LANCE Ultra cAMP: A New, Two-Component TR-FRET cAMP Assay for HTS of Gs- and Gi-Coupled Receptors Nancy Gauthier, Julie Blouin, Mireille Caron, Philippe Roby, Lucille Beaudet & Jaime Padrós cAMP Detection + 5 µL Compound(s) + 5 µL Cells - -12-11-10 -9 -8 -7 -6 -5 -4 -3 -2 Read on the EnVision® 500 0 5 LANCE Ultra Norepinephrine Company C Company D S/B EC50 (nM) S/B EC50 (nM) S/B EC50 (nM) Isoproterenol 18.8 0.29 7.1 0.41 2.2 1.9 Norepinephrine 20.7 3.0 6.4 4.6 2.1 15.4 10,000 Formoterol 19.4 9.7 6.3 11.9 2.0 53.3 5,000 BRL 37344 5.3 54.9 2.3 101 1.3 464 Salmeterol 9.2 335 3.6 375 1.5 752 25,000 Formoterol 20,000 BRL 37344 15,000 - -12-11-10 -9 -8 -7 -6 -5 -4 -3 -2 Salmeterol - -12-11-10 -9 -8 -7 -6 -5 -4 -3 -2 Log [Agonist] (M) 3,000 0 - -11 -10 -9 -8 -7 -6 -5 2,000 1,500 1,000 500 Log [Agonist] (M) 6 -10 -9 -8 -7 CXCL11 CXCL10 CXCL9 80,000 60,000 2,000 1,500 1,000 500 0 - -11 -10 -9 -8 -7 -6 -5 -4 Incubate 1h 20,000 -6 - -11 -5 -10 -9 -8 -7 -6 -9 -8 -7 -6 -5 7 1,500 AM 251 SR141716 LY 320135 1,250 1,000 750 LANCE Ultra Company C 500 Log [Antagonist] (M) S/B EC50 (nM) S/B EC50 (nM) S/B EC50 (nM) CXCL11 6.3 1.7 3.7 3.6 2.9 3.4 CXCL10 4.7 27.3 3.5 47.7 2.3 11.1 CXCL9 5.3 130 4.3 924 1.8 51.8 S/B IC50 (nM) S/B IC50 (nM) AM 251 5.0 11.9 3.5 11.2 SR141716 4.4 36.6 3.7 35.9 LY 320135 2.4 149 2.3 191 Agonist Responses in CHO Cells Expressing Gs-hMC4 Receptors -8 -7 -6 -5 -4 -3 Company C (dynamic 2 kit) 5,000 cells, 2.5 µM Forskolin + 1 µM WIN55,212-2 AM 251 SR141716 LY 320135 2,500 2,000 1,500 1,000 500 0 - -10 -9 -8 -7 -6 30 -5 -4 Log [Antagonist] (M) -3 40 50 60 Cells Agonist Agonist + Antagonist Detection time Mean SD %CV Mean SD %CV Mean SD %CV 1h 15518 952 6.1 1505 83 5.5 10080 945 9.4 O/N 12412 746 6.0 1363 111 8.1 8506 709 8.3 Cells/Agonist S/B 10.3 Z' 0.78 9.1 0.77 Agonist + Antagonist/ Agonist S/B Z' 6.7 0.64 6.2 0.66 LANCE Ultra Company C IC50 (nM) IC50 (nM) AM 251 120 55.9 SR141716 163 161 LY 320135 277 382 9 Standard Curve Stability Over Time LANCE Ultra cAMP kit Company C (dynamic 2 kit) 25,000 20,000 15,000 10,000 5,000 0 - -11 -10 -9 -8 -7 -6 -5 4,500 4,000 3,500 3,000 2,500 2,000 1,500 1,000 500 0 1h O/N - -11 Log [cAMP] (M) -10 -9 -8 -7 -6 -5 Log [cAMP] (M) LANCE Ultra Company C Detection time S/B IC50 (nM) S/B IC50 (nM) 1h 68.1 1.4 19.5 4.3 O/N 68.1 1.5 12.7 4.1 15,000 Melanotan II NDP--MSH -MSH 12,500 10,000 7,500 10 5,000 Conclusions 2,500 • The LANCE Ultra cAMP kit showed the highest assay sensitivity and signal window: IC50 value of 28 fmoles and S/B ratio of ~70 in a cAMP standard curve. These two assay parameters were stable following overnight incubation. 0 - -13 -12 -11 -10 -9 -8 -7 -6 Log [Agonist] (M) 0 - -10 -9 20 LANCE Ultra cAMP kit 1,000 cells 250 Log [Antagonist] (M) -4 Company D Log [Agonist] (M) 3,000 - -11 -10 Company C -5 Company C (dynamic 2 kit) 5,000 cells, 2.5 µM Forskolin + 1 µM WIN55,212-2 LANCE Ultra cAMP kit 2,500 cells, 2.5 µM Forskolin + 1 µM WIN55,212-2 900 800 700 600 500 400 300 200 100 0 LANCE Ultra 40,000 Antagonist Responses in CHO Cells Expressing Gi-hCB1 Receptors 2,500 10 100,000 Log [Agonist] (M) LANCE Ultra cAMP kit 2,500 cells, 2.5 µM Forskolin + 1 µM WIN55,212-2 0 1h O/N 0 0 - -11 NDP--MSH (0.5 nM) + SHU9119 (30 µM) Detection time TR-FRET Signal (665 nm) 6,000 NPD--MSH (0.5 nM) # Wells Tr-FRET Signal (665 nm) 9,000 2,500 Luminescence (cps) 12,000 TR-FRET Signal (665 nm) 15,000 Cells 15,000 12,500 10,000 7,500 5,000 2,500 0 Company D (XS+ kit) 5,000 cells, 20 µM Forskolin Company C (dynamic 2 kit) 5,000 cells, 1 µM Forskolin LANCE Ultra cAMP kit 2,000 cells, 300 nM Forskolin 20,000 17,500 Log [Agonist] (M) Agonist Responses in CHO Cells Expressing Gi-hCXCR3 Receptors Log [Antagonist] (M) Each kit was used with the cell density giving the highest S/B ratio. All reagents were prepared and dispensed according to manufacturer’s recommendations. Experiments with all kits were performed side-by-side with the same batch of frozen cells and using the same serially diluted solutions of the cAMP standard, forskolin, agonists and antagonists. For antagonist assays, agonist (Gs-GPCR) or forskolin/agonist (Gi-GPCR) were used at their EC90 (fluorescence values). All assays were conducted manually in 384well format. Signal was detected with the EnVision reader in TRFRET or luminescence mode. Isoproterenol 30,000 0 Log [Agonist] (M) + 5 µL Eu-cAMP + 5 µL ULight-anti-cAMP Incubate 30 min 1,000 35,000 TR-FRET Signal (665 nm) 0 Luminescence (cps) 3,000 TR-FRET Signal (665 nm) TR-FRET Signal (665 nm) 6,000 In the presence of free cAMP Protocol (384-well Format) Cell Stimulation 9,000 1,500 LANCE Ultra cAMP kit 1,000 cells TR-FRET Signal (665 nm) 3 12,000 2,000 cAMP (fmoles) In the absence of free cAMP 15,000 Company D (HS+ kit) 1,000 cells 2,500 TR-FRET Signal (665 nm) LANCE Ultra cAMP Assay Principle Company C (dynamic 2 kit) 7,500 cells 18,000 TR-FRET Signal (665 nm) 2 LANCE Ultra cAMP kit 2,000 cells TR-FRET Signal (665 nm) Guanosine triphosphate binding protein-coupled receptors (GPCRs) represent one of the largest and most important classes of pharmaceutical drug targets. We have developed a secondgeneration LANCE® time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay designed to measure cAMP produced upon modulation of adenylyl cyclase activity by activated GPCRs. The homogeneous two-component LANCE Ultra cAMP assay kit is based on the competition between europium chelate-labeled cAMP and cellular cAMP for binding to high-affinity anti-cAMP monoclonal antibodies labeled with the ULight™ dye. Here we present data comparing the performance in 384-well format of the new LANCE Ultra cAMP kit with that of alternative cAMP assay technologies, namely a TR-FRET assay (dynamic 2 kit from Company C) and two Enzyme Fragment Complementation assays (XS+ and HS+ kits from Company D). These cAMP assay technologies were evaluated for their ability to detect agonist- or antagonist-induced cAMP responses in suspension cells expressing endogenous (β-adrenergic) or recombinant receptors (Gs-MC4; Gi-CXCR3; Gi-CB1). The LANCE Ultra kit yields a cAMP assay with the highest sensitivity and signal window than any tested: IC50 value of 28 fmoles and signal-to-background (S/B) ratio of 70 in a cAMP standard curve. These features allow using fewer cells compared to other cAMP detection platforms. Results of cell-based assays indicated that all four cAMP platforms provide comparable assay pharmacology with the expected rank order of agonist or antagonist potency. However, in every application, the LANCE Ultra outperformed the other cAMP platforms in terms of S/B ratio. This key advantage, combined to a stable pharmacology, simple assay protocol and unique kit format for every application, makes the LANCE Ultra cAMP assay a superior HTS technology suitable for both Gs- and challenging Gi-coupled receptors. Z’ Study in CHO Cells Expressing Gs-hMC4 Receptors 8 Agonist Responses in SK-N-MC Cells Expressing Endogenous Gs-coupled β-adrenergic Receptors TR-FRET Signal (665 nm) 4 Abstract cAMP (fmoles) 1 Company C (dynamic 2 kit) 5,000 cells 2,000 Melanotan II NDP--MSH -MSH 1,500 1,000 • The LANCE Ultra technology enabled the development of robust cAMP assays suitable for HTS, as indicated by the high and stable Z’ values obtained. 500 0 - -13 -12 -11 -10 -9 -8 -7 -6 Log [Agonist] (M) LANCE Ultra Melanotan II NDP-α-MSH α-MSH S/B 10.9 10.8 9.0 EC50 (nM) 0.02 0.11 0.16 Company C S/B 4.4 4.0 3.8 • Expected pharmacological profiles and ranking of compound potency were obtained with all four cAMP kits tested. However, the LANCE Ultra technology consistently provided higher S/B ratios while using fewer cells. EC50 (nM) 0.04 0.15 0.20 • These key advantages, combined with a simple assay protocol and unique kit format, make the LANCE Ultra cAMP assay a superior HTS technology suitable for both Gs- and Gi-coupled GPCRs. PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com