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Rapid hit confirmation and early hERG liability testing using the IonWorks HT system


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 earl y human ether-a-go-go-related gene (hERG) liability testing for ion channel targets. Twentytwo compounds from LOPAC640 (Library 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 sodium channel
  • 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 (in 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. #430641)
  • Cell culture media: Dulbeccos Modified Eagle Medium (Gibco, Cat. #11965-092)
  • 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)

    Cell culture
    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)
    Aliquots 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 on the IonWorks HT instrument just prior to beginning the experimental run.

    Data analysis
    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, 47-57.

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