US 200802749 13A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0274913 A1 Lee (43) Pub. Date: Nov. 6, 2008 (54) MULTIPLEX ARRAYUSEFUL FOR (86). PCT No.: PCT/US2O06/020810 ASSAYING PROTEIN-PROTEIN INTERACTION S371 (c)(1), (2), (4) Date: Jun. 20, 2008 (75) Inventor: Kevin Lee, New York, NY (US) (30) Foreign Application Priority Data Correspondence Address: May 27, 2005 (US) .................................... 60685565 INVITROGEN CORPORATION O O CFO INTELLEVATE Publication Classification P.O. BOX S2OSO (51) Int. Cl. MINNEAPOLIS, MN 55402 (US) C40B 40/02 (2006.01) (52) U.S. Cl. .......................................................... 506/14 (73) Assignee: ision Corporation, Carlsbad (57) ABSTRACT The described invention shows how multiple interactions (21) Appl. No.: 11/915,689 between two proteins of interest can be determined by observ ing activation or lack thereof of intracellular proteins, follow (22) PCT Filed: May 30, 2006 ing interaction of ligand and receptor. 7TM Receptor 2222 Nuclear Localization Transcription Reporter Gene induction Factor tTA Protease Cleavage Site Arrestin TEV Protease Patent Application Publication Nov. 6, 2008 Sheet 1 of 13 US 2008/02749 13 A1 Ø uo?onpu]eues)d??Jode>+ Patent Application Publication Nov. 6, 2008 Sheet 2 of 13 US 2008/02749 13 A1 Epinephrine and isoproterenol Dose Responses 110 100 90 80 70 60 -o- Epinephrine EC50 E 227 nM 50 a soproterenol i 40 EC50 = 51.1 nM 30 20 10 Drug FIG. 2A Alprenolol Dose inhibition Curve (1 uM Epinephrine) 03-06-03 i Log Alprenolol FIG. 2B Patent Application Publication Nov. 6, 2008 Sheet 3 of 13 US 2008/02749 13 A1 Vasopressin Dose Response Curve 110 100 -o- Vasopressin 50 EC50 E 3.3 nM i No Ligand 10 uMDopamine D2 / B-arr 2 D21B-arr 2 D21 B-arr 1 D21B-arr 1 (A383) (A383) FIG. 4 Patent Application Publication Nov. 6, 2008 Sheet 4 of 13 US 2008/02749 13 A1 BetaAR-> lacz (Clone 41) 8OOO.O BetaAR -> lacz 7OOO.O 6OOOO 5000.0 4000.0 3OOOO 2000.0 - - 1 OOOO No Ligand +lso +Dopa +lso-Dop F.G. 5A D2 -> luc (Clone 1H10) 6OOOO.O 5OOOOO 40000.0 3OOOOO 2 O O O O. 10OOO.O 0.0 No Ligand +lso +Dopa +lso--Dop F.G. 5B Patent Application Publication Nov. 6, 2008 Sheet 5 of 13 US 2008/02749 13 A1 OPRK-AVPR2 LaCZ Multiplex Data ADRB2-AVPR2 120.00 1OOOO 80.00 60.00 40.00 20.00 O.OO No Ligand U69,593 lso 10 SO + 10 uM uM U69,593 F.G. 6 Patent Application Publication Nov. 6, 2008 Sheet 6 of 13 US 2008/02749 13 A1 EGFR(L) I SH2 Tev Patent Application Publication Nov. 6, 2008 Sheet 7 of 13 US 2008/02749 13 A1 Effect of IFNalpha and IFNbeta on IFN Type I Receptors in HTL5B8.1 No Ligand 300 250 200 150 100 50 Treatments FIG. 8 Patent Application Publication Nov. 6, 2008 Sheet 8 of 13 US 2008/02749 13 A1 Dose Response Curve for IFNalpha in HTL5B8.1 350 3OO 250 200 1. g 150 C 1OO EC50: 7621 u/ml 50 O O 1 2 3 4. 5 6 Log10 IFNalpha FIG. 9 Effect of IFNalpha on IFN Type I Receptors in CHO-K Cells -FN + 5000 u/ml IFNalpha d - na g 4. pFRLuc-R1(L)-GAL4-NF-KB +R2.2-TeV--STAT1+STAT2 pFRLuc-pCDNA3 Treatments FIG. 10 Patent Application Publication Nov. 6, 2008 Sheet 9 of 13 US 2008/02749 13 A1 ADRB1 (9029) pro293a, Alprenolol I Yohimbine 05.18.05 400 -o- Alprenolol IC50 = 1.50 nM 100 -- Yohimbine IC50 = n.a. - 12 - 11 - 10 -9 -8 -7 -6 -5 -4 logantagonist, M FIG 11A ADRB2 (374) pro293a, Alprenolol I Yohimbine 05.18.05 -o- Aprenolol IC50 = 115 pM -- Yohimbine IC50 F n.a. -12 -11 -lo -9 8 7 6 5 4 logantagonist, M) FIG. 11B Patent Application Publication Nov. 6, 2008 Sheet 10 of 13 US 2008/02749 13 A1 ADRA2A (9027) Pro293a, Alprenolol I Yohimbine 05.18.05 -o- Alprenolol IC50 = n.a. -- Yohimbine IC50 = 1.69 nM 300 100 - 12 -11 - 10 -9 -8 -7 -6 -5 -4 Logantagonist, M FIG. 11C ADRA2B (9019) pro293a, Alprenolol I Yohimbine 05.18.05 225 -o- Alprenolol IC50 = n.a. 2OO -- Yohimbine IC50 = 4.62 mm 175 150 125 i 100 75 50 25 -12 -11 -10 -9 -8 -7 -6 -5 -4 logantagonist, MI FIG. 11D Patent Application Publication Nov. 6, 2008 Sheet 11 of 13 US 2008/02749 13 A1 ADRA2C (9025) Pro293a, Alprenolol I Yohimbine 05.18.05 -- Alprenolol IC50 = n.a. -- Yohimbine IC50 = 11.5 nM -12 - 11 - 10 -9 -8 -7 -6 -5 -4 Logantagonist, M F.G. 11E ADRB1 (9029) UK 14,304 || US 13 Isoproterenol 05.06.05 EC50 = n.a. 350 -v- isoproterenol EC50 = 30.5 nM 300 -12 -11 -10 -9 -8 7 6 5 4 LogLigand, M F.G. 11 F Patent Application Publication Nov. 6, 2008 Sheet 12 of 13 US 2008/02749 13 A1 ADRB2 (374) UK 14,304. 1 isoproterenol 05.06.05 350 31. 200 • US50 13 F .a. 150 -- isoproterenol EC50 = 37.3 nM 1 OO 50 O -12 -11 - 10 -9 -8 -7 -6 -5 -4 LogLigand, MI F.G. 11G ADRB2A (9027) UK 14,304 f isoproterenol 05.06.05 7O -- UK 14,304 EC50 = 4.12 nM 60 -- isoproterenol EC50 = 28.8 uM 50 up 40 D - a 30 2O 10 - O -12 -11 -10 -9 -8 -7 -6 -5 4 LogLigand, M F.G. 11H Patent Application Publication Nov. 6, 2008 Sheet 13 of 13 US 2008/02749 13 A1 ADRA2B (9019) UK 14,3041 isoproterenol 05.06.05 -o- UK 14,304 EC50 = 188 nM -- isoproterenol EC50 = 13.0 uM () O - 1. -12 -11 - 10 -9 -8 -7 -6 -5 -4 LogLigand, M FIG. 11 ADRA2C (9025) UK 14,304 Isoproterenol 05.06.05 70 -- UK 14,304 EC50 = 94.4 nM -- isoproterenol 60 EC50 = 9.0 uM 50 n 40 O - 2 30 2O 1O O -12 -11 - 10 -9 -8 -7 -6 -5 -4 LogLigand, M FIG 11 US 2008/02749 13 A1 Nov. 6, 2008 MULTIPLEX ARRAYUSEFUL FOR ligand and receptor stimulates guanine nucleotide exchange ASSAYING PROTEIN-PROTEIN and dissociation of the G protein heterotrimer into C. and By INTERACTION Subunits. 0005. Both the GTP-bound C. subunit and the By dimer can FIELD OF THE INVENTION act to regulate various cellular effector proteins, including 0001. This invention relates to methods for determining adenylyl cyclase and phospholipase C (PLC). In conventional interaction between molecules of interest. More particularly, cell based assays for GPCRs, receptor activity is monitored it relates to determining if a particular Substance referred to as by measuring the output of a G-protein regulated effector the test compound modulates the interaction of two or more pathway, such as the accumulation of cAMP that is produced specific proteins of interest, via determining activation of a by adenylyl cyclase, or the release of intracellular calcium, reporter gene in a cell, where the activation, or lack thereof, which is stimulated by PLC activity. results from the modulation or its absence. The determination 0006 Conventional G-protein based, signal transduction occurs using transformed or transfected cells, which are also assays have been difficult to develop for Some targets, as a a feature of the invention, as are the agents used to transform result of two major issues. or transfect them. More particularly, the inventions relates to 0007 First, different GPCRs are coupled to different G what will be referred to as “multiple arrays, which permit an protein regulated signal transduction pathways, and G-pro investigator to screen test compounds against a plurality of tein based assays are dependent on knowing the G-protein proteins, such as receptors, GPCRs in particular. specificity of the target receptor, or require engineering of the cellular system, to force coupling of the target receptor to a BACKGROUND AND RELATED ART particular effect or pathway. Second, all cells express a large 0002 The study of protein/protein interaction, as exem number of endogenous GPCRs, as well as other signaling plified, e.g., by the identification of ligands for receptors, is an factors. As a result, the effector pathways that are measured area of great interest. Even when a ligand or ligands for a may be modulated by other endogenous molecules in addi given receptor are known, there is interest in identifying more tion to the target GPCR, potentially leading to false results. effective or more selective ligands. GPCRs will be discussed 0008 Regulation of G-protein activity is not the only hereinas a non-exclusive example of a class of proteins which result of ligand/GPCR binding. Luttrell, et al., J. Cell Sci., can be studied in this way. 115:455-465 (2002), and Ferguson, Pharmacol. Rev., 53:1- 0003. The G-protein coupled receptors, or “GPCRs' here 24 (2001), both of which are incorporated by reference, after, are the largest class of cell Surface receptors known for review other activities which lead to termination of the GPCR humans. Among the ligands recognized by GPCRs are hor signal. These termination processes prevent excessive cell mones, neurotransmitters, peptides, glycoproteins, lipids, stimulation, and enforce temporal linkage between extracel nucleotides, and ions. They also act as receptors for light, lular signal and corresponding intracellular pathway. odors, pheromones, and taste. Given these various roles, it is 0009. In the case of binding of an agonist to GPCR, serine perhaps not surprising that they are the Subject of intense and threonine residues at the C terminus of the GPCR mol research, seeking to identify drugs useful in various condi ecule are phosphorylated.