«2 United States Patent Zoller et al. 'US007906328B2 US 7,906,328 B2 *Mar. 15, 2011 (10) Patent No. 4s) Date of Patent: ‘CELL LINES THAT EXPRESS HETERO OLIGOMERIC TASTE RECEPTORS, 6 (75) Inventors: Mark Zoller, San Diego, CA (US); ‘Xinodong Li San Diego, CA (US): ‘Lena Staszewski, San Diego, CA (US) Shawn O°Connell, Encinitas, CA (US): Sergey Zozulya, San Diewo, CA (US): on Eliot Adler, San Diego. CA (US): Hong Xu, San Diego, CA (US) Fernando Eeheverel, Chula Vis ws) ca a) o Assignee: Senomyx, Ine., San Diego. CA (US) Subject to any diselsimer, the term of this patent is extended or adjusted under 35 TSC. 134%b) by 190 days, ‘This patent is subject to a terminal dise climes, Notice: an @y Appl. Nos 127270,824 Now. 13,2008 (ander 37 CFR 1.47) Filed 9) Prior Publication Data US 200910221067 AI Sep. 32009 Related US. Application Data Continnation of application No, 11/939,438, fled on Nov. 13, 2007, now abandoned, which is a ‘continuation of application No. 101725,489, filed on Dee. 3, 2003, now Pat. No. 7.364.908, which is a division of application No, 10/179.373, filed on Jun, 26, 2002, now Pat. No. 7.368.285, which is a ‘continuaton-in-part of application No. 10°035,045, ‘led on Jan. 3, 2002, now Pat. No. 7.241.880, and a ‘continiationin-part of application No. 09/897,427, ‘ed on Jul. 3, 2001, now Pat. No, 6.955.887, and a ‘contimiation-in-part of application No. 09/799,629, filed on Ma. 7, 2001, now Pat. No. 7.244.835. Provisional application No. 60/300,434, filed on Jun 26, 2001, provisional application No. 601308,749, filed on Jul. 13, 2001, provisional application No. 60310.493," filed on Aug. 8, 2001, provisional ‘application No, 60/331,771, filed on Now. 21, 2001, provisional application No. 61339,472, filed en Dee 14, 2001, provisional application No. 60/372,090, filed on Apr. 15, 2002, provisional application No. 600374,143, fled on Apr, 22, 2002 (1) Ime CRN 500 CrN 502 CRN 120 CRN 1500 CRN 1874 Cn2P 2106 cork m0 CO7K 14/00 cork 1700 (60) (60) (2006.01), (2006.01) (2006.01), (2006.01), (2005.01) (2006.01) (2006.01) (2006.01) (2006.01), (2) US.Cl 4957925; 435%69.1; 435/252.3; 435/320.1; 435/471; 530/350 (58) Field of Classification Seareh None ‘See application ile for complete search history. 60) References Cited US. PATENT DOCUMENTS 5998,778 A 11/1999 Festa et a 638378 BL '52002. Zuker et a ‘GSH0978 BL 42003, Mangoes al. GOSS) BD 102005 Alereral Fo2248s 242006 Servant etal 7208290 8243007 Leta Toov381 Bo $2007 Ader etal Toavsso B2 72007 Ader eta Toakset B2 72007 Zucker etal Toads m2 72007 Ader etal Toodana 211007 Zoller el 7297543 B2 102007 Zoller eal. Toor B1L007 Zoller ea. Toto 2 11007 Zoller ea TBORERS B2 122007 Zoller eal. (Continued) POREIGN PATENT DOCUMENTS. Wo Wossioxe7 ——ig9s (Continued) OTHER PUBLICATIONS Ader Fetal, A Novel Family of Mammalian Taste Receptors”, (Cell, yo 100, No.6. 693-702, Mar. 17, 2000, (Continued) Primary Examiner — Robert Landsman, (74) Auornes, Agent, oF Firm — Hunton & Williams LLP on ABSTRACT ‘The present inveation relates to the discovery that dhe TR receptors assemble 10 form finetional taste receptors. Pate ticularly it has heen diseovered that co-expression of TIRL and TRS results ina taste receptor that responds to umami taste stimuli, including monosodium glutamate. Also, it has been discovered that co-expression ofthe TIR2 and TIR3 recepiors results in a taste receptor that sponds to sweet taste stimuli including naturally occuring and artificial sweeteners, Also the present invention relates tothe use of hetero-oligomeric taste receptors comprising, TTRI/TIR3 and T1R2VT1R3 in assays identify compounds that respe- tively respond to mami taste simian sweet taste stim. unr, the invention relate to the constitutive of eel ines that stably o transiently co-express a combination of TIRI ‘and T1350 TIR2 ad TRS; ner constitutive or inducible conditions. The useot thse cll ines in cell-based assays 10 identify umami and sweet taste modulatory compounds is also provided, particularly high throughput sereening assays that detect receptor aetvity by use of Nuorometeie imaging 36 Claims, 16 Drawing SheetsUS 7,906,328 B2 Page 2 USS. PATENT DOCUMENTS 7300877 B2 122007 Zoller eal F3ae859 Br 32008 Zolleret a. 7Rot908 2 4D008 Zoller a. ‘Temas BD S008 Zoller a al 7402900 2 72008 Zeckeret a FaI9701 B2 92008 Adler ta. 738552 B2 10008 Adler etal Tagen BD 12008 Adler etal 765.550, 108. Zucker ta Tsurisaa m2 '¥2009 Aer eta aonaionsiog? AL $4002 Zuker sta dongoep4sst AL 7/2002 Areal. 20020181059 AL 10.2002 Chaathar ea. dooooteoia4 AL 102002 sleet a. aogovessas AL 112m02 Zuker eta aosoooxsat AL ‘1/2003 Ader ea boos0012278 AL 1/2003 Zuker a donvons22e8 AL 2003 Zuker a ao0s0n%60s9 AL 22003 Water. aoosooaooss AL 22003 Zoker ta donvonsieas AL 3/2003 Adler eta. dovso1esis? AL 92003 Zuker etal aoosoowest? AL $008 Leta dooso1oes7L AL $2005 Erlenoch etal aonvovosis9 AL $2007 Evlenbach eta FOREIGN PATENT DOCUMENTS. Wo woonass2 2.2000 WO — Woon0ss9s 12000 Wo Wooten 5 2001 (O WooLe6set a2 9.2001 Wo woork rant WO — wOnD%64631 42 ‘82002 WO Woosoois7s a2 12008 Wo Wousorsty? 42003 WO WO200S0I5158 a2 22005 OTHER PUBLICATIONS ‘Alexander, al, “Alerng the Antigens of protsins”, Pr, Nal Acad Se USA. vo. 89, 3352-3355, Ap. 1992 ‘Monk, W, etal, "The Naive Rat Olfictry Cystic Nucleotide: Gated Channels Composed of Tree Distnt Subunits The oul ‘of Neuroscience, vl 19, No. 136 p. S332-8347- Iu, 1999 Bowie, tal. "Deciphering tne Message in Protein Sequences: Tol ‘ranceto Amino Aes Sbtttions, Sens, 247,p 13061310, Mar 16,1990 Dama S, "Dts of Sct and Unani Taste inthe Absonce of “Tse Recepor T8301 pp 85083, 2005 Guo, et als “Proein Tolerance to Random Amino Acid change” PNAS, vol LOL, No 25, p. 92059210, Jum 22,2008, Hoon, ct al.Patative Mammalian Taste Resepors A Cas of Tse= ‘Specie GPCRS with Distinet Topographic Seestivity” Cel ol. 96, .Stt-s51, Feb 19,1900, Sohnson, Cet al, “The Best of the Sweetness Inibior 2(-- ‘methoxyphenoxy) propane aid (Sodium salt) (Nt-PMP) othe tase of bitraweet stan”, Cheical Sense, vol 19, No.4 30.386, 1094 ‘Kinumon, 8. and Cummings, T. “Chemosensory ‘Tansduction ‘Mechanisms in Taste, Ann. Rew. Physi, vol 4, p. 718731, 1092 ‘Kitagawa, M, etal, “Molecular Genetic Mentfiation of a Candi date Recepor Gone for Sweat Taste" Biochemical and Biophysical Research Comminications vol 283, 236-242 2001 ‘Krauts et al, “Tentfiation of pans for Olfactory Receptors by Functional Expression ofa Recepor Libary" Call vol. 98, 517-926, Dee. 23 1998 Li, Xtal “Homan Recepors for Sweet and umami tase”, Pro- ceding of the National Academy of Science, ¥al 99, No.7. 4602-4606, Ape 2, 2002, indemann, "A Taste for Umami", Natre Neuroscieneo.3,No.2, 9-100, Fi 2000, ‘Max, M, etal, "Tasle, encoding ne candidate taste receptors allelic othe set responsvenes locus Sac”, Nature Genet el, Dep S86, May 2001 Montmayer JP, etal, “A Canidae Taste Receptor Gene Near 2 ‘Sweet Tht Loot” Nature Neuroscience, vo. No.5, May 2001 Nelson.etal, "An Aminowacid Taste Receptor" Nase, vl 416,p, 199-202, Mat. 14,2002, ‘elon cal, "Mammalian wee Taste Reeepors, Call 0 106. 381-390, Aug. 10.2001 Pearucco, etal, “Possite Roe for gustduin in Taste Transduction ido Medicinalis", Society for Neuroscience Abstracts Ol. 2, No. 13, Abstract No, 66.15, 2000 Sain Eta, "Hentfieaion of Novel Member othe T1R fily of pal tase receptors" Joumal of Neurochemistry, vol 77, 8 ‘90.903, 2001 ‘emus: Petal “The history of vee tse: not exactly apiece of «ake, Journal of Molecular Recognition, 2006, 19 188-19. ‘Waterson, fH. "Direct Submission”, Genome Sequencing Centr, Apr 21,2000 XP002262036, Database EMBL "Onlin! cmblhsdebers: Aco: AcO62024, Jun, 21,2000, {Gentlank Acceision No, AASOTO22, May 19,1998 GenBank Accession No. ALISO267, clone RPS$9003, Feb, 13 avo, ‘European Search Report for Puropean Application No. 081536825, dtd an 21, 2009 "Extended European Search Report and Opinion for European Appl cation No OM0ST48 7, dated Now 31, 2008U.S. Patent Mar. 15,2011 Sheet 1 of 16 US 7,906,328 B2