IONWORKS HT APPLICATION NOTE #4
By James Costantin, Ph.D., Yen-Wen Chen, Ph.D., Jinfang Liao, Ph.D.,
Jeff Quast, Andrew Wittel, Shawn Handran, Ph.D. and Naibo Yang, Ph.D.
Molecular Devices Corporation, 3280 Whipple Road, Union City, CA 94587.
Voltage-dependent ion channels represent a
largely untapped portion of the potential drug
target market.1 Until recently, voltage-dependent
ion channels have been studied during the drug
discovery process either using indirect methods
of measuring the presence or activity of ion
channels or by the gold standard patch clamp
technique. Some of the higher throughput,
lower content techniques include binding and
flux assays, and the use of fluorescent dyes.2
While these assays have proven useful, they
lack the ability to maintain a population of ion
channels in a particular conformational state due
to the lack of voltage control. The traditional
patch clamp technique is labor intensive but
offers direct control of the membrane potential
and a direct measurement of ionic current.
Thus, a population of channels can be driven
to a particular conformational state, which
may affect drug binding. The low throughput
conventional patch clamp technique creates a
severe bottleneck in following up on the hits
from upstream indirect ion channel assays.3
This application note illustrates how the IonWorks
HT system can be used to rapidly confirm hits
from higher-throughput assays as well as perform
y human ether-a-go-go-related gene (hERG)
liability testing for ion channel targets. Twentytwo
compounds from LOPAC640
of Pharmacologically Active Compounds) were
identified with apparent inhibitory activity on
the cardiac sodium channel (Nav1.5) using the
FLIPR Membrane Potential Assay Kit. These 22
compounds were then assayed at a single dose using
the IonWorks HT system and one compound
(rimcazole) was confirmed as having inhibitory
activity on the Nav1.5 channel. The dose response curves of rimcazole
were subsequently obtained
on the IonWorks HT as well as by conventional
patch clamp electrophysiology. A literature review
did not reveal any prior publications demonstrating
activity of rimcazole on Na+ channels. A counterscreen
against the hERG channel revealed that
rimcazole exhibited inhibitory activity with an IC50
of approximately 5 M. In this study we utilized
the IonWorks HT system to identify an active
compound against our target ion channel as well as
identifying activity against the hERG channel.
Cells: Chinese hamster lung (CHL) cells expressing the Nav1.5
Reagents and buffers: LOPAC640 library (Sigma-RBI Cat.
#SC001), Veratridine (Sigma Cat. #V5754), Tetrodotoxin (Sigma Cat. #T-5651),
Rimcazole dihydrochloride (Tocris Cat. #1497); Amphotericin (Sigma Cat.
#A-4888), DMSO (Sigma Cat. # D-2650); Versene (Gibco Cat. #15050);
Internal Buffer (in mM): 140 KCl, 2 MgCl2 5 EGTA, 10 HEPES pH to 7.2 with
KOH (Sigma Cat. #s P-9333, M-1028, E-0396, H-7523, P-5958); External Buffer
mM): 137 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10
HEPES, 10 Glucose, pH to 7.4 with NaOH (Sigma Cat. #S-7653, #P-9333, #M-1028,
Fluka Cat. #21115, Sigma Cat. #s H-7523, G-7528, Fisher Cat. #SS266-1)
Tissue culture flasks: Cells grown in T-75 flasks (Corning Cat.
Cell culture media: Dulbeccos Modified Eagle Medium (Gibco,
10% Fetal Bovine Serum (FBS, Irvine Cat. #3000), 1% Penicillin-Streptomycin
(Irvine Cat. #9366) and 1% mL Geneticin (G418, Gibco Cat. #10131)
PatchPlate consumables (Molecular Devices Cat. #9000-0688)
Compound plates (IonWorks HT assay): Costar 96-well plate
(Corning Cat. #3355)
Cells were cultured in T-75 flasks and passaged
every 23 days at 1:3 or 1:6 dilutions. Cells were also maintained
at a lower seeding density (1:50) and passaged every 35 days. For
these lower density passages, flasks that were nearly confluent were frequently
used (i.e., 12 weeks) as a source for the higher density passages.
Electrophysiology: IonWorks HT system
Currents were elicited
by a voltage step from a holding potential of -90 mV to -20 mV for 40
ms. Compounds were incubated for 330430 seconds between the pre-
and post-compound reads. Electrophysiology: conventional patch clamp Experiments
were performed using a Multiclamp 700A (Molecular Devices) at room temperature.
Currents were elicited by a voltage step from a holding potential of -90
mV to -20 mV for 40 ms; pulses were delivered every 5 seconds. Leak subtraction
and series resistance compensation were not used. Patch Pipettes were
35 MO and were pulled from borosilic
ate tubing (World Precision
Instruments Cat. #TW150F-4) using a vertical puller (Kopf instruments).
Preparation of antibiotic solution (IonWorks HT assay)
of amphotericin (5.0 0.3 mg) were pre-weighed and stored at 4C.
Prior to cell preparation, 180 l DMSO was added to an aliquot of
amphotericin. Amphotericin/DMSO solution was sonicated until soluble (~1
minute), added to a 50 mL conical tube of Internal Buffer and vortexed
for ~1 minute. The solution was stored in the dark until ready for use.
Preparation of cells (IonWorks HT assay)
Step 1: Cells were
grown to 7090% confluence in a T-75 flask and removed from the
incubator (37C, 5% CO2) 12 days after plating.
Step 2: Growth media was aspirated from the
culture flasks using a 2 mL aspirating pipette
by vacuum. Cells were gently rinsed with
2.5 mL Versene solution for approximately
10 seconds before the solution was aspirated.
Step 3: The cells were again immersed in 2.5 mL
Versene solution at 37C. After 45 minutes,
visibly rounded cells were easily dislodged
from the bottom of the flask with a few brief
taps on a solid surface. PBS in the amount
of 20 mL was added to the flask and the
resulting solution was used to wash the sides
of the flask; the cell suspension was divided
equally into two 15 mL conical tubes.
Step 4: The two 15 mL tubes were centrifuged at
800 rpm for 4 minutes. The cell supernatant
was decanted, 1.5 mL of PBS was added per
tube, the cell suspensions were combined, and
the cells were gently triturated for 1 minute
using a p200 pipettor.
Step 5: A 3 mL volume of cell suspension was added to the cell boat
the IonWorks HT instrument just prior to beginning the experimental run.
Concentration-response curves for tetrodotoxin
(TTX) and rimcazole were fitted to a 4-parameter logistic equation:
% of control = 100 (1 + ([drug]/IC50)p)-1,
where IC50 is the concentration of drug required
to inhibit current by 50% and p is the Hill slope.
A single-point screen was performed on the IonWorks HT system
of 22 LOPAC640 compounds identified in a preliminary FLIPR screen. The
compounds were randomly distributed on a 96-well compound plate at concentrations
between 1040 M depending on their molecular weight. (See
Figure 1, Panel A.) The location of rimcazole [10 M] in 2 wells
on the compound plate is shown in addition to the location of 8 wells
that contained TTX positive controls at 10 or 30 M. The concentration
of all compounds was then diluted threefold when transferred to the PatchPlate
wells. The compound plate was assayed in one 45-minute experiment with
the results shown in Figure 1, Panel B. One compound was identified as
having confirmed activity against the Nav1.5 channel using the IonWorks
HT system. The potency of freshly prepared rimcazole solution was subsequently
tested on the IonWorks HT system and by conventional patch clamp electrophysiology.
(See Figures 23.) The compound plate layout that was used is shown
in Figure 2, Panel A, and the dose response curve is shown in Figure 2,
Panel B. Raw data traces of control and rimcazole inhibited currents in
the same cell are plotted in Figure 2, Panel C. An IC50 value of 0.9 M
was obtained which showed a threefold great
er potency of the freshly obtained
rimcazole compared to the potency in the LOPAC640 collection.
Conventional patch clamp techniques were then used to obtain a dose response
curve for rimcazole against the Nav1.5 channel. Raw traces
of control and blocked currents in the presence of rimcazole are shown
in Figure 3, Panel A. An IC50 value of 0.6 M was obtained.
(See Figure 3, Panel B.) Rimcazole was then counter-screened against the
hERG potassium channel using the IonWorks HT system. (See Figure 4.) Rimcazole
did have an inhibitory effect on hERG currents (Figure 4, Panel A); the
dose response curve is plotted in Figure 4, Panel B, and an IC50
value of 5.2 M was obtained.
The results demonstrate that primary screening using the FLIPR
system followed by secondary screening using the IonWorks HT system may
be a useful paradigm in identifying compounds which exhibit activity on
voltage-dependent ion channels. In this study, a preliminary screen on
the FLIPR instrument proved to be useful in identifying 22 potential hits
out of a 640- compound library. Subsequent follow-up assays on the IonWorks
HT instrument identified one of those compounds to be a confirmed hit
and similar IC50 values were obtained by using the IonWorks HT system,
as well as conventional patch clamp electrophysiology. The IonWorks HT
instrument was also used to determine whether rimcazole had any activity
against the hERG channel. Rimcazole blocked hERG channels with a potency
of approximately 5 M, indicating potential hERG liability. This
study demonstrates the usefulness of the IonWorks HT system in secondary
screening, hit confirmation and early
hERG liability testing in the drug
discovery process for ion channel targets.
1. Owen, D. and Silverthorn, A. (2002). Channeling drug discovery:
current trends in ion channel drug discovery research. Drug Discovery
World 3, 48-61.
2. Xu, J., Wang, X., Ensign, B., Li, M., Wu,
L. and Guia, A. (2001). Ion-channel assay
technologies: quo vadis? Drug Discovery
Today 6, 1278-1287.
3. Comley, J. (2003). Patchers vs. screeners, divergent opinion on
high-throughput electrophysiology. Drug Discovery World Fall 2003,