hERG K+ Channel Membranes: For Fast, Robust & Sensitive hERG Safety Screening

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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©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
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