United States Patent 11») Grandy et al. [54] MAMMALIAN METHADONE-S OPIOID RECEPTOR [75] Inventors: David K. Grandy; James R. Bunzow, both of Portland, Oreg.; Olivier Civelli, Aesch, Switzerland (73) ‘Oregon Health Science University, Portland, Oreg, [21] Appl. No-: 09/170,331 [22] Filed: Oct. 13, 1998 Related U. Application Data [62] _ivision of application No. 08°911,245, Aug. 15, 1997, Pa No. 5{821,007, which isa division ‘of application’ No. (G8/149,093, Now 8, 1993, Pa. No. 5688750 (51) CO7K 14/708 [52] 530/350; 435/69.1 [58] 530/350; 435/69.1 [56] References Cited USS. PATENT DOCUMENTS 4,683,195 4,683,202 7/1987 Malls ta 7/1987 Nilsen eta OTHER PUBLICATIONS Gluzman et a., (1981) Cell, 23:175~182. Walters, (1993) “Computer-Assisted Modeling of Drugs,” Pharmaceutical Biotechnology, pp. 165-174. Thomas & Capecchi, (1987) Cell, 51:503-512. Bertling, (1987) Bioscience Reports, 7: 107-112. Smithies et al. (1985) Nature, 317: 230-234, Kieffer et al, (1992) Proc. Nail. 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Natl Acad, Sci., 90: 6736-6740, USON6028175A 6,028,175 Feb. 22, 2000 (11) Patent Number: (45) Date of Patent: Yasuda et al., (1993) “Cloning and functional comparison of and 8 opioid receptors from mouse brain.” Proc. Natl Acad. Sci, 90: 6736-6740. Budega et al., (1993) “Regional expression and chromo- somal location of the 8 opiate receptor gene.” Proc. Natt. Acad. Sei, 90: 9305-9309. Brownstein etal, (1993) “A brief history of opiates, opioid peplides and opioid receptors.” Proc. Natl. Acad. Sci, 5391-5393, DiChara & Non, (1992) “Neurobiology of opiate abuse.” Trends in Phamacol. Sci, 13:185-193. Maneckji and Minna (1992) “Nonconventional opioid bind- ing sites medliate growth inhibijtory effects of methadone on human lung cancer cells." Proc, Nal, Acad. Sci, 89: 1169-1173, McKnight & Rees, (1991) “Opioid Receptors and their Ligands." Neurotransmissions, 7: 1-6. Goldstein (1987) “Binding selectivity profiles for ligands of multiple receptor types: focus on opioid receptors.” Trends in Pharmacol. Sei., 8: 456-459, Kristensen et al, (1995) Life Sciences, vol. 56, p. 56. Fukuda et al, (1993) FEBS Letters, vol. 327, p. 311 ‘Wang et al, (1993) PNAS, vol. 90, p. 10230. Reeck et al., (1987) Cell, vol. 50, p. 667. Lewin, (1987) Science, vol. 23. Primary Examiner—Sally Teng, Attorney, Agent, or Firm-—MeDonnell Bochnen Hulbert & Berghoit [57] ABSTRACT The present invention relates to a novel mammalian methadone-specific opioid receptor protein and genes that encode such a protein. The invention is directed toward the isolation, characterization and pharmacological use of mam= malian methadone-specifie opioid receptor proteins. The invention specifically provides isolated complementary DNA copies of mRNA corresponding to the rat homologue ‘ofthe mammalian methadone-specitie opioid receptor gene. ‘Also provided are recombinant expression constructs capable of expressing the mammalian methadone-spec ‘opioid receptor genes of the invention in cultures of trans- formed prokaryotic and eukaryotic cells, as well as such ‘cultures of transformed cells that synthesize the mammalian methadone-specific opioid receptor proteins encoded therein, The invention also provides methods for sereening compounds in vitro that ate capable of binding to the mammalian methadone specific opioid receptor proteins of the invention, and further characterizing the binding prop- erties of such compounds in comparison with known opioid receptor agonists and antagonists 1 Claim, 10 Drawing Sheets6,028,175 Sheet 1 of 10 Feb. 22, 2000 U.S. Patent 9¥9 BLS zos ocb BSE 987 biz Zyl TL IIt wuridutoIvA kK ’YGCA SHWE IT Lopet soica DOOLODOLYLIDDIWIOOLOLWLIOILOLWLOOIOWOWLOIOVDLWIIOIDWOLILOVILLLIWIOWIOWLIL @ tT WorACE MoO Tv Xo 4 ad wa OT TT II o RGR ae ee Se ge TN aN DHOWIVINDOWIILLIOOOILOWOVWLIOLLOLOOLODIWIWOLOOOLOWOOOLOLWWLLLYLWOWLL LW Uy II I Lwoby awd Ld t PAR WATT ON OUT 1 99 TT DOWLIOWIWOWWO LWOWWIODWOWINWOLIOLWILOLVLOLYILOILIONLOWYIOIOLIOLIOIOOOOI LY I Y San S test POCO ALM I od 1 ag vw S HS YN DOLOLOLIDOLLILYILIOOOLODLWIDWILOOWWILIVIOLLIOIIOLIOLLIDOOOWIWILOWLIOLYY Y Y 1 77H Hd ALAN TTS TN 9 04 HS OH TA DLOIOLIDLIDWIIWIDOOWLOIOWOVOLWWWLIILIDOLOLIOWWOOOWWILLLOWIOOWODILWIILLILD aak a@waa7s aw WDD LOWIWOOLOOLIOLLLILIOOLIWOOLWIOWIWDLOIOWIOLLLIOOLOUOLOWLOLIWWOOWIOLL DID LOLOLOLIOOVOWWOLILLIOVIIIONOLLLLWLOLOOOILIVIOWWLIOLILLLII9IIIOLIOO“ DOLIOLILIWIOLIWILIOLLWOLIWOWOLOWOWLOLWWIDDDWOWIOWVIOIIOWIIOLLWIIOWOIWIII VI ‘Old6,028,175 Sheet 2 of 10 Feb. 22, 2000 U.S. Patent OSIT BLOT 900T bee 798 06L BIL i 4 LOWWOWDLWDOLIOLLLIOIVLOLILIWOOOLWWLILOLLOWOWWOLDIW1299919009VOWOOLOLL IA utTiwvwaw 2 9S 9d OAD 19 O AHR UA EK 290913014909 L SUODLIWOYDLOULDDNODOVOLLSLODOLOVDDWWoudsosaaLILIOISONOIS IA Ud Pee Oo OA aA ORO NP ACER a a lb 1 we tN ud LIDDOWIOLIOLIOOOLOLLLILILIOOLOO.LOWLOOLIOLIVLOWOILOWOLWLIOIOOOLIOWWWIIOWO A Maus 9 S$ T1714 AD YT 8 WOT WT somo as 2 AI wdI@amd94 139 384 4a A OWS D WET OW AUK DDDLIDILWOYIOLIOLIOOLOYIILWOWOWWOLWOWWOOLOWWIWOOWOLLOOOLWOLYOONLLOLOOLLD A S Wop vow DW AUNTA Y OW us Sau aaa LODLLO9L9VILLI9DL00099 90109999 LOLWWLL9.LOD9WIDIOWWYOWIOLWOWIOILLDIWOLLD al Old