1. AbstractDrug discovery relies upon screening technologies capable of delivering high throughput data generation of

repeatable accuracy. Owing to the limitations of screening technologies currently in use (e.g. FLIPR, VIPR),

ion channels as drug targets have received comparatively little attention compared to, say, GPCRs. These

limitations are, in part, due to the slow response times of the instruments with respect to the very rapid

kinetics of the channels being screened, and the indirect nature of the readout.

Ionic flux is an alternative generic, high throughput assay that is widely used to monitor ion channel activity.

The requirement for high-energy radioisotopes, lack of voltage control, and low temporal resolution suggest

such assays are inappropriate for certain ion channel types. However, flux assays are a direct measure of

channel conductance. Non-radioactive ionic flux assays have been developed at Xention using atomic

absorption spectroscopy. In this study we compare the assay sensitivity of ionic flux to both conventional

patch clamp recording and AutoPatch for a Kv channel, using 72 known ion channel modulators (BioMol).

The integration of a 96-well plate-based drug application system into AutoPatch has significantly increased

its capacity and throughput by removing the limitation on number of possible test solutions per experiment.

In this study the majority of ion channel modulators were undetectable by ionic flux compared to

electrophysiology. Furthermore, AutoPatch-determined IC50s were similar to those obtained using

conventional electrophysiology. AutoPatch is thus capable of finding hits that are otherwise undetectable

through flux. Moreover, as opposed to flux, AutoPatch-derived data are comparable to data obtained with

conventional WCR, thereby demonstrating the successful use of a HTS ion channel screening platform with

no data compromise.

2. MethodsCompounds were dissolved in 100% DMSO to 10mM and diluted in the assay buffer to the appropriate

concentration prior to the experiment. Stable CHO-K1 cell line expressing Kv1.1 was routinely used in

experiments.

Electrophysiology: Whole-cell patch-clamp electrophysiological recordings were carried out using an EPC-

9 amplifier controlled by Pulse software (v8.54, HEKA, Germany) using Xention’s automated patch-clamp

software. The external bathing solution contained (in mM): 150 NaCl, 5 KCl, 3 MgCl2, 1 CaCl2, 10 HEPES, pH

7.4. Patch pipettes were filled with an electrode solution of composition (in mM): 150 KCl, 3 MgCl2, 10 HEPES,

5 EGTA-KOH, pH 7.2 and had resistances of 3-5MΩ. All experiments were conducted at room temperature

(22-24°C). Cells having a series resistance greater than 15MΩ were excluded from the study.

Electrophysiology voltage-protocols and analysis of data was performed as follows. Data was sampled at

10kHz, and filtered with a –3 dB bandwidth of 2.5kHz. Cells were held at a voltage of –80mV. Currents were

evoked to a voltage step applied for 500ms in duration from –80 to +30mV every 5s. Currents were analysed

using Pulsefit software (v8.54, HEKA, Germany), with the peak peak amplitude measured at +30mV.

Biochemical Flux: Looking at the cellular efflux of these potassium channel permeable ions allows you to

directly monitor potassium channel activity. Cells (50,000 cells/well) were incubated overnight with growth

media containing 5mM RbCl. After an overnight incubation unloaded Rb+ was aspirated and each well

washed 4x250µl with Earls Balanced Salt Solution (EBSS) which contained (in mM) 5 KCl, 140 NaCl, 2 CaCl2,

1 MgSO4, 10 HEPES, and 5 glucose, pH 7.4) on a Biotek ELX405 plate washer. These cells were then pre-

incubated with 50µl of EBSS +/- test compounds for 10 minutes at room temperature. After the 10 minute

incubation, 50µl of modified EBSS containing 140mM KCl was added, and the cells were incubated for a

further 10 minutes at room temperature. After the final incubation 80µl/100µl of the reaction from each well

was transferred to a fresh plate and diluted with 320µl 0.1% (w/v) CsCl and assayed for Rb+ ThermoElemental

Solaar S4 Atomic Spectrophotometer at 705nm.

3. Results - AutoPatch

Figure 1a: Interface Patch Method Figure 1b: AutoPatch HTS Family

The AutoPatch provides a fully automated de-skilled process for patching cells. The proprietary “Interface Patch

Method” works by allowing suspended cells to sediment to the liquid-air interface in a glass capillary tube. A glass

patch pipette then moves unidirectionally towards the liquid-air Interface and patches a cell. True Ω seals are

formed before going whole cell.

5. Results-Biochemical Flux

Figure 5: A Collection of Known Ion Channel Modulators Were Screened at 10µM by Non-Radiometric

Biochemical Flux against Kv1.1. Bepridil was the only compound to show significant inhibition (Green). False

negatives Verapamil and Nicardipine (red) are hits that were detected in the AutoPatch assay and not detected in

the flux assay. Verapamil, Bepridil, Nicardipine progressed to IC50 determination. Inactive compounds are

highlighted grey and TEA positive control as black.

Figure 6: Biochemical Flux IC50 Determination of Verapamil (Inactive), Bepridil (20µM), Nicardipine (Inactive).

6. ConclusionsDrug Target Therapeutic Use Autopatch Flux IC50 (µM) Electrophysiology Additional Ion References

IC50 (µM) IC50 (µM) Channel

Pharmacology

Verapamil Calcium Antianginal, 4.60 Inactive 8.82 HERG, Kv1.1, Kv1.3, 1, 2, 3, 4,

Channel Blocker Antiarrythmic, Kv1.4, Kv1.5, Kv3.1, 5, 6, 8

Antihypertensive Kv3.2, Kv4.2, BK

Bepridil Calcium Antianginal, 4.88 20 4.93 Kv1.5, HERG, 7, 8

Channel Blocker Chronic Obstructive KvLQT1-IsK, NaV

Lung Disease

Nicardipine Calcium Antianginal, 1.39 Inactive 3.27 BK, Kv4.2, Kv4.3, 9, 10

Channel Blocker Antiarrythmic, Kv1.4

Antihypertensive

This study confirms that AutoPatch produces high quality data equivalent to conventional electrophysiology. Both

methods are superior to non-electrophysiological methods such as biochemical flux. Conventional electrophysiology

suffers from low throughput and large volume compound requirements. Notably, in this study the biochemical flux

assay was unable to detect any significant compound-mediated ion channel modulating activity.

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Figure 2: A Collection of Known Ion Channel Modulators were Screened at 10µM on the Autopatch Against the

Target Kv1.1, a Shaker type Voltage-Gated Potassium Channel. Verapamil, Bepridil, Nicardipine, AM 92016, and

SDZ-201106 were “hits” showing >75% inhibition of Kv1.1 current. Verapamil, Bepridil, Nicardipine progressed to

IC50 determination whereas AM 92016, and SDZ-201106 were identified as false positives. Inactive compounds are

highlighted grey and TEA postive control as black.

Figure 3: Autopatch IC50 Determination of Verapamil (4.60µM), Bepridil (4.88µM), Nicardipine (1.39µM).

4. Results-Conventional Electrophysiology

Figure 4: Conventional Electrophysiology IC50 Determination of Verapamil (8.820µM), Bepridil (4.93µM),

Nicardipine (3.27µM).

CapillaryHolding

Suspension ofCells

Liguid Air Interface

ExtracellularSolution

GlassPatch-Pipette

A COMPARISON OF THE EFFECTS OF KNOWN ION CHANNEL MODULATORS AGAINST A K+ CURRENTRECORDED USING AUTOPATCH & ‘CONVENTIONAL’ ION CHANNEL SCREENING TECHNOLOGIES

J. Ford; A. Boxall; G. Clark; R Davies; J. Hutchings; A. Lawrence; D. Owen; V. Piotrowski; G. Redgrave; R. Tang. Xention Discovery Ltd, Cambridge, United Kingdom