Automated 1,536-well cAMP Assay for the Identification and Characterization of Agonists and Antagonists of a Gi-coupled Receptor Using the New LANCE Ultra cAMP Kit. Julie Blouin, Mireille Caron, Nancy Gauthier, Dianne Brazzill, Philippe Roby, Lucille Beaudet & Jaime Padrós 4 2 LANCE Ultra cAMP kit Company C (dynamic 2 kit) LANCE Ultra cAMP kit 1,500 S/B: 13.8 IC50: 1.1 nM 1,250 1,000 750 500 250 0 - -11 -10 -9 -8 -7 384-well Format 1,536-well Format -6 -5 1,200 S/B: 10.1 IC50: 1.2 nM 1,000 800 600 400 200 0 - -11 -10 Log [cAMP] (M) -9 -8 -7 -6 -5 1,536-well Format 800 S/B: 6.9 IC50: 4.8 nM 600 400 200 0 - -11 -10 -9 -8 -7 -6 -5 TR-FRET Signal (665 nm) 384-well Format 600 S/B: 5.1 IC50: 3.4 nM 500 400 300 200 100 800 Cells 300 nM Forskolin + 300 nM 8-OH-DPAT 600 300 nM Forskolin 400 300 nM Forskolin + 300 nM 8-OH-DPAT + 10 µM Spiperone 200 - -11 -10 -9 -8 -7 -6 0 10 20 30 40 50 # Wells -5 Log [cAMP] (M) Cells Detection time 5 Forskolin + Agonist/ Forskolin + Agonist/ Forskolin + Agonist Detection Forskolin + Antagonist time S/B Z' S/B Z' 0 0 Log [cAMP] (M) Log [cAMP] (M) TR-FRET Signal (665 nm) 1,536-well-Format 1,000 cells TR-FRET Signal (665 nm) The LANCE® Ultra cAMP assay is a second-generation LANCE time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay designed to measure cAMP produced upon modulation of adenylyl cyclase activity by activated guanosine triphosphate binding protein-coupled receptors (GPCRs). The homogeneous two-component assay 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. The LANCE Ultra cAMP assay was miniaturized and automated to 1,536-well format for the identification and characterization of agonists and antagonists of a model Gi-coupled GPCR (human Serotonin 5-HT1A). The performance of the miniaturized assay was compared to that of a commercially available TR-FRET cAMP assay (dynamic 2) for assessing the suitability of the two technologies for HTS. Automated assays were conducted in 8-μL total assay volume using the JANUS® Automated Workstation, and plates were read on ViewLux® ultraHTS Microplate Imager. Both assay technologies exhibited comparable assay pharmacology for agonist and antagonist responses. EC50/IC50 values determined in 1,536-well format were comparable to those of the 384-well plate assays. However, regardless of the reader used, cAMP assays performed using the LANCE Ultra technology exhibited significantly higher signal-tobackground ratio for both agonist and antagonist assays compared to the alternative technology. In addition, Z’-factor values for the LANCE Ultra cAMP assay remained stable throughout the entire incubation period tested (1 hour, 4 hour and overnight incubation; Z’ = 0.64-0.71). In contrast, the alternative TRFRET assay showed lower Z’-factor values after 1 and 4 hours of incubation (Z’ = 0.49-0.55) and a lack of assay robustness following overnight incubation (Z’ < 0.3). These results indicate that the LANCE Ultra technology is a superior TR-FRET cAMP assay technology for use as a primary and/or secondary assay in 1,536-well format for Gi-coupled GPCRs. Z’ Study in CHO Cells Expressing Gi-h5-HT1A Receptors 7 cAMP Standard Curves (384 and 1,536-well Format) TR-FRET Signal (665 nm) Abstract TR-FRET Signal (665 nm) 1 Agonist Response in CHO Cells Expressing Gi-h5-HT1A Receptors Forskolin 1h 3.0 0.64 2.9 0.66 4h 3.1 0.67 3.0 0.70 O/N 3.0 0.68 2.9 0.71 Forskolin + Agonist Forskolin + Agonist + Antagonist Mean SD %CV Mean SD %CV Mean SD %CV Mean SD %CV 1h 647 22 3.5 154 9 5.8 469 29 6.2 161 5 3.3 4h 608 21 3.4 146 9 6.5 456 25 5.4 152 6 3.7 O/N 647 22 3.5 165 11 6.7 489 24 4.9 172 7 4.1 LANCE Ultra cAMP Assay Principle 1,536-well Format 1,000 cells, 300 nM Forskolin 500 250 0 - -10 -7 -6 -5 -4 600 S/B: 4.4 EC50: 59.5 nM 400 200 0 - -10 -9 -8 -7 -6 -5 -4 S/B: 2.3 EC50: 83.4 nM 200 100 0 - -10 Log [8-OH-DPAT] (M) -9 -8 -7 -6 -5 1,536-well-Format 1,000 cells 1,536-well Format 1,000 cells, 700 nM Forskolin 400 300 Company C (dynamic 2 kit) -4 500 400 S/B: 1.5 EC50: 57.3 nM 300 200 100 0 - -10 Log [8-OH-DPAT] (M) -9 -8 -7 -6 -5 500 Cells 400 700 nM Forskolin + 300 nM 8-OH-DPAT 300 700 nM Forskolin 200 700 nM Forskolin + 300 nM 8-OH-DPAT + 10 µM Spiperone 100 Forskolin + Agonist/ Forskolin + Agonist/ Forskolin + Agonist Detection Forskolin + Antagonist time S/B Z' S/B Z' 0 0 10 -4 20 30 40 50 # Wells Log [8-OH-DPAT] (M) cAMP Detection Cell Stimulation + Compound(s) + Cells + Eu-cAMP + ULight-anti-cAMP The Janus Automated Workstation with a 384-tip modular arm was used to dispense reagents into 1,536-well plates. Automation and miniaturization to 1,536-well format were conducted by maintaining final concentration of reagents in the assay. Identical pipetting protocols were used for the alternative TR-FRET assay. 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 both 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. Forskolin and agonist were used at their EC90 (fluorescence values). Assays in 384-well plate format were conducted manually, whereas assays in 1,536-well plate format were automated using the JANUS Automated Workstation, except for the cell dispensing step (conducted manually). Signal was detected with the ViewLux Imager in TR-FRET mode. LANCE Ultra cAMP kit 384-well Format 2,000 cells, 500 nM Forskolin + 1 µM 8-OH-DPAT S/B: 5.0 IC50: 82.5 nM 750 500 250 0 - -10 -9 -8 -7 -6 -5 Log [Spiperone] (M) -4 S/B: 3.9 IC50: 79.5 nM 600 500 400 300 200 100 0 -9 -8 -7 -6 -5 Log [Spiperone] (M) 1,536-well Format 1,000 cells, 700 nM Forskolin + 300 nM 8-OH-DPAT 384-well Format 2,000 cells, 2.5 µM Forskolin + 1 µM 8-OH-DPAT 700 - -10 Forskolin 1h 1.7 0.50 1.7 0.55 4h 1.6 0.49 1.7 0.54 O/N 1.6 0.28 1.5 0.19 Forskolin + Agonist Forskolin + Agonist + Antagonist Mean SD %CV Mean SD %CV Mean SD %CV Mean SD %CV 1h 389 15 4.0 189 8 4.2 314 13 4.2 186 6 3.2 4h 360 13 3.5 172 7 4.3 283 11 4.0 169 6 3.7 O/N 416 40 9.7 173 10 5.6 269 13 4.9 175 12 6.8 Company C (dynamic 2 kit) 1,536-well Format 1,000 cells, 300 nM Forskolin + 300 nM 8-OH-DPAT 1,000 Cells Detection time Antagonist Response in CHO Cells Expressing Gi-h5-HT1A Receptors -4 400 S/B: 2.4 IC50: 111 nM 300 200 100 0 - -10 -9 -8 -7 -6 -5 Log [Spiperone] (M) -4 TR-FRET Signal (665 nm) 384-well plate 1,536-well plate Manual assay Automated assay 5 µL 2 µL 5 µL 2 µL 5 µL 2 µL 5 µL 2 µL 20 µL 8 µL 6 TR-FRET Signal (665 nm) Pipetting Protocols for the LANCE Ultra cAMP Assay Incubate 1h TR-FRET Signal (665 nm) Incubate 30 min Cells Compound(s) Eu-cAMP ULight-anti-cAMP Total assay volume -8 800 Log [8-OH-DPAT] (M) Read on the ViewLux Reagents -9 384-well Format 2,000 cells, 2.5 µM Forskolin TR-FRET Signal (665 nm) S/B: 3.8 EC50: 82.0 nM TR-FRET Signal (665 nm) Protocol 1,000 TR-FRET Signal (665 nm) 3 TR-FRET Signal (665 nm) 384-well Format 2,000 cells, 500 nM Forskolin 750 Company C (dynamic 2 kit) TR-FRET Signal (665 nm) LANCE Ultra cAMP kit In the Presence of Free cAMP TR-FRET Signal (665 nm) In the Absence of Free cAMP 8 500 • The LANCE Ultra cAMP assay can be easily automated and miniaturized from 384- to 1,536-well format without compromising assay performance. S/B: 1.8 IC50: 70.6 nM 400 Conclusions • Similar receptor pharmacology was obtained with the LANCE Ultra and alternative TR-FRET cAMP kits in both plate formats. 300 200 • Regardless of the plate format, S/B ratios obtained with the LANCE Ultra kit were consistently higher than those obtained with the alternative TR-FRET cAMP kit. 100 0 - -10 -9 -8 -7 -6 -5 Log [Spiperone] (M) -4 • The miniaturized LANCE Ultra cAMP assays are robust and suitable for HTS with Z’ values superior to that of the alternative TR-FRET cAMP assay. • The LANCE Ultra cAMP assay is a superior HTS technology for use as a primary or secondary assay in 1,536-well format for Gi-coupled GPCRs. PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com