US20190277845A1

US20190277845A1 - Screening assays to identify compounds which modulate t1r associated taste modalities which eliminate false positives

Info

Publication number: US20190277845A1
Application number: US16/363,106
Authority: US
Inventors: Guy Servant; Nicole Servant
Original assignee: Firmenich Inc
Current assignee: Firmenich Inc
Priority date: 2013-03-15
Filing date: 2019-03-25
Publication date: 2019-09-12
Also published as: US9804157B1; US20180106801A1; US10281467B2

Abstract

This invention relates to assays which screen for compounds that modulate taste elicited by the T1R2/T1R3 sweet taste receptor which include a novel counter screen to eliminate false positives. In addition, the invention contemplates assays which screen for compounds that modulate taste elicited by the T1R1/T1R3 umami taste receptor which include a novel counter screen to eliminate false positives. Preferably the assays are conducted in high throughput format thereby enabling the screening of many hundreds of different compounds whereby the counter screen significantly improves assay efficiency. Further, the invention relates to the use of the compounds identified in the subject screening assays to modulate T1R associated taste perception.

Description

This application claims priority to U.S. Ser. No. 61/789,993 filed Mar. 15, 2013. The contents of said application are incorporated by reference in its entirety herein.

FIELD OF THE INVENTION

This invention relates to assays which screen for compounds that modulate the T1R2/T1R3 sweet taste receptor which include a novel counter screen to eliminate false positives. In addition, the invention contemplates assays which screen for compounds that modulate the T1R1/T1R3 umami taste receptor which include a novel counter screen to eliminate false positives. Preferably the assays are conducted in high throughput format thereby enabling the screening of many hundreds of different compounds whereby the counter screen significantly improves assay efficiency. Further, the invention relates to the use of the compounds identified in the subject screening assays to modulate T1R associated taste perception.

BACKGROUND OF THE INVENTION

Researchers affiliated with the Assignee Senomyx as well as a research group at the University of California have previously reported the identification and functionalization of a family of G-protein coupled receptors involved in mammalian taste referred to as the T1R family. This family of taste receptors consists of 3 members, T1R1, T1R2 and T1R3. These receptors are expressed in different tissues including the tongue, organs in the digestive system as well as in other types of tissues and modulate sweet and umami taste.
In particular, these entities have demonstrated using mammalian cells that express the T1R1 and T1R3 as well as a suitable G protein, e.g., a promiscuous G protein or a chimeric G protein, that the T1R1 and T1R3 receptors dimerize to form a heteromeric taste receptor comprising T1R1 and T1R3 polypeptides that responds to umami (savory) taste compounds. These same entities have similarly demonstrated that when the T1R2 and T1R3 polypeptides are expressed in a cell that comprises a suitable G protein, e.g., a promiscuous G protein or a chimeric G protein, that the T1R2 and T1R3 polypeptides dimerize to form a heteromeric taste receptor comprising the T1R2 and T1R3 polypeptides that responds to sweet taste compounds. This research is reported in various scientific articles including Li et al., Proc. Natl. Acad. Sci, USA 2002, April 12 99(7)4692-4696; Xu et al., Proc. Natl. Acad. Sci, USA 2004, September 28, 101(36)14652863; as well as numerous patents assigned to Senomyx and the University of California.
Also, these entities and others have disclosed the use of recombinant and endogenous cells which co-express either T1R2 and T1R3 polypeptides or T1R1 and T1R3 polypeptides to respectively identify compounds that modulate sweet or umami taste. Also, T1R polypeptides have been suggested to be involved in glucose transport, food sensing and motility, and insulin responses.
Assays using T1R2/T1R3 and T1R1/T1R3 heteromers have resulted in the identification of numerous compounds that modulate sweet or umami taste some of which have been approved for use in foods for human consumption. While these assays are predictive as to the potential effect of the identified compounds on a particular taste modality associated with T1R-associated taste, i.e., sweet or umami taste, one recurring problem which has been observed is that the “hits”, i.e., the group of identified T1R2/T1R3 or T1R1/T1R3 modulatory compounds often contain “false positives”. That is to say, while the compounds demonstrably modulate the activity of the sweet or umami heteromeric taste receptor in vitro, they do not elicit a demonstrable or desired effect on sweet or umami taste in humans.
One way of potentially alleviating such “false positives” is the use of cells that endogenously express the T1R2/T1R3 or T1R1/T1R3 receptors rather than recombinant cells as these cells may be less susceptible to interacting with non-physiologically relevant compounds. The present invention provides another means for eliminating such “false positives” in screening assays using the T1R2/T1R3 sweet receptor which has been demonstrated with numerous compounds to be highly reliable.

BRIEF DESCRIPTION AND OBJECTS OF THE INVENTION

It is an object of the invention to improve the efficiency of screening assays for identifying compounds that modulate sweet taste in humans and potentially other animals such as rodents, dogs, cats, and other animals used in agriculture.
More specifically, is an object of the invention to provide improved screening assays for identifying compounds that modulate sweet taste in humans and potentially other animals such as rodents, dogs, cats, and other animals used in agriculture, wherein the improvement substantially reduces the number of “false positives”.
More specifically, it is an object of the invention to conduct screening assays to identify compounds that modulate sweet taste wherein the assay comprises (i) conducting a binding or functional assay to identify compounds that bind to or modulate the activity of the T1R2/T1R3 receptor, (ii) further conducting a counter-screen to assess whether the positive “hits” in step (i) bind to and/or modulate the activity of homomeric T1R2; (iii) if said compounds bind to and/or modulate the activity of homomeric T1R2, identifying such compounds as likely “false positives” which will not elicit a desired effect on sweet taste.
It is also an object of the invention to conduct screening assays to identify compounds that modulate umami taste wherein the assay comprises (i) conducting a binding or functional assay to identify compounds that bind to or modulate the activity of the T1R1/T1R3 receptor, (ii) further conducting a counter-screen to assess whether the positive “hits” in step (i) bind to and/or modulate the activity of homomeric T1R1; (iii) if said compounds bind to and/or modulate the activity of homomeric T1R1, identifying such compounds as likely “false positives” which will not elicit a desired effect on umami taste.
It is also an object of the invention to use the compounds identified by the present screening assays for the development of flavorants for use in different foods, beverages, medicaments or comestibles for human or animal consumption, as well as to synthesize optimized variants or derivatives of these compounds for use in different foods, beverages, medicaments or comestibles for human or animal consumption.

SUMMARY OF THE INVENTION

The present invention provides a novel use of hT1R2 to identify compounds which while activating the T1R2/T1R3 taste receptor in in vitro screening assays, may exhibit poor effects in human taste tests. Particularly, the present invention provides the use of homomeric T1R2 in screening assays to eliminate or reduce the number of false positives identified in screening assays using heteromeric T1R2/T1R3 taste receptors.
Also, the present invention provides a novel use of hT1R1 to identify compounds which while activating the T1R1/T1R3 taste receptor in in vitro screening assays, may exhibit poor effects in human taste tests. Particularly, the present invention contemplates the use of homomeric T1R1 in screening assays to eliminate or reduce the number of false positives identified in screening assays using heteromeric T1R1/T1R3 taste receptors.
As noted above, the human sweet taste receptor requires two subunits, named T1R2 and T1R3, to function and be activated by known sweeteners. Recently, it was allegedly reported in U.S. Pat. No. 8,124,360, by Slack, assigned to Givaudan, that homomeric T1R2 (defined as a monomer, dimer or oligomer of the T1R2 polypeptide) functionally responds to Perillartine, a synthetic oxime that is approximately 370-times more potent/sweet than sucrose). The reference does not contain any data indicating other sweet compounds activate T1R2. Based on their purported results with Perillartine, the patent suggests that T1R2 monomers activated by Perillartine may be used in direct functional screens to identify compounds that modulate sweet taste signaling and which modulate sweet taste in humans.
The present inventors recently screened cells that express T1R2 and which do not express T1R3 and discovered several compounds that effectively activate the hT1R2 subunit when expressed in the absence of T1R3 in heterologous cells. Based on this activation, these compounds were evaluated in human taste tests. As disclosed infra in the working examples, when tested the identified compounds which activated hT1R2 expressed in the absence of T1R3 behaved poorly in human taste tests. Particularly, they were much less potent then what would have been predicted from their potency measured with cells expressing the sweet taste receptor hT1R2/hT1R3.
Based on these results, the inventors hypothesized that cells expressing hT1R2 and hT1R3 likely express at least two types of functional receptors, most likely both dimers, but potentially an active monomer or other heteromer or oligomer. Whereas the heterodimer hT1R2/hT1R3 responds to compounds that are physiologically relevant, it was theorized that the homodimer form of T1R2 (i.e., hT1R2/hT1R2 or potentially a T1R2 monomer or oligomer or heteromer comprising T1R2 subunits) responds to compounds that are not physiologically relevant and based thereon will not elicit a favorable response in human or other animal taste tests. Based on these hypotheses it was further theorized that an hT1R2 homomeric assay, instead of purportedly being useful to identify sweet taste modulatory compounds, instead may be useful in identifying compounds, discovered using an hT1R2/hT1R3 assay that will ultimately behave poorly in human taste tests. That is to say the assay using the homomeric form of T1R2 will function as a counter-screen to identify “false positives” in the T1R2/T1R3 screening assays, i.e., compounds which apparently modulate T1R2/T1R3 in in vitro screening assays, but which elicit poor properties in taste tests.
In fact, the inventors' hypotheses were confirmed. As taught in Examples 1-7 infra, compounds identified as agonists in a fluorometric cell-based hT1R2/hT1R3 assay which detects receptor activity by detecting levels of intracellular calcium were screened using the same fluorometric cell-based assays, but instead using cells that only express T1R2. Seventeen compounds or sweeteners which had been identified as agonists in the hT1R2/hT1R3 assay and showing a good correlation between taste data and assay data (within a 95% confidence interval) were inactive in the hT1R2 assay. Conversely, six compounds identified as agonists in the hT1R2/hT1R3 assay were found to behave poorly in human taste tests or to be tasteless were active in the hT1R2 assay. Based on these results homomeric T1R2 assays may be used in screening assays as an adjunct to T1R2/T1R3 screening assays as a counter-screen, i.e., to eliminate false positives, e.g., compounds which are active in the T1R2/T1R3 screens, but which elicit poor results in taste tests.
Based thereon, the present invention is preferably directed to improve T1R2/T1R3 modulator screening assays, preferably cell-based functional or binding T1R2/T1R3 screening assays, wherein the improvement includes use of a counter-screen that eliminates false positives. The counter screen comprises conducting cell-based screening assays with compounds identified to modulate T1R2/T1R3 using cells that express T1R2 in the absence of T1R3. Preferably these assays are fluorometric assays using the FLIPR system. However, it is contemplated that other T1R binding assays or functional assays used to identify potential T1R modulators may be used. Such assays are disclosed herein. The use of this counter-screen should minimize false positives based on their binding or modulation of the activity of the homomeric T1R2 and thereby facilitate the discovery and development of compounds that modulate sweet taste.
In addition, the present invention encompasses improved T1R1/T1R3 modulator screening assays, preferably cell-based functional or binding assays screening for T1R1/T1R3 modulators, wherein the improvement includes use of a counter-screen that eliminates false positives. Similarly, the T1R1 counter screen will comprise conducting cell-based screening assays with compounds identified to modulate T1R1/T1R3 using cells that express T1R1 in the absence of T1R3. Preferably these assays are fluorometric assays using the FLIPR system. Such assays are disclosed herein. However, it is contemplated that other T1R binding assays or functional assays used to identify potential T1R1/T1R3 modulators. Similarly, this counter-screen should minimize false positives based on their binding or modulation of the activity of the homomeric T1R1 and thereby facilitate the discovery and development of compounds that modulate umami taste.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound A, a sweet taste receptor agonist known to bind to the transmembrane domain of hT1R2.

FIG. 2 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound B, a sweet taste receptor agonist.

FIG. 3 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound C, a sweet taste receptor agonist.

FIG. 4 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound D, a sweet taste receptor agonist.

FIG. 5 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by Perillartine, a sweet taste receptor agonist.

FIG. 6 contains the results of an experiment wherein cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound P-4000, a sweet taste receptor agonist.

FIG. 7 contains potency data at different molar ratios in comparison to sucrose for 17 compounds or sweeteners identified as agonists in the hT1R2/hT1R3 assay in comparison to their relative sweetness to sucrose in human sweet taste tests.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed supra, the present invention describes a novel use of hT1R2 to identify compounds which while activating the T1R2/T1R3 taste receptor in in vitro screening assays, exhibit poor effects in human taste tests. Particularly, the present invention provides the use of homomeric T1R2 in screening assays to eliminate or reduce the number of false positives identified in screening assays using heteromeric T1R2/T1R3 taste receptors.
Also, based on the results with T1R2, the present invention further contemplates a novel use of hT1R1 to identify compounds which while activating the T1R1/T1R3 taste receptor in in vitro screening assays, exhibit poor effects in human taste tests. Particularly, the present invention provides the use of homomeric T1R1 in screening assays to eliminate or reduce the number of false positives identified in screening assays using heteromeric T1R1/T1R3 taste receptors.
As noted above, the human sweet taste receptor requires two subunits, named T1R2 and T1R3, to function and be activated by known sweeteners. Likewise, the human umami taste receptor requires two subunits, named T1R1 and T1R3, to function and be activated by known umami compounds. Recently, it was reported in U.S. Pat. No. 8,124,360, by Slack, and assigned to Givaudan that homomeric T1R2 (defined as a monomer, dimer or oligomer of the T1R2 polypeptide) functionally responds to Perillartine, a synthetic oxime that is approximately 370-times more potent/sweet than sucrose). The reference does not contain any data indicating other sweet compounds activate T1R2. However, based on their purported results with Perillartine, the patent suggests that T1R2 monomers activated by Perillartine may be used in direct functional screens to identify compounds that modulate sweet taste signaling and which modulate sweet taste in humans. Also, it has been reported that homomeric T1R3 binds to some sweet compounds.
The present inventors recently screened cells that express T1R2 and which do not express T1R3 and discovered several compounds that effectively activate the hT1R2 subunit when expressed on its own in heterologous cells. Based on this activation, these compounds were evaluated in human taste tests. It was initially predicted that such compounds might elicit sweet taste. However, when tested the identified compounds which activated hT1R2 expressed in the absence of T1R3 behaved poorly in human taste tests, being much less potent then what would have been expected from their potency measured with cells expressing the sweet taste receptor hT1R2/hT1R3.
Based on these results, the inventors hypothesized that cells expressing hT1R2 and hT1R3 likely express at least two types of functional receptors, most likely both dimers, however potentially an active monomer, oligomer or other heteromer. It was predicted that whereas the heterodimer hT1R2/hT1R3 responds to compounds that are physiologically relevant; that the homodimer form of T1R2 (i.e., hT1R2/hT1R2 or potentially a T1R2 monomer or oligomer comprising T1R2 subunits) responds to compounds that are not physiologically relevant and based thereon will not elicit a favorable response in human or other animal taste tests.
Based on these hypotheses it was further theorized that an hT1R2 homomeric assay, instead of being useful to identify sweet taste modulatory compounds, instead may serve an opposite purpose. Particularly, the homomeric T1R2 may be useful in identifying compounds, discovered using an hT1R2/hT1R3 functional or binding assay, and that will ultimately behave poorly in human taste tests. That is to say the assay using the homomeric form of T1R2 will function as a counter-screen to identify “false positives” in the T1R2/T1R3 screening assays, i.e., compounds which apparently modulate T1R2/T1R3 in in vitro screening assays, but which elicit poor properties in taste tests.
As substantiated by the examples, the inventors' hypotheses were confirmed. Compounds identified as agonists in a fluorometric cell-based hT1R2/hT1R3 assay which detects receptor activity by detecting levels of intracellular calcium were screened using the same fluorometric cell-based assays, but instead using cells that only express T1R2. Seventeen compounds or sweeteners identified as agonists in the hT1R2/hT1R3 assay and showing a good correlation between taste data and assay data (within a 95% confidence interval) were inactive in the hT1R2 assay. Conversely, six compounds identified as agonists in the hT1R2/hT1R3 assay were found to behave poorly in human taste tests or to be tasteless were active in the hT1R2 assay. Based on these results homomeric T1R2 assays may be used in screening assays as an adjunct to T1R2/T1R3 screening assays as a counter-screen, i.e., to eliminate false positives, e.g., compounds which are active in the T1R2/T1R3 screens, but which elicit poor results in taste tests.
Based thereon, the present invention is preferably directed to improve T1R2/T1R3 modulator screening assays, preferably cell-based functional or binding T1R2/T1R3 screening assays, wherein the improvement includes use of a counter-screen that eliminates false positives. The counter screen comprises conducting screening assays, preferably cell-based, with compounds identified to modulate activity in T1R2/T1R3 screening assays, using T1R2 in the absence of T1R3. Preferably these assays are cell-based fluorimetric assays using the FLIPR system. However, it is contemplated that other T1R binding assays or functional assays used to identify potential T1R modulators may be used. Suitable examples of such assays are disclosed herein and are known in the art. The use of this T1R2 counter-screen should minimize false positives based on their binding or modulation of the activity of the homomeric T1R2 and thereby facilitate the discovery and development of compounds that modulate sweet taste.
In addition, the present invention encompasses improved T1R1/T1R3 modulator screening assays, preferably cell-based functional or binding assays screening for T1R1/T1R3 modulators, wherein the improvement includes use of a counter-screen that eliminates false positives. Similarly, the counter screen will comprise conducting cell-based screening assays with compounds identified to modulate activity in T1R1/T1R3 screening assays, using T1R1 in the absence of T1R3. Preferably these assays are cell-based fluorimetric assays using the FLIPR system. Such assays are disclosed herein. However, it is contemplated that other T1R binding assays or functional assays used to identify potential T1R1/T1R3 modulators. Similarly, this counter-screen should minimize false positives based on their binding or modulation of the activity of the homomeric T1R1 and thereby facilitate the discovery and development of compounds that modulate umami taste.
More specifically, the present invention also provides assays, preferably high throughput assays, which include a counter screen to identify molecules that interact with and/or modulate T1R2/T1R3 or T1R1/T1R3 polypeptide hetero-oligomeric complexes. These assays typically may use intact T1R polypeptides and heteromers containing. However, it is further contemplated that the initial heteromer screening assays and potentially the counter screen assays may be conducted with monomers or heteromeric T1Rs comprised of chimeric T1R subunits, i.e., comprising the extracellular domain of a T1R and the transmembrane region of another GPCR such as the calcium sensing receptor, or potentially another species T1R or the extracellular domain of a particular GPCR, potentially another species T1R or that of another GPCR such as the calcium sensing receptor and the transmembrane region of a T1R. It has been shown that heteromers comprised of T1R2/T1R3 heteromers and T1R1/T1R3 heteromers comprised of chimeric T1R2/T1R3 subunits or chimeric T1R1/T1R3 subunits are functional and may be used to isolate ligands, agonists, antagonists, or any other molecules that can bind to and/or modulate the activity of a T1R polypeptide. Also, the present invention contemplates assays, preferably high throughput assays, which include a counter screen to identify molecules that interact with and/or modulate T1R2/T1R3 or T1R1/T1R3 polypeptide hetero-oligomeric complexes, wherein the T1R subunits may comprise variants or fragments of a particular T1R, preferably human or rodent T1R2, T1R3 or T1R1, which are functional (bind or functionally respond to sweet or umami compounds). These T1R variants include T1R1, T1R2 and T1R3 polypeptides that are at least 80, 90, 95, 96, 97, 98, or at least 99% identical to particular native T1R sequences including those disclosed in the Sequence Listing and known in the art. For example. The nucleic acid and polypeptide sequences of different species T1R1, T1R2 and T1R3 including human, mouse, rat, cat and dog are in the public domain.

Definitions

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
“Taste cells” herein include any cell, which taste cell may be an endogenous T1R expressing cell or may comprise a recombinant cell that expresses at least one T1R1, T1R2 or T1R3 subunit, preferably human or rodent T1R subunits and typically a G protein that functionally couples to the T1R subunit or T1R subunits expressed in the taste cell. This includes by way of example neuroepithelial cells that are organized into groups to form taste buds of the tongue, e.g., foliate, fungiform, and circumvallate cells (see, e.g., Roper et al., Ann. Rev. Neurosci. 12:329-353 (1989)). Taste cells are also found in the palate and other tissues, such as the esophagus and the stomach as well as being expressed in other tissues and organs including by way of example those comprised in other tissues of the gastrointestinal system, the urinary system, the nervous system, the reproductive system, the skin and others.
“T1R” refers to one or more members of a family of G protein-coupled receptors that are expressed in taste cells such as foliate, fungiform, and circumvallate cells, as well as cells of the palate, and esophagus (see, e.g., Hoon et al., Cell, 96:541-551 (1999), herein incorporated by reference in its entirety). The current nomenclature for the T1R family members is T1R1, T1R2 and T1R3. Members of this family are also referred to as GPCR-B3 and TR1 in WO 00/06592 as well as GPCR-B4 and TR2 in WO 00/06593. T1R1 has also been referred to as GPCR-B3, and T1R2 as GPCR-B4. T1R family members are involved in sweet and umami taste perception.
“T1R's” are comprised in a family of GPCRs with seven transmembrane regions that have “G protein-coupled receptor activity,” e.g., they may bind to G proteins in response to extracellular stimuli and promote production of second messengers such as IP3, cAMP, cGMP, and Ca²⁺ via stimulation of enzymes such as phospholipase C and adenylate cyclase (for a description of the structure and function of GPCRs, see, e.g., Fong, supra, and Baldwin, supra). Like some other GPCRs the T1Rs dimerize to produce functional taste receptors, i.e., the T1R1 and T1R3 subunits dimerize to produce the umami taste receptor and the T1R2 and T1R3 subunits dimerize to produce the sweet taste receptor.
The term “T1R” herein includes polymorphic variants, alleles, mutants, and interspecies homologs of T1R1, T1R2 and T1R3 that: (1) have at least about 70-80% amino acid sequence identity, optionally about 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity to any of the T1R polypeptide sequences contained in the Sequence Listing or known in the art over a window of about 25 amino acids, optionally 50-100 amino acids, and more typically over at least 200, 300, 400, 500, 600 amino acids, or the entire length of the particular T1R polypeptide or at least the extracellular or transmembrane region thereof; (2) polypeptides which are encoded by a nucleic acid molecule encoding a T1R nucleic acid contained in the Sequence Listing or known in the art; or (iii) a T1R polypeptide encoded by a nucleic acid which specifically hybridizes under stringent hybridization conditions to the complement of any of the T1R nucleic acid sequences contained in the Sequence Listing or known in the art or a fragment thereof which is at least about 100, optionally at least about 200, 300, 400, or at least 500-1000 nucleotides and (3) chimeric polypeptides that comprise a polypeptide which is at least 90% identical to the extracellular region of a particular T1R and the transmembrane region of another GPCR other than said T1R and chimeric polypeptides that comprise a polypeptide which is at least 90% identical to the transmembrane region of a T1R and the extracellular region of another GPCR other than said T1R.
“Homomeric T1R2” herein refers to a T1R2 polypeptide defined as above, or a chimeric T1R2 polypeptide, that is expressed in the absence of a T1R3 polypeptide or which is in a composition that does not contain a T1R3 polypeptide. This potentially includes T1R2/T1R2 dimers, monomeric T1R2, as well as other oligomers of T1R2 or heteromers of T1R2 not comprising T1R3. Preferably such homomeric T1R2 will functionally respond to Perillartine and/or P-4000. Homomeric T1R2 is preferably obtained by the expression of a T1R2 in a cell that does not express T1R3, and which expresses a G protein such as a promiscuous G protein, e.g., Gα15 or Gα16, or transducin, gustducin, or a chimera of any of these G proteins, preferably a chimera of Gα15 or Gα16 and transducin or gustducin wherein the C-terminal 25-44 amino acids are the same as in gustducin or transducin.
“Homomeric T1R1” herein refers to a T1R1 polypeptide defined as above, or a chimeric T1R1 polypeptide, that is expressed in the absence of a T1R3 polypeptide or which is in a composition that does not contain a T1R3 polypeptide. This potentially includes T1R1/T1R1 dimers, monomeric T1R1, as well as other oligomers of T1R1 or heteromers of T1R1 not comprising T1R3.
Topologically, chemosensory GPCRs including T1Rs have an “N-terminal domain;” “extracellular domains;” “transmembrane domains” comprising seven transmembrane regions, and corresponding cytoplasmic, and extracellular loops; “cytoplasmic domains,” and a “C-terminal domain” (see, e.g., Hoon et al., Cell, 96:541-551 (1999); Buck & Axel, Cell, 65:175-187 (1991)). These domains can be structurally identified using methods known to those of skill in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains (see, e.g., Stryer, Biochemistry, (3rd ed. 1988); see also any of a number of Internet based sequence analysis programs, such as those found at dot.imgen.bcm.tmc.edu). Such domains are useful for making chimeric proteins and for in vitro assays of the invention, e.g., ligand-binding assays.
“Extracellular domains” therefore refers to the domains of T1R polypeptides that protrude from the cellular membrane and are exposed to the extracellular face of the cell. Such domains generally include the “N terminal domain” that is exposed to the extracellular face of the cell, and optionally can include portions of the extracellular loops of the transmembrane domain that are exposed to the extracellular face of the cell, i.e., the loops between transmembrane regions 2 and 3, between transmembrane regions 4 and 5, and between transmembrane regions 6 and 7.
The “N-terminal domain” region starts at the N-terminus and extends to a region close to the start of the transmembrane domain. More particularly, in one embodiment of the invention, this domain starts at the N-terminus and ends approximately at the conserved glutamic acid at amino acid position 563 plus or minus approximately 20 amino acids. These extracellular domains are useful for in vitro ligand-binding assays, both soluble and solid phase. In addition, transmembrane regions, described below, can also bind ligand either in combination with the extracellular domain, and are therefore also useful for in vitro ligand-binding assays.
“Transmembrane domain,” which comprises the seven “transmembrane regions,” refers to the domain of T1R polypeptides that lies within the plasma membrane, and may also include the corresponding cytoplasmic (intracellular) and extracellular loops. In one embodiment, this region corresponds to the domain of T1R family members which starts approximately at the conserved glutamic acid residue at amino acid position 563 plus or minus 20 amino acids and ends approximately at the conserved tyrosine amino acid residue at position 812 plus or minus approximately 10 amino acids. The seven transmembrane regions and extracellular and cytoplasmic loops can be identified using standard methods, as described in Kyte & Doolittle, J. Mol. Biol., 157:105-32 (1982)), or in Stryer, supra.
“Cytoplasmic domains” refers to the domains of T1R polypeptides that face the inside of the cell, e.g., the “C-terminal domain” and the intracellular loops of the transmembrane domain, e.g., the intracellular loop between transmembrane regions 1 and 2, the intracellular loop between transmembrane regions 3 and 4, and the intracellular loop between transmembrane regions 5 and 6. “C-terminal domain” refers to the region that spans the end of the last transmembrane domain and the C-terminus of the protein, and which is normally located within the cytoplasm. In one embodiment, this region starts at the conserved tyrosine amino acid residue at position 812 plus or minus approximately 10 amino acids and continues to the C-terminus of the polypeptide.
The term “ligand-binding region” or “ligand-binding domain” refers to sequences derived from a chemosensory receptor, particularly a taste receptor that substantially incorporates at least the extracellular domain of the receptor. In one embodiment, the extracellular domain of the ligand-binding region may include the N-terminal domain, the transmembrane domain, and portions thereof such as the extracellular loops of the transmembrane domain and in particular amino acids located in the membrane which may bind ligands. The ligand-binding region may be capable of binding a ligand, and more particularly, a tastant.
The phrase “heteromer” or “hetero-oligomeric complex” in the context of the T1R receptors or polypeptides of the invention refers to a functional combination of at least two T1R receptors or polypeptides, at least one T1R receptor or polypeptide and another taste-cell-specific GPCR, or a combination thereof, to thereby effect chemosensory taste transduction. For instance, the receptors or polypeptides may be co-expressed within the same taste receptor cell type, and the two receptors may physically interact to form a hetero-oligomeric or heteromeric taste receptor.
The phrase “functional effects” in the context of assays for testing compounds that modulate T1R family member mediated taste transduction includes the determination of any parameter that is indirectly or directly under the influence of the receptor, e.g., functional, physical and chemical effects. It includes ligand binding, changes in ion flux, membrane potential, current flow, transcription, G protein binding, GPCR phosphorylation or dephosphorylation, signal transduction, receptor-ligand interactions, second messenger concentrations (e.g., cAMP, cGMP, IP3, or intracellular Ca²⁺), in vitro, in vivo, and ex vivo and also includes other physiologic effects such as increases or decreases of neurotransmitter or hormone release.
By “determining the functional effect” in the context of assays is meant assays for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a T1R family member, e.g., functional, physical and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g. fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte T1R gene expression; tissue culture cell T1R expression; transcriptional activation of T1R genes; ligand-binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, and the like.
“Inhibitors,” “activators,” “enhancers” and “modulators” of T1R proteins include any compound that binds to or which modulates (increases or decreases) the activity of a T1R or T1R heteromer or homomeric T1R or which modulates (increase or decrease) the binding of another compound to a particular T1R monomer or a T1R heterodimer or T1R homodimer. These compounds may be identified in in vitro or in vivo assays, preferably cell-based assays that assay the effect of a compound on the activity of a particular T1R polypeptide or heteromer, typically T1R1/T1R3 or T1R2/T1R3 heteromers, and preferably human T1R1/T1R3 or T1R2/T1R3 heteromers. This includes agonists, antagonists, and their homologs and mimetics. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate T1R taste transduction, e.g., antagonists. Activators or enhancers are compounds that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize, or up regulate T1R taste transduction, e.g., agonists. T1R modulators can include genetically modified versions of T1R family members, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Assays for identifying T1R inhibitors and activators include, e.g., expressing T1R family members in cells or cell membranes, applying putative modulator compounds, in the presence or absence of tastants, e.g., sweet tastants, and then determining the functional effects on taste transduction, as described above. Samples or assays comprising T1R family members that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of modulation. Control samples (untreated with modulators) are assigned a relative T1R activity value of 100%. Inhibition of a T1R is achieved when the T1R activity value relative to the control is about 80%, optionally 50% or 25-0%. Activation of a T1R is achieved when the T1R activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
“Counter-screen” herein refers to an assay using a homomeric T1R, preferably homomeric T1R2 or T1R1, more preferably human homomeric T1R2 (T1R2 expressed in the absence of T1R3), that is designed to identify potential “false positives”, i.e., compounds which modulate the activity of T1R2/T1R3 in vitro or T1R1/T1R3 in vitro, but which do not elicit a desired effect on taste, e.g., sweet or umami taste. Such counter-screen assay will evaluate the effect of a compound shown to bind to and/or modulate the activity of heteromeric T1R2/T1R3 or a compound shown to bind to and/or modulate the activity of heteromeric T1R1/T1R3. In a preferred embodiment the counter-screen assay will fluorimetrically detect homomeric T1R2 activity by detecting intracellular calcium using a FLIPR system.
The terms “purified,” “substantially purified,” and “isolated” as used herein refer to the state of being free of other, dissimilar compounds with which the compound of the invention is normally associated in its natural state, so that the “purified,” “substantially purified,” and “isolated” subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample. In one preferred embodiment, these terms refer to the compound of the invention comprising at least 95% of the mass, by weight, of a given sample. As used herein, the terms “purified,” “substantially purified,” and “isolated,” when referring to a nucleic acid or protein, of nucleic acids or proteins, also refers to a state of purification or concentration different than that which occurs naturally in the mammalian, especially human, body. Any degree of purification or concentration greater than that which occurs naturally in the mammalian, especially human, body, including (1) the purification from other associated structures or compounds or (2) the association with structures or compounds to which it is not normally associated in the mammalian, especially human, body, are within the meaning of “isolated.” The nucleic acid or protein or classes of nucleic acids or proteins, described herein, may be isolated, or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.
The term “nucleic acid” or “nucleic acid sequence” refers to a deoxy-ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones (see e.g., Oligonucleotides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the N.Y. Academy of Sciences, Vol. 600, Eds. Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36:1923-1937 (1993); Antisense Research and Applications (1993, CRC Press), WO 97/03211; WO 96/39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997); Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, Antisense Nucleic Acid Drug Dev, 6:153-156 (1996)).
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating, e.g., sequences in which the third position of one or more selected codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
The term “plasma membrane translocation domain” or simply “translocation domain” means a polypeptide domain that, when incorporated into a polypeptide coding sequence, can with great efficiency “chaperone” or “translocate” the hybrid (“fusion”) protein to the cell plasma membrane. For instance, a “translocation domain” may be derived from the amino terminus of the bovine rhodopsin receptor polypeptide, a 7-transmembrane receptor. However, rhodopsin from any mammal may be used, as can other translocation facilitating sequences. Thus, the translocation domain is particularly efficient in translocating 7-transmembrane fusion proteins to the plasma membrane, and a protein (e.g., a taste receptor polypeptide) comprising an amino terminal translocating domain may be transported to the plasma membrane more efficiently than without the domain.
The “translocation domain,” “ligand-binding domain”, and chimeric receptors compositions described herein also include “analogs,” or “conservative variants” and “mimetics” (“peptidomimetics”) with structures and activity that substantially correspond to the exemplary sequences. Thus, the terms “conservative variant” or “analog” or “mimetic” refer to a polypeptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the polypeptide's (the conservative variant's) structure and/or activity, as defined herein. These include conservatively modified variations of an amino acid sequence, i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity.
More particularly, “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): ala/gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979)). One of skill in the art will appreciate that the above-identified substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative. In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered “conservatively modified variations.”
The terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the polypeptides, e.g., translocation domains, ligand-binding domains, or chimeric receptors of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogs of amino acids, or may be a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity.
As with polypeptides of the invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond (“peptide bond”) linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, a helix conformation, and the like. A polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g., —C═O—CH2- for —C═O)—NH—, aminomethylene (CH2-NH), ethylene, olefin (CH═CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY (1983)). A polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature.
A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
A “labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
As used herein a “nucleic acid probe or oligonucleotide” is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are optionally directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
A “promoter” is defined as an array of nucleic acid sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
As used herein, “recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide. “Recombinant means” also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of a fusion protein comprising a T1R protein according to the invention.
The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10 degrees C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60 degrees C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42 degrees C., or, 5×SSC, 1% SDS, incubating at 65 degrees C., with wash in 0.2×SSC, and 0.1% SDS at 65 degrees C. Such hybridizations and wash steps can be carried out for, e.g., 1, 2, 5, 10, 15, 30, 60; or more minutes.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially related if the polypeptides which they encode are substantially related. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 degrees C., and a wash in 1×SSC at 45 degrees C. Such hybridizations and wash steps can be carried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An “anti-T1R” antibody is an antibody or antibody fragment that specifically binds a polypeptide encoded by a T1R gene, cDNA, or a subsequence thereof.
The term “immunoassay” is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
The phrase “specifically (or selectively) binds” to an antibody or, “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to a T1R family member from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the T1R polypeptide or an immunogenic portion thereof and not with other proteins, except for orthologs or polymorphic variants and alleles of the T1R polypeptide. This selection may be achieved by subtracting out antibodies that cross-react with T1R molecules from other species or other T1R molecules. Antibodies can also be selected that recognize only T1R GPCR family members but not GPCRs from other families. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
The phrase “selectively associates with” refers to the ability of a nucleic acid to “selectively hybridize” with another as defined above, or the ability of an antibody to “selectively (or specifically) bind to a protein, as defined above.
The term “expression vector” refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell. The term includes recombinant expression “cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.
By “host cell” is meant a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, U2OS, U2OS, NIH3T3 or MDCK cell, BHK cells, and the like, e.g., cultured cells, explants, and cells in vivo.

Detection of T1R Modulators

Exemplary compositions and methods for determining whether a test compound specifically binds to a T1R2/T1R3 or T1R2/T1R3 heteromer or a homomeric T1R, e.g., T1R1, T1R2 or T1R3, are described below. Many aspects of cell physiology can be monitored to assess the effect of ligand binding to a T1R polypeptide of the invention. These assays may be performed on intact cells expressing at least one T1R receptor, on permeabilized cells, or on membrane fractions produced by standard methods.
Taste receptors bind tastants and initiate the transduction of chemical stimuli into electrical signals. An activated or inhibited G protein will in turn alter the properties of target enzymes, channels, and other effector proteins. Some examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by the stimulatory G protein, phospholipase C by Gq and other cognate G proteins, and modulation of diverse channels by Gi and other G proteins. Downstream consequences can also be examined such as generation of diacyl glycerol and IP3 by phospholipase C, and in turn, for calcium mobilization by IP3.
The T1R proteins or heteromers used in these assays will typically comprise a human or rodent T1R or heteromer, most typically human T1R2/T1R3 or T1R1/T1R3 or a homomeric T1R1 or T1R2 as defined herein. As noted, this includes T1R polypeptide fragments, chimeras, and conservatively modified variants thereof. These T1R polypeptides may be expressed in recombinant or endogenous cells, e.g., isolated human cells.
Alternatively, the T1R proteins or polypeptides of the assay can be derived from a eukaryote host cell and can include an amino acid subsequence having amino acid sequence identity to a T1R polypeptide contained in the Sequence Listing or known in the art or fragments or conservatively modified variants thereof. Generally, the amino acid sequence identity will be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Optionally, the T1R proteins or polypeptides of the assays can comprise a domain of a T1R protein, such as an extracellular domain, transmembrane region, transmembrane domain, cytoplasmic domain, ligand-binding domain, and the like. Further, as described above, the T1R protein or a domain thereof can be covalently linked to a heterologous protein to create a chimeric protein used in the assays described herein.
Modulators of T1R receptor activity are tested using T1R proteins or polypeptides as described above, either recombinant or naturally occurring. The T1R proteins or polypeptides can be isolated, co-expressed in a cell, co-expressed in a membrane derived from a cell, co-expressed in tissue or in an animal, either recombinant or naturally occurring. For example, tongue slices, dissociated cells from a tongue, transformed cells, or membranes can be used. Modulation can be tested using one of the in vitro or in vivo assays described herein.
In Vitro Binding Assays
Taste transduction can also be examined in vitro with soluble or solid state reactions, using hetero-oligomeric complexes of the T1R polypeptides of the invention. In a particular embodiment, hetero-oligomeric complexes of T1R ligand-binding domains can be used in vitro in soluble or solid state reactions to assay for ligand binding.
Ligand binding to a T1R2/T1R3 or T1R1/T1R3 heteromer or a homomeric T1R polypeptide can be tested in solution, in a bilayer membrane, optionally attached to a solid phase, in a lipid monolayer, or in vesicles. Binding of a modulator can be tested using, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index) hydrodynamic (e.g., shape), chromatographic, or solubility properties. Preferred binding assays of the invention are biochemical binding assays that use recombinant soluble T1R polypeptides.
Receptor-G protein interactions can also be examined. For example, binding of the G protein to the receptor complex, or its release from the receptor complex can be examined. More particularly, in the absence of GTP, an activator will lead to the formation of a tight complex of a G protein (all three subunits) with the receptor. This complex can be detected in a variety of ways, as noted above. Such an assay can be modified to search for inhibitors, e.g., by adding an activator to the receptor and G protein in the absence of GTP, which form a tight complex, and then screen for inhibitors by looking at dissociation of the receptor-G protein complex. In the presence of GTP, release of the a subunit of the G protein from the other two G protein subunits serves as a criterion of activation. An activated or inhibited G protein will in turn alter the properties of target enzymes, channels, and other effector proteins.
In another embodiment of the invention, a GTP γ³⁵S assay may be used. As described above, upon activation of a GPCR, the G a subunit of the G protein complex is stimulated to exchange bound GDP for GTP. Ligand-mediated stimulation of G protein exchange activity can be measured in a biochemical assay measuring the binding of added radioactively labeled GTP γ³⁵S to the G protein in the presence of a putative ligand. Typically, membranes containing the chemosensory receptor of interest are mixed with a complex of G proteins. Potential inhibitors and/or activators and GTPγ 35S are added to the assay, and binding of GTP γ³⁵S to the G protein is measured. Binding can be measured by liquid scintillation counting or by any other means known in the art, including scintillation proximity assays (SPA). In other assays formats, fluorescently labeled GTPγ35S can be utilized.

Fluorescence Polarization Assays

In another embodiment, Fluorescence Polarization (“FP”) based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels. This section describes how fluorescence polarization can be used in a simple and quantitative way to measure the binding of ligands to the T1R polypeptides of the invention.
When a fluorescently labeled molecule is excited with plane polarized light, it emits light that has a degree of polarization that is inversely proportional to its molecular rotation. Large fluorescently labeled molecules remain relatively stationary during the excited state (4 nanoseconds in the case of fluorescein) and the polarization of the light remains relatively constant between excitation and emission. Small fluorescently labeled molecules rotate rapidly during the excited state and the polarization changes significantly between excitation and emission. Therefore, small molecules have low polarization values and large molecules have high polarization values. For example, a single-stranded fluorescein-labeled oligonucleotide has a relatively low polarization value but when it is hybridized to a complementary strand, it has a higher polarization value. When using FP to detect and monitor tastant-binding which may activate or inhibit the chemosensory receptors of the invention, fluorescence-labeled tastants or auto-fluorescent tastants may be used. For example, fluorescence polarization has been used to measure enzymatic cleavage of large fluorescein labeled polymers by proteases, DNases, and RNases. It also has been used to measure equilibrium binding for protein/protein interactions, antibody/antigen binding, and protein/DNA binding.

Solid State and Soluble High Throughput Assays

In yet another embodiment, the invention provides soluble assays using T1R heteromers or homomeric T1R polypeptides; or a cell or tissue co-expressing T1R polypeptides or only one T1R polypeptide. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the T1R polypeptides, or cell or tissue expressing the T1R polypeptides is attached to a solid phase substrate.
In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 1000 to about 1500 different compounds. It is also possible to assay multiple compounds in each plate well. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds are possible using the integrated systems of the invention. Also, microfluidic reagent manipulation may be used.
The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non-covalent linkage, e.g., via a tag. The tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest (e.g., the taste transduction molecule of interest) is attached to the solid support by interaction of the tag and the tag binder.
A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo.).
Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherein family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.
Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth., 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron, 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et al., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry, 39(4):718-719 (1993); and Kozal et al., Nature Medicine, 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.

Cell-Based Binding Assays

T1R proteins or polypeptides are expressed in a eukaryotic or non-eukaryotic cell, preferably a mammalian cell or an oocyte. The subject T1R polypeptides can be expressed in any eukaryotic cell, such as U2OS, U2OS, NIH3T3 or MDCK cells or other mammalian cells. Preferably, the cells comprise a functional G protein, e.g., Gα15, or a chimeric G protein such as a chimera of G16 or G15 and gustducin or transducin that is capable of coupling the chimeric receptor to an intracellular signaling pathway or to a signaling protein such as phospholipase C. Activation of such chimeric receptors in such cells can be detected using any standard method, such as by detecting changes in intracellular calcium by detecting FURA-2 dependent fluorescence in the cell.
Activated GPCR receptors become substrates for kinases that phosphorylate the C-terminal tail of the receptor (and possibly other sites as well). Thus, activators will promote the transfer of 32P from γ-labeled GTP to the receptor, which can be assayed with a scintillation counter. The phosphorylation of the C-terminal tail will promote the binding of arrestin-like proteins and will interfere with the binding of G proteins. The kinase/arrestin pathway plays a key role in the desensitization of many GPCR receptors. For example, compounds that modulate the duration a taste receptor stays active would be useful as a means of prolonging a desired taste or cutting off an unpleasant one. For a general review of GPCR signal transduction and methods of assaying signal transduction, see, e.g., Methods in Enzymology, vols. 237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature, 10:349:117-27 (1991); Bourne et al., Nature, 348:125-32 (1990); Pitcher et al., Annu. Rev. Biochem., 67:653-92 (1998).
T1R modulation may be assayed by comparing the response of T1R polypeptides treated with a putative T1R modulator to the response of an untreated control sample. Such putative T1R modulators can include tastants that either inhibit or activate T1R polypeptide activity. In one embodiment, control samples (untreated with activators or inhibitors) are assigned a relative T1R activity value of 100. Inhibition of a T1R polypeptide is achieved when the T1R activity value relative to the control is about 90%, optionally 50%, optionally 25-0%. Activation of a T1R polypeptide is achieved when the T1R activity value relative to the control is 110%, optionally 150%, 200-500%, or 1000-2000%.
Changes in ion flux may be assessed by determining changes in ionic polarization (i.e., electrical potential) of the cell or membrane expressing a T1R polypeptide. One means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques (see, e.g., the “cell-attached” mode, the “inside-out” mode, and the “whole cell” mode, e.g., Ackerman et al., New Engl. J. Med., 336:1575-1595 (1997)). Whole cell currents are conveniently determined using the standard. Other known assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol., 88:67-75 (1988); Gonzales & Tsien, Chem. Biol., 4:269277 (1997); Daniel et al., J. Pharmacol. Meth., 25:185-193 (1991); Holevinsky et al., J. Membrane Biology, 137:59-70 (1994)). Generally, the compounds to be tested are present in the range from 1 pM to 100 mM.
The effects of the test compounds upon the function of the polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects GPCR activity can be used to assess the influence of a test compound on the polypeptides of this invention. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca²⁺, IP3, cGMP, or cAMP.
Other assays for GPCRs include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G protein-coupled receptors as negative or positive controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g. agonists, antagonists), changes in the level of ions in the cytoplasm or membrane voltage will be monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that may be employed are those disclosed in the Molecular Probes 1997 Catalog. For G protein-coupled receptors, promiscuous G proteins such as Gα15 and Gα16 or the afore-mentioned chimeric G proteins can be used in the assay of choice (Wilkie et al., Proc. Nat'l Acad. Sci., 88:10049-10053 (1991)). Such promiscuous G proteins allow coupling of a wide range of receptors.
Receptor activation typically initiates subsequent intracellular events, e.g., increases in second messengers such as IP3, which releases intracellular stores of calcium ions. Activation of some G protein-coupled receptors stimulates the formation of inositol triphosphate (IP3) through phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge & Irvine, Nature, 312:315-21 (1984)). IP3 in turn stimulates the release of intracellular calcium ion stores. Thus, a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as IP3 can be used to assess G protein-coupled receptor function. Cells expressing such G protein-coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it may be desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.
Other assays can involve determining the activity of receptors which, when activated, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymes such as adenylate cyclase. There are cyclic nucleotide-gated ion channels, e.g., rod photoreceptor cell channels and olfactory neuron channels that are permeable to cations upon activation by binding of cAMP or cGMP (see, e.g., Altenhofen et al., Proc. Nat'l Acad. Sci., 88:9868-9872 (1991) and Dhallan et al., Nature, 347:184-187 (1990)). In cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it may be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e.g., forskolin, prior to adding a receptor-activating compound to the cells in the assay. Cells for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-crated ion channel, GPCR phosphatase and DNA encoding a receptor (e.g., certain glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors, and the like), which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.
In a preferred embodiment, T1R polypeptide activity is measured by co-expressing T1R2 and T1R3 nucleic acids or T1R1 and T1R3 nucleic acids in a heterologous cell with a promiscuous or chimeric G protein that links the receptor to a phospholipase C signal transduction pathway (see Offermanns & Simon, J. Biol. Chem., 270:15175-15180 (1995)). Optionally the cell line is U2OS, U2OS, NIH3T3 or MDCK cell line or other mammalian cell that does not naturally express T1R genes and the G protein is Gα15 or Gα16 (Offermanns & Simon, supra), gustducin, transducin, or is a chimeric G protein containing any of the foregoing. Modulation of taste transduction is assayed by measuring changes in intracellular Ca²⁺ levels, which change in response to modulation of the T1R signal transduction pathway via administration of a molecule that associates with T1R polypeptides. Changes in Ca²⁺ levels are optionally measured using fluorescent Ca²⁺ indicator dyes and fluorimetric imaging. Preferably this is effected using a FLIPR detection system.
In one embodiment, the changes in intracellular cAMP or cGMP can be measured using immunoassays. The method described in Offemmanns & Simon, J. Bio. Chem., 270:15175-15180 (1995), may be used to determine the level of cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol., 11:159-164 (1994), may be used to determine the level of cGMP. Further, an assay kit for measuring cAMP and/or cGMP is described in U.S. Pat. No. 4,115,538, herein incorporated by reference.
In another embodiment, phosphatidyl inositol (PI) hydrolysis can be analyzed according to U.S. Pat. No. 5,436,128, herein incorporated by reference. Briefly, the assay involves labeling of cells with 3H-myoinositol for 48 or more hrs. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol-water after which the inositol phosphates were separated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation is determined by calculating the ratio of cpm in the presence of agonist, to cpm in the presence of buffer control. Likewise, fold inhibition is determined by calculating the ratio of cpm in the presence of antagonist, to cpm in the presence of buffer control (which may or may not contain an agonist).
In another embodiment, transcription levels can be measured to assess the effects of a test compound on signal transduction. A host cell containing T1R polypeptides of interest is contacted with a test compound for a sufficient time to effect any interactions, and then the level of gene expression is measured. The amount of time to effect such interactions may be empirically determined, such as by running a time course and measuring the level of transcription as a function of time. The amount of transcription may be measured by using any method known to those of skill in the art to be suitable. For example, mRNA expression of the protein of interest may be detected using northern blots or their polypeptide products may be identified using immunoassays. Alternatively, transcription based assays using reporter gene may be used as described in U.S. Pat. No. 5,436,128, herein incorporated by reference. The reporter genes can be, e.g., chloramphenicol acetyltransferase, luciferase, 3′-galactosidase and alkaline phosphatase. Furthermore, the protein of interest can be used as an indirect reporter via attachment to a second reporter such as green fluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology, 15:961-964 (1997)).
The amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test compound, or it may be compared with the amount of transcription in a substantially identical cell that lacks the T1R polypeptide of interest. A substantially identical cell may be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Any difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the T1R polypeptides of interest.
The invention is further described by the following examples.

EXAMPLES

In all of the examples below, assays were conducted using mammalian cells stably expressing hT1R2 and hT1R3, as well as mammalian cells transiently expressing hT1R2 alone or a control Mock vector using the FLIPR system to evaluate T1R2/T1R3 or homomeric T1R2 receptor activation. All of the cells express the chimeric G protein G16gust25. The production of cells stably expressing hT1R2 and hT1R3, and G16gust25 has been disclosed in earlier Senomyx patents. The hT1R2 gene was transduced into cells expressing G16gust25 and 48 hours later, test compounds or sweeteners were evaluated the same way as assays conducted using cells expressing hT1R2 and hT1R3, as well as Mock transfected cells using the FLIPR system.

Example 1

Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound A, a sweet taste receptor agonist known to bind to the transmembrane domain of hT1R2. Compound A was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay producing kinetics of calcium mobilization that are typical of GPCR activation. These results are contained in FIG. 1.

Example 2

Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound B, a sweet taste receptor agonist. Compound B was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 2.

Example 3

Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound C, a sweet taste receptor agonist. Compound C was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 3.

Example 4

Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by compound D, a sweet taste receptor agonist. Compound D was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 4.

Example 5

Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by Perillartine, a sweet taste receptor agonist. Perillartine was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 5.

Example 6

Cells stably expressing hT1R2 and hT1R3 and cells transiently expressing hT1R2 alone or a control Mock vector were evaluated on FLIPR for activation by P-4000, a sweet taste receptor agonist. P-4000 was not only active in the hT1R2/hT1R3 assay but also in the hT1R2 assay. These results are contained in FIG. 6.

Example 7

Seventeen compounds or sweeteners identified as agonists in the hT1R2/hT1R3 assay and showing a good correlation between taste data and assay data (within a 95% confidence interval) were inactive in the hT1R2 assay. Conversely, six compounds identified as agonists in the hT1R2/hT1R3 assay but ultimately found to behave poorly in human taste tests or being tasteless were active in the hT1R2 assay.
FIG. 7 summarizes the potency of these 17 compounds at different molar ratios relative to sucrose in the hT1R2/hT1R3 assay. Based on these results it can be seen that assays conducted using cells that express T1R2 alone (i.e., in the absence of T1R3), e.g., FLIPR based assays with compounds identified as putative sweet taste modulators based on their modulation of the activity of T1R2/T1R3 is an effective means for potentially minimizing or even eliminating false positives. Given the time and expense associated with identifying and producing optimized compounds suitable for use in products for human consumption this is a significant improvement that should lead to the development of novel sweet taste modulators.
The exemplified counter-screen assay in addition may be further optimized to use as a counter-screen, on a regular basis, to eliminate false positives, such as by producing and using cells that stably express T1R2 alone (i.e., in the absence of T1R3) and a suitable G protein such as G16gust25, G15gust25, G16trans44 or G16trans44.

Claims

1-27. (canceled)

28. A method of screening for a compound that putatively modulates umami taste wherein such method reduces or eliminates “false positives”, the method comprising the steps of:

(i) screening one or more compounds in an assay that identifies compounds which specifically bind or modulate the activity of a T1R1/T1R3 heteromeric taste receptor or which modulate the binding or the activation of a T1R1/T1R3 heteromeric taste receptor by another compound;

(ii) further conducting a counter-screen using one or more of the identified putative T1R1/T1R3 modulatory compounds identified n step (i), wherein said counter-screen detects whether said one or more identified putative T1R1/T1R3 modulatory compounds specifically bind or modulate the activity of a homomeric T1R1 polypeptide or whether said one or more identified putative T1R1/T1R3 modulatory compounds modulate the binding or the activation of a homomeric T1R1 taste receptor polypeptide by another T1R1 agonist compound; and

(iii) based on the results of the counter-screen identifying whether the identified compound or compounds are “false positives” or are compounds that putatively modulate umami taste.

29-59. (canceled)

60. A high throughput screening method for identifying compounds that putatively modulate sweet taste wherein such method reduces or eliminates “false positives”, the method comprising the steps of:

(i) screening a thousand or more compounds in an assay that identifies compounds which specifically bind to or modulate the activity of a human T1R2/T1R3 heteromeric taste receptor or which modulate the binding or the activation of a human T1R2/T1R3 heteromeric taste receptor by another compound;

(ii) further conducting a counter-screen using the identified putative human T1R2/T1R3 modulatory compounds identified from the thousand or more compounds screened in step (i), wherein said counter-screen detects whether said one or more identified putative human T1R2/T1R3 modulatory compounds specifically bind to or modulate the activity of a homomeric human T1R2 polypeptide or whether said one or more identified putative human T1R2/T1R3 modulatory compounds modulate the binding or the activation of a homomeric human T1R2 polypeptide by another human T1R2 agonist compound; and

(iii) assessing the effect of the compounds which test positive in (i) and not in (ii) in human taste tests which assay the effect of said compounds on sweet taste, wherein said screening method does not include any other binding or functional assays using T1R taste receptor polypeptides and further does not screen using a cell which endogenously expresses said human T1R2/T1R3 taste receptor or a cell which endogenously expresses said homomeric human T1R2 polypeptide.

61. The method of claim 60 which further comprises and producing variants or derivatives of the compounds that are identified as eliciting or modulating sweet taste in said human taste tests and identifying those variants and derivatives which are suitable for use as flavorants in foods, beverages, medicaments or comestibles for human or animal consumption in the development of variants or derivatives.

62. The method of claim 60, wherein step (i) of the method uses eukaryotic cells which stably or transiently express human T1R2/T1R3 and the counter-screen uses cells that stably or transiently express a human T1R2 polypeptide and which do not express any T1R3 polypeptide.

63. The method of claim 60, wherein step (i) uses a eukaryotic cell membrane comprising said human T1R2/T1R3 receptor.

64. The method of claim 60, wherein said human T1R2/T1R3 receptor in step (i) and said human T1R2 homomeric receptor are both expressed by a eukaryotic cell.

65. The method of claim 64, wherein said cell is a yeast, fungus, insect, oocyte, worm, or mammalian cell.

66. The method of claim 65, wherein the mammalian cell is a human or rodent cell.

67. The method of claim 64, wherein said eukaryotic cell is a CHO, COS, U2OS, NIH3T3 or MDCK cell, BHK, HeLa, or STO cell or comprises a Xenopus oocyte.

68. The method of claim 60, wherein the human T1R2/T1R3 heteromer and/or the homomeric human T1R2 receptor is linked to a solid phase.

69. The method of claim 66, wherein said eukaryotic cell further expresses a G protein that couples to said human T1R2/T1R3 heteromer and the homomeric human T1R2 polypeptide.

70. The method of claim 69, wherein said G protein is Gα15, Gα16, transducin, gustducin or a chimera of any of the foregoing G proteins.

71. The method of claim 70, wherein said G protein is a chimera of Gα15 or Gα16, and gustducin.

72. The method of claim 70, wherein said G protein is a chimera of Gα15 or Gα16, and transducin.

73. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by detecting changes in intracellular Ca2+ levels.

74. The method of claim 73, wherein Ca2+ levels are detected using an ion sensitive dye or a membrane voltage fluorescent indicator.

75. The method of claim 60, which uses a FLIPR detection system.

76. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by monitoring changes in fluorescence polarization.

77. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by detecting changes in second messenger levels.

78. The method of claim 77, wherein said second messenger is IP3.

79. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by detecting changes in intracellular cyclic nucleotides.

80. The method of claim 79, wherein said cyclic nucleotide is cAMP or cGMP.

81. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric T1R2 polypeptide are measured by measuring changes is Ca2+ levels by fluorescence imaging.

82. The method of claim 60, wherein the activity of said human T1R2/T1R3 heteromeric taste receptor and/or the homomeric human T1R2 polypeptide are measured by detecting changes in G protein binding of GTPδS.

83. The method of claim 60, wherein the human T1R2 polypeptide is at least 90% identical to a T1R2 polypeptide having or encoded by a sequence selected from SEQ ID NO:3, 7, 13, and 14.

84. The method of claim 60, wherein the human T1R2 polypeptide is at least 95% identical to a human T1R2 polypeptide having or encoded by a sequence selected from SEQ ID NO:3, 7, 13, and 14.

85. The method claim 60, wherein the human T1R3 polypeptide is at least 90% identical to a human T1R3 polypeptide having or encoded by a sequence selected from SEQ ID NO:1, 4, 6, and 8.

86. The method claim 60, wherein the human T1R3 polypeptide is at least 95% identical to a human T1R3 polypeptide having or encoded by a sequence selected from SEQ ID NO: 1, 4, 6, and 8.