hERG K+ Channel Membranes For fast, robust & sensitive hERG safety screening Assess potential cardiotoxicity of compounds early during the drug development cycle with a high-throughput hERG binding assay. The literature indicates that binding assay results for hERG strongly correlate with electrophysiological data.1-3 hERG K+ Channel binding assays with [3H]-astemizole A 25 + 5000 Total Specific Non-specific Bound (fmol) 20 4000 15 3000 10 2000 5 1000 0 0.0 2.5 5.0 7.5 Bound (DPM) hERG binding assays using PerkinElmer’s hERG K Channel membranes are an ideal complement to whole cell patch clamp for the detection and initial charac+ terization of hERG channel blockers. hERG K Channel membranes show excellent expression and performance when tested with different radiolabeled hERG blockers, both in filtration and homogenous binding assays. 0 10.0 [ 3H]-Astemizole (nM) Perform faster, easier safety tests using + PerkinElmer’s hERG K Channel membranes • Easy to automate – simple binding assay set-up • Precise – Z’ of 0.7 obtained in a FlashBlue™ GPCR homogeneous binding assay with [125I]-BeKm-1 • HTS-compatible – for 96- and 384-well formats • Choice of radioligands – chose between PerkinElmer’s [3H]-astemizole and [125I]-BeKm-1 radioligands B [3H]-Astemizole Bound (% of Control) • High expression – membranes derived from a hERG1 HEK-293 cell line with Bmax value for [3H]-astemizole between 5-10 pmol/mg protein 150 125 100 75 Astemizole Dofetilide E4031 Terfenadine Quinidine 50 25 0 -∞ -10 -9 -8 -7 -6 -5 -4 -3 -2 Competitor (Log M) • Read on any scintillation counter, such as PerkinElmer’s MicroBeta® or TopCount® Radioligands for hERG binding assays Compound PerkinElmer offers two complementary radioligands for hERG binding assays that bind preferentially to distinct conformations of the hERG channel. PerkinElmer can also custom label your proprietary hERG blocker. Astemizole 18 Dofetilide 33 3 Astemizole, [O-Methyl- H]-: This small molecule + binds to open/inactivated hERG K channels. Typical binding assay results obtained with tritiated astemizole are illustrated in Figure 1. The pharmacology of [3H]-astemizole binding has been shown to correlate well with patch clamp electrophysiological measurements1, and with the binding of another radioligand, [3H]-dofetilide2. w w w. p e r k i n e l m e r. c o m E-4031 K i (nM) 134 Terfenadine 445.5 Quinidine 40.000 3 Figure 1A. [ H]-Astemizole saturation binding assay performed + using hERG K Channel membranes. Bmax value of 6 pmol/mg and Kd value of 3 nM were obtained. Figure 1B. In competition experiments, the radioligand was used at the Kd concentration. Ki values are the average of two independent experiments. A quantity of 2.5 µg of hERG K+ Channel membranes was used per well. The assay was incubated for 1 h at room temperature. Signal was detected with a MicroBeta after filtration of the samples. BeKm-1, [125I-Tyr11]-: This peptidic toxin from the scorpion + Buthus eupeus binds principally to closed hERG K channels with an affinity in the sub-nanomolar range4. Results from filtration and homogeneous hERG binding assays obtained using iodinated BeKm-1 are illustrated in Figures 2 and 3, respectively. Ordering information Membrane Target Systems Product + hERG K Channel Cat. No. Size RBHERGM400UA RBHERGM000UA 400 assay units* Bulk *One assay unit is defined as the amount of membranes per well in a 96-well plate in a filtration assay. + hERG K Channel binding assays with [125I]-BeKm-1 Complementary products Radioligands A Total Specific Non-specific 1.0 Cat. No. Size 7000 BeKm-1, [125I-Tyr11]- NEX412 10 and 25 µCi 6000 Astemizole, [O-Methyl-3H]- NET1140 25 µCi, 250 µCi, 1 mCi 5000 4000 3000 0.5 2000 Bound (DPM) Bound (fmol) 1.5 Product Note: [125I-Tyr11]-BeKm-1 is used for QC validation of the membrane product. Microplates & Filtration 1000 0.0 0.0 0.1 0.2 0.3 0 0.5 0.4 [ 125I] BeKm-1 (nM) B 125 [ I]-BeKm-1 Bound (DPM) 5000 4000 3000 BeKm-1 2000 Dofetilide E4031 1000 Product Cat. No. Size OptiPlate™-96 White opaque 96-well microplate 6005299 200/box OptiPlate™-384 White opaque 384-well microplate 6007299 200/box IsoPlate-96 White 96-well Microplate with Clear Well 1450-515 100/box UniFilter-96, GF/C 6005174 50/box Filtermat A, GF/C 1450-421 100/PK Filtermat A, 24-well 1450-422 100/PK MeltiLex for MicroBeta® filters 140-441 100/PK Product Cat. No. Size FlashBlue™ GPCR FBB001500MG FBB001002G 500 mg 2g Wheat Germ Agglutinin FlashPlate® PLUS, 96-well SMP105A001PK 20/PK Wheat Germ Agglutinin FlashPlate® HTS PLUS, 384-well SMP411A001PK 10/PK RMP111 40/PK Terfenadine 0 -∞ -14 -12 -10 -8 -6 -4 -2 Competitor (Log M) Assay Platforms Compound BeKm-1 K i (nM) 0.13 Dofetilide 23 E-4031 61 Terfenadine 174 125 Figure 2A. [ I]-BeKm-1 saturation binding assay performed using hERG + K Channel membranes. Bmax value of 0.5 pmol/mg and Kd value of 0.13 nM were obtained. Figure 2B. In competition experiments, the radioligand was used at the Kd concentration. Ki values are the average of two independent experiments. + A quantity of 2.4 µg of hERG K Channel membranes was used per well. The assay was incubated for 1 h at room temperature. Signal was detected with a MicroBeta after filtration of the samples. 2 ® Image FlashPlate WGA coated, 384-well + FlashBlue hERG K Channel binding assay with [125I]-BeKm-1 Compatible Instrumentation Product Cat. No. Size Wallac MicroBeta TriLux, 12 Detector, 32-shelf Model 1450-030 1/EA TopCount® 12 detector, 96 and 384 format C384V01 1/EA ® 125 [ I]-BeKm-1 Bound (DPM) 4000 3000 Z'= 0.71 2000 Total Binding ViewLux™ ultraHTS Microplate Imager 1430-0010 1/EA Non-Specific Binding (100nM BeKm-1) UniFilter-96 Harvester C961961 1/EA UniFilter-96 Harvester (stainless steel) C961962 1/EA MicroBeta Filtermate-96 Harvester D961962 1/EA MicroBeta® Filtermate-24 Harvester D961241 1/EA 1000 ® 0 0 6 12 18 24 Wells References Figure 3. A homogeneous FlashBlue assay was developed with the radioligand [125I]-BeKm-1. The Z’ value was calculated according to the formula of Zhang et al.5 A Z’ value of 0.71 was obtained, demonstrating excellent performance. In this assay, a quantity of 5 µg of hERG K+ Channel membranes and 125 µg FlashBlue GPCR beads were added per well. The radioligand was used at the Kd concentration. The assay was performed manually in a PerkinElmer® Isoplate™ 96-well in a total volume of 80 µL. The plate was incubated for 1 h at room temperature and spun for 5 min at low speed prior to reading with the MicroBeta. 1. Chiu PJS et al. (2004) J. Pharmacol. Sci. 95: 311-319. 2. Finlayson K et al. (2001) Eur. J. Pharmacol. 430: 147-148. 3. Diaz GJ et al. (2004) J. Pharmacol. Toxicol. Methods. 50: 187-99. 4. Angelo K et al. (2003) Pflugers Arch. 447: 55-63. 5. Zhang JH et al. (1999) J. Biomol. Screen. 4: 67-73. 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PerkinElmer Life and Analytical Sciences 710 Bridgeport Avenue Shelton, CT 06484-4794 USA Phone: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices ©2005 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. FlashBlue, IsoPlate, OptiPlate and ViewLux are trademarks and FlashPlate, MicroBeta, PerkinElmer and TopCount are registered trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries that are depicted herein are the property of their respective owners. PerkinElmer reserves the right to change this document at any time without notice and disclaims liability for editorial, pictorial or typographical errors. 007468_01 Printed in USA