Reliable Identification of hERG Liability in Drug Discovery byAutomated Patch Clamp
Michael George1, Rodolfo Haedo2, Nina Brinkwirth1, Nadine Becker1, Claudia Haarmann1, Alison Obergrussberger1, Ronald
Knox2, Martin Hampl1, Niels Fertig1
1 Nanion Technologies, Munich, Germany, 2Nanion Technologies Inc, Livingston, NJ, USA. Email: info@nanion.deHigh throughput screening on the SyncroPatch 384i

Introduction

Slow inhibitors need an extended incubation time
Dofetilide

Terf.

Dofet.

8nM

8nM

8nM

8nM

24nM

24nM

24nM

24nM

74nM

74nM

Cpd
DofetilideTerfenadine

IC50
Literature*(CiPA)

IC50

0.036

0.018

0.052

0.092

74nM

74nM

0.2uM

0.2uM

0.2uM

0.2uM

Wells
included
176
169

IC50 plots and averaged time course

Voltage protocol

Averaged traces

Success
Rate [%]

176

92
88

Fig. 3 Terfenadine and Dofetilide were tested as 6pt-IC50 across the plate on HEK hERG using a multi hole 4xchip and perforated patch mode. To reach a steady state at lower concentrations an extended
compound incubation time was needed (>12min). If this is not done, the potency of these compounds isunderestimated.

D

Average Timeplot~24minevery 8s

500ms
ramp
-80mV -80mV
100ms
100ms 100ms-90mV
100ms2) standard

7min-80mV
100ms

60mV
500ms
-40mV
500ms

-80mV
100ms

every15s

4min-80mV
100ms

3) “longdepolarization”
60mV
2s
-40mV
80ms

-40mV
6s

-80mV
1s

every
10s

4min-80mV
920ms

Fig. 1 Diverse hERG voltage
protocols, example traces
and corresponding plots ofcurrent amplitude vs. time incontrol(grey),vehicle
control (4 times, cumulative
for protocols 2, and 3,; singleaddition in 1,; shades ofblue) and full block (darkgrey).

Seal Resistance (median of 333 cells)

ECHO hERG DUO
1x single hole, WC configuration
Recording time ~21min
3.0
2.5

Seal Resistance[GΩ]40mVΣ 19min1) CiPA

2.0
1.5
1.0
0.5
0.0
0200

400

Cpd

IC50 [uM]
Literature* CiPAIC50 [uM]
SyncroPatch

Success
Rate

Astemizole

0.004

0.037

78%

Bepridil

0.159

0.242

78%

Cisapride

0.024

0.029

94%

Dofetilide

0.036

0.021

94%

0.020

800

10001200Conclusions

The SyncroPatch 384i platform allows for unattended runs in a fully automatedmanner. Independent movements of gripper and pipette head and aselective tip option for serial compound dilution enable a workflow that fits anHTS screening environment.

• The 384-well APC system used in our study(SyncroPatch 384i) is well suited to perform
safety pharmacological studies within theguidelines of the CiPA initiative.

Table 1: The non-specific binding of compound to labware was investigated.
IC50 values of astemizole were evaluated after storing the compound
between 5 min and 3 h in teflon reservoirs, Eppendorf cups and glass vials. IC50
values were recorded on the Patchliner and the SyncroPatch 384PE.

• Different voltage protcols can be used torecordhERG,experiments
arestable
regardless of protocol used.

Instrument

LabwareTime between
cpd prep andapplication

384PE

Teflon

~5min

30nM

180

78%

384PE

Teflon~30min

83nM

177384PE

Teflon~2h151nM

168

Patchliner

Eppi

~5min

37nM

5IC50nFlecainide

1.49

0.920

100%

Quinidine

0.716

0.761

94%

Ranolazine

10.7

5.1991%

Terfenadine

0.052

0.020

69%

Patchliner

Eppi~2h63nM

4Verapamil

0.250

0.350

78%

Patchliner

Eppi~3h209nM

3Patchliner

glass vial

~5min35nM

6Patchliner

glass vial

~2h37nM

2Patchliner

glass vial

~3h37nM

4Fig. 4 Success rates and IC50 values compared withliterature values published by Chantest within the CiPA
Initiative*. Data was recorded using the CiPA Protocol (1)shown above in Fig. 1.

600

Fig. 2 A Screenshot of 5 cumulativereference additions followed by a full
block to study the assay stability ofHEK hERG (Chantest) in terms ofsealing and rundown. B Screenshotof 10 cumulative additions to study
the assay stability of CHO hERG DUO(B’sys). C Averaged time course of333 cells (from B). D Averaged tracesof 333 cells. E Stable seal resistanceof > 1.5GΩ for > 20min.

Time [s]SyncroPatch 384i liquid handling of sticky compounds and the effect of labware material on
calculated IC50 values

E4031

Datapoints
included169

hERG recordings using different
voltage protocols

GuidedLabware Setup!

TerfenadineCBΣ 33min

Here, we investigated non-specific binding using hydrophobic(“sticky”) compounds to assess the reliability of our experimentalroutine by analyzing IC50 values of CiPA compounds. We used a stateof-the-art liquid handling robot encompassing a 384-channel patch
clamp module for compound preparation and high throughput APC
recordings. Glass-coated compound plates were used to prevent
absorption of hydrophobic compounds. After compound preparation,
the user-unattended experiments were executed and IC50 values of
the compounds were measured and compared to literature values.

ANanion´s patch clamp module (left) for
the SyncroPatch 384i, enabling 384parallel recordings. The robotic system isbased on the Biomek i5 state-of-the-art
liquid handler (right).

Σ 30min

The Comprehensive in Vitro Proarrhythmia Assay (CiPA) was initiated in2013 to improve pre-clinical drug safety paradigms. The aims of theinitiative are not only to ensure that drugs which are reach the market
are safe, but also to reduce the number of false positives to ensure that
more potentially useful (and safe) drugs reach the market. As part ofthis initiative, compounds representing high, intermediate, and low proarrhythmic risk categories were tested across multiple sites andautomated patch clamp (APC) platforms. Resulting IC50 values varied
between platforms, which could be due to non-specific binding ofcompounds in well-based platforms since they don’t maintain aconstant flow of compound-containing solution to replenish losses tofluidics and well walls.

hERG – reliable assay due to stability of recordings*Ref for literature values: http://www.onemillionsolutionsinhealth.org/wp-content/uploads/2014/11/ChanTest-Data-Analysis-Summary.pdf• Variability of IC50 values of drugs in different
platforms within the CiPA study could havebeen caused by non-specific binding ofcompounds• Caution should be taken on the handling andstorage conditions of the compounds, as well
as exposure times of the compounds during
the measurement.
• When using teflon or plastic labware,experiments
shouldbecarriedout
immediately after compound preparation.