(19) TZZ _¥¥ _T (11) EP 2 137 322 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) A23L 2/52 (2006.01) 27.02.2013 Bulletin 2013/09 A23G 4/00 (2006.01) C07C 53/134 (2006.01) (21) Application number: 08714784.9 (86) International application number: PCT/CH2008/000134 (22) Date of filing: 27.03.2008 (87) International publication number: WO 2008/119195 (09.10.2008 Gazette 2008/41) (54) METHODS TO IDENTIFY TAS2R MODULATORS VERFAHREN ZUR IDENTIFIZIERUNG VON TAS2R MODULATOREN PROCÉDÉ D’IDENTIFICATION DE MODULATEURS TAS2R (84) Designated Contracting States: • BEHRENS MAIK ET AL: "Members of RTP and AT BE BG CH CY CZ DE DK EE ES FI FR GB GR REEP gene families influence functional bitter HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT taste receptor expression" JOURNAL OF RO SE SI SK TR BIOLOGICAL CHEMISTRY, vol. 281, no. 29, July 2006(2006-07), pages 20650-20659, XP002494217 (30) Priority: 30.03.2007 US 909143 P ISSN: 0021-9258 30.07.2007 US 962549 P • KUHN CHRISTINA ET AL: "Bitter taste receptors for saccharin and acesulfame K" JOURNAL OF (43) Date of publication of application: NEUROSCIENCE, vol. 24, no. 45, 10 November 30.12.2009 Bulletin 2009/53 2004 (2004-11-10), pages 10260-10265, XP002494218 ISSN: 0270-6474 (73) Proprietor: Givaudan SA • BUFE BERND ET AL: "The human TAS2R16 1214 Vernier (CH) receptor mediates bitter taste in response to beta- glucopyranosides" NATURE GENETICS, (72) Inventors: NATURE PUBLISHING GROUP, NEW YORK, US, • BRUNE, Nicole, Erna, Irene vol. 32, no. 3, 1 November 2002 (2002-11-01), San Diego, CA 92122 (US) pages 397-401, XP002269573 ISSN: 1061-4036 • SLACK, Jay, Patrick • DATABASE Geneseq [Online] 4 November 2004 Loveland, OH 45140 (US) (2004-11-04), "Taste receptor modulation- related • UNGUREANU, Ioana, Maria human T2R64 gene sequence SeqID182." Cincinnati, OH 45215 (US) XP002494220 retrieved from EBI accession no. • GRAY, Kimberley GSN:ADR29243 Database accession no. Loveland, OH 45140 (US) ADR29243 & WO 2004/069191 A (SENOMYX INC • SIMONS, Christopher, Todd [US]; SERVANT GUY [US]; OZECK MARK [US]; Wyoming, OH 45215 (US) BRUST PAUL [US];) 19 August 2004 (2004-08-19) • EVANS PENNIMPEDE, Jenny, Ellen • DATABASE EMBL [Online] 13 June 2003 Cincinnati, OH 45241 (US) (2003-06-13), "Sequence 36 from patent US 6558910." XP002494221 retrieved from EBI (74) Representative: Givaudan Patents accession no. EMBL:AR310322 Database Givaudan Schweiz AG accession no. AR310322 & US 2002/051997 A1 Ueberlandstrasse 138 (ZUKER CHARLES S [US] ET AL ZUKER 8600 Dübendorf (CH) CHARLES S [US] ET AL) 2 May 2002 (2002-05-02) (56) References cited: WO-A-2004/055048 US-A1- 2005 032 158 US-B2- 7 105 650 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 137 322 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 137 322 B1 • DATABASE EMBL [Online] 13 June 2003 • DATABASE Geneseq [Online] 9 March 2006 (2003-06-13), "Sequence 36 from patent US (2006-03-09), "Taste receptor type 2 member 16 6558910." XP002494222 retrieved from EBI T2R16/TAS2R16 encoding DNA." XP002494227 accession no. EMBL:AR310322 Database retrieved from EBI accession no. GSN: AEF19624 accession no. AR310322 Database accession no. AEF19624 • DATABASE Geneseq [Online] 7 April 2005 • BEHRENS M ET AL: "Bitter taste receptors and (2005-04-07), "Human bitter taste receptor human bitter taste perception" CELLULAR AND haplotype protein T2R44 Seq 196." XP002494224 MOLECULAR LIFE SCIENCES, vol. 63, no. 13, retrievedfrom EBI accession no. GSP: ADW74584 July 2006 (2006-07), pages 1501-1509, Database accession no. ADW74584 & WO XP002494219 ISSN: 1420-682X 2005/007891A (US GOVERNMENT [US]; DRAYNA • KIM UNKYUNG ET AL: "Worldwide haplotype DENNIS [US]; KIM UN-KYUNG [US]) 27 January diversity and coding sequence variation at 2005 (2005-01-27) human bitter taste receptor loci" HUMAN • DATABASE EPO Proteins [Online] 28 April 2004 MUTATION, vol. 26, no. 3, September 2005 (2004-04-28), "Sequence 25 from Patent (2005-09), pages 199-204, XP008095937 ISSN: WO2004029087." XP002494225 retrieved from 1059-7794 EBI accession no. EPOP:CQ800021 Database • SHAMIL S ET AL: "PHYSICO-CHEMICAL AND accession no. CQ800021 & WO 2004/029087 A PSYCHOPHYSICAL STUDIES OF 4 1’ 6’ (DEUTSCHES INST FUER ERNAEHRUNG [DE]; TRICHLORO-4 1’ 6’- BUFE BERND [DE]; HOFMANN THOMAS [) 8 April TRIDEOXYGALACTOSUCROSE SUCRALOSE" 2004 (2004-04-08) LEBENSMITTEL WISSENSCHAFT UND • DATABASE Geneseq [Online] 7 April 2005 TECHNOLOGIE, ACADEMIC PRESS, LONDON, (2005-04-07), "Human bitter taste receptor GB, vol. 25, no. 2, 1 January 1992 (1992-01-01), haplotype protein T2R46 Seq 208." XP002494226 pages 192-196, XP008095932 ISSN: 0023-6438 retrievedfrom EBI accession no. GSP: ADW74596 Database accession no. ADW74596 2 EP 2 137 322 B1 Description [0001] Provided are methods to identify modulators of the bitter aftertaste associated with sucralose. [0002] Sucralose is a sweetener that provides a bitter off-note/aftertaste, and thereby limits the use of sucralose in 5 food. Therefore, methods that are able to identify compounds or ingredients that are able to modulate, and in particular to inhibit or mask, this bitter aftertaste are of interest. [0003] Bitter taste is perceived via taste receptors, and a family of 25 functional bitter taste receptors (TAS2R or T2R) is known. None of these receptors had previously been shown to be activated by sucralose. [0004] Applicant found that four of these receptors are activated by sucralose. 10 [0005] In particular, applicant identified sucralose as a specific agonist of taste receptor type 2 member 44 (TAS2R44), member 1 (TAS2R1), member 10 (TAS2R10), and member 46 (TAS2R46). These four receptors are referred to herein below as "sucralose activated bitter taste receptors" or "SABTR". This finding allows to provide methods that employ the identified SABTR and their agonist sucralose to identify ingredients that modulate the response of a SABTR to sucralose, for example, antagonists (blockers, inhibitors or masking agents) 15 of the sucralose- dependentSABTR activation. The methodstherefore allowto identifymodulators including bitter masking agents for sucralose, as was demonstrated for (E)- 4-(2,2,3-trimethylcyclopent-3-enyl)but-2-enoic acid, which was shown to inhibit the response of a SABTR to sucralose, and to significantly reduce the bitter aftertaste of sucralose in human sensory evaluations. 20 SUMMARY [0006] Provided is the following: (1) A method to identify an agent that modulates the taste of sucralose, the method comprising: 25 (i) contacting cells that express a TAS2R bitter taste receptor that is able to be activated by sucralose with sucralose in the presence of at least one agent; and (ii) determining whether the at least one agent affects binding to sucralose or a functional response thereto of said TAS2R bitter taste receptor, 30 with the proviso that the cells are not unmanipulated cells in their natural environment. (2) The method as described herein, including under (1), wherein the TAS2R bitter taste receptor comprises one or more sequences selected from 35 a TAS2R bitter taste receptor substantially homologous to a polypeptide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, with a sequence identity of at least 90%; a TAS2R bitter taste receptor which is encoded by a nucleotide sequence selected from the group consisting of 40 a nucleic acid substantially homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, SEQ ID NO:7, as determined by sequence identity, a nucleic acid substantially homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, SEQ ID NO:7, as determined by hybridisation, wherein the substantially homologous nucleic acid as determined by sequence identity has a sequence identity 45 of at least 90%; wherein the substantially homologous nucleic acid as determined by hybridisation hybridises under stringent hybridization conditions at a temperature of 42° C in a solution consisting of 50% formamide, 5 3SSC, and 1% SDS, and washing at 65° C in a solution consisting of 0.2 3SSC and 0.1 % SDS; wherein the nucleic acid optionally comprises SEQ ID NO:12 (HSV tag) at or near its end to form the C-terminus 50 in the corresponding protein, and optionally comprises a membrane export tag, optionally selected from rat somatostatin (RSS) and rhodopsin, optionally selected from SEQ ID NO: 11 (RSS tag), at or near its end to form the N terminus, and wherein the TAS2R bitter taste receptor polypeptide sequence optionally comprises SEQ ID NO:12 (HSV tag) at or near its end to form the C-terminus, and optionally comprises a membrane-export tag, optionally 55 selected from rat somatostatin (RSS) tag and rhodopsin tag, optionally selected from SEQ ID NO: 11 (RSS), at or near its end to form the N terminus. (3) The method as described herein including under (1) and (2) wherein the TAS2R bitter taste receptor comprises 3 EP 2 137 322 B1 a conservative functional variant able to be activated by sucralose. (4) The method as described herein including under (1), (2) or (3) wherein the cells also express a G-Protein, optionally a chimeric G-protein substantially homologous to Gaq-Gustducin, or substantially homologous to the 5 chimeric G-protein G alpha 16-gustducin 44.