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hERG ASSAY
Screening methods
Applications
Urvashi Shakarwal
M Pharm Pharmacology
Presented by-
Urvashi Shakarwal
M Pharm Pharmacology
Amity Institute of pharmacy
Amity University
Lucknow
 hERG = ‘human ether-a-go-go
related gene’
 hERG is a gene that codes for a
protein.
 Can lead to fatal arrhythmias
hERG
Q
P
S
R
T
Q
P
S
R
T
Normal heart beat Activation of hERG
‘T’ wave is delayed
Urvashi Shakarwal
M Pharm Pharmacology
 . Arrhythmia is a lack of rhythm in the heart beat. A delay of the T wave by 5-10
milliseconds can cause lack of control of the heartbeat, which may lead to a fatal
arrhythmia.
 In humans, the hERG channel is expressed widely, including in the brain, adrenal gland,
thymus, retina, and in cardiac and smooth muscle tissues.
hERG
 hERG, or the ‘human ether-a-go-go gene’ was
identified in the late 1980s in a mutant fruit fly. The
presence of the gene was indicated by leg-shaking in the
flies when anaesthetised with ether
Urvashi Shakarwal
M Pharm Pharmacology
Structure of hERG
 A detailed atomic structure for hERG based on X-ray crystallography
is not yet available, but structures have recently been solved by
electron microscopy.
 In the laboratory the heterologously expressed hERG potassium
channel comprises 4 identical alpha subunits, which form the
channel's pore through the plasma membrane.
 Each hERG subunit consists of 6 transmembrane alpha helices,
numbered S1-S6, a pore helix situated between S5 and S6, and
cytoplasmically located N- and C-termini.
Urvashi Shakarwal
M Pharm Pharmacology
 The S4 helix contains a positively charged arginine or lysine amino
acid residue at every 3rd position and is thought to act as a voltage-
sensitive sensor, which allows the channel to respond to voltage
changes by changing conformations between conducting and non-
conducting states (called 'gating').
 Between the S5 and S6 helices, there is an extracellular loop (known
as 'the turret') and 'the pore loop', which begins and ends
extracellularly but loops into the plasma membrane.
 The pore loop for each of the hERG subunits in one channel face into
the ion-conducting pore and are adjacent to the corresponding loops of
the 3 other subunits, and together they form the selectivity filter region
of the channel pore.
Urvashi Shakarwal
M Pharm Pharmacology
Screening of hERG
 In the heart, hERG channels are the molecular correlate of the IKr current which,
together with other potassium currents, is involved in action potential repolarization.
 Reduced function of hERG causes action potential prolongation, which in rare cases
can lead to the potentially fatal ventricular tachyarrhythmia.
 In a body surface electrocardiogram (ECG), ventricular action potential prolongation
manifests itself as a prolongation of hERG assays.
Urvashi Shakarwal
M Pharm Pharmacology
Medium and High Throughput Assay
 The ideal hERG assay provides a linear measure of channel activity under physiologically
relevant conditions.
 However, such a study is extremely laborious and only amenable to the detailed
characterization of very few selected compounds.
 It is advantageous to screen compounds for hERG activity early on in the lead evaluation
and optimization process. However, this approach requires testing of hundreds and
potentially thousands of compounds within a single drug discovery program.
Urvashi Shakarwal
M Pharm Pharmacology
Electrophysiology
 The development of automated electrophysiology technologies has
improved the throughput of electrophysiological methods
 Electrophysiology can provide detailed and quantitative information on
the potency and mechanism of hERG block by a test compound.
 One of the unique advantages of such voltage clamp recordings is the
ability to control membrane potential.
 Since activation and inactivation of hERG is dependent on membrane
potential, voltage clamp recordings can differentiate between compounds.
 Limitation the high cost of the instruments and consumables.
Urvashi Shakarwal
M Pharm Pharmacology
Flux Assay
 An alternative to either manual or automated electrophysiology is a
functional assay that measures ion flux across cell or vesicle membranes.
 This assay offers advantage of the ability of Rubidium ion i.e. Rb+ to
permeate through hERG channels.
 Typically, cells are loaded with Rb+ overnight.
 hERG-dependent Rb+ efflux is initiated by an addition of high (50–60
mM) extracellular potassium concentrations to depolarize the cell and
open hERG channels.
 The amount of Rb+ efflux can be calculated by using 86Rb+ as a
radioactive tracer or by flame atomic absorption spectrometry (FAAS).
Urvashi Shakarwal
M Pharm Pharmacology
Fluorescence-based Assay
 The development of improved fluorescent dyes and plate readers has provided
another approach to high throughput screening of ion channel activities.
 Fluorescent dyes which are sensitive to changes in membrane potential have
proved.
 However, studying hERG by this approach presents a challenge since this channel
does not typically control a cell’s resting membrane potential.
 It has been possible, however, to select HEK-293 and CHO-K1 cell lines stably
expressing recombinant hERG channels.
Urvashi Shakarwal
M Pharm Pharmacology
Radio Ligand Binding Assay
 Radio ligand binding assays have been used extensively to screen for
interaction with the hERG channel..
 They do not provide a direct measure of IKr blockade, such binding assays
can test 50,000 to 100,000 compounds per day and are relatively
inexpensive, which is why they are commonly used in most large
pharmaceutical companies.
 It can be effective for the treatment of tachycardia.
 Radio ligand binding assays are manageable to a range of assay conditions
which may impact on the binding ability of test compounds.
Urvashi Shakarwal
M Pharm Pharmacology
Advantages of hERG
 The hERG channel has been shown to be the target for class III antiarrhythmic
drugs such as amiodarone, which reduce the risk of re-entrant arrhythmias by
prolonging the action potential.
 It can be used in drug development process of new small molecule drugs with
improved cardiovascular safety profiles.
 It can also be used as a diagnostic marker in treatment of diseases like Cancer,
Epilepsy, Schizophrenia.
Urvashi Shakarwal
M Pharm Pharmacology
Reference
 Sanguinetti MC, Tristani-Firouzi M (March 2006). "hERG potassium channels
and cardiac arrhythmia". Nature. 440 (7083): 463–9.
 Hedley PL, Jørgensen P, Schlamowitz S, Wangari R, Moolman-Smook J, Brink
PA, Kanters JK, Corfield VA, Christiansen M (2009). "The genetic basis of
long QT and short QT syndromes: a mutation update". Hum. Mutat. 30 (11):
1486–511.
 https://en.wikipedia.org/wiki/HERG.
Urvashi Shakarwal
M Pharm Pharmacology
THANK YOU

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Herg assay,Structure, Various screening methods and Advantages

  • 1. hERG ASSAY Screening methods Applications Urvashi Shakarwal M Pharm Pharmacology Presented by- Urvashi Shakarwal M Pharm Pharmacology Amity Institute of pharmacy Amity University Lucknow
  • 2.  hERG = ‘human ether-a-go-go related gene’  hERG is a gene that codes for a protein.  Can lead to fatal arrhythmias hERG Q P S R T Q P S R T Normal heart beat Activation of hERG ‘T’ wave is delayed Urvashi Shakarwal M Pharm Pharmacology
  • 3.  . Arrhythmia is a lack of rhythm in the heart beat. A delay of the T wave by 5-10 milliseconds can cause lack of control of the heartbeat, which may lead to a fatal arrhythmia.  In humans, the hERG channel is expressed widely, including in the brain, adrenal gland, thymus, retina, and in cardiac and smooth muscle tissues. hERG  hERG, or the ‘human ether-a-go-go gene’ was identified in the late 1980s in a mutant fruit fly. The presence of the gene was indicated by leg-shaking in the flies when anaesthetised with ether Urvashi Shakarwal M Pharm Pharmacology
  • 4. Structure of hERG  A detailed atomic structure for hERG based on X-ray crystallography is not yet available, but structures have recently been solved by electron microscopy.  In the laboratory the heterologously expressed hERG potassium channel comprises 4 identical alpha subunits, which form the channel's pore through the plasma membrane.  Each hERG subunit consists of 6 transmembrane alpha helices, numbered S1-S6, a pore helix situated between S5 and S6, and cytoplasmically located N- and C-termini. Urvashi Shakarwal M Pharm Pharmacology
  • 5.  The S4 helix contains a positively charged arginine or lysine amino acid residue at every 3rd position and is thought to act as a voltage- sensitive sensor, which allows the channel to respond to voltage changes by changing conformations between conducting and non- conducting states (called 'gating').  Between the S5 and S6 helices, there is an extracellular loop (known as 'the turret') and 'the pore loop', which begins and ends extracellularly but loops into the plasma membrane.  The pore loop for each of the hERG subunits in one channel face into the ion-conducting pore and are adjacent to the corresponding loops of the 3 other subunits, and together they form the selectivity filter region of the channel pore. Urvashi Shakarwal M Pharm Pharmacology
  • 6. Screening of hERG  In the heart, hERG channels are the molecular correlate of the IKr current which, together with other potassium currents, is involved in action potential repolarization.  Reduced function of hERG causes action potential prolongation, which in rare cases can lead to the potentially fatal ventricular tachyarrhythmia.  In a body surface electrocardiogram (ECG), ventricular action potential prolongation manifests itself as a prolongation of hERG assays. Urvashi Shakarwal M Pharm Pharmacology
  • 7. Medium and High Throughput Assay  The ideal hERG assay provides a linear measure of channel activity under physiologically relevant conditions.  However, such a study is extremely laborious and only amenable to the detailed characterization of very few selected compounds.  It is advantageous to screen compounds for hERG activity early on in the lead evaluation and optimization process. However, this approach requires testing of hundreds and potentially thousands of compounds within a single drug discovery program. Urvashi Shakarwal M Pharm Pharmacology
  • 8. Electrophysiology  The development of automated electrophysiology technologies has improved the throughput of electrophysiological methods  Electrophysiology can provide detailed and quantitative information on the potency and mechanism of hERG block by a test compound.  One of the unique advantages of such voltage clamp recordings is the ability to control membrane potential.  Since activation and inactivation of hERG is dependent on membrane potential, voltage clamp recordings can differentiate between compounds.  Limitation the high cost of the instruments and consumables. Urvashi Shakarwal M Pharm Pharmacology
  • 9. Flux Assay  An alternative to either manual or automated electrophysiology is a functional assay that measures ion flux across cell or vesicle membranes.  This assay offers advantage of the ability of Rubidium ion i.e. Rb+ to permeate through hERG channels.  Typically, cells are loaded with Rb+ overnight.  hERG-dependent Rb+ efflux is initiated by an addition of high (50–60 mM) extracellular potassium concentrations to depolarize the cell and open hERG channels.  The amount of Rb+ efflux can be calculated by using 86Rb+ as a radioactive tracer or by flame atomic absorption spectrometry (FAAS). Urvashi Shakarwal M Pharm Pharmacology
  • 10. Fluorescence-based Assay  The development of improved fluorescent dyes and plate readers has provided another approach to high throughput screening of ion channel activities.  Fluorescent dyes which are sensitive to changes in membrane potential have proved.  However, studying hERG by this approach presents a challenge since this channel does not typically control a cell’s resting membrane potential.  It has been possible, however, to select HEK-293 and CHO-K1 cell lines stably expressing recombinant hERG channels. Urvashi Shakarwal M Pharm Pharmacology
  • 11. Radio Ligand Binding Assay  Radio ligand binding assays have been used extensively to screen for interaction with the hERG channel..  They do not provide a direct measure of IKr blockade, such binding assays can test 50,000 to 100,000 compounds per day and are relatively inexpensive, which is why they are commonly used in most large pharmaceutical companies.  It can be effective for the treatment of tachycardia.  Radio ligand binding assays are manageable to a range of assay conditions which may impact on the binding ability of test compounds. Urvashi Shakarwal M Pharm Pharmacology
  • 12. Advantages of hERG  The hERG channel has been shown to be the target for class III antiarrhythmic drugs such as amiodarone, which reduce the risk of re-entrant arrhythmias by prolonging the action potential.  It can be used in drug development process of new small molecule drugs with improved cardiovascular safety profiles.  It can also be used as a diagnostic marker in treatment of diseases like Cancer, Epilepsy, Schizophrenia. Urvashi Shakarwal M Pharm Pharmacology
  • 13. Reference  Sanguinetti MC, Tristani-Firouzi M (March 2006). "hERG potassium channels and cardiac arrhythmia". Nature. 440 (7083): 463–9.  Hedley PL, Jørgensen P, Schlamowitz S, Wangari R, Moolman-Smook J, Brink PA, Kanters JK, Corfield VA, Christiansen M (2009). "The genetic basis of long QT and short QT syndromes: a mutation update". Hum. Mutat. 30 (11): 1486–511.  https://en.wikipedia.org/wiki/HERG. Urvashi Shakarwal M Pharm Pharmacology