hERG Assay

hERG ASSAY
B K Mody Govt Pharmacy College, Rajkot 1
PREPARED BY : VANARAJ RABARI
(M.PHARM PHARMACOLOGY)
GUIDED BY : DR. JIGNESH PATEL
(MPHARM, PhD)

 hERG is the name of a human gene
 stands for human ether-a-go-go related gene.
 a gene found in Drosophila melanogaster
 new nomenclature, hERG is KCNH2.
 hERG channel is a voltage-gated potassium
channel
 Highly selective for potassium is a tetramer
formed of four subunits, each containing six
transmembrane domains

3B K Mody Govt Pharmacy College, Rajkot
hERG channel structure
Per- Arnt -Sim
domain

 Two alternative splice forms
1) hERG1a
2) hERG1b
 hERG channels are involved in action potential
repolarization.
 hERG channel is expressed widely, including in the
 brain,
 adrenal gland,
 thymus,
 Retina,
 in cardiac and smooth muscle tissues.

 Reduced function of hERG causes action potential
prolongation, it lead to the potentially fatal
ventricular tachyarrhythmia (Torsades de
Pointes).
 In a body surface electrocardiogram (ECG),
ventricular action potential prolongation manifests
itself as a prolongation of the QT interval or the
period between the beginning of the QRS complex
and end of the T wave.
 A particular form of inherited long QT syndrome
(LQTS), LQTS2, has been linked to loss-of-function
mutations in hERG.

NORMAL ECG

ABNORMAL ECG

 In 1989, overdoses of terfenadine were shown to
prolong the QT interval
 terfenadine was subsequently shown to inhibit the
delayed rectifier potassium current in isolated
myocytes, and hERG channels expressed in Xenopus
oocytes.
 antihistamines terfenadine and astemizole to the
antipsychotic droperidol, have been withdrawn from
the market.

hERG ASSAY
1) Electrophysiological Assay
2)FluxOR Thallium Assay
3) 35S-MK-499 Binding Assay

The CHO or HEK hERG cell line was used
CHO hERG cell line improved seal rates, seal resistances,
and current stability on the Qpatch

T175 flask with hERG cells was rinsed three times with 10 mL
of Dulbecco’s phosphate buffered saline (DPBS) (Ca/Mg free).
6mL of accutase was added to the cells for 10 min at 37̊C

Centrifugation at 1,400 rpm for 3 min 45 s
Recover for 30 min,
Cells were then re-suspended in 10 mL of growth media
Pelleted again at 1,400 rpm for 3 min 45 s.

Supernatant was discarded and cells were resuspended in
10 mL of saline.
Cells were subjected to centrifugation one final time at 1,100
rpm for 4 min,
resuspended in 500 mL of saline, and placed in the
QPatch for use
Whole-cell patch-clamp experiments were carried out on
the QPatch-HT automated platform
Utilizing an external solution containing (in mM): 151 NaCl, 4
KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES (hydroxyethyl
piperazineethane sulfonic acid) (pH 7.4
An internal solution containing (in mM) 120 KCl, 1.5 MgCl2, 10
EGTA( egtazic acid,), 10 HEPES, and 4 ATP

Peak tail currents were measured using Sophion’s Assay Software and
further data analyses were performed using IgorPro

 For determining compound 50% inhibition
concentration (IC50) values in the QPatch assay,
averaged percent control values were plotted as a
function of compound concentration and fitted using
a four parameter logistic equation with the minimum
fixed at zero and the maximum fixed to 100% of
control.
 The inhibitor concentration against the percent
activity is plotted ([I]-Activity % graph). Using the
linear (y=mx+n) or parabolic (y=ax2+bx+c) equation
on this graph for y=50 value x point becomes IC50
value.

The membrane potential was voltage clamped at -80 mV, and
stepped to -50mV for 100 ms and then to +20mV for 3 s
Cells were then stepped back down to -50mV for 2 s to elicit tail
currents, and finally stepped back down to -80 mV.
This voltage protocol was repeated once every 20 s.
Current measured at the initial step to -50mV was used as a leak
subtraction for the peak tail currents
Currents elicited using this voltage protocol and the resulting inhibition
produced by application of 0.03 and 0.3 mM cisapride

 Multiple control saline additions were applied initially
to monitor hERG current stability.
 Each concentration was applied 2–3 times to allow
more time for reaching steady state inhibition.
 QPatch assay can be used to accurately detect
inhibitors of hERG, and provide a higher throughput
electrophysiological assay for generating additional
validation data for the thallium flux assay.

 limitation of all automated Electrophysiology
platforms is the high cost of the instruments and
consumables.
 automated platforms is the potential for results that
are inconsistent with those from conventional voltage
clamp assays.
 however, some compounds are known to appear less
potent when tested by automated electrophysiology.
Discrepancies typically arise from hydrophobic
compounds being adsorbed to the surface of the plate
used to hold the compound or to the patch plate itself.

Assay that measures ion flux across cell or vesicle membranes
This assay offers higher throughput than the automated voltage clamp
Flux assays, which can operate in 96- and 384-well formats
They offer a robust measure of channel activity, but lack
voltage control and temporal resolution.
IC50 values determined in a Thallium flux assay were approximately
10-fold higher than those determined by electrophysiology

HEK293 cells stably transfected with hERG were plated using a
Thermo Scientific Matrix WellMateat 30,000 cells/well on a
BioCoatflatbottom
poly- Dlysine – coated black-wall, clear-bottom, 384-well plate
in 50 mL growth medium, and incubated overnight (16–20 h) at
37°C in a 10% CO2 atmosphere
All liquid handling was done on a Thermo Scientific Matrix PlateMate
2 × 3

Cell growth medium was removed and cells incubated with 0.025
mL of a solution containing FluxOR dye (Fura-2-acetoxymethyl
ester) loading reagent, prepared Hank’s buffered saline solution
(HBSS) containing CaCl2 and MgCl2
After incubation in the dark for 60 min at 25°C cells were washed
once with 0.040 mL of assay buffer solution containing (in mM) 1.3
CaCl2, 0.9 MgCl2, 5.5 Glucose, and 20 HEPES NaOH (pH 7.4)
incubated in the dark for 30 min at 25⁰C with 0.025 mL of the same
assay buffer solution containing 300 mM ouabain, in the absence or
presence of test compound

At the end of the 30 min incubation period, the plate was placed in a
FLIPRTETRA illuminated at 490 nm, and fluorescence emission was
recorded at 525 nm
After a 30 s baseline reading, 0.00625 mL of a 5× solution containing
15mM thallium sulfate, with or without 50mM K2SO4, prepared in the
FluxOR chloride-free buffer was added
Fluorescence emission was recorded for an additional 3–6 min,
with an exposure time of 0.4 s and a read interval of 3–5 s.

FLIPRTETRA

The change in fluorescence emission (F/F0) was calculated
F= averaging the three readings just before the signal reaching a
plateau level, usually from 90 to 120 s
F0= the baseline calculated by averaging the initial four readings usually
from 1 to 10 s

 IC50 values for inhibition in the thallium flux binding
assays were determined according to the Hill equation
from 10 point dose–response curves, where all
parameters were left unconstrained.
 Quinidine (100 mM) was added at the end of each
experiment as a positive control.
 evaluate the quality of the thallium flux data, the Z¢-
factor was calculated using the following

 where SDc and SDn are the standard deviation of the
control group (c)
 and the group in the presence of 1 mM MK-499 (n),
and C and N are the means of the two groups,
respectively
Z= 1-
3(SDc + SDn)
|C−N|

 HEK cells expressing the hERG channel were plated in
384- well plates, loaded with the FluxOR dye reagent,
incubated with hERG standard inhibitors under
normal physiological conditions (i.e., HBSS) for 30
min, placed in the FLIPRTETRA instrument
 And assayed for activity after addition of a
thallium/high potassium solution to provide final
concentrations of 3 and 20 mM, respectively

The interaction of 35S-MK-499 with membranes prepared from
HEK293 cells expressing hERG
The 35S-MK-499 tracer was prepared at 50 pM in assay buffer
containing (in mM): 70 NaCl, 60 KCl, 1 CaCl2, 2 MgCl2 and 10
HEPES-NaOH (pH 7.4)
0.25 mL of the diluted tracer was dispensed into a 2 mL, 96-square-
well polypropylene deep-well block.
Nonspecific binding was defined in the presence of 1 mM unlabeled
MK-499
MK-499 [(+)-N-[1'-(6-cyano-1, 2, 3, 4-tetrahydro-2(R)-naphthalenyl)-3,
4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2, 4'-piperidin)-6-yl]
methanesulfonamide]

HEK-hERG membranes diluted in assay buffer were added
to the tracer/ compound solution and incubation took
place for a minimum of 90 min at room temperature.
The reaction was quenched and the assay block was washed
twice with 0.5 mL of quench buffer containing (in mM) 130
NaCl, 1 CaCl2, 2 MgCl2, and 10 HEPES-NaOH (pH 7.4).
Radioligand bound to hERG was captured on a UniFilter-96
GF/C white , 96-well filtered microplate
Presoaked with 0.3% bovine serum albumin, using a Filter Mate
Universal Harvester
The filter plate was allowed to dry completely before
sealing the bottom

Each well was treated with 0.025 mL Microscint 0 before
sealing the top of the plate
Then analyzed for 1 min/well in a TopCount NXT
scintillation counter

Data Analysis
 IC50 values for inhibition in the 35S-MK-499 binding
assays were determined according to the Hill equation
from 10 point dose–response curves, where all
parameters were left unconstrained.

 mean control fluorescence traces from 32 wells and
traces from the same 384-well plate from 32 wells in
which the cells had been preincubated with 1 mM MK-
499
 Control signals are large and reproducible, and
significantly attenuated in the presence of MK-499.
 The small signal observed in the presence of saturating
concentrations of MK-499 likely represents the
presence of endogenous potassium pathways, such as
potassium transporters or other potassium channel
subtypes.

 Z’-factor values for this plate
 were 0.91, suggesting that the assay is adequate for
HTS operation.
 Next, seven hERG standards, cisapride, E-4031,
haloperidol, MK-499,
 pimozide, risperidone, and verapamil, were evaluated
to determine
 the assay sensitivity to pharmacological inhibition of
the channel

 Concentration–response curves for each of the
standards were generated and IC50 values were
calculated

Inhibition of Human Ether-`a-go-go Related Gene Channel
compound Ic50 value
QPatch Thallium
Flux
MK-499
Binding
Cisapride 0.037 0.054 – 0.017 0.031
E-4031 0.039 0.680 – 0.084 0.006
Haloperidol 0.032 0.127 – 0.036 0.028
MK-499 0.029 0.066 – 0.011 0.001
Risperidone 0.361 1.515 – 0.493 0.485
Verapamil 0.358 2.542 – 1.117 1.037

 Birgit Priest, Ian M. Bell & Maria Garcia (2008) Role of hERG
potassium channel assays in drug development.
 William A. Schmalhofer,1,*,{ Andrew M. Swensen, Brande S. Thomas,1,
John P. Felix, Rodolfo J. Haedo,Kelli Solly, Laszlo Kiss, Gregory J.
Kaczorowski, and Maria L. Garci A Pharmacologically Validated, High-
Capacity, Functional Thallium Flux Assay for the Human Ether-`a-go-
go Related Gene Potassium Channel.
 Tao Jin, Bingxue Hu, and Zhao Zhang An in Vitro Assay of hERG K
Channel Potency for a New EGFR Inhibitor FHND004.

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