i. further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to extracellular loop 2 and extracellular loop 3 of the human transmembrane domain of the T1R3 subunit of T1R2/T1R3.

2. The method of claim 1 , wherein another binding assay is conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to a T1R2/T1R3 receptor composed of rT1R2/hT1R3.

3. The method of claim 1 , wherein another binding assay is conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically binds to the N-terminal extracellular domain of T1R2 of the hT1R2/hT1R3 receptor.

4. The method of claim 2 , wherein another binding assay is conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically binds to a T1R2/T1R3 receptor composed of rT1R2/hT1R3 but not hT1R2/rT1R3.

5. The method of claim 1 , which includes further binding assays conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to hT1R2/hT1R3 and rT1R2/r3-h3 but not to rT1R2/rT1R3 or to hT1R2/h3-r3.

6. The method of claim 1 , which includes further binding assays conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to hT1R2/hT1R3 and r2-h2/rT1R3 but not to rT1R2/rT1R3 or to h2-r2/hT1R3.

7. The method of claim 1 , which includes further binding assays conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the Venus Flytrap Domain (VFD) of T1R2 of the hT1R2/hT1R3 and hT1R2/rT1R3 receptor.

8. The method of claim 1 , which includes further binding assays conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to amino acid residues 144 and 302 of the human N-terminal Venus flytrap domain of the T1R2 subunit of the T1R2/T1R3 receptor.

9. The method of claim 1 , which includes further binding assays conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the Transmembrane Domain (TM) of T1R2 of the hT1R2/hT1R3 receptor.

10. The method of claim 1 , which includes further binding assays conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the human N-terminal extracellular domain of the T1R3 subunit of the T1R2/T1R3.

11. The method of claim 1 , which includes further binding assays conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the Venus Flytrap Domain (VFD) of T1R3 of the hT1R2/hT1R3 receptor.

12. The method of claim 1 , which includes further binding assays conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to the to the Transmembrane Domain (TM) of T1R3 of the hTIR2/hTIR3 receptor.

13. The method of claim 1 , which includes further binding assays conducted to assay whether the selected non-naturally occurring compounds of (iii) specifically bind to extracellular loop 2 and extracellular loop 3 of the human transmembrane domain of the T1R3 subunit of T1R2/T1R3.

14. The method of claim 1 , wherein assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) demonstrate compound-dependent increase in fluorescence with an activity compared to the maximal activity for fructose of at least 25% in a fluorescence-based assay using a FLIPR (Molecular Devices) instrument.

15. The method of claim 1 , wherein further binding assays are conducted to assay whether the selected non-naturally occurring compounds of (iii) demonstrate a compound-dependent decrease in the EC 50 for a sweetener by at least two-fold in a fluorescence-based assay using a FLIPR (Molecular Devices) instrument.

16. The method of claim 1 , wherein in step (iv) one or more of the identified non-naturally occurring compounds are assessed in a human taste test.

17. The method of claim 1 , wherein one or more of the identified compounds are added to a comestible composition.

Assignees

• Senomyx Inc

Inventors

• Li Xiaodong
• Luukkonen Lena
• Xu Hong

Classifications

• A23L27/202

  Aliphatic compounds · CPC title

• A23L27/2054

  having nitrogen as the only hetero atom · CPC title

• C07D307/52

  Radicals substituted by nitrogen atoms not forming part of a nitro radical · CPC title

• C07D333/38

  Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals · CPC title

• C07D405/12

  linked by a chain containing hetero atoms as chain links · CPC title