S. Wu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5859–5863
5863
7. Stump, G. L.; Wallace, A. A.; Regan, C. P.; Lynch, J. J.,
Jr. J. Pharmacol. Exp. Ther. 2005, 315, 1362.
8. Castle, N. A.; Hughes, P. F.; Mendoza, J. S.; Wilson, J.
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et al.23 HERG is stably overexpressed in HEK cells.
Microelectrodes are pulled from borosilicate glass
(TW150) and heat polished (tip resistance, 1.5–3 MX).
The external solution is standard Tyrode’s solution. The
internal (microelectrode) solution contained: 110 mM
KCl, 5 mM K2APT, 5 mM K4BAPT, 1 mM MgCl2, and
10 mM Hepes, adjusted to pH 7.2 with KOH. Command
potentials are applied for 2 s to +20 mV from a holding
potential of À80 mV using Axon software (pClamp 8.1)
and hardware (Axopatch 1D, 200B). Tail currents are
generated by returning to À40 mV for 2 s. Compounds are
prepared as 10–20 mM DMSO stocks and diluted to
appropriate test concentrations. After stable currents are
achieved, compounds are perfused onto the cells and the
cells are pulsed every 20 s until no further changes in
current are evident at a given compound concentration.
Inhibition of HERG is measured at the peak of the tail
currents and expressed relative to controls. Initial HERG
activity is estimated by single point determinations run at
10 lM. Concentration–response curves are generated for
appropriate compounds utilizing at least four concentra-
tions and an n = 3. Curve fitting and IC50 estimating were
done using Graphpad software (Ver. 4).
9. Gross, M.; Castle, N. A. PCT Int. Appl. 1999, WO 99/
37607.
10. Gross, M.; Beaudoin, S.; Reed, A. D. U.S. Patent 6458794
B2, 2002; PCT Int. Appl. 2001, WO 01/46155 A1.
11. Castle, N. A.; Gross, M.; Mendoza, J. S.; U.S. Patent
6174908 B1, 2001; U.S. Patent, 6395730 B1, 2002; PCT
Int. Appl. 1999, WO 99/62891.
12. Peukert, S.; Brendel, J.; Hemmerle, H.; Kleemann, H-W.
PCT Int. Appl. (2002), WO 02/48131 A1. WO 02/44137
A1. WO 02/446162 A1.
13. Peukert, S.; Brendel, J.; Pirard, B.; Bruggemann, A.;
Below, P.; Kleemann, H.-W.; Hemmerle, H.; Schmidt, W.
J. Med. Chem 2003, 46, 486.
14. Brendel, J.; Schmidt, W.; Below, P. PCT Int. Appl. 2001,
WO 01/25189.
15. Brendel, J., Pirard, B. U.S. Patent 0193422 A1, 2002.
16. Brendel, J.; Bohme, T.; Peukert, S.; Kleemann, H.-W.
U.S. Patent 0114499 A1, 2003.
22. HL-1 cells expressing endogenous L-type calcium channels
are removed from culture flasks using trypsin, plated on
fibronectin/gelatin-coated, clear-bottomed, black-walled
96-well microplates in Claycomb media (JRH Biosciences
#51800) containing 10% fetal bovine serum, 4 mM
L-glutamine, and 10 lM norepinephrine, and grown to
confluency overnight. The next day, growth medium is
aspirated from confluent cell monolayers and replaced
with 100 lL per well Tyrode’s solution (in mM: 130 NaCl,
4 KCl, 1.8 CaCl2, 1.0 MgCl2, 20 Hepes, and 10 glucose,
pH 7.35) and 50 lL per well FLIPR Calcium Assay kit,
component A (#R-8033, Molecular Devices Corporation)
and incubated for 60 min. in a 5% CO2 37 °C incubator.
50 lL per well test compounds is added to the plates and
further incubated for 15 min. in a 5% CO2 37 °C incuba-
tor. All final solutions contain the anion exchange
inhibitor, probenecid (2.5 mM). The 96-well plates are
then placed in the center position of the FLIPR 1(Fluo-
rometric Imaging Plate Reader, Molecular Devices Cor-
poration). Cell monolayers in each well are simultaneously
illuminated at 488 nm with an Argon ion laser, and
fluorescence emission is monitored using a 510–570 nm
bandpass filter and a cooled CCD camera. To depolarize
the plasma membrane and activate L-type calcium chan-
nels, 50lL per well of 20 mM KCl (final concentration) is
dispensed simultaneously to all 96 wells using the FLIPR’s
automatic 96-well pipettor. Fluorescence measurements
are captured for 5 min. following KCl addition. Calcium
influx, expressed as % control, is calculated for each
concentration of test compound and concentration–re-
sponse curves and IC50 values are generated using
GraphPad Prism 4.0.
17. Wirth, K. J.; Paehler, T.; Rosenstein, B.; Knobloch, K.;
Maier, T.; Frenzel, J.; Brendel, J.; Busch, A. E.; Bleich, M.
Cardiovasc.Res. 2003, 60, 298.
18. Peukert, S.; Brendel, J.; Pirard, B.; Strubing, C.; Klee-
mann, H-W.; Bohme, T.; Hemmerle, H. Bioorg. Med.
Chem. Lett. 2004, 14, 2823.
19. Wu, S.; Janusz, J. 227th ACS National Meeting, Anaheim,
CA. March 28, 2004.
20. Kv1.5 currents are recorded by the whole cell mode of
patch clamp electrophysiology. Kv1.5 is stably over
expressed in either HEK or LTK-cells. Microelectrodes
are pulled from borosilicate glass (TW150) and heat
polished (tip resistance, 1.5–3 MX). The external solution
is standard Tyrode’s solution. The internal (microelec-
trode) solution contained: 110 mM KCl, 5 mM K2APT,
5 mM K4BAPTA, 1 mM MgCl2, and 10 mM Hepes,
adjusted to pH 7.2 with KOH. Command potentials are
applied for 1 s to +60 mV from a holding potential of
À70 mV using Axon software (pClamp 8.1) and hardware
(Axopatch 1D, 200B). Compounds are prepared as 10–
20 mM DMSO stocks and diluted to appropriate test
concentrations. After stable currents are achieved, com-
pounds are perfused onto the cells and the cells are pulsed
every 5 s until no further changes in current are evident at
a given compound concentration. Inhibition was mea-
sured at the end of the 1 s pulses and expressed relative to
controls. Concentration–response curves are generated for
appropriate compounds utilizing at least four concentra-
tions and an n = 3. Curve fitting and IC50 estimating were
done using Graphpad software (Ver. 4).
21. HERG currents are recorded by the whole cell mode of
patch clamp electrophysiology as described by Hamill
23. Hamill et al. Pflugers Archiv. 1981, 391, 85.