hERG Assay

hERG Assay and their important role in drug evaluation.

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

2. 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 h ERG ASSAY B K Mody Govt Pharmacy College, Rajkot 2

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

4. Two alternative splice forms hERG1a 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. hERG ASSAY B K Mody Govt Pharmacy College, Rajkot 4

5. hERG ASSAY 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. B K Mody Govt Pharmacy College, Rajkot 5

6. 6 B K Mody Govt Pharmacy College, Rajkot NORMAL ECG

7. B K Mody Govt Pharmacy College, Rajkot 7 ABNORMAL ECG

8. 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 antihistamines terfenadine and astemizole to the antipsychotic droperidol , have been withdrawn from the market.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 B K Mody Govt Pharmacy College, Rajkot 8

9. 9 B K Mody Govt Pharmacy College, Rajkot hERG ASSAY 1) Electrophysiological Assay 2)FluxOR Thallium Assay 3) 35S-MK-499 Binding Assay

10. 1) Electrophysiological Assay B K Mody Govt Pharmacy College, Rajkot 10 The CHO or HEK hERG cell line was used CHO hERG cell line improved seal rates, seal resistances, and current stability on the Qpatch

11. 11 B K Mody Govt Pharmacy College, Rajkot T175 flask with hERG cells was rinsed three times with 10 mL of Dulbecco’s phosphate buffered saline (DPBS) (Ca/Mg free). Electrophysiological Assay 6mL of accutase was added to the cells for 10 min at 37̊C

12. 12 B K Mody Govt Pharmacy College, Rajkot 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.

13. B K Mody Govt Pharmacy College, Rajkot 13 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 U tilizing 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

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16. Electrophysiological Assay B K Mody Govt Pharmacy College, Rajkot 16 Peak tail currents were measured using Sophion’s Assay Software and further data analyses were performed using IgorPro

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19. 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=ax 2 +bx+c) equation on this graph for y=50  value  x point becomes  IC50 value . 19 B K Mody Govt Pharmacy College, Rajkot Data Analysis

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21. RESULTS B K Mody Govt Pharmacy College, Rajkot 21 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

22. 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. RESULTS B K Mody Govt Pharmacy College, Rajkot 22

23. 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 . Electrophysiology B K Mody Govt Pharmacy College, Rajkot 23

24. FluxOR Thallium Assay (384-Well Plates ) B K Mody Govt Pharmacy College, Rajkot 24 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

25. FluxOR Thallium Assay (384-Well Plates) B K Mody Govt Pharmacy College, Rajkot 25 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

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27. FluxOR Thallium Assay (384-Well Plates) B K Mody Govt Pharmacy College, Rajkot 27 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

28. FluxOR Thallium Assay (384-Well Plates) B K Mody Govt Pharmacy College, Rajkot 28 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.

29. 29 B K Mody Govt Pharmacy College, Rajkot FLIPRTETRA

30. FluxOR Thallium Assay (384-Well Plates) B K Mody Govt Pharmacy College, Rajkot 30 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

31. 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 Data Analysis B K Mody Govt Pharmacy College, Rajkot 31

32. 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-   Data Analysis B K Mody Govt Pharmacy College, Rajkot 32

33. 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 RESULTS B K Mody Govt Pharmacy College, Rajkot 33

34. 35S-MK-499 Binding Assay B K Mody Govt Pharmacy College, Rajkot 34 The interaction of 35S-MK-499 with membranes prepared from HEK293 cells expressing hERG T he 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 ] 

35. B K Mody Govt Pharmacy College, Rajkot 35 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 P resoaked with 0.3% bovine serum albumin, using a Filter Mate Universal Harvester The filter plate was allowed to dry completely before sealing the bottom

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38. B K Mody Govt Pharmacy College, Rajkot 38 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

39. 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 . 35S-MK-499 Binding Assay B K Mody Govt Pharmacy College, Rajkot 39

40. 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. RESULTS B K Mody Govt Pharmacy College, Rajkot 40

41. 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 RESULTS B K Mody Govt Pharmacy College, Rajkot 41

42. Concentration–response curves for each of the standards were generated and IC50 values were calculated RESULTS B K Mody Govt Pharmacy College, Rajkot 42

43. 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 43 B K Mody Govt Pharmacy College, Rajkot

44. 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. 44 B K Mody Govt Pharmacy College, Rajkot References